The Enhanced Effect Of Lysophosphatidic Acid On Endothelial Progenitor Cells Treatment In Acute Respiratory Distress Syndrome Rat

endothelial progenitor cells(EPCs)is a kind of precursor stem cells,which can directly differentiate to endothelial cells.EPCs were not only involved in the formation of the early embryonic blood vessels,but have a closest relationship with angiogenesis and vascular formation in adult individuals.EPCs have a good therapeutic potential on angiogenesis with a key role in vascularization and repair in vivo.However,there are some problems during the process of stem cells cultured in vitro,although a good effect of EPCs transplantation for vascular repair is proved in preclinical animal experiments and clinical trials,leading to greatly weakened the ability of EPCs on angiogenesis treatment.Our previous study found that Lysophospholipids acid(LPA)improved the umbilical cord mesenchymal stem cell proliferation through LPAR1 mediated way and suppress lipopolysaccharide induced cell apoptosis.LPA is a bioactive lysophospholipids small molecules without immunogenicity.In recent years,LPA was attracted wide attention as a drug application in tissue engineering.LPA mainly associate with specific G protein coupled receptors,and regulate a variety of biological effects,such as cell proliferation,migration and differentiation.We also found that LPAR1 was remarkably high expressed in EPCs.Therefore,we hypothesized that LPA and LPAR1 have a same effect in EPCs.In this study,it is aimed to confirm the effect of LPA on proliferation,migration,differentiation,and cytokines of EPCs and its action mechanism,to explore the improvement effect of LPA on EPCs transplantation in acute respiratory distress syndrome rat.The EPCs was successfully isolated from femoral bone marrow of SD rats by lymphocyte separation liquid,The mononuclear cells was isolated from rat femur bone marrow and confirmed by detecting the specific surface marker CD34,CD133 and KDR,vWF,acLDL-DiI and FITC-UEA-I expression by flow cytometry and laser confocal microscope.LPA has no influence on the cell surface marker of EPCs,indicating LPA has not induced differentiation in EPCs.LPA promotes the clone formation of EPCs.LPA promotes proliferation of EPC mediated by LPAR1 coupling Gi protein and activation of ERK1/2.LPA promotes EPC migration mediated by LPAR1 coupling with Gq/11 and Gi/o proteins.Bio-plex suspension chip method was used to detect the content of cytokines in cellular supernatant.LPA inhibites the secretion of pro-inflammatory factor IL-1 a and IL-6(LPS induced)through activation of NF-κB/p65 and promotes the anti-inflammatory factor IL-4 and IL-6(TNF-a induced)secretion in EPCs.Fluorescence in situ hybridization experiment proves that LPA pretreatment can improve the number of EPCs in lung after transplantation in ARDS rat.Bio-plex suspension chip method was used to detect the content of cytokines in plasma.EPCs transplantation significantly reduce the lung injury induced by lipopolysaccharide through decreasing edema and the expression of pro-inflammatory factors in plasma.LPA pretreatment can improve the effect of EPCs on injury repair,and increase the IL-10 and IL-7 content in plasma.There are no difference between LPA pretreatment of EPC group and untreated EPC group on pulmonary edema and pro-inflammatory factor expression.In this study,the vitro experiments prove that the LPA regulate the proliferation and migration of EPCs,without differentiation.LPA promotes proliferation of EPC mediated by LPAR1 coupling Gi protein and activation of ERK1/2,and promotes EPC migration mediated by LPAR1 coupling with Gq/l1 and Gi/o proteins.LPA decreases the secretion of pro-imflamatory factor and increases the secretion of anti-imflamatory factor through inhibition of NF-kappa B/p65 phosphorylation in EPCs.LPA promotes secretion of VEGF under inflammation in EPCs.The animal experiment further prove that LPA pretreatment can increase EPCs number in lung after transplantation,decreases the expression of pro-inflammatory factor,promotes the anti-inflammatory factor and M-CSF,and then reduces the degree of lung injury,promoted the therapeutic effect of EPC on ARDS.Our study indicate that LPA shows a potential protective role on EPC transplantation in the treatment of ARDS and other cardiovascular disease,having good application value on clinical therapy.