Intimal hyperplasia (IH) is an abnormal response to vessel injury characterized by the dedifferentiation, migration, and proliferation of quiescent vascular smooth muscle cells (VSMC) to form a neointima layer. Vascular connexins (Cx) are involved in the pathophysiology of various vascular diseases, and Cx43, the main Cx expressed in VSMC, has been shown to promote VSMC proliferation and IH. The aim of this study was to investigate the participation of another Cx, namely Cx37, in the formation of the neointima layer.Wild-type (WT) and Cx37-deficient (Cx37-/-) C57BL/6J mice were subjected to carotid artery ligation (CAL), a model of vessel injury and IH. The neointima developed linearly in WT until 28 days post surgery. In contrast, the neointima layer was almost absent 14 days after surgery in Cx37-/- mice, and twice as more developed after 28 days compared to WT mice. This large neointima formation correlated with a two-fold increase in cell proliferation in the media and neointima regions between 14 and 28 days in Cx37-/- mice compared to WT mice. The CAL triggered Cx43 overexpression in the media and neointima layers of ligated carotids in WT mice, and selectively up-regulated Cx37 expression in the media layer, but not in the neointima layer. The de novo expression of Cx37 in human primary VSMC reduced cell proliferation and P-Akt levels, in association with lower Cx43 levels, whereas Cx43 overexpression increased P-Akt levels.The presence of Cx37 in the media layer of injured arteries restrains VSMC proliferation and limits the development of IH, presumably by interfering with the pro-proliferative effect of Cx43 and the Akt pathway.
ABSTRACT Objective Hydrogen sulfide (H 2 S) is a proangiogenic gas produced primarily by the transsulfuration enzyme cystathionine-gamma-lyase (CGL). CGL-dependant H 2 S production is required for neovasculariation in models of peripheral arterial disease. However, the benefits of increasing endogenous CGL and its mechanism of action have yet to be elucidated. Methods 10 weeks old male whole-body CGL overexpressing mice (CGL Tg ) and wild type littermates (C57BL/6J) were subjected to the hindlimb ischemia model. Functional recovery was assessed through treadmill exercise endurance testing, while ischemic leg perfusion recovery was measured by laser Doppler perfusion imaging and tissue immunohistochemistry. To examine angiogenic potential, aortic ring sprouting assay and post-natal mouse retinal vasculature development studies were performed. Lastly, comparative metabolomics, NAD + /NADH analysis, and quantitative real-time PCR were performed on WT and CGL Tg gastrocnemius muscles. Results The restoration of blood flow upon femoral ligation occurred more rapidly in CGL Tg mice. CGL Tg mice were able to run further and for longer compared to WT mice. In ischemic gastrocnemius, capillary density was increased in mice overexpressing CGL. Endothelial cell sprouting was increased in aorta isolated from CGL Tg mice, especially when cultured in VEGF-only media. Metabolomics analysis demonstrated an increased presence of niacinamide, a precursor of nicotinamide adenine dinucleotide (NAD + / NADH) in the muscle of CGL Tg mice. Finally, CGL overexpression and NMN supplementation improved endothelial cell migration in vitro . Conclusions Taken together, our results demonstrate that CGL overexpression improves the neovascularization of skeletal muscle upon hindlimb ischemia. These effects are mediated by changes in the NAD pathway, which improves endothelial cell migration.
