Autophagy Is Involved In The Differentiation Of Epicardial Progenitor Cells Into Vascular Smooth Muscle Cells In Mice

To study the development of coronary artery and clarify the origin and differentiation mechanism of coronary smooth muscle cells(CoSMCs)will provide important information for the prevention and treatment of congenital heart disease and coronary regeneration after myocardial infarction.At present,it is considered that epicardial progenitor cells are the main origin of coronary smooth muscle cells,but the mechanism of epicardial progenitor cells differentiation to CoSMCs remians clear.Autophagy is the decomposition process of proteins and organelles mediated by lysosomes,which is related to the clearance of aged organelles and proteins destroyed and the maintenance of the stability of intracellular environment.Previous studies have confirmed that autophagy is with great significance in the process of heart development and remodeling of tissues and cell differentiation in mammals,but there is no evidence that autophagy can be involved in the regulation of the differentiation of epicardial progenitor cells into coronary smooth muscle cells.In this study,we successfully cultured mouse epicardial progenitor cells in vitro,and observed the effect of autophagy on the differentiation of epicardial progenitor cells into smooth muscle cells through the induction and inhibition experiments of autophagy.At the same time,we further observed the effect of autophagy on the development of coronary smooth muscle cells in vivo in order to confirm that autophagy is involved in regulating the differentiation of epicardial progenitor cells into coronary smooth muscle cells.Part ? Culture and identification of primary EPCsObjective: To explore the construction method of cultivation of high purity of epicardial progenitor cells,and lay the foundation for the in vitro differentiation mechanism of EPCs.Methods: The embryonic hearts of E11.5 mice were isolated and cultured in vitro using tissue adherence method.The self-characteristics of cultured cells were observed under microscope,and the expression of Tbx18 and WT1 genes was detected by qRT-PCR and immunofluorescence techniques to obtain the purity of cells.Results: primary EPCs were successfully cultured.Morphological observation confirmed that the primary cell group had typical epithelial morphology,and the morphological changes of the differentiation of the cell group were confirmed after 72 hours of continuous observation.Furthermore,myocardial cells were used as the control group,and it was confirmed by qPCR and immunofluorescence that the myocardial cells had multiple marker genes of epicardial progenitor cells.Conclusion: EPCs derived from primary culture by tissue adherence method were relatively simple and that could simultaneously express multiple marker genes of epicardial epithelium with high purity.The morphology and arrangement of primary epicardial progenitor cells cultured in vitro showed typical epithelial morphology,and further differentiation was observed in morphology,which laid a foundation for subsequent studies on differentiation of EPCs.Part ? Experiments in vitro revealed the effect of autophagy on the differentiation of EPCs into SMCsObjective: to investigate the effect of autophagy on the differentiation of EPCs into smooth muscle cells and the function of SMCs derived from EPCs.Methods: on the basis of primary epicardial progenitor cell model,autophagy induction and inhibition models were established with autophagy inducer RAPA and inhibitor 3-ma.The morphological effects of autophagy induction/inhibition on the differentiation of epicardial progenitor cells were observed,and the mRNA levels of autophagy related Akt and mTOR were measured by qPCR.Furthermore,MDC staining,LC3 B protein fluorescence staining and Westernblot assay were used to reveal whether the differentiation process of epicardial progenitor cells was autophagy.After 72 h culture,qPCR and immunofluorescence assay were used to detect the levels of smooth muscle marker proteins a-sma and MYH11,and MTT assay was used to detect the cell proliferation ability of each group.96 h cells were collected and cultured,and their contractile function was evaluated by collagengel contraction test,and cell migration ability was evaluated by scratch test.Resurts: our experiment observed that RAPA significantly induced the up-regulation of autophagy level of epicardial progenitor cells within 72 h,resulting in significantly slower proliferation of epicardial progenitor cells,but increased elongated spindle mass allogeneic cells.The results of immunofluorescence staining showed that the a-sma and MYH11 peaks in the RAPA group were earlier than those in the NORMAL group and 3-MA group,suggesting that autophagy could induce the early differentiation of epicardial progenitor cells.After 48 h of cell culture,a large number of spindle shaped and heteromorphic differentiated cells appeared.In 3-MA group,the autophagy level was significantly down-regulated,but cell proliferation and migration were up-regulated,and differentiation was delayed.These results suggest that inhibition of autophagy may inhibit the differentiation of EPCs into SMCs and promote their proliferation and migration.Conclusion: Autophagy may regulate the differentiation of EPCs into smooth muscle cells.Upregulation of autophagy may induce the early differentiation of EPCs,enhance the contractility and decrease the proliferation and migration of smooth muscle cells.Inhibition may inhibit the differentiation of EPCs into smooth muscle cells and promote their proliferation and migration.PART ? Experiments In vivo reveal the effects of autophagy on the development of coronary artery in miceObjective: to determine the effect of epicardial progenitor cells on the development of coronary vascular smooth muscle and the spatial localization relationship between autophagy marker signaling molecules and smooth muscle marker genes at the tissue level,so as to explore the effect of autophagy on the development of coronary artery by epicardial progenitor cells in vivo.Methods: the application of amniotic cavity injection drug delivery way autophagy induction/inhibition in mice model was established.The effects of autophagy induction/inhibition on the structure of mice E12.5-E14.5 coronary artery development were observed by HE staining.And the effects of autophagy on the RNA and protein expression of a-SMA during the differentiation of coronary smooth muscle in mice were observed by q-PCR and immunofluorescence.Then the autophagy marker protein LC3 B and smooth muscle cell marker were used.Protein MYH11 was CO-stained to observe the spatial localization of the two proteins.Results: after the induction of autophagy,the heart almost failed to form mature coronary arteries in the lumen,but gradually appeared sinus-like changes under the endocardium,suggesting that the mouse heart failed to develop mature coronary arteries and establish effective coronary circulation.At the same time,ventricular outflow tract development disorder,cardiac circulatory defects and ventricular wall thinning were observed after autophagy induction.QPCR and histofluorescence results suggested that autophagy of mouse E12.5-14.5 could occur in both epicardial and myocardial tissues.In the autophagy induction group,the expression of a-SMA in the epicardial myocardial tissue was slightly higher than that in the normal group at E12.5 days,but the expression of a-sma in RAPA group was significantly lower than that in the normal group at E14.5 days,and failed to develop into a mature coronary lumen.During E12.5-14.5 days,the expression level of a-SMA in the autophagy inhibition group was lower than that in the normal group at all time periods,and the final expression level of a-SMA around the coronary artery was also lower than that in the normal group.Fluorescence co-staining experiments confirmed that MYH11 positive cells in multiple epicardial and subepicardial MYH11 positive cells could be co-expressed with LC3 B protein.Conclusion: autophagy requires appropriate intensity in coronary development.Excessive activation of autophagy may lead to increased differentiation and migration of smooth muscle cells in advance,which may result in misrecruitment of smooth muscle cells around endothelial cells and failure to form an effective coronary lumen.Overall,coronary artery development is inhibited.Autophagy inhibition may delay the differentiation of epicardial progenitor cells,thereby reducing the number of coronary artery formation.The differentiation of epicardial progenitor cells into coronary smooth muscle requires appropriate autophagy intensity.