Abstracts From the 35th Annual Scientific Meeting of the HBPRCA and the 39th Annual Scientific Meeting of the AAS
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EXCESS PRENATAL CORTICOSTERONE EXPOSURE PROGRAMS HYPOTENSION, VASCULAR REMODELLING AND ALTERS THE RESPONSE TO RESTRAINT STRESS IN ADULT MALE MICE
O’Sullivan L, Cuffe JSM, Koning A, Singh RR, Moritz KM, Paravicini TM
School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia
Background: Exposure to excess glucocorticoids in utero is known to program susceptibility to cardiovascular disease in later life. Endogenous levels of glucocorticoids are increased during periods of stressThe long-term cardiovascular implications for the offspring of women who suffer from stress during pregnancy are, however, poorly understood.
Aim: To examine the direct effects of prenatal corticosterone (CORT) exposure on cardiovascular and renal outcomes in adult offspring.
Methods: Pregnant C57Bl6 mice received CORT (33 g/kg/h) for 60 h from embryonic day 12.5 and allowed to litter naturally. Nephron number was determined in the offspring at postnatal day 30, and radiotelemetry was used to measure blood pressure and heart rate (both basal and during restraint stress) at 12 months. Pressurized myography was used to assess vascular function, structure and mechanics.
Results: Excess prenatal CORT reduced nephron endowment by 33% and 20% in male and female offspring (respectively) compared to untreated controls (n=6–7; P<0.05). At 12 months, CORT-exposed male offspring were hypotensive: MAP 104.3 11.0 vs 115.6 5.5 mmHg in untreated controls (n=8–9; P<0.05). These mice also showed a blunted tachycardiac response to restraint stress. Power spectral density analysis of heart rate suggested that this may be due to an altered vagal/sympathetic balance. Prenatal CORT reduced activity levels of female offspring at night, but did not alter any cardiovascular parameters. In male offspring (but not females), prenatal CORT caused inward remodeling of mesenteric arteries (luminal diameter = 202 7 m vs 249 9 m in controls) without affecting vascular reactivity or the elasticity of vascular wall components.
Conclusion: Prenatal CORT exposure programs hypotension in male offspring, despite the reduced nephron number and inward vascular remodeling in these animals. In contrast, CORT-exposed female offspring have reduced nocturnal activity levels (possibly indicating anxiety-like behaviour) and fewer nephrons, with no other cardiovascular alterations. Together, these data demonstrate that excess prenatal CORT causes long-term alterations to the renal and cardiovascular systems, as well as central control mechanisms.
THE INTRARENAL RENIN-ANGIOTENSIN SYSTEM (RAS) IN HUMAN PREGNANCY
Lumbers ERa,b, Pringle KGa,b, Sykes SDa,b, Weatherall Lb,c, Clausen DCd, Rae Ka,b, Smith Rb
aSchool of Biomedical Sciences and Pharmacy, University of Newcastle, Newcastle, New Soth Wales; bMothers and Babies Research Centre, Hunter Medical Research Institute, University of Newcastle, Newcastle, New South Wales; cGomeroi gaaynggal Centre, Tamworth, New South Wales; dPathology New England, Tamworth, New South Wales, Australia.
Background: Components of the renin-angiotensin system (RAS) are found in the renal tubules. Angiotensin II plays an important role in tubular sodium reabsorption. In human pregnancy increased salt reabsorption is required to maintain maternal blood volume.
Aims: To determine whether the activity of the intrarenal renin-angiotensin system (IRAS), measured by the urinary angiotensinogen/creatinine ratio, is increased in pregnancy and to determine whether alterations occur in levels of other IRAS components.
Methods: Levels of urinary angiotensinogen (uAGT), active renin, prorenin and angiotensin converting enzyme (uACE) were measured in 10 non-pregnant and 16 pregnant non-Indigenous women in late gestation (9 healthy, 4 with gestational hypertension and 3 with proteinuria) and 51 Indigenous pregnant women throughout gestation using ELISAs. Active renin was measured enzymatically.
Results: uACE and uAGT were correlated in pregnant Indigenous women (rho=0.526; P<0.001). uAGT/creatinine ratios were higher in both healthy and hypertensive non-Indigenous pregnant women than in non-pregnant non-Indigenous women (18.2 ± 3.2 μg/mmol compared with 1.1 ± 0.3 μg/mmol; P=0.001). uAGT levels in pregnant non-Indigenous women with proteinuria were suppressed and similar to levels measured in non-pregnant non-Indigenous women. Indigenous pregnant women also had suppressed uAGT/creatinine, these women were subdivided into 2 groups based on their uAGT/creatinine being normal (>2 μg/mmol) or low (<2 μg/mmol). Most Indigenous pregnant women who had microalbuminuria had a low uAGT/creatinine. A low uAGT/creatinine occurred in 37/51 pregnant Indigenous women, including those with microalbuminuria, possibly indicative of renal dysfunction.
Conclusions: Failure to activate the IRAS in pregnancy may reduce maternal blood volume predisposing to intrauterine growth retardation and/or preeclampsia.
MATERNAL STRESS DURING PREGNANCY PROGRAMS NEPHRON DEFICITS AND GENDER SPECIFIC HYPERTENSION IN SECOND GENERATION OFFSPRING
Vickneswaran Ca, Jefferies AJa, Anevska Ka, Cheong JNa, Hanvey A, Singh RRb, Moritz KMc, Wlodek MEa
aDepartment of Physiology, The University of Melbourne, Parkville, Victoria, Australia; bDepartment of Physiology, Monash University, Clayton, Victoria, Australia; cSchool of Biological Sciences, University of Queensland, St. Lucia, Queensland, Australia
Background: Being born small increases the risk of cardiovascular disease, especially in males. Evidence suggests that disease risks are not limited to the first, directly exposed generation (F1) but can be transmitted to the next generation (F2). Stress during pregnancy adversely impacts fetal development.
Aims: To characterize nephron endowment and blood pressure in F2 offspring born to normally grown and growth restricted (F1) mothers and to further assess the impact of maternal stress during late pregnancy.
Methods: Late gestation rat uteroplacental insufficiency was induced by bilateral uterine vessel ligation (restricted) or sham (control) surgery in F0 females. F1 females were mated and allocated to unstressed or late pregnancy stressed (24 h metabolic cage, tail cuff blood pressure, glucose tolerance test) groups. F2 body weights (birth to 12 months), nephron number (day 35) and blood pressure (6, 9 and 12 months) were measured.
Results: F2 offspring born to mothers exposed to maternal stress had reduced weight at birth (–5%, P<0.05), but no differences thereafter. There were no differences in heart, left ventricle or kidney weights. Nephron number was reduced in offspring exposed to maternal stress with males more affected (–20%; P<0.05) than females (–14%; P<0.05), with no effect on renal function. Restricted unstressed males had high blood pressure at 6 and 12 months compared to control unstressed (+7 mmHg; P<0.05). Control male offspring exposed to maternal stress during pregnancy had elevated blood pressure at 6, 9 and 12 months compared to control unstressed (+10–13 mmHg; P<0.05). This maternal stress pregnancy response in control males resulted in an elevated blood pressure that was not different to the high blood pressure of male offspring born to unstressed mothers who were born small.
Conclusion: Maternal stress in pregnancy reduced nephron endowment regardless of maternal birth weight. Mothers, born of normal birth weight, but exposed to stress during pregnancy, produced males that developed adult hypertension to a similar extent to that of males whose mothers were born small but not exposed to stress. Maternal stress in pregnancy has profound cardio-renal effects on the next generation offspring regardless of whether the mother was born small or born of normal birth weight.
ALTERATIONS IN RENAL FUNCTION OCCUR PRIOR TO THE INCREASE IN ARTERIAL PRESSURE IN RAT OFFSPRING FOLLOWING SHORT-TERM EXPOSURE TO MATERNAL CORTICOSTERONE
Fong Da, Brown RDa, Singh RRa, Flower RLa, Evans RGa, Moritz KMb, Denton KMa
aDepartment of Physiology, Monash University, Melbourne, Victoria; bSchool of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia
Background: An acute exposure of the fetus to elevated levels of maternal corticosterone (CORT; natural glucocorticoid in rodents) results in reduced nephron number and hypertension in rat offspring in adulthood. At birth, each nephron must rapidly adapt to take over the role of maintaining extracellular fluid (ECF) homeostasis from the placenta. In a kidney with fewer nephrons, hypertrophy of the glomerulus and tubule occurs to enable the remnant nephrons to handle a larger share of ECF. We hypothesize that these structural adaptations are accompanied by adaptations in glomerular and tubular function, which in the short-term will normalize renal function, but in the long-term will increase the risk of renal and cardiovascular disease.
Aim: To examine the changes in arterial pressure, renal function and tubuloglomerular feedback (TGF) during the postnatal period in rat offspring exposed to elevated levels of maternal CORT in utero.
Methods: In male offspring (n=8; Sprague-Dawley rats) of mothers treated with CORT 0.8 mg/kg/day i.p. bid) or vehicle (VEH; 0.2 ml/kg/day) on days 14 and 15 of gestation (term 21 days), mean arterial pressure (MAP) was recorded via radio-telemetry from 3–6 weeks of age. TGF was assessed via renal micropuncture at 3 and 10 weeks of age under basal conditions and during intra-tubular nitric oxide (NO) blockade with L-NAME.
Results: MAP was not significantly different between the offspring of VEH and CORT groups at a postnatal age of 25 days. MAP increased with age (PAge<0.002) in both groups. However, the increase in MAP was enhanced in the offspring of the CORT-treated group (PGroupxAge<0.001). There was a leftward shift of the TGF response curve in offspring of the CORT group at 3 weeks of age, which was associated with a reduced contribution of NO.
Conclusion: In offspring with a congenital nephron deficit induced by maternal glucocorticoid administration, TGF is sensitized at a postnatal age of 3 weeks to allow increased reabsorption of sodium and fluid and so reducing single nephron glomerular filtration rate. These changes occur prior to the increase in arterial pressure suggesting that the alterations in TGF early in life maybe a potential mechanism that could drive the development of hypertension later in life.
DOES OBESITY ALTER THE NORMAL CARDIOVASCULAR AND RENAL ADAPTATIONS OF PREGNANCY?
Cai X, Kett MM
Department of Physiology, Monash University, Australia
Background: Obesity is associated with complications during pregnancy, including hypertensive disorders of pregnancy, and increased risk of cardiovascular and renal disease later in life. Surprisingly, little is known about the impact of obesity on the normal cardiovascular and renal adaptations that occur during pregnancy.
Aim: To measure mean arterial pressure (MAP), heart rate (HR) and renal function across pregnancy in a mouse model of diet-induced obesity.
Methods: Four-week old female C57BL6J mice were placed on control (7% fat, 3.85 kCal/g) or high fat (HF; 23.5% fat, 4.54 kCal/g) chow for 10 weeks. Baseline glucose tolerance (n=10, 16), MAP and HR (radio-telemetry; n=12, 16), and 24 h urinary albumin excretion (n=6, 6) were measured. MAP and HR were measured throughout gestation and albumin excretion was reassessed at gestational age (GA) 13.
Results: After 10 wks of the diet, body weight of HF mice was 44% greater than control mice (32.7 ± 0.9 vs 22.7 ± 0.4g; P<0.001). Obese mice were hyperglycaemic (9.1 ± 0.4 vs 7.6 ± 0.5 mmol/L; P<0.05), glucose intolerant (716 ± 43 vs 490 ± 41 AUC; P<0.001), had elevated 24 h MAP (105.7 ± 1.3 vs 98.1 ± 1.4 mmHg; P<0.001) and HR (548 ± 8 vs 492 ± 9 bpm; P<0.001) compared to control mice. MAP and HR of obese mice remained elevated compared to control mice throughout pregnancy (Pdiet<0.001). The adaptations of MAP and HR in obese mice during first half of pregnancy, including the mid-gestation fall in MAP at GA9, were consistent in timing and magnitude with control mice. However, the rise in MAP in the later phase of pregnancy, near term, was blunted compared to control mice (Pdietxtime<0.05 night-time MAP). The marked rise in HR observed in control mice during the second half of pregnancy was also significantly blunted in obese mice such that HR was no longer different between the two groups from GA11 (Pdietxtime<0.05). Both groups had increased albuminuria at GA13 (Ppreg<0.001). This increase was, however, ~3-fold greater in obese mice (P<0.01).
Conclusion: Obesity results in higher MAP and HR that persists throughout pregnancy. Obese mice demonstrated, however, a blunted increase in HR and to a lesser extent MAP in the later stages of pregnancy. As the high cardiac output (CO) in the second half of pregnancy is dependent on an increase in HR, our data suggests that obesity may limit the increase in CO late in pregnancy, increasing the risk of maternal and fetal complications. Further, pregnancy exacerbates albuminuria in obese females, suggesting that obesity may increase the risk of renal complications during pregnancy and lead to renal disease in later life.
MATERNAL STRESS DURING PREGNANCY PROGRAMS SECOND GENERATION MALE CARDIO-RENAL DYSFUNCTION
Pre AIa, Jefferies AJa, Soeding Pb, Gerace ELa, Moritz KMc, Wlodek MEa
aDepartment of Physiology, The University of Melbourne, Parkville, Victoria, Australia; bDepartment of Pharmacology, University of Melbourne, Parkville, Victoria, Australia; cSchool of Biological Sciences, University of Queensland, St. Lucia, Queensland, Australia
Background: Being born small increases the risk of cardiovascular disease, especially in males. Evidence suggests that disease risks are not limited to the first, directly exposed generation (F1), but can be transmitted to the next generation (F2). Stress during pregnancy adversely impacts fetal development.
Aims: To characterize cardio-renal function in F2 males at 16 months of age born to normally grown and growth restricted (F1) mothers, and assessed the impact of maternal stress during late pregnancy.
Methods: Late gestation rat uteroplacental insufficiency was induced by bilateral uterine vessel ligation (restricted) or sham (control) surgery in F0 females. F1 females were mated and allocated to unstressed or late pregnancy stressed (24 h metabolic cage, tail cuff blood pressure, glucose tolerance test) groups. F2 male body weights, heart function (echocardiography) and basal and restraint stress blood pressure (telemetry) were measured.
Results: F2 offspring born to mothers exposed to maternal stress had reduced weight at birth (–5%, p<0.05) but no differences thereafter. Maternal stress, regardless of birth weight, programmed decreased left ventricular fractional area change (systolic dysfunction; –4%, P<0.05), increased relative left ventricle wall thickness (+7%, p<0.05) and enhanced pulse pressure response to restraint stress (+2–3 fold, P<0.05). 24 h albumin (not Na+ or K+) excretion was increased (no change in creatinine clearance) in control stressed males (+60%, P<0.05), which was associated with left ventricular hypertrophy (post mortem; +6%, P<0.05). Restricted unstressed males had high blood pressure at 9 months compared to control unstressed (P<0.05). Control males exposed to maternal stress during pregnancy had elevated blood pressure at 9 months compared to control unstressed males (P<0.05), which was the same as the high blood pressure of males born to unstressed mothers who were born small. Blood pressure was unaffected at 16 months.
Conclusion: Maternal stress in pregnancy caused left ventricular concentric remodeling regardless of maternal birth weight. Mothers, born of normal birth weight but exposed to stress during pregnancy, produced males who developed concentric hypertrophy. Maternal stress during pregnancy programmed cardio-renal dysfunction in F2 males; the response, at 16 months in the absence of a high fat/salt diet, was exacerbated if the mother was born of normal weight and was relatively protected if she was born small.
ROLE OF TISSUE-INFILTRATING T CELLS DURING ANGIOTENSIN II-INDUCED HYPERTENSION
Wei Z, Diep H, Welungoda I, Widdop RE, Vinh A
Department of Pharmacology, Monash University, Clayton, Victoria, Australia
Background: T cells and the adaptive immune system have been implicated recently in the development of experimental hypertension. Like several peripheral autoimmune/inflammatory disorders, hypertension is associated with increased T cell infiltration into blood pressure-controlling organs such as the aorta and kidney. The precise function and mediators that are released from these infiltrates remains undefined. Identification of cytokine/chemokines or reactive oxygen species (ROS), whose release is increased by these infiltrating T cells during hypertension, would indicate the existence of hypertension-specific T cell phenotype(s) that may be targeted for treatment of inflammation associated with hypertension.
Aims: To define T cell-derived cytokine/chemokine and ROS production from aortic, renal and central infiltrating T cells during angiotensin II-induced hypertension.
Methods: C57BL/6J mice received 14-day vehicle or angiotensin (Ang) II (490 ng/kg/min, s.c.) infusion via osmotic minipump. Following infusions, blood spleen, aorta, kidney and brains were harvested from all mice and T cells were isolated from each tissue using a magnetic microbead isolation kit (Miltenyi Biotec). Purity of enriched T cells was confirmed to be >90% using flow cytometry. T cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin (1ug/ml) or anti-CD3/anti-CD28 for T cell-derived cytokine analysis. ROS formation was measured by L-012 enhanced chemiluminescence following phorbol 12,13-dibutyrate stimulation.
Results: T cell infiltration was increased in aorta, brain, and kidneys from hypertensive mice. Cytokine production from T cells of all organs indicated an overall Th1 pro-inflammatory phenotype, although there was no difference in the proportion (flow cytometry) and quantity (cytometric bead array) of IFN-γ and TNF-α released from T cells from vehicle- and Ang II-treated groups. Strikingly, we observed increased T cell-derived production of a chemokine, CCL-2, in aorta and kidneys of hypertensive mice compared to vehicle-treated mice (~4 fold) (P<0.05, n=5–16), which was absent in the spleen or blood. Moreover, aortic T cell-derived ROS production was significantly elevated in Ang II-infused mice compared to vehicle-treated mice (~3-fold; P<0.05; n = 7–14).
Conclusions: Ang II-induced hypertension does not affect the systemic or infiltrating T cell cytokine profile. We identified, however, a unique hypertension-specific phenotype of aortic infiltrating T cells that may specifically promote local oxidative stress and leukocyte recruitment during the development of hypertension.
NEW GERM-LINE MUTATIONS AND A RARE CODING SNP WITHIN KCNJ5 IN “APPARENTLY SPORADIC” PRIMARY ALDOSTERONISM: A POSSIBLE ROLE IN PATHOGENESIS
Xu Sa, Murthy Mb, Massimo Gb, Wolley Ma, Gordon RDa, Stowasser Ma, O’Shaughnessy Kb
aEndocrine Hypertension Research Centre, University of Queensland School of Medicine, Brisbane, Queensland, Australia; bClinical Pharmacology Unit, Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK
Background: Primary aldosteronism (PA) is the most common specifically-treatable cause of hypertension. The KCNJ5 potassium channel is of great interest because both germ-line mutations (familial hyperaldosteronism type 3) and somatic mutations within aldosterone-producing adenomas have been described recently. PA usually appears to be sporadic rather than familial.
Aim: To seek germ-line variants which might contribute to the pathophysiology of the common sporadic PA.
Methods: The coding sequence and flanking regions of the gene, KCNJ5, were resequenced in 251 Caucasian subjects with “apparently sporadic” PA. Functional change in mutated channels was studied in vitro by expressing them in Xenopus oocytes and human adrenocortical carcinoma cells. Resting membrane potentials and current-voltage (I-V) plots for clamped Xenopus oocytes expressing either wild-type (WT) or mutant KCNJ5 channels were recorded. Basal and angiotensin II (Ang II)-induced aldosterone release from H295R cells transiently transfected with either the control (empty GFP vector) or one of the mutant KCNJ5 channels was observed. Clinical features of patients affected and unaffected by mutations were compared.
Results: Three “new” heterozygous missense mutations (R52H, E246K and G247R) were identified, and 12 (5% of the cohort) were carriers for the rare non-synonymous single nucleotide polymorphism (SNP) rs7102584 causing a E282Q substitution in KCNJ5. On expression of the channels in Xenopus oocytes and human adrenal H295R cells, the R52H, E246K and E282Q substitutions were functional, but the G247R mutation was indistinguishable from WT. Although the functional substitutions are remote from the selectivity filter, they affect (1) inward rectification, (2) the selectivity of the KCNJ5 channels and (3) Ang II-induced aldosterone release from the H295R cell line. Clinically, the rs7102584 SNP was associated with less florid PA.
Conclusion: These data suggest that germ-line variations in the KCNJ5 gene play a role in the common sporadic as well as in the rare syndromic forms of PA. Further studies are needed to confirm these findings and explore roles of other germ-line variants in KCNJ5 in various forms of PA.
REGULATION OF ALDOSTERONE SYNTHASE (CYP11B2) BY miR-125a-5p AND miR-125b
Robertson Sa–c, MacKenzie SMc, Bursill Ca,b, Davies Ec
aHeart Research Institute, Sydney, New South Wales, Australia; bFaculty of Medicine, University of Sydney, New South Wales, Australia; cBritish Heart Foundation Glasgow Cardiovascular Research Centre, University of Glasgow, UK
Background: Essential hypertension is known to have a large genetic component. Single nucleotide polymorphisms (SNPs) in the gene CYP11B2, which encodes the aldosterone synthase enzyme, are associated with excess aldosterone production and hypertension. Currently the causative mechanism remains elusive. We propose a regulatory role for microRNA (miRNA). miRNAs are small, endogenous RNA molecules that negatively regulate mRNA abundance by binding the 3’-untranslated region (3’UTR) of their targets. Augmented miRNA expression and function have been implicated in various pathologies.
Aim: To investigate wether miRNAs expressed in the human adrenal gland regulate CYP11B2 mRNA, and to determine the impact of CYP11B2 SNPs on miRNA function.
Methods: miRNAs predicted to target CYP11B2 were identified using four bioinformatics algorithms, then cross-referenced with miRNA microarray expression data from normal human adrenal glands and aldosterone-producing adenomas (APA). Four miRNAs were tested in vitro for regulation of CYP11B2.
Results: Microarray and bioinformatic data identified four adrenal miRNAs (miR-125a-5p, miR-125b, miR-134 and miR-495a) having putative binding sites in CYP11B2. All four miRNAs were tested using a CYP11B2 3’UTR reporter plasmid that was co-transfected alongside a synthetic miRNA (pre-miR) or miRNA inhibitor (anti-miR). Both miR-125a-5p and miR-125b pre-miRs significantly decreased luciferase activity when over-expressed. Conversely, their anti-miRs significantly increased luciferase activity. Furthermore, CYP11B2 mRNA levels were significantly altered in H295R adrenal cortical cells following modulation of miR-125a-5p and miR-125b levels, consistent with canonical miRNA binding and repression. Modifyication of miR-495a and miR-134 levels did not alter luciferase activity significantly. Comparison of miRNA expression levels showed that miR-125a-5p and miR-134 are significantly down regulated in APAs. Mapping of CYP11B2 intronic and 3’UTR SNPs indicated that genetic variation is unlikely to have an impact on miRNA expression or function
Conclusion: We have identified two miRNAs, miR-125a-5p and miR-134, that exhibit aberrant expression in APA samples and that have putative binding sites in the CYP11B2 gene. We also confirmed that miR-125a-5p and miR-125b, but not miR-134, can repress CYP11B2 by directly targeting the 3’UTR. The regulation of CYP11B2 mRNA by miRNAs and altered expression in adrenal adenomas may asssit in development of novel therapeutic targets for the treatment of essential hypertension.
SINGLE NUCLEOTIDE POLYMORPHISM, EPISTATIC AND SEX-DEPENDENT ASSOCIATION ANALYSES OF GENES OF THE RENIN-ANGIOTENSIN SYSTEM AND BLOOD PRESSURE IN FAMILIES
Scurrah KJa, Lamantia Aa, Ellis JAb, Harrap SBa
aDepartment of Physiology, University of Melbourne; bMurdoch Childrens Research Institute, Melbourne, Victoria, Australia
Background: Genes encoding key elements of the renin-angiotensin system (RAS) cascade have been previously, but inconsistently, associated with blood pressure. Comprehensive gene-wide association analyses of RAS genes have not typically been performed, nor have epistatic or sex interactions been investigated thoroughly.
Aim: To assess variation in the genes encoding renin (REN), angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II type 1 receptor (AGTR1) and aldosterone synthase (CYP11B2) and determine whether individual single nucleotide polymorphisms (SNPs), interactions between SNPs or sex-SNP interactions are associated with systolic blood pressure (SBP).
Methods: 90 SNPs from the 5 RAS genes were genotyped using leucocyte DNA from 2,969 individuals (comprising over 800 two-generation adult families) from the Victorian Family Heart Study. Variance components models were used that adjusted for measured covariates and within-family correlation due to shared (but unmeasured) genes and environmental effects.
Results: Initial analyses found that two SNPs (rs8075924 in ACE and rs12721297 in AGTR1) were inversely associated with SBP (β=–1.67 mmHg and β=–2.44 mmHg, respectively; both P<0.005), both individually and when adjusted for each other. No interactions with sex or with other SNPs were observed for either SNP. Both SNPs were also associated with diastolic blood pressure (P<0.02 and P<0.001, respectively), but not with body mass index. There was also weak evidence of interaction between 3 pairs of SNPs that were less strongly associated with SBP individually (P=0.01–0.05). Initial results also indicated that three SNPs show strong evidence of differential association with SBP for males and females (sex-SNP interaction, P=0.005–0.015).
Conclusion: Our analyses confirm that several SNPs in and around key RAS genes are associated with SBP. They suggest that epistatic interactions between these RAS genes are at most weakly associated with blood pressure, but that sex interactions may be important and should be investigated further.
EFFECT OF AGING ON ASCENDING AORTIC FLOW MEASURED BY NON-INVASIVE MRI
Adji Aa,b, Kachenoura Nc, Bollache Ec,d, Avolio APa, O’Rourke MFb,d, Mousseaux Ec,e
aAustralian School of Advanced Medicine, Macquarie University, Australia; bSt Vincent’s Clinic, Sydney, Australia; cLaboratoire d’Imagerie Fonctionnelle, Paris, France; dInstitut Jean le Rond d’Alembert, Paris, France; eUniversity of New South Wales, Victor Change Cardiac Research Institute, Sydney, New South Wales, Australia; fCardiovascular Imaging Department, Hôpital Européen Georges Pompidou, AP-HP, Paris, France
Background: Limited invasive (electromagnetic) ascending aortic (AA) flow wave contour indicates impaired left ventricular (LV) function in the elderly. However, conventional non-invasive (Doppler) recordings have not shown similar aging change.
Aim: To illustrate changes in AA flow and carotid pressure waves in an apparently normal cohort, using cardiac magnetic resonance imaging (CMRI).
Methods: Fifty subjects (aged 21–70 years; 28 males) underwent velocity-encoded CMRI of the thoracic aorta using a 1.5T system (Signa, GEMS, Waukesha, USA). AA flow was measured non-invasively by subtracting simultaneous forward and backward flow velocity across the AA cross-section. DR⅓ was determined as ratio of difference in flow velocity between systolic peak and one-third of deceleration phase to peak flow velocity. Comparisons were made between young (≤50 years; n=30) and older subjects (>50 years; n=20).
Results: Peak AA flow velocity decreased substantially (P<0.0001) from young (66.7 ± 17.7 cm/s) to older subjects (47.7 ± 12.7 cm/s). This could be associated with the age-related increase in aortic diameter. The substantial differences between CMRI flow waveforms and Doppler are due to the use of the outer flow envelope (highest particle flow) to estimate flow across the aortic cross-section with Doppler. However, late systolic flow was relatively lower in the older than in the younger subjects, with flow concavity in older persons (corresponding to greater augmentation of the pressure waveform), represented as DR⅓, which declined from 78% (≤50 years) to 69% (>50 years) (P<0.001). These features are explicable on the basis of LV impairment from early and increased wave reflection during systole, and suggest reduction of wave reflection as a strategy for treating heart failure in the elderly.
Conclusion: AA flow waves recorded by CMRI showed aging changes, which are not apparent in conventional Doppler flow patterns. Our finding warrants further use of AA CMRI flow and pressure waveforms to characterize aging changes and ill-effects of stiffened central arteries. Findings support predictions of Westerhof and O’Rourke (J Hypertens 1995;13:943–952) and the force/velocity interpretations of Miyashita et al. (Heart Vessels 1994;9:30–39) and Chiu et al. (Circ Res 1992;70:530–535).
NON-INVASIVE DETERMINATION OF ASCENDING AORTIC IMPEDANCE
Kachenoura Na, Bollache Ea,b, Adji Ac,d, Avolio APc, O’Rourke MFd,e, Mousseaux Ea,f
aLaboratoire d’Imagerie Fonctionnelle, Paris, France; bInstitut Jean le Rond d’Alembert, Paris, France; cAustralian School of Advanced Medicine, Macquarie University, Sydney, New South Wales, Australia; dSt Vincent’s Clinic, Sydney, New South Wales, Australia; eUniversity of New South Wales and Victor Chang Cardiac Research Uni, Sydney, New South Wales, Australia; fCardiovascular Imaging Department, Hôpital Européen Georges Pompidou, AP-HP, Paris, France
Background: Non-invasive studies of ascending aortic (AA) impedance, using Doppler flow and carotid tonometry have not confirmed the aging changes demonstrated with invasive electromagnetic flow/micromanometer catheters in humans.
Aim: To determine AA impedance from cardiac magnetic resonance imaging (CMRI) flow recorded non-invasively and carotid pressure tonometry, and compare results with previously reported invasive data and realistic arterial model (O’Rourke and Avolio, Circ Res 1980;46;363–372).
Methods: Fifty apparently normal subjects (aged 21–70 years; 28 males) underwent velocity-encoded CMRI of the thoracic aorta using a 1.5T system (Signa, GEMS, Waukesha, USA). AA flow was measured non-invasively by subtracting simultaneous forward and backward flow velocity across the AA cross-section. Impedance was determined by relating in modulus and phase, corresponding frequency components of the AA flow waveforms with tonometric carotid pressure waveforms (used as surrogate of AA pressure) and recorded sequentially after CMRI. Data were presented in 5 age groups: 21–30, 31–40, 41–50, 51–60 and 61–70 years.
Results: Values of impedance modulus and phase were similar to those previously reported for invasive studies, and with impedance modulus being higher in older than younger subjects up to 3 Hz (reflecting higher peripheral resistance in older subjects, and impedance phase crossed from negative to positive values between 3 and 4.5 Hz. Characteristic impedance (taken as average modulus 5–6 Hz) was 710 dyne.s.cm-3. Values of AA impedance fall within the range calculated for a realistic model of the arterial system between age 20 and 80 years.
Conclusion: AA impedance values calculated non-invasively from AA CMRI flow waveforms and carotid pressure waveforms show the same pattern as seen with invasive studies in older and younger subjects and lie within the same range as predicted form models at age 20 to 80 years. Better correspondence is expected when AA pressure waveforms can be measured in the magnet room simultaneously with AA flow acquisition.
TRACKING OF AORTIC SYSTOLIC PRESSURE OVER THE ENTIRE LIFE COURSE
Adji Aa,b, O’Rourke MFb,c, Avolio APa
aAustralian School of Advanced Medicine, Macquarie University, Sydney, New South Wales, Australia; bSt Vincent’s Clinic, Sydney, New South Wales, Australia; cUniversity of New South Wales and Victor Change Cardiac Research Institute, Sydney, New South Wales, Australia
Background: Recent studies clarify the tracking of brachial cuff systolic blood pressure (BP) over the extremes of age: from birth to adolescence (NHANES and OLAF) and early adulthood to old age (FHS). Principal features are rapid increase in infancy, less rapid increase in childhood, relative plateau in late adolescence and slow increase from age 40. Gender differences become apparent at age 14, whereafter systolic BP is higher in males than females. No such studies are available for central aortic pressure.
Aim: To describe change in central aortic systolic pressure over the whole lifespan from published papers and to compare with brachial cuff pressure tracking.
Methods: From the three brachial BP studies (NHANES. Pediatrics. 2004;114(2S):555–576; OLAF. J Hypertens. 2012;30:1942–1954; Framingham. Hypertension. 2012;60:1393–1399), we estimated central aortic systolic pressure using their respective cuff brachial values. For adults (age >18 years), we applied a validated generalized transfer function in a normal adult population. For children up to age 18 years, we used the “second systolic shoulder” method from a normal pediatric population to calculate central aortic pressure. Near-identity of the two methods was established in adults and children. Neonatal central and peripheral pressures were based on invasive measurements.
Results: While there was no difference between central and estimated brachial pressure in neonates, the difference became apparent by age of 4 years, and extreme at 18 years when aortic systolic pressure was 15–20 mmHg less than brachial. There was no gender difference between central pressure up to age 14, whereafter central and brachial pressure and body height in males continued to increase up to age 18.
Conclusion: Findings suggest that systolic aortic BP increases progressively throughout life, with the major changes in brachial values attributable to bodily growth, and variable distortion in the pulse wave during transmission from aorta to periphery of the body. Gender differences become apparent in early adult life, and lessen in later adult life. At the extremes of age, there are no gender-differences in systolic BP between aorta and brachial artery. Approximate asymptotic values are 50 mmHg at birth and 150 mmHg at age 100.
SALINE SUPPRESSION TESTING PERFORMED IN SEATED PATIENTS IS SUPERIOR TO RECUMBENT AND COMPARABLE TO FLUDROCORTISONE SUPPRESSION TESTING IN DIAGNOSING PRIMARY ALDOSTERONISM
Ahmed AH, Cowley D, Wolley M, Gordon RD, Taylor PJ, Stowasser M
Endocrine Hypertension Research Centre, University of Queensland School of Medicine, Greenslopes and Princess Alexandra Hospitals, Brisbane, Queensland, Australia.
Background: Demonstrating aldosterone (aldo) production, which is relatively autonomous of its normal chronic regulator, angiotensin II, confirms primary aldosteronism (PA). This is done by demonstrating lack of aldosterone (also) suppression when plasma renin has been suppressed by sodium loading manoeuvers such as fludrocortisone suppression testing (FST) and saline suppression testing (SST). We have found previously that recumbent SST lacks sensitivity. Because aldo levels are higher in the upright (e.g., seated) position compared to recumbency in most patients with PA, and upright aldo levels are used for diagnosis during FST, we hypothesized that SST would be more sensitive when performed on upright than on recumbent patients.
Methods: Of 42 patients who underwent FST (plasma aldo measured at 10 am after 2–3 h upright posture basally and after 4 days administration of fludrocortisone 0.1 mg 6 hourly and oral salt loading) between April 2012 and Jun 2013, 22 agreed to participate in the study and underwent SST (aldo measured basally at 8 am and at completion of an infusion of 2 liters of normal saline over 4 h) both (1) recumbent (infusion commenced at least 30 min after assuming recumbency) and (2) seated, with SSTs spaced at least 2 weeks apart, and in randomized order.
Results: FST confirmed PA (day 4 upright aldo >165 pmol/l) in 16 patients, excluded PA in 3 and was inconclusive in 3 (one of whom subsequently lateralized on adrenal venous sampling, confirming PA). Of the 17 with confirmed PA (5 unilateral, 8 bilateral and 4 undetermined subtype), all tested positive (plasma aldo >165 pmol/l at 4 h) by seated SST. Recumbent SST was positive (plasma aldo >140 pmol/l at 4 h) in only 5 of the 17, missing 1 with unilateral, all 8 bilateral and 3 with undetermined subtype of PA. All 3 patients without PA (FST negative) tested negative by seated and recumbent SST.
Conclusion: Seated SST appears to be superior to recumbent SST in detecting PA and may be a reliable alternative to FST.
HYPERTENSION AUGMENTS PLAQUE INSTABILITY IN A NOVEL HYPERTENSIVE ATHEROSCLEROTIC MOUSE MODEL
Andrews KLa, Michell DLa,b, Lumsden NGa, Sampson AKa, Jackson KL, Head GAa, Chin-Dusting JPFa,b
aBaker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia; bDepartment of Medicine, Monash University, Clayton, Victoria, Australia
Background: Hypertension is a significant risk factor for heart disease and the single biggest contributing factor for ischemic stroke. We have shown previously that an acute increase in pressure induces leukocyte adhesion and inflammation, but whether chronic high blood pressure affects plaque pathology is unknown.
Aim: In the current study, we explored the chronic effect of high blood pressure on plaque progression and examined whether blocking inflammation with the P-selectin blocking antibody RB40.34 can reduce plaque development in a novel spontaneously hypertensive, diet-induced atherosclerotic mouse model.
Methods: Spontaneously hypertensive BPH/2J mice were crossed with Apoe–/– mice to develop a fixed line of hypertensive mice with a deficiency of the Apoe gene (BPHxApoe–/–). 8-week-old BPHxApoe–/– or Apoe–/– mice were fed a high fat rodent diet (HFD) 21% and 0.15% cholesterol for 12 weeks.
Results: 24-hour averages of arterial pressure (AP), systolic AP (SAP), diastolic AP (DAP) and mean AP (MAP), determined with telemetry, were higher in BPHxApoe–/– compared to Apoe–/–- mice (13–15 mmHg; n=5; P<0.05). Total plaque area was similar in BPHxApoe–/–- and Apoe–/– mice (n=4–6; P>0.05). However, aortic sinucs lesions from BPHxApoe–/– mice had greater lipid deposition (Oil Red O; 18.1 ± 2.4% vs 29.3 ± 3.4%; n=4–5; P<0.05) and macrophage content (CD68; 13.9 ± 2.0% vs 31.4 ± 4.6%; n=3–5; P<0.05) compared to Apoe–/– mice. Collagen content trended towards a reduction in BPHxApoe–/– compared to Apoe–/– mice (11.7 ± 2.4% vs 6.1 ± 1.2%; n=3–4; P=0.09). Collectively, these results suggest BPHxApoe–/– mice have reduced plaque stability. Treatment with two injections of RB40.34 (100 µg, i.p.) at week 1 and 3 did not affect total or regional plaque area, lipid deposition or macrophage content (n=5–11; P>0.05). However, whilst there was no change in collagen content in Apoe–/– mice (n=3–6; P>0.05), BPHxApoe–/– mice demonstrated increased collagen with RB40.34 treatment (control: 5.1 ± 1.0% vs RB40.34: 20.5 ± 4.2%; n=4–5; P<0.01). Plaque stability, as measured by the collagen/lipid ratio, was also improved with RB40.34 treatment in the BPHxApoe–/– mice (control: 0.20 ± 0.06 vs RB40.34: 0.93 ± 0.20; n=4–5; P<0.01) but not in Apoe–/– mice.
Conclusion: Chronic high blood pressure reduces plaque stability, which in the presence of hypertension, is partially improved by anti-inflammatory therapy. Our data suggest that targeting leukocyte recruitment may be beneficial for the long-term management of heart disease in hypertensive patients.
ADENOSINE AND LIDOCAINE COMBINATION IMPROVES AORTIC RING PRESERVATION AFTER 6-DAY COLD STORAGE
Arsyad MA, Dobson GP
School of Veterinary & Biomedical Sciences, James Cook University, Townsville, Queensland, Australia
Background: Vessel allograft is often stored in cold medium for few hours to several days for revascularization or replacement of infected vessels. Maintenance of vascular quality and function, however, following extended storage for 24–48 hours remains unsatisfactory. Adenosine-lidocaine (AL) with melatonin (M) and insulin (I) in low Ca2+/high Mg2+ Krebs-Henseleit (modified KH) solution has been shown to improve heart function after prolonged preservation.
Aim: To examine the effect of AL with or without MI combination in modified KH on rat aorta physiological functions after 6 days of cold storage.
Methods: Thoracic rat aortic rings (3 mm) stored in cold (4oC) KH, modified KH, modified KH+AL, and modified KH+ALMI solutions for 6 days. At the end of cold storage, all segments were transferred to KH-filled organ bath and rewarmed to 37°C. The contractility and relaxation functions of preserved rings were measured and compared with freshly harvested rings (controls) using isometric force transducer. Values were expressed as the percentage of tension/relaxation of controls.
Results: After 6 days of cold storage, aortic rings preserved in standard KH lose ~55% and ~65% of their contractile response to NE and KCl, respectively; while those in modified KH only experienced ~10% and 25% loss. AL or ALMI addition in modified KH, fully restored the contractile response of the rings to NE (100%), but were unaltered with KCl (~80%). Relaxations to acetylcholine (Ach) were 42%, 80%, 93% and 70% for KH, modified KH, modified KH+AL and modified KH+ALMI, respectively. All rings except those in KH could achieve 100% relaxation with nitric oxide.
Conclusion: After 6-day cold storage, low Ca2+/Mg2+ ratio of KH (modified KH) somewhat improved vascular reactivity. Adding AL and ALMI seemed to further increase vascular contractility, especially in response to NE. Yet, only AL treatment significantly improved Ach-induced relaxation compared to standard KH, which may indicate improved endothelial preservation.
POTENT VASODILATORY EFFECT OF ADENOSINE-LIDOCAINE (AL) INDEPENDENT OF INTACT ENDOTHELIUM: POSSIBLE ROLE OF Kv AND mitoKATP CHANNELS
Arsyad MA, Dobson GP
School of Veterinary & Biomedical Sciences, James Cook University, Townsville, Queensland, Australia
Background: Vasodilator therapy has been used in attempt to prevent or to dilate graft spasm. Some vasodilators, however, require functional endothelium and become ineffective if the endothelial layer is damaged during graft harvesting. Adenosine and lidocaine are both vasoactive agents and the combination (AL) has been shown to induce coronary dilation.
Aims: (1) To examine the vasodilatory effect of AL solution compared with adenosine or lidocaine in endothelium intact and denuded rat aortic rings. (2) To investigate the possible involvement of potassium channels in AL vasodilatory action.
