Hydrogen Sulfide On The Endothelial Cell Migration And Ischemic Process Of Angiogenesis, The Role Of Research

Hydrogen sulfide(H₂S) is well known as a toxic gas with the characteristic smell of rotten eggs.But it is becoming increasingly clear that mammalian cells also produce H₂S.The H₂S concentration in rat serum is about 46μmol/L.Endogenous H₂S is synthesized naturally in the body from L-cysteine mainly by the activity of two enzymes,cystathionineβ-synthase(CBS) and cystathionineγ-1yase(CSE).CBS seems to be main HES-forming enzyme in the brain and nervous system,whereas CSE is the main H₂S-synthesizing enzyme in the cardiovascular system.It is reported that in cardiovascular system,HES can relax smooth muscle cells by activating ATP-dependent K⁺ channels(KATP) and decrease the blood pressure.The deficit of HES/CSE system may be responsible for the development of spontaneous hypertension.The administration of exogenous H₂S can effectively prevent myocardial ischemia injury.Since H₂S is a small molecule of gas,freely permeable to membrane,endogenously and enzymatically generated,has physiological functions,it is regarded as third gasotransmitters together with nitric oxide(NO) and carbon monoxide(CO).Angiogenesis,the formation of new blood vessels from pre-existing vessels, occurs during embryonic development and reparative processes.Persistent and uncontrolled angiogenesis is observed in tumour development.So regulation of angiogenesis is important for clinical appliance.Our laboratory has recently demonstrated that endothelial cells express CSE,physiologically relevant dosages of HES promote a highly robust angiogenic response that is largely dependent on activation of Akt.This paper reported the angiogenic effect of HES for the first time, and my role in this paper is focus on endothelial cell migraion.So we will discuss those findings already puhlished and some new obserbvations.Recent investigations also implied that HES can promote angiogenesis in mice matdgel plug model and chicken chorioallantoid membranes(CAM).That brings about the hypothesis of the present study,i.e.HES may regulate the process of angiogenesis in ischemic animal muscles and therefore improve blood supply to these regions.The purpose of the present study is to assess the role of HES in regulation of angiogenesis in ischemic conditions.First,various methods of endothelial cell migration were established and the influence of HES on endothelial cell migration was investigated from different aspects. Three kinds of models:scratch wound healing,transmembrane migration and time-lapse single cell migration were successively used and we observed that 10 and 20μmol/L NariS(H₂S donor) can promote scratch wound healing,transmembrane migration and time-lapse single cell migration ability.We also found that relatively low concentrations of NariS(1 and 10μmol/L) enhanced endothelial cell skeleton reorganazion and stress fiber formation using F-actin fluorescence staining method. Furthermore,we observed that KATP channel blocker Glibenclamide(10μmol/L) and CSE inhibitor DL-propargylglycine(PAG,10 mmol/L) both prevented H₂S-induced endothelial cell transmembrane migration and actin polymerization.These data suggested that H₂S stimulates endothelial cell migration by opening KATP channel. Endogenous H₂S participates in the regulation of endothelial cell migration process.Then we used rat unilateral hindlimb ischemia model to investigate the effect of H₂S on in vivo angiogenesis and blood supply recovery.The therapeutic effect of NaHS(10,50 and 200μmol/kg/d) and CSE plasmid injection was assessed 28 days after induction of ischemia by 1) regional blood flow analysis,2)histological study of hindlimb muscles,3) quantification of capillary density,and 4) iliac blood flow and maximal vasodilatory reserve.Both 50μmol/kg/d NariS and CSE plasmid injection improved residual muscle blood flow(1.073±0.153 and 1.089±0.178,respectively) compared with the control group(0.558±0.0524,p＜0.05) by a fluorescent microsphere assay.These results were confirmed by HE staining,showing relatively normal muscle fiber and capillary arrangement in rats treated with NariS and CSE plasmid.Capillary density count increased from 0.847±0.0365 CD31 positive cells/myofiber in untreated ischemic controls to 1.567±0.119 with 50μmol/kg/d Naris and 1.546±0.0953 with CSE plasmid injection(p＜0.01).Maximal vasodilatory reserve tended to increase from 0.313±0.0134 ml/min in untreated ischemic controls to 0.824±0.128 ml/min with 50μmol/kg/d Naris and 0.657±0.105 ml/min with C SE plasmid injection(p＜0.01 and p＜0.05,respectively).These data suggested that H₂S promotes in vivo angiogenesis in ischemic conditions and improve blood supply recovery.Furthermore,we studied three dimentional culture method for endothelial cells and cardiac explants,which provides series of in vitro models to mimic the in vivo conditions for further investigation.In summary,the present study provides the first evidence of H₂S on endothelial cell actin polymerization.H₂S can promote in vivo angiogenesis in ischemic conditions,which leads to blood supply recovery.