Study On The Role And Mechanism Of PRMT4 In Post-ischemic Angiogenesis

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Expression and localization of PRMT4 in post-ischemic angiogenesisBackground: Blood vessels are responsible for the delivery of oxygen and nutrition to the whole body.During local tissue ischemia,angiogenesis and reconstruction of vasculature are very important for the recovery of local tissue blood supply.As an arginine methyltransferase,many studies have shown that PRMT4 acts as a transcriptional co-activator to regulate the occurrence and development of diseases by interacting with its substrates.However,the role of PRMT4 in post-ischemic angiogenesis is still unclear.This study mainly discusses whether PRMT4 is involved in post-ischemic angiogenesis.Methods: The expression of PRMT4 was detected by western blot and immunohistochemistry.Then the co-localization of CD31,VE-cadherin,CD45 and Myosin with PRMT4 was observed by tissue immunofluorescence staining,and the expression of PRMT4 in endothelial cells and non-endothelial cells was detected by CD31 magnetic beads.Finally,HUVECs were isolated to detect whether the expression of PRMT4 was affected by VEGF stimulation.Results: In the hindlimb ischemia model,the protein expression level of PRMT4 increased significantly and reached the peak on the 7th day,which was positively correlated with the change of VEGF.Immunohistochemistry showed that PRMT4 increased remarkably in ischemic tissues,and mainly in interstitial cells.Immunofluorescence showed that PRMT4 was mainly co-localized with endothelial cells,but less expressed in skeletal muscle cells and immune cells.VEGF stimulated HUVECs in vitro showed an increase in PRMT4 protein level,but no significant change in PRMT4 m RNA level.Conclusion: PRMT4 is highly expressed in ischemic diseases,mainly located in vascular endothelial cells,and VEGF can induce the increase of VEGF protein level,suggesting that PRMT4 may be involved in post-ischemic angiogenesis.Part ? Role of PRMT4 in post-ischemic angiogenesisBackground: Post-ischemic angiogenesis is mainly caused by VEGF produced after ischemia and hypoxia inducing endothelial cell proliferation,migration and tube formation,forming a new capillary network,and then improving the blood supply of ischemic area.In the previous part,we proved that PRMT4 is involved in the process of post-ischemic angiogenesis,but the role of PRMT4 in angiogenesis is still unclear.In this part,we mainly study the role of PRMT4 in post-ischemic angiogenesis.Methods: In vivo,we used adenovirus local intramuscular injection to mediate the overexpression and knockdown of PRMT4 in local tissue,and established hindlimb ischemia model at the same time,and then studied the effects of PRMT4 overexpression and knockdown on hindlimb blood flow recovery and capillary density after HLI;in vitro,we infected HUVECs with adenovirus mediated PRMT4 overexpression or knockdown to detect whether PRMT4 influenced proliferation(Ed U staining),migration(transwell test)and tube formation(tube formation test)of endothelial cells.Results: In vivo,overexpression of PRMT4 could promote the recovery of blood perfusion and increased the capillary density,while knockdown of PRMT4 could inhibit the recovery of blood perfusion and decreased the capillary density.In vitro,overexpression of PRMT4 in endothelial cells could significantly promote the proliferation,migration and tube formation,while knockdown result in the opposite phenotype.Conclusion: PRMT4 plays a positive role in the recovery of blood perfusion after hindlimb ischemia,and promotes post-ischemic angiogenesis by promoting endothelial cell proliferation,migration and tube formation.Part ? Mechanism of PRMT4 in post-ischemic angiogenesisBackground: PRMT4 is usually involved in gene regulation as a transcription co-activator.The VEGF-VEGFR2 signaling pathway is a very important signaling pathway in the process of angiogenesis,which is regulated by multiple levels,including ligand receptor expression level,dephosphorylation of protein tyrosine phosphatases(PTPs),receptor endocytosis rate and signal pathway crosstalk.Our in vivo and in vitro experiments have proved that PRMT4 plays a positive role in post-ischemic angiogenesis,but how PRMT4 affects angiogenesis is unclear.In this part,we will explore the specific molecular mechanism of PRMT4 on angiogenesis.Methods: First,we overexpressed or knocked down PRMT4 in HUVECs to detect the expression level of VEGF.Then we detected whether the VEGF-VEGFR2 signaling pathway was activated by detecting the phosphorylation level of VEGFR2 and ERK after VEGF stimulation for different time while overexpressing PRMT4 or knockdown PRMT4.Next,we confirmed whether knockdown VEGF could reverse the effect of PRMT4 on proliferation,migration and tube formation of HUVECs.In addition,we screened PTPN1,PTPN2,Ephrin B2,VE-cadherin,NRP1,Notch and NICD which affect the VEGF-VEGFR2 signaling pathway to explore whether they may affect this signaling pathway from these aspects.At the same time,we considered whether PRMT4 affects the expression of VEGF from the transcriptional level,so we searched for its interacting protein through co-IP,observed the intracellular localization of PRMT4 and the interacting protein by immunofluorescence,confirmed the effect of PRMT4 on the transcriptional activity of VEGF promoter by luciferase reporter gene while intervening the interacting protein,and finally confirmed whether intervening the interacting protein can reverse phenotypic changes in HUVECs that mediated by PRMT4.Results: Overexpression or knockdown of PRMT4 in HUVECs can up-regulate or down-regulate VEGF m RNA and protein levels,subsequently activate or inhibit VEGF-VEGFR2 signaling pathway;knockdown of VEGF can eliminate PRMT4 mediated endothelial cell proliferation,migration and tube formation phenotype;in addition,overexpression of PRMT4 can enhance Ephrin B2 and inhibit NICD protein levels.Co-IP showed that PRMT4 interacted with YB1,and both co-located in endothelial cells.Knockdown of YB1 could inhibit the effect of PRMT4 on the transcription activity of VEGF promoter.Finally,cell experiment showed that knockdown of YB1 could eliminate the effect of PRMT4 on endothelial cell proliferation,migration and tube formation.Conclusion: On the one hand,PRMT4 activates VEGF expression through YB1 transcription,on the other hand,it may activate VEGF-VEGFR2 signaling pathway by affecting the endocytosis efficiency of receptor and Notch signal crosstalk.In addition,the up regulation of VEGF expression further increases prmt4 level,forming a positive feedback loop,which can subsequently promote angiogenesis.