Methods: Endothelium intact or denuded thoracic rat aortic rings (3 mm) was equilibrated in a Krebs Henseleit-filled organ bath, bubbled with 95% O2 and 5% CO2 at 37°C. After precontracted with norepinephrine (NE), concentration relaxation curves to adenosine, lidocaine or AL (100–1000 μM) were recorded using an isometric force transducer. Another series of aortic rings was examined to observe AL relaxation with and without specific Kv and mitoKATP channel blockers. Relaxant responses were expressed as percentage of maximal relaxation to 100 µM papaverine.
Results: In intact rings, lidocaine produced 65.6% dilation at maximal concentration of 1000 μM compared to 100% and 97.8% relaxation with adenosine and AL, respectively. Adenosine relaxation, however, was attenuated after endothelial removal (89.6%), whereas lidocaine-induced relaxation remained unchanged (65.4%). Interestingly, maximal (100%) relaxation was achieved with AL regardless of the absence of endothelium. The relaxation effect of AL was significantly inhibited by Kv and mitoKATP channel blockers in endothelium-denuded aortic ring, but not when endothelium was intact.
Conclusion: AL and adenosine are potent vasodilators in rat aortic rings, while lidocaine is less potent. Unlike adenosine, AL relaxation was endothelium independent. Endothelium independent relaxation of AL may involve Kv and mitoKATP channel activation.
CENTRAL AORTIC PRESSURE AND ASSESSMENT OF BARORECEPTOR FUNCTION
Avolio A, Kouchaki Z, Qasem A, Butlin M
Australian School of Advanced Medicine, Macquarie University, Sydney, New South Wales, Australia
Background: Non-invasive assessment of baroreflex sensitivity (BRS) is conventionally performed by relating changes in heart rate (HR) and systolic pressure (SBP), where continuous beat-to-beat changes in blood pressure are measured in a peripheral location (e.g., finger). The pressure pulse is, however, amplified between the aorta and periphery, with amplification depending on HR due to the frequency dependency of the brachial transfer function. In addition, the degree of pulse amplification reduces with increased aortic stiffness as occurs with age.
Aim: To assess BRS using changes in SBP and pulse interval (PI) and to compare values of BRS computed from peripheral (finger) SBP (pSPB) and corresponding central aortic SBP (cSBP).
Methods: Continuous blood pressure and ECG signal were acquired in 6 adult subjects (mean age 42 ± 16 years) over periods of 5–20 minutes. The spontaneous sequence technique was used for computation of BRS from the slopes of linear relationships of SBP and PI of contiguous cardiac cycles, where SBP and PI change in the same direction. Central aortic pressure was determined from the finger pressure pulse (Finometer) by applying a mathematical transfer function. BRS was computed for a lag of 0, 1, 2 and 3 cardiac cycles between PI and SBP.
Results: The relationship between BRS computed from cSBP (y) and from pSBP (x) was y = 0.98x + 0.19 (r2=0.84). However, for all computed BRS values (n=24), BRS using cSBP were higher (by 21.5 ± 28.9SD %) in 62.5% of measurements (15/24) and lower (by 23 ± 12.8 %) in 35.5% (9/24). Across subjects, there was a trend for the difference to be positively related to heart rate (r2=0.21) and negatively related to age (r2=0.24) and mean arterial pressure (r2=0.11).
Conclusions: BRS computed from changes in HR and cSBP show a relation to values computed from pSBP. The difference between the two can, however, be positive or negative, and is related to hemodynamic parameters and age. Further studies are required to assess underlying mechanisms of the differences and potential clinical significance.
ETHANOL IMPAIRS EXPRESSION OF ESTROGEN RECEPTORS AND INCREASES ICAM-1 AND GALECTIN-3 IN HUMAN UMBILICAL VEIN AND CEREBRAL MICROVASCULAR ENDOTHELIAL CELLS
Gangoda SVSa, Jang SAb, Jufri NFa, Koo HJb, Butlin Ma, Avolio APa, Sohn EHc
aAustralian School of Advanced Medicine, Macquarie University, Sydney, New South Wales, Australia; bDepartment of Life Science, Gachon University, Seongnam, South Korea; cDepartment of Herbal Medicine Resources, Kangwon National University, Samcheok, South Korea
Background: Endothelial cells play a pivotal role in maintaining vascular homeostasis and endothelial dysfunction (ED) is an underlying mechanism of cardiovascular disease. Estrogen deficiency is one of the causes of ED that precedes the changes in vasomotor tone, inflammation and arterial stiffness, leading to vascular structural changes and increased blood pressure.
Aim: To determine the effects of ethanol (EtOH) on the expression of estrogen receptors (ERs) and inflammatory markers (ICAM-1, galectin-3) using two different types of endothelial cells: human umbilical vein (HUVEC), as a model of large vessel endothelial cells, and cerebral microvascular endothelial cells (hCMEC), which are associated with the blood-brain barrier.
Methods: Cells were treated with EtOH and ED and were then evaluated for the expression of ERs (ERα, ERβ, G protein coupled ER (GpER)), as well as for the regulation of ICAM-1, galectin-3, and the phosphorylation of IκBα using Western blot assay.
Results: Exposure to EtOH decreased the expression of ERα and GpER, but did not change ERβ (ERα was not detected in the HUVEC line used in this study). In terms of inflammatory factors, EtOH increased ICAM-1 and galectin-3 in both cell lines and activated p-IκBα in hCMEC.
Conclusions: These findings suggest that alcohol accelerates ED by decreasing estrogenic effects and increasing inflammatory response in both the systemic and cerebral vasculature.
THE PROTECTIVE EFFECT OF APOLIPOPROTEIN A-I ON MEAN ARTERIAL PRESSURE IN AN IN VIVO MODEL OF CYTOKINE-INDUCED HYPERTENSION IN PREGNANCY
Charlton FSa, Bobek Gb, Mirabito Kb, Xu Bab, Makris Aa, Rye Ka, Hennessy Aa,b
aThe Heart Research Institute, Newtown, Sydney, New South Wales, Australia; bSchool of Medicine, University of Western Sydney, Campbelltown, Sydney, New South Wales, Australia
Background: Failure of the trophoblast to appropriately invade uterine spiral arteries is an initiating event in preeclampsia (hypertension in pregnancy). The pro-inflammatory cytokine TNF-α inhibits trophoblast invasion. Previous work has demonstrated a protective effect of apolipoprotein A-I (apoA-I) against the deleterious effect of TNF-α on trophoblast-endothelial cell interactions in vitro. The present study investigated the protective effect of apoA-I on mean arterial pressure (MAP) via radiotelemetry in vivo.
Aims: To determine if apoA-I can reverse the deleterious effects of TNF-α on MAP using the cytokine-induced model of hypertension in pregnancy in the mouse.
Methods: Mice were time-mated and randomly assigned to treatment groups (n=3 per group). Saline or lipid-free apoA-I was administered at a dose of 40 mg/kg via the tail vein on gestation days (gd) 10.5 and 12.5. A mini-osmotic pump that released either saline or TNF-α (500 ng/kg/day) was implanted on gd13.5. Mice were implanted with radio-telemeters prior to pregnancy and their MAP recorded throughout gestation.
Results: The animals administered lipid-free apoA-I, prior to the TNF-α insult, showed a significantly smaller rise from baseline (0.99 ± 0.79SD mmHg) in their mean arterial pressure than the TNF-α treated animals (8.84 ± 0.65SD mmHg; P<0.05) upon infusion of TNF-α on gd13.5. On gd17, the rise in MAP from baseline of TNF-α treated animals (9.08 ± 1.30SD mmHg) was significantly higher than that of animals administered with lipid-free apoA-I (5.4 ± 0.72SD mmHg; P<0.05).
Conclusion: Although the lipid-free apoA-I did not fully normalize their MAP, these animals showed a significantly smaller rise in MAP than the TNF-α-only treated animals. Lipid-free apoA-I confers at least a partially protective effect against cytokine-induced hypertension in a whole animal in the presence of a fetus.
DOES STEM CELL COMBINATION THERAPY ENHANCE CORONARY VESSEL NUMBER IN A RODENT HEART FAILURE MODEL MORE THAN MONOTHERAPY?
Chen Y-Ca, Shirai Mb, Kainuma Sc, Fujii Yb, Inagaki Tb, Umetani Kd, Evans RGa, Tsuchimochi Hb, Pearson JTa,e,f
aDepartment of Physiology, Monash University, Clayton, Victoria, Australia; bNational Cerebral and Cardiovascular Center Research Institute, Osaka, Japan; cGraduate School of Medicine, Osaka University, Japan; dJapan Synchrotron Radiation Research Institute, Japan; eMonash Biomedical Imaging Facility, Monash University, Australia; fAustralian Synchrotron, Clayton, Victoria, Australia
Background: Transplantation of various stem cells and angiogenic stimulation have both been shown to improve heart function after myocardial infarction (MI). While stem cells are being used to regenerate cardiac muscle and contractile function, including skeletal myoblast sheets, most therapies do not promote revascularization. Omentum provides a rich source of endogenous angiogenic factors for revascularization.
Aim: To investigate if myoblast-omentum combination therapy improves the number and endothelial function of coronary arterial vessels within and adjacent to the infarct zone of rat hearts 3–6 weeks post-treatment in rats more than monotherapy with either treatment alone.
Methods: Lewis male rats (10 weeks old) were allocated to four treatment groups 2 weeks after inducing MI under anesthesia: control, no treatment (n=4), combined treatment (O+M, n=6), myoblast sheets (n=5) and omentum (n=6). All rats were compared with synchrotron coronary microangiography in vivo during baseline, acetylcholine, sodium nitroprusside and dobutamine infusions a further 3–6 weeks later.
Results: Total visible arterial vessel number (P<0.01) and microvessel number (P<0.01) were significantly greater in the O+M group (Figure). All treatment groups maintained basal perfusion, but none showed normal endothelial responses to vasodilator or dobutamine simulation.
Conclusion: We found the combined treatment group maintained better basal perfusion in the short term. While omentum treatment groups showed more widespread perfusion of new vessels under a dobutamine stress test, none of the treatments examined completely prevented vasoconstriction associated with heart failure. Further work is needed to determine if normal endothelial function develops in the long term.
BRACHIAL-TO-RADIAL SYSTOLIC BLOOD PRESSURE AMPLIFICATION IS SIGNIFICANTLY BLUNTED IN PATIENTS WITH TYPE 2 DIABETES: UPPER LIMB HEMODYNAMICS HAVE AN INFLUENTIAL ROLE
Climie RED, Picone DS, Keske M, Sharman JE
Menzies Research Institute Tasmania, University of Tasmania, Hobart, Tasmania, Australia
Background: We found recently significant age-related increases in brachial-to-radial systolic blood pressure amplification (Bra-Rad-SBPAmp). This has implications for correct central SBP estimation. Patients with type 2 diabetes (T2D) have vascular irregularities that may alter Bra-Rad-SBPAmp.
Aim: To determine the magnitude and correlates of Bra-Rad-SBPAmp in T2D.
Methods: Twenty T2D (64 ± 8 years; 50% male) and 20 controls (60 ± 8 years; 50% male) underwent simultaneous cuff auscultation and two-dimensional ultrasound imaging of the brachial and radial arteries. The 1st Korotkoff sound (denoting SBP) at each arterial site was identified from the first inflection point of Doppler flow during BP cuff deflation. Bra-Rad-SBPAmp was calculated by radial minus brachial SBP. Local and systemic hemodynamics were recorded by tonometry and ultrasound.
Results: Radial SBP was higher than brachial SBP for both T2D (136 ± 16 vs 127 ± 17; P<0.001) and controls (135 ± 12 vs 121 ± 11; P<0.001), and Bra-Rad-SBPAmp was significantly lower in T2D (9 ± 8 mmHg vs 14 ± 7 mmHg; P=0.042). Central SBP was significantly higher in both controls and T2D when radial pressure waveforms were calibrated using radial, compared with brachial SBP (both P<0.001). The product of brachial artery flow velocity and diameter was significantly increased in T2D (213 ± 108 vs 315 ± 144 cm/s/mm; P=0.023), and this was inversely correlated with Bra-Rad-SBPAmp (r=–643; P=0.003) even after adjustment for age and sex (β=-0.031, adjusted R2=0.366; P=0.002).
Conclusions: Patients with T2D have higher radial SBP than brachial SBP, but compared with controls, overall Bra-Rad-SBPAmp is significantly blunted. Local hemodynamics influence the magnitude of Bra-Rad-SBPAmp and overall these findings have implications regarding correct estimation of central BP.
AN UNEXPECTED DICHOTOMY IN WHOLE HEART AND CARDIOMYOCYTE FUNCTION IN THE HYPERTROPHIC HEART RAT – A MODEL OF HYPERTROPHY IN THE ABSENCE OF HYPERTENSION
Curl CL, Raaijmakers AJA, Chandramouli CC, Harding TW, Harrap SB, Delbridge LMD
Department of Physiology, University of Melbourne, Melbourne, Victoria, Australia
Background: The transition from hypertrophy to heart failure is associated with the development of subnormal myocardial contractile function. The underlying cardiomyocyte characteristics of this transition have not been well defined – and the load-dependent and load-independent elements of cellular contractility changes are yet to be established. The hypertrophic heart rat (HHR), a model of genetically determined cardiac enlargement, provides a novel opportunity to study the effect of long-term hypertrophy and the transition from hypertrophy to failure in the absence of confounding hypertension.
Aim: To compare ex vivo cardiomyocyte and in vivo myocardial function prior to transition-to-failure stage in a hypertensive-independent genetic setting of primary cardiac hypertrophy.
Methods: Echocardiography (B-mode, M-mode and apical 4 chamber axis, GE Vivid 9) was performed in adult (28–30 week) male HHR and normal heart rat (NHR) with a 15 mHz i13L linear array transducer (EchoPac analysis software). Collagenase digested isolated cardiomyocytes were fura 2-AM loaded, and contractility and [Ca2+]i were measured by edge-detection and microfluorimetry under basal conditions (3 Hz, 2.0 mM Ca2+, 37oC).
Results: HHR had significantly greater cardiac weight index (4.14 ± 0.2 vs 3.39 ± 0.1 mg/g), cardiomyocyte length (163 ± 2.3 vs 133 ± 1.0 µm) cardiomyocyte width (35.8 ± 0.7 vs 29.7±0.3µm) and cardiomyocyte volume (44.3 ± 1.3 vs 29.8 ± 0.4 pL) when compared with NHR. Septal wall thickness was reduced in the HHR (1.64 ± 0.04 vs 1.75 ± 0.04 mm) coupled with increased chamber diameter (8.87 ± 0.46 vs 7.98 ± 0.09 mm) and reduced systolic function (72.0 ± 1.89 vs 80.1 ± 0.94 ejection fraction %; all P<0.05). Significantly shorter deceleration time (32.2 ± 3.15 vs 50.0 ± 3.21 ms) and increased E/E’ ratio (31.0 ± 3.4 vs 21.7 ± 2.5) indicative of diastolic dysfunction was also apparent in the HHR. In contrast, HHR cardiomyocytes had substantially higher indices of contractile function compared with NHR (94% increase in relative myocyte shortening) along with higher amplitude (91% increase) and increased (22% faster) rate of decay of Ca2+ transient.
Conclusion: These findings indicate that with load-independent progression to failure in vivo heart function is diminished, even though cardiomyocyte hypercontractility is observed. The reduced myocyte population in the HHR hypertrophic myocardium may elicit a maladaptive hypercontractile compensatory response.
CARDIAC STABILITY AND HEMODYNAMIC RECOVERY WITH ADENOSINE-LIDOCAINE/MG2+ (ALM) FOLLOWING ASPHYXIAL CARDIAC ARREST DURING HYPOTHERMIA AND REWARMING
Djabir YY, Dobson GP
Heart and Trauma Laboratory, School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland, Australia
Background: Cardiac arrest claims around 17 million lives per year globally and the survival rate remains low (<10%). While therapeutic hypothermia followed by rewarming continue to gain popularity in resuscitation protocols, the efficacy of the standard drug, epinephrine, has been questioned due to potential risk of post-arrest myocardial dysfunction. Adenosine-lidocaine/Mg2+ (ALM) has been shown to provide cardio-protection and resuscitation following electrical-induced cardiac arrest and hemorrhagic shock.
Aim: To examine the effects of ALM compared to epinephrine on return of spontaneous circulation (ROSC) and post-ROSC hemodynamics following 8 min asphyxial cardiac arrest during hypothermia and rewarming.
Methods: Non-heparinized Sprague-Dawley rats (n=24) were randomly assigned to ALM and epinephrine (0.01 mg/kg BB) groups. Asphyxial cardiac arrest (MAP<10 mmHg) with hypothermia (33oC) was induced for 8 minutes followed by resuscitation (0.5 ml i.v bolus and chest compressions) and rewarming. Arterial pressures (systolic (SAP), diastolic (DAP) and mean (MAP)) and heart rate (HR) during and post resuscitation were recorded.
Results: Despite lower arterial pressures during chest compressions, all rats in the ALM group were resuscitated, while 25% of epinephrine-treated rats did not achieve ROSC due to persistent ventricular fibrillation (VF). None of ALM rats experienced VF during and post resuscitation. Immediately after ROSC, the epinephrine group had elevated MAP and HR, whereas those in the ALM group MAP and HR were moderately low (MAP 129 ± 7.0 vs 59 ± 5.0 mmHg; HR 151 ± 13.6 vs 59 ± 5.5 bpm). Hemodynamics in the ALM group gradually increased, leading to significantly higher MAP with similar HR after 120 min post ROSC.
Conclusion: Epinephrine increased arterial pressures, HR and VF incidence during resuscitation. In contrast, ALM lowered arterial pressures and HR to a certain level, leading to improved cardiac stability and hemodynamic recovery after 2 hours of ROSC compared to epinephrine.
INVESTIGATION OF THE ROLE OF RENAL LONG NON-CODING RNA IN ESSENTIAL HYPERTENSION THROUGH THE USE OF RNA-SEQ
Doogan Ca, Rana Ia, Deniff Mb, Tomaszewski Mb, Charchar FJa
aSchool of Health Sciences, University of Ballarat, Ballarat, Victoria, Australia; bDepartment of Cardiovascular Sciences, University of Leicester, Leicester, UK
Background: The kidney has long been thought to be involved in the etiology of essential hypertension (EH). The cause may involve genetic variants that are classified as long non-coding (lnc) RNAs, which can act over long distances and across chromosomes to activate transcription of genes that can have consequences for various disease.
Aim: To perform genome-wide RNA sequencing of renal RNAs to determine which lncRNA are differentially expressed between kidneys of 15 untreated EH and 15 normotensive (NT) white European subjects.
Methods: Stranded 100 bp pair end mRNA libraries were sequenced on an Illumina HiSeq 2000 platform following polyA purification and ribosomal depletion. Bioinformatic analysis for differential expression involved demultiplexing, alignment, RNA counting and normalization. Reads were aligned against the human genome (build version HG19) using the tophat aligner (http://tophat.cbcb.umd.edu/). Transcripts were assembled using reference-based annotation by Partek software using refseq from the UCSC genome browser. lncRNA were counted, normalized and analysed for the identification of differential expression.
Results: The expression of lncRNA was very low compared to other RNAs. We found 10 transcripts that were differentially expressed between normotensive and hypertensive subjects. Some of these were located in EH quantitative trait loci from the hypertension GWAS meta-analyses. These were LOC100130417, LINC00152, LOC339975, LOC100507584, LOC401463, LINC00639, LOC100507217, LOC283683, LOXL1-AS1, LOC389895 and LOC388849.
Conclusion: We identified 10 lncRNAs that were differentially expressed in EH kidneys. This is the first study to show that lncRNA may be involved in the etiology of EH in humans.
THE ROLE OF THE AT2 RECEPTOR IN MONOCYTE ACTIVATION AND MACROPHAGE POLARIZATION
Dragoljevic Da,b, Andrews KLa,b, Moore XL, Sampson AKa, Widdop REb, Chin-Dusting JPFa, Irvine JC
aBaker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia; bDepartment of Pharmacology, Monash University, Clayton, Victoria, Australia
Background: Monocyte activation and macrophage infiltration into the vessel wall are pivotal to the development of atherosclerosis. Macrophages polarize into either M1 (classically-activated) or M2 (alternatively-activated) phenotypes, with higher levels of M1 macrophages present in unstable regions of human plaque. The angiotensin type 2 receptor (AT2R) can exert anti-inflammatory effects in atherosclerosis.
Aim: To determine whether AT2R stimulation influences monocyte activation and macrophage polarization.
Methods: Whole human blood was pre-treated with the AT2R agonist Compound 21 (C21, 1 µM) prior to monocytic activation with LPS (100 ng/mL; 15 min). Flow cytometry analysis (CD14+/CD11b+) showed that LPS-induced monocyte activation (223 ± 15.8% vs control 100%; n=4; P<0.05) was unchanged following C21 pre-treatment. In separate experiments, human primary monocytes from buffy coat were differentiated into macrophages via M-CSF (100 ng/ml) for 7 days, and polarized using IFNγ (20 ng/mL)/LPS (100 ng/mL) to induce M1 macrophages, and IL-4 (20 ng/mL) to induce M2 macrophages, in the presence and absence of C21 (1 µM). Macrophage phenotype was determined via flow cytometry after 24 h and gene expression after 6 h.
Results: IFNγ/LPS increased CD64 expression 1.5 ± 0.1-fold compared to unstimulated macrophages (M0; n=8; P<0.001), and IL-4 increased CD206 and CD200R expression 1.2 ± 0.0- and 2.1 ± 0.2-fold compared to M0 control, respectively (n=8; P<0.01). C21 pre-treatment, however, had no effect on either M1 or M2 cell surface marker expression. IFNγ/LPS increased M1 marker TNFα and IL-6 gene expression to 43.8 ± 10.2 and 235 ± 60-fold compared to M0 control, respectively (n=5–8; P<0.01). C21 significantly reduced TNFα expression by 15.2 ± 4.1-fold compared to M0 control (n=8; P<0.05) and had a tendency to reduce IL-6 expression (by 93.2 ± 50.0-fold compared to M0 control; n=5; P=0.11). Gene expression of the M2 marker SRB1 was increased 8.1 ± 2.2-fold compared to M0 control (n=8; P<0.05) in response to IL-4. C21 pre-treatment had no effect.
Conclusion: Direct AT2R activation does not influence monocyte activation nor macrophage surface marker phenotype, but may reduce M1 gene expression, warranting further exploration of the role of AT2R stimulation on macrophage function.
EFFECT OF AN AORTIC ELASTIC WRAP ON SYSTEMIC IMPEDANCE
Giudici Fa, Segers Pb, O’Rourke Mc, Qian Ya, Avolio Aa
aAustralian School of Advanced Medicine, Macquarie University, Sydney, New South Wales, Australia; bGhent University, IBiTech-bioMMeda, Ghent, Belgium; cUniversity of New South Wales and Victor Change Cardiac Research Unit, Sydney, New South Wales, Australia
Background: Stiffening and dilatation of the ascending aorta (AA) with age is a major determinant of increased aortic pulse pressure (PP), leading to isolated systolic hypertension. The aortic wrap (AW) is our proposed method to restore higher distensibility in the stiffened AA. By wrapping the AA with a highly distensible synthetic material and decreasing the AA diameter it is possible to shift the pulsatile load from the AA walls to the AW, thus reducing PP and systolic pressure.
Aim: To investigate the effects of the AW on input impedance (Zin) and characteristic impedance (Zc) in a bench model of the arterial circulation.
Methods: A hydraulic loop model of the circulation was used. This is composed by a silicon model of the arterial tree connected to a pulsatile pump, which mimicks physiological conditions (3 heart rates (HRs) of 50, 65 and 80 bpm; and 3 stroke volumes (SVs) of 60, 70 and 80 ml/s). The AA segment of the model was wrapped with a distensible elastic band (3.5 cm long), achieving a 30% diameter reduction of the AA at 0 mmHg. Flow waves were recorded at the inlet via a transonic flowmeter, and pressure waves were recorded at 10 locations along the model with a Millar microtip pressure catheter. Zin (= P(ω)/Q(ω), where ω=frequency) and Zc (initial slope of the P-Q curve) were calculated for all the possible combinations of HR and SV.
Results: After wrapping, there was a consistent reduction of Zc, with the exception of the 50 bpm HR–80 ml SV configuration. The maximum reduction (–41.9% compared with the corresponding unwrapped value) was achieved at the lowest HR (50 bpm) and lowest SV (50 ml). Zin modulus was reduced consistently by the AW, the 1st harmonic component showing a decrease (varying from –14.5% to –30.1% of the corresponding unwrapped value) for all the low (50 bpm) and medium (65 bpm) HR configurations.
Conclusion: Our in vitro modelling study shows that the AW procedure consistently decreases Zin and Zc for most HR and SV configurations, resulting in a concomitant decrease of PP. This suggests a potential use of the AW as a non-pharmacological treatment of isolated systolic hypertension. The dependency of the impedance reduction on HR and SV indicates a potential patient-specific optimization approach.
MEASUREMENT OF RETINAL ARTERY PULSE WAVE VELOCITY IN THE RAT USING HIGH SPEED IMAGING
Golzan SM, Butlin M, Kouchaki Z, Avolio AP, Graham SL
Australian School of Advanced Medicine, Macquarie University, Sydney, New South Wales, Australia
Background: Arterial stiffness, as measured by arterial pulse wave velocity (PWV) in large conduit arteries, is being increasingly accepted as a significant prognostic index of cardiovascular function. The significance of PWV in arteries in the microvasculature is not yet established, due mainly to the difficulty in obtaining reliable non-invasive measurements in vivo. The eye, as the only organ providing direct non-invasive imaging access to its internal anatomical structure, has the potential to be used to assess arterial stiffness at the level of the microcirculation.
Aim: To develop signal processing and high-speed imaging techniques to measure pulse transit time (PTT) to determine retinal artery PWV (rPWV) in the rat eye.
Methods: Measurements were performed in nine Wistar-Kyoto (280 ± 30 g, 12 weeks) anesthetized male rats. Due to the high heart rate of rats, high-speed imaging was used to record retinal arterial pulsations without aliasing. Retinal video images were captured using a high speed camera (Optronis, Germany) at a rate of 250 frames per second for 10 seconds with a 50o field of view. A simultaneously recorded electrocardiogram was used in subsequent waveform analysis and heart rate (HR) calculation. Recorded video images were analysed with custom algorithms to measure arterial diameter pulsations at two sites 0.5 mm apart. PTT between the sites was calculated using the time delay of a fiducial point at 20% of the waveform height.
Results: The average rPWV was 11.5 ± 6.0 cm/s at a HR of 322 ± 37 bpm, mean BP of 89 ± 10 mmHg, and average retinal artery diameter of 54 ± 11 µm. A positive correlation was obtained between arterial diameter and pulse amplitude (r2=0.52). The correlation between rPWV and HR was r2=0.32.
Conclusion: This study confirmed the feasibility of a novel approach to measure rPWV in animal models. This technique is a promising accessible tool for microvascular assessment of PWV. The inter-individual variability is likely related to variations in depth of anesthesia and blood pressure which needs to be controlled to improve reproducibility. Future studies are required to assess the significance of PWV in the retinal microvasculature in rat models of cardiovascular disease.
THE ROLE OF T CELLS AND MACROPHAGES IN PERIVASCULAR SYMPATHETIC NERVE GROWTH AND THE DEVELOPMENT OF NEUROGENIC HYPERTENSION IN OBESITY
Eccles Institute of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital Territory, Australia
Introduction: Hypertension in obesity occurs as a result of abnormal increases in sympathetic nerve density over arteries controlling blood pressure; the causative factors underlying these changes are unknown.
Aim: To examine whether T cells drive obesity-induced sympathetic hyperinnervation and the development of neurogenic hypertension in obesity.
Methods: C57BL/6 and Rag1–/– mice, which lack mature B and T cells, were fed normal chow or a high fat diet (HFD). Mesenteric arteries were isolated and pressure myography, electrophysiology and videomicroscopy was used to measure intraluminal pressure, membrane potential and vessel diameter. Nerves were activated by electrical field stimulation. Immunohistochemistry was performed using antibodies against nerve growth factor (NGF), CD3+ (T cells) and F4/80 (macrophages). Sympathetic innervation was examined over both mesenteric and renal arteries using synaptophysin (synaptic vesicles). Images were collected using confocal microscopy and analysed using Image J.
Results: Obese C57BL/6 mice were hypertensive. Nerve-mediated contractions and excitatory junction potentials (EJP) were augmented and the perivascular sympathetic nerve plexus was denser. Numbers of perivascular NGF-producing immune cells (T cells and macrophages) were increased. Infiltration of these cells was shown to precede expansion of the nerve plexus. Rag1–/– mice fed a HFD also became obese. Numbers of perivascular NGF-producing immune cells were not, however, increased, arteries were not hyperinnervated and mice remained normotensive.
Conclusion: Nerve growth factor producing T cells and macrophages drive sympathetic hyperinnervation and neurogenic hypertension in obese C57BL/6 mice.
HYPERTENSION, WHITE COAT HYPERTENSION AND MASKED HYPERTENSION: AMBULATORY BLOOD PRESSURE FINDINGS FROM THE AUSDIAB COHORT (WAVE III)
Head GA, Shaw JE, Dunstan DW, Marks K, Owen N, Magliano DJ
Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia
Background: The Australian Diabetes, Obesity and Lifestyle Study (AusDiab) is an Australian national, population-based study of over 11,000 adults from all states and the Northern Territory of Australia that began in 1999–2000 and was designed to assess the risks of all-cause and CVD mortality. The third wave of assessment carried out in 2011–2012 provided the opportunity to compare office and out-of-office blood pressure (BP) using ambulatory BP monitoring (ABPM) in an opportunistic sub-group drawn from this well described Australian cohort.
Aim: To compare clinic BP and ABPM in the AusDiab cohort at wave III.
Methods: During the wave III on-site assessment of 4614 AusDiab participants, 838 were approached and 586 agreed to undergo 24 hour ambulatory monitoring. Of these assessments, 491 were conducted successfully (>15 readings, >15 hours recording).
Results: Mean age was 59.7 years, mean BMI 27.5 kg/m2, and 53% were women. The mean clinic systolic over diastolic BP was 127/73 mmHg and mean 24 hour BP was 120/72 mmHg; 39% were hypertensive based on 14% having ABPM mean 24 h >130/80 mm Hg, or were on antihypertensive therapy (25%). Both office and ambulatory BP were higher in men (office +6.5/6.5, 24 h +6.4/5.2 mmHg). These differences persisted after adjustment for age. The prevalence of white-coat hypertension was 12% and masked hypertension was 4%. Based on ABPM, 14% had untreated hypertension and 11% of those treated were still hypertensive, which was 3 times more common in men than in women (19% vs 6%).
Conclusions: 24 hour ABPM findings in this sub-group of the AusDiab cohort at wave III showed that one in three have blood pressure above target, despite a high prevalence of antihypertensive treatment. One in 8 have white coat hypertension, but the level of masked hypertension appears to be relatively low. These findings highlight the importance of out-of-office BP assessments in hypertension management.
DEPLETION OF B CELLS BY AN ANTI-CD20 MONOCLONAL ANTIBODY INHIBITS ANGIOTENSIN II-INDUCED HYPERTENSION IN MICE
Chan CTa, Sobey CGa, Diep Ha, Kim HAa, Tipping Pb, Kyaw TSc, Toh BHb, Bobik Ac, Drummond GRa
aVascular Biology & Immunopharmacology Group, Department of Pharmacology, Monash University, Clayton, Victoria; bCentre for Inflammatory Diseases, Department of Medicine, Southern Clinical School, Monash University, Clayton, Victoria; cVascular Biology & Atherosclerosis Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia
Background: Lymphocyte-deficient RAG1–/– mice are protected from angiotensin II (Ang II)-induced hypertension. Adoptive transfer studies suggest that this protective effect may be due to the lack of T cells, and not to a lack of B cells. Two major subsets of B cells exist. B2 cells produce antigen-specific antibodies and thus represent an important component of the adaptive immune response. By contrast, B1 cells generate non-specific (natural) antibodies, thus forming part of the innate immune response.
Aims: To re-assess the role of B cells in Ang II-induced hypertension in mice by their depletion with anti-CD20 monoclonal antibody.
Methods: Mice were infused with Ang II (0.7 mg/kg/day) for 28 days and B1, B2 and T cells were measured in aorta, kindey and spleen by flow cytometry.
Results: Subcutaneous infusion of Ang II (0.7 mg/kg/day) for 28 days resulted in a markedly higher systolic BP than that in saline-treated controls (175 ± 5 mmHg versus 119 ± 3 mmHg, respectively; P<0.05; n≥9). Flow cytometric analysis revealed the presence of B2 cells (CD19+CD22+) in the aorta, kidney and spleen, and B1 cells (CD19+CD11b+CD5+) in the peritoneal cavity of Ang II-treated mice. The numbers of B cells in these locations were not, however, different from the number seen in the saline-treated controls. Treatment of mice with an anti-mouse CD20 antibody (5 mg/kg, i.v.) one day prior to commencement of Ang II infusion and then 14 days later, reduced both B1 and B2 cell numbers by >95% in the aorta, kidney, spleen and peritoneal cavity (P<0.05; n≥9) without affecting T cell (CD3+) levels. Importantly, B cell depletion blunted Ang II-induced increases in systolic BP by ~40% (i.e., 156 ± 8 mmHg in the Ang II+anti-mouse CD20 antibody group versus 177 ± 5 mmHg in the Ang II+control antibody group; P<0.05; n≥9).
Conclusion: B cells are crucial for the development of Ang II-induced hypertension in mice. Future studies to elucidate the specific subset of B cells and immune mechanisms involved (e.g., antigen presentation, antibody and/or cytokine production) may uncover targets for novel anti-hypertensive therapies.
AMBULATORY AND CENTRAL HEMODYNAMICS ARE ELEVATED DURING PROGRESSIVE TREKKING ASCENT TO HIGH-ALTITUDE AND ASSOCIATED HYPOXIA
Schultz MG, Climie RED, Sharman JE
Menzies Research Institute Tasmania, University of Tasmania, Hobart,Tasmania, Australia
Background: High-altitude hypoxia may cause temporary increases in resting brachial blood pressure (BP), but the effect on more sensitive measures of BP control (24 hour ambulatory BP and resting central BP) is largely unknown.
Aim: To determine 24 hour ambulatory BP and resting central BP, as well as haemodynamic correlates of acute mountain sickness (AMS) during a progressive ascent to high-altitude.
Methods: Measures of oxygen saturation (pulse oximetry), 24 hour ambulatory BP (A&D), resting brachial and central BP (Pulsecor) were recorded in 10 adults aged 27 ± 4 years (30% male) during a 9-day trek to Mt. Everest base camp, Nepal. Data were recorded at sea level (stage 1; <450 m above sea level (ASL)) and at progressive ascension to 3440 m ASL (stage 2), 4350 m ASL (stage 3) and 5164 m ASL (stage 4). The Lake Louise Score (LLS) was used to quantify AMS symptoms.
Results: Total LLS increased step-wise from sea level to stage 4 (0.3 ± 0.7 vs 4.4 ± 2.0; P=0.012), whilst oxygen saturation decreased to 77 ± 9% (P=0.001). The highest recordings of 24 hour ambulatory, daytime, night-time, brachial and central systolic BP and diastolic BP were achieved at stage 3. These were significantly greater than at sea level (P<0.005 for all). 24 hour ambulatory heart rate and night heart rate correlated with oxygen saturation (r=–0.741 and –0.608; both P<0.001) and total LLS (r=0.648 and r=0.493; both P<0.001).
Conclusion. 24 hour ambulatory BP, central BP and heart rate are elevated during high-altitude hypoxia, but AMS symptoms appear only related to tachycardia.
VASCULAR FUNCTION IN THE STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RAT IS Y CHROMOSOME DEPENDENT
Khan SI, Jefferis AM, Jennings GJ, Chin-Dusting JPF, Andrews KL, Sampson AK
Baker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia
Background: The origin of the Y chromosome accounts for 15–20 mmHg difference in arterial pressure. The gene(s) and mechanism(s) underlying this effect, however, remain unknown. As vascular dysfunction is a hallmark of hypertension, we hypothesize that vascular dysfunction in the stroke-prone spontaneously hypertensive rat (SHRSP) may be influenced by the Y chromosome.
Aim: Using the Y consomic approach, which exclusively replaces the Y chromosome of the SHRSP with the normotensive WKY Y chromosome (SP.WKYGlaY) and vice versa (WKY.SPGlaY), we aimed to examine the influence of the origin of the Y chromosome on vascular function.
Methods: Thoracic aortas were excised from rats and mounted in organ baths.
Results: Concentration vasorelaxation curves to acetylcholine (ACh), and sodium nitroprusside (SNP) revealed impaired endothelium-dependent (ACh), but not endothelium-independent (SNP), vasorelaxation in the SHRSP aorta compared to the WKY (ACh: EC50; SHRSP: 7.3 ± 0.08 vs WKY: 7.8 ± 0.16; n=7–8; P=0.02). Replacement of the SHRSP Y chromosome with the WKY Y chromosome (SP.WKYGlaY) significantly improved ACh-induced vasorelaxation (EC50 of SP.WKYGlaY = 7.7 ± 0.1; n=6; P<0.001). Importantly, prostanoid blockade with indomethacin (10 µM) abolished differences in endothelium-dependent vasodilation, implicating a role for the origin of the Y chromosome in determining constrictor prostanoid release. Furthermore, contractions to L-NAME were impaired in the SHRSP (16.4 ± 16.2SD % of maximum; n=5) compared to WKY (70.5 ± 19.2SD % of maximum; n=3) and SP.WKYGlaY aortas (57.8 ± 11SD % of maximum; n=8; P=0.05), suggesting the origin of the Y chromosome also modulates basal NO levels. Responses to the vasoconstrictor angiotensin II (100 nM) were similar, despite differences in circulating angiotensin II levels (WKY 44.3 ± 4.7SD %, SHRSP 30.2 ± 8.2 SD %, SP.WKYGlaY 38.3 ± 7.9SD %, contraction from baseline; n=5–9; P>0.5).
Conclusion: We provide evidence that the origin of the Y chromosome influences endothelium-dependent vasodilatation by regulating constrictor prostanoid release and basal NO levels.
BRACHIAL-TO-RADIAL-SYSTOLIC-BLOOD-PRESSURE-AMPLIFICATION AND THE IMPACT ON NON-INVASIVELY DERIVED CENTRAL SYSTOLIC BLOOD PRESSURE
Picone DSa*, Climie REDa*, Ahuja KDKb, Keske MAa, Sharman JEa
aMenzies Research Institute Tasmania, University of Tasmania, Hobart, Tasmania, Australia; bSchool of Human Life Sciences, University of Tasmania, Launceston,Tasmania, Australia(*Equal first authors)
Background: The reference standard for non-invasive central blood pressure (BP) measurement is radial tonometry calibrated using brachial BP. Brachial-to-radial-systolic-BP-amplification (Bra-Rad-SBPAmp) may introduce error into central BP measurement, but the magnitude of Bra-Rad-SBPAmp in healthy people is uncertain.
Aims: To determine the magnitude of Bra-Rad-SBPAmp as well as the effect of ageing and the impact of Bra-Rad-SBPAmp on estimated central SBP.
Methods: Forty healthy younger (28 ± 5 years; 50% male) and 20 older (60 ± 8 years; 50% male) participants underwent brachial and radial artery ultrasound to identify SBP (1st Korotkoff sound) from the 1st Doppler flow inflection during BP cuff deflation. Hemodynamics were recorded by ultrasound and tonometry. Bra-Rad-SBPAmp was calculated by radial minus brachial SBP.
Results: Radial SBP was significantly higher than brachial SBP in younger (118 ± 12 mmHg versus 110 ± 10 mmHg) and older (135 ± 12 mmHg versus 121 ± 11 mmHg; P<0.001 both) participants. However, the magnitude of Bra-Rad-SBPAmp was higher in older age (14 ± 7 mmHg versus 8 ± 7 mmHg; P=0.004), independent of heart rate, mean arterial pressure and sex. Radial waveform recalibration using radial versus brachial SBP produced higher central SBP in younger and older age groups (P<0.001 both). Bra-Rad-SBPAmp caused significantly greater underestimation of central SBP in older participants (P<0.001). Irrespective of the magnitude of Bra-Rad-SBPAmp, high central augmentation index was highly correlated with greater underestimation of central SBP (r=0.751; P<0.001).
Conclusion: Major Bra-Rad-SBPAmp occurs in healthy people and is significantly increased with age. This has implications for correct central SBP estimation using radial tonometry and brachial BP calibration, whereby greater underestimation of central SBP is likely with advancing age.
THE EFFECTS OF POSITIVE ALLOSTERIC MODULATION OF GABAA RECEPTORS UPON STRESS AND HYPERTENSION IN SCHLAGER HYPERTENSIVE MICE
Stevenson ERa,b, Jackson KLa,b, Abegaz Ba, Moretti J-La, Gueguena Ca, Davern PJa, Head GAa,b
aBaker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia; bMonash University, Clayton, Victoria, Australia
Background: An exaggerated pressor response to stress has been shown to be a predictor of the subsequent development of hypertension. Hypertensive Schlager mice (BPH/2J strain) have neurogenic hypertension associated with abnormal reactivity of neurons in the forebrain integrating the response to aversive stress. Recent studies suggest they also have functional and molecular differences in GABAA receptors compared with their normotensive counterparts (BPN/3J strain). Allopregnanolone is an endogenous neurosteroid reduced by chronic stress and, when administered, decreases anxiety by positive allosteric modulation of GABAA receptors.
Aim: To determine if allopregnanolone reduces the pressor effects of stress and basal MAP in BPH/2J mice.
