Heparin Cofactor II Protects Against Angiotensin II-Induced Cardiac Remodeling Via Attenuation of Oxidative Stress in Mice
Heparin cofactor II (HCII), a serine protease inhibitor, inhibits tissue thrombin action after binding with dermatan sulfate proteoglycans in the extracellular matrix of the vascular system. We previously reported that heterozygous HCII-deficient (HCII+/−) humans and mice demonstrate acceleration of vascular remodeling, including atherosclerosis. However, the action of HCII on cardiac remodeling never has been determined. HCII+/+ and HCII+/− mice at age 25 weeks were infused with angiotensin II (Ang II; 2.0 mg/kg/d) for 2 weeks by an osmotic mini-pump. Echocardiography revealed acceleration of cardiac concentric remodeling in HCII+/− mice and larger left atrial volume in HCII+/− mice than in HCII+/+ mice. Histopathologic studies showed more prominent interstitial fibrosis in both the left atrium and left ventricle in HCII+/− mice than in HCII+/+ mice. Daily urinary excretion of 8-hydroxy-2′-deoxyguanosine, a parameter of oxidative stress, and dihydroethidium-positive spots, indicating superoxide production in the myocardium, were markedly increased in Ang II-treated HCII+/− mice compared to those in HCII+/+ mice. Cardiac gene expression levels of atrial natriuretic peptides and brain natriuretic peptides, members of the natriuretic peptide family, Nox 4, Rac-1, and p67phox as components of NAD(P)H oxidase, and transforming growth factor-β1 and procollagen III were more augmented in HCII+/− mice than in HCII+/+ mice. However, administration of human HCII protein attenuated all of those abnormalities in Ang II-treated HCII+/− mice. Moreover, human HCII protein supplementation almost abolished cardiac fibrosis in Ang II-treated HCII+/+ mice. The results indicate that HCII has a protective role against Ang II-induced cardiac remodeling through suppression of the NAD(P)H oxidase-transforming growth factor-β1 pathway.
Thrombin not only acts as a coagulation key enzyme but also is involved in tissue repair and remodeling, embryogenesis, angiogenesis, and development of atherosclerosis.1,2 These biological functions of thrombin appear to be mediated by specific thrombin receptors, particularly protease-activated receptor-1 (PAR-1).1,2 Because it has been reported that PAR-1 is expressed in cardiomyocytes and cardiac fibroblasts3,4 and that PAR-1 contributes to cardiac remodeling and hypertrophy,5 there is a possibility that modulation of the thrombin-PAR-1 axis affects cardiac remodeling. Thrombin is inactivated by 2 major coagulation modulators: antithrombin and heparin cofactor II (HCII).6,7 In circulating blood of the intravascular lumen, antithrombin binding to heparan sulfate inhibits thrombin action, whereas in the subendothelium HCII binding to dermatan sulfate inhibits thrombin action. Therefore, we hypothesized that HCII exerts antivascular remodeling effects through inactivation of the thrombin-PAR-1 axis. We previously reported that high-plasma HCII activity is associated with reduced incidence of in-stent restenosis after percutaneous coronary intervention and that plasma HCII activity was negatively correlated with carotid maximum plaque thickness and prevalence of peripheral arterial disease in humans.8–11 In experimental animal studies, we and Tollefsen et al12,13 demonstrated that HCII-deficient mice manifest prominent intimal hyperplasia with increased cellular proliferation after tube cuff and wire vascular injury. Conversely, the intimal hyperplasia in HCII+/− mice with vascular injury was abrogated by human HCII supplementation.12 These results indicated that HCII plays a protective role against the progression of vascular remodeling. Because it has been recognized that development of vascular remodeling, including atherosclerosis, is closely associated with cardiac remodeling in humans and experimental animal models,14–18 we hypothesized that HCII is involved in the process of development of cardiac remodeling and vascular remodeling. To clarify this issue, we investigated cardiac remodeling in angiotensin II (Ang II)-infused mice with and without HCII deficiency, and we found that HCII protects against Ang II-induced cardiac remodeling with suppression of oxidative stress.
