The goal of the current study was to investigate the role of exogenous and endogenous hydrogen sulfide (H 2 S) on neovascularization and wound healing in vitro and in vivo. Incubation of endothelial cells (ECs) with H 2 S enhanced their angiogenic potential, evidenced by accelerated cell growth, migration, and capillary morphogenesis on Matrigel. Treatment of chicken chorioallantoic membranes (CAMS) with H 2 S increased vascular length. Exposure of ECs to H 2 S resulted in increased phosphorylation of Akt, ERK, and p38. The K ATP channel blocker glibenclamide or the p38 inhibitor SB203580 abolished H 2 S-induced EC motility. Since glibenclamide inhibited H 2 S-triggered p38 phosphorylation, we propose that K ATP channels lay upstream of p38 in this process. When CAMs were treated with H 2 S biosynthesis inhibitors dl-propylargylglycine or beta-cyano-L-alanine, a reduction in vessel length and branching was observed, indicating that H 2 S serves as an endogenous stimulator of the angiogenic response. Stimulation of ECs with vascular endothelial growth factor (VEGF) increased H 2 S release, while pharmacological inhibition of H 2 S production or K ATP channels or silencing of cystathionine gamma-lyase (CSE) attenuated VEGF signaling and migration of ECs. These results implicate endothelial H 2 S synthesis in the pro-angiogenic action of VEGF. Aortic rings isolated from CSE knockout mice exhibited markedly reduced microvessel formation in response to VEGF when compared to wild-type littermates. Finally, in vivo, topical administration of H 2 S enhanced wound healing in a rat model, while wound healing was delayed in CSE −/− mice. We conclude that endogenous and exogenous H 2 S stimulates EC-related angiogenic properties through a K ATP channel/MAPK pathway.

Background and Purpose Hydrogen sulfide (H2S) is a signalling molecule that belongs to the gasotransmitter family. Two major sources for endogenous enzymatic production of H2S are cystathionine β synthase (CBS) and cystathionine γ lyase (CSE). In the present study, we examined the selectivity of commonly used pharmacological inhibitors of H2S biosynthesis towards CSE and CBS.Experimental Approach To address this question, human CSE or CBS enzymes were expressed and purified from Escherichia coli as fusion proteins with GSH-S-transferase. After purification, the activity of the recombinant enzymes was tested using the methylene blue method.Key Results β-cyanoalanine (BCA) was more potent in inhibiting CSE than propargylglycine (PAG) (IC50 14 ± 0.2 μM vs. 40 ± 8 μM respectively). Similar to PAG, L-aminoethoxyvinylglycine (AVG) only inhibited CSE, but did so at much lower concentrations. On the other hand, aminooxyacetic acid (AOAA), a frequently used CBS inhibitor, was more potent in inhibiting CSE compared with BCA and PAG (IC50 1.1 ± 0.1 μM); the IC50 for AOAA for inhibiting CBS was 8.5 ± 0.7 μM. In line with our biochemical observations, relaxation to L-cysteine was blocked by AOAA in aortic rings that lacked CBS expression. Trifluoroalanine and hydroxylamine, two compounds that have also been used to block H2S biosynthesis, blocked the activity of CBS and CSE. Trifluoroalanine had a fourfold lower IC50 for CBS versus CSE, while hydroxylamine was 60-fold more selective against CSE.Conclusions and Implications In conclusion, although PAG, AVG and BCA exhibit selectivity in inhibiting CSE versus CBS, no selective pharmacological CBS inhibitor is currently available.