Methods: Male BPN/3J (n=7) and BPH/2J (n=5) mice received vehicle or allopregnanolone (5 mg/kg/day) via subcutaneous minipumps for a period of two weeks. Prior implantation of telemetric probes enabled recording of mean arterial pressure (MAP), heart rate (HR) and activity before and 7 and 14 days after minipump implantation. The cardiovascular response to aversive (dirty cage switch and restraint) and non-aversive (feeding) stress tests, as well as ganglionic blockade with 5 mg/kg pentolinium, were recorded before and 7–14 days after minipump implantation. Mice were perfused following stress and brains were removed for immunohistochemistry.
Results: In BPH/2J mice, 2 weeks of allopregnanolone reduced systolic arterial pressure (–8.8 mmHg; P=0.01) and attenuated the depressor response to pentolinium, whereas no effect on MAP or HR was observed in BPN/3J mice. Allopregnanolone produced marked reductions in the pressor response to both cage switch and feeding stress (–20%; P<0.01) in BPH/2J mice, whilst increasing the pressor response to aversive stress in BPN/3J mice (+33–48%; P<0.001). Stress-induced Fos counts within the medial amygdala and paraventricular nucleus were higher in untreated BPH/2J compared to BPN/3J mice. Allopregnanolone reduced Fos expression and abolished the difference between strains.
Conclusions: The selective antihypertensive and stress inhibitory effects of allopregnanolone in BPH/2J hypertensive mice suggests that allosteric modulation of GABAA receptors at the level of the hypothalamus and amygdala may be a major cause of hypertension in this model and may offer a possible new area for the development of therapy.
EFFECTS OF HEART RATE ON ARTERIAL STIFFNESS, CENTRAL HAEMODYNAMICS, CARDIAC OUTPUT AND TOTAL PERIPHERAL RESISTANCE
Tan Ia, Butlin Ma, Kiat Ha,b, Barin Ec, Avolio APa
aAustralian School of Advanced Medicine, Macquarie University, Sydney, New South Wales, Australia; bCardiac Health Institute, Sydney, New South Wales, Australia; cMacquarie Heart, Sydney, New South Wales, Australia
Background: Increased arterial stiffness is a marker for cardiovascular disease. Whilst numerous cross-sectional studies have found a pressure-independent effect of heart rate (HR) on arterial stiffness, others have found increased arterial stiffness with increased HR in the presence of mean arterial pressure (MAP) changes. The effect of HR on arterial stiffness has not, however, been studied previously in conjunction with cardiac output (CO) and total peripheral resistance (TPR) measurements.
Aim: To investigate the effects of acute, sympathetic-independent changes in HR on central pressures, CO and TPR.
Methods: 28 subjects aged 81 ± 6SD years (4 females) with in situ cardiac pacemakers or implanted cardioverter defibrillators were studied. Each subject was paced in a random order from 60 to 100 beats per min (bpm), with 10 bpm increments. Brachial cuff-based pulse wave analysis was used to measure central (c) systolic (SBP), diastolic (DBP) and MAP, and pulse wave velocity (PWV) was measured with a thigh cuff and carotid tonometry (SphygmoCor® XCEL). TPR and stroke volume (SV) were derived from measured finger arterial pressure waveform (Finometer®), and CO was calculated as the product of SV and HR as measured from the electrocardiogram.
Results: All parameters measured, except cSBP, changed significantly with HR (Table; mean±SEM). Despite a decrease in both SV and TPR, CO and MAP increased with increases in HR. PWV also increased with HR, but the significance was lost once corrected for changes in MAP (Table).
Conclusion: Acute, sympathetic-independent increases in HR raises CO and cDBP, which in turn increases MAP despite decreases in SV and TPR, and results in increased arterial stiffness.
IS IT TIME FOR A NEW LOOK AT THE “WATCH AND WAIT” APPROACH — MID-LIFE ANTIHYPERTENSIVE TREATMENT MAY NOT NORMALIZE LEFT VENTRICULAR MASS IN SPITE OF CONTROLLED BLOOD PRESSURE
Ghosh AKa,b, Hardy RJb, Francis DPa, Chaturvedi Na, Pellerin Dc, Deanfield Jd, Kuh Db, Mayet Ja, Hughes ADa
aInternational Centre for Circulatory Health, Imperial College, National Heart and Lung Institute, London, UK; bMedical Research Council Unit for Lifelong Health and Ageing, London, UK; cThe Heart Hospital, University College London Hospitals NHS Trust, London, UK; dDepartment of Vascular Physiology, Institute of Child Health, University College London, London, UK
Background: In cross-sectional studies, elevated blood pressure (BP) is associated with increased left ventricular mass (LVM), which leads to increased cardiovascular morbidity and mortality. Current practice involves a “watch and wait” approach in younger individuals with borderline-high BP. We investigated if this was a correct assumption.
Methods: 1,653 participants in the 1946 birth cohort underwent echocardiography at age 60–64 years and LVM, indexed to body-surface-area (LVMI), was measured. The relationship between repeated measures of SBP and antihypertensive treatment (HTT-measured at 4 time points: 60–64 (current), 53, 43 and 36 years) and LVMI at 60–64 years was examined using adjusted multiple regression models. Multilevel models of SBP were then used to estimate person-specific intercepts (SBP at 36 years) and slopes (rate of change in SBP between 36–60/64 years). The intercepts and slopes were included in sex-adjusted linear regression models with LVMI as the outcome.
Results: Individuals on HTT, from 43 years onwards, had higher mean LVMI than those who were not on treatment, irrespective of level of BP at the same age (Table). The effect of mid-life rate of change in BP (36–53 years) on LVMI at age 60–64 years was 10 times greater than the effect of more recent rate of change (53 years–current age).
Conclusions: HTT does not normalize LVMI in older individuals. This may be due to irreversible cardiac damage occurring in mid-life in poorly-controlled hypertensives. Early identification and treatment of individuals with rapidly increasing BP in mid-life may be key to preventing such damage. A review of current guidelines on monitoring and screening of BP may be required.
THE EFFECT OF BLOOD PRESSURE VARIABILITY ON THE RISK OF DEATH OR DEPENDENCY IN THE ACUTE PHASE OF INTRACEREBRAL HAEMORRHAGE
Hirakawa Y, Arima H, Manning L, Wang X, Robinson TG, Anderson C, Chalmers J
The George Institute for Global Health, Royal Prince Alfred Hospital and University of Sydney, Sydney, New South Wales, Australia; The University of Leicester, Leicester, UK
Background: The prognostic value of visit-to-visit blood pressure variability (BPV) has been confirmed in a wide spectrum of clinical populations. Increased BPV following acute intracerebral hemorrhage (ICH) may also be a predictor of short term outcome though data are very limited.
Aim: The observational analysis of the INTERACT2 Trial examined the effect of systolic blood pressure (SBP) variability on the risk of death or dependency in patients with acute ICH.
Methods: INTERACT2 was an international, multicentre, blinded endpoint, randomized controlled trial to investigate the effect of rapid lowering of elevated BP on the outcome in 2,839 hypertensive patients with acute ICH. Among them, 2,645 patients without missing values of SBP were included in the analysis of hyperacute phase (first 24 hours), and 2,347 in the analysis of acute phase (2 to 7 days). Variability in SBP was defined using the standard deviation (SD-SBP) of 5 measurements in hyperacute phase, and 12 measurements in acute phase. Maximum SBP was also analysed for both periods. Outcome was death or dependency at 90 days. Logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) with adjustment for age, sex, region, high NIHSS, hematoma volume, mean SBP, and randomized treatment.
Results: There was a strong and continuous association between hyperacute SD-SBP and death or dependency at 90 days: ORs (95% CIs) were 1.0 (reference), 1.11 (0.83–1.48), 1.16 (0.87–1.56), 1.26 (0.94–1.69) 1.41 (1.05–1.9) for 5 groups defined by fifths of SD-SBP, respectively (P=0.02 for trend). Likewise, acute SD-SBP was associated with death or dependency (P=0.01 for trend). Maximum SBP was also associated with death or dependency in the hyperacute phase (P=0.03 for trend), and in the acute phase (P=0.02 for trend).
Conclusion: These analyses of INTERACT2 Trial data revealed that SBP variability and maximum SBP in the hyperacute and acute phase of stroke are both associated with increased risks of death or dependency. This highlights the need for both the very rapid and sustained control of SBP in the first 7 days following ICH.
ASSOCIATION BETWEEN MAGNITUDE OF BLOOD PRESSURE REDUCTION AND CLINICAL OUTCOMES AFTER ACUTE INTRACEREBRAL HEMORRHGE: THE INTERACT2 TRIAL
Chalmers J, Wang X, Arima H, Heeley E, Delcourt C, Hirakawa Y, Anderson C
The George Institute for Global Health, Sydney, New South Wales, Australia
Background: The main phase of the INTEnsive blood pressure Reduction in Acute Cerebral haemorrhage Trial (INTERACT 2) indicated that early intensive lowering of blood pressure (BP) was safe and improved functional outcomes in patients with acute intracerebral haemorrhage (ICH).
Aim: To examine whether larger BP reductions improve clinical outcomes in acute ICH.
Methods: INTERACT2 was an international, open, blinded endpoint, randomized controlled trial. Eligible patients with spontaneous ICH within 6 h of onset and elevated systolic BP (SBP; 150–220 mmHg) were allocated to receive intensive (target SBP<140 mmHg within 1 h using intravenous agents) or guideline-recommended (SBP <180 mmHg) BP lowering treatment. BP reduction was defined as baseline SBP minus average of achieved SBP levels during 3 periods after randomization (15–60 minutes, 1–24 hours, and 2–7 days). Outcome was death or major disability at 90 days.
Results: Larger reductions in SBP between 15 and 60 minutes were associated with lower risks of death or major disability (P trend <0.01). Similar associations were also observed for SBP reductions between 1 and 24 h (P trend <0.01) and between 2 and 7 days (P trend =0.01). The association of SBP reductions and death or major disability appeared to be stronger among patients with baseline SBP levels of ≥180 mmHg than those with baseline SBP <180 mmHg (P homogeneity=0.07 for 15–60 minutes, 0.04 for 1–24 hours and 0.02 for 2–7 days).
Conclusion: Optimal protection against death or major disability after ICH was observed in patients who achieved the greatest SBP reductions in the first hour and maintained these consistently over 7 days.
COST EFFECTIVENESS OF HYPERTENSION MANAGEMENT GUIDED BY CENTRAL BLOOD PRESSURE MEASUREMENT: HEALTH ECONOMIC EVALUATION OF THE BPGUIDE STUDY
O’Malley SPa, Marwick THb, Abhayaratna WPc, Stowasser Md, Sharman JEb
aMacquarie University, Sydney, New South Wales, Australia; bMenzies Research Institute Tasmania, University of Tasmania, Hobart, Tasmania, Australia; cAustralia National University, Canberra, Australian Capital Territory, Australia; dUniversity of Queensland, Brisbane, Queensland, Australia
Background: The BPGUIDE study was a clinical trial in 286 hypertensive patients randomized to treatment decisions guided by best-practice usual care or in addition by central BP (SphygmoCor) over twelve months.
Aims: To present a health economic assessment from the BPGUIDE study results.
Methods: The primary finding of BPGUIDE was that significantly less antihypertensive medication was used to achieve BP control in patients randomized to central BP guided care. The savings from reductions in 5 antihypertensive medication classes over time were used to determine the cost-effective fee for service. This amount was compared to the actual cost of central BP measurement in order to gauge financial viability. Health system savings were calculated from Australian Pharmaceutical Benefits Scheme (PBS) costs.
Results: Savings from less use of medication (based on actual daily dose) were calculated at $17.91/person/3 months. This saving is sufficient to cover the cost of measuring central BP based on capital costs of $10,000 (for SphygmoCor), 5 years capital life, 5% discount rate, patient throughput of 50/year and, labour costs up to $250/hour. Therefore, a fee for service to measure central BP of ≈$17 is both economically and financially feasible. The 2012–2013 PBS costs for antihypertensive drugs in the study was ≈$317 million. Based on the percentage reduction with central BP guided care, the potential annual saving was calculated at ≈$56 million.
Conclusions: Management of hypertension using central BP has cost-savings relating to decreased medication and may be regarded as cost-neutral when factoring in a fee for central BP measurement.
ALDOSTERONE-INDUCED OXIDATIVE STRESS AND INFLAMMATION IN THE BRAIN: ROLE OF THE ENDOTHELIAL CELL MINERALOCORTICOID RECEPTOR
Dinh QNa, Drummond GRa, Young MJb, Sobey CGa, Chrissobolis Sa
aVascular Biology & Immunopharmacology Group, Department of Pharmacology, Monash University, Clayton, Victoria, Australia; bPrince Henry’s Institute and Departments of Physiology & Medicine, Monash University, Clayton, Victoria, Australia
Background: Elevated aldosterone levels are associated with stroke risk that is independent of blood pressure and other risk factors. Aldosterone acts on the mineralocorticoid receptor (MR) in the kidney to regulate fluid and electrolyte homeostasis and blood pressure, but can also act on other tissues such as the brain and its vasculature, and cause detrimental effects such as oxidative stress and inflammation.
Aim: To examine whether aldosterone causes endothelial cell MR-dependent oxidative stress in cerebral vessels, and increases pro-inflammatory markers in the brain.
Methods: Male mice were anesthetised with ketamine (100 mg/kg)/xylazine (10 mg/kg) i.p. and treated with vehicle or aldosterone (0.28 or 0.72 mg/kg/day) by osmotic minipumps for 2 weeks. Endothelial cell MR-deficient (MRflox/flox/Tie2Cre/+) and wild-type (MRflox/flox) mice, and C57Bl/6 mice pre-treated with spironolactone (25 mg/kg/day, i.p.), an MR antagonist, were used to test for MR involvement. Systolic blood pressure (SBP) was measured using tail cuff plethysmography, superoxide levels using L012 chemiluminesence, and chemokine ligand (CCL7, CCL12) and receptor (CCR2) mRNA expression using real-time PCR.
Results: In C57Bl/6 mice, aldosterone (0.72 mg/kg/day) moderately increased SBP when compared to vehicle (SBP, vehicle-treated=102mmHg; n=10; SBP, aldosterone-treated=16.32 mmHg; n=11, P<0.05). Nox2-dependent increases in superoxide were ~60% greater in cerebral arteries from aldosterone- vs vehicle-treated mice (n=7–8; P<0.05). Pretreatment with spironolactone prevented aldosterone-induced increases in Nox2-dependent superoxide (n=5; P>0.05), suggesting this effect was MR-dependent. In wildtype (MRflox/flox) mice, Nox2-dependent increases in superoxide were ~50% greater in cerebral arteries from aldosterone- vs vehicle-treated mice (n=7–8; P<0.05), and this effect was abolished in endothelial cell MR-deficient (MRflox/flox/Tie2Cre/+) mice (n=7–8 P>0.05). Aldosterone increased expression of CCL7 mRNA in the brain compared to ~1.8-fold of vehicle, (n=11–12; P=0.07), but had no effect on CCL12 or CCR2 mRNA expression (n=11–12; P=0.13 and P=0.14, respectively).
Conclusion: Chronic aldosterone administration increases cerebrovascular superoxide levels by a mechanism involving endothelial cell MR. This treatment also increases expression of CCL7 mRNA in brain. Endothelial cell-specific MR antagonism may represent a novel approach to treat cerebrovascular disease.
EXERCISE-INDUCED ALBUMINURIA IS INDEPENDENTLY RELATED TO EXERCISE AORTIC RESERVOIR FUNCTION IN PATIENTS WITH TYPE 2 DIABETES
Climie REDa, Srikanth Va,b, Keith LJa, Davies JEc, Sharman JEa
aMenzies Research Institute Tasmania, University of Tasmania, Hobart, Tasmania, Australia; bStroke and Ageing Research Group, Monash Medical Centre, Monash University, Clayton, Victoria, Australia; cInternational Centre for Circulatory Health, Imperial College, London, UK
Background: Patients with type 2 diabetes (T2D) are susceptible to exercise-induced albuminuria even at submaximal exercise, but the mechanisms are unknown. Recent data indicates that T2D patients have raised central blood pressure (BP) during submaximal exercise and this could contribute to renal dysfunction independent of upper arm BP.
Aim: To determine the relationship between exercise central hemodynamics and exercise-induced albuminuria in T2D.
Methods: Forty T2D patients (aged 62 ± 9 years; 50% male) and 40 healthy controls (aged 53 ± 9 years; 50% male) were examined at rest and during a 20 minute bout of light cycle exercise (40 W; 50 rpm). Hemodynamics recorded included aortic reservoir function (excess pressure integral (xsP) and aortic reservoir pressure), aortic stiffness, augmented pressure (AP), brachial and central BP. Albuminuria was assessed by albumin/creatinine ratio (ACR) at rest and within 20 minutes after exercise.
Results: There was no difference between groups in resting ACR (P>0.05). Exercise induced a significant rise in ACR in T2D patients but not controls (0.39 ± 0.89 vs 1.05 ± 1.38 mg/mol; P=0.017). All central hemodynamic variables indicative of systolic stress were significantly higher during exercise in T2D participants (i.e., xsP, systolic BP and AP; all P<0.01). For T2D patients, exercise xsP was associated with increased ACR (β=0.003; P=0.001), independent of age, sex, body mass index, and 24 hour ambulatory SBP.
Conclusion: Aortic reservoir function, as determined by excess pressure during submaximal exercise, is independently associated with exercise-induced albuminuria in T2D patients. These novel findings suggest that aortic reservoir function could be important for appropriate renal function in patients with T2D.
INFLAMMASOME ACTIVITY IS ESSENTIAL FOR DEOXYCORTICOSTERONE ACETATE/SALT-INDUCED HYPERTENSION IN MICE
Krishnan SMa, Sobey CGa, Kemp-Harper Ba, Chan Ca, Diep Ha, Dowling Jb, Pinar Ab, Mansell Ab, Drummond GRa
aDepartment of Pharmacology, Monash University, Clayton, Victoria, Australia; bCentre of Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Clayton, Victoria, Australia
Background: Inflammasomes are signaling complexes comprised of a NOD-like receptor protein (NLRP), an adapter protein (ASC) and caspase-1. Inflammasomes detect host-derived danger signals and cause an inflammatory response via activation of caspase-1, which in turn processes the pro-inflammatory cytokines pro-interleukin (IL)-1β and pro-IL-18 into their active forms. Hypertension is associated with chronic renal inflammation, but the role of the inflammasome in this inflammatory process has not been assessed.
Aim: To investigate whether hypertension in mice is associated with increased expression and activation of the inflammasome and to assess the impact of genetic inhibition of the inflammasome on blood pressure (BP) and renal inflammation during hypertension.
Methods: Male C57BL6/J (wild type), NLRP3–/– and ASC–/– mice aged 10–12 weeks had their left kidney surgically removed, a deoxycorticosterone acetate (DOCA) pellet implanted subcutaneously (2.4 mg/d) and drinking water replaced with 0.9% saline (1K/DOCA/salt). Control mice also had their left kidney removed but received a placebo pellet and normal drinking water (1K/placebo). Tail cuff plethysmography was used to monitor systolic BP. After 21 days, mice were killed via isoflurane inhalation and the remaining kidney was excised for measurement of mRNA for NLRP3, ASC, caspase-1, pro-IL-1β and pro-IL-18, along with markers of renal inflammation (IL-6, IFN-γ, CCL2, CCL5, ICAM1, VCAM1) by real-time PCR. Inflammasome activation was assessed by Western blotting for the active caspase-1 p10 subunit (10 kDa).
Results: 1K/DOCA/salt-treated mice had elevated systolic BP (147 ± 4 mmHg) compared to 1K/placebo-treated mice (115 ± 2 mmHg; n≥13; P<0.05). Expression of NLRP3, ASC, caspase-1 and pro-IL-1β mRNA and protein expression of activated caspase-1 were all significantly increased in kidneys of 1K/DOCA/salt-versus 1K/placebo-treated mice. Inflammasome-deficient NLRP3–/– and ASC–/– mice displayed blunted hypertensive responses to 1K/DOCA/salt-treatment (149 ± 4 and 141 ± 4 mmHg, respectively) compared to wild-types (165 ± 5 mmHg; n≥5; P<0.05). Surprisingly, this reduction in BP was independent of an effect of inflammasome-deficiency on expression of inflammasome components or renal inflammatory markers.
Conclusion: 1K/DOCA/salt hypertension in mice is critically dependent on a functional inflammasome, although the mechanism by which inflammasome activity regulates BP remains to be determined.
POTENTIAL VASCULAR MECHANISMS OF RAMIPRIL-INDUCED INCREASES IN WALKING ABILITY IN PATIENTS WITH INTERMITTENT CLAUDICATION
Ahimastos AAa, Latouche Ca, Natoli AKa, Reddy-luthmoodoo Ma, Golledge Jb, Kingwell BAa
aBaker IDI Heart and Diabetes Institute and Department of Cardiovascular Medicine, Alfred Hospital, Melbourne, Victoria, Australia; bQueensland Research Centre for Peripheral Vascular Disease, James Cook University, Townsville, Queensland, Australia
Backgound: We recently reported that ramipril more than doubled walking times in peripheral artery disease patients with intermittent claudication (Ahimastos et al. JAMA. 2013:2013;309:453–460).
Aim: To conduct exploratory analyses of the effects of ramipril therapy on circulating biomarkers of angiogenesis/arteriogenesis, thrombosis, inflammation and leukocyte adhesion in patients with intermittent claudication.
Methods: 165 patients with intermittent claudication (65.3 ± 6.7SD years), were administered ramipril 10 mg/day (n=82) or matching placebo (n=83) for 24 weeks, in a randomized, double-blind study. Plasma biomarkers of angiogenesis/arteriogenesis – vascular endothelial growth factor (VEGF); fibroblast growth factor (FGF2), thrombosis (D-dimer); von Willebrand Factor (vWF); thrombin-antithrombin III (TAT), inflammation (high sensitivity C-reactive protein (hsCRP)); osteopontin (OPN), and leukocyte adhesion (soluble vascular cell adhesion molecule-1 (sVCAM-1); soluble intracellular adhesion molecule-1 (sICAM-1) – were measured at baseline and at 24 weeks.
Results: Relative to placebo, ramipril was associated with increases in VEGF by 38% (95% CI, 34–42) and FGF2 by 64% (95% CI, 44–85; P<0.001 for both), and reductions in D-dimer by 24% (–30 to –18), vWF by 22% (–35 to –9), TAT by 16% (–19 to –13), hsCRP by 13% (–14 to –9), OPN by 12% (–14 to –10), sVCAM-1 by 14% (–18 to –10), and sICAM-1 by 15% (–17 to –13; all P<0.001). These changes correlated significantly with the change in maximum walking time (P<0.001).
Conclusions: Ramipril is associated with an increase in biomarkers of angiogenesis/arteriogenesis and reduction in markers of thrombosis, inflammation and leukocyte adhesion. This study informs strategies to improve mobility in patients with intermittent claudication.
IMPLICATIONS OF DIET MODIFICATION ON SYMPATHOINHIBITORY MECHANISMS AND HYPERTENSION IN OBESITY
Sartor DM, Sfrantzis KD, How JMY
University of Melbourne, Department of Medicine, Austin Health, Heidelberg, Victoria, Australia
Background: Over 50% of cardiac output is directed towards the gut and kidney postprandially, highlighting the importance of blood flow regulation to these regions. The gastrointestinal hormone cholecystokinin (CCK) acts at vagal afferents to induce renal and splanchnic sympathoinhibition and vasodilation, via reflex inhibition of a subclass of cardiovascular-controlling neurons in the rostroventrolateral medulla. We have demonstrated that sympathoinhibitory, vasodilator and central neuronal responses to CCK are blunted in obese hypertensive rats fed a moderately high fat diet (MHFD), possibly impacting on cardiovascular homeostatic mechanisms and contributing to the etiology of obesity-related hypertension.
Aim: To determine whether swapping a MHFD for a low fat diet (LFD) would reverse the signs of hypertension and restore sympathoinhibitory reflexes in obese hypertensive rats.
Methods: Male Sprague-Dawley rats were placed on a LFD (controls; n=8) or a MHFD (n=24) for 11 weeks; the latter animals exhibited either an obesity-prone (OP) or obesity-resistant (OR) phenotype as determined by weight gain falling into the upper or lower tertile, respectively. All animals were then placed on the LFD for a further 6 week period after which they were anesthetised with isoflurane and artificially ventilated for evaluation of resting arterial pressure (AP) and renal nerve responses to CCK (0.1–4 µg/kg).
Results: Weight gain in OP animals remained higher than OR or controls following diet switch (P<0.05 for both). Resting AP was not significantly different between OP (103 ± 4 mmHg), OR (102 ± 3 mmHg) or control (104 ± 3 mmHg) animals and sympathoinhibitory responses to CCK were not significantly different between the groups (P>0.05 for all concentrations).
Conclusion: These results further implicate sympathoinhibitory mechanisms in the etiology of obesity-related hypertension, and demonstrate that diet modification can have beneficial effects on sympathetic function and restore normotension without the need for weight reduction.
DROPLET DIGITAL PCR MEASUREMENT OF COPY NUMBER VARIATION IN GENOME-WIDE ASSOCIATION REGIONS FOR BLOOD PRESSURE REVEAL ASSOCIATION WITH ESSENTIAL HYPERTENSION
Marques FZa, Prestes PRa, Pinheiro LBb, Scurrah Kc, Emslie KRb, Tomaszewski Md, Harrap SBc, Charchar FJa
aSchool of Health Sciences, University of Ballarat, Ballarat, Victoria, Australia; bNational Measurement Institute, Lindfield, New South Wales, Australia; cDepartment of Physiology, University of Melbourne, Melbourne, Victoria, Australia; dDepartment of Cardiovascular Science, University of Leicester, Leicester, UK
Background: The role of copy number variation (CNV) has been poorly explored in essential hypertension in part due to technical difficulties in accurately assessing absolute numbers of DNA copies. Droplet digital PCR (ddPCR) provides a powerful new approach to CNV quantification.
Aims: Using ddPCR to investigate whether CNVs located in regions previously associated with blood pressure (BP) variation in genome-wide association studies were associated with essential hypertension.
Methods: Using a “power of extreme” approach, we quantified nucleic acids using ddPCR in white subjects from the Victorian Family Heart Study with extremely high (n=96) and low (n=92) SBP, providing power equivalent to 1,714 subjects selected at random.
Results: A deletion of the CNVs 3306 and 64617 on chromosome 1p13.2 was significantly more prevalent in subjects with extremely high BP after adjustment for age, body mass index and sex (12.6% vs 2.2%; P=0.013). Subjects with 3 copies of the CNV 4094 on chromosome 20p12.2 had 5.5 mmHg lower diastolic BP in the groups with extremely high BP than those with 2 copies in the same group (P=0.024).
Conclusions: These data suggest that CNVs within regions identified in previous GWAS of BP variation may play a role in human essential hypertension.
CENTRAL AND SYSTEMIC ADMINISTRATION OF THE NOVEL ANCIOTENSIN TYPE 2 RECEPTOR AGONIST, COMPOUND 21, EVOKES NEUROPROTECTION IN A CONSCIOUS MODEL OF STROKE IN RATS
McCarthy CAa, Facey LFa, Miller AAb, Vinh Aa, 1Widdop REa
aDepartment of Pharmacology, Monash University, Clayton, Victoria, Australia; bDepartment of Pharmacology, RMIT University, Melbourne, Victoria, Australia
Background: Systemic angiotensin type 1 (AT1) receptor blockade is beneficial in stroke. Additionally, central angiotensin type 2 (AT2) receptor stimulation using the agonist CGP42112 is neuroprotective.
Aim: To examine the neuroprotective properties of a novel nonpeptide AT2R agonist, C21, to verify a class effect of AT2R agonists as neuroprotective agents.
Methods: Spontaneously hypertensive rats (SHR) were administered C21 (580 μg/kg) into the intracerebral ventricle, either alone or in combination with AT2R antagonist PD123319 (144 ng/kg i.c.v.) commencing 6 hours after stroke. C21 (1 mg/kg/day, i.p.) and candesartan (0.5 mg/kg/day, i.p.) were given systemically, with drug administration starting 24 hours prior to stroke. A focal reperfusion model of ischemia was induced in conscious SHR by injecting endothelin-1 to the middle cerebral artery (MCA) through a surgically implanted cannula. Motor coordination was measured at 1 and 3 days after stroke and post mortem analyses of cortical and striatal infarct volumes, microglia activation and neuronal survival were performed at 72 hours post MCA occlusion. The vascular affects of direct AT2R stimulation were assessed in isolated basilar arteries. The relationship between the AT2R and microglial polarization was examined using flow cytometric analysis. Additionally, PC12 cells were treated for 6 consecutive days with angiotensin II (10–7 M) or C21 (10–7,–8 M) alone or in combination with PD123319 (10–5 M) and the percentage of cells exhibiting neurites was calculated.
Results: Following stroke, regardless of the drug administration protocol, C21 decreased cortical infarct volume, preserved neuronal integrity and improved motor deficit to a similar degree as candesartan. Microglia obtained from untreated stroke brains revealed a positive relationship between BDNF production and AT2R expression. In vitro, C21 evoked AT2R-mediated vasorelaxation in basilar arteries, while both angiotensin II and C21 evoked neurite outgrowth in cultured PC12 cells.
Conclusion: Central and systemic administration of C21 confers neuroprotection. This benefit is likely to involve various mechanisms that conserve brain function, including microglial activation of the endogenous repair pathways, enhanced cerebroperfusion, and direct activation of neurotrophic pathways. Thus, we have confirmed the neuroprotective effect of AT2R stimulation using a nonpeptide compound. The results indicate a class effect and highlight the clinical potential of the AT2R agonists for future development.
VASCULAR ENDOTHELIAL GROWTH FACTOR-MEDIATED SIGNALOSOME FORMATION AND S-NITROSYLATION OF CELL CYCLE-RELATED PROTEINS IN PROLIFERATING HUMAN ENDOTHELIAL CELLS
Gentile Ca,b,c, Muise-Helmericks RCa, Davies MJb,c, Drake CJa
aDepartment of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, South Carolina, USA; bThe Heart Research Institute, Newtown, Sydney, New South Wales, Australia; cFaculty of Medicine, University of Sydney, Sydney, New South Wales, 2006, Australia
Background: In previous studies we established the role played by the VEGF/Akt1/eNOS/NO signaling pathway in mediating cell proliferation of angioblasts and embryonic endothelial cells (ECs) during murine development (Gentile et al., Dev Biol 2013;373:163–175). These findings, together with the emerging role(s) played by HSP90 in mediating the formation of the signalosome (the multimolecular complex comprising pAkt1, peNOS, HSP90 and the molecular target of NO) in human endothelial cells, led us to investigate the relevancy of the VEGF/eNOS signaling pathway in human ECs.
Aim: To investigate the role(s) played by the VEGF/eNOS signaling pathway in mediating signalosome formation in proliferating human ECs.
Methods: Levels of eNOS phosphorylation at S1177 (peNOS) were evaluated by western blot analysis in serum-starved (SS) human umbilical vein endothelial cells (HUVECs) and HUVECs synchronized in the G2/M phase by the addition of nocodazole. We also compared proliferation between HUVECs overexpressing wild type eNOS (eNOS HUVECs) and HUVECs that express a mutated, unphosphorylatable eNOS (eNOSΔS1177A HUVECs).
Results: peNOS levels were increased in mitotic HUVECs compared to SS HUVECs. An increase in proliferation observed in eNOS HUVECs compared to HUVECs overexpressing a scrambled plasmid was dependent on the phosphorylation of eNOS on Ser1177. Furthermore, VEGF mediated the S-nitrosylation of cyclin B1 (controlling the G2/M transition) in HUVECs by western blot analysis. There was a correlation between HSP90 binding and S-nitrosylation of cyclin B1 following VEGF treatment. Finally, VEGF-mediated S-nitrosylation of cyclin B1 was substantially reduced in the presence of 17-AAG, an inhibitor of HSP90, demonstrating that the VEGF-mediated S-nitrosylation of cyclin B1 is dependent on HSP90 in human ECs.
Conclusions: These studies have demonstrated for the first time that VEGF-mediated proliferation in human ECs is dependent on the S-nitrosylation of cyclin B1 following signalosome formation. Current studies are focusing on evaluating whether VEGF mediates proliferation via S-nitrosylation of cell cycle-related proteins in other cell types, such as cardiomyocytes.
EXERCISE SYSTEMIC VASCULAR RESISTANCE AND RAISED TRIGLYCERIDES INDEPENDENTLY PREDICT A HYPERTENSIVE RESPONSE TO EXERCISE
Nikolic SBa, Adams MJb, Otahal Pa, Edwards LMc, Sharman JEa
aMenzies Research Institute Tasmania, University of Tasmania, Hobart, Tasmania, Australia; bSchool of Human Life Sciences, University of Tasmania, Launceston, Tasmania, Australia; cCentre of Human & Aerospace Physiological Sciences, King’s College London, London, UK
Background: A hypertensive response to moderate intensity exercise (HRE) is associated with increased cardiovascular mortality risk. Preliminary data suggests this relationship may be explained by hemodynamic, metabolic and/or hematological factors. The present study investigated these postulates.
Methods: Eighty participants aged 57 ± 10 years (71% male) with indication for exercise stress testing underwent comprehensive hemodynamic assessment including brachial and central blood pressure (BP), aortic stiffness and systemic vascular resistance (SVR) at rest and during moderate intensity exercise. Various metabolic and hematological markers were determined from blood samples taken at rest and following exercise (including triglycerides and von Willebrand factor (vWF)).
Results: A total of 24 participants were identified with HRE (exercise systolic BP ≥170 mmHg in men and ≥160 mmHg in women). At rest, HRE participants had higher body mass index (BMI), brachial and central BP, aortic stiffness, triglycerides and vWF compared to normal BP responders (P<0.05 for all), but had no difference in SVR (P>0.05). During exercise, however, HRE participants had significantly increased SVR (1777 ± 239 vs. 1579 ± 232 dyne/s/cm-5; P=0.002). Exercise SVR, triglycerides and vWF (but not other hematological and metabolic markers) were significantly related to exercise brachial systolic BP (r=0.444, r=0.396 and r=0.276, respectively; P=0.05 for all). After multiple regression analysis, exercise SVR and triglycerides predicted exercise brachial systolic BP, independent of age, sex, BMI and vWF (adjusted R2=0.298; P<0.001).
Conclusions: Increased exercise SVR and triglycerides are independently related to increased exercise systolic BP at moderate intensity. Thus, peripheral vascular function and hyperlipidemia may be mechanisms explaining exercise hypertension.
EMERGING VISUAL METHODOLOGIES TO BETTER UNDERSTAND THE PROCESS OF CARE IN HYPERTENSION MANAGEMENT IN AUSTRALIAN GENERAL PRACTICE
Howes FSa, Nash Mb, Walters Jc, Nelson MRa
aMenzies Research Institute, University of Tasmania, bSchool of Social Sciences, University of Tasmania, cSchool of Medicine, University of Tasmania, Hobart, Tasmania, Australia
Background: Despite high blood pressure being a leading cause of morbidity and mortality, evidence-practice gaps persist. Modifying physician behaviour by introducing evidence and clinical guidelines into routine daily practice can be challenging, with few studies examining the basis for provider behaviour. General practitioners (GPs) view the management of hypertension as their area of speciality. In Australia the majority of blood pressure care occurs within general practice, with 9% of consultations related to hypertension.
Aim: To formulate a methodology for a mixed methods investigation titled: Understanding clinical decision-making in the management of hypertension in Australian general practice (ACTRN12613000056796).
Methods: A systematic literature review of videorecording consultations in general practice was conducted with the practical objectives of describing the aim of previous video-methodology studies and distilling practical guidance on how to design and conduct such studies. Ethical issues including those of consent and the occurrence of any problems encountered were also extracted.
Results: Qualitative visual methodologies were illustrated with previously published work and emerging data from the study underway. Searches were undertaken of PubMed, Embase, Cinahl and the CDSR combining MeSH terms for Video and Method and terms for General Practice. 87 papers met the inclusion criteria. The number of GPs recruited ranged from 12–42, with subsequent numbers of patients recruited ranging from 46–385. Most often, patients were recruited by researchers consecutively prior to their consultation. The video method was rarely detailed and the majority of authors adopted a quantitative analytic technique.
Conclusion: Qualitative visual methodologies allow for deeper understanding of the barriers to optimal blood pressure management.
THE MEDIAL AMYGDALA CONTRIBUTES TO OVERACTIVITY OF THE SYMPATHETIC NERVOUS SYSTEM AND RENIN-ANGIOTENSIN SYSTEM IN GENETICALLY HYPERTENSIVE BPH/2J MICE
Jackson KLa,b Moretti J-La, Abegaz Ba, Stevenson ERa, Davern PJa, Head GAab
aNeuropharmacology Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia; bDepartment of Pharmacology, Monash University, Clayton, Victoria, Australia
Background: We have shown that lesions of the medial amygdala (MeAm) reduce blood pressure (BP) in BPH/2J mice but not in BPN/3J mice, suggesting that neurons located within the MeAm contribute to hypertension. Hypertension in BPH/2J mice also appears to involve a sympathetically mediated enhancement of the peripheral renin-angiotensin system (RAS).
Aim: To determine whether the MeAm influences BP in hypertensive BPH/2J mice via enhancement of the sympathetic nervous system (SNS) and RAS.
Methods: Pre-implanted radiotelemetry probes were used to measure BP before and three weeks after microinjection of ibotenic acid into the MeAm to create bilateral lesions in normotensive BPN/3J and hypertensive BPH/2J mice. Acute cardiovascular responses induced by ACE inhibitor enalaprilat (1.5 mg/kg, i.p.), followed thirty minutes later by ganglion blocker pentolinium (5 mg/kg, i.p.) were measured in BPH/2J mice.
Results: MeAm lesions did not influence BP in BPN/3J mice (P=0.8; n=7), but reduced average 24 h BP in BPH/2J mice by 13 ± 2 mmHg (P<0.001; n=7). In BPH/2J mice, after pre-treatment with enalaprilat, injection of pentolinium caused marked depressor responses during the light (–592 mmHg) and dark (–552 mmHg) periods. Following MeAm lesions, the depressor responses to pentolinium were attenuated by 29% and 33% during the light and dark periods, respectively (Plesion<0.003). Treatment with enalaprilat alone did not influence BP during the light period, but reduced BP from baseline during the dark period (–11 ± 7 mmHg; P<0.001). MeAm lesions abolished the depressor response induced by enalaprilat during the dark period (Plesion=0.04).
Conclusion: Taken together with previous findings the results suggest that neurons located within the MeAm contribute to hypertension in BPH/2J mice via enhancement of the SNS and RAS.
EFFECTS OF CONTINUOUS FIELD ACTIVATION OF CAROTID BARORECPTORS ON CHANGES OF ARTERIAL PRESSURE IN THE RAT
Kouchaki Za, Butlin Ma, Georgakopuolos Db, Avolio Aa
aAustralian School of Advanced Medicine, Macquarie University, Sydney, New South Wales, Australia; bCVRx Inc., Minneapolis, Minnesota, USA
Background: Electric field activation of carotid baroreceptors is used for treatment of resistant hypertension. Although baroactivation is generally associated with reduction of blood pressure, there is, however, a marked variability in the response across individuals. The present study addresses the effect of baroactivation in a rat model in which blood pressure was modulated by pharmacological agents affecting peripheral resistance.
Aim: To assess the effects of carotid baroactivation in the presence of pharmacologically induced changes in blood pressure.
Methods: Male WKY rats (aged 12 weeks; n=6) were anesthetized (urethane, 1.3 g/kg) and implanted with a field stimulation electrode (1 kHz, pulse width of 0.53 ms) surrounding the common carotid artery, immediately proximal to the carotid bifurcation. Mean arterial pressure (MAP) was measured using two pressure sensors located in the proximal and distal descending aorta. Vasoactive agents (10 µg phenylephrine (PE) or 30 µg sodium nitroprusside (SNP), intravenous bolus) were used to alter MAP to quantify the induced changes in blood pressure before and during continuous field activation of carotid baroreceptors.
Results: MAP decreased significantly with stimulation (14 ± 7.9 mmHg) accompanied by a simultaneous increase in heart rate (HR) (25 ± 6.2 bpm). These new baselines for MAP and HR were maintained during the entire stimulation period (30–60 minutes) with injection of vasoactive agents. The injection of PE and SNP caused significant (P<0.01) changes in MAP during stimulation (58 ± 12 mmHg and 26 ± 18 mmHg, respectively), as well as changes in HR (43 ± 29 bpm and 18 ± 8 bpm, respectively). These changes did not show any significant difference with injection of drugs in the absence of baroreceptor stimulation, showing that field activation of carotid baroreceptors does not reduce the capacity for cardiovagal control of blood pressure.
Conclusion: The findings show a consistent decrease in arterial blood pressure during field activation of carotid baroreceptors in the presence of large pressure changes due to pharmacological manipulation of peripheral resistance. Another significant result was stability of baroreceptor sensitivity during stimulation.
NITRIC OXIDE DEFICIENCY PROMOTES THE ENHANCED AGE-RELATED DECLINE IN RENAL FUNCTION IN 5-YEAR OLD FEMALE SHEEP FOLLOWING FETAL UNINEPHRECTOMY
Lankadeva YRa, Singh RRa, Tare Ma, Moritz KMb, Denton KMa
aDepartment of Physiology, Monash University, Clayton, Melbourne,Victoria, Australia; bSchool of Biomedical Sciences,University of Queensland, Brisbane, Queensland, Australia
Background: A congenital reduction in renal mass is associated with an increased risk of renal impairment and hypertension in adulthood. To understand the underlying mechanisms we have established a model of fetal uninephrectomy (uni-x) in sheep, in which arterial pressure is chronically elevated and renal blood flow (RBF) declines with age in uni-x female sheep. We hypothesized that the age-dependent decline in RBF in 5-year old female uni-x sheep is due to a reduction in input from nitric oxide (NO).
