P23 The angiogenic and vasorelaxant effect of H2S require the endogenous production of NO, while the angiogenic and vasorelaxant effect of NO require the endogenous production of H2S

Abstract

Background Hydrogen sulfide (H2S) and nitric oxide (NO) are recognized as essential endogenous gaseous signaling molecules involved in a variety of homeostatic and disease processes. Recent studies suggest that multiple interactions exist between H2S and NO, and indicate that these interactions are crucial in regulating biological responses of vascular cells. However, the precise nature and mechanisms of the interactions between these two gasotransmitters remain incompletely understood. The goal of the current study was to investigate whether angiogenesis and vasodilation require the simultaneous production of H2S and NO. Methods Cultured murine bEnd.3 endothelial cells were used for the studies. CSE was silenced using siRNA. eNOS and PKG were inhibited by pharmacological inhibitors. H2S production was measured by the methylene blue method. Burn-induced wound healing was measured in rats by planimetry. Results Exposure of endothelial cells to a H2S donors (NaHS) increased intracellular cGMP in a NO-dependent manner and activated protein kinase G (PKG) as evidenced by phosphorylation of its downstream substrate, VASP. Inhibition of eNOS or PKG abolished the H2S-stimulated angiogenic response. Inhibition of eNOS also attenuated H2S-stimulated vasorelaxation. These findings demonstrate the requirement of NO in vascular H2S signaling, and the convergence of the vascular actions of H2S and NO to the cGMP/PKG pathway. Conversely, silencing of the H2S-producing enzyme cystathionine-gamma-lyase (CSE) abolished NO-stimulated cGMP accumulation and angiogenesis and attenuated the NO-induced vasorelaxation, indicating the requirement of H2S in the vascular actions of NO. Moreover, acetylcholine-induced, endothelium-dependent relaxations were attenuated after silencing of CSE. VEGF, a pro-angiogenic and vasorelaxant hormone is associated with the production of both NO and H2S: VEGF-induced angiogenic and vasorelaxant responses are attenuated either by the inhibition of NO or by the silencing of CSE. A key site of the cooperative interaction between NO and H2S involves the cGMP/PKG pathway: H2S exerted a potent inhibitory effect on phosphodiesterase 5 (PDE5) activity in vitro leading to cGMP elevation and PKG activation. Another site of the interaction between H2S and NO is Akt: H2S increased the phosphorylation (activation) of Akt, as well as the phosphorylation of eNOS at Ser1177. The in vivo relevance of the current findings is highlighted by the fact that H2S-induced wound healing in rats and angiogenesis in the matrigel plug model in mice, were suppressed by pharmacological inhibition (L-NAME) or genetic ablation of eNOS (eNOS−/− mice). Conclusions In conclusion, NO and H2S are mutually required for the control of angiogenesis and vasorelaxation.