VEGFR₂ Mediates H₂S Relaxing Cerebrovascular Effects In Rats And Its Mechanism

Hydrogen sulfide,a chemical compound with the formula H₂S,is regarded as a pollutant and hazardous toxic gas for a long time.As a third member of gasotransmitter family along with carbon monoxide?CO?and nitric oxide?NO?,H₂S exerts a wide variety of molecular and cellular actions in our body.Previous studies have shown that H₂S could promote the relaxation of vascular smooth muscles and H₂S-induced vascular relaxation and the other smooth muscle preparations might contribute to its relaxation of the smooth muscle cells.Endogenous H₂S in vasculature is primarily produced by cystathionine-?-lyase?CSE?,and regarded as a prominent endothelium-derived relaxation factor.The experimental model of hypertension in mice and deficiency in endothelium-dependent vasorelaxation could be made via knocking out of CSE.H₂S has been shown to function as an EDHF through activation of potassium ion channels,The ion channel targets of H₂S appear to vary according to tissue and cells,such as activating large conductance calcium-activated potassium(BK_Ca)intermediate conductance calcium-activated potassium(IK_Ca),and small conductance calcium-activated potassium(SK_Ca)channels in endothelial cells,K_ATPchannels in mesenteric arteries and cerebral arterioles;and BK_Cachannels in mesenteric arteries and cerebral arterioles.Our previous studies have demonstrated that endothelium-derived H₂S mediated the hyperpolarization and dilation of rat cerebral arteries including the basilar artery?BA?and the middle cerebral artery?MCA?.Endogenous H₂S has been found to act as endothelium-derived hyperpolarizing factor?EDHF?in cerebral arteries Although H₂S has been reported to be involved in various physiological and pathological processes,little is known about the possible molecular target of H₂S in its actions.Hence,we tested the hypothesis in present research,whether mammalian cells and tissues express a �receptor�for H₂S.VEGFR₂is one of tyrosine kinase receptors,mediates most of the biological effects of VEGF in endothelial cells,such as permeability,proliferation,invasion and migration Besides the presence in vascular endothelial cells,the VEGFR₂also has been found in hematopoietic stem cells and smooth muscle cell,In vascular endothelial cells,VEGFR₂has been reported to be directly activated by H₂S suggesting that VEGFR₂could act as a direct target molecule for H₂S.Furthermore,it has been pointed out that VEGFR₂-mediated vascular dilation is the mechanism of VEGF-induced mobilization in anemia and bone marrow cell.All of above findings lead us to hypothesize that VEGFR₂may be involved in H₂S-mediated vasodilation,or VEGFR₂can act as a direct receptor to mediate H₂S to exert vascular relaxation.Hence,in present study,we sought to characterize the�receptor�of H₂S and investigate how H₂S interacts with its receptor in vascular smooth muscle cells.Aims:This study was undertaken to demonstrate the mechanism of cerebrovascular dilation of hydrogen sulfide?H₂S?and further explore the potential�receptor�of hydrogen sulfide?H₂S?in H₂S-induced cerebrovascular relaxation.Materials and methods:The relaxation effect of H₂S on rat basilar artery?BA?was measured by in vitro measurement of microvascular pressure-diameter.The vascular endothelial growth factor receptor-2 inhibitor SU5416,vascular endothelial growth factor?VEGF?,VEGFR₂knockdown at the whole-body and cellular levels were used to evaluate whether VEGFR₂participates in H₂S-mediated cerebrovascular relaxation.The changes of intracellular calcium ion concentration([Ca²⁺]_i)were detected to assess the underlying mechanism of H₂S-induced cerebral vasodilation via VEGFR₂.Results:1. Effect of VEGFR₂inhibitor SU5416 on H₂S-mediated vasodilation of BA.Na HS,exogenous H₂S donor and VEGFR₂inhibitor SU5416 were used to investigate the role of VEGFR₂on H₂S-mediate BA vasodilation.As shown in Fig.1,10^-6?10^-3mol/L Na HS induced a concentration-dependent relaxation in the isolated rat BA.But the dilation of BA to Na HS was markedly attenuated by pretreatment with 10^-5mol/L SU5416,a relatively