Materials and Methods
We used HCII+/+ male mice and HCII+/− male mice (HCII+/−; The University of Tokushima, Graduate School of Health Biosciences, Tokushima, Japan) that we previously generated at 25 weeks of age.12 The mice underwent sham operation or were infused with Ang II (WAKO) at a rate of 2.0 mg/kg per day for 2 weeks by an osmotic mini-pump (Alzet model 1002; Alza) as previously described.16–18 One group of Ang II-treated HCII+/− mice was administered human purified HCII protein 3 times per week for 2 weeks as previously reported.12 And 1 group of Ang II-treated HCII+/+ mice was also administered human purified HCII protein 3 times per week for 2 weeks. All experimental procedures were performed in accordance with the guidelines and approval from the Animal Research Committee of The University of Tokushima Graduate School. We performed the following experimental procedures: echocardiographic analysis, histological analysis and immunohistochemistry, analysis of urinary excretion of 8-hydroxy-2′-deoxyguanosine, superoxide detection in myocardial tissues, evaluation of plasma superoxide dismutase activity, quantitative real-time polymerase chain reaction analysis, and Western blot analysis as detailed in the online data supplement (please see http://hyper.ahajournals.org).
All data are expressed as means±SEM. For comparisons among groups, statistical significance was assessed using a 1-way analysis of variance, and the significance of each difference was determined by post hoc testing using the Tukey-Kramer method. Statistical significance was considered at P<0.05.
Prominent Concentric Cardiac Remodeling in Ang II-Treated HCII+/− Mice
Although systolic blood pressure levels were elevated to ≈40 mm Hg beyond baseline blood pressure by Ang II infusion, HCII deficiency did not affect levels of systolic blood pressure and heart rate during the experimental period, regardless of Ang II infusion in the mice (Figure 1A-C). There was no difference in fractional shortening percentage among the groups regardless of Ang II infusion (Figure 2A, D). Augmented relative wall thickness, but not left ventricular mass index, indicating prominent concentric cardiac remodeling was observed in Ang II-treated HCII+/− mice compared to that in Ang II-treated HCII+/+ mice (Figure 2A-C and Figure 3A). When human HCII protein was administered to HCII+/− mice, the Ang II-induced alteration was attenuated (Figure 2A, B and Figure 3A).
Increased Left Atrial Volume in Ang II-Treated HCII+/− Mice
Echocardiographic analysis showed that Ang II stimulation increased left atrial volume in HCII+/+ mice as well as in HCII+/− mice (Figure 2E, F). The enlarged left atrial volume was much greater in HCII+/− mice than in HCII+/+ mice (Figure 2E, F). However, HCII supplementation attenuated left atrial volume enlargement in Ang II-treated HCII+/− mice to almost the same level as that in Ang II-treated HCII+/+ mice (Figure 2E, F). These observations were consistent with macroscopic findings as shown in Figure 3A.
Exacerbation of Cardiac Fibrosis in Ang II-Treated HCII+/− Mice
There were no morphological differences in left ventricular mass, left atrial tissues, and ventricular tissues between HCII+/+ mice and HCII+/− mice without Ang II infusion (Figure 3A). Ang II stimulation caused not only prominent concentric change in left ventricular mass but also exacerbation of atrial and ventricular interstitial fibrosis in HCII+/− mice compared to those in HCII+/+ mice (Figure 3A-C). These unfortunate changes in hearts of HCII+/− mice were also restored by human HCII protein supplementation (Figure 3A-C).
Augmented Oxidative Stress in Ang II-Treated HCII+/− Mice
To evaluate Ang II-induced superoxide production, we analyzed dihydroethidium (DHE) staining of the atria and ventricles of the mice by fluorescence microscopy. Ang II stimulation increased cardiac superoxide production to a greater extent in HCII+/− mice than in HCII+/+ mice (Figure 4C-E). Next, we estimated urinary excretion of 8-hydroxy-2′-deoxyguanosine as an oxidative stress marker. In parallel with the results of cardiac DHE staining, Ang II treatment caused a more notable increase of urinary 8-hydroxy-2′-deoxyguanosine excretion in HCII+/− mice than in HCII+/+ mice (Figure 4A). Human HCII protein treatment significantly attenuated the Ang II-induced augmentation of cardiac superoxide production and elevation of urinary 8-hydroxy-2′-deoxyguanosine excretion in HCII+/− mice (Figure 4A). Although we also evaluated plasma superoxide dismutase (SOD) activity, indicating antioxidant capacity, there was no significant difference among the mice groups regardless of Ang II infusion (Figure 4B).
