Study On Signal Transduction Mechanism Of Small G Protein Rac1 Mediated By Hydrogen Sulfide In Promoting Angiogenesis

Hydrogen sulfide (H2S), well known as a toxic gas with the characteristic smell of rotten eggs, which is now considered as the third member of the gaseotransmitter family along with nitric oxide (NO) and carbon monoxide (CO). During recent years, endogenous H2S has been recognized as an important signaling molecule of the nervous system, the cardiovascular system, and the inflammatory system. In the nervous system, it is reported that physiological concentrations of H2S specifically potentiate the activity of the N-methyl-D-aspartate (NMDA) receptor, facilitate the induction of hippocampal long-term potentiation, and then take it effect in the physiological process of memory. In cardiovascular system, H2S can relax smooth muscle cells by activating ATP-dependent K+channels (KATP) and decrease the blood pressure. The deficit of H2S/CSE system is responsible for the development of spontaneous hypertension. H2S can also act as a cytoprotectant to protect myocardial cells from oxidative stress and anti-apoptosis by preserving the function of mitochondrial during ischemia-reperfusion. In inflammatory system, H2S has both pro-and anti-inflammatory effects depending on the different situation. Besides H2S has nociceptive effects, the regulatory function of insulin release, and is even involved in longevity.While in our previous study, we have provided the first piece of evidence regarding the proangiogenic effect of exogenous H2S, and our results also manifest that he proangiogenic effect is dependent on the phosphorylation of Akt in cultured RF6A endothelial cells. The proangiogenic effect of H2S was both evidenced by in vitro tube formation assay and in vivo matrigel plug model on mice. And then the proangiogenic effect of H2S was also demonstrated by Szabo C on chicken chorioallantoid membranes (CAM). Using the in vivo hindlimb ischemia model, we have also demonstrated that H2S is a proangiogenic factor, and the effects of H2S may be mediated by interaction between up-regulated VEGF in the skeletal muscle cells and the VEGFR2in the vascular endothelial cells. Later studies by Papapetropoulos et al indicate that endogenous H2S is a stimulator of angiogenesis and it promotes angiogenesis through a KATP channel/MAPK pathway. However, the pro-angiogenic effect of H2S and its concrete underlying mechanisms are still poorly understood. 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 of angiogenesis. In the present study, to study the mechanisms of H2S promoted angiogenesis, we mainly focused on the pro-migration effect of H2S on endothelial cells and its concrete molecular mechanisms.Firstly, the pro-angiogenic effect of H2S was explored. Exposure of HUVECs to50μM NaHS promoted cell migration rate with nearly2-fold increase in both scratch wounding assay and transwell boy den chamber assay. Furthermore,50μM NaHS significantly enhanced capillary-like structure formation of HUVECs cultured on reduced-growth factor matrigel. But the cell proliferation assay manifested that50μM NaHS significantly inhibited endothelial cell growth. All these results indicate that the pro-angiogenic effect of H2S is mainly dependent on the pro-migration effect of H2S.Secondly, the effects of H2S on actin cytoskeleton and paxillin-contained focal adhesions were studied. Following the prolongation of NaHS treatment period, the typical lamellipodia were markedly generated in the leading edge of the cell, and the characteristic membrane ruffles were apparently shaped. The paxillin-contained focal adhesion had no evident turnover transmission and redistribution change after NaHS treatment. In the western blot analysis, the phosphorylation levels of paxillin on the site Ser126and site Tyr118were not changed either. But remarkably, the phosphorylation of FAK on the site of Tyr576/577was significantly increased in a time-dependent manner following the treatment. These results demonstrate that H2S induce the actin cytoskeleton reorganization on HUVECs, which might mediate the pro-migration effect of H2S; and the phosphorylation of FAK might also play a role in the pro-migration effect of H2S.Thirdly, the effects of H2S on Rho GTPases were evidenced. Pull down assays and G-LISA assays were conducted and the results demonstrated that Racl was quickly activated after being treated with NaHS. But there was no observation that Rho A and Cdc42were activated by H2S in the time points we have chosen. To ascertain whether H2S directly interacts with Rac1, purified recombinant proteins Rac1were used. The results showing no significance between groups revealed that H2S did not interact with Racl in vitro cell free system. And this implicate that Rac1may be activated by upstream molecule which was probably directly activated by H2S and then in turn activated Racl. Fourthly, we studied whether activated Racl mediated the effects of H2S. Studies on actin cytoskeleton indicated the typical lamellipodia caused by H2S in the leading cell edge was prevented by the dominate negative Rac1expression. These results demonstrated that Rac1mediated the actin cytoskeleton reorganization induced by H2S. The fact that Racl mediated the pro-migration effect and the pro-angiogenic effect caused by H2S was also evidenced by RNA interference targeting Racl and transfection of dominate negative Rac1.Fifthly, the upstream and downstream signaling of Racl was explored. Inhibition of Racl by NSC23766significantly reduced NaHS induced Racl activation, while inhibition of VEGFR with SU5416also significantly blocked the activation of Racl. Similar results were obtained using specific inhibitors of PI3K (LY2924002). On the basis of these results, it was suggested that the Racl activation induced by H2S was regulated by VEGFR-PI3K signal transduction. While the observation that both of SU5416and LY2924002had no effect on the activation of ERK elevated by50μM NaHS signify that ERK acted independently of VEGFR-PI3K pathway. The result that the dominate negative Akt cannot inhibit the activated Racl by H2S indicated that the activation of Racl was not dependent on Akt. Transfecting with dominate negative plasmid of Rac1and RNA interference of Rac1both markedly inhibit the augmentation of phosphorylation on cofilin (Ser3) induced by50μM NaHS. These results demonstrated that cofilin acts as the effector of Racl and might directly mediate the remodeling of actin cytoskeleton triggered by H2S.Lastly, the expression of CSE and CBS on HUVECs and rat thoracic aorta was investigated. Using RT-PCR, both the fragments of CSE and that of CBS were detected on HUVECs, which suggested that HUVECs express CSE mRNA and CBS mRNA. The result that mRNA level of CBS on HUVECs was64fold that of CSE was manifested by Real-Time PCR. We also detected the protein expression level of CSE and CBS on HUVECs. The expression and distribution of CSE and CBS on thoracic aorta were detected by immunohistochemistry, and the results shows that both CSE and CBS are expressed on endothelial cell in tunica intima of artery.In conclusion, H2S activates Rac1and thus inactivates its downstream effector cofilin, which in turn influences actin dynamics by triggering lamellipodia formation. In addition, our data support that the VEGFR-PI3K is upstream pathway which regulates Rac1in H2S induced signaling pathway. These observations demonstrate that H2S activates Racl indirectly and ascribe a novel function to H2S in actin cytoskeleton regulation and cell migration. Induction of the VEGFR/PI3K/Racl/cofilin pathway by H2S contributes to the regulation of cell motility, which are processes required for angiogenesis induced by H2S. Moreover, ERK were also involved in action of H2S on cell migration but independently of Racl pathway. In cardiovascular system, both CSE and CBS are expressed and distributed on endothelial cell in tunica intima of artery.