Angiotension Ⅱ Induces The Differentiation Of Mouse Epicardial Progenitor Cells Into Vascular Smooth Muscle-like Cells

At present,coronary heart disease,a serious threat to human health,is still the most common cause of death in the world.Understanding the origin and differentiation mechanism of vascular smooth muscle cells is very important to prevent and treat the coronary heart disease and protect the health of human.Epicardial progenitor cells(EpiCs)derived from the proepicardium,an epithelial layer on the surface of the heart,play a crucial role in coronary myocardial development and vasculature formation and have the capacity for differentiation into vascular smooth muscle cells,fibroblasts,and sinoatrial node cells.For now,EpiCs expressing specific transcription factors Wt-1,Tbx18,and Tcf21 are receiving increasing interest in recent years as possible tools for myocardial regeneration.Increasing evidence has demonstrated that differentiated coronary vascular smooth muscle cells(SMC)originate from the embryonic epicardium[5-7].However,the mechanism controlling the development and differentiation of these cells is not well understood.Angiotensin II(Ang II)is the most important effector peptide of the renin–angiotensin system,which works through its receptors,including Ang II receptor Type 1(AT1)and Ang II receptor Type 2(AT2).In addition to regulating body fluid homeostasis,Ang II is an important growth regulator that participates in cellular proliferation,apoptosis,differentiation and so on.A number of studies haveshown that Ang II plays an important role in the differentiation of stem cell/progenitor cells.In addition,it has been proposed that Ang II can stimulate the differentiation of mononuclear cells derived from bone marrow and mesenchymal stem cells derived from adipose tissue into the smooth muscle-like cells.However,the potential effect of Ang II on the differentiation of Epi Cs is still unknown.Part I: culture and identification of embryonic epicardial progenitor cells.Objective: To culture embryonic epicardial progenitor cells(EpiCs)in vitro using C57BL/6 mice and observe the mechanism of the differentiation of EpiCs into vascular smooth muscle cells.Method: The EpiCs were cultured from embryonic hearts dissected from the mouse at pregnant 12.5 day.The specific markers of Epi Cs Wt-1,Tbx18 were stained by the immunofluorescence technique.Result: We successfully cultured EpiCs in consideration of the origin,morphology and the expression of Wt-1 and Tbx18.Conclusion:The EpiCs were successfully cultured from embryonic hearts dissected from the C57BL/6 mouse at pregnant 12.5 day.Part II: Ang II can induce the differentiation of EpiCs to Smooth Muscle-like cells through the AT1 receptor.Objective: To observe the effect of angiotension II in inducing the differentiation of Epi Cs into vascular smooth muscle cells.Method: The Ang II receptors AT1,AT2 were stained by the immunofluorescence technique.Cultured EpiCs were randomly divided into Ang II group,Ang II+losartan group,Ang II+PD123319 group and the control group.The immunofluorescence was used to detected theexpression of SMCs specific markers α-SMA and Myh11 in the four groups and qRT-PCR was applied to analyze the mRNA expression level ofα-SMA and Myh11.The contractile of AngII-induced cells was measured by collagen gel contraction assay.Result: Here we detected the expression of Angiotensin II(Ang II)receptors AT1 and AT2 on EpiCs and demonstrated that AngII could increase the expression of smooth muscle specific markers,includingα-smooth muscle actin(α-SMA)and myosin heavy chain 11(Myh11)in EpiCs.Moreover,the expression of α-SMA and Myh11 induced by Ang II was blocked by pretreatment of EpiCs with the AT1 receptor antagonist losartan,but not the AT2 receptor antagonist.We further showed that the AngII-induced cells showed significant contractile responses to carbachol.Conclusion:AngII could effectively induce EpiCs to differentiate into vascular smooth muscle-like cells through the AT1 receptor.