Accelerated Gene Expression of Natriuretic Peptide and Procollagen III But Not PAR-1 in Cardiac Tissue of Ang II-Treated HCII+/−Mice
It has been well-known that natriuretic peptides, including atrial natriuretic peptides and brain natriuretic peptides, and procollagen III and PAR-15 are upregulated during cardiac remodeling, leading to cardiac failure. Therefore, we evaluated cardiac expression of those genes in the present study. There was no significant difference in mRNA levels of those genes in HCII+/+ and HCII+/− mice without Ang II infusion. Atrial natriuretic peptides, brain natriuretic peptides, and procollagen III, but not PAR-1, mRNA levels were prominently augmented in Ang II-treated HCII+/− mice compared to the levels in Ang II-treated HCII+/+ mice. Human HCII supplementation abrogated the increased expression of atrial natriuretic peptides, brain natriuretic peptides, and procollagen III genes in Ang II-treated HCII+/− mice (Figure 5).
Enhanced NAD(P)H Oxidase-Transforming Growth Factor-β Pathway in Ang II-Treated HCII+/− Mice
Because the major source of Ang II-induced superoxide production in the cardiovascular system is thought to be the NAD(P)H oxidase system,19,20 we examined the mRNA levels of NAD(P)H oxidase components, including Nox subunits, p22phox for membrane subunits, p67phox and p47phox for cytosol subunits, and the small GTP-binding protein Rac-1 in the heart. Although no difference was observed between the levels of NAD(P)H oxidase expression in the mice without Ang II infusion, Ang II-treated HCII+/− mice showed higher mRNA expression levels of Nox 4, p67phox, and Rac-1, but not p22phox and p47phox, than those in Ang II-treated HCII+/+ mice (Figure 5). Ang II plays a pivotal role in tissue fibrosis, partly through increasing production of transforming growth factor (TGF)-β1, which is a potent accelerator in extracellular matrix remodeling. Therefore, we evaluated expression levels of TGF-β1 mRNA and protein in the mice with and without Ang II stimulation. In this study, Ang II-treated HCII+/− mice showed prominent TGF-β1 mRNA and protein expression compared with that in Ang II-treated HCII+/+ mice (Figures 5, 6⇓A). Moreover, immunohistochemistry of left ventricular tissues showed a larger number of TGF-β1-stained spots in cardiomyocytes and interstitial areas with fibrotic change in Ang II-treated HCII+/− mice than in Ang II-treated HCII+/+ mice (Figure 6B). Conversely, human HCII protein administration abolished the enhancement of the cardiac NAD(P)H oxidase-TGF-β1 pathway in Ang II-infused HCII+/− mice (Figures 5, 6⇓A, B).
Excess of Human HCII Protein Attenuates Cardiac Fibrosis Even in Ang II-Infused HCII+/+ Mice
To clarify the dose-dependency of the cardiac-protective action of HCII against Ang II excess, we quantified fibrosis areas in cardiac tissues of Ang II-infused HCII+/+ mice with and without human HCII supplementation. As shown in Figure 6C, HCII supplementation significantly reduced the cardiac fibrosis area, even in Ang II-infused HCII+/+ mice, suggesting that greater plasma HCII activity exerts a greater cardioprotective action against cardiac stress.
The present study demonstrated that HCII has protective actions against Ang II-induced cardiac remodeling, including left atrium enlargement, left ventricular concentric change, and cardiac fibrosis, with activation of the NAD(P)H oxidase-TGF-β1 signaling pathway. Recent studies have indicated that NAD(P)H oxidases are one of major sources of superoxide generation in cardiomyocytes compared with xanthine oxidase, arachidonic acid metabolism, or mitochondrial oxidases,21 and because increased oxidative stress with acceleration of superoxide production are known to be involved in high incidences of acute myocardial infarction, left ventricular hypertrophy, and heart failure, activation of NADPH oxidase components by Ang II plays a crucial role in the development of cardiovascular diseases.
We previously demonstrated that HCII deficiency causes exaggeration of atherosclerotic region formation with increased oxidative stress in apolipoprotein E-null mice.12 Therefore, there is a possibility that HCII can counteract not only vascular remodeling but also cardiac remodeling through attenuation of oxidative stress.
