Study On Angiogenesis Mechanism Of Hydrogen Sulfide

Hydrogen sulfide (H2S), well known as a toxic gas with the characteristic smell of rotten eggs, has been newly understanded from1990s. In1989, scientists detected the endogenous production of H2S in rat and human brain, suggesting that H2S may have some physiological role. In recent years, it is becoming increasingly clear that mammalian cells also produce H2S. The H2S concentration in rat serum is about46μmol/L, while it reaches100μmol/L in their brain. Endogenous H2S is generated naturally in the body from L-cysteine mainly by the activity of two enzymes, cystathionine y-lyase (CSE) and cystathionine β-synthase (CBS). CSE is the main H2S-synthesizing enzyme in the cardiovascular system, whereas CBS seems to be main H2S-forming enzyme in the nervous system. Endogenous H2S is involved in physiological regulation of cardiovascular system, nervous system, digestive system and respiratory system. In addition, H2S is a small molecule of gas, freely permeable to membrane, so in recent years, it is regarded as the third gasotransmitters together with nitric oxide (NO) and carbon monoxide (CO).In cardiovascular system, H2S can relax smooth muscle cells by activating ATP-dependent K+channels (KATp) and decrease the blood pressure. H2S can inhibit the abnormal proliferation of smooth muscle cells and induce apoptosis, to ease the vascular structural remodeling. Additionally, it has negative inotropic activity and decreases the severity of ischemic/reperfusion-induced myocardial injuries. In2007, our group provided the first evidence showing the proangiogenic effect of H2S, and the effect was both evidenced by in vitro tube formation assay on RF/6A and in vivo matrigel plug model on mic. In2009, using the hindlimb ischemia model, we also demonstrated that H2S is a proangiogenic factor. And the proangiogenic effect of H2S was also demonstrated by Szabo C on chicken chorioallantoid membranes (CAM).Angiogenesis is the process of new cappilaries growing from the original blood vessels, including extracellular matrix degradation, endothelial cell proliferation, migration, the formation of arteries. It can occur in embryonic development, wound healing, tumorigenesis and metastasis, diabetes, and other physiological, pathological processes. A variety of growth factors regulate the process of angiogenesis, and the signal pathway of Vascular Endothelial Growth Factor-2(VEGFR-2), activated by Vascular Endothelial Growth Factor(VEGF), is the most important in angiogenesis and can promote survival,proliferation and migration of vascular endothelial cells.The process of angiogenesis is closely related with vascular endothelial cell proliferation and cell migration. The migration of endothelial cells plays an important role in the process. In our previous research, we observed that H2S also had a proangiogenic effect on human umbilical vein endothelial cells (HUVECs). NaSH (lOμmol/L) stimulated endothelial cell growth. NaSH (30-200μmol/L) promoted wound healing ability and tube formation. These effects were not depended on the increase of VEGF expression, but VEGFR-2/PI3K/Akt was involved. In addition, H2S could directly elevate the activity of VEGFR-2, but had no effect on PI3K/Akt. This suggests that the target molecule of H2S in endothelial cells may be VEGFR-2. Therefore, at present, to study the mechanisms of H2S promoted angiogenesis, we mainly focused on how H2S can directly activate VEGFR-2.Firstly, we further studied the role of H2S on VEGFR-2. There are many tyrosine sites in VEGFR-2, including Tyr951, Tyr996, Tyr1054, Tyr1059, Tyr1175and Tyr1214. Results showed that VEGF(10ng/ml) can prominently increase the phosphorylation of all these tyrosine sites, but NaHS(50μmol/L) can only increase Tyr1175phosphorylation. This indicates that the mechanisms of VEGFR-2activation by hydrogen sulfide might be quite different from that of VEGF. The siRNA for VEGFR2caused a partial knockdown in protein expression of VEGFR2in vascular endothelial cells, and a significant decrease in H2S-induced cell migration in the VEGFR2siRNA cells was observed as compared to the cells transfected with nonsense siRNA. This further indicates that VEGFR-2plays a vital role in the process of H2S promoting endothelial cell migration. In addition, the migration-promoting effect of H2S was blunted by the neutralizing antibodies administered24h before. However, this effect was not blocked by the VEGF neutralizing antibodies administered0.5h before. This indicates that in the vascular endothelial cells, the H2S effects were dependent on basal VEGF.Secondly, ESI-CID-MS-MS analysis of VEGFR-2revealed a novel S-S bond located between Cys1045and Cys1024within its structure, which can be be broken by treatment with H2S. And we have not found any other forms of modification. ESI-MS analysis of major functional peptides in VEGFR-2, hexapeptide, basic animo acids and cystine showed that NaSH can break the S-S bond in hexapeptide and cystine, but exerts no modification on any of the functional peptides or any of the basic animo acids. These results confirmed that breaking of the disulfide bond is the only chemical modification induced by hydrogen sulfide. Kinase activity of the mutant VEGFR-2(C1045A) devoid of the Cys1045-Cys1024disulfide bond was significantly higher than wild type VEGFR-2, indicating that the Cys1045-Cys1024disulfide complex functions as a novel intrinsic inhibitory motif. Further wound healing experiments showed that transfection with vectors expressing VEGFR-2(C1045A) caused a significant increase in cell migration. However, though H2S still exerted a migration-promoting effect in the cells transfected with control vectors, this H2S effect was inhibited in the cells transfected with VEGFR-2(C1045A). These data not only indicate that the Cys1045-Cys1024disulfide bond serves as an intrinsic inhibitory motif, but also demonstrate that the Cys1045-Cys1024disulfide complex functions as a target structure in VEGFR-2to mediate H2S-induced cell migration.Additionally, intracellular reactive oxygen species (ROS) were identified in both migrating and motionless vascular endothelial cells. Quantification of ROS showed that total intracellular ROS levels were significantly increased in migrating cells as compared with that of motionless cells. Immunofluorescent double staining showed that ROS were colocalized with VEGFR-2in migrating vascular endothelial cells. This indicates that VEGFR2is subjected to a transient intracellular oxidizing environment during cell migration.Thirdly, we explored the mechanism of H2S interrupting disulfide bonds. Ascorbic acid, a potent reducing agent, was not able to break the S-S bond at a concentration that showed equal reducing potency as H2S, suggesting that the mechanism underlying H2S induced S-S bond breakage is not solely due to its reducing nature. H2S in solution is composed of a mixture of H2S gas and the HS-anion that are in a dynamic equilibrium. This equilibrium is pH sensitive with acidification reducing the concentration of the HS-anion and increasing that of H2S gas. Cleavage of the S-S bond occurred at pH values≥7.0, while that at pH values≤5.5it was abolished. This finding suggesting that the S-S bond breaking ability is largely due to the HS-anion and not H2S gas. In the study of the breakout of S-S bond, we found Cys-SSH was transiently appeared, which provides some information for us to further study the mechanism of the breakout of S-S bond by NaSH.Lastly, the expression of CSE and CBS on HUVECs was investigated. Both CSE and CBS were present in vascular endothelial cells. CSE was mainly localized to subcellular areas near cell membrane, while CBS was distributed within the cytoplasm. Interestingly, double staining showed that both CSE and CBS were colocalized with VEGFR-2in vascular endothelial cells, suggesting that endogenous H2S might be generated in these cells where VEGFR-2resides.In conclusion, the present study provides first piece of evidence about the "receptor" of H2S and reveals a new intrinsic inhibitory S-S bond in VEGFR2that serves as a target motif for H2S-induced modification and factional regulation. The S-S bond breaking ability is largely due to the HS-anion and not H2S gas. The HS-anion acts as a nucleophile during the breakng of the S-S bond.