The current strategies to reduce intimal hyperplasia (IH) principally rely on local drug delivery, in endovascular approach. The oral angiotensin converting enzyme inhibitor (ACEi) Zofenopril has additional effects compared to other non-sulfyhydrated ACEi to prevent intimal hyperplasia and restenosis. Given the number of patients treated with ACEi worldwide, these findings call for further prospective clinical trials to test the benefits of sulfhydrated ACEi over classic ACEi for the prevention of restenosis in hypertensive patients. Abstract Objectives Hypertension is a major risk factor for intimal hyperplasia (IH) and restenosis following vascular and endovascular interventions. Pre-clinical studies suggest that hydrogen sulfide (H2S), an endogenous gasotransmitter, limits restenosis. While there is no clinically available pure H2S releasing compound, the sulfhydryl-containing angiotensin-converting enzyme inhibitor Zofenopril is a source of H2S. Here, we hypothesized that Zofenopril, due to H2S release, would be superior to other non-sulfhydryl containing angiotensin converting enzyme inhibitor (ACEi), in reducing intimal hyperplasia in the context of hypertension. Materials Spontaneously hypertensive male Cx40 deleted mice (Cx40-/-) or WT littermates were randomly treated with Enalapril 20 mg (Mepha Pharma) or Zofenopril 30 mg (Mylan SA). Discarded human vein segments and primary human smooth muscle cells (SMC) were treated with the active compound Enalaprilat or Zofenoprilat. Methods IH was evaluated in mice 28 days after focal carotid artery stenosis surgery and in human vein segments cultured for 7 days ex vivo. Human primary smooth muscle cell (SMC) proliferation and migration were studied in vitro. Results Compared to control animals (intima/media thickness=2.3±0.33), Enalapril reduced IH in Cx40-/- hypertensive mice by 30% (1.7±0.35; p=0.037), while Zofenopril abrogated IH (0.4±0.16; p<.0015 vs. Ctrl and p>0.99 vs. sham-operated Cx40-/-mice). In WT normotensive mice, enalapril had no effect (0.9665±0.2 in control vs 1.140±0.27; p>.99), while Zofenopril also abrogated IH (0.1623±0.07, p<.008 vs. Ctrl and p>0.99 vs. sham-operated WT mice). Zofenoprilat, but not Enalaprilat, also prevented intimal hyperplasia in human veins segments ex vivo. The effect of Zofenopril on carotid and SMC correlated with reduced SMC proliferation and migration. Zofenoprilat inhibited the MAPK and mTOR pathways in SMC and human vein segments. Conclusion Zofenopril provides extra beneficial effects compared to non-sulfhydryl ACEi to reduce SMC proliferation and restenosis, even in normotensive animals. These findings may hold broad clinical implications for patients suffering from vascular occlusive diseases and hypertension.
The saphenous vein is the conduit of choice for bypass grafting. Unfortunately, the hemodynamic stress associated with the arterial environment of the bypass vein graft leads to the development of intimal hyperplasia (IH), an excessive cellular growth and collagen deposition that results in restenosis and secondary graft occlusion. Hydrogen sulfide (H2S) is a ubiquitous redox-modifying gasotransmitter that inhibits IH. H2S is produced via the reverse trans-sulfuration pathway by three enzymes: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). However, the expression and regulation of these enzymes in the human vasculature remains unclear. Here, we investigated the expression of CSE, CBS and 3-MST in segments of native human saphenous vein and large arteries. Furthermore, we evaluated the regulation of these enzymes in vein segments cultured under static, venous (7 mmHg pressure) or arterial (100 mmHg pressure) pressure. CSE was expressed in the media, neointima and intima of the vessels and was negatively regulated by arterial shear stress. Adenoviral-mediated CSE overexpression or RNA interference-mediated CSE knock-down revealed that CSE inhibited primary human VSMC migration but not proliferation. We propose that high shear stress in arteriovenous bypass grafts inhibits CSE expression in both the media and endothelium, which may contribute to increased VSMC migration in the context of IH.
Therapies to accelerate vascular repair are currently lacking. Pre-clinical studies suggest that hydrogen sulfide (H2S), an endogenous gasotransmitter, promotes angiogenesis. Here, we hypothesized that sodium thiosulfate (STS), a clinically relevant source of H2S, would stimulate angiogenesis and vascular repair. STS stimulated neovascularization in WT and LDLR receptor knockout mice following hindlimb ischemia as evidenced by increased leg perfusion assessed by laser Doppler imaging, and capillary density in the gastrocnemius muscle. STS also promoted VEGF-dependent angiogenesis in matrigel plugs in vivo and in the chorioallantoic membrane of chick embryos. In vitro, STS and NaHS stimulated human umbilical vein endothelial cell (HUVEC) migration and proliferation. Seahorse experiments further revealed that STS inhibited mitochondrial respiration and promoted glycolysis in HUVEC. The effect of STS on migration and proliferation was glycolysis-dependent. STS probably acts through metabolic reprogramming of endothelial cells toward a more proliferative glycolytic state. These findings may hold broad clinical implications for patients suffering from vascular occlusive diseases.
