Soluble Guanylate Cyclase Stimulation on Cardiovascular Remodeling in Angiotensin II–Induced Hypertensive Rats
It is unknown whether long-term pharmacological stimulation of soluble guanylate cyclase (sGC), elevating intracellular cGMP levels, has a beneficial effect on hypertension. The purpose of this study is to investigate the effects of BAY41-2272, an orally available sGC stimulator, on cardiovascular remodeling in hypertensive rats. Eight-week-old male Wistar rats with hypertension induced by angiotensin II infused subcutaneously at 250 ng/kg per minute were treated orally with a low ([L] 2 mg/kg per day) or high ([H] 10 mg/kg per day) dose of BAY41-2272 for 14 days. BAY41-2272-H partially suppressed the rise in blood pressure and reduced the heart weight (4.20±0.34 versus 3.68±0.20 mg/g; P<0.01), whereas BAY41-2272-L had no effect. However, both doses decreased the angiotensin II–induced left ventricular accumulation of collagen in the perivascular area (L, −20%, P<0.05; H, −30%, P<0.01) and myocardial interstitium (L, −21%, P<0.05; H, −38%, P<0.01), reducing the number of activated fibroblasts surrounding coronary arteries (L, −74%; H, −79%; P<0.05). BAY41-2272 downregulated the angiotensin II–induced left ventricular gene expression of type 1 collagen (L, −41%, P<0.05; H, −49%, P<0.01) and transforming growth factor-β1 (L, −49%, P<0.05; H, −65%, P<0.01). cGMP levels were elevated by BAY41-2272 not only in the left ventricle, but also in cultured cardiac fibroblasts, resulting in reduced thymidine incorporation into the cells. Thus, stimulation of sGC by BAY41-2272 attenuates fibrosis of the left ventricle in rats with angiotensin II–induced hypertension partly in a pressure-independent manner, suggesting an important role for sGC generating cGMP in inhibiting cardiovascular remodeling.
Hypertensive heart disease is characterized histologically by left ventricular (LV) hypertrophy and fibrosis. LV hypertrophy is recognized as a risk factor for cardiovascular morbidity and death,1 and fibrosis surrounding coronary arteries and myocardial fibers decreases the supply of oxygen and nutrients to the myocardium.2 These histological changes, termed “cardiac remodeling,” impair the diastolic function of the LV, often leading to overt heart failure or fatal arrhythmia.2–5 Cardiac remodeling is caused by hemodynamics and various neurohumoral factors, such as catecholamine, angiotensin II (Ang II), aldosterone, and endothelin-1.6,7 Notably, activation of the renin—angiotensin–aldosterone system accelerates the process of cardiac remodeling, and, indeed, inhibiting the system has been demonstrated to have beneficial effects.8 Therefore, not only the reduction of blood pressure but also the suppression of these humoral factors would be important to regulate cardiac remodeling in hypertensive heart disease.
Natriuretic peptides–cGMP signaling has been reported to inhibit cardiac remodeling: atrial natriuretic peptide (ANP) evoked potent antihypertrophic effects on cardiac ventricles,9,10 and brain natriuretic peptide (BNP) inhibited cardiac fibrosis in vitro11,12 and in vivo through a cGMP-dependent pathway.13 Thus, cGMP signaling seems to play a critical role in attenuating cardiac remodeling. Soluble guanylate cyclase (sGC [GC]), a heterodimeric haem protein consisting of α- and β-subunits, is an intracellular effector for NO,14 converting guanosine triphosphate to cGMP. However, it remains unknown whether pharmacological stimulation of sGC attenuates cardiac hypertrophy and fibrosis in hypertension.
BAY41-2272, developed recently as an orally active sGC stimulator,15 has been shown to have beneficial effects on hemodynamics in systemic hypertension,15 heart failure,16 and pulmonary hypertension.17 In the present study, to examine whether continuous stimulation of sGC inhibits cardiac hypertrophy and fibrosis, we administered BAY41-2272 to rats with hypertension induced by Ang II. The goal of this study was to better understand the role of sGC in cardiovascular remodeling in Ang II–induced hypertension.