Aims: To examine the role of NO in age-related decline in cardiovascular and renal responses in sheep.
Methods: Cardiovascular and renal responses were examined before and after an acute saline load (2.5% body weight) during systemic NO synthase inhibition via L-NAME (40 mg/kg bolus + 20 mg/kg/h i.v) or vehicle (saline) treatment in conscious female uni-x (n=7) and sham (n=7) sheep at 5 years of age.
Results: Basal mean arterial pressure (MAP) was ~15 mmHg higher, whilst glomerular filtration rate (GFR), RBF and urinary sodium excretion (UNa+V) were all ~30% lower (all PGROUP<0.001) in uni-x sheep. L-NAME increased basal MAP by ~17 mmHg in both groups (PGROUP<0.001). The reduction in basal GFR, RBF and UNa+V in response to L-NAME was, however, attenuated in uni-x compared with sham sheep (all PGROUPXTREAT<0.01). Saline loading during vehicle treatment induced rises in RBF, GFR and UNa+V in both groups. These responses were, however, all significantly attenuated in uni-x sheep (PGROUPXTREAT<0.01). In sham sheep, in the presence of L-NAME, the increases in RBF and GFR to a saline load were significantly blunted, whilst the natriuresis was markedly augmented (all PGROUPXTREAT<0.05). In contrast, in uni-x sheep NO inhibition did not alter the RBF, GFR or UNa+V responses to saline loading (all PGROUPXTREAT>0.05).
Conclusion: Renal dysfunction in aged uni-x female sheep was associated with impaired NO modulation of renal hemodynamic and sodium excretory function. Thus, renal NO deficiency may accelerate decrements in cardiovascular and renal function in aged subjects born with a reduced renal mass. Therefore, increasing NO bioavailability in individuals with a congenital reduction in renal mass may be an effective therapeutic strategy to prevent further loss of renal function with age.
THE ALTERED DOWNSTREAM REGULATION OF LEPTIN SIGNALING IN HIGH FAT DIET FED RABBITS
Lim Ka, Barzel Ba,b, Tassone Fa,c, Burke Sa, Head Ga
aNeuropharmacology Laboratory, Baker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia; bDepartment of Anatomy & Developmental Biology and cDepartment of Physiology, Monash University, Clayton, Melbourne, Victoria, Australia
Background: A high fat diet (HFD) leads to an increase in blood pressure (BP) and renal sympathetic nerve activity (RSNA) in rabbits. We suggest that there is an altered downstream sensitivity to leptin in the hypothalamus and that this involves an altered responses to α-melanocyte stimulating hormone (α-MSH) and neuropeptide Y (NPY) when rabbits are fed a HFD.
Aim: To investigate the role of α-MSH and NPY in the hypothalamus of HFD-fed rabbits.
Methods: New Zealand white rabbits were implanted with a bilateral guide cannula (into the dorsomedial hypothalamus (DMH) or ventromedial hypothalamus (VMH)) and with an RSNA recording electrode, and were then placed on a normal or a 13.3% HFD for 3 weeks. After 3 weeks on the diet, rabbits were administered α-MSH (0.1 and 0.3 nmol), NPY (0.1 and 0.5 nmol) or Ringer’s solution directly into the DMH or VMH.
Results: Compared to controls, rabbits fed a HFD exhibited higher BP, HR and RSNA (n=6–8). α-MSH injection into the DMH increased BP by 5.7% with 0.1 nmol (P<0.05) and 8.3% with 0.3 nmol (P<0.01), and increased RSNA by 40% with 0.3 nmol (P<0.05) in HFD rabbits. When α-MSH was injected into the VMH, BP was unchanged, but RSNA was increased by 28% (P=0.01) and 38% (P<0.01) in HFD rabbits. NPY injection had no effect in either control or HFD rabbits.
Conclusion: Elevated blood pressure and sympatho-excitation in HFD rabbits are due to increased sensitivity of the leptin signaling pathway. This involves a dorsal and ventral hypothalamic projection of α-MSH activated second-order neurons.
A NOVEL POPULATION OF CARDIAC RESIDENT STEM CELLS ISOLATED FROM HUMAN ATRIA
Sivakumaran Pa, Newcomb AEb, Liu GSc, Elliott DAd, Dusting GJa,c, Lim SYa
aO’Brien Institute and bDepartment of Cardiothoracic Surgery, St Vincent’s Hospital, Melbourne; cCentre for Eye Research Australia, Melbourne; dMurdoch Childrens Research Institute, Melbourne, Victoria, Australia
Background: Stem cell-based therapies to repair and regenerate lost myocardium have potential to revolutionize modern medicine for treatment of myocardial infarction and heart failure. We have recently identified a novel subclass of human cardiac resident stem cells (CRSCs) that are positive for W8B2 antigen.
Aim: To characterize the human W8B2+ CRSCs isolated from adult human atrial appendages.
Methods: W8B2+ CRSCs were isolated from explants of human atrial appendages. Cytokines were screened by microarray using culture media and cell lysate harvested from confluent W8B2+ CRSCs. For tissue engineering, 3x106 W8B2+ CRSCs were implanted into an in vivo vascularized tissue engineering chamber in immunocompromised rats. Tissue constructs were harvested 4 weeks later for histological analysis.
Results: Human W8B2+ CRSCs can self-renew (population doubling time of ~27 hours) and are highly clonogenic (cloning efficiency of 50%). Immunophenotyping showed they were CD29+ (100%), CD73+ (87.4%), CD90+ (65.7%), CD105+ (68.6%), CD106+ (85.6%), HLA-ABC+ (83.3%), were largely negative for HLA-DR+ (0.03%), CD31+ (1.7%), CD34+ (1.3%), CD45+ (0.2%), CD133+ (0.2%), and Lin+ (0.8%) (n=4). RT-PCR analysis of these cells failed to detect Islet-1 or Nkx2.5 mRNA expression, and immunostaining showed that W8B2+ CRSCs expressed GATA4, CD271, vimentin and nestin, but not Wilm’s tumor gene-1 (a specific marker of epicardial progenitors) or discoidin domain receptor-2 (a specific marker of fibroblasts). Of 274 human cytokines analysed, W8B2+ CRSCs were found to secret various cytokines implicated in angiogenesis (angiogenin, angiopoietin, bFGF), chemotaxis (MCP-1, eotaxin, RANTES, etc), inflammation (IL-6, GDF-15, etc), extracellular matrix remodelling (decorin, MMP-10, etc), growth and survival (DPP-4, HGF, follistatin, etc). W8B2+ CRSCs can differentiate into cardiogenic cells that were responsive to electrical stimulation, into endothelial and smooth muscle cells, as well as being able to undergo adipogenesis, osteogenesis and chondrogenesis. W8B2+ CRSCs survived through in vivo implantation and were found to occupy the perivascular spaces.
Conclusion: W8B2+ CRSCs are distinct from currently known CRSCs found in human hearts. W8B2+ CRSCs may be an ideal cardiovascular cell source for tissue engineering and for autologous cell therapies in patients with cardiovascular diseases.
RAISED SOLUBLE P-SELECTIN MODULATES PLAQUE INSTABILITY IN AN EXPERIMENTAL MODEL OF VULNERABLE PLAQUE
Lumsden NGa, Woollard KJb, Andrews KAa, Aprico Aa, Irvine JCa, Jefferis AMa, Fang KLa, Kanellakis Pa, Bobik Ab, Chin-Dusting JFPb
aBaker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia; bDivision of Immunology & Inflammation, Department of Medicine, Imperial College London, London, UK
Background: We have previously shown that sP-selectin increases leukocyte recruitment to immobilized platelets and the resting endothelium. We therefore hypothesize that chronically raised sP-selectin will hasten the progression of atherothrombosis in a genetically susceptible mouse model of the disease, the SM22α-hDTR Apo–/– mouse.
Aim: To determine whether sP-selectin alters plaque phenotype in SM22α-hDTR Apoe–/– mice.
Methods: SM22α-hDTR Apoe–/– mice and Apoe–/– littermate controls were placed on a high fat diet (0.15% cholesterol, 21% fat) and treated for 16 weeks with recombinant murine sP-selectin or sE-selectin (150 ng/ml for each administered s.c. by daily injections). Five ng/ml of diphtheria toxin was injected 3 times per week from week 12–16 to induce VSMC apoptosis. Six-micrometer cross-sections of aortic sinus were examined for macrophage and leukocyte accumulation via CD68 and CD45, respectively, lipid deposition via oil red O and collagen content via picrosirius red staining. Plasma was analysed for cytokine/chemokine content.
Results: Plaque analysis indicated that sP-selectin treatment was associated with: significantly higher macrophage accumulation (sP-selectin: 11.7 ± 2.3SE %; sE-selectin: 5.0 ± 1.07 %; n=6–8; P=0.01); significantly higher CD45 content (sP-selectin: 9.3 ± 1.4 %, sE-selectin: 5.8 ± 0.52 %; n=7–8; P=0.03) and significantly lower collagen content (sP-selectin: 5.8 ± 0.72 % vs sE-selectin: 11.3 ± 1.7 %; n=5–7; P=0.04). Lipid deposition was not significantly different between groups. After 16 weeks of treatement, plasma MCP-1 and RANTES were increased and decreased, respectively, in sP-selectin treated mice.
Conclusion: These results suggest that chronically raised sP-selectin favours progression of an unstable atherosclerotic plaque phenotype.
T CELL INFILTRATION IN THE AORTA AND KIDNEY OF HYPERTENSIVE RATS IS Y CHROMOSOME DEPENDENT
Memon Ba, Chin-Dusting JPFa, Jennings GLa, Vinh Ab, Sampson AKa
aBaker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia; bDepartment of Pharmacology, Monash University, Clayton, Victoria, Australia
Background: The immune system is essential for the development of hypertension. We have shown whether the Y chromosome from a hypertensive or normotensive rat strain accounts for 12–15 mmHg difference in blood pressure. The copy of the Y chromosome in humans associated with an increased risk of CVD is also associated with an upregulation of inflammatory gene expression, suggesting that the origin of the Y chromosome may influence CVD risk by augmentation of the immune response. Whether the origin of the Y chromosome directly influences immune responses in the context of hypertension, remains unknown.
Aim: To examine the influence of the origin of the Y chromosome on immune cell infiltration – specifically T cells (CD45+CD3+), T helper cells (CD45+CD3+CD4+), cytotoxic T cells (CD45+CD3+CD8+) and regulatory T cells (CD45+CD3+CD4+Foxp3+).
Methods: Immune cell number in the aorta, kidney, spleen and blood was quantified using flow cytometry in 16 week-old normotensive (WKY), hypertensive (SHRSP) and two Y consomic rat strains, one comprising the WKY background with the hypertensive Y chromosome (WKY.SPGlaY) and the other the opposite (SP.WKYGlaY).
Results: We confirmed via radiotelemetry that systolic blood pressure (SBP) was Y chromosome dependent. SP.WKYGlaY had lower SBP than SHRSP (195 ± 5 mmHg vs 227 ± 8 mmHg; P<0.05) and SBP was higher in WKY.SPGlaY compared to WKY (157 ± 3 mmHg vs 148 ± 3 mmHg; P<0.05). We observed a greater number of infiltrated T cells in the aorta and kidney of SHRSP compared to WKY, attributable to a greater infiltration of T helper cells in the aorta and a greater infiltration of T helper cells, cytotoxic T cells and regulatory T cells in the kidney. Replacement of the hypertensive Y chromosome with the normotensive Y chromosome (SP.WKYGlaY) significantly reduced T cell infiltration to WKY levels in the aorta and kidney. There were no differences in circulating plasma or splenic immune cell levels between any of the strains, suggesting that the origin of the Y chromosome influences T cell function and infiltration rather than production.
Conclusion: Our data demonstrates that the increased T cell infiltration observed in hypertensive rats in target cardiovascular organs, such as the kidney and aorta, is Y chromosome-dependent. We suggest that there are genes located on the Y chromosome that influence blood pressure regulation via augmentation of immune responses and that they may predispose certain males to the development of CVD.
A CCR2 ANTAGONIST, INCB3344, REDUCES MACROPHAGE ACCUMULATION AND AORTIC FIBROSIS AND LOWERS BLOOD PRESSURE IN ANGIOTENSIN II-INDUCED HYPERTENSION IN MICE
Moore JPa, Krishnan Sa, Samuel CSa, Kemp-Harper Ba, Sakkal Sb, Ricardo SLc, Sobey CGa, Drummond GRa
aVascular Biology and Immunopharmacology Group, Department of Pharmacology, Monash University, Clayton, Victoria, Australia; bCollege of Health and Biomedicine, Victoria University, Melbourne, Victoria, Australia; cSchool of Biomedical Sciences, Monash University, Clayton, Victoria, Australia
Background: Vascular fibrosis and stiffening are hallmarks of hypertension and contribute to elevated systolic blood pressure (SBP) and end organ damage. Hypertension is also associated with macrophage accumulation in the vessel wall. Macrophages are attracted into tissues by chemokines and can adopt pro-inflammatory (M1) or anti-inflammatory/pro-fibrotic (M2) phenotypes. Thus, macrophages could conceivably contribute to the vascular changes that occur in hypertension.
Aim: To determine the polarization state of macrophages in the vessel wall during hypertension and to investigate if pharmacological antagonism of the chemokine receptor-ligand interactions responsible for their accumulation reduces vascular fibrosis and stiffening, and SBP in hypertension.
Methods: Angiotensin II (Ang II) (0.7 mg/kg/day, s.c.) was infused for 28 days into male C57BL6/J mice. Macrophage markers were mearured by flow cytometry and real-time PCR. The effect of a CCR2 antagonist, INCB3344 (30 mg/kg/d, i.p.), on the response to Ang II was examined.
Results: Ang II infusion caused a sustained increase in SBP (158 ± 3 mmHg) compared to saline-infusion (122 ± 3 mmHg; n≥24; P<0.0001). There was a 2-fold increase in total macrophage (F4/80+) numbers in Ang II-infused mice. These cells were M2 polarized based on expression of the M2 marker, CD206. Real-time PCR confirmed that CD206 expression was elevated in aortas from Ang II-treated mice, along with other markers of M2 polarization, arginase-1 and FCRLS1 (n≥9; P<0.05). By contrast, expression of M1 markers iNOS, CXCL2 and TNF was unchanged. A PCR array to identify potential chemokine receptor targets identified a 2-fold increase in aortic expression of CCR2 and its ligand CCL8 in aortas from Ang II-treated mice (n≥9; P<0.01). INCB3344, administered 7 days after Ang II, reduced aortic macrophage numbers by 55% (n≥11; P<0.01). INCB3344 also reduced Ang II-induced collagen deposition (picrosirius red) by 20% (n≥5; P<0.05) and tended to improve ex vivo aortic compliance. Importantly, INCB3344 prevented Ang II-induced increases in heart weight (mg) to body weight (g) ratio from 6.5 ± 0.3 to 5.5 ± 0.1 (n=7; P<0.05), and profoundly reduced SBP from 159 ± 3 mmHg to 138 ± 3 mmHg (n≥14; P<0.0001).
Conclusion: CCR2 may represent a promising target for future therapies aimed at reducing macrophage influx, vascular fibrosis and stiffening, and elevated SBP during hypertension.
INSULIN/INSULIN-LIKE GROWTH FACTOR-1 SIGNALING PATHWAY GENE POLYMORPHISMS: AN ASSOCIATION STUDY OF HEALTHY AGING AND LONGEVITY
Morris BJa–c, Donlon TAa, He Qa, Grove JSa,d, Masaki KHa,b, Elliott Aa, Willcox DCa,e, Willcox BJa,b
aThe Hawaii Lifespan and Healthspan Study, Kuakini Medical Center and bDepartment of Geriatric Medicine, John A. Burns School of Medicine, University of Hawaii, Kuakini Medical Center, Honolulu, Hawaii, USA; cBasic & Clinical Genomics Laboratory, School of Medical Sciences, University of Sydney, Sydney, New South Wales, Australia; dPublic Health Sciences, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, USA; eOkinawa International University, Ginowan, Okinawa, Japan
Background: Evolutionarily conserved nutrient/energy-sensing pathways influence common conditions of aging and lifespan. The hyperfunction theory of aging states that growth promoting pathways operating early in life become pathogenic in later life by causing overstimulation, growth and hypertrophy as seen in diseases of aging. One of these is the insulin/insulin-like growth factor signaling pathway (IIS). The Hawaii Lifespan/Healthspan Study was the first to show that genetic variants of a crucial gene in the IIS pathway, FOXO3A, are associated with extreme old age (defined as living beyond 95 years) in the HHP/HAAS cohort (Willcox et al. PNAS 2008;105:13987–13992). This finding has been replicated by others in further cohorts aged 95 years and over worldwide. Here we explore single nucleotide polymorphisms (SNPs) of other important IIS pathway genes for association with extreme old age.
Aim: To test SNPs of IIS pathway genes that may be influenced by the transcription factor FOXO3A for association with human longevity and clinically relevant aging phenotypes.
Methods: Subjects comprised a homogeneous population of American men of Japanese ancestry, well-characterized for aging phenotypes and that have been followed for 48 years. We used a nested-case control study design involving 213 subjects aged ≥95 years (range 95–106) followed to Aug 2007 and 402 controls (age at death 73–81). Genotypes were determined for 3 tagging SNPs for ATF1, 6 for CBL, 4 for JUN, as well as single SNPs for CDKN2B and EXO1. Association analyses involved χ2 and ANOVA.
Results: Long-lived cases were healthier than controls at examination 4 (in 1991–1993) when aged 71–93. They had lower glucose, log insulin, HOMA, systolic and diastolic blood pressure, less hypertension, stroke, cancer, difficulty waking 0.8 km, better grip strength and self-rated health. After correction for multiple testing we found no significant association of any of the 15 SNPs with longevity, nor with hypertension or any of 19 other phenotypes relevant to aging in this population.
Conclusion: This study failed to implicate additional IIS pathway gene variants in human longevity. (Morris et al., J Gerontol A Biol Sci Med Sci 2014;69:270-273)
HUMAN TOR COMPLEX COMPONENTS IN GENETICS OF LONGEVITY AND HYPERTENSION
Morris BJa–c, Donlon TAa, He Qa, Grove JSa,d, Masaki KHa,b, Elliott Aa, Willcox DCa,e, Allsopp Rf, Willcox BJa.b
aThe Hawaii Lifespan and Healthspan Study, Kuakini Medical Center and bDepartment of Geriatric Medicine, John A. Burns School of Medicine, University of Hawaii, Kuakini Medical Center, Honolulu, Hawaii, USA; cBasic & Clinical Genomics Laboratory, School of Medical Sciences, University of Sydney, Australia; dPublic Health Sciences, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii, USA; eOkinawa International University, Ginowan, Okinawa, Japan; fInstitute for Biogenesis Research, University of Hawaii, Honolulu, Hawaii, USA
Background: Evolutionarily conserved nutrient/energy-sensing pathways influence common conditions of aging and lifespan. Each of these likely invoke the hyperfunction theory of aging. Mechanistic target of rapamycin (mTOR) is at the hub of one such pathway. mTOR inhibition extends mammalian lifespan. mTOR is a critical regulator of cardiac hypertrophy in the SHR. Although genetic factors play major roles in chronic conditions of aging and attainment of extreme old age, few genes have been implicated.
Aim: To test tagging single nucleotide polymorphisms (tagSNPs) that captured most of the genetic variation across the key TOR complex 1 (TORC1) and TOR complex 2 (TORC2) genes MTOR, RPTOR and RICTOR and the important downstream effector gene RPS6KA1 for association with human longevity (defined as attainment of at least 95 years of age) and clinically relevant phenotypes of aging.
Methods: Subjects comprised a homogeneous population of American men of Japanese ancestry, well characterized for aging phenotypes and that have been followed for 48 years. We used a nested-case control study design involving 440 subjects aged ≥95 years (range 95–106) and 374 controls (age at death 73–81). Genotypes were determined for 6 tagging SNPs for MTOR, 61 for RPTOR, 7 for RICTOR, as well as 5 for RPS6KA1. Associations were examined for 40 phenotypes. Analyses involved χ2 and ANOVA.
Results: Data showed long-lived cases were healthier than controls, especially after adjusting for age. They had lower blood pressure and less hypertension at age 71–93 years. There was no significant association of any of the 79 SNPs with longevity. Phenotypes related to blood pressure, essential hypertension (EHT), isolated systolic hypertension (ISH) and body weight showed genotypic associations with 23 tagSNPs of RPTOR. The associations remained after correction for multiple testing.
Conclusion: TOR complex genes showed no genetic association with longevity, but an association of RPTOR SNPs with hypertension and related phenotypes was apparent in the Kuakini cohort. (Morris et al., J Gerontol A Biol Sci Med Sci. 2014:e-pub ahead of print Mar 3)
A QUANTITATIVE ANALYSIS OF THE FACTORS INFLUENCING OXYGEN DIFFUSION IN THE VICINITY OF ARTERY-VEIN PAIRS IN THE KIDNEY
Ngo JPa, Kett MMa, Pearson JTa, Gardiner BSb, Smith DWb, Abdelkader Aa, Kar Sb, Bertram JFc, Evans RGa
aDepartment of Physiology, Monash University, Clayton, Melbourne, Victoria, Australia; bSchool of Computer Science and Software Engineering, University of Western Australia, Perth,Western Australia; cDepartment of Anatomy and Developmental Biology, Monash University, Clayton, Melbourne, Victoria, Australia.
Background: Diffusion of oxygen from arteries to veins in the kidney (AV oxygen shunting) acts to limit oxygen delivery to renal tissue. We recently employed computational modeling to identify two factors critical to determination of the quantity of AV oxygen shunting within the renal circulation. These were (i) the distance between the artery and the vein, and (ii) the degree to which the vein wraps around the wall of the artery.
Aim: To quantify how the factors in (i) and (ii) above change along the course of the renal circulation.
Methods: The renal vasculature of Sprague Dawley rats (n=6) was perfusion fixed and filled with Microfil®. A section from each kidney was chosen and the shortest arterial/arteriolar diameter, distance to the nearest vein, and the degree to which a vein wraps an artery were measured.
Results: The diffusion distance between arteries and veins increased with decreasing arterial diameter (Figure 1). The proportion of the arterial wall surrounded by the vein (wrapping) decreased as arterial diameter decreased (Figure 2).
Conclusions: The spatial relationships (separation and wrapping) between arteries and veins that promote AV oxygen shunting are more prominent in the larger vessels than the smaller vessels of the kidney. These observations challenge the conventional notion that most AV oxygen shunting occurs in the smaller cortical vessels (e.g., interlobular arteries) after the divergence of the cortical and medullary circulations. Thus, AV oxygen shunting may limit oxygen delivery to the renal medulla as well as the renal cortex.
BLOOD-BRAIN BARRIER LEAKAGE IN THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS OF RENOVASCULAR HYPERTENSIVE RATS
Nishi EEa, May CNb, Camposz RR, Bergamaschi CTa, Yao ST
aUniversidade Federal de Sao Paulo, Sao Paulo, Brazil; bFlorey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Victoria, Australia
Background: An intact blood-brain barrier (BBB) is important for normal brain function. Whilst the integrity of the BBB is thought to be affected in a number of diseases, its permeability has not yet been investigated in hypertensive animals.
Aim: To evaluate whether, in an angiotensin II-dependent model of hypertension, two-kidney one clip (2K1C) hypertension, the BBB is disrupted in the paraventricular nucleus of the hypothalamus (PVN), a nucleus that plays a central role in cardiovascular regulation.
Methods: Renovascular hypertension was induced by placing a constricting clip around the left renal artery to reduce renal blood flow in male Sprague-Dawley rats (180–200 g). At 42 days after renal clip placement (n=6) or sham (n=5) surgery, the femoral artery was catheterized under isofluorane (1.8–2.5% in 100% oxygen) anesthesia and, following recovery, direct measurement of arterial blood pressure and Evans Blue (EB) 2% (2 ml/kg) intra-arterial infusion (over approximately 20 s) were performed in conscious rats. Rats were decapitated 30 min after the EB infusion and the brains were collected for histological analysis. To assess the permeability of BBB, coronal sections (20 µm thick) containing the PVN were cut and EB labeling intensity was quantified using fluorescent microscopy and image analysis software (Image J, NIH).
Results: Hypertensive rats (systolic blood pressure 188 ± 4 vs. control 109 ± 5 mmHg; P<0.0001) showed greater EB leakage in the PVN compared with control normotensive rats. EB intensity was 31% higher in the parvocellular subdivision (P<0.05) and 39% higher in the magnocellular subdivision (P<0.05) of the PVN in the 2K1C group. No significant change of EB intensity was, however, found in the cortex.
Conclusion: Our data show that in the PVN of 2K1C rats the BBB is compromised, suggesting that in hypertensive states this cardiovascular region might be susceptible to the actions of substances that do not normally cross the BBB.
RENAL TISSUE HYPOXIA IN A RAT MODEL OF POLYCYSTIC KIDNEY DISEASE
Ow CPCa, Abdelkader Aa, Phillips JKb, Evans RGa
aDepartment of Physiology, Monash University, Clayton, Melbourne, Victoria, Australia; bThe Australian School of Advanced Medicine, Macquarie University, Sydney, New South Wales, Australia
Background: Polycystic kidney disease (PKD) is characterized by the development of numerous fluid-filled cysts. Hypoxia has been identified as a final common pathway in the progression of chronic kidney disease. Histological analysis of kidney sections from animals with PKD have provided qualitative evidence of tissue hypoxia. To date, no direct measurements of tissue PO2 have been made.
Aim: To directly measure renal tissue PO2 in the Lewis rat model of PKD (LPK) and to determine the relative contributions of altered renal oxygen delivery and oxygen consumption in driving tissue hypoxia.
Methods: Experiments were performed in 11–13 week-old Lewis and LPK rats. Rats were anesthetized with sodium thiobarbital and artificially ventilated. Renal tissue oxygenation was measured using the Clark electrode (10 μm diameter). Tissue PO2 was determined within multiple sites in the renal parenchyma and in cysts in the superficial cortex (n=12 Lewis; n=11 LPK). Arterial and renal venous oxygen content were determined by direct blood oximetry.
Results: In LPK rats, tissue PO2 was higher within the cysts (32.8 ± 4.0 mmHg) than in the superficial cortical tissue itself (18.3 ± 3.5 mmHg), but still lower than the tissue PO2 of the superficial cortex of Lewis rats (46.0 ± 3.1 mmHg). Renal tissue oxygen delivery was 78.5% lower in LPK rats than Lewis rats. Total sodium reabsorption was 98.0% less in LPK rats than Lewis rats, but renal oxygen consumption did not differ significantly between LPK and Lewis rats.
Conclusion: In this model of PKD, the superficial renal cortex is severely hypoxic. Tissue hypoxia in the kidney of LPK rats is driven in part by the compromised tissue oxygen delivery. Our inability to detect a significant deficit in renal oxygen consumption in LPK rats, despite a marked deficit in sodium reabsorption, suggests that inefficient utilization of oxygen for sodium reabsorption may also contribute to the development of renal hypoxia in PKD.
BLOOD PRESSURE VARIATION AND END ORGAN DAMAGE IN PATIENTS WITH UNCOMPLICATED HYPERTENSION: INFLUENCE OF ANTIHYPERTENSIVE MEDICATION TITRATION
Veloudi Pa, Blizzard La, Marwick THa, Lukoshkova EVb, Head GAc, Sharman JEa
aMenzies Research Institute Tasmania, University of Tasmania, Hobart, Tasmania, Australia; bNational Cardiology Research Centre, Moscow, Russian Federation; cBaker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia
Background: There are limited data regarding effects of antihypertensive medication titration on blood pressure variation (BPV).
Aim: To investigate the effect of antihypertensive medication titration on BPV and the relation to end organ damage associated with hypertension.
Methods: Data were analysed from 219 patients with uncomplicated hypertension (age 64 ± 8; males 54%) who participated in a clinical trial over 12 months in which doses of antihypertensive medication were altered but BP remained controlled. Patients were stratified based on decreasing (n=86), maintained (n=98) or increasing (n=35) medication over time. Daytime rate of systolic BP variation (BPV) was calculated from 24–hour ambulatory BP. Left ventricular mass index (LVMI) was derived from three-dimensional echocardiography. Changes in BPV and LVMI were calculated by the difference between base-line and 12-month measurements.
Results: Increasing medication reduced BPV compared with steady state or decreasing medication (–8.3 ± 15.2 vs –1.1 ± 13.7 and –1.6 ± 14.5 mmHg/h, respectively; P<0.05), but no significant change in LVMI was observed between groups. The change in BPV was significantly related to the change in LVMI only in the group with decreasing medication (r=–0.302; P=0.01). This relationship was maintained after multiple regression analysis adjusting for age, body mass index and sex (unstandardized β=–0.062; P=0.018).
Conclusions: Changes in antihypertensive medications appear to have differential effects on BPV, as well as the relationship between BPV and end organ damage. These findings have potential implications regarding appropriate medical therapy of patients with uncomplicated hypertension.
ROLE OF ANDROGENS IN SEX DIFFERENCES IN CARDIAC DAMAGE DURING MYOCARDIAL INFARCTION
Le TYLa,b, Ashton AWa,c, Mardini Ma,b,d, Stanton PG, Funder JWe, Handelsman DJf, Mihailidou ASa,b
aKolling Institute of Medical Research, bDepartment of Cardiology and cDivision of Perinatal Research Royal North Shore Hospital and The University of Sydney, Sydney, New South Wales, Australia; dDepartment of Cardiology, Westmead Hospital, Sydney, New South Wales, Australia; ePrince Henry’s Institute, Clayton, Victoria, Australia; fAnzac Research Institute, University of Sydney, Sydney, New South Wales, Australia
Background: Age-specific incidence of ischemic heart disease in men is higher than in women. The molecular mechanism(s) are poorly understood since most studies focus on estradiol (E2) actions mediated via estrogen receptors (ER), with less attention to androgen receptor (AR)-mediated androgen actions. We have previously reported larger infarct size and aggravated apoptosis in hearts from intact males than female hearts following ischemia-reperfusion (I-R).
Aim: To determine whether androgens have a role in cardiac damage during myocardial infarction (MI) in both males and females.
Methods: Mature age-matched male and female Sprague Dawley rats – intact or surgically gonadectomized (Gx) – received testosterone (T) or estradiol (E2) via subdermal silastic implants. A subset of male rats received dihydrotestosterone (DHT). After 21 days, hearts were subjected to ex vivo regional I-R.
Results: In Gx males, androgens (DHT, T) and E2 aggravated I-R induced cardiac damage, whereas in Gx females, T had no effect and E2 reduced infarct area. Increased circulating T levels upregulated AR and receptor for advanced glycation end products (RAGE), aggravating cardiac damage in both males and females by preventing progression of autophagy and decreasing levels of anti-apoptotic proteins.
Conclusions: We demonstrate a novel and key role for testosterone during myocardial I-R to increase levels of androgen receptors and RAGE, which contributes to aggravated cardiac damage in both males and females. This could be relevant for postmenopausal women receiving T. Our results provide a platform for new potential treatment strategies for reperfusion injury in both females and males.
LEPTIN REDUCES FOOD INTAKE BUT FAILS TO RAISE BLOOD PRESSURE IN MICE WITH DEFICIENCY OF INSULIN RECEPTOR SUBSTRATE (IRS2) IN THE ENTIRE BRAIN OR SPECIFICALLY IN POMC NEURONS
do Carmo JM, da Silva AA, Hall JE
University of Mississippi Medical Center, Jackson, Mississippi, USA
Background: Insulin receptor substrate (IRS2) is one of three major leptin receptor signaling pathways. Its role in mediating the chronic effects of leptin on appetite, blood pressure and glucose regulation is, however, unclear.
Aim: To test whether deletion of IRS2 in the whole brain or specifically in proopiomelanocortin (POMC) neurons attenuates the chronic cardiovascular and metabolic responses to leptin.
Methods: Mice with IRS2 deletion in the entire brain (IRS2flox/flox/Nestin-Cre; n=7), specifically in POMC neurons (IRS2flox/flox/Pomc-Cre; n=7) and control IRS2flox/flox (n=9) mice were instrumented with telemetry probes for measurement of 24 h mean arterial pressure (MAP) and heart rate (HR) and venous catheters for saline or leptin infusions. After a 5-day control period, mice received leptin infusion (2 μg/kg/min, i.v.) for 7 days.
Results: Compared to IRS2flox/flox mice, IRS2flox/flox/Pomc-Cre mice had similar body weight and food intake (33 ± 1 vs 35 ± 1 g and 3.6 ± 0.5 vs 3.8 ± 0.2 g/day) and higher MAP and HR (110 ± 2 vs 102 ± 2 mmHg and 641 ± 9 vs 616 ± 5 bpm). IRS2flox/flox/Nestin-Cre mice were heavier (38 ± 1.5 g), had increased food intake (4.5 ± 1.0 g/day), and higher MAP and HR (108 ± 1.5 mmHg and 659 ± 9.0 bpm) compared to control mice. Leptin infusion for 7 days in control IRS2flox/flox mice gradually increased MAP by 5 mmHg despite decreasing food intake by 31%. In contrast, leptin infusion did not change MAP in IRS2flox/flox/Nestin-Cre and IRS2flox/flox/Pomc-Cre mice. The anorexic effect of leptin was not, however, attenuated in IRS2flox/flox/Pomc-Cre or IRS2flox/flox/Nestin-Cre mice (–35% and –34%, respectively). In addition, deletion of IRS2 in the whole brain or POMC neurons did not impair the ability of leptin to reduce plasma glucose levels (IRS2flox/flox: 170 ± 15 to 130 ± 12; IRS2flox/flox/Pomc-Cre: 180 ± 20 to 150 ± 15; IRS2flox/flox/Nestin-Cre: 160 ± 10 to 131 ± 20 mg/dl).
Conclusion: These results indicate that activation of IRS2 signaling in the CNS, and particularly in POMC neurons, is essential for the long-term actions of leptin to raise MAP and HR, but not for its anorexic or antidiabetic effects.
THE EFFECT OF CHRONIC ANGIOTENSIN TYPE 2 RECEPTOR STIMULATION ON THE ADHESION CASCADE AND PLAQUE DEPOSITION IN APOE–/– MICE FED A HIGH FAT DIET
Shihata Wa, Lumsden NGa, Irvine JCa, Widdop REb, Chin-Dusting JPFa, Sampson AKa
aBaker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia; bDepartment of Pharmacology, Monash University, Clayton, Victoria, Australia
Background: The adhesion cascade, involving the recruitment and adhesion of leukocytes to the endothelium, is a critical early event in the development of atherosclerosis. Activation of the angiotensin II type 1 receptor (AT1R) stimulates vasoconstrictive, pro-fibrotic and pro-inflammatory effects. Conversely, the angiotensin II type 2 receptor (AT2R) induces vasodilation and anti-fibrotic effects. Importantly, the AT2R is upregulated in disease states such as atherosclerosis, with increased expression reported in atherosclerotic plaques. We reported previously that acute stimulation of the AT2R reduces leukocyte to endothelial adhesion in cell culture as well as ex vivo in harvested vessels.
Aim: To examine whether chronic AT2R activation affects the adhesion cascade and plaque progression in diet-induced atherosclerosis.
Methods: The study involved ApoE knockout (Apoe–/–) mice fed for 10 weeks with either a high fat diet (HFD 21%, 0.15% cholesterol) to induce inflammation, or a normal chow diet. In the final 4 weeks, HFD treated mice received either saline or the AT2R agonist Compound 21 (C21; 100ng/kg/min, s.c.) via osmotic minipumps.
Results: We observed greater leukocyte adhesion to the endothelium of harvested mouse aortae from HFD fed Apoe–/– mice compared to normal chow fed Apoe–/– mice (3 ± 1 vs 7 ± 1 leukocytes/field of view/vessel; n=9–10; P<0.05). Thi was attenuated consistently in mice treated with C21 (4 ± 1 leukocytes/field of view/vessel; n=5). There was no difference in total plaque area. HFD-induced increases in circulating LDL/vLDL (HFD vs control: 503 ± 11 vs 250 ± 62 mg/dL; n=6; P<0.05) and HDL levels (258 ± 40 vs 56 ± 20 mg/dL; n=6; P<0.05). Plasma levels of both lipoproteins were unexpectedly reduced in C21 treated Apoe–/– mice (311 ± 34 mg/dL and 65 ± 10 mg/dl, respectively; n=6).
Conclusion: Chronic AT2R agonism reduces HFD-induced vascular inflammation in the setting of atherosclerosis and reduces atherogenic circulating lipid profiles. This study highlights a novel role for direct AT2R stimulation in the treatment of patients with hypercholesterolemia.
THE ANTI-INFLAMMATORY ROLE OF NITROXYL (HNO) ON THE ENDOTHELIUM
Andrews KLa*, Sampson AKa*, Lim CXEa, Lumsden NGa, Kemp-Harper BKb, Chin-Dusting JPFa
aBaker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia; bDepartment of Pharmacology, Monash University, Clayton, Victoria, Australia(*Equal first authors)
Background: The biological gas nitric oxide (NO) impedes important events in plaque development, including endothelial-leukocyte adhesion. The use of the NO donor, glyceryl trinitrate (GTN) as a treatment for cardiovascular disease (CVD) is, however, limited due to its clearance by superoxide and propensity for tolerance to develop with long-term use.
Aim: Since the reduced congener of NO, nitroxyl (HNO) is resistant to scavenging by superoxide and tolerance, the aim of the present study was to examine the anti-inflammatory potential of HNO and compare this with NO.
Methods: The anti-inflammatory effects of the HNO donors Angeli’s salt (AS; 10 µM) and isopropylamine NONOate (IPA/NO; 10 µM) were compared to GTN (10 µM).
Results: AS, IPA/NO and GTN attenuated endothelial-leukocyte adhesion of monocytes (THP-1) to TNFα-activated (10 ng/ml) HUVECs in a concentration-dependent manner and at the highest concentration (10 µM) reduced adhesion by 57%, 39% and 25%, respectively (n=4–8; P<0.001). The effects of AS and IPA/NO were blocked by the soluble guanylate cyclase (sGC) inhibitor, ODQ (10 µM), and GTN by the NO scavenger, hydroxocobalamin (100 µM; n=4–5; P<0.05). Importantly, when HUVECs were treated with either AS or GTN for 24 h to induce tolerance, the adhesion response to AS, but not GTN, was preserved (n=3–4). Flow cytometry analysis revealed that the protein expression of the adhesion molecule, ICAM-1, on TNFα-stimulated HUVECs, was reduced by AS (P<0.001), but not by GTN (P>0.05), whilst the cytokines MCP-1 and IL-6 were reduced by both AS and GTN (P<0.05). In proof of concept experiments using mouse aorta stimulated with TNFα, both AS (10 µM) and GTN (10 µM) attenuated TNFα-induced adhesion of leukocytes after 10 min (TNFα: 18 ± 2; TNFα+AS: 4 ± 1; TNFα+GTN: 5 ± 1 leukocytes/field; n=5–10; P<0.001). The effects of AS were abolished by the HNO scavenger, L-cysteine (3 mM; n=3; P<0.001), and ODQ (n=3; P<0.01) and GTN by hydroxocobalamin (n=3; P<0.001) but not ODQ. Expression of the transcription factor NFκB was increased with TNFα and both AS and GTN reduced NFκB fluorescence intensity by 36% and 31%, respectively. The reduction in NFκB fluorescence by AS was inhibited by ODQ (n=4–5; P<0.05).
Conclusions: These results show for the first time that HNO reduces inflammation, without the development of tolerance, through an endothelial sGC-dependent activation of NFκB, adhesion molecule expression and cytokine release. Therefore, HNO donors may be a viable therapeutic agent for the treatment of CVD.
INFLUENCE OF APOLIPOPROTEIN E, AGE AND AORTIC SITE ON CALCIUM PHOSPHATE INDUCED ABDOMINAL AORTIC ANEURYSM IN MICE
Wang Y, Moxon J, Dinh TN, Krishna SM, Jose RJ, Golledge J
The Vascular Biology Unit, Queensland Research Centre for Peripheral Vascular Disease, School of Medicine and Dentistry, James Cook University, Townsville, Queensland, Australia
Background: Abdominal aortic aneurysm (AAA) affects ~5% men aged >65 years and is an important cause of morbidity and mortality. The study of AAA pathogenesis in humans is limited. The use of appropriate animal models could potentially have an important role in furthering the understanding of the pathogenesis of human AAA and in targeting the development of new therapies for the management of AAA.
Aim: To assess relevant features of AAA induced by calcium phosphate in a mouse model.
Methods: AAA was induced by perivascular application of calcium phosphate to the infra-renal aorta of 3 and 7 month old male mice. Infra-renal aortic diameter was assessed prior to AAA induction and 2 weeks later. AAA induction was assessed by calculating expansion of the infra-renal aortic diameter over 2 weeks. Blood pressure was measured by the tail cuff method, and plasma concentrations of total cholesterol, low density lipoprotein and very low density lipoprotein cholesterol, pro-inflammatory cytokines and matrix metalloproteinase 9 were measured using commercially available kits.
Results: The median expansion of the infra-renal aorta 2 weeks after AAA induction was significantly greater in mice that were deficient in apolipoprotein E than that in the age- and gender-matched wild-type controls (276% versus 94.7%; P=0.02). Plasma low density lipoprotein/very low density lipoprotein cholesterol concentrations 2 weeks after AAA induction were positively correlated with the expansion of the infra-renal aorta induced by calcium phosphate (Spearman r=0.661; P=0.04). The median expansion of the infra-renal aorta 2 weeks after AAA induction was similar in 3 and 7 month-old wild-type mice (121% versus 95%; P=0.33). The local administration of calcium phosphate was associated with an increase in the mean maximal diameter of distant aortic segments, but not associated with changes in the concentrations of circulating pro-inflammatory markers.