In fact, the present study demonstrated that Ang II-treated HCII+/− mice had a larger number of DHE-stained spots in the heart, indicating increased superoxide production, and HCII supplementation reduced the amount of oxidative stress in the heart. Because the present study revealed that HCII is involved in mRNA levels of Nox 4, Rac-1, and p67phox in the Ang II-infused murine heart, HCII action on modulation of oxidative stress under the condition of Ang II excess is partly through regulation of gene expression of those NAD(P)H oxidase components. Because previous studies showed that thrombin activates NAD(P)H oxidase, leading to increase oxidative stress in vascular endothelial cells and vascular smooth muscle cells,22,23 HCII may contribute to reducing cardiac superoxide production via inhibition of thrombin-induced NAD(P)H oxidase stimulation.
TGF-β1 is a crucial mediator of cardiac adaptation to hemodynamic overload and is closely associated with the pathogenesis of cardiac remodeling such as cardiac hypertrophy and heart failure.24,25 TGF-β1 has been shown to be a powerful initiator of cellular hypertrophy and interstitial fibrosis in the heart25 and to be involved in NAD(P)H oxidase activation.26 These findings concerning the interplay between NAD(P)H oxidase and TGF-β1 are consistent with the molecular phenotypes of accelerated cardiac remodeling in Ang II-treated HCII+/− mice.
Recent studies have shown that cardiac fibrosis is a process characterized by massive remodeling of the myocardial extracellular matrix and subsequent substitution of functional tissue by inelastic fibrotic tissue leading to failing heart.27,28 Vanhoutte et al29 demonstrated that syndecan-1, which is a transmembrane (type I) heparan sulfate proteoglycan and is a member of the syndecan proteoglycan family, plays a pivotal role in protection against exaggerated inflammation and adverse infarct healing after myocardial infarction in mice. Moreover, Hong et al30 showed that supplementation with dermatan sulfate, a glycosaminoglycan, improved cardiac function and maintained viability of cardiac myocytes with complement activation. Based on these observations and the fact that HCII has the ability to inhibit thrombin action by formation of a bimolecular complex with dermatan sulfate,7,31 the bimolecular complex with HCII and dermatan sulfate might cooperatively have beneficial effects in the heart against cardiac stress, including ischemia and Ang II excess.
Because the enhanced expression of PAR-1, in turn, is expected to further promote cardiac remodeling,5 we speculated that the organ-protective action of HCII is exerted on cardiomyocytes in which the thrombin-PAR-1 axis is activated to stimulate cardiac remodeling. However, in the present study, Ang II-treated HCII+/− mice showed no difference in mRNA levels of cardiac PAR-1 (Figure 5) compared to those in Ang II-treated HCII+/+ mice. Moreover, it is unlikely that inactivation of the thrombin-PAR-1 axis by HCII is the only mechanism for reduction of oxidative stress against Ang II infusion. Therefore, there is a possibility that HCII independently and directly modulates expression of NAD(P)H oxidase components in cardiac tissue conditioned with an excess of Ang II (Figure 6D). Further examinations are needed to clarify this issue.
All of these observations in the present study were further corroborated by results of a clinical study about cardiac remodeling and plasma HCII activities (T. Ise, K. Aihara, Y. Sumitomo-Ueda, S. Yoshida, Y. Ikeda, S. Yagi, T. Iwase, H. Yamada, M. Akaike, M. Sata, T. Matsumoto, unpublished observations, 2010). In that study, we investigated the relationship between plasma HCII activity and cardiac remodeling surrogate markers measured by echocardiography, and we found that plasma HCII activity is independently and inversely associated with the development of cardiac remodeling, including cardiac concentric change, left atrial enlargement, and left ventricular diastolic dysfunction determined by increased E-to-Ea (peak E velocity to early diastolic mitral annulus velocity) ratio. Therefore, the observations in our animal model might be applicable to human hypertensive heart disease in HCII deficiency.
HCII counteracts the development of cardiac remodeling, including cardiac concentric changes, left atrium enlargement, and cardiac fibrosis associated with regulating oxidative stress. These results indicate that treatment with HCII might be a novel and valuable therapeutic approach to prevent cardiac remodeling and atherosclerosis.
The authors thank Tomoko Mori for technical help and Akimasa Oomizu and Minoru Tsukada for providing human purified HCII protein.
Sources of Funding
This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, a grant from the Mitsui Life Social Welfare Foundation, a grant from the Takeda Science Foundation, and a grant for a Study Group on Aseptic Femoral Neck Necrosis from the Ministry of Health, Labour, and Welfare of Japan.
- Received February 21, 2010.
- Revision received March 1, 2010.
- Accepted July 2, 2010.
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