Eight-week-old male Wistar rats (Charles River Laboratories Japan, Yokohama, Japan) weighing 200 to 250 g were housed in a temperature and light-controlled room (25±1°C; 12/12-hour light/dark cycle) for 1 week before use, with free access to normal rat chow and water. Control rats not infused with Ang II were divided into 3 groups given either a placebo (n=7) or a low or high dose (2 and 10 mg/kg per day) of BAY41-2272 (10 for each). Similarly, rats infused with Ang II were divided into a placebo-treated group (n=13) and low-dose (n=9) and high-dose (n=13) treatment groups. Ang II was infused at 250 ng/kg per minute subcutaneously by miniosmotic pumps (Alzet model 2002) for 14 days as described previously.18 The BAY41-2272 compound, kindly supplied by Bayer HealthCare, was given orally twice a day for 14 days. Blood pressure was measured while the animal was conscious ≥9 times by tail-cuff plethysmography (Softron, BP-98A) at 2:00 to 3:00 pm.
The doses of BAY41-2272 used in this study were determined based on our preliminary study, where oral administration at a single dose of 1, 5, or 10 mg/kg was tested in Ang II–infused rats (Figure I, available online at http://hyper.ahajournals.org). The blood pressure–lowering effect of 1 mg/kg BAY41-2272 was minimal and insignificant, but we observed similar hypotensive actions lasting for 12 hours after the administration at 5 and 10 mg/kg. Meanwhile, the primary purpose of the present study was to test inhibitory actions of BAY41-2272 on cardiovascular remodeling, so we therefore chose the experiment period of 14 days based on our previous study, in which sufficient cardiac hypertrophy and fibrosis occurred with significant changes of LV gene expressions in this model of hypertension.18
At day 14, rats were anesthetized with pentobarbital sodium, and a 2F micromanometer–tipped catheter (Model SPC-721, Millar Instruments) was inserted into the LV through the right carotid artery. LV end-diastolic pressure (LVEDP) was measured using a transducer control unit (Model TCB-500, Millar Instruments) connected to a PowerLab system (ADInstruments Pty Ltd). Then the rats were euthanized by drawing blood from the abdominal aorta. After the whole heart was weighed, LV was frozen in liquid nitrogen or fixed in 4% paraformaldehyde and embedded in paraffin wax.
The present study was performed in accordance with the Animal Welfare Act and with approval of the University of Miyazaki Institutional Animal Care and Use Committee (2006-014). This investigation also conformed with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996).
Histology and Immunohistochemistry
Immunohistochemical staining was performed as described previously.18 For determining myofibroblastic differentiation, slides were stained with an anti-α-smooth muscle actin (α-SMA) monoclonal antibody (Clone 1A4, DAKO) at a dilution of 1:200 overnight at 4°C. For the detection of collagen fibers, slides were incubated with 0.1% picrosirius red (Direct Red 80, Sigma) dissolved in saturated picric acid for 10 minutes as described previously.18
The morphological evaluation and cell counting of myofibroblasts surrounding intramyocardial coronary arteries were performed in the middle portion of the LV by a single observer in a blinded manner as described previously.18 Each section immunostained with the antibody against α-SMA was scanned at a magnification of ×400. The number of cells positive for α-SMA surrounding the intramyocardial coronary artery was counted and normalized to the coronary vessel area, encircled by the external elastic lamella. To evaluate the magnitude of perivascular fibrosis, medium-sized intramyocardial coronary arteries with a diameter of 100 to 200 μm were randomly selected from ≥3 different sites, and the ratio of the perivascular fibrotic area to the coronary vessel area was determined using WinROOF (Mitani Co.). Collagen volume fraction in the interstitial space of myocardial fibers was determined by calculating the ratio of the collagen area to the entire area of an individual section. To measure cardiocyte size, cross-sectional areas of ≥50 myocardial fibers were measured at the level of nuclei while omitting longitudinally or obliquely sectioned cells as described previously.18
Gene expression for type 1 collagen and transforming growth factor (TGF)-β1 in the LV was measured by real-time quantitative RT-PCR (ABI Prism 7700 Sequence Detector, Applied Biosystems) as described previously.18 cDNA reverse transcribed from total RNA was amplified with oligonucleotide primers, forward and reverse, and with probes labeled with 6-carboxy-fluorescencein as reporter fluorescence and 6-carboxy-tetramethyl-rhodamine as quencher fluorescence. The oligonucleotide sequences used are detailed in previous reports.18,19 The PCR products were of the expected molecular size, and the gene expression levels were normalized relative to the level of 18S ribosomal RNA.