Conclusion: This study suggests that apolipoprotein E deficiency, but not age, predisposes to AAA induced in the calcium phosphate model. AAA induction is related to circulating concentrations of low-density lipoprotein/very low-density lipoprotein cholesterol. Local application of calcium phosphate also promotes dilation of distant aortic segments.
THE CARDIOPROTECTANT 3’,4’-DIHYDROXYFLAVONOL INHIBITS THE MITOCHONDRIAL PERMEABILITY TRANSITION PORE
Woodman OLa, Long Rb, Pons Sb, Eychenne Nb, Berdeaux Ab, Morin Db
aSchool of Medical Sciences, RMIT University,Melbourne, Victoria, Australia; bINSERM U955 and Universite Paris Est, Facultie de Medecine, Paris, France
Background: 3’,4’-Dihydroxyflavonol (DiOHF) reduces injury caused by myocardial ischemia and reperfusion (IR) in association with a reduction in oxidative stress. Oxidative stress contributes to the opening of the mitochondrial permeability transition pore (mPTP), a key event in myocardial IR injury.
Aim: To determine the effect of DiOHF on mPTP opening, mitochondrial respiration and the generation of reactive oxygen species (ROS) by cardiac mitochondria, after IR in anesthetised rats.
Methods: Male Wistar rats were anesthetised with pentobarbitone (70 mg/kg, i.v.) and ventilated. The left main coronary artery was occluded for 30 min and reperfused for 15 min. The heart was rapidly removed and the area at risk of ischemia was homogenized and centrifuged to isolate the mitochondria. Sham rats were anesthetized but not subjected to ischaemia. DiOHF (10 mg/kg, i.v.) or vehicle (DMSO) was administered 5 min before reperfusion. mPTP opening was measured by mitochondrial Ca2+ retention capacity. Mitochondrial O2 consumption was measured in the presence of pyruvate (5 mM) and malate (5 mM) with a Clark electrode and ROS generation was measured as the rate of H2O2 production using Amplex red.
Results: Treatment of sham rats with DiOHF significantly increased the concentration of Ca2+ required to stimulate mPTP opening (sham 87 ± 6; sham+DiOHF 120 ± 9 µM). This was accompanied by an increase in state 3 O2 consumption (sham 352 ± 55; sham+DiOHF 572 ± 21 nmol O2/min/mg protein) and a decrease in H2O2 release (sham 0.028 ± 0.002; sham+DiOHF 0.019 ± 0.002 nmol/min/mg protein). IR significantly decreased the concentration of Ca2+ required to stimulate mPTP opening (IR 44 ± 5 µM), decreased state 3 O2 consumption (IR 232 ± 15 nmol O2/min/mg protein) and increased H2O2 release (IR 0.034 ± 0.001 nmol/min/mg protein) compared to sham. Treatment with DiOHF prevented IR-induced changes in mPTP opening (IR+DiOHF 78 ± 7 µM), state 3 O2 consumption (IR+DiOHF 375 ± 30 nmol O2/min/mg protein) and H2O2 release (IR 0.028 ± 0.002 nmol/min/mg protein) so that there was no difference compared to sham.
Conclusion: In normal rats DiOHF inhibits mPTP opening and decreases mitochondrial ROS production. Importantly, DiOHF administration before reperfusion prevents IR-induced mPTP opening, impairment of state 3 respiration and increases in ROS production. The beneficial actions of DiOHF on mitochondria are likely to make a major contribution to its cardioprotective actions.
BILIRUBIN: A NOVEL ENDOGENOUS HYPOLIPIDEMIC AND HYPOTENSIVE AGENT PREVENTING CARDIOVASCULAR DISEASE
Bulmer ACa, Bakrania Ba, Boon A-Ca, Headrick JPa, Wagner K-Hb, Du Toit Ea
aHeart Foundation Research Centre, Griffith University, Gold Coast Campus, Southport, Queensland, Australia; bDepartment of Nutritional Science, University of Vienna, Vienna, Austria
Background: A clear relationship exists between circulating bilirubin and coronary atherosclerotic disease, with mildly elevated bilirubin being protective. Despite bilirubin’s well-known antioxidant effects possibly contributing to atheroprotection, the effect of bilirubin on lipid status and systolic heart function remains unknown.
Aim: To determine whether elevated bilirubin is associated with hypotension and hypolipidemia in mutant hyperbilirubinemic (Gunn) rats and in patients with benign hyperbilirubinemia.
Methods: Eight Gunn rats and normo-bilirubinemic controls underwent cardiac ultrasound and catheterization for assessment of the aortic and left ventricular pressures that developed. Hearts were subsequently mounted and perfused on a Langendorff perfusion apparatus for assessment of ex vivo heart function. 44 human volunteers, half of whom possessed mild hyperbilirubinaemia (Gilbert’s Syndrome), were matched to controls. Blood pressure and serum lipid status were assessed using sygmomanometry and biochemistry, respectively.
Results: Aortic Doppler analysis revealed Gunn rats experienced a marked reduction in peak blood velocity and rate of velocity development (AoVTI peak/slope; P<0.001) whilst maintaining stroke volume, fractional shortening and ejection fraction. Millar catheterization indicated the rate of aortic systolic pressure development (ΔpΔt) was reduced in Gunn rats, which was mirrored by reduced developed pressures and rate of pressure development ex vivo (all P<0.05). No difference in systolic or diastolic pressures were noted in Gilbert’s Syndrome versus control subjects. Hyperbilirubinaemic rodents and humans demonstrated, however, significantly reduced circulating total cholesterol and lipoprotein concentrations (P<0.01).
Conclusion: Hyperbilirubinemia may induce negative inotropic effects in the heart, reducing the rate of pressure development in vivo. This, combined with the lipid lowering effects of bilirubin, provides a new hypothesis to explain protection from ischemic heart disease in hyperbilirubinemic individuals.
THE AORTIC RESERVOIR IS A GENUINE PHYSIOLOGICAL PARADIGM: FIRST STUDY IN HUMANS
Schultz MGa, Davies JEb, Hardikar Ac, Pitt Sc, Hughes ADb & Sharman JEa
aMenzies Research Institute Tasmania, University of Tasmania, Hobart, Tasmania, Australia; bInternational Centre for Circulatory Health, Imperial College London, London, UK; cRoyal Hobart Hospital, Hobart, Tasmania, Australia
Background: Central (aortic) blood pressure (BP) predicts mortality, but the physiological mechanisms underlying aortic BP waveform morphology are debated. The “aortic reservoir” is proposed as a component of aortic BP, but this relationship has only been assessed using a mathematically-derived aortic reservoir-excess pressure model (ARderived). For the first time, the present study aimed to directly measure the aortic reservoir (ARdirect) by cyclic change in aortic volume and determine the relationship with ARderived and aortic BP.
Methods: Ascending aortic BP and Doppler flow velocity were recorded via intra-arterial wire in 10 males (aged 62 ± 12 years) during coronary artery bypass surgery. Simultaneous ascending aortic transesophageal echocardiography was used to measure ARdirect. Published mathematical formulae were used to calculate ARderived.
Results: ARdirect was strongly and linearly related with ARderived during systole (r=0.988; P<0.001) and diastole (r=0.985; P<0.001). Peak cross-correlation (r=0.98) occurred at a phase lag of 0.004 seconds into the cardiac cycle, suggesting close temporal agreement between waveforms. The relationship between aortic BP and ARdirect was qualitatively similar to the cyclic relationship between aortic BP and ARderived, with peak cross-correlations occurring at almost identical phase lags (ARdirect vs. aortic BP, r=0.96 at 0.062 seconds and ARderived vs. aortic BP, r=0.98 at 0.057 seconds).
Conclusion: Mathematically-derived aortic reservoir pressure is highly correlated with changes in proximal aortic volume, consistent with its physiological interpretation as corresponding to the instantaneous volume of blood stored in the aorta. Thus, the aortic reservoir paradigm has a genuine physiological basis and should be considered when interpreting central BP waveform morphology.
LOSARTAN NORMALIZES PERIPHERAL CHEMOREFLEX CONTROL OF SYMPATHETIC NERVE ACTIVITY IN CHRONIC KIDNEY DISEASE
Yao Ya, Hildreth CMa, Farnham MMa,b, Phillips JKa
aAustralian School of Advanced Medicine, Macquarie University, Sydney, NSW, Australia; bHeart Research Institute, University of Sydney, Newtown, Sydney, NSW, Australia
Background: Impaired peripheral chemoreflex sensitivity has been suggested to contribute to sympathetic overdrive and hypertension in chronic kidney disease (CKD). The role of the renin-angiotensin system (RAS) in generating the sympathetic responses to activation of the peripheral chemoreflex in CKD remains unknown.
Aim: To determine whether the peripheral chemoreflex function is affected by an acute inhibition of RAS in CKD using the Lewis polycystic kidney (LPK) rat model of CKD.
Methods: Under urethane anesthesia (1.3 g/kg. i.p.), 12 week-old male LPK or Lewis control rats (n=8–10) were vagotomized, paralysed and artificially ventilated. Baseline values of arterial pressure (AP), renal (rSNA), lumbar (lSNA) and/or splanchnic (sSNA) sympathetic nerve activity were recorded. The peripheral chemoreflex was activated by ventilating the animal with 100% N2 inhalation for 12 seconds. The reflex was then repeated after administration of the RAS inhibitor losartan (3 mg/kg, i.v.).
Results: Baseline values of AP (82 ± 2 vs 93 ± 4 mmHg), rSNA (3.1 ± 0.6 vs 8.5 ± 1.6 µV), lSNA (2.3 ± 0.4 vs 6.9 ± 1.6 µV) and sSNA (3.7 ± 0.7 vs 6.6 ± 1.2 µV) were all significantly higher in the LPK (P<0.05). Activation of the peripheral chemoreflex increased AP (13 ± 3 mmHg) and all sympathetic outflows (rSNA 1.6 ± 0.6 µV; lSNA 1.0 ± 0.3 µV; and sSNA 1.9 ± 0.7 µV) in the Lewis, but in the LPK it reduced AP (–13 ± 3 mmHg) and both rSNA and lSNA (–1.6 ± 0.8 µV and –1.0 ± 0.4 µV, respectively), but produced no change in sSNA. Losartan administration did not affect baseline SNA in either strain, but reduced AP significantly in both Lewis and LPK (–36 ± 3 and –16 ± 4 mmHg, respectively). After losartan, the AP response to activation of peripheral chemoreflex was not altered in either strain, nor were the SNA responses in the Lewis strain. Losartan normalized the sympathetic peripheral chemoreflex response in the LPK, however, converting it into a sympathoexcitatory response (rSNA 2.8 ± 1.3 µV; lSNA 1.8 ± 0.5 µV; and sSNA 1.6 ± 0.7 µV)] that was of comparable magnitude to that seen in the Lewis controls.
Conclusion: These data indicate a role for the RAS in the altered sympathetic responses to activation of the peripheral chemoreflex observed in the LPK model of CKD.
TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL CIRCUITRY IN THE BRAIN AFTER TRANSIENT AND PERMANENT ISCHEMIA
Tabet Fa, Lee Sb, Cuesta Torres La, Levin Mc, Drummond GRb, Remaley Ac, Rye KAa, Vickers Kc,d, Sobey CGb
aCentre for Vascular Research, University of New South Wales, Sydney, New South Wales, Australia; bDepartment of Pharmacology, Monash University, Clayton, Victoria, Australia; cNational Institutes of Health, USA; dVanderbilt University School of Medicine, Nashville, Tennessee, USA
Background: Stroke is a major neurovascular condition and a leading cause of mortality and long-term disability. Within cells of the brain, short non-coding microRNAs (miRNAs) serve to modulate gene expression and likely contribute to most, if not all, neurological processes. Changes in miRNA in brain tissue in response to stroke have not, however, been reported.
Aim: To investigate the functional roles of brain miRNAs and gene regulatory networks in stroke injury.
Methods: Adult (8–12 weeks of age) male C57Bl/6 mice underwent intraluminal filament-induced middle cerebral artery (MCA) occlusion. Permanent ischemia (ischemia no reperfusion, InoR; n=8) was achieved by occlusion for 24 h, and ischemia with reperfusion (IR; n=8) was completed after 30 min of MCA followed by 23.5 h reperfusion. Sham-operated mice (n=8) were used as controls. Total RNA was isolated from mouse brains and gene arrays (Affymetrix) and miRNA arrays (TaqMan OpenArray microRNA) were carried out. Validation studies were performed using RT-PCR and TaqMan individual assays.
Results: InoR significantly altered (P≤0.05; fold-change ≥1.5) the levels of 471 mRNAs in the brain, as compared to sham mice. By contrast, IR resulted in only 114 significant gene expression changes at 24 h. We found 7 mRNAs to be down-regulated and 1 mRNA to be up-regulated by reperfusion. Brain miRNAs were also very sensitive to both ischemia and reperfusion. We found 28 brain miRNAs (11 downregulated, 17 upregulated) to be significantly altered with InoR compared to Sham. Likewise, 12 brain miRNAs (3 down, 9 up) were significantly altered with reperfusion compared to sham. Interestingly, we found 10 miRNAs to be significantly altered (5 upregulated, 5 downregulated) with ischemia (InoR/sham), but were also significantly corrected towards normal sham levels by 23.5 h reperfusion (IR/InoR). Validation studies confirmed that levels of multiple miRNAs were significantly altered with InoR. Interestingly, we found that reperfusion significantly corrected (i.e., increased) the levels of all of these miRNAs. Of the mRNAs that were altered 48% (327/680) were predicted targets of miRNAs that were significantly altered, and our results showed inverse directional changes, consistent with regulatory effects of the miRNAs on the mRNAs.
Conclusion: This study shows a potential role for specific miRNAs and post-transcriptional circuits in both adaptive and maladaptive responses to ischemic stroke and reperfusion.
UNDERSTANDING DISEASED NETWORKS OF THE HEART USING PI3K AND CARDIAC DISEASE MOUSE MODELS
Waardenberg AJa, McMullen JRb, Lin RCYc
aCardiac Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Sydney, New South Wales, Australia; bCardiac Hypertrophy Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia; cRamaciotti Centre for Genomics, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia
Background: Cardiovascular disease is underpinned by modifications in the cellular gene expression programme. Transcription Factors (TFs), representing the end points of often converging signaling pathways, whereby signaling is finally converted into a DNA binding event for the regulation of gene expression, represent attractive disease identifiers for either direct targeting or targeting of the associated pathways.
Aim: To determine the specificity of TFs in whole hearts from mouse models with distinct forms of cardiac remodeling, and from different heart regions.
Methods: We performed a meta-analysis of high-throughput gene expression data obtained from (i) 16 left ventricular (LV) samples from mice with constitutively active [ca] or dominant negative [dn] phosphoinositide 3-kinase (PI3K) mice under sham or myocardial infarction conditions, and (ii) 32 atrial samples from genetically modified mice with dnPI3K, dilated cardiomyopathy (DCM) or atrial fibrillation (AF). Application of mixture distribution model-based clustering across 10 conditions (4×LV: caPI3K, dnPI3K sham or in the presence of myocardial infarction; 6×left/right atria: DCM, AF, dnPI3K), each relative to non-transgenic controls, was performed to discriminate patterns of TFs.
Results: PI3K and DCM models demonstrated specific patterns from 71 TFs (gene expression F-statistic <1x10−4), including a reciprocal expression of TFs between caPI3K and dnPI3K sham and MI mice. DCM models demonstrated a much larger number and degree of TFs differentially expressed. Most strikingly, TF expression patterns specific to DCM also clustered together with enrichment of biological processes known to be associated with these TFs, suggesting that these TFs are aggressively locked into a feed-forward loop. For example, we found a specific up-regulation of HIF1α (a TF involved in hypoxic conditions) in DCM mice that also coincided with a genome-wide gene expression cluster enriched with down-regulation of mitochondrial processes (e.g., oxidative phosphorylation – corrected P=8x10−43).
Conclusion: This study demonstrated that specific TF expression signatures are associated with either atrium or ventricle in different cardiac disease settings. The underlying dysregulated signaling pathways did not, however, converge on a small number of TFs, instead revealing a complexity of diseased gene regulatory networks. Construction of these disease networks also implicated TF cross-talk between atrium and ventricle which may impact therapeutic targeting of TFs and signaling pathways in a clinical setting.
PRESSOR RESPONSIVENESS TO ANGIOTENSIN II IN ADULT FEMALE MICE IS ENHANCED WITH AGE: INHIBITORY ROLE OF THE ANGIOTENSIN TYPE 2 RECEPTOR
Mirabito KMa, Hilliard LMa, Head GAb, Widdop REc, Denton KMa
aDepartment of Physiology, Monash University, Clayton, Victoria, Australia; bBaker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia; cDepartment of Pharmacology, Monash University, Clayton, Victoria, Australia
Background: The prevalence of hypertension increases in women post menopause. We, and others, have demonstrated that the pressor response to angiotensin II (Ang II) is attenuated in adult females as compared to males. Furthermore, this response was mediated via an angiotensin type 2 receptor (AT2R)-dependent pathway. We hypothesized that estrogen provides protection against Ang II-induced hypertension by counter-balancing the pressor actions of the angiotensin type 1 receptor (AT1R) via an enhanced AT2R mediated pathway. A corollary of this hypothesis is that with age this pathway may no longer operate in females.
Aim: To determine if the AT2R-mediated attenuated pressor response to Ang II is present in aged females.
Methods: Mean arterial pressure (MAP) was measured via telemetry in adult (20 week-old) and aged (65 week-old) FVB/N wild-type (WT) and AT2R knock-out (KO) female mice during baseline and 14 day infusion of vehicle (saline) or Ang II (600 ng/kg/min). Renal expression of the AT1R and AT2R was determined using real time RT-PCR.
Results: Basal MAP was similar between the adult females (WT 93 ± 1 mmHg; n=13; AT2R-KO 93 ± 1 mmHg; n=12). With age, there was no change in basal MAP (aged WT 93 ± 1 mmHg; n=11; aged AT2R-KO 93 ± 1 mmHg; n=14). In the 20-week old adult females, the pressor response to Ang II was significantly attenuated in the WT as compared to the AT2R-KO mice (29 ± 3 mmHg vs 10 ± 4 mmHg, respectively, on day 14; P<0.01). The pressor response to Ang II was, however, augmented in the aged WT as compared to the 20-week old adult WT mice (P<0.01). Consequently, the increase in MAP in response to Ang II was similar between aged WT and AT2R-KO females (34 ± 3 mmHg vs 31 ± 4 mmHg, respectively, on day 14; P>0.05). In WT females, aging was associated with an increase in the renal AT1R/AT2R ratio.
Conclusion: The augmented pressor response to Ang II in the aged female WT mice demonstrates that the protective role of the AT2R depressor pathway is lost with age. Loss of this mechanism may contribute to the sharp rise in arterial pressure post menopause. Consequently, targeting deficits in AT2R expression and/or signaling represents a novel therapeutic approach for postmenopausal hypertension.
ALISKIREN REDUCES MYOCARDIAL ISCHEMIA-REPERFUSION INJURY BY A BRADYKININ B2 RECEPTOR- AND ANGIOTENSIN TYPE 2 RECEPTOR-MEDIATED MECHANISM
Koid SSa,b,c, Ziogas Jb, Campbell DJa,c
aSt Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia; Departments of bPharmacology and cMedicine, The University of Melbourne, Parkville, Victoria, Australia
Background: Angiotensin-converting enzyme inhibitors and angiotensin type 1 (AT1) receptor blockers reduce myocardial ischemia-reperfusion (I/R) injury via bradykinin B2 and/or angiotensin type 2 (AT2) receptor-mediated mechanisms. The renin inhibitor aliskiren increases cardiac tissue kallikrein and bradykinin levels, both of which are important for cardioprotection. Importantly, the increase in cardiac bradykinin levels raises the possibility of cardioprotection by aliskiren.
Aims: To investigate the effect of aliskiren on myocardial I/R injury and the roles of B2 and AT2 receptors in this effect.
Methods: Female Sprague-Dawley rats were treated for 4 weeks with vehicle, aliskiren (10 mg/kg/day, s.c.), valsartan (30 mg/kg/day. p.o.), or the combination of aliskiren and valsartan. We examined the roles of the B2 and AT2 receptors by co-administration of vehicle, the B2 receptor antagonist icatibant (0.5 mg/kg/day, s.c.) or the AT2 receptor antagonist PD123319 (30 mg/kg/day, s.c.). Myocardial I/R injury was elicited by 30 min occlusion of the left anterior descending coronary artery followed by 120 min reperfusion in anesthetised rats. Myocardial I/R injury, as indicated by infarct size, was expressed as the percentage of the area at risk.
Results: Aliskiren increased cardiac bradykinin levels and attenuated valsartan-induced increases in plasma angiotensin II levels. In vehicle-treated rats, myocardial infarct size (mean±SEM; n=7–13) was 43 ± 3%. This was reduced to a similar extent by aliskiren, valsartan, and their combination to 24 ± 3%, 25 ± 3% and 22 ± 2%, respectively. Icatibant reversed the cardioprotective effects of aliskiren and the combination of aliskiren plus valsartan, but not valsartan alone, indicating that valsartan-induced cardioprotection was not mediated by the B2 receptor. PD123319 reversed the cardioprotective effects of aliskiren, valsartan and the combination of aliskiren plus valsartan.
Conclusion: Aliskiren protects the heart from myocardial I/R injury via a B2 and AT2 receptor-mediated mechanism, whereas cardioprotection by valsartan is mediated via the AT2 receptor. In addition, aliskiren attenuates valsartan-induced increases in angiotensin II levels, thus preventing AT2 receptor-mediated cardioprotection by valsartan.
IMPACT OF IN VITRO CULTURE AND EMBRYO TRANSFER ON IGF-PHOSPHATIDYL-3 KINASE (PI3K) MEDIATED SIGNALING PATHWAY IN THE HEART OF THE POSTNATAL SHEEP
Padhee Ma, McMillen ICa, MacLaughlin SMa, Zhang Sa, Kleeman DOb, Walker SKb, Morrison JLa
aSansom Institute of Health Research, University of South Australia, Adelaide, South Australia; bTurretfield Research Centre, South Australian Research and Development Institute, Adelaide, South, Australia, Australia
Background: Previous studies have demonstrated that embryo transfer and in vitro embryo culture, which are important steps in assisted reproductive technologies, result in an increased weight of the heart relative to the body due to changes in the IGF/PI3K signaling pathway in the heart of the sheep fetus.
Aim: To investigate whether the increase in heart weight as well as the alteration of IGF/PI3K signalling pathway persists in postnatal life.
Methods: Embryos were either transferred to an intermediate ewe (ET) or cultured in vitro in the absence (IVC) or presence of human serum (IVCHS) and a methyl donor (IVCHS+M) for 6 days. Naturally mated (NM) ewes acted as controls. At 24 weeks after birth, hearts were weighed, extracted and mRNA and protein expression of molecules involved in IGF-PI3K pathway were measured.
Results: There was no difference in the heart weight relative to body weight in any of the treatment groups compared to NM. There was no difference in the expression of IGF1R mRNA and IGF2R mRNA in any of the treatment groups compared to the NM group. There was no difference in the abundance of protein kinase B (PKB)/AKT or its phosphorylated form, mammalian target of rapamycin (mTOR) and phosphorylated mTOR, as well as S6 and its phosphorylated form between the treatment groups.
Conclusion: This study suggests that the increase in heart weight in fetal life that is associated with embryo transfer or in vitro embryo culture do not persist in postnatal life. In addition, embryo transfer and in vitro embryo culture do not result in an activation of the IGF/PI3K (P110α) signaling pathway in the heart in postnatal life.
IMPACT OF IN VITRO CULTURE AND EMBRYO TRANSFER ON CARDIAC CONTRACTILITY IN THE HEART OF SINGLETON AND TWIN SHEEP FETUSES
Padhee Ma, IC McMillena, SM MacLaughlina, S Zhanga, DO Walkerb, KJ Bottinga, JL Morrisona
aEarly Origins of Adult Health Research Group, Sansom Institute of Health Research, University of South Australia, Adelaide, South Australia;, bTurretfield Research Centre, South Australian Research and Development Institute, Adelaide, South, Australia, Australia
Background: Studies have shown that assisted reproductive technologies are associated with abnormal cardiac development.
Aim: To investigate the effect of in vitro culture and transfer of the embryo on signaling molecules involved in cardiac contractility in singleton and twin sheep fetuses.
Methods: Embryos were collected 24 h after artificial insemination of superovulated donor ewes, and were either transferred to an intermediate ewe until day 6 (ET group; singletons=6, twins=6) or cultured in vitro in the absence (IVC group; singletons=5, twins=7) or presence of human serum (IVCHS group; singletons=6, twins=4) for 6 days before transfer to recipient ewes. Naturally mated (NM) ewes were used as controls (singletons=4, twins=6). At 144/145 days gestation, ewes were killed humanely, hearts were dissected and tissues were snap frozen in liquid nitrogen. Protein abundance was measured by Western blotting.
Results: There was no change in abundance of protein kinase C-α (PKCα) or troponin I in all treatment groups compared to controls in both singletons and twins. The protein abundance of phospho-PKCα and phospho-troponin I did not change in any treatment group in singleton fetuses. There was, however, a decrease in the protein abundance phospho-PKCα (P<0.05) and phospho-troponin (P<0.01) in the ET and IVC groups, in twins only. Culture and transfer of the embryo did not alter the protein abundance of SERCA in any treatment group in either singletons or twins.
Conclusion: The present findings suggest that in vitro embryo culture and transfer may affect the contractility of cardiomyocytes in twins, but not singletons. If a decrease in contractility persists, the individual may be at an increased the risk of cardiovascular disease in later life.
WIDESPREAD CORONARY ENDOTHELIAL DYSFUNCTION IN THE ABSENCE OF HDL RECEPTOR SRB1 IN A MOUSE MODEL OF ISCHEMIC CARDIOMYOPATHY
Pearson JTa–c, Yoshimoto Md, Chen Y-Ca, Sultani Ra, Nakaoka He, Nishida Me, Yamashita Se, Umetani Kf, Tsuchimochi Hd, Shirai Md
aDepartment of Physiology and bMonash Biomedical Imaging Facility, Monash University, Clayton, Victoria, Australia; cAustralian Synchrotron, Clayton, Victoria, Australia; dNational Cerebral and Cardiovascular Center Research Institute, Japan; eGraduate School of Medicine, Osaka University, Osaka, Japan; fJapan Synchrotron Radiation Research Institute, Kansai, Japan
Background: Occlusive coronary artery disease is a leading cause of morbidity and mortality. Reduced clearance of lipoproteins by HDL scavenger receptor class B1 (SRB1) plays an important role in the progression of atherosclerosis. It is unclear how SRB1 influences coronary endothelial function in the face of high levels of oxidized LDLs. Here we utilized mice with hypomorphic ApoE lipoprotein (hypo) that lacked SRB1 (–/–) or were heterozygous for SRB1(–/+) to investigate coronary control in atherogenic state.
Aim: To determine the extent of endothelial dysfunction in vivo in the coronary circulation of male mice after exposure to a Paigen high fat diet for 7 days and normal chow for a further 2 weeks.
Methods: 8 hypo–/–and 10 hypo–/+ mice were compared with synchrotron microangiography cine recordings during baseline, acetylcholine (ACh), sodium nitroprusside (SNP) infusions and vehicle or ACh infusion after NOS and COX inhibition with L-NAME (50 mg/kg) and sodium meclofenamate (3 mg/kg).
Results: Significant endothelium-dependent or independent dilator responses were absent in hypo–/– mice across the 1st to 4th branching order arterial segments (ACh and SNP). Partially occlusive stenoses were clearly observed in angiograms. Vessel segment adjacent stenoses displayed constriction following NOS/COX blockade, while globally non-stenotic segments did not differ significantly from baseline. Although hypo–/– mice did not show dilation of vessel caliber there were significant increases in visualized vessel number during infusions of ACh, SNP and ACh post blockade, in both small and large vessels (P=0.01–0.05).
Conclusion: A high fat diet induced widespread coronary endothelial dysfunction ahead of occlusive plaque formation. The absence of caliber changes suggest that SRB1 is essential for nitric oxide-mediated coronary dilation. Interestingly, in the absence of SRB1 the role of EDHF as a coronary dilator was variable between animals, but important for the maintenance of coronary flow through the opening of medium to small epicardial and penetrating transmural arteries and arterioles in mice after exposure to the Paigen diet. Enhanced EDHF production is likely to be important for the maintenance of larger vessel calibres in mice lacking SRB1 or when SRB1 expression is downregulated by oxidative stress.
CHANGES IN MICRO-RNA EXPRESSION IN THE AGING HUMAN HEART
Skommer JM, Rana I, Prestes PR, Marques FZ, Ghandi A, Charchar FJ
School of Health Sciences, University of Ballarat, Ballarat, Victoria, Australia
Background: Aging is associated with disproportionate prevalence of cardiovascular diseases, even in cohorts with low cardiovascular risk. The anatomic, functional and molecular changes that occur in the heart over the course of aging render this organ more vulnerable to various stressors, favoring the development of cardiac disease. MicroRNAs (miRNAs), a class of small non-coding RNA molecules of ~22–23 nucleotides in length, regulate the expression of protein-coding genes by post-transcriptional mechanisms and play a role in the cardiac remodeling that occurs during the postnatal period and in aging. Importantly, the profile of cardiac miRNA expression was shown to change during aging in animal models. A systematic study combining the analysis of global miRNA expression in aging human hearts with functional assessment of their role in cardiomyocyte sensitivity to stress has not to date been conducted.
Aim: To determine the role of miRNAs in regulation of survival and cell death signaling pathways in the aging human heart.
Methods: We used miRNA 3.0 arrays (Affymetrics) and Partek Genomic Suite for data analysis to compare the expression profiles of miRNAs isolated from hearts of human fetuses (age 20–31 weeks; n=3), young dults (24–32 years old; n=3) and elderly adults (66–76 years old; n=3).
Results: The comparison of fetal vs. young and elderly groups identified 6 miRNAs with significantly altered expression (FDR<0.05, and fold change threshold of ±1.5). Of these, 3 miRNAs (miR-106b-3p, miR-29b-2* and miR-664*) were also differentially expressed between the young and the elderly groups. These miRNAs have been linked to regulation of autophagy and fibrosis, and associated with aging and/or atrial fibrillation in animal models.
Conclusion: Aging is associated with changes in the expression of human cardiac miRNAs. In future studies we aim to confirm expression profiles of these miRNAs in an extended sample set, and conduct functional studies in human cardiomyocytes derived from induced pluripotent stem cells.
ROLE OF MICRO-RNA IN CARDIOPROTECTION BY A RENIN-ANGIOTENSIN SYSTEM INHIBITOR IN AN ANIMAL MODEL OF RENAL INJURY
Rana Ia, Velkoska Eb, Skommer Ja, Marques FZa, Quarrell Sa, Burrell LMb, Charchar FJa
aSchool of Health Sciences, University of Ballarat, Ballarat, Victoria, Australia; bDepartment of Medicine, The University of Melbourne, Austin Health, Melbourne, Victoria, Australia
Background: Cardiac hypertrophy and structural abnormality are key pathologies that contribute to the high rate of cardiac death in humans with kidney disease. Elevated renin-angiotensin system (RAS) activity plays an important role in the pathophysiology of heart complications in kidney disease. Thus RAS inhibitors are commonly used to minimize heart pathology in these patients. MicroRNAs (miRs) are small endogenously transcribed regulatory RNAs. They modulate gene expression by binding to 3’ or 5’ untranslated regions (UTR) of mRNAs, and have been shown to act as circulating biomarkers of heart damage following myocardial infarction. Animal studies have reported that dysregulated levels of miR-1, miR-133b, miR-133a, miR-208a, miR-208b, miR-499 and miR-21 in the heart can cause cardiac hypertrophy, apoptosis or fibrosis of cardiomyocytes. It is not, however, known if these miRs are involved in cardiac abnormality in the context of kidney disease and pharmacothereapy with RAS inhibitors.
Aim: To investigate expression of miR-1, miR-133b, miR-133a, miR-208a, miR-208b, miR-499 and miR-21 in heart tissue collected from an animal model of renal injury.
Method: Heart tissues collected from rats 10 days after subtotal nephrectomy (STNx), where one kidney was removed and the other partially ligated (n=7), was compared with sham (n=7) rats or STNx rats (n=7) treated with the ACE inhibitor ramipril. RNA was extracted from hearts collected from the rats and real-time quantitative PCR (qPCR) was used to measure miR expression levels.
Results: We found that miR-208a, miR-208b and miR-21 were significantly (P<0.05) up-regulated in heart in STNx rats. There was a trend towards miR-1 up-regulation, but this did not reach statistical significance (1.5 fold increase; P=0.09). There was no change in miR-133b levels in hearts collected from STNx compared to the respective sham rats. Compared to vehicle-treated rats, treatment of STNx rats with ramipril did not prevent the observed increase in levels of miR-208a, miR-1, miR-208b and miR-21 in heart, but significantly increased the expression of miR-499 and miR-133a (P<0.01).
Conclusion: The cardioprotective action of ACE inhibition in an animal model of acute kidney injury could be mediated, at least in part, by up-regulation of miR-499 and miR-133a expression.
DEVELOPMENT OF A CARDIOVASCULAR MORTALITY RISK PREDICTION MODEL FOR PARTICIPANTS IN THE OHASAMA STUDY
Huq MMa, Head GAa,b, Owen Aa, Ademi Za,c, Ohkubo Td, Imai Ye, Reid CMa
aDepartment of Epidemiology and Preventive Medicine, Monash University, Clayton, Victoria, Australia; bBaker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia; cMelbourne EpiCentre, University of Melbourne, Melbourne, Victoria, Australia; dSchool of Medicine, Teikyo University, Tokyo, Japan and Department of Clinical Pharmacology and Medicine, Tohoku University, Sendai, Japan
Background: Ambulatory blood pressure monitoring provides a more accurate blood pressure (BP) assessment than isolated clinic measurements, but can be more intensive and expensive than clinic measures. The extent to which novel BP indices from ambulatory monitoring improve cardiovascular risk prediction is less clear.
Aim: To develop a cardiovascular mortality risk prediction model for participants in the Ohasama Study in Japan, examining conventional and novel indices of ambulatory blood pressure.
Methods: The study used 16.6 years of follow-up data from 1,535 participants in the Ohasama study. Our risk model was developed using univariate and multivariable Cox proportional hazards models. Variable selection was done using bootstrap methods on 1000 bootstrap samples. After development the model was validated using a 500 fold bootstrap validation method.
Results: Ambulatory and conventional blood pressure values were obtained from 1,535 participants with average age of 61.7 ± 10.7SD years, of whom 63% were women. In this population, 8.2% experienced a fatal cardiovascular event. The final cardiovascular risk prediction model included risk factors such as age, smoking status, number of antihypertensive medications, mean daytime ambulatory systolic arterial blood pressure and difference between day and night ambulatory systolic arterial blood pressure (C statistic was 0.822). The ambulatory variables predicted cardiovascular mortality better than conventional BP variables. The internal bootstrap validation provided us with a C statistic of 0.817.
Conclusion: This risk prediction model incorporating novel indices from ABPM provides a direct risk assessment of cardiovascular mortality in Ohasama study participants. It could be externally validated and compared with other relevant cardiovascular mortality risk prediction models.
THE IMPACT OF AN EDUCATION PROGRAM TO REDUCE THERAPEUTIC INERTIA IN PRIMARY CARE MANAGEMENT OF HYPERTENSION
Reid CM, Owen AJ, Krum H
Centre of Cardiovascular Research & Education in Therapeutics, Monash University, Clayton, Melbourne, Victoria, Australia.
Background: Therapeutic inertia is a term used to describe the situation in which there is reluctance to modify/intensify a treatment regimen when target treatment goals remain unmet. Therapeutic inertia has been identified as a significant contributor to the burden of uncontrolled blood pressure, and it has been suggested that uncertainty surrounding measurement and interpretation of blood pressures by primary care physicians may be a major factor.
Aim: To determine whether delivery of an education intervention focussing upon guideline-driven hypertension management to Australian General Practitioners (GPs) could reduce therapeutic inertia.
Methods: GPs enrolling in a clinical audit program were randomized to either control or intervention groups. All GPs undertook two audits of hypertension management in at least 10 patients, separated by a period of 9 months. A therapeutic inertia score was calculated for each patient audited. In the intervening period the intervention group completed an education program focussing on the challenges of, and strategies for, blood pressure management, with five of the six modules being interactive face-to-face workshops and one being a self-guided learning activity.
Results: Both the control and education groups demonstrated improvements in therapeutic inertia score from the first to second audit.
Conclusion: The results of this study suggest that participation in an audit of hypertension management was as effective as an intensive face-to-face program of education in addressing therapeutic inertia in general practice.
ENDOTHELIAL CELL MINERALOCORTICOID RECEPTORS REGULATE DEOXYCORTICOSTERONE/SALT-MEDIATED CARDIAC REMODELING AND VASCULAR REACTIVITY, BUT NOT BLOOD PRESSURE
Rickard AJ*a, Morgan J*a, Chrissobolis Sb, Miller AAb, Sobey CGb, Young MJa,c,d
aPrince Henry’s Institute of Medical Research, bDepartment of Pharmacology, cDepartment of Physiology, and dDepartment of Medicine, Monash University, Clayton, Victoria, Australia(*Equal 1st authors)
Background: Recent studies have described cell-specific roles for mineralocorticoid receptor (MR)-signaling in the cardiovascular system in terms of blood pressure regulation, recruiting and activation of immune cells, and promotion of cardiac tissue remodeling.
Aims: Firstly, to identify the role of endothelial cell MR in the vascular function of vehicle and aldosterone-treated mice, and secondly, to define the contribution of endothelial cell MR to deoxycorticosterone (DOC)/salt-mediated inflammation and cardiac damage.
Methods: The vascular function of wild-type (WT) and endothelial cell MR-null (EC-MRKO) mice treated with vehicle or aldosterone (0.72 mg/kg/day) for 2 weeks was assessed ex vivo. MR-mediated inflammation and cardiac damage was evaluated in WT and EC-MRKO mice treated with DOC/salt for 8 days or 8 weeks.
Results: Endothelial nitric oxide function was impaired in the thoracic aorta and mesenteric arteries of aldosterone-treated WT mice. While endothelial nitric oxide function was equivalently impaired in the mesenteric arteries of aldosterone-treated EC-MRKO mice, endothelial function was unaffected in the aorta, suggesting a differential role for endothelial cell MR depending on the vascular bed. At 8 days, loss of endothelial cell MR prevented DOC/salt-induced macrophage infiltration and the increased expression of proinflammatory genes in the myocardium. Cardiac collagen content was equivalent between genotypes at 8 days, although mRNA levels of profibrotic genes were significantly lower in EC-MRKO mice versus WT mice. At 8 weeks DOC/salt treatment increased macrophage recruitment and proinflammatory gene expression in WT mice but not EC-MRKO mice. Cardiac collagen deposition and CTGF mRNA levels were significantly reduced in EC-MRKO mice versus WT mice. Interestingly, systolic blood pressure was equivalently elevated in DOC/salt-treated WT and EC-MRKO mice at 8 weeks.
Conclusion: Our data demonstrate that endothelial cell MR signaling contributes to vascular nitric oxide function in large conduit arteries but not resistance vessels. We have, moreover, highlighted an important and independent role for endothelial cell MR signaling in the cardiovascular proinflammatory and profibrotic response to DOC/salt.
PATIENTS WITH AT LEAST THIRTY PERCENT OF HOME SYSTOLIC BLOOD PRESSURES ELEVATED ARE LIKELY TO HAVE UNCONTROLLED BLOOD PRESSURE: A PRAGMATIC METHOD FOR DOCTORS TO ASSESS BLOOD PRESSURE CONTROL FROM PATIENT BLOOD PRESSURE DIARIES
Sharman JE, Blizzard L, Kosmala W, Nelson MR
Menzies Research Institute Tasmania, University of Tasmania, Hobart, Tasmania, Australia
Background. Home blood pressure (HBP) is a method that is superior to clinic BP for the measurement of usual BP. A pragmatic barrier to using HBP is the requirement of doctors to calculate mean BPs from patient diaries.
Aims. To develop a timely and pragmatic method for determining the optimal ratio of HBP readings above threshold (systolic BP ≥135 mmHg) that would best predict elevated ambulatory systolic BP.
Methods. HBP (2× morning and evening diary readings over 7 days), 24-hour ambulatory BP and left ventricular mass index (LVMI; using 3D-echocardiography) were measured in 286 patients with uncomplicated treated hypertension (aged 64 ± 8 years; 53% female). Uncontrolled BP was defined as 24-hour SBP ≥130 mmHg. Indices of model calibration (deviance) and classification (area under the curve, AUC; net reclassification index and integrated discrimination improvement) were used to determine the optimal number of systolic HBP readings ≥135 mmHg (from the last 10 recorded) to predict those with uncontrolled BP.
Results. Having ≥3 of the last 10 systolic HBP readings ≥135 mmHg provided the best prediction of systolic BP above treatment/target threshold (AUC=0.71, 80% specificity and 62% sensitivity). These individuals also had LVMI that was 1.28 g/m2.7 (95% CI 0.05–2.61) higher that those who did not meet this criterion.
Conclusions. To facilitate uptake of HBP monitoring we propose that doctors can determine the percentage of the last 10 systolic HBP values above 135 mmHg and manage their patient accordingly.
