Sphingosine 1-Phosphate Induces Epicardial Progenitor Cells Differentiation Into Smooth Muscle Cells

Coronary artery disease?CAD?is the leading cause of cardiovascular death worldwide,while effective therapy remains few.The pathophysiology of CAD may involve the aberrant proliferation of coronary artery smooth muscle cells?CoSMCs?.Besides vascularization,CoSMCs also participated in the composition and function of coronary artery.Therefore,unravel the CoSMCs origin will shed light on the therapy for CAD.Epicardial progenitor cells?EpiCs?is a progenitor pool which expressed transcriptor factors such as Tbx18,WT1 and Tcf21.EpiCs compensate for the first and the second heart-forming field,as the third heart-forming field.EpiCs played an important role in the heart development and neovascularization.Current studies identified that EpiCs is one of the origins of CoSMCs.In addition,it is proven that EpiCs have the potential to differentiated into fibroblasts,endothelial cells,sinus atrial nod cells and so on.The sphingosine 1-phosphate?S1P?is a highly hydrophobic zwitterionic lysophospholipid which usually functions via its 5 receptors belonged to the class A family G protein-coupled receptors?GPCRs?.Indeed,S1PRs include 5 receptors which expressed widely on different types of cells.S1PRs expressing in different organs/tissues regulate many events in health and disease.As in the heart,S1PRs are correlated with smooth muscle cell function regulation,including proliferation,migration and differentiation.Previous studies identified the involvement of S1P in the differentiation of adipose derived mesenchymal stem cells into SMCs.Therefore we assumed that S1P is involved in the EpiCs-CoSMCs differentiation.In the present study,S1PRs in the epicardial progenitor cells were identified and their function in the EpiCs differentiation into coronary vasculature SMCs were determined,thus providing a therapeutic strategy in coronary artery rebuilding.Part 1:The extraction and identification of EpiCsAim:The in vitro cell culture system of EpiCs was established on E11.5day and the expression of S1P receptors was detected to further research of EpiCs differentiation.Methods:To establish the EpiCs in vitro culture system,E11.5 hearts were dissected and then the EpiCs migrated from the ventricle onto the dish and formed an epithelial monolayer.To identify the specificity of EpiCs,transcription factors Tbx18 and WT1 were detected by PCR and cell immunofluorescence.The expression of S1P receptors was detected by PCR.Results:The transcript factors Tbx18 and WT1 were highly expressed in the extracted cells,together with a low expression of cTnT and the specific cobblestone-like morphology.The extracted cells mainly contained the S1P₁,S1P₂ and S1P₃ receptors,rather than S1P₄ and S1P₅ receptors.Part 2:S1P stimulates the differentiation of EpiCs into smooth muscle cells.Aim:To investigate the mechanism of the differentiation of EpiCs into smooth muscle cells induced by S1P.Methods:After establishing the in vitro culture system of EpiCs,different concentrations of S1P?0.1uM?0.5uM?1uM?2uM?5uM?were used to stimulate the cells,then the expressions of?-SMA and MYH11 were identified by qRT-PCR to confirm the proper concentration of S1P.To further confirm the major subtype of S1PRs in the EpiCs involving the differentiation,the EpiCs were randomly divided into the S1P group?0.5uM S1P??S1P+JTE013 group?0.5uM S1P+1uM JTE013??S1P+VPC23019 group?0.5uM S1P+1uM VPC23019?.The expressions of?-SMA and MYH11 were identified by qRT-PCR and immunofluorescence to determine the effects of S1P on the EpiCs differentiation after the inhibition of S1P₁,S1P₂ and S1P₃ receptors respectively.In order to determine the effects of S1P1 receptor on the EpiCs differentiation,the EpiCs were randomly divided into the control group,S1P group?0.5uM S1P??SEW2871 group?30uM SEW2871?and S1P+W146 group?0.5uM S1P+10uM W146?.The expressions of?-SMA and MYH11 were identified by qRT-PCR and immunofluorescence to determine the effects of S1P₁ on the EpiCs differentiation.Furthermore,three-dimensional collagen gel assay was performed to determine the contraction of treatment by different S1P receptor agonists and antagonists.Results:Expressions of?-SMA and MYH11 reached the peak after the treatment of S1P for 6 days with the concentration of 0.5 uM,which means the proper S1P concentration is at 0.5 uM.With the pretreatment of S1P₂antagonist JTE013 and the S1P₁/S1P₃ antagonist VPC23019,the S1P induced increase of?-SMA and MYH11 mRNA levels were significantly attenuated,which were also abrogated more in VPC23019 treatment.And similar results were found in the immunofluorescence assays.While in the presence of S1P₁ agonist SEW2871,the mRNA levels of?-SMA and MYH11 was not changed compared with the control group.In the presence of S1P₁ antagonist W146,the mRNA levels of?-SMA and MYH11 was not changed compared with the S1P group.And similar results were found in the immunofluorescence assays.Furthermore,the contraction of differentiated VSMCs conducted by carbachol were significantly increased by S1P pretreatment determined by the gel-contraction assay,same as the S1P+W146 pretreatment.While the S1P+JTE013 or S1P+VPC23019 or SEW2871 pretreatment did not induce the gel contraction.Conclusion:These results collectively suggested that S1P,principally through its receptor S1P₃,induces EpiCs differentiation into SMCs,while S1P₂ plays a secondary role.