Cinaciguat prevents neointima formation after arterial injury by decreasing vascular smooth muscle cell migration and proliferation

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Abstract

Aims

Vascular smooth muscle cell (VSMC) migration, proliferation and remodeling of the extracellular matrix contribute to lumen loss after arterial injury leading to restenosis. Several studies indicated the role of the cyclic guanosine monophosphate signaling in neointimal formation. Cinaciguat, the novel soluble guanylate cyclase activator, currently being in phase IIb clinical trial, has been shown to exert antiplatelet and anti-remodeling effects in animal models of vascular pathology. In this study we investigated the effects of cinaciguat on post-injury arterial stenosis.

Methods and Results

Male Sprague-Dawley rats (n = 100) underwent endothelial denudation by wire injury of the right common carotid artery. Cinaciguat (10 mg/kg/day orally) were administered to 50 rats (1-, 2-, 3-day and 1-, 3-week treatment time), while 50 rats received placebo. A 3-week treatment resulted in a significantly reduced vascular stenosis (17.53 ± 10.84% in the treatment group vs. 43.25 ± 30.83% in the control wire injury group) and neointima/media area ratio (0.45 ± 0.32 in the treatment group vs. 1.09 ± 0.69 in the control wire injury group). By using quantitative real-time PCR, Western blot and immunohistochemistry, matrix-metallopreoteinase-9 (MMP-9) was found to be upregulated in the control-injured carotids over the whole follow-up, and cinaciguat significantly decreased MMP-9 expression by 3 weeks. As assessed by protein immunoblot, injury-induced local decrease of soluble guanylate cyclase β1 subunit could be recovered by cinaciguat. In vitro wound healing assay with VSMCs revealed dose-dependent antimigratory and antiproliferative effects of cinaciguat. Plasma level of cyclic guanosine monophosphate was significantly elevated after 3 weeks of treatment.

Conclusion

Our results show that cinaciguat prevents injury-induced neointimal hyperplasia by decreasing VSMC migration and proliferation through the regulation of MMP-9.

Introduction

Despite significant progress in reducing recurrent stenosis following angioplasty and stent implantation, restenosis remains a common complication that limits the long-term benefits of revascularization procedures causing recurrent angina, or even acute coronary syndrome [1]. Drug-eluting stents effectively decreased the prevalence of restenosis however their use is associated with a higher risk for stent thrombosis [2]. Restenosis is characterized by four overlapping stages: the initial endothelial injury and platelet activation is followed by inflammation of the vessel wall, vascular smooth muscle cell (VSMC) migration, proliferation and extracellular matrix (ECM) remodeling [3]. The latter two processes are responsible for neointimal hyperplasia that leads to luminal re-narrowing. In the last decade, several studies indicated the pathophysiological role of decreased nitric-oxide (NO) - cyclic guanosine monophosphate (cGMP) - protein kinase G (PKG) signaling in the development of neointimal hyperplasia [4], [5], [6]. In agreement with these findings, the activation of the NO-cGMP-PKG pathway resulted in a reduced neointima formation after arterial injury [5], [6], [7], [8], [9], [10]. cGMP signal enhancement can take place through the activation of the soluble guanylate cyclase (sGC), or through selective inhibition of cGMP-degrading phosphodiestersae-5 [11]. Two classes of NO-independent, direct activators of guanylate cyclase are described in the literature. The hem-dependent stimulators -like YC-1 or BAY 41-2272- require an intact hem in the cyclase [12]. Recently a new hem- and NO-independent sGC activator was introduced: cinaciguat (BAY 58–2667) that activates sGC in its hem-deficient or hem-oxidized form more potently [13], [14], [15]. Cinaciguat is currently in clinical development for acute decompensated heart failure, which is characterised by endothelial dysfunction, decreased bioavailability of NO and chronic vasoconstriction leading to increased preload and afterload. Cinaciguat is able to induce vasodilation even in diseased vessels; the first clinical trials showed its efficacy and good tolerability in patients with heart failure [16], [17].

We presumed that the NO-independent sGC activator cinaciguat may increase cGMP signaling and reduce neointimal hyperplasia in vessels with previous endothelial injury. This hypothesis may also be supported by a recent study showing efficacy of cinaciguat in an experimental model of pulmonary hypertension. Cinaciguat reduced the pulmonary hypertension associated structural changes, like VSMC migration, proliferation and extracellular matrix accumulation; this was partially a direct effect of the drug [18]. Therefore we aimed to evaluate for the first time the effects of the novel sGC activator cinaciguat in an in vivo model of injury-induced arterial stenosis.

Section snippets

Rat carotid artery wire injury

The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology. The protocol was approved by the Regional Ethical Committee for Laboratory Animal Use and conformed with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. 85–23, revised 1996). One hundred male Sprague-Dawley rats (250 to 300 g body weight) underwent wire injury to

Attenuation of neointimal hyperplasia by cinaciguat

Wire injury of the rat carotid artery causes a gentle removal of the endothelial layer without damaging the underlying media, in contrast to balloon injury or stent deployment. This induces neointimal hyperplasia as early as 1–2 weeks after injury, but allows a better quantitative evaluation of the concentric, homogeneous, aSMA positive neointima at later time. Therefore we evaluated the stenoses three weeks after the injury. In the de-endothelized arteries of non-treated rats we observed 43.25%

Discussion

In the present study we showed for the first time the efficacy of the newly developed NO- and hem-independent sGC activator cinaciguat in the reduction of injury-induced arterial stenosis. sGC activation in injured rat carotid arteries lead to downregulation of MMP-9, and to decreased proliferation and migration of vascular smooth muscle cells. Finally, the three-week treatment with cinaciguat resulted in a reduced neointimal hyperplasia. Cinaciguat is orally bioavailable, with a long-lasting

Conflict of interest

None declared.

Acknowledgements

The expert technical assistance of Heike Ziebart, Patricia Kraft, Rosa Eurich and Annika Berg is gratefully acknowledged. The authors acknowledge Bayer HealthCare AG for the donation of cinaciguat.

The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology.

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    Sources of funding: This work was supported by the Land Baden-Württemberg. K. Hirschberg and S. Korkmaz are supported by the Medical Faculty of the University of Heidelberg. Sz Páli is supported by the Garduate Academy 1126 of the University of Heidelberg.

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