PREVALENCE OF KLHL3 AND CUL3 MUTATIONS IN FAMILIAL HYPERKALEMIC HYPERTENSION
Wolley Ma, Glover Mb, Ware Jc,d, Henry Ab, Walsh Rc, Wain Ld, Xu Sa, Ahmed Aa, Van’t Hoff We, Tobin Md, Hall Ib, Cook Sd,f,g, Gordon Ra, O’Shaughnessy Ka, Stowasser Ma
aEndocrine Hypertension Research Unit, Greenslopes Hospital and University of Queensland School of Medicine, Brisbane, Queensland, Austraia; bDivision of Therapeutics and Molecular Medicine, University of Nottingham, Nottingham, UK; cNIHR Royal Brompton Cardiovascular Biomedical Research Unit, Imperial College, London, UK; dNational Heart and Lung Unit, Imperial College, London, UK; eGenetic Epidemiology Group, University of Leicester, Leicester, UK; fGreat Ormond Street Hospital for Children, London, UK; gDuke National University of Singapore, Singapore; hNational Heart Centre Singapore, Singapore; iClinical Pharmacology Unit, University of Cambridge, Cambridge, UK
Background: Familial hyperkalemic hypertension (FHHt, Gordon’s syndrome) is an inherited form of salt dependent hypertension caused by mutations in genes encoding proteins regulating the NaCl co-transporter (NCC) in the distal tubule. Mutations have been documented previously in ‘“with no lysine (K)” kinases (WNK1 and WNK4). More recently mutations in two more genes, Kelch-like 3 (KLHL3) and Cullin 3 (CUL3), have been implicated in the causation of this condition.
Aim: To examine whether mutations in KHLH3, CUL3 or SLC4A8 (an alternative thiazide sensitive sodium transporter) are present in our FHHt pedigrees previously screened and found negative for WNK1 and WNK4 mutations.
Methods: 25 affected individuals from 16 families with unexplained FHHt underwent genetic analysis by next generation sequencing. Validation of results was by Sanger sequencing.
Results: Affected individuals from 10 of 16 families were found to have CUL3 or KLHL3 variants not reported in the general population. Eight pedigrees carried variants previously associated with FHHt; two in CUL3 (change: c.1377+1G>C, c.1207-1G>A) and four in KLHL3 (c.1499G>T, c.1160T>C in three pedigrees, c.1019C>T, c.1480G>A). Two pedigrees had previously unreported variants in CUL3 (c.1377+1G>T, c.1207-12T>A) and one individual was homozygous for a previously reported heterozygous KLHL3 variant (c.1499G>T). We found no evidence for disease causing variants in SLC4A8.
Conclusion: Overall, 63% of our WNK1 and WNK4 mutation-negative pedigrees now have a genetic diagnosis, implying mutations in other, as yet unknown, regulators of the NCC are likely to exist.
CHRONIC MID-LATE GESTATIONAL HYPOXIA LEADS TO HYPERTENSION IN MALE AND FEMALE MOUSE OFFSPRING
Walton SLa, Singh RRa, Paravicini TMa, Wilkinson Lb, Little MHb, Moritz KMa
aSchool of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia; bInstitute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia
Background: Fetal hypoxia is a common gestational insult characterized by the redistribution of cardiac output to favour the brain and heart. This occurs at the expense of peripheral organ development and frequently leads to growth restriction. We hypothesized that these hypoxia-induced alterations to the fetal cardiovascular system may persist into adulthood and increase the risk of hypertension and cardiovascular disease.
Aim: To examine whether mid to late gestational hypoxia can increase the risk of hypertension and cardiovascular disease in aged mouse offspring.
Methods: Pregnant CD-1 mice were placed in a hypoxic chamber (12.0% O2; n=11) or control (21% O2; n=11) environment from embryonic day (E) 14.5 to birth (E19.5). Systolic blood pressure (SBP) and diastolic blood pressure (DBP) was assessed in the offspring (control: male n=6, female n=5; hypoxia: male n=6, female n=6) at 14 months (mo) of age via radiotelemetry recording over three days. Mean arterial pressure (MAP), pulse pressure (PP) and heart rate (HR) were calculated from these parameters.
Results: Offspring exposed to maternal hypoxia were growth-restricted (PTREATMENT=0.01) at postnatal day 1. No differences in body weights were observed at 14 mo. Male and female offspring exposed to maternal hypoxia had significantly higher MAP throughout both the light and dark cycle compared to control counterparts (males: PTREATMENT=0.01; females: PTREATMENT<0.0001). In male offspring, this was driven by an increase in DBP (PTREATMENT<0.0001) with no change in SBP. Maternal hypoxia-exposed male offspring also had lower PP compared to control (PTREATMENT=0.02). In contrast, female offspring had an increase in both SBP (PTREATMENT=0.0001) and DBP (PTREATMENT<0.0001), with no change in PP. No change in HR was observed between groups in male or female offspring.
Conclusion: Fetal hypoxia led to growth restriction, but catch-up growth was observed later in life. Both male and female offspring developed hypertension, but in male offspring this was associated with a low pulse pressure. This may reflect decreased cardiac function in male offspring, and potentially an increased risk of cardiovascular disease and mortality. Examination of vessel function and structure is currently underway to determine whether changes can be attributed to adulthood hypertension.
THE FUNCTION OF ErbB4 RECEPTOR IN THE POSTNATAL MOUSE HEART
Wang Z, Chan HW, Thomas WG, Paravicini TM
School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia
Background: ErbB receptors are a subfamily of tyrosine kinase receptors that regulate cell proliferation and differentiation. Of the four subtypes of ErbB receptor (ErbB1–ErbB4), ErbB4 is the most abundant subtype in the postnatal heart. Upon activation by growth factors such as neuregulin 1 (NRG1), ErbB4 forms homodimers or heterodimers to initiate downstream signaling. NRG1-ErbB4 signaling is critical for cardiac development, and deletion of either gene causes embryonic mortality. The role of ErbB4 in the adult heart, however, remains poorly understood.
Aims: To examine the role of the ErbB4 receptor in mediating hypertrophic growth of cardiomyocytes in vitro, and maintenance of the adult heart in vivo.
Methods: ErbB4 expression was suppressed using RNAi (shRNA and siRNA) in neonatal rat ventricular cardiomyocytes, and NRG1 (10 nM) was used to induce hypertrophy. Promoter activity of hypertrophic genes was measured using luciferase reporter assays, ERK1/2 activation was measured by western blot, and hypertrophy was determined by an increase in protein:DNA ratio. Cre-lox recombination using a tamoxifen-inducible Cre recombinase driven by a Myh6 promoter was used to selectively delete the ErbB4 receptor from cardiomyocytes in adult mice. ErbB4 deletion was confirmed using qPCR, and echocardiography was used to measure cardiac function (fractional shortening, FS) at various time points after gene deletion.
Results: NRG caused ERK1/2 phosphorylation and robust activation of pro-hypertrophic genes (myosin light chain 2v, atrial natriuretic peptide, and cyclin D). This activation was significantly reduced by silencing of ErbB4 (n=3–5; P<0.01), confirming that ErbB4 activation contributes to hypertrophic signaling. Furthermore, NRG1-induced hypertrophy (protein:DNA ratio) was reduced by the downregulation of ErbB4. PCR confirmed selective deletion of ErbB4 in the heart after tamoxifen administration. Echocardiographic analysis at 3 months after tamoxifen treatment showed no differences in FS between groups. At 7 months post-tamoxifen there was a tendency for reduced FS in the cardiac ErbB4 knockouts (25.52.6 vs 31.84.3 % in non-floxed controls; n=5–8). This was not, however, statistically significant.
Conclusion: Activation of the ErbB4 receptor is required for NRG1-induced cardiomyocyte hypertrophy. Using Cre/Lox recombination we have successfully established a model of cardiac ErbB4 deletion in the adult. Deletion of ErbB4 from cardiomyocytes in adult mice tends to impair cardiac function. We are now examining whether ErbB4 deletion also affects cardiac hypertrophy, apoptosis and fibrosis.
NITRIC OXIDE (NO) REVERSED TNF-α INHIBITION OF TROPHOBLAST INTEGRATION INTO ENDOTHELIAL CELLULAR NETWORKS
Xu Ba,c, Charlton Fc, Makris Ab,c, Hennessy Aa,c
aSchool of Medicine, University of Western Sydney, Sydney; bRenal Unit, Liverpool Hospital, Sydney; cVascular Immunology Research Laboratory, The Heart Research Institute, University of Sydney, Sydney, New South Wales, Australia
Background: The interaction between trophoblast cell and maternal uterine endothelium is important for placental vascular modeling. Nitric oxide (NO) is a potent vasorelaxant that regulates systemic blood pressure. Reduced NO has been seen in preeclampsia.
Aim: To examine whether NO has a role in regulating TNF-α-induced inhibition of trophoblast cell integration into endothelial cellular networks in vitro.
Methods: Red fluorescent-labeled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on Matrigel. After endothelial cellular networks appeared, green fluorescent-labeled HTR-8/SVneo trophoblast cells were co-cultured with endothelial cells, together with or without TNF-α (0.5 ng/ml) and/or the NO donor, sodium nitroprusside dihydrate (SNP) (100 µM). Images were captured after 24 hours. The effects of NO on HTR-8/SVneo cell integration were quantified by image analysis software (Image J). The cells were then recovered from Matrigel to extract mRNA. Quantitative PCR was performed to evaluate the expression of eNOS, VCAM and E-selectin. The concentration of VCAM and E-selectin in the conditioned medium was measured by ELISA.
Results: TNF-α inhibited HTR-8/SVneo trophoblast cell integration into endothelial cellular networks, as well as decreasing eNOS mRNA expression. An increase in VCAM and E-selectin mRNA and protein concentrations in the conditioned medium was also seen. NO reversed the inhibitory effect of TNF-α on trophoblast integration and increased eNOS mRNA expression. NO also reduced the expression of E-selectin and VCAM that were increased by TNF-α in the conditioned medium.
Conclusion: Our data suggest that the inhibitory effect of TNF-α on trophoblast integration may be mediated by NO, via a reduction in endothelial cell activation.
BLOOD-BRAIN BARRIER DISRUPTION FACILITATES INTRA-CAROTID ACTIVATION BY ANGIOTENSIN II OF TYROSINE HYDROXYLASE POSITIVE NEURONS IN THE RAT ROSTRAL VENTROLATERAL MEDULLA
Yao ST, May CN
Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Victoria, Australia
Background: Angiotensin II (Ang II) plays an important role in blood pressure control in both the periphery and brain. With the exception of the circumventricular organs, circulating angiotensin is excluded from the brain by the blood-brain-barrier (BBB).
Aim: To investigate whether BBB disruption leads to increased activation by circulating Ang II of tyrosine hydroxylase (TH)-containing neurons in the rostral ventrolateral medulla (RVLM).
Methods: Male SD rats were anaesthetized (Pentobarbitone, 60 mg/kg, i.p.), prior to BBB disruption with intracarotid hypertonic mannitol (1.6 M, 2 ml/kg) followed by subsequent intracarotid infusion of a subpressor dose of Ang II (5 ng/kg). Rats were then perfused and the brains were processed for TH and Fos immmunohistochemistry.
Results: Ang II activated ~24% of TH-containing RVLM neurons, whereas only ~8% of TH neurons were activated in the saline group. Intracarotid pretreatment with the Ang II receptor type 1 (AT1R) blocker, losartan (20 µg/kg), significantly reduced the number of TH cells activated to ~11% (P<0.05 by one-way ANOVA; n=5).
Conclusions: Disruption of the BBB resulted in increased activation by circulating Ang II of TH containing cells in the RVLM. This was blocked by losartan, indicating a specific action on AT1Rs. These results suggest that disruption of the BBB allows entry of circulating Ang II into the RVLM, which might act to increase sympathetic outflow.
DOWN-REGULATED Ca2+/CALMODULIN-DEPENDENT KINASE II ACTIONS IN THE HYPERTROPHIC FEMALE HEART – A LIABILITY FOR ISCHEMIA/REPERFUSION ARRHYTHMIAS?
Bell JR, Curl CL, Raaijmakers AHA, Harding TW, Harrap SB, Delbridge LMD
Cardiac Phenomics Laboratory, Department of Physiology, University of Melbourne, Melbounre, Victoria, Australia
Background: Women are less susceptible to ischemia-related lethal arrhythmias than men, yet female (but not male) risk increases substantially with an underlying hypertrophic pathology. With a continuing rise in obesity, diabetes and contingent hypertension, the impact of hypertrophy on ischemia-related arrhythmias/mortality in women would be expected to increase. Ca2+/calmodulin-dependent kinase II (CaMKII) is a key regulator of myocardial Ca2+-handling proteins, which mediates cardiac hypertrophy, failure, and atrial/ventricular arrhythmias, yet its role in generating reperfusion arrhythmias in hypertrophic hearts has not been investigated.
Aims: To determine the mechanistic relationship in female cardiac hypertrophy linking CaMKII, phospholamban phosphorylation at the CaMKII-specific residue (PLB-Thr17) and arrhythmias, using the female hypertrophic heart rat (HHR) model of primary cardiac hypertrophy.
Methods: Cardiomyocytes were isolated from female HHR and normal heart rat control (NHR) and were superfused (2.0 mM Ca2+, 37°C). Spontaneous contractions were measured in the presence of 10 nM isoproterenol. Female HHR/NHR isolated hearts (n=7–8) were subjected to ischemia/reperfusion, and arrhythmias were measured with an intraventricular balloon. Isoproterenol-challenged HHR cardiomyocytes exhibited more frequent spontaneous activity (HHR vs NHR beats: 13.4 ± 1.9 vs 8.5 ± 1.2; P<0.05). HHR hearts were more susceptible to ventricular tachycardia and/or fibrillation in early reperfusion (duration, 10 min reperfusion: 199±65 vs 19±14 s; P<0.05), which was associated with a suppression of CaMKII-mediated phospholamban phosphorylation (PLB-Thr17: 0.37±0.05 vs 1.03±0.29 arbitrary units; P<0.05).
Conclusions: These data challenge the canonical view of CaMKII as a pro-arrhythmic mediator, and suggest its actions on PLB may improve diastolic function through facilitated cytosolic Ca2+ removal and suppression of Ca2+-mediated arrhythmias. Ongoing studies imply specific upregulation of the auto-phosphorylated CaMKIIδB splice variant may provide a highly selective female anti-arrhythmic intervention target in the presence of an underlying hypertrophic pathology.
NEURONAL ACTIVATION IN THE HYPOTHALAMUS, MIDBRAIN AND MEDULLA FOLLOWING MYOCARDIAL INFARCTION IS INHIBITED BY MINOCYCLINE
Dworak Ma, Stebbing Ma, Kompa Ab, Rana Ia,c, Krum Hb, Badoer Ea
aSchool of Medical Sciences and Health Innovations Research Institute, RMIT University, Australia; bDepartment of Epidemiology and Preventative Medicine, University of Melbourne, Melbourne, Victoria, Australia: cCurrent address: School of Health Sciences, University of Ballarat, Ballarat, Victoria, Australia
Background: Following myocardial infarction, neuronal activity is elevated in the hypothalamic paraventricular nucleus (PVN) suggesting this nucleus contributes to the autonomic reflex responses observed in ventricular dysfunction. There is little is known about effects on other brain nuclei.
Aims: To investigate (i) whether the rostral ventrolateral medulla (RVLM), the nucleus tractus solitaries (NTS) and the periaqueductal gray (PAG), regions known to have important cardiovascular regulatory functions, also show increased neuonal activation; (ii) the effect of administering the anti-inflammatory drug, minocycline into the brain ventricles on neuronal activation and heart function.
Methods: Sprague Dawley rats were infused with either saline (0.09% NaCl) or minocycline (172 ng/ml, 0.3 µl/h) into the lateral ventricle of the brain. The rats then underwent either a myocardial infarction (MI) or sham procedure. Cardiac function was determined by echocardiography 12 weeks post MIThe rats were then killed and the brains processed for immunohistochemiistry to detect changes in neuronal activity using the presence of the marker proteins Fos related antigens (FRA).
Results: MI elicited a significant increase in FRA in the PVN, RVLM, NTS and PAG (P<0.001; n=3). Minocycline significantly attenuated the responses by at least 50% in the PVN and almost completely in the PAG, RVLM and NTS (n=5). Cardiac function was significantly reduced by 55% following MI, but this was not ameliorated by minocycline.
Conclusion: Following MI there is increased neuronal activity in brain nuclei that play key roles in cardiovascular regulation. Attenuation of this response may not be sufficient to improve cardiac function.
SELECTIVE CARDIOVASCULAR ADAPTATION TO CHRONIC STRESS IN MICE
Abegaz Ba, Stevenson ERa,b, Jackson KLa,b, Gueguen Ca, Moretti J-La, Head GAa,b, Davern PJa
aBaker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia; bMonash University, Clayton, Melbourne, Victoria, Australia
Background: The cardiovascular effects of acute emotional stress are well known and include increases in blood pressure (BP) and heart rate (HR). There is habituation with chronic stress leading to less cardiovascular effects. However, the cardiovascular effects of a novel stress may be similar or possibly enhanced.
Aim: To develop a model of chronic stress in conscious mice in order to investigate BP as well as cardiovascular responses to repeated and novel stressors.
Methods: Male C57Bl6 mice were implanted with telemetry probes and recordings of mean arterial pressure (MAP) and HR were made in non-stressed (n=4) and chronically stressed mice (n=10; 2 hours stress per day for 3 weeks). The daily stress included a random combination of 60 min of restraint and 2×30 min of placement in a cage that had previously been occupied by another male mouse. Shaker stress was chosen as the novel stress and involved placing the mice in its home cage on to a slowly rotating orbital platform for 5 min.
Results: Exposure to chronic stress had no effect on basal levels of BP or HR, but attenuated the pressor (14.0 vs 21.2 mmHg; P<0.01) and tachycardic (95 vs 219 bpm; P<0.001) responses to dirty cage switch. No differences were observed in the cardiovascular response to restraint stress. There was, however, a marked increase in the response to the novel shaker stress in that MAP increased by +7.1 mmHg (P<0.01) and HR increased by +70 bpm (P=0.05) compared with responses from non-stressed mice.
Conclusion: The findings shows that cardiovascular responses to chronic stressors do not always lead to habituation. Responses to novel stressors can be enhanced and adaptation to long-term stressful situations may be non-uniform and may depend on the type of stress involved. Furthermore, the non-uniform adaptation suggests that the modulation likely involves higher brain regions such as the amygdala, rather than lower common autonomic pre-sympathetic pathways.
IMPACT OF CHRONIC HYPOXEMIA ON RAS CONTROL OF SYMPATHETIC REGULATION OF CARDIAC DEVELOPMENT ACROSS GESTATION
Zhang Sa, McMillen ICa, Botting KJa, Tie Ma, Snell Aa, MacLaughlin SMa, Gentili Sa, Head Rb, Bennet L3, Morrison JL1
aEarly Origins of Adult Health Research Group; bSansom Institute for Health Research, University of South Australia, Adelaide, South Australia, Australia; cFetal Physiology & Neuroscience Group, Department of Physiology, University of Auckland, Auckland, New Zealand
Background: The development of sympathetic innervation begins at 80–90 days gestation (term, 150 days) in the sheep and is regulated by the expression of nerve growth factor (NGF) in the target tissues. Studies in the spontaneously hypertensive rat have demonstrated that increased angiotensin II results in upregulation of NGF expression and in sympathetic hyperinnervation in a range of target tissues. There is an increase in circulating norepinephrine concentrations in the IUGR fetus and we hypothesized that NGF would be elevated in the heart of the IUGR fetus due to changes in the renin-angiotensin system (RAS).
Aim: To determine whether there is a RAS-dependent change in cardiac NGF in the IUGR fetus.
Methods: IUGR was induced by removing endometrial caruncles (placental implantation sites) in the non-pregnant ewe before mating. Ewes were killed humanely at 55, 90 and 140 days gestation, the fetal heart was dissected, weighed and ventricular samples collected. Gene expression was determined relative to the geometric mean of 3 housekeeper mRNAs using real-time PCR. Data were analysed using 2-way ANOVA (age and treatment).
Results: Body weight and in heart weight was reduced in IUGR fetuses at all ages. Cardiac NGF gene expression was higher in the hearts of the IUGR fetuses compared to controls at 90 days gestation. ACE1 gene expression was lower in IUGR fetuses at 140 days gestation., There was, however, a delay in the rise of ACE2 gene expression at 90 days gestation in IUGR fetuses. There was no effect of IUGR on cardiac expression of adrenergic receptor-β1 (ADRB1) mRNA, but interestingly adrenergic receptor-α2 (ADRA2) was higher at 55 days, 90 days and 140 days gestation in the IUGR group compared to controls.
Conclusions: These data show that NGF expression is higher in the heart of the IUGR fetus corresponding to sympathetic innervation in the sheep fetus. This may be related to the changes in local angiotensin II expression (to be measured) resulting in altered sympathetic innervation of the heart. These effects may be amplified by changes in cardiac expression of AR and underlie an increased vulnerability to cardiovascular disease.
FUNCTIONAL REGROWTH OF SYMPATHETIC NERVES IS INCOMPLETE FOLLOWING LONG-TERM RENAL DENERVATION IN THE SPONTANEOUSLY HYPERTENSIVE RAT
Tjongue M, Tare M, Denton KM
Department of Physiology, Monash University, Clayton, Melbourne, Victoria, Australia
Background: Renal denervation (RDN) can attenuate the development, or delay the onset, of hypertension. Ongoing clinical trials using RDN have shown a sustained reduction in arterial pressure in humans with resistant hypertension. The mechanisms, however, contributing to this sustained decrease in arterial pressure remain unclear.
Aim: To examine mean arterial pressure (MAP) and functional sympathetic reinnervation of the renal vasculature in spontaneously hypertensive rats (SHR) following RDN.
Methods: 8 week-old male SHR were implanted with a radiotelemetry probe to measure MAP. At 10 weeks of age and following a 6-day basal MAP recording, rats underwent surgical RDN (n=7) or a sham operation (n=6) and MAP was recorded until 22–26 weeks of age (12–16 weeks post-RDN). At the end of the recording period, segments of the renal lobar artery were isolated and mounted on a wire myograph and responses to perivascular nerve stimulation were examined. Smooth muscle membrane potential was measured using intracellular microelectrodes. Thus membrane potential and tension were recorded simultaneously. Smooth muscle constriction to α1- and α2-adrenoceptor agonists was also assessed.
Results: Basal MAP was not different between sham and RDN groups. MAP was significantly attenuated in RDN SHR, with the RDN group, being 16 ± 7 mmHg lower than the shams at 12 weeks post-RDN (P<0.04). Perivascular nerve stimulation with single pulses evoked excitatory junction potentials (ejps). Ejp amplitude was reduced in arteries from RDN SHR animals (P<0.01). Repetitive stimulation (1–8Hz, 5 s) evoked slow depolarization and contraction. At 5 Hz 5 s, slow depolarization amplitude was markedly smaller in RDN SHR arteries (12 ± 2 mV vs 8 ± 1 mV; P<0.05), with contractions being ~45% smaller in RDN arteries compared with sham (22 ± 3 % vs 12 ± 2 % of the contraction to 100 mM potassium; P<0.03). There was no difference in smooth muscle sensitivity to phenylephrine, methoxamine or clonidine between the groups.
Conclusion: Our results suggest that there is a sustained decrease in MAP following RDN. The renal vasculature is reinnervated following RDN. The functional responses are not, however, restored even after 12–16 weeks. Smaller ejps indicate that fewer neuromuscular junctions are reformed. Incomplete reinnervation of the renal vasculature may be a contributing factor to the sustained effect of RDN on blood pressure.
WHOLE GENOME DNA SEQUENCING OF A GENETIC MODEL OF CARDIAC HYPERTROPHY IDENTIFIES TWO CANDIDATE GENES IN CARDIAC MASS QTL 22
Prestes PRa, Marques FZa, Lopez-Campos Gb, Harrap SBc, Charchar FJa
aSchool of Health Sciences, University of Ballarat, Ballarat, Victoria, Australia; bHealth and Biomedical Research Unit, Melbourne Medical School, University of Melbourne, Melbourne, Victoria, Australia; cDepartment of Physiology, University of Melbourne, Melbourne, Victoria, Australia
Background: Cardiac hypertrophy (CH) is a silent condition that involves thickening of heart muscle, so reducing functionality and increasing risk of cardiac disease and morbidity. Genetic factors are known to be involved, but their contribution is poorly understood.
Aim: To unveil genetic contribution to CH independent of blood pressure.
Methods: We sequenced the whole genome of the hypertrophic heart rat (HHR), a normotensive genetic model of CH, and its control, the normal heart rat (NHR) using the Illumina HiSeq 2000 platform. Clean reads were aligned and compared to the Rat Genome Database v3.4. We analysed four types of variants: single nucleotide polymorphisms (SNPs), copy number variations (CNVs), insertions and deletions (InDels) and structural variants (SVs).
Results: Overall, we found around 5.7 and 5.0 million variants in HHR and NHR, respectively. The majority of those variants (80%) were SNPs. Unique variants represented 27% and 16% of HHR and NHR mutations observed in all four types. HHR had around 4.5 million SNPs (25% unique), 100 thousand CNVs (all unique), one million InDels (29% unique) and 12 thousand SVs (99% unique), while NHR had almost 4 million SNPs (13% unique), 26 thousand CNVs (all unique), 960 thousand InDels (25% unique) and 11 thousand SVs (99% unique). In further analyses we identified variants in a locus we previously reported to control heart size independent of blood pressure on chromosome 2 (cardiac mass 22, former Lvm1) and correlated those with gene expression observed in whole-genome gene expression analyses in neonatal rats. Although two genes were differentially expressed, we only found mutations (19) in one of them. These mutations could contribute to the hypertrophic phenotype.
Conclusion: We have identified polymorphisms at the whole genome level in a genetic model of CH and pinpointed potential candidate genes for hypertrophy development located in the cardiac mass 22 QTL.
INTRARENAL PERFUSION AND OXYGENATION IN AN OVINE MODEL OF SEVERE SEPSIS WITH HYPOTENSION AND KIDNEY INJURY
Calzavacca Ca, Evans RGb, Bellomo Rc, May CNa
aFlorey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Victoria, Australia; bDepartment of Physiology, Monash University, Clayton, Victoria, Australia; cDepartments of Intensive Care and Medicine, Austin Health, Melbourne, Victoria, Australia
Background: The pathophysiology of septic acute kidney injury (AKI) is poorly understood. It has been proposed that septic AKI may result from decreased perfusion and oxygenation of the renal medulla.
Aim: To establish the ability of chronically implanted optic fibre probes to monitor intrarenal perfusion and oxygenation in conscious sheep, and to determine the changes in perfusion and oxygenation in the renal cortex and medulla in an ovine model of severe sepsis.
Methods: Mean arterial pressure (MAP), cardiac output (CO), renal blood flow (RBF), and renal cortical and medullary tissue perfusion and oxygen partial pressure were measured in conscious sheep (n=8). Renal blood flow was reduced by 20% and 50% for 30 min with a vascular occluder on the renal artery. After 24 hours of baseline data collection, sepsis was induced with live Escherichia coli infusion for 24 hours.
Results: In the renal cortex and medulla, a 20% reduction in RBF decreased perfusion (14.6 ± 8.6 and 41.2 ± 8.5%, respectively) and oxygenation (48.1 ± 8.5 and 72.4 ± 8.5%, respectively). Following E. coli, all sheep developed a hyperdynamic state with a doubling in heart rate and CO, a 50% increase in RBF, and a 15 mmHg decrease in MAP. Urine output halved, serum creatinine doubled and creatinine clearance decreased by one third. Cortical perfusion and oxygenation did not change significantly, but medullary flow showed an early trend towards reduction, while medullary pO2 progressively decreased by 53 ± 11% at 24 hours of sepsis. Calculated total renal oxygen consumption did not change (33 vs 32 ml O2/min).
Conclusions: Changes in RBF induced in this study predicted changes in intrarenal perfusion and oxygenation that returned to control levels after the occlusion was released, indicating responsiveness and stability of the measurements by the optic fibre probes. In a conscious ovine model of septic AKI, medullary oxygenation decreased, possibly due to reduced perfusion with unchanged oxygen consumption, or to intrarenal shunting. This reduction in medullary oxygenation may contribute to the development of septic AKI.
IS THE HEART OF THE IUGR FETUS HYPOXIC IN EARLY GESTATION?
Tie M, Botting KJ, Zhang, McMillen IC, Gentili S, MacLaughlin SM, Morrison JL
Early Origins of Adult Health Research Group, Sansom Institute for Health Research, University of South Australia, Adelaide, South Australia, Australia
Background: Intrauterine growth restriction (IUGR) and chronic hypoxemia cause a decrease in cardiomyocyte endowment in late gestation, in the absence of a change in the expression of hypoxia inducible factors (HIF1α, 1β and 2α) or genes with hypoxia response elements (HRE). We hypothesized that the decreased cardiomyocyte endowment in late gestation is due to either a decrease in proliferation or an increase in cardiomyocyte death in early gestation in the IUGR fetus.
Methods: A sheep model of IUGR was induced by removing endometrial caruncles (placental implantation sites). At 55 days gestation, control and IUGR ewes were killed humanely and fetuses were weighed. The heart was weighed and ventricular samples were frozen and fixed. Gene expression was determined relative to the geometric mean of 3 housekeeping mRNAs using real-time PCR. The percent proliferating cardiomyocytes was determined with Ki67 staining. Protein expression was determined relative to a loading control using western blotting. Data were analysed using a Students’ t-test.
Results: The IUGR fetuses were smaller (control 0.034 ± 0.001 kg; n=17; IUGR 0.027 ± 0.001 kg; n=19), but there was no difference in relative heart weight. There was no difference in expression of HIF1α, 1β and 3α mRNAs or of genes with HRE including those for VEGF, IGF2, IGF2R and GLUT1 in the IUGR group compared to controls. There was no change in the expression of mRNAs for proteins that promote cell cycle entry (Chek1, CDC2A, cyclinD1, cyclinD2 and Cdk9), or in the percent of Ki67 positive cardiomyocytes or PCNA protein abundance between the IUGR and control fetuses. There was, however, a decrease in the protein expression of Bcl-xl, an anti-apoptotic protein, and an increase in the protein expression of LC3B, a marker of autophagy in IUGR fetuses.
Conclusions: These data suggest that the heart of the IUGR fetus may not be hypoxic and that there is no change in cardiomyocyte proliferation in early gestation, there may be an increase in apoptosis or autophagy of cardiomyocytes. A small decrease in cardiomyocyte number early in gestation may thus result in reduced cardiomyocyte endowment in the IUGR fetus in late gestation.
A POTENTIAL ROLE FOR RHO KINASE IN EARLY LEFT VENTRICULAR DYSFUNCTION IN DIABETES
Waddingham MTa, Edgley AJa,b, Astolfo Ac, Inagaki Td, Tsuchimochi Hd, Yagi Ne, Kelly DJa, Shirai Md, Pearson JTb,f,g
aDepartment of Medicine, St. Vincent’s Hospital, University of Melbourne, Melbourne, Victoria, Australia; bDepartment of Physiology, Monash University, Clayton, Victoria, Australia; cSwiss Light Source, Switzerland; dNational Cerebral and Cardiovascular Center Research Institute, Osaka, Japan; eJapan Synchrotron Radiation Research Institute, Hyogo, Japan; fMonash Biomedical Imaging Facility, Victoria; gAustralian Synchrotron, Clayton, Victoria, Australia
Background: Patients with diabetes have a 2–5 fold increased incidence of heart failure. Using synchrotron x-ray diffraction in the in situ beating heart, we have shown that cross-bridge (CB) dynamics are impaired in the left ventricle (LV) of rats with early diabetes. Rho kinase (ROCK) plays an important role in regulating the phosphorylation state of proteins myosin light chain 2 (MLC-2) and myosin binding protein–C (MyBP-C), which are involved in controlling myosin head leverage and CB dynamics in the myocardium. Furthermore, increased ROCK activity results in LV contractile dysfunction in animal models of diabetes.
Aim: To examine if early diabetes is associated with altered phosphorylation of proteins involved in regulating CB dynamics and if ROCK inhibition prevents impaired CB dynamics in early diabetes.
Methods: Type 1 diabetes was induced in rats by streptozotocin (65 mg/kg, i.p.). Control rats received citrate vehicle. One week later, diabetic rats were further randomized to receive fasudil (10 mg/kg/day) or saline for 2 weeks. Rats underwent cardiac catheterization to assess LV function or were subjected to x-ray diffraction experiments at the SPring-8 Synchrotron, Japan to assess cardiac CB dynamics in situ.
Results: In comparison to control, 3 weeks of diabetes in rats resulted in prolonged LV relaxation times (P<0.05) and impaired systolic function (P<0.05). Fasudil mildly improved systolic function in diabetic rats. A trend for a 20% reduction of MyBP-C phosphorylation in diabetic rats compared to controls was shown and fasudil partially prevented this reduction in diabetic rats. MLC-2 phosphorylation was increased in diabetic rats, but fasudil did not affect MLC-2 phosphorylation. Furthermore, myosin head extension was impaired at end diastole in diabetic rats and was improved with fasudil treatment in the subendocardial layer of the LV wall.
Conclusion: Our results suggest that ROCK contributes to impaired LV contractility and CB dynamics in early diabetes, possibly by altering phosphorylation of proteins involved in regulating myosin head extension.
B CELL SUBSETS AND PATHOGENESIS OF ATHEROSCLEROSIS
Bobik Aa,b, Kyaw TSa,b, Tay C, Hosseini Ha,c, Tipping Pc, Toh B-Hc
aVascular Biology and Atherosclerosis Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, bDepartment of Immunology, Monash University, Melbourne, Victoria, cCentre for Inflammatory Diseases, Department of Medicine, Monash University, Clayton, Victoria, Australia
Background: Atherosclerosis is a chronic inflammatory disorder responsible for the majority of deaths due to myocardial infarctions and strokes. Atherosclerotic lesions that develop in the vessel wall of medium/large arteries are the result of complex interactions between accumulated LDL-cholesterol, endothelial and smooth muscle cells and cells of the innate and adaptive immune system. Multiple immune cells accumulate in atherosclerotic lesions including macrophages and dendritic cells, NK and NKT cells, CD4+ and CD8+ T cells and B cells. Early studies indicated that B cells are protective against atherosclerosis by producing low affinity IgM antibodies against oxidised LDL (low density lipoprotein). However, recent advances in B cell immunobiology indicate multiple subtypes of B cells, suggesting a more complex role in the pathogenesis of atherosclerosis.
Aim: To reinvestigated the role of B cells in atherosclerosis using fat fed ApoE−/− mice
Methods and Results: Using an anti-CD20 B cell depleting antibody, we demonstrated that B cell depletion decreases development and progression of murine atherosclerosis. Adoptive transfer approaches demonstrated that the B2 B cell subtype was proatherogenic. By targeting the B2 B cell survival factor BAFF we demonstrated their important role in atherosclerotic lesion inflammation and development/progression of atherosclerosis. Unlike B2 B cells, B1a B cells produce natural IgM antibodies as well as interleukin-10. In contrast to B2 B cells, deletion of peritoneal and splenic B1a B cells aggravates atherosclerosis. These effects were accompanied by marked reductions in anti-oxidized LDL IgM antibodies, lesion apoptotic cell numbers and necrotic core development. Administration of anti-TIM (RMT1-10) mAb to hyperlipidemic ApoE−/− mice expanded the B1a B cell population including TIM-1+IgM+ and TIM-1+IgM+IL-10+ B1a B cell subsets and attenuates atherosclerosis.
Conclusion: B2 B cells are proatherogenic, whereas B1a B cells are athorprotetive.
THE ROLE OF HIGH-DENSITY LIPOPROTEINS IN POLARIZING HUMAN MACROPHAGE PHENOTYPES
Lee MKSa,b, Moore XL, Fu Yc, Fang La, Dart AMa,d, Sviridov Da, Murphy AJa, Chin-Dusting Ja,b
aBaker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia, bDepartment of Medicine (Alfred Hospital), Monash University, Clayton, Australia, cPeking University Health Science Center, Beijing, China, dAlfred Hospital, Melbourne, Victoria, Australia
Background: Macrophages play a critical role in the development and progression of atherosclerosis. Macrophages can polarize into two subtypes with distinctive phenotypes; inflammatory (M1) and anti-inflammatory (M2). High-density lipoproteins (HDLs) have many cardio-protective properties including potent anti-inflammatory effects, largely through the removal of cholesterol from cells. It is currently not known if this extends to influencing human macrophage phenotype.
Aims: To investigate the effect of HDL on polarizing human macrophages to distinct phenotypes.
Methods: Human blood monocyte-derived macrophages were polarizåed to either an M1-phenotype by incubation with lipopolysaccharide (LPS: 100 ng/mL) and interferon-γ (IFN-γ: 20 ng/mL) or interleukin-4 (IL-4: 20 ng/mL) to induce M2-phenotype. Polarization was performed in the presence or absence of human HDL (400 μg/mL) or apoA-I (280 μg/mL). We examined cell surface markers, phagocytosis and ROS using flow cytometry and mRNA expression using real-time PCR. Downstream signaling pathways were also assessed by western blot analysis.
Results: We discovered that HDL inhibited polarization to M1 as evidenced by a decrease in the expression of the M1 surface marker CD192. This was accompanied by a decreased expression of the M1 inflammatory genes TNFA, IL6 and MCP1. HDL also inhibited M1 inflammatory function, with reduced phagocytic capacity as well as hydrogen peroxide and superoxide production. Similarly, apoA-I was also able to suppress M1 polarization (CD192) and inflammatory mRNA expression. We next explored the signaling cascades involved and found a decrease in ERK1/2 phosphorylation in M1 macrophages treated with HDL, suggesting cholesterol efflux inhibits the MAPKs during macrophage polarisation to the M1 phenotype. However, HDL did not affect macrophage polarization to M2 phenotype.
Conclusion: We provide evidence that HDL not only reduces macrophage polarization to the inflammatory M1 phenotype, but also inhibits phagocytosis and ROS production in these cells. These data provide a new dimension to our understanding of the protective role of HDL and suggest that HDL may act to resolve inflammation in the atherosclerotic lesion.
THE ROLE OF NATIVE LOW-DENSITY LIPOPROTEINS (nLDL) IN MACROPHAGE POLARIZATION FROM DIFFERENT MONOCYTE SUBSETS
Al-Sharea A, Andrews KL, Fang L, Lee MKS, Murphy AJ, Moore XL, Chin-Dusting JPF
Baker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia
Background: Phenotypic changes in monocytes and macrophages (M0) alter the progression of atherosclerosis. Differences in the three monocyte subsets (classical: CD14++CD16-, intermediate: CD14+CD16+ and non-classical: CD14dimCD16+) have been associated with cardiovascular disease outcome. Monocyte derived M0 can polarize into either the classically activated (M1) phenotype or an alternatively activated (M2) phenotype, depending on their microenvironment. Extracellular lipids can influence the heterogeneity of monocytes and M0, yet the manner in which this occurs is unclear.
Aim: We aimed to determine if the different monocyte subsets have an innate ability to differentiate and polarize to M0 and whether plasma lipoproteins can influence the outcome.
Methods: Primary monocytes or monocyte subsets were differentiated into macrophages (M0) by macrophage-colony stimulating factor (M-CSF; 100 ng/mL) for 6 days in the absence and presence of nLDL (200 ng/mL). M0 were treated with lipopolysaccharide (LPS; 100 ng/mL) and interferon (IFN-γ; 20 ng/mL) or interleukin-4 (IL-4; 20 ng/mL) for 6–24 hours to induce polarisation to a M1 and M2 phenotype, respectively.
Results: Treatment with nLDL reduced the M2 surface markers, CD206 (M2: 320 ± 66 vs M2+nLDL: 159 ± 19%; n=6; P<0.05) and CD200R (M2: 167 ± 20 vs M2+nLDL: 79 ± 12%; n=8; P<0.05) expression in M2 macrophages. As well, mRNA expression of the cytokines TGFβ and IL-10 was blunted in M2 macrophages treated with nLDL (n=3–4; P<0.05). M0 differentiated into M2 from classical and intermediate, but not non-classical, monocytes expressed increased TGFβ mRNA levels, which were reduced 1.5-fold in M2 macrophages from classical monocytes only by nLDL treatment (n=4; P<0.05). Alternately, nLDL does not affect the expression of the M1 surface marker, CD64 (n=5; P>0.05), but does increase TNFα and IL-6 gene expression in M1 macrophages by 28- and 95-fold, respectively (n=3; P<0.05). Classical, intermediate and non-classical monocytes that were polarized to the M1 phenotype all had increased gene expression of TNFα and IL-6 (n=3–4; P<0.05) and nLDL only increased TNFα and IL-6 mRNA levels in classical and intermediate macrophages, respectively (n=3–4; P<0.05).
Conclusion: nLDL reduces M2 macrophage polarization and enhances gene expression of the inflammatory cytokines TNFα and IL-6. Furthermore, both M1 and M2 macrophages derived from the differing monocyte subsets respond differently to polarization stimuli and the effects of nLDL are subset specific.
POLARIZATION OF MONOCYTE SUBSETS TO AN M1 PHENOTYPE IN ATHEROSCLEROSIS
Williams H, Cassorla G, Pertsoulis N, Marmash N, Hitos K, Fletcher J and Medbury H
VBRC, Department of Surgery, University of Sydney, Westmead Hospital, New South Wales, Australia
Background: Monocytes and macrophages are integral to atherosclerotic plaque development, progression and, most importantly, rupture. The presence of macrophages in general (CD68), or a polarization towards an inflammatory (M1) phenotype (CD86/CD163), is associated with plaque instability.
Aim: To determine whether monocytes likewise display an inflammatory M1 phenotype in donors with atherosclerosis (or increased risk) and importantly, how this varies for the different monocyte subsets.
