The HIF-1?-dependent Neuroprotection Of Lyophilized Powder Of Catalpol And Puerarin On Ischemic Rat In The Aspects Of Apoptosis And Neovascularization

Background HIF-1?,a transcription factor widely expressed in penumbral areas in ischemic brain,is essential for cell survival and neovascularization.Altho?gh its significance in neovascularization is certain,if and how it regulates tip cells and EPCs in the aspect of neovascularization are still unknown.The lyophilized powder of catalpol and puerarin(CP)developed by our team is a promissing candidate drug for cerebral ischemia.It consists of catalpol and puerarin,the bioactive ingredients from Rehmannia glutinosa and Lobed Kudzuvine Root,respectively.CP has been shown promissing protection on cerebral ischemia including reduced neurological deficiency,infarct volume,and brain edema.As vessel is a key therapitc target for cerebral ischemia,vascular protection in the early stage and promoting neovascularization in the late stage of stroke would be beneficial for the recovery from the ischemic damage.Altho?gh our previous studies have showed the protection of CP on cerebral ischemia,the details of if and how it protecting vessels from ischemia and promoting post-ischemic neovascularization are still unknown.Objectives(1)To study the role of HIF-1? in tip cell formation and EPCs homing;(2)To study the neuroprotection of CP on cerebral ischemia;if and how HIF-1? contributes to the neuroprotection.Methods and Results1.The role of HIF-1? in post-ischemic neovascularization.Methods(1)MCAO rats were prepared by electrocoagulation.A successful MCAO rat was considered with a more than 80% reduction of r CBF in ischemic core and a score of m NSS between 3 and 8.(2)Lentivirus sh RNA was used to inhibit HIF-1?,and then neurological function,infarct volume,and r CBF were evaluated on the 14 th day after the MCAO.(3)On the 14 th day after the occlusion,the effect of loss of HIF-1? on vascular arcticture and vessel density were evaluated by staining endothelial cells via VIII,CD31,and CD105.(4)On the 7th day and 14 th day after the occlusion,the vascular branches and microvessels were measured by immunostaining.On the 7th day,tip cells were observed via immunostaining.(5)On the 7th day after thr occlusion,homing EPCs in ischemic brain was measured by CD34/Flk1,CD133/Flk1 immunostaining.Whether the loss of HIF-1? in bm EPCs or in ischemic brain influences the homing of exogenous bm EPCs was studied on the 7th day of occlusion;on the 10 th day,the incorporation of these exogenous bm EPCs into native vessel and the expression of Dll4 in exogenous bm EPCs were evaluated.Tube formations as well as expressions of Dll4,NRP1,Flk1,VEGF were observed in bm EPCs under hypoxia with control sh RNA or HIF-1? sh RNA.(6)The proteins relative to vascular sprouting and EPC homing and the number of reactive astrocytes were evaluated on the 7th day of occlusion.Additionally,the regulative role of HIF-1? in the expressions of HMGB1,CXCL12 in astrocytes and CXCR4 in bm EPCs were studied under hypoxia.Results(1)Loss-of-HIF-1? resulted in deterioration of ischemic damage.The loss of HIF-1? in ischemic brain from 5th day to 14 th day resulted in impaired recovery of neurological function,enlarged infarct volume,and reduced r CBF in peripheral area of ischemic core.(2)Loss-of-HIF-1? impaired post-ischemic neovascularization.Loss-of-HIF-1? resulted in deficient sprouting angiogenesis and vasculogenesis.HIF-1? was successfully inhibited by sh RNA from the 5th-14 th day after MCAO.Simultaneously,vascular arcticture and vessel density in peripheral area of infarct in cortex and SVZ were impaired and decreased.(3)Loss-of-HIF-1? impaired sprouting angiogenesis.On the 7th day and the 14 th day,knocking down HIF-1? respectively reduced the numbers of vascular branches and microvessels(diameter below 20 mm)in ischemic brain.On the 7th day after occlusion,knocking down HIF-1? resulted in the decrease in the tip cells identified by Dll4/VIII,Flk1/Dll4 or NRP1/Dll4.