Important Role of Erythropoietin Receptor to Promote VEGF Expression and Angiogenesis in Peripheral Ischemia in Mice

Abstract

Abstract-We have recently demonstrated that endogenous erythropoietin (Epo)/Epo receptor (EpoR) system plays an important protective role in hypoxia-induced pulmonary hypertension. However, it remains to be examined whether vascular EpoR system contributes to angiogenesis in response to ischemia. We examined angiogenesis in EpoR Ϫ/Ϫ -rescued mice that lack EpoR in most organs including cardiovascular system except erythroid-lineage cells. Two weeks after femoral artery ligation, blood flow recovery, activation of VEGF/VEGF receptor system, and mobilization of endothelial progenitor cells were all impaired in EpoR Ϫ/Ϫ -rescued mice as compared with wild-type (WT) mice. Bone marrow (BM) transplantation with WT-BM cells in EpoR Ϫ/Ϫ -rescued mice partially but significantly improved blood flow recovery after hindlimb ischemia. The extent of VEGF upregulation and the number of BM-derived cells in ischemic tissue were significantly less in EpoR Ϫ/Ϫ -rescued mice compared with WT mice even after BM reconstitution with WT-BM cells. Similarly, the recovery of blood flow was significantly impaired in recipient EpoR Ϫ/Ϫ -rescued mice that had been transplanted with WT-BM or EpoR Ϫ/Ϫ -rescued-BM as compared with recipient WT mice. Furthermore, the Matrigel implantation assay and aortic ring assay showed that microvessel growth in vitro was significantly reduced in EpoR Ϫ/Ϫ -rescued mice as compared with WT mice. These results indicate that vascular EpoR system also plays an important role in angiogenesis in response to hindlimb ischemia through upregulation of VEGF/VEGF receptor system, both directly by enhancing neovascularization and indirectly by recruiting endothelial progenitor cells and BM-derived proangiogenic cells. Key Words: angiogenesis Ⅲ ischemia Ⅲ progenitor cells Ⅲ VEGF Ⅲ erythropoietin P rognosis of patients with severe peripheral artery disease (PAD) still remains poor when there is no indications of revascularization therapies such as bypass surgery or percutaneous transluminal angioplasty. 1 Angiogenesis is a promising new therapeutic strategy for severe PAD, however, the effects of angiogenic therapies to improve ischemia are not durable or stable. 2-4 Hypoxia inducible factor-1 (HIF-1) is one of the important factors to induce angiogenesis, 5,6 which upregulates both erythropoietin (Epo) and VEGF. 7,8 These angiogenic cytokines play an important role in recruitment of bone marrow (BM)-derived cells to ischemic tissue, enhancing endothelial cell proliferation and migration, synthesis of extracellular matrix and resultant angiogenesis. 9 -11 Epo is a hypoxia-induced hormone that exclusively stimulates proliferation and differentiation of erythroid progenitor cells and endothelial cells. 12-15 Furthermore, systemic administration of Epo mobilizes endothelial progenitor cells (EPCs) and recruits them to ischemic tissue, 16,17 where EPCs produce abundant cytokines including VEGF and promote postnatal vasculogenesis. 18,19 Although Epo receptor (EpoR) is known to be expressed abundantly not only in BM but al...

Revisiting the role of erythropoietin for treatment of ocular disorders

“…Evidence from the study hypothesized that increase in the expression and activation of EPOR can be triggered by VEGF-A, present in the vascular endothelium, upon binding to VEGF receptor-2 (VEGFR-2). 34,36,51 Phosphorylated VEGFR-2 then interacts with phosphorylated EPOR to enhance angiogenesis via a STAT3 signaling pathway 51 and this was equally reported by Nakano et al, 52 Sautina et al, 53 and Yang et al 54 In conclusion, the EPO also modulates angiogenesis by inducing the secretion of VEGF, which then binds and activates VEGFR-2. [51][52][53][54] Apart from VEGFR-2, Sautina et al 53 also demonstrated that the stimulation of EPO is dependent on the βcR.…”

“…39,53 This study proposed that NO stimulation was regulated by the heterotrimeric interaction between EPOR/βcR/VEGF-R2 upon binding of EPO to EPOR/βcR. 39,[51][52][53][54][55] Meanwhile, studies have reported that genetic polymorphism in the EPO gene may be a pathogenic factor for acquiring risk of proliferative DR. 56 Katsura et al 55 showed that vitreous EPO was present in significantly higher amount in proliferative DR patients than in macular hole patients who had no retinopathy. The authors further showed the high EPO concentration was not induced by systemic anemia.…”

Hypertension Promotes Phosphorylation of Focal Adhesion Kinase and Proline-Rich Tyrosine Kinase 2 in Rats: Implication for the Pathogenesis of Hypertensive Vascular Disease

“…We also used rabbit anti-human integrin β -1 (M-106) and goat anti-human integrin β -3 (N-20) polyclonal antibody (Sigma, St. Louis, MO, USA) as primary antibodies. We also used alkaline phosphatase-conjugated goat anti-mouse secondary antibody (American Qualex, San Clemente, CA, USA), alkaline phosphatase-conjugated swine anti-rabbit secondary antibody (Dako, Glostrup, Denmark), and alkaline phosphatase-conjugated rabbit anti-goat secondary antibody (Sigma, St. Louis, MO, USA) (Nakano et al 2007;Saji et al 2007;Do e et al 2009). …”

“…The membrane was developed with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. Signals were visualized by the ECL detection system (Amersham Biosciences, Uppsala, Sweden) (Nakano et al 2007;Do e et al 2009). …”

Kidney Ischemia and Reperfusion Injury – Field of Glory or Warterloo for Erythropoietin?

“…It was found that it also has antiapoptotic, antioxidative and angiogenetic effects that can be used to avoid and treat tissue damage in general. 5,6 This is possible, because of the widespread distribution of the EPO receptor that can mediate non-hematopoietic effects.…”