Methods: Blood samples were collected from controls and atherosclerotic patients. Donor lipid profiles were measured and flow cytometry was used to examine the inflammatory profile of the major (CD16–) and minor (CD16+) monocyte populations. Expression of M1 (CD86) and M2 (CD163) surface markers as well as intracellular levels of inflammatory cytokines (IL-1β, IL-6 and TNFα) were quantified and compared between monocyte subsets, donor groups and relative to donor’s lipid profiles.
Results and Discussion: In control donors, the CD16+ subset was more inflammatory as seen by a higher CD86/CD163 ratio (P<0.05). However, for CD16-, but not CD16+, monocytes there was a correlation between CD86/CD163 expression and donor’s ApoB/A ratio (P<0.05), suggesting that the CD16- subset is becoming more inflammatory with increased atherosclerotic risk. In the patient group, CD16+ monocytes were similarly found to have a higher CD86/CD163 ratio than CD16- monocytes (P<0.05). However notably, the CD86/CD163 ratio for the CD16+ subset was the same for patients and controls, while CD86/CD163 in the CD16- subset was higher in the patient group (P<0.05). As such, when controlling for standard variables, donors with the highest (top 50%) CD86/CD163 on CD16- monocytes were ~6 times more likely to be a patient than a control. M1 skewing of the CD16- subset with increased atherosclerotic risk was also seen by a positive correlation between ApoB and LDL with IL-1β, IL-6 and TNFα production.
Conclusion: With atherosclerosis or increased atherosclerotic risk, the CD16– monocyte subset displays a skewed M1 marker profile (with the CD86/CD163 ratio being a potential biomarker for atherosclerosis). The M1 skewing of the CD16– subset suggests that a greater number of monocytes, not just the minor CD16+ proportion, circulate in an inflammatory state in donors with increased atherosclerotic risk/atherosclerosis. The potential impact of this on atherosclerosis progression is now being explored.
WHY DO CLINICAL TRIALS OF LIPID MODIFYING THERAPIES FAIL?
South Australian Health & Medical Research Institute, Adelaide, South Australia, Australia
Background: The residual risk of cardiovascular events despite widespread use of statins supports the need to develop additional therapeutic strategies to achieve greater reductions in vascular risk. Over the course of the last decade several large clinical trials of lipid-modifying therapies have failed to demonstrate further lowering of adverse cardiovascular outcomes. Increasing scrutiny of these studies has revealed a number of important limitations that may have influenced the ability of these studies to demonstrate clinical efficacy.
Aim: To review the lessons learned from these studies and potential implications for the design of future studies.
EFFICACY AND SAFETY OF AMG 145, A FULLY HUMAN MONOCLONAL ANTIBODY TO PCSK9: DATA FROM FOUR PHASE 2 STUDIES IN OVER 1200 PATIENTS
Sullivan Da, Raal Fb, Giugliano RPc, Koren MJd, Roth EMe, Weiss Rf, Kim JBg, Somaratne RSg, Yang Jg, Liu Tg, Albizem Mg, Scott Rg, Wasserman SMg, Stein EAh
aCarbohydrate & Lipid Metabolism Research Unit, University of Witwatersrand, Johannesburg, South Africa; bTIMI Study Group, Brigham and Women’s Hospital, Boston, MA; cJacksonville Center For Clinical Research, Jacksonville, Florida; dDepartment of Clinical Biochemistry, Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia; eSterling Research Group, Cincinnati, OH, USA; fMaine Research Associates, Auburn, ME; gAmgen Inc., Thousand Oaks, CA, USA; hMetabolic and Atherosclerosis Research Center, Cincinnati, Ohio, USA
Background: In 4 recent randomized phase 2 trials, AMG 145, a fully human monoclonal antibody to PCSK9, administered subcutaneously (s.c.) every 2 or 4 weeks (Q2W or Q4W), demonstrated marked reductions in LDL cholesterol (LDL-C), with favorable changes in other lipids.
Aim: To assess the efficacy and safety of AMG 145 from a prespecified pooled analysis of data from 4 phase 2 trials.
Methods: The 4 trials enrolled 1359 patients. The pooled efficacy analysis included 1252 patients: 951 received AMG 145 and 301 received placebo s.c.; 107 patients who received ezetimibe were excluded. The pooled safety analysis included 1314 patients; 981 received AMG 145, alone or with ezetimibe; 333 received s.c. placebo, alone or with ezetimibe; 45 who received ezetimibe alone were excluded. In each trial, treatment duration was 12 weeks; primary end point was percentage change in LDL-C by ultracentrifugation (UC) from baseline to week 12. Three of 4 trials permitted stable background statin therapy. Serious adverse events (SAEs) and other safety data were collected.
Results: Mean age (±SD) was 56 ± 12 years, 56% were women, and 60% were on statins. Mean pre-treatment LDL-C by UC (±SD) was 138 ± 37 mg/dL. Mean changes in LDL-C (SE) from baseline at week 12 ranged from –40% (1.6%) to –59% (1.6%) across AMG 145 doses vs 0.1% (1.6%) to 0.5% (1.4%) for placebo (P<0.001 for all dose groups). Favorable changes were also observed in Apo B, Lp(a), triglycerides, HDL-C, and Apo A1. The highest doses, AMG 145 140 mg Q2W (n=123) and 420 mg Q4W (n=213) vs placebo Q2W (n=123) and Q4W (n=178), produced the greatest efficacy (Table 1). AEs were more frequent with AMG 145 vs placebo (57% vs 49%; Table 2). SAEs occurred in 2% of AMG 145 patients and 1% of placebo patients. None were considered treatment-related. No relationship between AEs and dose/frequency was observed (data not shown). Muscle-related AEs occurred in 6.0% vs 3.9% and CK elevations in 1.4% vs 0.9% of patients receiving AMG 145 vs placebo. Injection-site reactions and ALT/AST elevations were similar between groups. No neutralizing antibodies were detected.
Conclusion: This large pooled analysis of four phase 2 studies demonstrated marked and significant reductions in LDL-C and favorable changes in other pro-atherogenic and anti-atherogenic lipids with AMG 145 dosed either Q2W or Q4W. The overall safety profile of AMG 145 was acceptable. Further development of AMG 145 in longer-term studies is ongoing.
This study was funded by Amgen Inc.
PCSK9 IN HETEROZYGOUS FH (HeFH) PATIENTS
Petrides Fa, Chatelais Mb, Marais Dc, Lambert Gb
aCentre for Vascular Research, in affiliation with the School of Medical Science, University of New South Wales, Australia; bINSERM U957, Université de Nantes, France; cUniversity of Cape Town, South Africa
Background: PCSK9 inhibition has been demonstrated recently to be an effective therapeutic approach to lower LDL in HeFH patients. The aim of the present study was to determine whether or not PCSK9 is similarly effective at inhibiting the LDL receptor (LDLR) in a wide range of HeFH molecular defects.
Aim: To asses the risk associated with elevated PCSK9 levels is greater in heterozygous FH patients.
Methods: PCSK9 levels were measured by ELISA in samples from normolipemic controls (n=35) and in FH patients carrying either a D200G (n=18), a G361V (n=9), a P664L (n=9), a ◻197 (n=23), a G361 (n=1), or a D157V (n=1) mutation on one of their LDLR alleles. These LDLR variants were cloned and transiently expressed in HEK293 cells. Skin fibroblasts were also obtained from our HeFH patients. Cells were grown with 20% serum or 0.5% serum and increasing doses of mevastatin (0–40 mg/mL). Increasing amounts of PCSK9 (0–1 mg/mL) were added to the cells for 0 to 6 hours. LDLR expression was assessed by western blot, flow cytometry and confocal microscopy.
Results: PCSK9 plasma level was increased 2–3 fold in FH patients compared with controls. The expression of the LDLR was increased similarly in all FH fibroblasts upon serum depletion and statin treatment, although to a lower extent than in control fibroblasts. PCSK9 dose and time dependently reduced the expression of the LDLR in wild-type (by up to 65%) and FH (by up to 85%) fibroblasts. In transfected cells, PCSK9 inhibited LDLR cell surface expression by up to 40–50% in wild-type as well as in D200G, G361V, P664L, Del197, G361, and D157V LDLR transfected cells, after normalization for base line LDLR cell surface expression
Conclusion: This study demonstrates that PCSK9 inhibits cell surface expression of wild-type LDLR as well as HeFH-LDLR variants to a similar extent in the presence of statins. This suggests that PCSK9 inhibition will lower LDL plasma levels in most HeFH patients, irrespective of their underlying LDLR molecular defect.
THE EFFECT OF A HIGH POTASSIUM DIET ON VASCULAR FUNCTION
Blanch Na, Clifton PM, Petersen KSa, Willoughby SR, Keogh JBa
aSchool of Pharmacy and Medical Science, University of South Australia, South Australia; bCentre for Heart Rhythm Disorders, University of Adelaide and Royal Adelaide Hospital, Adelaide, South Australia, Australia
Background: Increased potassium intake has been related to reduced blood pressure (BP) and improved vascular function. Interventional studies have reported inconsistent findings, which may be explained by a threshold required to detect an effect of increased potassium intake on vascular function. The effect of increased dietary potassium on endothelial function remains unknown.
Aim: To determine the effect of increased dietary potassium (70 mmol/day) on endothelial function.
Methods: Thirty-nine healthy men and women (age 32 ± 12 y) completed a randomized cross over study of 2×6 day diets. The dietary interventions were high potassium (150 mmol/day) diet and usual potassium (80 mmol/day). Measurements of flow mediated dilatation (FMD), BP, pulse wave velocity (PWV), augmentation index (AI) and a fasting blood sample for analysis of intercellular adhesion molecule-1 (ICAM-1), E-selectin and asymmetric dimethylarginine (ADMA) were taken on completion of each intervention. Participants completed 6 day weighed food diaries and a 24 hour urine sample during each diet to estimate adherence to the intervention.
Results: There was no significant difference in FMD between the diets (high potassium 7.02 ± 1.94% vs low potassium 6.56 ± 1.78%, P=0.06). Mean potassium excretion was 92 ± 35 mmol/d for the high potassium diet and 50 ± 30 mmol/day for the low potassium diet (P<0.001). When analysis was completed to include compliers to the protocol (n=35, as defined by an increase in urinary potassium excretion greater than 0 mmol/day) FMD was significantly improved following the high potassium diet compared to the low potassium diet (P=0.03). There were no significant differences in systolic BP (P=0.85), diastolic BP (P=0.09), mean arterial pressure (P=0.16), PWV (P=0.67), AI (P=0.31), ICAM-1 (P=0.73), or ADMA (P=0.86) between the interventions. There was a significant reduction in E-selectin following the high potassium diet (Mdn=5.96 ng/ml) vs the low potassium diet (Mdn=6.22 ng/ml; z=-2.64, P=0.008). Potassium intake and potassium excretion were positively correlated following the high potassium and low potassium dietary interventions (r=0.48, P=0.002; and r=0.76, P<0.001, respectively).
Conclusion: An increase in dietary potassium improves endothelial function as assessed by FMD within 1 week in healthy men and women when an increase in potassium is achieved. This suggests potassium intake may have protective effects on vascular function, although the mechanisms for this effect remain unclear.
DIET QUALITY AND ARTERIAL COMPLIANCE IN PEOPLE WITH TYPE 1 AND TYPE 2 DIABETES
Petersen KS, Blayney RH, Clifton PM, Keogh JB
School of Pharmacy and Medical Sciences, University of South Australia, South Australia, Australia
Background: People with diabetes are at higher risk of cardiovascular disease (CVD). There is evidence that better diet quality may be protective against CVD.
Aim: To investigate the association between indices of dietary quality and pulse wave velocity (PWV) in people with type 1 and type 2 diabetes.
Methods: Participants (age 59±13 years) were 66 adults with type 1 (n=5) or type 2 (n= 61) diabetes recruited from the community. Dietary intake was measured using the 74 item electronic version of the Dietary Questionnaire for Epidemiological Studies (version 2) Food Frequency Questionnaire. Diet quality was assessed according to the a priori defined Australian Recommended Food Score (ARFS) and Healthy Eating Index (HEI). A SphygmoCor® XCEL (Sydney, Australia) was used to measure carotid-femoral PWV.
Results: The mean ARFS and HEI scores were 31.1±7.6 (score out of 74) and 62.1 ± 13.0 (score out of 100) respectively. Mean PWV was 9.6±2.0 m/s. No correlation was observed between the ARFS or HEI and PWV. However, when individual components of the HEI were considered, there was a significant inverse association between the dairy score and PWV (r= –0.460; P<0.05). Total dairy intake (grams/day) was inversely associated with PWV after adjustment for age (r= –0.35; P<0.05). The observed correlation was attributable to milk consumption (r= –0.282; P<0.05). Yoghurt and cheese consumption were not significantly associated with PWV.
Conclusion: In this cohort of subjects with type 1 and type 2 diabetes dairy consumption, particularly milk intake, was inversely associated with arterial compliance. No association was observed between indices of diet quality and PWV.
FENUGREEK EXTRACT IS MORE EFFECTIVE THAN ITS ACTIVE COMPOUND, SAPONINS, IN ATTENUATING ENDOTHELIAL ACTIVATION IN HUMAN ARTERIAL ENDOTHELIAL CELLS
Ahmad Ra,b, Rahman Ta, Froemming GRAb, Nawawi Ha
aThe Centre for Pathology Diagnostic and Research Laboratories (CPDRL) and bInstitute of Medical Molecular Biotechnology (IMMB), Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Malaysia.
Background: Inflammation and endothelial dysfunction are key events in the pathogenesis of atherosclerosis. There have been recent advancees in identifying cardioprotective properties in natural products. Trigonella foenum graecum, more commonly known as fenugreek, is one compound that has attracted interest. However, to date, there have been few studies on the potential atheroprotective properties of fenugreek and its active class of compounds, saponins.
Aim: To determine the effects of fenugreek crude extract and its saponins on protein and gene expression of endothelial activation biomarkers in stimulated human coronary artery endothelial cells (HCAECs).
Methods: LPS stimulated confluent HCAECs (Lonza, USA) were treated with fenugreek and saponins (concentration range: 46.9–375; 3.2–25.0 µg/ml). At 16 hours post-incubation supernatants were tested by enzyme-linked immunosorbent assays (ELISA) (eBioscience, North America) to measure sICAM-1 and sVCAM-1 protein expression. RNA was extracted from the cells for analysis of gene expression.
Results: Treatment with fenugreek reduced sICAM-1 and sVCAM-1 protein: 46.9 vs. 93.8 µg/ml (P=0.020) and 46.9 vs. 93.8 µg/ml (P=0.021), respectively, and gene expression: 93.8, 187 and 375 µg/ml (P=0.03) and 46.9, 93.8, 187 and 375 µg/ml (P<0.04) respectively. In contrast, saponin treatment increased protein expression of sICAM-1: 3.2, 6.3 and 12.5 (P<0.03) with no change in gene expression (P>0.05). There was no effect on sVCAM-1 protein (P>0.05), but a reduction in sVCAM-1 gene expression at high concentration (25.0 µg/ml; P=0.020).
Conclusion: Fenugreek crude extract is more effective than the active class of compound, saponins, in attenuating endothelial activation in HCAEC. This suggested that fenugreek in its natural form has better potential than the active constituent, saponins, as an anti-atherosclerotic agent.
Y CHROMOSOME-LINKED LONG NON-CODING RNA: FUNCTION AND INVOLVEMENT IN DIABETES WITH IMPLICATIONS FOR CORONARY ARTERY DISEASE
Molina E, Myers S, Chew G, Charchar FJ
School of Health Sciences, University of Ballarat, Victoria, Australia
Background: Diabetes is a major risk factor for coronary artery disease (CAD), which is responsible for substantial morbidity and mortality among people with diabetes. The diabetic metabolic environment predisposes to aggressive obstructive CAD that causes heart attacks, heart failure and death. However, the pathogenesis of the vascular and myocardial complications of diabetes is not well understood. In this context, studies have demonstrated a positive correlation between increased hepatic free fatty acids (FFAs) in atherosclerosis and CAD. Recently, studies have shown that the human Y chromosome is associated with a greater risk of CAD in men. Moreover, long non-coding RNAs (lncRNAs) have gained attention as a new class of regulatory RNAs involved in cardiovascular function and associated disease. However, the molecular mechanisms implicated are not well defined.
Aims: We aim to identify the Y-specific lncRNAs involved in atherosclerosis and to investigate their role in this disease in an in vitro liver model of insulin resistance.
Methods: Primers for ten IncRNA transcripts from the Y chromosome annotated sequence were designed and analysed by quantitative PCR (qPCR) in a human tissue panel for differential expression of lncRNA. To create an insulin resistant model, human liver HepG2 cells were treated with 0.3 mM of the FFA palmitate for 24 hours and qPCR was performed on the transcripts.
Results: qPCR measured six lncRNA transcripts expressed in untreated liver tissues and in Hep G2 cells. In HepG2 cells treated with palmitate, statistical analysis (Student’s t-test) determined a significant increase in the expression of two lncRNA transcripts, lnc-KDM5D-4:1 (P=0.019) and lnc-ZFY-1:1 (P=0.032).
Conclusion: Given the role of FFA in the pathogenesis of CAD, we propose that lncRNAs in the liver may play a role in regulating metabolic processes implicated in atherosclerosis and thereby participate to the risk of CAD in men.
SAXAGLIPTIN AND CARDIOVASCULAR OUTCOMES IN PATIENTS WITH TYPE 2 DIABETES
Raz Ia, Scirica BMb, Braunwald Ec, Mosenzon Oa, Hirshberg Bd, Wiviott SDb, Frederich Re, Davidson Jf, Leiter LAg, Bhatt DLc
aDiabetes Unit, Department of Medicine, Hadassah University Hospital, Jerusalem, Israel; bTIMI Study Group, Cardiovascular Division, Brigham and Women’s Hospital, and Harvard Medical School, Boston, Massachusetts, USA; cTIMI Study Group, Cardiovascular Division, Brigham and Women’s Hospital, VA Boston Healthcare System, and Harvard Medical School, Boston, Massachusetts, USA; dAstraZeneca Research and Development, Wilmington, Delaware, USA; eBristol-Myers Squibb, Princeton, New Jersey, USA; fDivision of Endocrinology, Department of Internal Medicine, university of Texas Southwestern Medical Center, Dallas, Texas, USA;. gDivision of Endocrinology and Metabolism, Keenan Research Centre in the Li Ka Shing Knowledge Institute of St. Michael’s Hospital, University of Toronto, Toronto, Canada
Background: Saxagliptin is a new oral hypoglycemic agent in the dipeptidyl peptidase-4 inhibito class of anti-diabetic drugs.
Aim: The aim of the SAVOR-TIMI 53 trial was to evaluate the long-term cardiovascular efficacy and safety of saxagliptin in patients with type 2 diabetes (T2D) at risk for cardiovascular events.
Methods: The study randomly assigned 16,492 patients with T2D who had a history of, or were at risk for, cardiovascular events to receive saxagliptin or placebo and followed them for a median of 2.1 years.
Results: Overall, saxagliptin neither increased nor decreased the risk of the primary endpoint of cardiovascular death, myocardial infarction, or ischemic stroke. The composite secondary end point was balanced, supporting the overall cardiovascular safety. However, there was an unexpected 27% increased relative risk (61 subject excess, 0.37/100 subject-years difference between saxagliptin and placebo) of hospitalization for heart failure (hHF), a component of the secondary endpoint, in patients assigned to saxagliptin. There were clinical features, including patients with a history of heart failure or elevated levels of NT-proBNP, that identified patients at an overall increased risk of hHF, although, even in those patients the primary and secondary endpoints were balanced. Saxagliptin improved glycemic control with significantly greater percentage of patients reaching target A1C without hypoglycemia in spite of a 30% decrease in initiation of insulin therapy and a 23% reduction of increase in oral hypoglycemic therapy. Saxagliptin significantly reversed or prevented deterioration of microalbuminuria after 1 and 2 years on therapy. Saxagliptin increased major hypoglycemic events in patients treated with sulfonylurea and baseline hba1c <7% without increasing the need for hospitalization or the primary or secondary endpoint. There was no excess hypoglycemia with other background antidiabetic treatments including insulin. Overall adverse events including adverse events of special interest for antidiabetic medications as well as incretin based therapies were similar between saxagliptin and placebo. This DPP4 inhibitor did not increase the overall risk or severity of pancreatitis or show signs of increased risk for pancreatic cancer.
Conclusions: Findings from the SAVOR study expand and clarify the saxagliptin safety profile established in the Phase 2b/3/3b clinical program, through greater exposure in an older population with longer diabetes duration and multiple risk factors for, or established, cardiovascular disease. The safety and tolerability of saxagliptin use in T2D is supported by the SAVOR results.
MECHANISMS OF DIABETES-ACCELERATED ATHEROSCLEROSIS
Department of Medicine, Division of Metabolism, Endocrinology and Nutrition, Department of Pathology, Diabetes and Obesity Center of Excellence, University of Washington, Seattle, Washington, USA
Background: Diabetes accelerates the formation and progression of atherosclerotic lesions, which likely explains the increased risk of cardiovascular disease in diabetic humans. The accelerated atherosclerosis is driven, at least in part, by the altered function and properties of myeloid cells, namely cells that display and contribute to an increased inflammatory vascular state in atherogenesis. Pro-inflammatory changes in endothelial cells also likely contribute to diabetes-accelerated atherosclerosis.
Aim: To identify cellular and molecular mechanisms of diabetes-accelerated atherosclerosis using mouse models.
Methods and Results: In mouse models of type 1 diabetes, initiation of atherosclerosis is accelerated due to increased accumulation of macrophages in the arterial wall. Based on our recent studies, the enzyme acyl-CoA synthetase 1 (ACSL1), which converts long-chain fatty acids into their acyl-CoA derivatives, has emerged as causal to the enhanced atherosclerosis associated with diabetes. ACSL1 is expressed at higher levels in myeloid cells from diabetic mice, and is induced by inflammatory mediators in these cells. Moreover, deletion of ACSL1 in myeloid cells results in complete protection of these cells from the inflammatory activation associated with diabetes and from early diabetes-accelerated atherosclerosis. Interestingly, ACSL1-deficiency appears to target a pathway that is selectively activated by diabetes. In endothelial cells, ACSL1 is induced by TNF-α, and contributes to TNF-α-induced secretion of the important chemokine CCL2, suggesting that endothelial ACSL1 might also promote atherosclerosis.
Conclusion: Myeloid ACSL1 specifically mediates diabetes-accelerated atherosclerosis in mice. An important question is whether ACSL1 and other factors identified in mouse models play equally important roles in diabetes-accelerated atherosclerosis in humans.
THE ROLE OF DOMINANT-ACTIVE IDOL IN DIET INDUCED HYPERCHOLESTEROLEMIA AND ATHEROSCLEROSIS
Calkin ACa,b, Goult BTb, Weissglas-Volkov Dc, Zhang Ld, Fairall Lb, Hong Cd, Pajukanta Pc, Schwabe JWRb, Tontonoz Pd
aDiabetes & Dyslipidaemia Group, Baker IDI Heart and Diabetes Institute; bDepartment of Biochemistry, University of Leicester, UK; cUniversity of California Los Angeles, Human Genetics, Los Angeles, California, USA; cDepartment of Human Genetics and dDepartment of Pathology and Laboratory Medicine, University of California Los Angeles, California, USA
Background: Dyslipidemia is a common feature of diabetes and the metabolic syndrome, and can manifest itself as an elevated level of LDL cholesterol. The E3 ubiquitin ligase, IDOL (inducible degrader of the LDL receptor, LDLR), is a novel regulator of LDLR-dependent cholesterol uptake, but its mechanism of action, and its influence on plasma cholesterol and atherosclerosis in vivo have not been examined.
Aim: We sought to define the IDOL-LDLR interaction and to examine the consequence of chronic liver-specific expression of a dominant active form of IDOL in mice.
Methods and Results: Through the use of mutational studies, we identified critical residues within the FERM domain of IDOL that bind a newly identified recognition sequence in the cytoplasmic tails of its lipoprotein receptor targets. Furthermore, we expressed a degradation-resistant, dominant-active form of IDOL (sIDOL) in C57Bl/6J mice from the liver-specific albumin promoter (L-sIDOL Tg). L-sIDOL transgenic (Tg) mice were fed a Western diet for 20 or 30 weeks and then analyzed for plasma lipid levels and atherosclerotic lesion formation. L-sIDOL Tg mice demonstrated substantial reductions in hepatic LDLR protein and increased plasma LDL cholesterol levels on both chow and Western diets. Moreover, L-sIDOL mice developed marked atherosclerotic lesions when fed a Western diet, with male mice showing a two-fold greater lesion burden compared to females. qPCR revealed elevated mRNA expression of LXR target genes such as ABCA1 and IDOL as well as pro-inflammatory genes such as STAT6 in aortas of Western diet-fed L-sIDOL mice.
Conclusion: Our data identify the IDOL–LDLR interaction and suggest that this could potentially be exploited for the pharmacologic modulation of lipid metabolism and atherosclerosis. Furthermore, liver-specific expression of dominant active IDOL is associated with hypercholesterolemia and a marked elevation in atherosclerotic lesions. Our results demonstrate that increased activity of the IDOL pathway in the liver can override other LDLR regulatory pathways leading to cardiovascular disease. L-sIDOL mice are a robust, dominantly-inherited, diet-inducible model for the study of atherosclerosis.
DIABETES INCREASES RETICULATED PLATELETS DUE TO ENHANCED PROLIFERATION AND EXPANSION OF BONE MARROW MEGAKARYOCYTE PROGENITORS
Kraakman MJa, Nagareddy PRb, Goldberg IJc, Murphy AJa
aHaematopoiesis and Leukocyte Biology, Baker IDI Heart and Diabetes Institute, Victoria, Australia; bDepartment of Internal Medicine, University of Kentucky, Lexington, Kentucky, USA; cDivision of Preventive Medicine and Nutrition, Columbia University, New York, New York, USA
Background: Diabetes (types 1 and 2) is a major risk factor for cardiovascular disease (CVD) and represents the major cause of mortality in affected patients. Even with current treatments, diabetic patients with CVD have worse outcome than patients with CVD alone. This is likely explained by the mechanisms contributing to accelerated atherosclerosis in diabetes being different from those without diabetes. We hypothesized that increased reticulated platelets, which play an important role in atherogenesis, are increased in diabetes by overproduction from the bone marrow (BM).
Aim: To investigate the mechanisms contributing to increased reticulated platelets in diabetes.
Methods: Wild-type mice were made diabetic with streptozotocin and the number of total and reticulated platelets was measured. To determine the mechanism for increased platelets we assessed the population of megakaryocyte progenitors (MkPs) in the BM by flow cytometry. We also examined the levels of thrombopoietin (TPO) in the plasma by ELISA. To determine if hematopoietic RAGE was playing a role we performed BM transplant studies comparing wild-type to Rag–/– BM.
Results: We found a significant increase in platelets and reticulated platelets in diabetic mice. This appeared to be due to an over-production as MkPs were expanded and proliferating more in the BM of hyperglycemic mice. This also resulted in more megakaryocytes in the BM. We also found an increase in plasma TPO levels, but no change in the TPO receptor, c-MPL, on any of the BM progenitor cells or circulating platelets, suggesting that the effect was due to increased TPO production from the liver. The expression of TPO is generally upregulated by Kupffer cell derived IL-6. We found more Kupffer cells in the liver of diabetic mice and these cells expressed higher levels of cell surface RAGE. Depletion of Kupffer cells using clodronate liposomes normalized the levels of total and reticulated platelets along with MkPs in the BM. Transplantation of Rage–/– BM into diabetic mice prevented diabetes-induced thrombocytosis in a non-cell autonomous manner.
Conclusion: Increased reticulated platelets in diabetes are due to enhanced proliferation and expansion of MkPs in the BM. This is driven by increased TPO production, likely due to IL-6 release by Kupffer cells downstream of RAGE signaling.
DELETION OF BONE MARROW OR NON-BONE MARROW DERIVED RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS ATTENUATES DIABETES-ASSOCIATED ATHEROSCLEROSIS
Koulis C, Kanellakis P, Pickering RJ, Tsorotes D, Thomas MC, Cooper ME, Jandeleit-Dahm KA, Allen TJ.
Diabetic Complications, Baker IDI Heart and Diabetes Research Institute, Melbourne,Victoria, Australia
Background: The receptor for advanced glycation end products (RAGE) is expressed on multiple cell types implicated in the progression of atherosclerosis, particularly in diabetes.
Aim: To determine the role of either bone marrow (BM)- or non-BM-derived RAGE in the pathogenesis of streptozotocin (STZ)-induced diabetes-associated atherosclerosis.
Methods: Seven week-old ApoE–/– and ApoE–/–:RAGE–/– mice were rendered diabetic with STZ. At eight weeks of age, ApoE–/– and ApoE–/–:RAGE–/– control and diabetic mice were lethally irradiated and transplanted with BM from either ApoE–/– or ApoE–/–:RAGE–/– mice to generate 4 different chimeras. After 10 and 20 weeks of diabetes, mice were sacrificed and gene expression and atherosclerotic lesion formation were evaluated, respectively.
Results: The presence of global RAGE (ApoE–/– mice transplanted with ApoE–/– BM) increased diabetes-associated atherosclerosis by 1.9-fold (plaque size 10.7 ± 1.7 mm2) and was accompanied with a significant increase in aortic accumulation of VCAM-1, CD68 as well as increased gene expression of NF-KB and VCAM-1 when compared to the ApoE–/– control mouse transplanted with ApoE–/– BM. Deletion of RAGE in either the BM or non-BM cells resulted in a significant attenuation in diabetes-associated atherosclerosis of approx 48% (plaque size 5.5 ± 1.1 mm2) and 55% (4.8 ± 0.5 mm2), respectively. Furthermore, the reduced plaque size and associated reduced VCAM-1 gene expression translated to reduced adhesion in vitro as assessed by a static adhesion assay.
Conclusion: Global, non-BM and BM deletion of RAGE attenuates diabetes-associated atherosclerosis. These findings emphasise the importance of utilising either non-BM- or BM-derived RAGE in the development of therapeutics to target the prevention and treatment of diabetes-associated atherosclerosis.
EFFECT OF NIACIN ON TRIGLYCERIDE-RICH LIPOPROTEIN APOLIPOPROTEIN B-48 KINETICS IN TYPE 2 DIABETIC SUBJECTS ON A STATIN
Pang J, Chan DC, Hamilton SJ, Tenneti VS, GF, Barrett PHR
School of Medicine & Pharmacology, University of Western Australia, Perth, Western Australia, Australia
Background: Type 2 diabetic subjects often have hypertriglyceridemia and an increased concentration of apolipoprotein B-48 (apoB-48) in the circulation, particularly in the postprandial period. There is an accumulating body of evidence to suggest that apoB-48 plays a central role in the development of atherosclerosis. Statins are the frontline therapy to reduce cardiovascular risk. However, a large residual risk still remains. This residual risk suggests that additional therapeutic interventions may be required to further reduce cardiovascular disease (CVD) risk.
Aim: To investigate the effect of niacin on the metabolism of triglyceride rich lipoprotein (TRL) apoB-48 in men with type 2 diabetes on a background of statin therapy.
Methods: Twelve type 2 diabetic men were recruited for this randomized, cross-over design study. Patients required a statin-treated low density lipoprotein (LDL) cholesterol of less than 2.5 mmol/L to enter the trial. Patients were then randomized to rosuvastatin alone or rosuvastatin plus niacin (titrated up from 1 to 2 g daily) for a period of 12 weeks and then were crossed over to the alternate therapy with a 3-week washout period in between. Metabolic studies were performed at the end of each treatment period. A bolus intravenous infusion of D3-leucine was administered as subjects consumed a standardized high-fat liquid meal. Blood samples were collected over 24 hours and TRL apoB-48 tracer/tracee ratios were measured using gas chromatography-mass spectrometry. Kinetic parameters, including fractional catabolic rate (FCR) and production rate (PR), were derived using a multicompartmental model.
Results: Niacin significantly reduced triglyceride, plasma cholesterol, LDL cholesterol and apoB (all P<0.005). TRL apoB-48 concentration was lower with niacin (8.24 ± 1.98 vs 5.48 ± 1.14 mg/L; P=0.03). ApoB-48 FCR was not altered with niacin (8.78 ± 1.04 vs 9.17 ± 1.26 pools/day; P=0.79). Basal apoB-48 PR (3.21 ± 0.34 vs 2.50 ± 0.31 mg/kg/day; P=0.04) and postprandial apoB-48 PR were significantly lower (1.35 ± 0.19 vs 0.84 ± 0.12 mg/kg; P=0.02) on niacin.
Conclusion: Niacin reduces TRL apoB-48 concentration by lowering basal and postprandial apoB-48 PR. This effect on apoB-48 metabolism may be beneficial for reducing atherogenic postprandial TRL particles and may provide CVD risk benefit to type 2 diabetic patients.
NON-LIPID CARDIOVASCULAR RISK FACTORS IN FAMILIAL HYPERCHOLESTEROLAEMIA: A CROSS-SECTIONAL STUDY IN WESTERN AUSTRALIA
Pang J, Chan DC, van Bockxmeer FM, Bates TR, Burnett JR, Bell DA, Watts GF
University of Western Australia, Royal Perth Hospital, Perth, Western Australia, Australia
Background: Familial hypercholesterolaemia (FH) is classically due to LDL receptor mutations that result in marked hypercholesterolemia and premature coronary artery disease (CAD). The contribution of other cardiovascular (CV) risk factors in FH is unclear.
Aim: To examine the relationship between non-lipid CV risk factors and CAD in FH.
Methods: A cross-sectional study of 384 phenotypically (20%) and genotypically (80%) defined FH (211 women and 173 men, age 47.8 ± 15.3 years, BMI 27.1 ± 5.1 kg/m2, Dutch Lipid Network Criteria score >8) from the Western Australia cascade screening program was conducted. The CV risk factors included smoking, diabetes mellitus, hypertension (BP >140/90 mmHg) and obesity (BMI >30 kg/m2). Logistic regression analysis was used to study the relationship between non-lipid CV risk factors and CAD in these patients. CAD was defined as a history of myocardial infarction, or coronary revascularization.
Results: The prevalence of CV risk factors were: current/ex-smokers 49%, obesity 21%, hypertension 26%, low HDL-cholesterol 29%, type 2 diabetes 9%, high LDL-cholesterol 94% and previous CAD 20% (myocardial infarction 12%, angioplasty 14%, bypass graft 8%). In univariate logistic regression analysis, history of hypertension (OR 5.25, P<0.0001) and type 2 diabetes (OR 5.05, P<0.0001) were significantly associated with history of CAD in FH patients. Hypertension and diabetes remained significant determinants of CAD in FH patients in multivariate logistic regression analysis adjusting for age and gender (OR 3.24 and 2.88; P<0.001 and P=0.01, respectively).
Conclusion: The spectrum of modifiable CV risk factors extends beyond hypercholesterolemia in patients with FH. Aggressive management of all CAD risk factors in FH patients is indicated.
ASSOCIATION BETWEEN SKELETAL MUSCLE FAT CONTENT AND VERY-LOW-DENSITY LIPOPROTEIN-APOLIPOPROTEIN B-100 TRANSPORT IN OBESITY: EFFECT OF WEIGHT LOSS
Chan DC, Gan SK, Wong ATY, Barrett PHR, Watts GF
School of Medicine & Pharmacology, University of Western Australia, Perth, Western Australia, Australia
Background: Ectopic deposition of fat in skeletal muscle is a feature of metabolic syndrome, but its specific association with dyslipidemia remains unclear. Although the oversecretion of very-low-lipoprotein (VLDL)-apolipoprotein (apo) B-100 may increase deposition of triglycerides in skeletal muscle, this metabolic process may vary with the degree of insulin resistance.
Aim: To examine the association between skeletal muscle fat content and VLDL-apoB-100 kinetics, and the corresponding responses to weight loss in obese subjects.
Methods: Fat content of liver, abdomen and skeletal muscle was measured by magnetic resonance techniques and VLDL-apoB-100 kinetics were assessed using stable isotope tracers in 25 obese subjects Kinetic parameters were derived using a multicompartmental model.
Results: In univariate analysis (n=25), skeletal muscle fat content was significantly (P<0.05 in all) associated with body weight (r=0.415), visceral fat area at L3 vertebra (r=0.531), energy intake (r=0.531), plasma non-esterified fatty acid (r=0.428) and glucose concentrations (r=0.477). In obese subjects who were insulin sensitive (HOMA score <2.5), skeletal muscle fat content was significantly associated with hepatic fat content (r=0.636), energy intake (r=0.694), plasma triglyceride (r=0.644), apoB-100 (r=0.529), glucose (r=0.622), VLDL-apoB-100 concentrations (r=0.860), VLDL-apoB-100 fractional catabolic rate (r=–0.581) and VLDL-apoB-100 secretion rate (r=0.607). These associations were not found in obese subjects who were insulin resistant (HOMA score >2.5). Of the 25 subjects, 10 underwent a 16-week weight loss program. A low fat diet achieved significant reduction (P<0.05) in body weight, BMI, visceral and subcutaneous fat areas, liver and skeletal muscle fat, energy intake, triglyceride, insulin, HOMA score, retinal binding protein-4, VLDL-apoB100 concentrations and VLDL-apoB100 secretion rate. There was a significant increase (P<0.05) in plasma adiponectin concentration. The percentage reduction of skeletal muscle fat with weight loss was significantly associated with a corresponding fall in VLDL-apoB100 concentration (r=0.770; P=0.009) and VLDL-apoB-100 secretion (r=0.682; P=0.030).
Conclusion: This study demonstrates for the first time the direct association between skeletal muscle fat content and VLDL-apoB-100 transport. Furthermore, with weight loss, the reduction in skeletal muscle fat was associated with lower rates of VLDL-apoB100 secretion.
PLASMA PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9: A MARKER OF APOLIPOPROTEIN B-48 CATABOLISM IN OBESITY
Chan DC, Wong ATY, Barrett PHR, Watts GF
School of Medicine & Pharmacology, University of Western Australia, Perth, Western Australia, Australia
Background: Postprandial lipemia, characterized by hypertriglyceridemia due to elevated plasma chylomicron apolipoprotein (apo) B-48 concentrations, contributes to the increased risk of cardiovascular disease in obesity. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protease that mediates degradation of low-density lipoprotein (LDL) receptor. Recent evidence also suggests that PCSK9 could play a critical role in the regulation of chylomicron metabolism.
Aim: To investigate the association between plasma PCSK9 concentration and apoB-48 metabolism in obesity.
Methods: Seventeen obese subjects (9 men and 8 women, age 59 ± 6 years, BMI 33 ± 6 kg/m2) were given an oral fat load. ApoB-48 tracer/tracee ratios were measured after intravenous d3-leucine administration using gas chromatography-mass spectrometry. ApoB-48 fractional catabolic rate (FCR) and secretion rate were derived using a multicompartmental model. Plasma total and incremental triglyceride and apoB-48 (0–10 h) area-under-the-curves (AUCs) were also determined. Plasma PCSK9 was determined using immunoassay.
Results: In univariate regression, plasma PCSK9 concentration was significantly (P<0.05 in all) and positively associated with plasma fasting triglyceride (r=0.718) and apoB-48 (r=0.531) concentrations, AUCs for plasma triglycerides and apoB-48 (r=0.514 and r=0.547, respectively) and inversely correlated with apoB-48 FCR (r=–0.589). The incremental AUC for plasma apoB-48 was also significantly associated with plasma PCSK9 concentration (r=0.486; P<0.05). Plasma PCSK9, however, was not significantly correlated with apoB-48 secretion rate in the fasted or postprandial state. The association between plasma PCSK9 and apoB-48 FCR remained significant (P<0.05 in all) after adjusting for age (r=-0.567), body mass index (r=–0.576), HOMA score (r=–0.575) or energy intake (r=–0.583).
Conclusion: In obese individuals, variation in plasma PCSK9 concentration may influence the catabolism of apoB-48. This association appears to be independent of age, obesity, insulin resistance and energy intake.
PLASMA APLOLIPOPROTEIN B-48 TRANSPORT IN OBESE MEN: A NEW TRACER KINETIC STUDY IN THE POSTPRANDIAL STATE
Wong ATY, Chan DC, Pang J, Watts GF, Barrett PHR
Background: The mechanisms responsible for raised chylomicron concentrations in obese subjects have not been adequately investigated.
Aim: To compare apolipoprotein (apo) B-48 metabolism in obese and lean men by developing a new model to describe the kinetics of apoB-48 particles in the postprandial state.
Methods: Seven obese and thirteen age-matched lean men were given an oral fat load. ApoB-48 tracer/tracee ratios were measured after intravenous d3-leucine administration using gas chromatography-mass spectrometry. Kinetic parameters were derived using a non-steady state multicompartmental model.
Results: Compared with lean men, fasting plasma triglyceride (+148%) and apoB-48 (+110%) concentrations, as well as plasma total and incremental triglycerides (+184% and +185%, respectively) and apoB-48 (+182% and 224%, respectively) area-under-the-curves AUCs (0–10 h) were significantly higher in obese men (P<0.05, for all). The obese men also had significantly (P<0.05, for all) higher secretion rates of apoB-48 in the fasted state (+145%), as well as at 3 h (+70%), 4 h (+82%), 5 h (+82%), 6 h (+76%) and 8 h (+61%) in response to the fat load. This was associated with a greater number of apoB-48-containing particles secreted over the 10 study hour period in the obese men, compared with lean men (+125%; P<0.01). The fractional catabolic rate of apoB-48 was significantly lower in the obese men compared with the lean men (–33%; P<0.05)
Conclusion: We demonstrate that postprandial hypertriglyceridemia in central obesity relates to an overproduction and impaired catabolism of apoB-48-containing lipoproteins. These findings are based on a new kinetic model that is more physiologically relevant and which describes the non-steady state post-prandial metabolism of apoB-48.