Cultured cardiac fibroblasts were isolated from ventricles of 1-day-old Wistar rats as described previously.20 The cells were treated with 1 μmol/L Ang II in the absence or presence of BAY41-2272 for 24 hours. The magnitude of their proliferation was assessed by measuring the amount of [3H]-thymidine incorporated into the cells.20
Assays for ANP, BNP, and cGMP
Blood samples were collected at day 14 with 1.5 mg/mL of di-sodium ethylenediamine tetraacetate and 500 kallikrein inactivator units per milliliter of aprotinin, centrifuged at 2000g for 15 minutes at 4°C and then stored at −80°C until use. Plasma levels of ANP were measured with a specific radioimmunoassay, as described previously,21 and those of BNP with a commercially available kit (Peninsula Laboratories Inc). To determine the effect of BAY41-2272 on cGMP levels in the LV, rats were infused with 250 ng/kg per minute of Ang II for 14 days and given orally 2 or 10 mg/kg per day of BAY41-2272 twice a day on days 13 and 14. After the animals were euthanized, the myocardial tissue was immediately collected and stored at −80°C. In the cell culture study, fibroblasts were treated with BAY41-2272 for 10 minutes and immediately collected as described previously.12 cGMP content was determined using a radioimmunoassay kit (YAMASA Cyclic GMP Assay Kit).
Values shown are expressed as mean±SEM. Differences between groups were assessed using the 1-way ANOVA followed by Scheffe’s test, and statistical significance was accepted at P<0.05.
Figure 1A and 1B illustrate the effects of Ang II and BAY41-2272 on the systolic blood pressure and heart rate. Continuous, subcutaneous infusion of Ang II significantly (P<0.01) raised systolic blood pressure from days 3 to 14. The high dose of BAY41-2272 significantly reduced systolic blood pressure during the first 10 days in the Ang II–infused rats; however, the reduction became insignificant at day 14, and the low dose of BAY41-2272 had no effect on blood pressure raised by Ang II (Figure 1A). Heart rates of the Ang II infusion groups without BAY41-2272 or with the low dose of BAY41-2272 were reduced, but the changes were statistically insignificant (Figure 1B). As shown in Figure 1C, LVEDP was significantly (P<0.05) raised by the infusion of Ang II and was lowered by the high dose of the drug (P<0.05) but not by the low dose.
Cardiac Hypertrophy and Collagen Deposition
As shown in Figure 2A and 2B, the infusion of Ang II significantly (P<0.01) increased the ratio of heart weight:body weight and cross-sectional area of myocardial fibers, compared with the control, at day 14. The low dose of BAY41-2272 had no effect on the Ang II–induced increase in heart weight:body weight and cardiocyte size, but the high dose significantly (P<0.01) reduced both. Figure 3A shows the effect of BAY41-2272 on collagen deposition in the perivascular area of intramyocardial coronary arteries. Ang II significantly (P<0.01) increased the deposition, but the low and high doses of BAY41-2272 significantly reduced it by 20% (P<0.05) and by 30% (P<0.01), respectively. Similarly, Ang II–induced collagen deposition in the myocardial interstitial area was reduced by both the low (−21%; P<0.05) and high (−38%; P<0.01) dose of BAY41-2272 (Figure 3B).