(4)Loss-of-HIF-1? impaired the EPC-dependent angiogeneisi.On the 7th day,homing EPCs(CD34/Flk1,CD133/Flk1)in ischemic brain were decreased as well.Additionally,loss-of-HIF-1? in exogenous EPCs or in ischemic brain resulted in decreases of homing,incorporated EPCs into the local vessels,and Dll4 expression in ischemic brain.Knocking down HIF-1? in primary bm EPCs decreased the tube formation and spheroid sprouts.Simultaneously,Loss-of-HIF-1? decreased expressions of VEGF,Dll4,NRP1,and Flk1 under hypoxia conditions.(5)Loss-of-HIF-1? impaired the micro-environment of EPCs homing and vascular sprouting.Knocking down HIF-1? in ischemic brain reduced the expressions of CXCL12/CXCR4 and HMGB1 in ischemic brain on the 7th day after MCAO.In vitro,deleting HIF-1? decreased the expressions of HMGB1 and CXCL12 in astrocytes and CXCR4 in bm EPCs.Knocking down HIF-1? impaired the micro-environment of vascular sprouting,including the decreased Flk1/VEGF-A positive cells and the activation of VEGF-A/ERK pathway in ischemic rat brains(p?0.05).2.The protection of CP on vascular endothelial cells damaged by ischemia and HIF-1?-dependent mechanism2.1 Estabolishment of penumbra model in vitroMethods(1)Cultured vascular endothelial cells from brian were identified using scanning electron microscope(SEM),transition electron microscope(TEM)to observe cell morphology and tight junction.Potential contaminated cell type was identified by immunostaining.TEER and tube formation were observed on the cultured cells.(2)In vitro penumbral model consisted of OGD+LON: after damaged by OGD,ECs were cultured in hypoxia(2% O2)and inadequate nutrition culture medium for 24 hours(F12/DMEM medium containing 20% FBS was diluted to four times of volume with deionized water).Results(1)The identity of vascular endothelial cells.The SEM,TEM,immunostaing of vascular EC markers and function tests identified the vascular endothelial characterizations of the cultured cells.And the endothelial purity was more than 99%.(2)OGD+LON recapitulates the feature of penumbra in vivo.Cell survival was decreased to 70% after BMECs was damaged by OGD for 20 hours.Then,cell survival was maintained about 70% while cells were cultured in LON for 12 hours.After cultured for 16 hours,cell survival decreased shapely,maintained around 60%.Then,cell survival decreased to 28% after cultured for 28 hours.This data s?ggested that OGD-damaged cells could live for a limited time,which is similar to the penumbral situation in vivo.2.2 The HIF-1?-dependent anti-apoptosis of CP on vascular endothelial cells damaged by ischemia in vivo and in vitroMethods(1)MCAO preparation is same to section 1.(2)Animal grouping: Sham,MCAO,CP 65.4 mg/kg,CP 32.7 mg/kg,and CP 16.4 mg/kg.r CBF and m NSS of MCAO rats in groups were analyzed to check if there was a statistical difference among groups.(3)Electrocoagulation-prepared MCAO rats were used.14 days after CP infusion(65.4 mg/kg,32.7 mg/kg,16.4 mg/kg),neurological function,infarct volume,and r CBF were detected.(4)On the 4 days after occlusion,improvement of CP on vascular morphology and endothelial apoptosis at doses of 65.4 mg/kg,32.7 mg/kg,16.4 mg/kg were observed.In vivo,the protection of CP on BMECs at doses of 24.5 ?g/m L,49 ?g/m L,and 98 ?g/m L were investigated under OGD+LON.(5)After 7 days of CP administration at doses of 65.4 mg/kg,32.7 mg/kg,16.4 mg/kg,the HIF-1? level in ischemic brains were detected;on the 1st day,7th day,and 14 th day,the HIF-1? level was assayed in groups.In vitro,the effect of CP on HIF-1? expression and nucleus enrichment were observed in BMECs in OGD+LON model.Further,HIF-1? knockdown BMECs were produced by sh RNA,and then the endothelial protection and anti-apoptosis of CP on HIF-1?-knockdown BMECs were evaluated.(6)Using pathway inhibitors,the involvement of ERK and m TOR in protection of CP on BMECs and regulation on HIF-1? were studied in OGD+LON model.Additionally,how CP activates m TOR was studied.Results(1)CP protecting brain from ischemia is relative to HIF-1?.CP at dose of 65.4mg/kg significantly reduced infarct volume on the 14 days after MCAO,and it increased r CBF at doses of 65.4mg/kg and 32.7mg/kg simultaneously.