The Cardioprotective Effects Of Erythropoietin In Myocardial Ischemic Injury Via Upregulation Of SDF-1 By JAK2/STAT3

Accumulating data showed that progenitor/stem cells were beneficial for restoring the function of the injured heart. However, adequate regulation of signaling between the bone marrow, the peripheral circulation and the infarcted myocardium is important in orchestrating the process of mobilization, homing, incorporation, survival, proliferation and differentiation of stem cells, which relies on some small protein. Thus, protein therapeutics for cardiac regeneration after MI has also been proposed. EPO played a crucial role in reduction of infarct size in experimental models of myocardial ischemic injury, which was associated with angiogenesis and antiapoptotic effects. Thus, EPO seems to be a promising cytokine for cardioprotection following MI, however, the detailed mechanisms were not yet fully elucidated. SDF-1 was shown to modulate the recruitment and homing of bone marrow-derived stem cells (BMSCs) to injured myocardium, and further to promote angiogenesis and vascularization. Meanwhile, investigations documented that SDF-1 possessed anti-apoptotic activity. Notably, recent studies indicated that the expression of SDF-1 was up-regulated in the EPO-treated ischemic myocardium, suggesting augmented SDF-1 expression was critical for the EPO-induced improvement of cardiac performance. By binding to its receptor (EPOR), EPO stimulates at least three signals, namely PI3K/Akt, ERK1/2 MAPK and JAK2-STAT3, and plays a crucial role in many physiological and pathological processes. And STAT3 has long been shown as one of cytoplasmic transcription factors that usually translocated to the nucleus to regulate various gene expressions. Based on the observation mentioned above, we speculate that protection of the ischemic heart by EPO predominantly relied on the enhanced function of SDF-1 via activation of the JAK2/STAT3 and consequent recruitment of progenitor cells, augmented neovascularization and antiapoptotic effect.Part ISDF-1 was involved in erythropoietin-induced cardioprotection after myocardium infarctionObjective:We intended to demonstrate the role of SDF-1 in EPO-induced cardioprotection after myocardium infarction (MI).Methods:Intramyocardial injections of EPO (3000U/kg) or equivalent doses of saline were administered immediately after MI induced by ligation of the left anterior descendens coronary artery (LAD). After LAD ligation, mice received anti-SDF-1 neutralizing antibody or corresponding normal mouse IgG through tail vein once every 2 days until 2 weeks post MI. ELISA, qRT-PCR, immunohistochemistry and immunofluorescent were performed to detect the expression of SDF-1. At day 3, apoptotic cells were detected by measuring nuclear DNA fragmentation by fluorescent TUNEL assay kit. At day 14, flow cytometry of cardiac cells was performed to investigate the effect of EPO on migrated subpopulations of BMSCs. The vessel densities in the peri-infarct zones, infarct size and hemodynamic measurement were examined at 4 weeks.Results:Immunostaining, qRT-PCR and ELISA analysis showed that SDF-1 was up-regulated and predominantly localized in cardiomyocytes of the infarct border zone, the expression level of SDF-1 was maintained up to at least 7 days after MI in EPO-treated mice, which was matched with an increase in vessel density, a decrease in cell apoptosis and an improvement of cardiac function. However, the beneficial effects of EPO were attenuated by anti-SDF-1 neutralizing antibody.Conclusion:Our results demonstrated that EPO restored myocardial functions following MI, which may attribute to the crucial role of SDF-1 in the recruitment of BMSCs, promotion of angiogenesis and prevention of apoptosis.Part IIEPO treatment induced SDF-1 expression via activation of JAK2/STAT3 pathwayObjective:To determine which type of cell is the target of EPO and which sigaling is involved in the EPO-induced SDF-1 expression.Methods:Ventricular cardiomyocytes, fibroblasts and endothelial cells were isolated from adult male C57BL/6 mice. EPO (10 IU/mL) and wortmannin (1 mM) or PD98059 (25 mM) or pPYLKTK-mts (1μM) were added to the medium before the induction of hypoxia (1% O2,5% CO2 and 94% N2). ELISA and qRT-PCR was performed to detect the expression of SDF-1. Apoptotic cell was detected by TUNEL assay. In order to confirm the role of STAT3 activation in EPO-induced SDF-1 expression, p-STAT3 was examined by Western blot.Results:After 8 h of hypoxia, EPO treatment led to a significant increase in SDF-1 expression and a decrease in apoptosis in cardiomyocytes, which was attenuated by administration with either anti-SDF-1 neutralizing antibody or SDF-1 siRNA. But the similar effect was not observed in fibroblasts and endothelial cells. In EPO-treated cells, STAT3 was activated, and pretreatment with pPYLKTK-mts (STAT3 inhibiting peptide) abrogated EPO-induced SDF-1 expression and consequently EPO-induced anti-apoptosis.Conclusion:Our data documented that JAK2/STAT3 signaling was involved in the EPO-induced SDF-1 expression under hypoxia.Taken together, in this study we reported the protection of the ischemic heart by EPO predominantly relies on the enhanced function of SDF-1 via activation of STAT3 and consequent recruitment of progenitor cells, augmented neovascularization and antiapoptotic effect.