RS12718465 SINGLE NUCLEOTIDE POLYMORPHISM IN EXON 3 OF APOA1 GENE IS ASSOCIATED WITH LOW HIGH-DENSITY LIPOPROTEIN SUBJECTS
Mokhsin Aa, Sakri FHa, Wan Ahmad WNHa, Hoh BPb, Rahman Ta, Mohd Nasir Na, Abdul Razak Sa, Mohd Ismail Aa, Md Yasin Ma, Nawawi Ha
aThe Center for Pathology and Diagnostic Research Laboratories (CPDRL) and bInstitute of Molecular Medicine and Biotechnology (IMMB), Faculty of Medicine, Universiti Teknologi MARA (UiTM), Sg. Buloh Campus, Selangor, Malaysia
Background: Coronary artery disease (CAD) is the major cause of death worldwide. High density lipoprotein cholesterol (HDL-c) is negative risk factor for CAD. Apolipoprotein A1 (ApoA1), the main apolipoprotein in HDL-c consists of 4 exons encoding for 243 amino acids. Genetic variation of ApoA1 may contribute to low HDL-c levels. However, its role in causing low HDL-c levels in South East Asian is unclear.
Aim: To identify and characterize ApoA1 single nucleotide polymorphisms (SNPs) in Malaysia.
Methods: Sixty-eight subjects (37 females, HDL-c ≤0.70 mmol/L and 31 males, HDL-c ≤0.65 mmol/L, based on the 2.5% lower end cut-off of normal local population distribution) and 68 age-, gender- and ethnic-matched controls (39 females, HDL-c ≥1.3 mmol/L and 29 males, HDL-c ≥1.0 mmo/L) were recruited for this study. Whole blood samples were collected for DNA extraction. ApoA1 gene amplification was carried out by polymerase chain reaction. Amplified DNA fragments were sequenced. Confirmation of the SNPs was analysed using Mega 5.1.
Results: DNA sequencing of the ApoA1 gene showed seven SNPs; one SNP (rs12718465) was found in 6/68 case but not in controls, there being a significant association of this SNP with low HDL-c levels (P=0.03) with an odds ratio of 6.58. This variant has been reported previously to affect HDL level.
Conclusion: The ApoA1 SNP rs12718465 is associated with low HDL levels in a Malaysian cohort. Further studies of larger cohorts are required to explore these associations since they could be responsible for enhanced CAD prevalence, especially among those without other risk factors.
TOCOTRIENOL-ENRICHED MIXED FRACTION SUPPLEMENTATION REDUCES INFLAMMATION AND PLAQUE INSTABILITY IN RABBITS WITH EXPERIMENTALY INDUCED EARLY ATHEROSCLEROSIS
Aishah NMa, Omar Eb, Nawawi Ha
aThe Centre for Pathology Diagnostic and Research Laboratories (CPDRL) and bInstitute for Molecular Medical Biotechnology (IMMB), Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh Campus, Selangor, Malaysia
Background: Inflammation plays a fundamental role in the pathogenesis of atherosclerosis, leading to plaque instability. Palm tocotrienol-enriched mixed fraction (TEMF) is a potent antioxidant. The effect of TEMF in the treatment of early atherosclerosis remains unclear.
Aim: To determine the in vivo effects of TEMF on inflammation and plaque stability in early atherosclerosis.
Methods: Ten New Zealand white rabbits were randomized into two groups and fed with 1% high cholesterol diet (HCD) for two weeks to induce early atherosclerosis, followed by normal diet for another eight weeks. Intervention with either (i) TEMF (15 mg/kg body weight; tocotrienol-tocopherol ratio = 70:30%) or (ii) placebo were given only after HCD commencement at week 2. At the end of the study, aortas were taken and examined for atherosclerotic lesions using Sudan IV staining. Further evaluation on inflammatory markers, i.e., interleukin-6 (IL-6), C-reactive protein (CRP), intercellular adhesion molecules-1 (ICAM-1), smooth muscle actin (SMA) and matrix metalloproteinase-12 (MMP-12) expression in the tunica intima were done by immunohistochemistry.
Results: There was lower CRP (3.0 ± 0.86 vs. 25.5 ± 10.8%; p=0.02), SMA (3.9 ± 1.3 vs. 16.2 ± 4.1%; P=0.04) and MMP-12 (1.8 ± 0.34 vs. 10.1 ± 3.3%; P=0.01) tissue expression in TEMF-treated groups compared to placebo. There was no difference in tissue expression of IL-6 and ICAM-1, and atherosclerotic lesions between TEMF and placebo groups.
Conclusion: TEMF treatment in early atherosclerosis reduces tissue inflammatory biomarkers and plaque instability, despite an absence of an effect on the atherosclerotic lesions.
AN EXPLORATORY STUDY OF SPHINGOLIPIDOMIC ALTERATIONS IN PLASMA AND LIPOPROTEINS IN OVERWEIGHT MEN WITH VARYING DEGREES OF DYSGLYCEMIA
Ng TW, Huynh K, Rasmiena A, Tan R, Wong G, Magliano D, Zimmet P, Shaw J, Meikle PJ
Metabolomics Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia
Background: Alterations in plasma sphingolipids have been associated with the development of insulin resistance and type 2 diabetes (T2D). The effects of different degrees of abnormal glycemia on plasma and lipoprotein sphingolipid composition have not been comprehensively examined.
Aim: To investigate changes in the sphingolipidomic profile in plasma and lipoproteins in dyslipidemic, overweight men with normoglycemia (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and T2D.
Methods: Sphingolipid profiling in plasma and lipoproteins were carried out in 24 dyslipidemic, overweight, and age-matched men with normal glucose tolerance (n=6), impaired fasting glucose (n=6), impaired glucose tolerance (n=6), or type-2 diabetes (n=6). Lipoproteins were isolated by fast performance liquid chromatography. Sphingolipids were quantified by tandem mass spectrometry. Plasma cholesterol efflux was assessed by 3H-cholesterol in THP-1 macrophages.
Results: Compared with NGT subjects, total (+43%; P=0.004) and individual plasma ceramides were significantly elevated in T2D subjects. Total and individual high-density lipoprotein (HDL)-sphingomyelin (SM) species were significantly lower in T2D subjects compared with NGT (total HDL-SM: –30%; P=0.02) and IGT subjects (total HDL-SM: –21%; P=0.03). No statistically significant group differences were found with sphingolipids in VLDL and LDL. After adjustment for HDL-cholesterol, cholesterol efflux in T2D subjects was significantly higher compared with non-T2D subjects (+27%; P=0.01) and with NGT (+43%; P=0.06).
Conclusion: Plasma- and HDL-sphingolipids are differentially altered with dysglycemia. Lowered HDL-SM enrichment was accompanied by increased plasma cholesterol efflux in T2D and may reflect a compensatory mechanism to counter the dyslipidemia associated with T2D.
DOSE-DEPENDENT EFFECTS OF ROSUVASTATIN ON THE PLASMA SPHINGOLIPIDOME IN THE METABOLIC SYNDROME
Ng TWa,b, Ooi EMa, Watts GF, Chan DCa, Meikle PJb, Barrett PHRa
aSchool of Medicine & Pharmacology, University of Western Australia, Perth, Western Australia; bMetabolomics Laboratory, Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia
Background: 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are established low densitiy lipoprotein (LDL)-cholesterol lowering agents. Statins may, however, also exert effects on the plasma sphingolipidome beyond LDL-cholesterol lowering.
Aim: To investigate the dose-dependent effects of rosuvastatin on plasma sphingolipids in men with the metabolic syndrome.
Methods: Men with the metabolic syndrome (n=12) were studied in a randomized, double-blind, triple-crossover trial involving a 5 week treatment period with placebo or rosuvastatin (10 or 40 mg/day) with 2 week wash-outs between treatments. Plasma sphingolipid profiling was determined by liquid chromatography electrospray ionization tandem mass spectrometry.
Results: Rosuvastatin at 10 mg/day (R10) and 40 mg/day (R40) significantly (all P<0.001 unless stated otherwise) lowered plasma cholesterol (–34% change with R10; –42% change with R40) and LDL-cholesterol (–49% with R10 and –57% with R40) and triglyceride (–24% with R10, P=0.03; and –42% with R40). Compared with placebo, R10 and R40 significantly decreased plasma levels of total ceramide (–33% with R10 and –37% with R40), sphingomyelin (–27% with R10 and –31% with R40), and monohexosylceramide (–40% with R10 and –47% with R40), dihexosylceramide (–31% with R10 and –34% with R40), trihexosylceramide (–29% with R10 and –31% with R40), and GM3 gangliosides (–29% with R10 and –26% with R40). Reduction in plasma ceramide was associated with a reduction in very-low-density lipoprotein (VLDL) apolipoprotein (apo) B-100 fractional catabolism independent of changes in LDL-cholesterol with high-dose rosuvastatin.
Conclusion: Rosuvastatin is effective at achieving reductions in total and individual plasma sphingolipids in metabolic syndrome men with evidence of dose-dependent effects. These changes relate to the fractional catabolism of VLDL apoB-100 and were independent of LDL-cholesterol lowering.
ENHANCED ENDOTHELIAL ACTIVATION IN HUMAN SUBJECTS WITH LOW CONCENTRATION OF HIGH DENSITY LIPOPROTEIN
Wan Ahmad WNH, Mokhsin A, Sakri FH, Rahman T, Mohd Nasir M, Abdul Razak S, Mohd Ismail A, Md Yasin M, Hoh BP, Nawawi H
The Center for Pathology and Diagnostic Research Laboratories (CPDRL), Faculty of Medicine, Universiti Teknologi MARA (UiTM), Sg. Buloh Campus, Selangor, Malaysia
Background: Soluble vascular cell adhesion molecule I (sVCAM-I), intercellular cell adhesion molecule I (sICAM-I), and E-selectin are biomarkers reflecting endothelial activation. Endothelial activation has been established as one of the key events in the initiation and progression of atherosclerosis. High-density lipoprotein cholesterol (HDL-c) is a negative risk factor for atherosclerosis, which plays a vital role in reverse cholesterol transport. The role of HDL-c in endothelial activation, however, remains unclear.
Aim: To compare the serum sVCAM-I, sICAM-I and E-selectin concentrations between subjects with low HDL and controls.
Methods: 177 subjects (72 males and 105 females aged 44.0 ± 11.3SD years) with low HDL-c levels (HDL-c <1.0 mmol/L and <1.3 mmol/L for males and females, respectively) and 209 age-, gender-, ethnicity-, smoking-, diabetes- and hypertension-matched normolipemic controls (75 males and 134 females aged 43.2 ± 12.0SD years (HDL-c ≥1.0 and ≥1.3 mmol/L for males and females respectively) were recruited. Fasting blood samples were collected. Lipid profiles were measured using automated analyser (Cobas Integra 400, Roche, Germany). Enzyme-linked immunosorbent assays (ELISA) were performed to measure the concentrations of sVCAM-I, sICAM-I and E-selectin.
Results: We found higher sVCAM-I, sICAM-I and E-selectin concentrations in low HDL-c patients than controls: 726 ± 42SE vs 535 ± 23SE ng/ml; (P<0.01); 798 ± 33SE vs 685 ± 21SE ng/ml (P<0.01); and 47.7 ± 7.0 vs 28.3 ± 2.3 ng/ml (P<0.01), respectively.
Conclusion: These findings suggest there is increased endothelial activation in subjects with low HDL-c levels and which in part may contribute to enhanced atherogenesis.
MODULATION OF ENDOTHELIAL CELL FUNCTION BY THE MYELOPEROXIDASE-DERIVED OXIDANT HYPOCHLOROUS ACID (HOCl) AND ITS CONTRIBUTION TO ATHEROSCLEROSIS
Rayner BS, Lloyd MM, Summers FA, Davies MJ, Hawkins CL
Heart Research Institute, 7 Eliza St, Newtown, Sydney, New South Wales, Australia; Faculty of Medicine, University of Sydney, Sydney, New South Wales, Australia
Background: Myeloperoxidase (MPO) is a haem enzyme released by activated phagocytes under inflammatory conditions. MPO forms reactive oxidants that play an important role in the human immune system. Hypochlorous acid (HOCl) is the major oxidant produced by MPO under normal physiological conditions. Although HOCl is a potent bactericidal agent, excessive or misplaced production of HOCl has been linked to tissue damage and the progression of many diseases, including atherosclerosis. MPO is also an independent risk factor for the development of coronary artery disease and a powerful prognostic agent predicting outcome in patients with chest pain or after myocardial infarction.
Aim: In this study, we examine the cellular pathways responsible for the induction of endothelial cell death and dysfunction on exposure of cells to HOCl.
Methods: Primary human coronary artery endothelial cells (HCAEC) were exposed to pathophysiological levels of HOCl. Cellular targets were assessed by a proteomics approach, molecular pathway expression was analysed by real-time PCR and Western blotting, and the extent of cell death was measured by flow cytometry.
Results: Exposure of HCAEC to HOCl resulted in a dose-dependent increase in cell death, with programmed cell death (apoptosis) observed at low doses and necrosis predominating at higher doses. Proteins containing free Cys (thiol) residues are important targets for HOCl, with protein disulfide isomerase (PDI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), thioredoxin-related protein and galectin-1 found to be particularly sensitive to oxidation. This results in modulation of enzyme function, with rapid inactivation of GAPDH observed after HOCl treatment. HOCl exposure also induced several key transcription factors involved in inflammatory pathways, including activating transcription factor 3 (ATF3), activator protein 1 (AP-1), early growth response 1 (Egr-1), and CCAAT/enhancer binding protein homologous protein (CHOP). Expression of these molecules occurred concurrently with the induction of mediators of inflammation such as TNFα, interleukin 8 (IL8), and monocyte chemotactic protein-1 (MCP-1).
Conclusion: Targeting cellular thiol-containing proteins and activation by HOCl of the transcription factors ATF3, AP-1, Egr-1, and CHOP results in endothelial cell death and the propagation of inflammation, which may accelerate atherosclerosis and contribute to the development of clinically-relevant lesions.
ATORVASTATIN PLUS OMEGA-3 FATTY ACID ETHYL ESTER DECREASES VERY-LOW-DENSITY LIPOPROTEIN TRIGLYCERIDE PRODUCTION IN INSULIN RESISTANT OBESE MEN
Ng TW, Ooi EM, Chan DC, Watts GF, Barrett PHR
School of Medicine & Pharmacology, University of Western Australia, Pert, Western Australia, Australia
Background: Abnormal very-low-density lipoprotein (VLDL)-triglyceride (TG) metabolism underscores the hypertriglyceridemia in obese, insulin resistant men and contributes to increased cardiovascular disease (CVD) risk. Statins reduce CVD risk by decreasing low-density lipoprotein (LDL)-cholesterol, but their effects on plasma TGs are modest. Supplementing with high dose omega-3 fatty acid ethyl esters (-3 FAEEs) may achieve a greater reduction in plasma TG concentration and potentially attenuate residual CVD risk.
Aim: To test the effect of atorvastatin and atorvastatin plus -3 FAEEs on VLDL-TG metabolism in obese, insulin resistant men.
Methods: We carried out a 6-week randomized, placebo-controlled study to examine the effect of atorvastatin (40 mg/day) and atorvastatin plus -3 FAEEs (4 g/day) on VLDL-TG metabolism in 36 insulin resistant, obese men. VLDL-TG kinetics were determined using d5-glycerol, gas chromatography-mass spectrometry, and compartmental modeling.
Results: Compared with placebo, atorvastatin significantly decreased VLDL-TG concentration (–40%; P<0.001) by increasing VLDL-TG fractional catabolic rate (FCR) (+47%; P<0.01). Atorvastatin plus -3 FAEEs lowered VLDL-TG concentration to a greater degree compared with placebo (–46%; P<0.001) or atorvastatin monotherapy (–13%; P=0.04). This was achieved by a reduction in VLDL-TG production rate (PR) compared with placebo (–32%; P=0.008) or atorvastatin (–20%; ˆ=0.03), as well as a reciprocal increase in VLDL-TG FCR (+42%; P<0.05) compared with placebo.
Conclusion: In insulin resistant, dyslipidemic, obese men, atorvastatin improves VLDL-TG metabolism by increasing VLDL-TG FCR. The addition of 4 g/day -3 FAEE to statin therapy provides further TG-lowering by reducing VLDL-TG PR.
THERAPEUTIC USE OF eNOS/CAVEOLIN-1 ANTAGONISTIC PEPTIDES FOR DIABETES-ASSOCIATED ATHEROSCLEROSIS
Sharma A1,2, Tan M2, Stefanovic N2, Bernatchez PN1, and de Haan JB2
1The UBC James Hogg Research Centre and Department of Anesthesiology, Pharmacology & Therapeutics, University of British Columbia, Vancouver, Canada; 2Oxidative Stress Laboratory, Diabetic Complications Division, BakerIDI Heart and Diabetes Institute, Melbourne, Victoria, Australia
Background: Diabetes is associated with an increased risk for the development of atherosclerosis, mainly due to the reduction in endothelial function, which is characterized by decreased nitric oxide (NO) bioavailability. Endothelial nitric oxide synthase (eNOS), the enzyme responsible for the production of NO, is negatively regulated through its association with caveolin-1, the major coat protein of caveolae. We have recently shown that a mutant Caveolin-1-derived peptide (CavNOxin) increases NO release and bioavailability by antagonizing the binding of eNOS to Caveolin-1. Since the upregulation of NO production is therapeutically relevant in atherosclerosis we hypothesized that CavNoxin can attenuate the progression of diabetes-associated atherosclerosis.
Aim: To examine whether antagonizing the eNOS/caveolin-1 interaction in diabetes might be a potentially novel and unexplored anti-atherosclerotic therapeutic strategy.
Methods: 8-week old apolipoprotein E (ApoE) knockout mice were rendered diabetic with the diabetogenic drug, streptozotocin. At 10 weeks of age, diabetic mice were injected with either CavNoxin (2.5 mg/kg and 5.0 mg/kg) or vehicle peptide once every 3 days for 14 weeks.
Results: Diabetes led to an increase in atherosclerotic lesions throughout the aorta (total plaque: 1.5% in non-diabetic vs 11% in diabetic mice; P<0.001) and aortic sinus, which was significantly attenuated with CavNOxin treatment (total plaque: 5%; P<0.01 vs diabetic). The reduction in atherosclerotic lesions was associated with decreased oxidative stress, as assessed by DHE and 4-HNE staining, and expression of pro-atherogenic mediators, such as VCAM-1 (mRNA fold induction: 2.8 in diabetic vs 1.5 in diabetic + CavNOxin; P<0.05) and MCP-1 (mRNA fold induction: 11 in diabetic vs 4.5 in diabetic + CavNOxin; P<0.05). In cultured endothelial cells grown in high glucose and diabetic aortas perfused with whole blood, CavNOxin treatment led to a significant reduction of leukocyte/monocyte adhesion (P<0.05 and P<0.001, respectively).
Conclusion: These data are the first to show a positive effect of eNOS/caveolin-1 antagonism on diabetes-associated atherosclerosis. Since superoxide is a NO scavenger, these data suggest that CavNoxin mediates its antiatherogenic effect by increasing NO bioavailability.
A NOVEL MOUSE MODEL THAT REFLECTS HUMAN ATHEROSCLEROTIC PLAQUE INSTABILITY IS A UNIQUE TOOL FOR DRUG TESTING AND MECHANISTIC DISCOVERIES
Chen Y-C, Peter K
Atherothrombosis and Vascular Biology, Baker IDI Heart & Diabetes Institute, Melbourne, Victoria, Australia
Background: The high morbidity/mortality of atherosclerosis is typically precipitated by plaque rupture and consequent thrombosis. However, research on underlying mechanisms and therapeutic approaches is significantly hampered by the lack of animal models that reproduce plaque instability observed in humans.
Aim: To generate a mouse model that reflects human plaque instability/rupture.
Methods: Typical hemodynamic conditions that contribute to the development of vulnerable, unstable atherosclerotic plaques are low shear stress and high tensile stress. Based on computational fluid dynamics modeling, we developed a mouse model that imitates these hemodynamic conditions. A tandem stenosis was applied to the carotid artery of ApoE–/– mice, which were fed a high fat diet.
Results: At 7 weeks postoperatively, we observed intraplaque hemorrhage in ~50% of mice, as well as disruption of fibrous caps, intra-luminal thrombosis, neovascularization and further characteristics typically seen in human unstable atherosclerotic plaques. Administration of atorvastatin was associated with plaque stabilization and down-regulation of MCP-1 and ubiquitin. Microarray profiling of mRNA and microRNA, in particular its combined analysis, demonstrated major differences in the hierarchical clustering of genes and microRNAs between non-atherosclerotic arteries, stable and unstable plaques and allowed the identification of distinct genes/microRNAs, potentially representing novel therapeutic targets for plaque stabilization. The feasibility of the animal model described as a discovery tool was established in a pilot approach, identifying ADAMTS4 and miR-322 as potential pathogenic factors of plaque instability in mice. The involvement in plaque instability was validated in human atherosclerotic plaques.
Conclusion: The newly described mouse model reflects human atherosclerotic plaque instability/rupture and represents a unique discovery tool that allows the identification of distinctly expressed genes and microRNAs that are linked to plaque instability. It holds promise towards the development and testing of therapeutic strategies aimed at preventing plaque rupture.
TRAIL-DEFICIENCY ACCELERATES VASCULAR CALCIFICATION IN ATHEROSCLEROSIS VIA MODULATION OF RANKL
Cartland SP1,3*, Di Bartolo BA1*, Harith HH1,2,4, Bobryshev YV2, Schoppet M5, Kavurma MM1,2,3
1Centre for Vascular Research and 2School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia; 3The Heart Research Institute, Sydney, New South Wales, Australia; 4Department of Biomedical Sciences, Universiti Putra Malaysia, Malaysia; 5Department of Internal Medicine and Cardiology, Philips University, Germany.(*Equal contribution)
Background: An increasingly recognized risk factor for cardiovascular disease is vascular calcification. The triad cytokine system of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL) and its receptor RANK control bone homeostasis. This system has recently been implicated in vascular calcification. Osteoclastogenesis is initiated by RANKL binding to RANK expressed on osteoclasts. RANKL is also able to bind its soluble decoy receptor, OPG, which inhibits differentiation, maturation and survival of osteoclasts, impeding bone mineralization. TNF–related apoptosis-inducing ligand (TRAIL) is a second ligand for OPG and is protective for atherosclerosis, yet the role of TRAIL in vascular calcification is unclear.
Aim: To identify the involvement of TRAIL in vascular calcification in arteries in vivo using TRAIL–/–ApoE–/– and ApoE–/– mice.
Methods: Calcification of vascular smooth muscle cells (VSMCs) was measured using an alizarin red-based assay. Calcification in vivo was assessed in TRAIL–/–ApoE–/– and ApoE–/– mice placed on a high fat diet for 12 and 20 weeks. mRNA and inflammatory markers were measured by standard techniques.
Results: In vitro, TRAIL dose-dependently inhibited calcium-induced calcification of human VSMCs. Correspondingly, murine TRAIL–/– VSMCs demonstrated accelerated calcification, compared to wild-type (WT) cells, when induced by multiple concentrations of calcium. Wild-type cells exposed to calcium had significantly elevated mRNA expression of RANKL, a pro-calcific factor, while OPG and TRAIL were inhibited. At 12 weeks on ahigh fat diet brachiocephalic arteries from TRAIL–/–ApoE–/– had increased numbers of chondrocyte-like cells, whereas by 20 week TRAIL–/–ApoE–/– aortas displayed significantly increased calcification. 12 week TRAIL–/–ApoE–/– aortas had increased expression of mRNA for the osteochondrogenic marker, collagen II, as well as cellular RANKL, with no change in circulating RANKL or OPG. They also displayed altered expression of inflammatory markers regulating bone, including increased IL-1β and PPAR-γ.
Conclusion: This study provides the first evidence that TRAIL deficiency leads to accelerated cartilaginous metaplasia and calcification in atherosclerosis, and that TRAIL plays an important role in the regulation of RANKL and inflammatory markers mediating bone turnover in the vasculature in vivo. We also demonstrate that TRAIL has a protective role in vascular calcification in vitro.
PHARMACOLOGICAL INHIBITION OF NADPH OXIDASE (NOX)-DERIVED ROS ATTENUATES IMMUNE-INFLAMMATORY RESPONSES AND ACCELERATED ATHEROSCLEROSIS IN DIABETES
Di Marco E1,2, Gray SP1, Chew P1, Szyndralewiez C3, Cooper ME1, Jandeleit-Dahm KA1,2
1Diabetic Complications Division, Baker IDI Heart & Diabetes Research Institute, Melbourne, Victoria, Australia; 2Department of Medicine, Monash University, Melbourne, Victoria, Australia; 3Genkyotex SA, Geneva, Switzerland.
Background: Enhanced infiltration of inflammatory-immune cells and heightened vascular oxidative stress are key processes driving atherosclerosis development in diabetes. Growing evidence demonstrates a central role for NADPH (Nox) isozymes in pathological production of reactive oxygen species (ROS) in the vasculature.
Aims: The aim of this study was to examine the role of Nox-derived ROS in pro-inflammatory and pro-oxidant responses involved in diabetes-associated atherosclerosis.
Methods: Diabetes was induced in ApoE-deficient (–/–) mice by 5 injections of streptozotocin (55 mg/kg/day). The Nox1/4 dual inhibitor, GKT137831, was administered to diabetic and non-diabetic groups at a dose of 60 mg/kg/day by gavage for 10 weeks. At study completion, aortic sinuses were isolated and analysed for atherosclerotic plaque area, immune cell infiltration and gene expression.
Results: Induction of diabetes in ApoE–/– mice significantly increased atherosclerotic plaque area in the aortic sinus (63%) and necrotic core size in association with elevated MCP-1 expression and increased lesional accumulation of CD4+, CD8+ and CD11c+ cells. In addition, diabetic mice displayed increased vascular superoxide as measured by dihydroethidium (DHE) fluorescence, which correlated with elevations in Nox1 and Nox4 gene expression in the aortic sinus. Pharmacological inhibition of Nox-derived ROS using GKT137831 attenuated the diabetes-mediated changes in atherosclerosis plaque size and phenotype in the aortic sinus of ApoE–/– mice.
Conclusions: Accelerated atherosclerosis development in the aortic sinus of ApoE–/– mice was associated with elevated production of Nox-derived ROS and increased accumulation of inflammatory cells typically involved in adaptive immune responses. Treatment with GKT137831 ameliorated these diabetes-induced pro-atherogenic responses, thus supporting a central role of Nox1 and Nox4 isozymes in diabetes-associated atherosclerosis.
VANIN-1, A NOVEL GENE THAT INFLUENCES CHOLESTEROL HOMEOSTASIS
Bozaoglu Ka, Lake NJa, Curran JEb, Taylor RLa, Hoang Aa, Low Ha, Johnson MPb, Dyer TDb, Göring HHHb, Abraham Lc, Moses EKc, Sviridov Da, Jowett JBMa, Blangero Jb
aBaker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia; bTexas Biomedical Research Institute, San Antonio, Texas, USA; cUniversity of Western Australia, Crawley, Western Australia, Australia
Background: A low serum level of high density lipoprotein cholesterol (HDL-C) is a strong independent risk factor for development of cardiovascular disease (CVD). There are, however, limited therapeutic options to increase HDL-C, leading to an urgent need for therapies. In our prior work, we combined genomic, transcriptomic and functional genetic analyses to identify the gene for vanin-1 (VNN1) as a novel gene whose expression shows a significant correlation with human HDL-C levels.
Aim: To validate the association between HDL-cholesterol and VNN1 expression using in vitro cellular models and to identify the mechanisms of action of vanin-1 on HDL cholesterol metabolism
Methods: To investigate how vanin-1 affects HDL cholesterol, experiments that included cholesterol efflux assays and confocal microscopy were performed on in vitro cellular models where VNN1 was either silenced or over-expressed.
Results: Over-expression of VNN1 significantly induced ApoA1-mediated cholesterol efflux in human liver (70% increase; P=0.015), colorectal (70% increase; P=0.015) and small intestine (60% increase; P=0.023) cells. In contrast, siRNA-mediated silencing of VNN1 mRNA significantly decreased ApoA1-mediated cholesterol efflux. Similar trends were observed with HDL-mediated cholesterol efflux. siRNA-mediated knockdown of ABCA1 mRNA prevented VNN1 overexpression from significantly increasing cholesterol efflux in HepG2 cells, suggesting that the effect of vanin-1 on cholesterol efflux is reliant on the ABCA1 transporter. Furthermore, suppression and over-expression of VNN1 mRNA affected the distribution of late endosomes within HepG2 cells.
Conclusion: Taken together, these data suggests that vanin-1 is an important in vivo regulator of cholesterol efflux in various human cell types. The study also suggests that the effects of vanin-1 on cholesterol efflux are dependent on the ABCA1 transporter. We propose that vanin-1 may influence cholesterol efflux and metabolism by regulating late endosome distribution. Our data suggest that vanin-1 may be an important regulator of HDL-C and could be a potential candidate for the development of new a HDL-raising agent.
IMAGING OF ADVANCED ATHEROSCLEROTIC PLAQUE BY HIGH RESOLUTION, MULTI-ENERGY X-RAY COMPUTER TOMOGRAPHY
Gieseg SPa,b, Janmale Ta, Panta Rb, Aamir Rb, Healy Ja, de Ruiter NJAb, Bateman CJb, Bell STe, Walsh MFb, Chernoglazov Aa, Roake Jb,c, Anderson NGb,d,e, Butler APHa,b,d,e, Butler PHa,d,e
aUniversity of Canterbury, Christchurch, New Zealand; bUniversity of Otago, Christchurch, New Zealand; cChristchurch Hospital, Christchurch, New Zealand; dCERN, European Organization for Nuclear Research, Geneva, Switzerland; eMARS Bioimaging Ltd, Christchurch, New Zealand
Background: Cardiovascular disease (CVD) is usually symptom free until atherosclerotic plaques have become large enough either to restrict blood flow or to rupture, triggering clotting and occlusion of the artery. Under ultrasound, MRI or CT imaging, preclinical atherosclerotic plaques are usually too small to be seen, since they are less than 2 mm thick. As a result, treatment of CVD often does not occur till after the plaque has become structurally complicated and is causing significant clinical harm. Clinicians treat the signs and symptoms of CVD, such as high plasma cholesterol, rather than the actual plaque growth. Imaging of preclinical plaque requires the development of high-resolution scanners with low radiation dose, which are capable of distinguishing between different types of tissue. One such approach is to measure the X-ray energy of the individual photons that have passed through the plaque tissue.
Aim: To describe a novel method for detection of preclinical plaque from human carotid arteries.
Methods: Using a MARS scanner designed and constructed at the Universities of Otago and Canterbury, we image excised plaque from human carotid arteries in the human CT energy range (30–120 keV). The X-ray camera within the MARS scanner contains a CERN-designed Medipix3.1 X-ray detector chip, which measures the location, timing and energy of each single X-ray photon striking any one of the 16,512 pixels on the sensor layer, which in these experiments was CdTe. This unique imaging system allowed us to simultaneously collect X-ray attenuation data from the plaque tissue in 4 separate energy bins. Images were reconstructed and regions of tissue were distinguished by material decomposition using the variation of X-ray attenuation within each voxel.
Results: Multi-energy imaging using the MARS scanner has allowed us to distinguish lipid-rich regions from water-rich and calcium-rich regions within carotid plaque. The chemical characteristics of these regions were confirmed by histological staining and comparisons to phantoms of known composition. The image analysis was able to show calcium deposition occurring in sheets near the basement membrane of the arterial wall.
Conclusion: High resolution, muti-energy X-ray computer tomography can distinguish preclinical plaque excised from carotid arteries. This technology is being further developed to allow rapid imaging of live small animals to enable the analysis of plaque development over time in cholesterol fed animal models.
TRAK2, A NOVEL REGULATOR OF CHOLESTEROL EFFLUX AND HDL BIOGENESIS
Lake NJa, Taylor RLa, Curran JEb, Hoang Aa, Low Ha, Meikle P, Johnson MPb, Dyer TDb, Göring HHHb, Moses EKc, Blangero Jb, Sviridov Da, Jowett JBMa, Bozaoglu Ka
aBaker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia; bTexas Biomedical Research Institute, San Antonio, Texas, USA; cUniversity of Western Australia, Crawley, Western Australia, Australia
Background: An improved understanding of the genes that influence HDL-cholesterol abundance is required urgently in order to enable development of effective HDL-cholesterol-raising therapies. We previously employed genetic and transcriptomic analyses of human cohorts to identify a novel association and negative correlation between TRAK2 and HDL-cholesterol levels. As TRAK2 had not been implicated previously in lipid metabolism, the mechanism underlying the association of TRAK2 with HDL-cholesterol was not known.
Aim: To characterize the biological mechanism underlying the in vivo association between TRAK2 and HDL-cholesterol abundance.
Methods: In vitro cellular models of siRNA-mediated silencing were used to investigate the role of TRAK2 in pathways that regulate HDL-cholesterol metabolism.
Results: siRNA-mediated silencing of TRAK2 mRNA significantly increased cholesterol efflux to apoA-I and HDL in human liver HepG2 cells by 60% and 40%, respectively. TRAK2 mRNA silencing in HepG2 cells significantly increased the expression of the key cholesterol transporter ABCA1 by 150% at the mRNA level, with ABCA1 protein levels being elevated by 80%. Cells treated with both TRAK2 and ABCA1 siRNA simultaneously displayed severely reduced cholesterol efflux relative to controls at levels comparable to treatment with ABCA1 siRNA alone, demonstrating that TRAK2 expression is dependent on ABCA1. Mass spectroscopy studies further revealed an in vitro and in vivo correlation between TRAK2 expression and multiple lipid species, implicating a role for TRAK2 in broader lipid metabolism.
Conclusion: TRAK2 appears to regulate hepatic cholesterol efflux and therefore HDL biogenesis through a pathway that is dependent on ABCA1. These studies validate the in vivo genetic and transcriptomic association between TRAK2 and HDL-cholesterol levels. The characterization of TRAK2 as a novel negative regulator of lipid metabolism has potential to progress the development of anti-atherosclerotic therapies.
STAINLESS STEEL-BOUND APOA-I AND RECONSTITUTED HIGH-DENSITY LIPOPROTEINS ARE ANTI-THROMBOTIC AND CAUSE ANTI-INFLAMMATORY EFFECTS IN HUMAN VASCULAR SMOOTH MUSCLE CELLS
Vanags La,b, Tan Ja,b, Michael Ja,b, Bilek Ma,c, Wise Sa,b, Bursill Ca,b
aThe Heart Research Institute; bDepartment of Medicine and cDepartment of Applied Physics, University of Sydney, Sydney, New South wales, Australia
Background: The clinical efficacy of current endovascular stents is limited by thrombosis and inflammation-induced restenosis. Plasma activated coating (PAC) facilitates covalent binding of proteins to stainless steel (SS) surfaces in their bioactive state. High density lipoprotein (HDL) and its main apolipoprotein constituent apoA-I regulate key biological processes involved in restenosis and thrombosis, highlighting their potential for immobilization on PAC coated SS surfaces.
Objective: To determine if apoA-I or reconstituted HDL (rHDL) can be covalently bound to PAC-coated SS, while retaining key biological properties that stand to improve stent biocompatibility.
Methods: Thrombosis was assessed on SS and PAC-coated SS by static assay and under flow using a modified chandler loop. Smooth muscle cell (SMC) and endothelial cell (EC) attachment was quantified by crystal violet staining. Chemokine expression (CCL2, CCL5 and CX3CL1) of TNF-α-stimulated SMCs grown on immobilized apoA-I and rHDL was measured by qPCR.
Results: The capacity of SS and PAC-coated SS to covalently retain apoA-I or rHDL was assessed using stringent SDS washing following protein incubation. On PAC-coated surfaces 58.1% of the original protein (before SDS washing) was retained, while on SS, only 16.2% was. Covalently retained apoA-I and rHDL on PAC-coated SS was 39.7 and 35.7 µg/cm2, respectively. When exposed to heparinized whole blood, PAC-coated SS strikingly reduced thrombus formation relative to SS controls. However, for PAC+apoA-I and PAC+rHDL samples, thrombosis was completely absent, at 10, 30 and 60 min time points. Under flow, thrombus weight was reduced by 98%, 97% and 94% on PAC, PAC+apoA-I and PAC+rHDL, respectively, relative to SS (P<0.001). ApoA-I and rHDL immobilized on PAC-coated SS reduced SMC attachment by 70% and 80%, respectively (P<0.001). Conversely, EC attachment was increased by 36% on PAC+apoA-I (P<0.05). Furthermore, SMCs plated on immobilized apoA-I and rHDL had significantly attenuated TNF-α-induced increases in CCL2 (40% and 47%), CCL5 (27% and 30%) and CX3CL1 (36% and 36%) mRNA expression (P<0.05).
Conclusion: ApoA-I and rHDL can be covalently immobilized on to SS and exhibit anti-thrombotic and anti-inflammatory properties. This may represent a novel site-directed approach to increase stent patency.
A LIPIDOMICS ASSESSMENT OF THE ANTI-OXIDATIVE CAPACITY OF HIGH-DENSITY LIPOPROTEINS
Rasmiena AAa,b, Ng TWa, Barlow CKa, Meikle PJa
aBaker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia; bDepartment of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria, Australia.
Background: Oxidized low-density lipoprotein (LDL) may promote atherosclerosis. High-density lipoprotein (HDL) can protect LDL against oxidative damage by inactivating oxidized lipids as well as removal of oxidized lipids from LDL. Under conditions of chronic oxidative stress, HDL may become dysfunctional and so may contribute to the atherosclerotic process.
Aims: (1) To measure the formation of oxidized lipids in LDL during oxidation in the presence of native or oxidized HDL, and (2) to quantify the transfer of oxidized lipids from oxidized LDL to HDL.
Methods: Lipoproteins were isolated from pooled plasma of healthy volunteers (n=5) by sequential ultracentrifugation. Lipoprotein oxidation was catalysed using copper chloride. To achieve Aim 1, LDL was oxidized in the presence of native, mildly, or strongly pre-oxidized HDL. To achieve Aim 2, LDL was strongly oxidized and co-incubated with HDL followed by re-isolation. Oxidized phosphatidylcholine (oxPC) and oxidized cholesterol esters (oxCE) were determined by electrospray ionization liquid chromatography tandem mass spectrometry.
Results: oxPC and oxCE in LDL were reduced by 58% (n=3; P<0.001) and 38% (n=3; P<0.01), respectively, when oxidation was carried out in the presence of native HDL. However, pre-oxidation of HDL attenuated this process and only achieved 45% (n=3; P<0.01) and 12% (n=3; P<0.05) reduction in the formation of LDL-derived oxPC with mild and strong oxidative conditions, respectively. Co-incubation of oxidized LDL with HDL led to 71% and 28% reductions in LDL-derived oxPC and oxCE, respectively.
Conclusion: HDL was effective at reducing the formation of oxidized PC and oxidized CE residing on LDL. However, oxidation of HDL attenuated this process.
RETARGETING GENETICALLY MODIFIED ADENOVIRUS 5 BY RGD4D INCLUSION IMPROVES VASCULAR TROPISM AND GENE TRANSFER
Robertson Sa–c, Clarke Cb, Nicklin SAb, Bursill Ca,b, Baker AHb
aHeart Research Institute, Sydney, New South Wales, Australia; bFaculty of Medicine, University of Sydney, New South Wales, Australia; cBHF Glasgow Cardiovascular Research Centre, University of Glasgow, UK
Background: Cardiovascular disease is the second most common application for gene therapy clinical trials, which most frequently employ adenovirus serotype 5 (Ad5)-based vectors as delivery vehicles. Whilst gene therapy has great potential as a cardiovascular therapeutic strategy, intravascular delivery of Ad5 vectors are currently limited since they predominantly target the liver and interact with circulating proteins that reduce their efficacy. The development of more efficient vectors for vascular gene transfer is therefore warranted. We have shown that integration of amino acid mutations at key residues within hexon hypervariable regions (HVRs) generates an Ad5 vector (Ad5T*) that lacks hepatic tropism. Further modification of the virus by inclusion of targeting peptides may facilitate improved tropism and gene transfer efficiency to vascular cells.
Aim: To identify compatible locations for targeting peptide inclusion in genetically engineered adenovirus to enhance levels of vascular transduction and transgene expression.
Methods: For hexon modifications a αvβ3-intergrin targeting peptide, RGD4C, was incorporated into either HVR 5 or 7 by an insertion (I) or replacement (R) strategy, while for fiber we inserted the peptide within the HI loop of the knob domain.
Results: RGD4C insertion within HVR7 proved incompatible with virus propagation, while insertion in HVR5 resulted in viral preparations with high viral particle to infectious particle ratios, suggesting packaging limitations when modifying certain regions of the virus capsid. In two αvβ3-integrin-positive cell lines, SKOV3 and A549, cell surface binding and viral transduction were significantly (P<0.001) increased for Ad5T*HVR7R RGD4C compared to control (Ad5T*). In cultures of human saphenous vein primary smooth muscle cells only Ad5T*HVR7R RGD4C increased cell surface binding (P<0.001), whereas transduction and transgene expression were significantly (P<0.001) increased with both Ad5T*HVR7R RGD4C and Ad5T*HI loop RGD4C compared to control (Ad5T*).
Conclusion: Both hexon HVR7 and fiber HI loop are viable locations for target peptide inclusion in Ad5T* which increases viral cell binding, uptake and transgene expression. These novel vectors with improved vascular transduction may provide vector platforms that enable the successful translation of human vascular gene transfer strategies.
- © 2014 American Heart Association, Inc.
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