selective inhibitor of Flk-1?VEGFR₂?,with the Emax being reduced from 119%�13.7%to 88.0%�12.7%when the VEGFR₂inhibitor was added prior to vasoconstrictor.The results suggested that blocking the VEGFR₂could decrease the H₂S-induced relaxation of rat BA.2. Role of VEGF on H₂S-mediated vasodilation of BA Compared with the vehicle group,VEGFR₂agonist VEGF(10^-9-10^-5mmol/L)caused concentration-dependent BA relaxation with Emax of 29.6%�10.1%.Fig.2B showed that the relaxation of BAs is profoundly increased to 94.7%�6.0%by co-application of the VEGF and Na HS,which is significantly more than VEGF but not Na HS alone.These findings indicated that the vasodilation of VEGF and H₂S may be related to the same target.3. Identification of VEGFR₂on vascular smooth muscle cells?VSMCs?.Primary cultured smooth muscle cells from SD rat were used to identify the VEGFR₂expression on VSMCs.We can observe the scattered cells around the tissue block climbing out into a spindle shape or a long spindle shape after about 4 days under the microscope.After two weeks,the cells grow more and show a"peak"and a"valley"growth.SMCs were identified by immunocytochemical staining with anti-?-actin antibody,green fluorescence of long spindle-shaped was observed in the cytoplasm,indicating that the primary cultured SMCs was successful.Subsequently,the VEGFR₂expression on SMCs was identified using immunochemical with anti-VEGFR₂antibody.As shown in Fig 3C VEGFR₂was found to express on smooth muscle cells,which provided a basis for studying whether VEGFR₂participates in H₂S-mediated cerebrovascular relaxation.4. Effect of VEGFR₂knockdown on H₂S-mediated relaxation of smooth muscle cells.The expression of VEGFR₂protein in VSMCs was detected using Western bolt method.In comparison with the control group,the expression of VEGFR₂protein was dramatically down-regulated by using VEGFR₂-si RNA1?p<0.05?.Hence,VEGFR-si RNA1 was selected as a valid interference sequence in VSMCs?*P<0.05 vs Blank group?to evaluate the effect of VEGFR₂knockdown on H₂S-mediated relaxation of VSMCs.the relaxation of the VSMCs after VEGFR₂knockdown was observed by giving a gradient concentration of exogenous H₂S donor,Na HS(10^-7-10^-3mol/L),and the results showed that Na HS(10^-5-10^-3mol/L)significantly caused relaxation of untreated VSMCs compared to the control group,while in the VEGFR₂knockdown group,the relaxation of VSMCs to Na HS was remarkably inhibited.5. Effect of H₂S on the changes of dilation of BA VEGFR₂knockdown rat.After 48 hours of transfection of VEGFR₂-si RNA1 in rats,the expression of VEGFR₂in rat basilar artery was measured by western blot.compared with the control group,there was no significant change in VEGFR₂in the negative si RNA group and the transfection reagent group,but the VEGFR₂expression in si RNA1 transfection group was significantly decreased by about 60%.These results indicated that transfection of VEGFR₂-si RNA1 in vivo can effectively reduce the expression of VEGFR₂in cerebral blood vessels.Na HS-induced BA relaxation from VEGFR₂knockdown rat significantly attenuated,These data further confirmed that VEGFR₂is involved in H₂S-mediated cerebrovascular dilation.6. Effect of H₂S on the concentration of calcium in VSMCs via VEGFR₂.Next,we investigated the effect of H₂S on intracellular calcium concentration in VSMCs via VEGFR₂.We measured the base ratio as 1.9�0.5,and a significantly increase of[Ca²⁺]_iwhen u46619(10^-7mol/L)was given.On the contrary,[Ca²⁺]_idecreased when Na HS(10^-4mol/L)was given.Compared with the control group,[Ca²⁺]_iwas reduced significantly when the smooth muscle cells were treated with Na HS(10^-4mol/L)before pre-incubation of SU5416(10^-5mol/L).Likewise,we followed the same approach to assess the effect of co-application of VEGF?10ng/ml?and Na HS on[Ca²⁺]_iin VSMCs,not surprisingly,we also recorded the decrease of[Ca²⁺]_iin VSMCs.Conclusion:These findings revealed that the mechanism of H₂S-induced cerebral vasodilation may be related to activating VEGFR₂and then reducing[Ca²⁺]i,and provided first piece of evidence of H₂S-targeting receptor protein kinase in H₂S-induced cerebrovascular relaxation.