Abstract 5643 The Erythropoietin Heterodimeric Beta Common Receptor is in a Complex With and Requires VEGFR2 for Downstream Signaling

Abstract

Background: Endothelial progenitor cells (EPC) circulate in the bloodstream and are capable of carrying out endothelial repair. Erythropoietin (EPO) has been shown to have stimulatory effects on EPC. We have previously demonstrated that EPO effects EPC mobility and increases nitric oxide (NO) levels within cells via the heterodimeric beta-common receptor (BCR) and EPO receptor (EPOR). We now test the hypothesis that not only does EPO induction of NO by the BCR/EPOR complex require vascular endothelial growth factor receptor 2(VEGFR2), but VEGFR2 signaling itself requires the BCR. Methods: EPC colonies isolated from human or mice were incubated with various kinase or VEGF inhibitors for 30 minutes prior to treating 50 mU/mL EPO and determining the bioavailable nitric oxide (NO) by DAF-FM-Diacetate. In addition we examined the ability of EPO and VEGF to promote an increase in NO in EPC isolated from the BCR −/− mice. Results: The EPO induced increase in NO could be completely blocked with a VEGFR2 blocking antibody but not by an antibody against VEGF itself. Blocking VEGFR2 also inhibited the EPO mediated increase in transcription of the RNA for endothelial and inducible NO synthase. Interestingly, the increase in NO was not blocked by a JAK-2 kinase inhbitor (Tyrphostin AG-490) confirming that the EPO induced NO effects are independent of the homodimeric EPO receptor-JAK kinase pathway. Most importantly EPC isolated from the BCR−/− mice had no NO stimulation in response to EPO, or VEGF but had a normal NO stimulation in response to bradykinin. We also demonstrate that the VEGFR2 co-immunoprecipitates and colocalizes with the BCR. Conclusion: We conclude that many of the tissue-protective effects of EPO demonstrated in models of ischemic, traumatic, and inflammatory injuries may be occurring through horizontal signaling of BCR with VEGFR2. In addition VEGFR2 signaling requires the expression of the BCR. These findings have profound implications for the potential side effects of EPO.