Hypoxia-induced Augmented Cardioprotective Effects Of Mesenchymal Stem Cells Is Due To Increased Expression Of Leptin

Objective:Nowdays, the morbidity and mortality caused by cardiovascular disease in the crowd is in a leading position. It has been proven stem cell transplantation have the potential to protect, repair and perhaps regenerate the myocardium and become the next generation therapy for myocardial infarction. Bone marrow-derived mesenchymal stem cells (MSCs) are adult stem cells and could differentiate into multiple types of cells. Because of their ability to self-renew, multipotency, immunosuppressive properties and without ethical issues, MSCs are a promising cell source for cellular therapy. Amounts of research have demonstrated hypoxic preconditioning, a treatment that involves pre-exposing stem cells to sever hypoxia (0.5%O2) prior to cell therapy could enhance the therapeutic effect of MSCs. However, the mechanisms are still unclear. In our research we explored the molecular mechanisms that support the enhanced therapeutic effect of hypoxic preconditioning.Methods:MSCs were isolated through whole bone marrow adherent and incubated under normoxia (21%O2,5%CO2) or hypoxia (0.5%O2,5%CO2) for24hours. Leptin expression at gene and protein level was respectively detected by RT-PCR and ELISA. In vitro, the protective effect of MSCs was tested by co-culture assay and TUNEL staining was used to identify the apoptosis ratio of cardiomyocyte. In vivo, apoptosis ratio of cardiac myocytes3days after MSCs transplantation was measured by TUNEL staining, cardiac function28days after MSCs transplantation was measured by Echocardiography and hemodynamic examination. Furthermore, Masson’s trichrome staining was used to measure myocardial infarct size28days after MSCs transplantation.Results:Recent research showed that paracrine effect play an important role in implantation of mesenchymal stem cells. Leptin is one of important paracrine factors. Lots of studies confirmed leptin could inhibit the apoptosis of cardiomyocytes and promote significant angiogenesis.1. In our previous study we did gene chip and observed the gene expression of leptin was upregulated after hypoxic preconditioning. In this research we tested the phenomenon and confirmed hypoxic preconditioning could induce leptin expression by RT-PCR and ELISA.2. In vitro, we used a co-culture assay to quantify protection of cardiac myocytes subjected to simulated ischemia in the presence and absence of leptin blockade. We found hypoxic preconditioned MSCs could significantly decreased the apoptosis ratio of cardiomyocytes. However, knockdown of leptin expression with a selective shRNA abolished the hypoxic preconditioning-mediated hMSCs protective effect. Similarly, we found the same phenomenon when we use MSCsob/ob to do the co-culture assay. These results suggest that leptin is required to realize the enhanced paracrine actions of MSCs conferred by hypoxic preconditioning.3. We established myocardial infarction models and compared the therapeutic effect of MSCWT and MSCob/ob to determine whether these results translated into a similar requirement of hypoxic preconditioning for leptin in vivo. Firstly, w found transplantation of hypoxic preconditioned MSCWT markedly decreased apoptotic nuclei in both the infarct and border zone as compared with the normoxic-treated MSCWT group at3days, while no significant difference in cardiac cell apoptosis was observed between hypoxic-and normoxic-preconditioned MSCob/ob groups. Secondly, We measured myocardial contractile parameters at28days after MI and cell therapy. Echocardiography revealed that transplantation of MSCWT after hypoxic preconditioning limited LV dilation and significantly improved systolic function. In contrast hypoxic preconditioning did not significantly improve cardiac functions in response to transplantation of MSCob/ob. Results from hemodynamic examination showed a similar effect. Therefore leptin is required for enhanced MSC therapy in this MI model. This conclusion is further supported by Masson’s trichrome staining of the myocardial infarct size.Conclusion:In summary, in our study we explored the molecular mechanisms that support the enhanced therapeutic effect of hypoxic preconditioned MSCs. We found that leptin expression is induced by severe hypoxia and is essential for the enhanced function and therapeutic benefit of MSCs subjected to hypoxic preconditioning.