Figure 4 illustrates numbers of fibroblasts positive for α-SMA, a marker for myofibroblastic differentiation, in the perivascular area surrounding intramyocardial coronary arteries. Ang II increased the number of α-SMA–positive cells (P<0.05) compared with controls; however, the low and high doses of BAY41-2272 similarly decreased the number by 74% and 79% (P<0.05), respectively.
LV Gene Expression
As shown in Figure 5A and 5B, Ang II significantly augmented the LV gene expression of type 1 collagen (P<0.01) and TGF-β1 (P<0.05). Coadministration of BAY41-2272 significantly reduced the Ang II–induced increases in mRNA for type 1 collagen (low dose: −41%; high dose: −49%) and TGF-β1 (low dose: −49%; high dose: −65%). Although statistically insignificant, slight elevations of both mRNA levels were observed in the high-dose treatment group without Ang II infusion.
Measurements of ANP, BNP, and cGMP
The Ang II infusion significantly (P<0.01) increased the plasma level of ANP, but this was not the case for those of BNP, where both the low and high doses of BAY41-2272 had no significant effects on their plasma levels (Table 1). As shown in Table 2, tissue cGMP concentrations in the LV were significantly increased by treatment with the low and high doses of BAY41-2272 with or without the infusion of Ang II.
Cell Culture Study
Figure 6A and 6B illustrate the effects of Ang II and BAY41-2272 on proliferation and intracellular cGMP in cultured cardiac fibroblasts. As shown in Figure 6A, BAY41-2272 significantly (P<0.01) attenuated not only basal but also Ang II–stimulated [3H]-thymidine incorporation. The inhibition of fibroblast proliferation was accompanied by a significant rise in the intracellular cGMP level (Figure 6B).
Both mechanical load and humoral activation have been shown to cause cardiac hypertrophy and fibrosis in hypertensive heart disease.22,23 Accordingly, not simply controlling systemic blood pressure but also pharmacological approaches to managing cardiovascular remodeling are necessary for treatment of hypertensive patients. The direct sGC stimulator BAY41-2272 has been shown to lower arterial pressure and peripheral resistance and to increase cardiac output and renal blood flow by raising intracellular cGMP levels15–17; however, it remained to be elucidated whether this compound affects cardiac remodeling. In the present study, the continuous stimulation of sGC with BAY41-2272 attenuated LV hypertrophy and fibrosis in rats with Ang II–induced hypertension, suppressing the phenotypic change of fibroblasts and the expression of extracellular matrix–related genes. Thus, this study suggests the importance of the activation of sGC and subsequent rise in the intracellular concentration of cGMP in attenuating adverse cardiac remodeling associated with hypertension.
Two isotypes of GC, particulate GC and sGC, are widely distributed in various tissues and organs including the heart and kidneys.24 Although 2 isotypes of the cGMP-generating enzymes share some structural homology, their enzymatic activity is regulated differentially: natriuretic peptides stimulate particulate GC, whereas NO evokes the activity of sGC.24 Most of the actions of natriuretic peptides, including the suppression of cardiac hypertrophy and fibrosis, are assumed to be mediated by intracellular cGMP. It has been shown that the direct sGC stimulator BAY41-2272 also exerts its effects by raising intracellular cGMP levels,15–17 and consistent with this, BAY41-2272 increased cGMP levels in the LV myocardium tissue and in cultured cardiac fibroblasts in the present study. Because a question may arise over activity of the natriuretic peptide particulate GC system during activation of sGC by BAY41-2272, we measured plasma ANP and BNP levels in the study groups. The subcutaneous infusion of Ang II indeed increased the plasma level of ANP, but BAY41-2272 had no effect, suggesting that natriuretic peptides were unlikely involved in the increased cGMP levels in the LV.
