Study Of The Effect Of FHL1 On Angiogenesis And Underlying Mechanism

Objective:To study the effect of FHL1 on angiogenesis,we took human umbilical vein endothelial cells(HUVEC)as the research object,which were overexpressed and knocked down of FHL1 separately.The effect of FHL1 on the proliferation,migration and tube formation of HUVEC cells in vitro was observed.Subsequently,the mouse model of endothelium-specific overexpression of FHL1(Tie2-FHL1)was successfully established.The Aortic ring neovascularization assay and retinal neovascularization assay were used to further explore the effect of FHL1 in vivo.The mechanism of FHL1 about angiogenesis is preliminarily elucidated,which provides an experimental basis for FHL1 to affect angiogenesis by affecting endothelial cells.In vitro experiments:1.Designed the model of HUVEC cells,which were overexpressed and knocked down of FHL1;2.By extracting protein and RNA of HUVEC cells,which were overexpressed and knocked down of FHL1,to identified the expression of FHL1 by Western-blot and q PCR test;3.Using CCK-8 assay to detect the effect of the proliferation of HUVEC cells,which were overexpressed and knocked down of FHL1;4.Using wound healing assay to detect the migration ability of HUVEC cells,which were overexpressed and knocked down of FHL1;5.Using tube formation assay to detect the tube formation ability of HUVEC cells,which were overexpressed and knocked down of FHL1,including the tube length and nodes;In vivo experiments:1.Established the C57BL/6 mouse model of endothelium-specific overexpression of FHL1(Tie2-FHL1);2.By extracting mice’ protein and RNA of the aorta and vein tissues to identified the expression of FHL1 in blood vessels by doing Western-blot and q PCR test;3.culturing the aorta rings of Ctrl and Tie2-FHL1 mice in vitro,and calculated the new blood vessels’ branches and lengths of these aorta rings;4.Calculating the mice’ new blood vessels’ of retinal by establishing the OIR(oxygen-induced retinopathy)models.Results:1.FHL1 promoted the ability of proliferation of HUVEC cells’ in vitro(1)Compared with the Ctrl group,the overexpression of FHL1 promoted the proliferation of HUVEC cells;(2)Compared with the Ctrl group,knockdown of FHL1 inhibited the proliferation of HUVEC cells;2.FHL1 promoted the ability of migration of HUVEC cells’ in vitro(1)Compared with the Ctrl group,the overexpression of FHL1 promoted HUVEC cells’ migration ability;(2)Compared with the Ctrl group,knockdown of FHL1 inhibited HUVEC cells’ migration ability;3.FHL1 promoted the ability of tube formation ability of HUVEC cells’ in vitro(1)Compared with the Ctrl group,the overexpression of FHL1 promoted the tube-like structure formation of HUVECs,and the tube length and nodes were increased;(2)Compared with the Ctrl group,knockdown FHL1 inhibited the tube formation of HUVECs,and the tube length and nodes were increased;4.FHL1 promoted the growth of neovascularization about mouse aortic ringsEndothelial-specific FHL1 overexpression mice showed increased neovascularization in aortic rings compared with Ctrl mice;5.FHL1 promoted retinal neovascularizationAfter establishing OIR model,retinal neovascularization was increased in endothelial-specific FHL1 overexpression mice compared with Ctrl mice;6.FHL1 regulated neovascularization through AKT-m TOR signaling pathwayConclusion:FHL1 promoted angiogenesis by promoting the ability of proliferation,migration and tube formation of HUVEC cells in vitro.In vivo,the aortic rings’ experiment and the retinal neovascularization demonstrated that FHL1 promoted angiogenesis,which may affect angiogenesis by affecting AKT/m TOR signal pathway.