The Role And Mechanism Of Pericytes VEGFR1 In Regulating Angiogenesis After Cerebral Infarction

Background and objective:Cerebral infarction refers to the ischemic necrosis of localized brain tissue caused by local blood supply disorder.Cerebral infarction is the most common type of cerebrovascular disease,with high morbidity,high recurrence rate,high mortality and high disability rate.Currently,anticoagulant,antiplatelet drug prophylaxis,thrombolysis and thrombolectomy in the acute phase are the main treatment methods,but there is still a lack of effective neuroprotective treatment methods.However,due to the short time window for thrombolysis and thrombolectomy,less than 10%of patients can benefit from this treatment,which brings a heavy burden to individuals,families and society.Therefore,it is meaningful to promote the recovery of nerve function in patients with cerebral infarction.Previous treatments for cerebral infarction have focused on neuroprotection,stem cell transplantation and nerve regeneration,but most of the therapeutic strategies have not made significant progress.Analysis of the causes indicated that the stem cells or newborn neurons in the infarcted area could not survive without sufficient energy and oxygen supply,suggesting that the good microcirculation is the prerequisite to provide oxygen and energy for the regeneration of neurons."Pro-angiogenesis therapy"aims to improve the microcirculation in the cerebral infarction area,provide a good microenvironment for neurogenesis and neuroplasticity,and achieve the goal of promoting the neurological function recovery of patients with cerebral infarction.This therapy is still in the exploratory stage at present,there is evidence that the new blood vessels after cerebral infarction play an important role in promoting the recovery of neurological function,but the precise mechanism of regulating the angiogenesis after cerebral infarction has not been fully clarified.Recently,"Pro-angiogenesis therapy"clinical trials for the treatment of cerebral infarction have used VEGF to promote angiogenesis,but it did not achieve satisfactory progress.Scholars have observed that although VEGF induced a large number of new blood vessels,it formed abnormal new blood vessels,which presented"mist".A large number of new blood vessels presented high permeability and a lack of coverage of pericytes.Therefore,in-depth study of the precise mechanism of angiogenesis after cerebral infarction is expected to provide new ideas for the treatment of cerebral infarction,with important clinical significance and social value.Pericytes are essential components of cerebral microvessels,covering the capillary wall and embedded in the basement membrane in direct contact with the vascular endothelium.Studies have shown that in the development of the central nervous system of mice,the absence of pericytes can lead to severe cerebrovascular malformations.In addition,pericytes can regulate the expression of tight junction protein of endothelial cells and the transport of vesicles,suggesting that pericytes play an indispensable role in the development and maturation of cerebral microvessels.In the process of brain microvascular development,endothelial cells sprout into tubes and secrete cytokines to act on pericytes and promote the recruitment of pericytes,which is an indispensable link in angiogenesis,However,what is the interaction between pericytes and endothelial cells in angiogenesis after cerebral infarction,and what signal pathways regulate their functions?Although some progress has been made,it has not been fully illustrated.VEGF is an important cytokine that regulates angiogenesis after cerebral infarction.After cerebral infarction,hypoxia induces increased VEGF expression in brain tissue,and activates VEGF/VEGFR2 signaling pathway of endothelial cells,promoting the proliferation and migration of endothelial cells.Studies have found that,in the process of angiogenesis,VEGFR1 of endothelial cells competitively binds VEGF to inhibit the VEGF/VEGFR2 signaling pathway of endothelial cells,thereby inhibiting the proliferation and migration of endothelial cells.However,the effect of pericyte VEGFR1 on angiogenesis remains unclear.Due to the role of selective splicing after transcription,VEGFR1 gene encodes membrane receptors and soluble receptors.Previous studies have suggested that cell membrane VEGFR1 plays a role in intracellular signal transduction,while soluble VEGFR1,as a secretory factor,plays an important role in intercellular communication.To this end,this study used induced knockout pericyte VEGFR1 mice to construct a cerebral