Hydrogen Sulfide And Promote Angiogenesis Mechanism Research

Research on hydrogen sulfide (H2S) has already transformed it from a kind of toxic gas with a characteristic smell of rotten eggs to the third gaseous signaling molecule, like nitric oxide (NO) and carbon monoxide (CO). Mammalian cells can often produce H2S. The H2S concentration is about50μmol/L in human and rat serum and can be150μmol/L in the brain. Endogenous H2S is synthesized from L-cysteine mainly by two enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). CBS mainly exists in the nervous system, whereas CSE in the cardiovascular system. Endogenous H2S is involved in physiological regulation of nervous system, cardiovascular system, digestive system and inspiratory system.H2S dilates blood vessels, lower the blood pressure. In the heart, It has negative inotropic activity both in vitro and in vivo. H2S decreases the severity of ischemia/reperfusion-induced myocardial injuries. Chronic treatment with H2S prevented medial thickening of intramyocardial arterioles and perivascular fibrosis. Novel biological effects of this new gas molecule have recently been identified. H2S has been shown by this group as a stimulator of angiogenesis.Angiogenesis is a process of forming capillaries from pre-exsiting blood vessels, which plays an important role in a variety of physiological and pathological conditions, such as embryonic development, wound healing and tumor development. The signaling pathway of VEGFR-2(Vascular Endothelial Growth Factor Receptor-2), activated by VEGF (Vascular Endothelial Growth Factor), is the most important in angiogenesis and can promote survival、proliferation and migration of vascular endothelial cells.H2S has been shown for the first time by our group that it is a stimulator of angiogenesis and the proangiogenic effect is dependent on the phosphorylation of Akt. Till now, there is no other report about it.The proangiogenic effect of H2S was found by using RF/6A. This cell line spontaneously transformed at an early age and has been passaged over540times. Is H2S also a stimulator of primary human umbilical vein endothelial cells (HUVECs)? The target molecule of H2S has never been reported and it is challenging for us to find it.First, the effect of H2S on HUVECs and angiogenesis in vitro was explored. NaHS was used as donor of H2S, and HUVECs were used within passage6. Cell growth was examined with BrdU incorporation assay and we found that NaHS (10μmol/L) stimulated endothelial cell growth. The effects of H2S on cell migration and tube formation were also studied. NaHS (30-200μmol/L) promoted wound healing ability and tube formation.These data suggested H2S can promote angiogenesis in vitro and VEGFR-2/PI3K/Akt and VEGFR-2/FAK signaling pathways may be involved.We investigated the mechanism of angiogenic effect of H2S on HUVECs. Interestingly, the VEGFR inhibitor SU5416, the PI3K inhibitor LY294002and HIF-1inhibitor CdCl2(Cadmium Chloride) prevented H2S-induced cell proliferation, cell migration and tube formation. It suggested that VEGFR-2/PI3K/Akt, VEGFR-2/FAK signalling pathways and HIF-1might be involved in the effect. NaHS (50μmol/L) treatment induced a time dependent increase in VEGFR-2, PI3K, Akt and FAK phosphorylation and HIF-1α protein level in endothelial cells.VEGFR-2is usually activated by VEGF. However, western blot and ELISA results showed the expression and secretion of VEGF was not increased by H2S, and there was less than5pg/ml of VEGF in cell culture supernates, suggesting VEGF might not be involved in the proangiogenic effect of H2S.It seemed that H2S could activate VEGFR-2signalling pathway independent of VEGF. It is possible that molecules in this pathway be target proteins of H2S. We tried to find the target molecule using a reaction system with only NaHS (0-200μmol/L), kinase fusion protein(signaling molecules), substrate, buffer and ATP. Interestingly, NaHS (30-200μmol/L) directly increased the phosphorylation level and kinase activity of VEGFR-2. H2S could also directly elevate the activity of VEGFR-1, but had no effect on PI3K and Akt.We have found the target molecule of H2S——VEGFR. In order to investigate the interaction between H2S and VEGFR, molecular docking of H2S and VEGFR was processed using AutoDock software. We found that the most probable site for H2S located nearby the activation loop of the VEGFR-2, and the most probable site for HS lied in the activation loop, nearby the autophosphorylation sites (Y1054) of the receptor.In summary, the present study provides the first evidence that VEGFR-1and VEGFR-2are direct target molecules of H2S in its proangiogenic effect. The activation loop of the receptor might be interacted with H2S, leading to the increase of phosphorylation level and kinase activity of the receptor, resulting in the activation of VEGFR-2/PI3K/Akt and VEGFR-2/FAK signaling pathways.