Background Intimal hyperplasia (IH) remains a major limitation in the long-term success of any type of revascularization. IH is due to vascular smooth muscle cell (VSMC) dedifferentiation, proliferation and migration. The gasotransmitter Hydrogen Sulfide (H2S), mainly produced in blood vessels by the enzyme cystathionine- γ-lyase (CSE), inhibits IH in pre-clinical models. However, there is currently no H2S donor available to treat patients. Here we used sodium thiosulfate (STS), a clinically-approved source of sulfur, to limit IH.Methods Hypercholesterolemic LDLR deleted (LDLR-/- ), WT or CSE-/- male mice randomly treated with 4g/L STS in the water bottle were submitted to focal carotid artery stenosis to induce IH. Human vein segments were maintained in culture for 7 days to induce IH. Further in vitro studies were conducted in primary human vascular smooth muscle cells (VSMC).Findings STS inhibited IH in mice and in human vein segments. STS inhibited cell proliferation in the carotid artery wall and in human vein segments. STS increased polysulfides in vivo and protein persulfidation in vitro, which correlated with microtubule depolymerization, cell cycle arrest and reduced VSMC migration and proliferation.Interpretation STS, a drug used for the treatment of cyanide poisoning and calciphylaxis, protects against IH in a mouse model of arterial restenosis and in human vein segments. STS acts as an H2S donor to limit VSMC migration and proliferation via microtubule depolymerization.Funding: This work was supported by the Swiss National Science Foundation (grant FN-310030_176158 to FA and SD and PZ00P3-185927 to AL); the Novartis Foundation to FA; and the Union des Sociétés Suisses des Maladies Vasculaires to SD, and the Fondation pour la recherche en chirurgie vasculaire et thoracique.Declaration of Interest: None to declare. Ethical Approval: The study protocols for organ collection and use were reviewed and approved by the Centre Hospitalier Universitaire Vaudois (CHUV) and the Cantonal Human Research Ethics Committee (http://www.cer-vd.ch/, no IRB number, Protocol Number 170/02)
Intimal hyperplasia (IH) remains a major limitation in the long-term success of any type of revascularisation. IH is due to vascular smooth muscle cell (VSMC) dedifferentiation, proliferation and migration. The gasotransmitter Hydrogen Sulfide (H2S), mainly produced in blood vessels by the enzyme cystathionine- γ-lyase (CSE), inhibits IH in pre-clinical models. However, there is currently no H2S donor available to treat patients. Here we used sodium thiosulfate (STS), a clinically-approved source of sulfur, to limit IH.Low density lipoprotein receptor deleted (LDLR-/-), WT or Cse-deleted (Cse-/-) male mice randomly treated with 4 g/L STS in the water bottle were submitted to focal carotid artery stenosis to induce IH. Human vein segments were maintained in culture for 7 days to induce IH. Further in vitro studies were conducted in primary human vascular smooth muscle cells (VSMCs).STS inhibited IH in WT mice, as well as in LDLR-/- and Cse-/- mice, and in human vein segments. STS inhibited cell proliferation in the carotid artery wall and in human vein segments. STS increased polysulfides in vivo and protein persulfidation in vitro, which correlated with microtubule depolymerisation, cell cycle arrest and reduced VSMC migration and proliferation.STS, a drug used for the treatment of cyanide poisoning and calciphylaxis, protects against IH in a mouse model of arterial restenosis and in human vein segments. STS acts as an H2S donor to limit VSMC migration and proliferation via microtubule depolymerisation.This work was supported by the Swiss National Science Foundation (grant FN-310030_176158 to FA and SD and PZ00P3-185927 to AL); the Novartis Foundation to FA; and the Union des Sociétés Suisses des Maladies Vasculaires to SD, and the Fondation pour la recherche en chirurgie vasculaire et thoracique.