infarction model,and combined with in vitro angiogenesis model,to explore the regulatory effect and mechanism of pericyte VEGFR1 in post-stroke angiogenesis.Method:Part?:Effect of pericyte VEGFR1 on angiogenesis after cerebral infarctionChanges of pericyte VEGFR1 expression after cerebral infarction were detected1.A cerebral infarction model was constructed.Mice were sacrificed 0,1,3,5,7,and14 days after infarction,respectively.qPCR was used to detect the mRNA expression of membrane VEGFR1 and soluble VEGFR1 in the infarcted tissues.2.Mice were sacrificed 7 days after cerebral infarction.The infarct area was harvested and PDGFR⁺cells and CD31⁺cells were sorted by flow cytometry.3.Changes in VEGFR1 expression 7 days after cerebral infarction were detected by immunofluorescence.To explore the effect of pericyte VEGFR1 on angiogenesis after cerebral infarction1.Induced knockout VEGFR1(Vegfr1^i?PC)mice was used to construct cerebral infarction model,and the cerebral infarction size was calculated 7,14 days after cerebral infarction;2.By immunofluorescence labeling CD31,the number and the length of new blood vessels was detected 7 days after cerebral infarction,and with the combination of biotinylated plant lectins perfusion,the new blood vessels function was detected.3.The fluorescent tracer molecule perfusion combined with immunofluorescence labeling NG2 were used to detect the vascular permeability and pericytes coverage after cerebral infarction.Part?:Pericyte VEGFR1 regulates the angiogenesis after cerebral infarctionTo study the effect of pericyte VEGFR1 on endothelial cell budding1.PDGFR⁺cells and CD31⁺cells in the peripheral area of cerebral infarction were sorted by flow cytometry 7 days after cerebral infarction,and the levels of VEGFR2 and Akt phosphorylation were detected by Western blot.2.VEGFR1 shRNA was used to interfere the human brain primary microvascular pericytes?HBPCs?VEGFR1 expression.microbeads were coated with parental human brain primary microvascular endothelial cells?HBECs?and then embedded in a gel matrix.HBPCs transfected with control shRNA?shCont-PCs?or shRNA were seeded on the top of the gel.Endothelial sprouting was quantified 5 days later,and the levels of VEGFR2 and Akt phosphorylation in HBECs were detected by Western blot.To investigate the regulatory effect of pericyte VEGFR1 on pericyte self-migration and recruitment1.Pericyte migration was evaluated by scratch assay and Tranwell chamber migration assay after.2.After stimulating HBPCs with PDGF,intracellular VEGFR1 was labeled with immunofluorescence,and Akt phosphorylation level in HBPCs was detected by Western blot.Results:Part?:1.The expression levels of membrane VEGFR1 mRNA and soluble VEGFR1 mRNA were significantly increased at 0,1,3,5,7 and 14 days after cerebral infarction in normal wild mice,and reached the highest level at 3 and 5 days after cerebral infarction,and then decreased.7 days after cerebral infarction,the expression levels of VEGFR1 on the cell membrane and soluble VEGFR1 in pericytes in the infarct area of brain tissue were significantly increased,but the changes were not obvious in the endothelial cells.2.After specific knockout of pericyte VEGFR1,the number of new blood vessels in mice after cerebral infarction was significantly reduced,the permeability of new blood vessels was significantly increased,the pericyte coverage rate was significantly reduced,and the pericyte morphology was abnormal and did not bind closely to endothelial cells.Part?:1.Pericytes inhibit the activation of VEGFR2 in endothelial cells by paracrine of soluble VEGFR1,thereby regulating endothelial cell sprouting.2.After interfering with the expression of VEGFR1 in pericytes,the migration ability of pericytes was significantly weakened,and the recruitment effect of endothelial cells on pericytes was also significantly weakened.In addition,Akt phosphorylation was significantly inhibited in pericytes.Conclusion:These studies suggest that pericyte VEGFR1 can promote angiogenesis and promote the structural and functional maturation of new blood vessels after cerebral infarction.In the process of angiogenesis after cerebral infarction,pericytes act on endothelial cells by paracrine of soluble VEGFR1 and regulate endothelial cells sprouting.Meanwhile,pericytes VEGFR1 promotes endothelial recruitment of pericytes by promoting intracellular phosphorylation of Akt.This study not only clarifies the new mechanism of pericyte regulation of angiogenesis after cerebral infarction,but also provides new ideas and targets for"Pro-angiogenesis therapy",which has important theoretical and practical significance.