The goal of the present studies was to investigate the role of changes in hydrogen sulfide (H 2 S) homeostasis in the pathogenesis of hyperglycemic endothelial dysfunction. Exposure of bEnd3 microvascular endothelial cells to elevated extracellular glucose (in vitro "hyperglycemia") induced the mitochondrial formation of reactive oxygen species (ROS), which resulted in an increased consumption of endogenous and exogenous H 2 S. Replacement of H 2 S or overexpression of the H 2 S-producing enzyme cystathionine-γ-lyase (CSE) attenuated the hyperglycemia-induced enhancement of ROS formation, attenuated nuclear DNA injury, reduced the activation of the nuclear enzyme poly(ADP-ribose) polymerase, and improved cellular viability. In vitro hyperglycemia resulted in a switch from oxidative phosphorylation to glycolysis, an effect that was partially corrected by H 2 S supplementation. Exposure of isolated vascular rings to high glucose in vitro induced an impairment of endothelium-dependent relaxations, which was prevented by CSE overexpression or H 2 S supplementation. siRNA silencing of CSE exacerbated ROS production in hyperglycemic endothelial cells. Vascular rings from CSE −/− mice exhibited an accelerated impairment of endothelium-dependent relaxations in response to in vitro hyperglycemia, compared with wild-type controls. Streptozotocininduced diabetes in rats resulted in a decrease in the circulating level of H 2 S; replacement of H 2 S protected from the development of endothelial dysfunction ex vivo. In conclusion, endogenously produced H 2 S protects against the development of hyperglycemia-induced endothelial dysfunction. We hypothesize that, in hyperglycemic endothelial cells, mitochondrial ROS production and increased H 2 S catabolism form a positive feed-forward cycle. H 2 S replacement protects against these alterations, resulting in reduced ROS formation, improved endothelial metabolic state, and maintenance of normal endothelial function.

Objective-Recent studies have demonstrated that hydrogen sulfide (H 2 S) is produced within the vessel wall from L-cysteine regulating several aspects of vascular homeostasis. H 2 S generated from cystathione ␥-lyase (CSE) contributes to vascular tone; however, the molecular mechanisms underlying the vasorelaxing effects of H 2 S are still under investigation. Methods and Results-Using isolated aortic rings, we observed that addition of L-cysteine led to a concentrationdependent relaxation that was prevented by the CSE inhibitors DL-propargylglyicine (PAG) and ␤-cyano-L-alanine (BCA). Moreover, incubation with PAG or BCA resulted in a rightward shift in sodium nitroprusside-and isoproterenol-induced relaxation. Aortic tissues exposed to PAG or BCA contained lower levels of cGMP, exposure of cells to exogenous H 2 S or overexpression of CSE raised cGMP concentration. RNA silencing of CSE expression reduced intracellular cGMP levels confirming a positive role for endogenous H 2 S on cGMP accumulation. The ability of H 2 S to enhance cGMP levels was greatly reduced by the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Finally, addition of H 2 S to a cell-free system inhibited both cGMP and cAMP breakdown. Conclusion-These findings provide direct evidence that H 2 S acts as an endogenous inhibitor of phosphodiesterase activity and reinforce the notion that this gasotransmitter could be therapeutically exploited. Key Words: endothelium Ⅲ hypertension Ⅲ signal transduction Ⅲ vascular muscle Ⅲ vasodilation Ⅲ cystathione ␥-lyase Ⅲ hydrogen sulfide Ⅲ cAMP Ⅲ cGMP Ⅲ phosphodiesterase N itric oxide (NO) is believed to account for most of the endothelium-derived relaxing factor activity released within the vessel wall, at least in some vessels. 1 On muscarinic stimulation, NO is produced following the conversion of L-arginine to NO by endothelial nitric oxide synthase (eNOS). 2 NO diffuses from the endothelium to the underlying smooth muscle cell layer, where it stimulates soluble guanylate cyclase to produce cGMP. cGMP in turn activates protein kinase G (PKG), which initiates a cascade of events leading to relaxation. 2,3 Hydrogen sulfide (H 2 S) is emerging as a new gaseous signaling molecule in the cardiovascular system. 4,5 Vascular endothelial cells express cystathionine ␥-lyase (CSE) and produce measurable amounts of this gasotransmitter. 6 Recent evidence suggests that H 2 S exhibits endothelium-derived relaxing factor activity. 6 In addition, it has been shown that muscarinic stimulation leads to CSE activation in the endothelium, triggering the conversion of L-cysteine to H 2 S and that CSE, like eNOS, is a calcium/calmodulindependent enzyme. Therefore, within the vascular wall, these 2 pathways coexist and serve a similar function. The relative amounts of NO versus H 2 S likely depend on the vascular bed studied 7 or on the state of the tissue, eg, healthy versus diseased. 5,8 Mice with targeted disruption of the CSE locus (CSE null mice) exhibit hypertension, similarly to what is observe...