Background-Recent studies have suggested that endogenous erythropoietin (Epo) plays an important role in the mobilization of bone marrow-derived endothelial progenitor cells (EPCs). However, it remains to be elucidated whether the Epo system exerts protective effects on pulmonary hypertension (PH), a fatal disorder encountered in cardiovascular medicine. Methods and Results-A mouse model of hypoxia-induced PH was used for study. We evaluated right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling in mice lacking the Epo receptor (EpoR) in nonerythroid lineages (EpoR Ϫ/Ϫ rescued mice) after 3 weeks of exposure to hypoxia. Those mice lack EpoR in the cardiovascular system but not in the hematopoietic system. The development of PH and pulmonary vascular remodeling were accelerated in EpoR Ϫ/Ϫ rescued mice compared with wild-type mice. The mobilization of EPCs and their recruitment to the pulmonary endothelium were significantly impaired in EpoR Ϫ/Ϫ rescued mice. By contrast, reconstitution of the bone marrow with wild-type bone marrow cells ameliorated PH in the EpoR Ϫ/Ϫ rescued mice. Hypoxia enhanced the expression of EpoR on pulmonary endothelial cells in wild-type but not EpoR Ϫ/Ϫ rescued mice. Finally, hypoxia activated endothelial nitric oxide synthase in the lungs in wild-type mice but not in EpoR Ϫ/Ϫ rescued mice. Conclusions-These results indicate that the endogenous Epo/EpoR system plays an important role in the recruitment of EPCs and prevents the development of PH during chronic hypoxia in mice in vivo, suggesting the therapeutic importance of the system for the treatment of PH.

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...

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Objective-Left ventricular (LV) remodeling after acute myocardial infarction still remains an important issue in cardiovascular medicine. We have recently demonstrated that low-intensity pulsed ultrasound (LIPUS) therapy improves myocardial ischemia in a pig model of chronic myocardial ischemia through enhanced myocardial angiogenesis. In the present study, we aimed to demonstrate whether LIPUS also ameliorates LV remodeling after acute myocardial infarction and if so, to elucidate the underlying molecular mechanisms involved in the beneficial effects of LIPUS. Approach and Results-We examined the effects of LIPUS on LV remodeling in a mouse model of acute myocardial infarction, where the heart was treated with either LIPUS or no-LIPUS 3 times in the first week (days 1, 3, and 5). The LIPUS improved mortality and ameliorated post-myocardial infarction LV remodeling in mice. The LIPUS upregulated the expression of vascular endothelial growth factor, endothelial nitric oxide synthase, phosphorylated ERK, and phosphorylated Akt in the infarcted area early after acute myocardial infarction, leading to enhanced angiogenesis. Microarray analysis in cultured human endothelial cells showed that a total of 1050 genes, including those of the vascular endothelial growth factor signaling and focal adhesion pathways, were significantly altered by the LIPUS. Knockdown with small interfering RNA of either β1-integrin or caveolin-1, both of which are known to play key roles in mechanotransduction, suppressed the LIPUS-induced upregulation of vascular endothelial growth factor. Finally, in caveolin-1-deficient mice, the beneficial effects of LIPUS on mortality and post-myocardial infarction LV remodeling were absent. Conclusions-These results indicate that the LIPUS therapy ameliorates post-myocardial infarction LV remodeling in mice in vivo, for which mechanotransduction and its downstream pathways may be involved. (Arterioscler Thromb Vasc