Mechanistic Studies On The Role Of TGF-β1 In Angiogenesis Through EndMT

During embryonic development and disease progression,endothelial cells(ECs)lose specific endothelial characteristics,obtain some of mesenchymal cell feature and lead to physiological or pathological change,which called EndMT.EndMT is an important pathological change in myocardial and renal fibrosis,and large parts of fibroblasts in the pathogenesis are converted from ECs via EndMT.EndMT exist in many diseases,including fibrosis of organs,invasion and metastasis of tumors,etc.Angiogenesis is an important process in tissue development.The first signs of angiogenesis appear in the early stages of embryonic development.EPCs from mesoblast differentiate into mature ECs,and after division,proliferate to initial vascular network.VEGF play a vital role in this process,inducing ECs proliferation and migration.This primitive vascular structure,also known as the primary capillary plexus,needs to be further remodeled and transformed into mature circulation network.Angiogenesis is not limited to embryonic development.In adults,angiogenesis mainly exists in ovarian cycle,damaged tissue and organ growth,inflammatory response,and malignant tumor.Most of the time,endothelial cells are stable and almost static.When tissues are stimulated by various cytokines,the balance between pro-angiogenesis and anti-angiogenesis is broken,and endothelial cells divide,proliferate,migrate and eventually form new blood vessels.Angiogenesis includes physiologic(including tissue damage repair,endometrial repair,etc.)and pathologic angiogenesis(inflammation,malignancy).Angiogenesis is associated with a variety of diseases.In-depth study on the mechanism of angiogenesis provides a new target for the diagnosis and treatment of clinically related diseases.This study is mainly divided into the following three partsPart Ⅰ:The experimental conditions and phenotypic identification of TGF-β1 induced EndMTObjective:To induce interstitial changes in HUVECs by TGF-β1 and verify the phenotypic changes,so as to provide a qualified cells for subsequent experiments.Methods:TGF-β1 is the most important factor in EndMT.We use TGF-β1 in vitro(5 days)to induce EndMT.In addition,we detect ECs marker CD31,VWF,and mesenchymal cell marker α-SMA through q-PCR and Western-blot to confirm the phenotype change.Results:The changes of cell morphology were observed by microscope.The normal HUVECs was like a paving stone with regular distribution and tight connection.HUVECs stimulated by TGF-β1 arranged irregularly,the morphology became slender,antennae increased,tight junction loosely and some cell similar as smooth muscle cells.P-smad3 is a detection indicator of TGF/smad signaling pathway activity.We detected p-smad3 induced by TGF-β1 at different concentrations by Western-blot.The results showed that p-smad3 most significantly increased when TGF-β1 concentration was 10ng/mL.Through q-PCR and Western-blot,decreased expression of CD31,VWF,and increased expression ofα-SMA at both RNA and protein levels were fonnd,which suggested that TGF-β1 promotes EndMT in HUVECs.Conclusion:TGF-β1(10ng/mL for 5 days)can successfully induce EndMT in HUVECs.Part Ⅱ The influence of TGF-β1 in endtothelial cell angiogenesis functionObjective:To explore the proliferation migration and angiogenic abiligy of HUVECs after adding TGF-β1.Methods:Using blood vessel formation,scratch test,Transwell and CCK8 experiment to detect the function of angiogenic,migration and proliferation function of ECs.Results:HUVECs can be divided and proliferated to form rope-shaped capillaries in matrix glue,which are shown as circular structures under the microscope.We implanted ECs in matrix and results showed that more annulus could be seen in HUVEC stimulated by TGF-β1,suggesting that HUVECs could generate more capillaries after added with TGF-β1.We observed the cell migration distance at 0h,12h and 24h respectively,and results showed that the migration ability of HUVECs increased after TGF-β1 stimulated.At the same time,transwell experiment also confirmed the results.The results of CCK8 showed that HUVECs stimulated by TGF-β1 was more absorbance at 490nm after the addition of CCK8 reagent.Conclusion:The angiogenic,migration and proliferation ability of HUVECs are significantly increased after stimulated by TGF-β1.Part Ⅲ Mechanism of EndMT induced by TGF-β1 in angiogenesisObjective:To investigate the molecular mechanism of EndMT in angiogenesisMethods:Vascular endothelial growth factor(VEGF)is the most important cytokine in angiogenesis.We detected VEGF and VEGF-R2 in HUVECs after TGF-β1 stimulation by q-PCR and Elisa.In order to investigate the role of EndMT in angiogenesis stimulated by TGF-β1,we used Oxymatrine to inhibit EndMT and detect the angiogenic ability of cells in vivo and vitro.Subsequently,p-AKT levels were detected by Western-blot to investigate the changes of phosphorylation of AKT after TGF-β1 stimulation.The angiogenesis ability were observed after AKT phosphorylation inhibitor HY15186 was added.Results.After TGF-β1 stimulated,the expression of VEGF and VEGF-R2 increased.Inhibiting EndMT weaken the promoting angiogenesis ability of TGF-β1.Akt is a serine/threonine protein kinase.Through Western-blot,we found that p-AKT expression significantly increased after TGF-β1 stimulated.At the same time,after the addition of AKT phosphorylation inhibitor HY15186,the angiogenesis function of HUVECs stimulated by TGF-β1 was significantly reduced.Conclusion TGF-β1 promotes angiogenesis by upregulation of VEGF.Inhibiting EndMT attenuates the angiogenesis effect of TGF-β1.AKT phosphorylation is involved in angiogenesis promoted by EndMT.