| Objectives:In present study,experiments were designed to investigate endothelial differentiation of rat MAPCs cultured in the condition without exogenous VEGF and the behavior of VEGF and TGFβ1 secretion by rat MAPCs.In addition,the effect of growth factor on rat MAPCs endothelial differentiation was observed too.Methods:Rat MAPCs,as the source of bone marrow stem cells, were cultured under differentiation condition without exogenous growth factors.We observed the cells morphology and investigate the endothelial markers,CD31,Flk1,vWF.VE-cadherin or stem cell marker,oct4 expression by Q-RT PCR as well as VEGF and TGFβ1 protein level by ELISA or immunofluorescence in the whole differentiation period.The endothelial function tests were performed by Dil-acLDL uptake and Matrigel tube formation.We further compared the effects of VEGF,anti VEGF neutralizing antibody and anti TGFβ1 neutralizing antibody on endothelial marker expression in rat MAPCs by Q-RT-PCR.Results:We showed here that rat MAPCs expressed endothelial marker CD31,Flk1,vWF and VE-cadherin,exhibited spindle and cobblestone morphology,took up Dil-acLDL and formed tube structures on the Matrigel after 12 days differentiation.In addition,these cells secret VEGF and TGFβ1.Exogenous VEGF or VEGF neutralizing antibody did not appear significantly stimulative or arrested effect on endothelial marker expression(P＞0.05),but TGFβ1 neutralizing antibody significantly decreased CD31 and Flk1 mRNA expression on day 3 in the differentiation(P＜0.05).Conclusions:The study presented here provides the first evidence that rat MAPCs do differentiate to the endothelial like cells even without exogenous growth factors.The data confirm previous findings and reinforce the potential of secreting growth factors VEGF and TGFβ1 in rat MAPCs.These findings also suggest it is TGFβ1,but not VEGF associated with the endothelial differentiation in rat MAPCs.(chapter two)Objectives:We investigated the effect of high glucose on rat MPACs proliferation,apoptosis,endothelial differentiation and growth factor secretion,and we also tried to partially clarify the correlative signaling pathways of VEGF expression under high glucose condition.Methods:Different D-glucose concentrations(20,30,40 and 50 mM)were applied to evaluate D-glucose effects on undifferentiated MAPCs apoptosis and proliferation which were performed by Annexin V apoptosis kit and Brdu incorporation colorimetric ELISA kit.Rat MAPCs were culture under normal glucose(5.5 mM D-glucose),high glucose(30 mM D-glucose)and same amount of mannitol was used as osmolarity control.VEGF,CD31 and Flk1 mRNA expression was quantitatively determined by Q-RT-PCR.VEGF and TGFβ1 content in the media were measured by ELISA.JAK2,STAT3 and AKT phosphorylation were analyzed by Western blotting.We also investigated the effects of in vivo blockade of JAK2/STAT3 pathway by the specific JAK2 blocker tyrphostin AG490 on VEGF production in differentiating rat MAPCs. Intracellular VEGF and nuclear translocation of phoho-STAT3 were visualized by immunofluorescence.Results:We showed that high glucose did not affect rat MAPCs proliferation and apoptosis(P＞0.05)except 50mM D-glucose significantly decreased cells Brdu incorporation in our experiment condition(P＜0.05).Q-RT-PCR assay showed high glucose dramatically decreased VEGF and Flk1 mRNA level after 1 day incubation, interestingly,those two genes rose up at the late differentiation time, although there was no significant difference compared with normal glucose condition(P＞0.05),but CD31 gene expression increased and came to the marked difference in the whole differentiation period(P＜0.05).We also found high glucose not only decreased VEGF mRNA and protein level,but also reduced phosphor-STAT3 level,which was analogous AG490 effect on rat MAPCs.On the other hand,we can not observe high glucose affect AKT phosphorylation in our experiments condition.Immunofluorescence data showed intracellular VEGF and phosphor-STAT3 nuclear translocation was decreased by high glucose. Furthermore,a significant difference was observed between normal and high glucose condition with regards to TGFβ1 mRNA expression and protein production.Conclusions:These results provide evidence that inhibition of the JAK2/STAT3 signaling pathway by HG may be of importance in the decreased VEGF expression and production in rat MAPCs and this effect is not due to decreased cells proliferation or increased cells apoptosis. High glucose itself pursues a positive effect on rat MAPCs endothelial differentiation which may affiliate with increased TGFβ1 expression and production by high glucose.
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