(2)CP protected BMECs from ischemia and anti-apoptosis.CP improved vascular morphology at doses of 65.4mg/kg,32.7 mg/kg,16.4 mg/kg.Among tested doses,the doses of 65.4 mg/kg and 32.7 mg/kg significantly reduced apoptotic BMECs(p?0.05,p?0.01).CP improved the survival ration of BMECs in both the penumbral model and the OGD model in vitro at doses of 98 ?g/m L,49 ?g/m L,and 24.5 ?g/m L(p?0.05,p?0.01).Simultaneously,it protected BMECs from apoptosis,which was relative to P53-dependent Cyt-C/Caspase-3 apoptotic pathway.(3)HIF-1? involved in CP protecting BMECs from apoptosis.CP increased HIF-1? expression in ischemic brain at doses of 65.4mg/kg and 32.7mg/kg on the 7th day after occlusion.Additionally,CP at dose of 65.4mg/kg increased HIF-1? in the brain of MCAO rat on the 7th day and the 14 th day.In vitro,CP at dose of 49 ?g/m L increased HIF-1? expression and nucleus location in the in vitro penumbral model(p?0.05,p?0.01).Further,loss-of-HIF-1? impaired the protection of CP on BMECs,including cell survival,apoptosis,and the release of Cyt-C from mitochondria.(4)ERK and PI3K/AKT/m TOR involved in CP increasing HIF-1? and protecting BMECs.In vitro,CP at doses of 65.4mg/kg and 32.7 mg/kg increased p AKT,p S6 K,and p ERK in ischemic brain on 7th day after MCAO.CP at dose of 49 ?g/m L increased p ERK,p S6 K,and p4 EBP in penumbral model,indicating the activation of ERK and m TOR by CP.Further,PD98059 and rapamycin the pathway inhibitors of ERK and m TOR reduced the enhancement of HIF-1?,cell survival ration,and anti-apoptosis induced by CP.Additionally,inhibiting ERK by PD98059 effected little on CP-activated m TOR,and inhibiting PI3K/AKT by LY249002 significantly repressed the activation of m TOR induced by CP.3.The HIF-1?-dependent promotion of CP on post-ischemic neovascularizationMethods(1)MCAO preparation was same to section 1.(2)Animal grouping was same to section 2.(3)On the 14 th day after occlusion,the promotion of CP at doses of 65.4 mg/kg,32.7 mg/kg,16.4 mg/kg on vessel density,the number of Brd U/VIII positive cells,VEGF and Ang2 expression were evaluated and tested on in ischemic brains.(4)The effect of CP at doses of 65.4 mg/kg,32.7 mg/kg,16.4 mg/kg on tip cell(Dll4/VIII)formation and EPC(CD133/Flk1)homing were tested by immunofluorescent stain on the 7th day of occlusion.Simultaneously,the VEGF positive cells and HMGB1 expression were assayed as well.(5)In intro,the effects of CP at dose of 49 ?g/m L on endothelial tube formation,migration,and proliferation were studied by LON.Further,the role of HIF-1? in CP promoting endothelial tube formation,migration,and proliferation was studied by sh RNA for inhibiting HIF-1? expression.Results(1)CP promoted post-ischemic neovascularization.CP increased the vessel density in peripheral area of infarct in cortex on the 14 th day of MCAO at doses of 65.4mg/kg and 32.7mg/kg(p?0.05,p?0.01).On the 7th day,CP increased Brd U+/VIII dual positive cells as well as enhanced the expressions of VEGF-A and Ang-2 in brain tissue.(2)CP stimulated the formation of tip cells and promoted the homing of EPCs to ischemic brain.On the 7th day,CP increased the number of tip cells which was labeled by Dll4/VIII at doses of 65.4mg/kg and 32.7mg/kg;simultaneously,CP at dose of 65.4mg/kg increased the homing EPCs which were marked by CD133/Flk1 and the expression of HMGB1 in ischemic brain.(3)CP promoting angiogenesis was relative to HIF-1?.In vitro,CP at dose of 49?g/m L promoted tube formation,migration,and proliferation(tested by Brd U)in endothelial cells under low oxygen and nutrient situation.Simultaneously,it enhanced expressions of VEGF-A and Ang-2.Loss-of-HIF-1? in endothelial cells impaired the promotion of CP on endothelial tube formation and migration in vitro,but had no effect on endothelial proliferation(p?0.05,p?0.01).Conclusions 1.Loss-of-HIF-1? impaired the neovascularization environment in the ischemic brian,resulting the deficient tip cell formation and EPC homing.2.CP protected BMECs from ischemic damage in vivo and in vitro.And its anti-apoptosis was dependent on PI3K/AKT-m TOR/HIF-1?.3.CP promoted post-ischemic neovascularization,tip cell formation,and EPCs homing,which was relative to HIF-1?.