Transcription Factor E2-2 Regulates The Proliferation Of Endothelial Progenitor Cells Via Mediating Autophagy

Background:Endothelial progenitor cells(EPCs)play a significant role in repairing of injured vascular endothelium,as the primary cellular source of endothelial cells.Recently researches shown E2-2 could negatively regulate proliferation of EPCs.E2-2 is an important subfamily member of the basic helix-loop-helix transcription regulation regulator,which belongs to the E protein family.The regulation mechanisms of E2-2 in the proliferation of EPCs are still poorly understood.Autophagy is a basic physiological activity in eukaryotes,which is essential for maintaining cellular homeostasis.E2-2 had been shown to suppress autophagy by activating Wnt/?-catenin signaling pathway in tumor cells.A recent study revealed that inhibition of basal autophagy prevented their differentiation into mature endothelial cells.We supposed that E2-2 could regulate the proliferation of EPCs via mediating autophagy.Therefore,our study will demonstrate the effect of E2-2 on proliferation of EPCs via regulating the level of autophagy to provide new insights for repairing injured vascular endothelium and treatment of coronary heart disease.Objective:To investigate the role of autophagy in the proliferation of EPCs via regulated by E2-2.Methods:Murine EPCs of spleen should be isolated,cultured and identified.Proliferation and autophagy were detected when EPCs were overexpressed and knocked down E2-2.Chloroquine(CQ)treated EPCs with different concentration and time.Then we added CQ in the group of E2-2 knocked-down,compared proliferation and autophagy with E2-2 knocked-down and control group.We detected the expression of ATG7 when knock-down E2-2 in EPCs.Lastly,we simultaneously and respectively knock-down E2-2 and ATG7 by transfecting small interfering RNA,observed the change of autophagy marker protein.CCK-8 assay was used to detect the capacity of proliferation of EPCs.Confocal fluorescence microscope was used to observe the autophagosomes and autolysosomes which were labeled with adenovirus harboring tandem fluorescent mRFP-GFP-LC3(Ad-mRFP-GFP-LC3).Western blotting was used to detect the expression of protein.PCR?Q-PCR was used to detect the expression of mRNA.Results:1.Overexpression of E2-2 could inhibit the proliferative capacity and downregulation of autophagy.2.Knock-down E2-2 could upregulate the level of autophagy and increase the capacity of proliferation in EPCs.3.Added CQ could disturb the upregulation of autophagy and capacity of proliferation from knock-down E2-2.4.ATG7 gene was upregulated when knock-down E2-2.5.Autophagy and proliferation of EPCs were decreased when Co-interferencing E2-2 and ATG7.Conclusion:E2-2 inhibits the proliferation of EPCs via downregulating the level of autophagy.Effects of E2-2 in autophagy of EPCs might partly be regulated via modulating transcription of ATG7.