BAY41-2272 has been reported to be quickly oxidized after oral administration, although Straub et al25 showed that the oxidized metabolite exerted a stronger and longer pharmacological effect than BAY41-2272 itself in vivo. In contrast to nitroglycerin, which activates sGC by releasing NO, drug tolerance has been reported to hardly occur for BAY41-2272.14 In the present study, the blood pressure–lowering effect of the high dose of BAY41-2272 in the Ang II–infused rats had been significant during the first 10 days, but it became insignificant at day 14. Despite the incomplete reduction of blood pressure, BAY41-2272 substantially alleviated cardiomyocyte hypertrophy and collagen accumulation surrounding the intramyocardial coronary arteries and in the myocardial interstitium, reducing LVEDP, in the Ang II–infused rats. The reduction in collagen deposition was accompanied by suppression of the phenotypic change of fibroblasts into myofibroblasts and by lowering of the mRNA levels of type 1 collagen and TGF-β1. Because the phenotypic change of fibroblasts to myofibroblasts by Ang II or TGF-β1 has been found critical in stimulating fibroblast proliferation and producing extracellular matrix,26 suppression of this process is important for attenuating cardiac fibrosis. Notably, the low dose of BAY41-2272 had no effect on blood pressure or cardiac hypertrophy but substantially suppressed fibroblastic activation, LV gene expression, and collagen deposition, raising the LV cGMP level.
Because the natriuretic peptides–particulate GC system has been shown to suppress cardiac hypertrophy and fibrosis independently of blood pressure,9,13,27 we further investigated whether BAY41-2272 has the direct effects in vitro on the cultured cardiac fibroblasts. In the cell culture study, BAY41-2272 inhibited the proliferation of cardiac fibroblasts, elevating the intracellular cGMP level, supporting a direct inhibitory action of this compound on cardiac fibrosis observed in vivo. On the other hand, alleviation of the cardiomyocyte hypertrophy was observed in the high-dose group but not in the low-dose group in the present study, and we found that BAY41-2272 had little effect on hypertrophy in the cultured cardiomyocytes (data not shown). Thus, we speculate that the improvement of cardiomyocyte hypertrophy observed in this study is largely dependent on mechanical load rather than a direct effect of BAY41-2272.
In conclusion, this study demonstrated that the continuous stimulation of sGC with BAY41-2272 for 2 weeks ameliorated Ang II–induced cardiac remodeling in rats, and the effects on the extracellular matrix may have been exerted partially via cGMP, independently of blood pressure. Thus, sGC generating cGMP would be a therapeutic target for reducing the adverse cardiovascular remodeling associated with hypertension.
Given the significance of myocardial fibrosis and hypertrophy in the process of cardiac remodeling in hypertensive subjects, the present findings may have important implications with regard to pharmacological stimulation of sGC for attenuating the remodeling process of the LV. In this study, we have shown that the orally available compound BAY41-2272, a direct sGC stimulator, would be useful not only in reducing blood pressure but also in attenuating cardiac remodeling. In addition, no adverse effects of BAY41-2272 on the liver or kidneys were detected at least in data of the serum aminotransferases and creatinine levels (Table I, available online at http://hyper.ahajournals.org). Because of the limited clinical use of human recombinant ANP and BNP because of their short half-lives, the present study suggests a potential usefulness for this compound in the treatment of hypertension, warranting further studies, such as administration to other models of hypertension or treatment for longer time periods.
We thank Ritsuko Sotomura for technical assistance.
Sources of Funding
This study was supported by Grants-in-Aid for Scientific Research on Priority Areas; by the 21st Century Centers of Excellence Program (Life Science) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; and by a grant from Bayer HealthCare.
H.M. and T.T. have received a research grant from Bayer HealthCare, and J-P.S. is an employee of that company. BAY41-2272 was synthesized at Bayer HealthCare as a research tool but not for use in humans.
The first 2 authors contributed equally to this work.
- Received May 18, 2006.
- Revision received June 6, 2006.
- Accepted August 8, 2006.
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