| Although mesenchymal stem cells (MSCs) have great promise in the recovery of damaged myocardium after myocardial infarction, several factors limit therapeutic effect of MSCs transplantation. One of them is low viability of MSCs transplanted into infarcted heart. MSCs apoptosis transplanted into infarcted heart might result from more than one factor besides hypoxia and serum deprivation (Hypoxia/SD). The increase of exogenous reactive oxygen species (ROS) in infracted heart might be another important factor to induce apoptosis of MSCs. Identification of such factors contributes to improvement of the cell viability.Transplanted MSCs without any modification might be another factor to limit the therapeutic effect. The therapeutic effects of MSCs results partly from release of paracrine factors, among which vascular endothelial growth factor (VEGF) is one of crucial mediators by regulating angiogenesis and protecting myocardium against ischemic injury. Increase of VEGF secreted from MSCs improves the therapeutic effects of MSCs. Lysophosphatidic acid (LPA), as an endogenous lipid messenger, is involved in diverse physiological and pathological processes. Our previous studies indicated that LPA enhanced capillary density after transplantation of MSCs in ischemic myocardium in vivo, and increase VEGF production under hypoxia/SD in vitro, but its mechanism is not known. DGPP is a novel phospholipid, and ofen used as one of LPA1/3 receptor antagonists, but the effect of DGPP on MSCs is not clear. In our previous study, we superisely found that DGPP promote VEGF secretion than LPA in MSCs, but its accurate effect and mechanism are not well understood.According to the background mentioned above, we investigated the role of reactive oxidative species (ROS) in transplanted MSCs, the effect of LPA and DGPP in paracrine of MSCs and their mechanism, and the regulation of LPA on MSCs proliferation. The study includes four sections.1. The role of hydrogen peroxides in the viability of MSCs and its mechanismTo identify the role of hydrogen peroxide, a model of oxidative stress was established to mimic the environment in vivo, and the mechanism of H2O2 on MSC apoptosis was investigated. Obvious apoptosis of MSCs resulted from a certain concentration of H2O2 (≧0.9 mM) and treatment time (≧12 h). H2O2 treatment resulted into loss of mitochondrialΔΨm, release of cytochrome C released from mitochondria to cytosol and bax translocation from the cytosol to the mitochondria in MSCs.Caspase-3 and caspase-12 were activated after H2O2 treatment, and their inhibitors prevented apoptosis of MSCs.However, the caspase-12 inhibitor had no effect on cytochrome C release and cleavage of caspase-3 caused by H2O2. Transient phosphorylation of JNK/p38 increased after H2O2 treatment. P38 inhibitor prevented the early apoptosis of MSCs, accompanied by the decrease of cytochrome C release and bax translocation, while JNK inhibitor inhibited the late apoptosis. In conclusion, H2O2 induced apoptosis of MSCs in a dose and time dependent way. Apoptosis of the cells involved both mitochondrial death pathway and ER stress which were parallel. H2O2 activated the JNK/p-38 pathway, among which p-38 contributed to the early apoptosis of MSCs by regulating mitochondrial pathway, and JNK was involved in the late apoptosis independent of mitochondrial pathway. 2.The effect of LPA on VEGF secretion in MSCs and its mechanismTo identify the effect of LPA on VEGF secretion, MSCs were treated with LPA, and the relative signaling pathways were detected. The results indicated that LPA promoted VEGF secretion in a dose and time manner in MSCs, but had no effect on expression of VEGF protein and VEGF mRNA in the cells. LPA inhibited Na+, K+-ATPase activity, upgraded the expression of ORP150, and decreased the resident of VEGF in endoplasmic reticulum(ER).Diazoxide, the mitochondrial KATP channel opener, inhibited both the expression of ORP150, the secretion of VEGF, and the resident of VEGF in ER induced by LPA. In conclusion, LPA promoted VEGF secretion in MSCs through the post-translation level. LPA stimulated VEGF secretion by upgrading ORP150 expression, and the increase of ORP150 expression resulted from inhibition of Na+, K+-ATPase activity and mitoKATP channels induced by LPA.3.The role of DGPP in VEGF secretion in MSCs and its mechanismTo investigate the role of DGPP in VEGF secrection, MSCs were treated with DGPP, VEGF secretion was detected, and the relative mechanism was investigated. The results indicated that DGPP promoted more VEGF production secreted to extracellular supernatant than LPA, and it did not change the amount of the apoptosis cells at the same condition, but high concentration of DGPP inhibited MSCs proliferation. DGPP had no effect on the expression of VEGF protein and VEGF mRNA in the cells.The mitochondrial KATP channel opener diazoxide failed to inhibit VEGF secretion induced by VEGF.DGPP increased the expression of GRP94 mRNA. The Sulfinator software predicted the tyrosine sulfation sites of VEGF.In conclusion, DGPP had better effect in VEGF secretion than LPA in MSCs, and the increase VEGF production did not result from the amount of the cells.The mitochondrial KATP channel was not involved in VEGF secretion induced by DGPP, thus, it was different between DGPP and LPA in the regulation of VEGF secretion. The roles of GRP94, N-glycosylation and tyrosine sulfation of VEGF in VEGF secretion were worth to further explore.4.The effect of LPA on MSCs proliferation and its mechanismMSCs were treated with LPA, and proliferation of the cells was detected and its mechanism was investigated. The results demonstrated that LPA promoted MSCs proliferation in a dose-dependent manner. The inhibitors for LPA1/3 receptor and ERK prevented the cell proliferation. LPA also increased the expression of c-myc,which was inhibited by the inhibitors of LPA1/3 receptor and ERK. In conclusion, LPA promoted proliferation of MSCs by binding LPA1/3 receptor, activating ERK pathway and increasing the expression of c-myc.In sum, the study identified the effect of ROS in MSCs viability and its signaling pathway, analyzed the role of the phospholipids in MSCs paracrine and proliferation and their mechanism. The data would improve the viability of transplanted MSCs, facilitate application of the phospholipids in cell transplantation, and enhance the therapeutic effects of transplanted MSCs. |
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