Lysophospholipids Protect Mesenchymal Stem Cells Against Ischemia Induced Apoptosis

Remarkable advances have been made in animal and clinical studies of stem cell-based therapy for ischemic cardiomyopathy.However,majority of engrafted cells die within hours that severely limits the effectiveness of transplantation therapy.The ischemic environment induced apoptotic death is believed as a major factor responsible for massive cell death.Several strategies have been explored to enhance graft cells survival.But several disadvantages hamper their clinical implications.The lysophospholipids(LPs) are simple phospholipids that have been recognized for decades as components in the biosynthesis of cell membranes.Lysophosphatidic acid (LPA) and sphingosine 1-phosphate(S1P),as two of the best characterized LPs have been revealed to participate in the regulation of many important physiological and pathophysiological processes via specific G protein-coupled receptors termed LPA1-5 and S1P1-5.We have previously shown that LPA antagonized the apoptosis of mesenchymal stem cells(MSCs) induced by hypoxia and serum deprivation(hypoxia/SD) mimicking ischemic myocardium microenvironment.Whether LPA has the same potentially beneficial effect on MSCs in vivo is unknown.In addition,it is unclear whether S1P could protect ischemia induced apoptosis of MSCs.MicroRNAs(miRNAs) are a recently discovered class of small,evolutionarily conserved,non-protein-coding RNA molecules that negatively regulate gene expression by inhibiting protein translation or by destabilizing target transcripts at the post-transcriptional level.MicroRNAs have been implicated in the control of many fundamental cellular and physiological processes such as tissue development,cellular proliferation and apoptosis,oncogenesis.Whether miRNA plays important roles in the process of ischemia induced apopotosis of MSCs and the mechanisms it involves are unkown.The present study explored the protective actions of LPA on transplanted MSCs in vivo and the antiapoptotic effects of S1P on MSCs in vitro and in vivo.In addition,we studied the mechanisms of miRNA mediated the apoptosis of implantated MSCs.All of these studies will contribute to the new strategies of promoting the survival of donor cells.1.Female rats were operated to induce coronary artery occlusion.Animals were then grouped to receive intramyocardial injection with DMEM(30μL,Groupl),male MSCs(2×10~6 per rat,Group2) or male MSCs pretreated with LPA(2×10~6 per rat, Group3),respectively.The results demonstrated that LPA treatment improved graft MSC survival in ischemic myocardium assessed in a gender-mismatched transplantation model by real time-PCR,as well as by TUNEL assay.Moreover, transplantation of LPA treated MSCs enhanced capillary density determined by immmunostaining for platelet endothelial cell adhesion molecule(PECAM)-1,and it is also found that LPA enhanced vascular endothelial growth factor(VEGF) release from MSCs under hypoxia/SD in vitro.We did not get an improvement in Left ventricular(LV) function at 1 weak after transplantation of LPA treated MSCs.These data suggest that LPA exerts both protective actions on MSC survival and enhancement on MSC paracine in vivo and may represent a novel and effective treatment strategy in cell transplantation.2.Rat MSCs were prepared and subjected to hypoxia and serum deprivation (hypoxia/SD for 6 hours with apoptotic cell death determined by flow cytometry. Addition of S1P significantly decreased the percentage of early apoptosis via Gi-coupled S1P1 receptor and activation of downstream Akt and ERK1/2 signaling pathways.In the in vivo study,the same experimental system was used as LPA.The results showed that S1P treatment significantly enhanced the survival of transplanted MSCs at 1 hour,1 day and 1 week after being injected into ischemic heart; transplantation of S1P treated MSCs enhanced angiogenesis 1 week after transplantation in ischemic myocardium.In addition,S1P increased the secretion of VEGF from MSCs under hypoxia and serum deprivation.All of these data suggest that S1P treatment may be a novel strategy to improve MSC survival and promote angiogenesis in ischemic hearts.3.To identify microRNAs that are differentially expressed during MSC apoptosis,we used miRNA microarray analysis of RNA from the MSCs subjected to hypoxia/SD at 3 different time points(Oh,1.5h,and 6h).We were excited to find that,compared with the control group,4 miRNAs were upregulated,3 miRNAs were upregulated first and then downregulated,4 miRNAs were downregulated first and then upregulated and only 2 was downregulated.Further studies were done to validate some microarray results by Realtime-PCR assay and we found that a new miR-337 was upregulated.The final statistical analysis revealed that the differential change of miR-337,miR-503 and miR-483 were statistically significant.Furthermore,we extended our studies to identify the target proteins and we found that API5 maybe a target ofmiR-337 and VEGF maybe regulated by miRNA.Our present study not only explored a new and effective strategy for enhancing graft cells survival but also a new probable mechanism about the apoptosis of transplanted MSCs mediated by microRNA,providing a new idea and theoretical basis for actual application of MSC-based therapy for ischemic cardiomyopathy.