Poster Sessions

CYT-4

Ruifeng Teng

R. F. Teng, B. Piknova , N. Sibmooh , D. J. Lefer, C. T. Noguchi

Exogenous erythropoietin cardioprotection in a mouse ischemia-reperfusion injury model requires nitric oxide synthase and endothelial cell response

Erythropoietin (Epo) is a glycoprotein produced in fetal liver and adult kidney, and is critical for erythropoiesis. It acts by binding to its receptor (EpoR) predominantly expressed on erythroid progenitor cells. The production of Epo is oxygen-dependent and can increase more than 50 fold in hypoxic or anemic conditions. Complete deletion of Epo or its receptor results in embryonic lethality due to severe anemia. Increasing evidence indicates that high level of serum Epo can improve ischemia/reperfusion injury in heart independent of red blood cell mass in experimental animal models and in preliminary clinical studies. We previously observed Epo induction in endothelial cells of endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO) by inducing phosphorylation of eNOS at S1177 residue. We now demonstrate Epo stimulation of NO production in human coronary endothelial cells (HCAEC) measured directly by real time gaseous NO release by chemiluminescence and bioimaging. Epo activates downstream PI3 kinase/AKT signaling pathway to increase NO production. In an animal model of myocardial ischemia/reperfusion injury, Epo treatment provided protection in wild type mice. Although eNOS and EpoR are expressed in cardiomyocytes, we did not observe any NO response and Epo protection in primary neonatal cardiomyocytes cultured under starvation and hypoxia conditions. In parallel with our results from endothelial cell cultures, we demonstrate that the endothelial Epo response to produce NO is necessary to provide cardioprotection in the mouse model for myocardial ischemia/reperfusion injury. The requirement for eNOS in Epo myocardial protection is shown by the loss of Epo protection in eNOS-/- mice. Selective rescue of EpoR in hematopoietic and endothelial tissue in EpoR-/- mice using a Tie2/cre transgene also rescues Epo myocardial protection to ischemia/reperfusion injury. Interestingly, we observe that in mice expressing EpoR only in hematopoietic tissue, without Epo treatment there is no difference in myocardial ischemia/reperfusion injury compared to wild type mice, indicating that endogenous Epo alone is unable to provide a protective effect. Therefore, cardioprotection by exogenous EPO requires eNOS expression and endothelial cell response. Our results provide evidence for Epo stimulation of eNOS activity in endothelial cells as a necessary and sufficient response for cardioprotection in this mouse model of ischemia-reperfusion injury.