doi: 10.1161/01.RES.0000261610.11754.b1
© 2007 American Heart Association, Inc.
Editorials |
Switching on Reparative Angiogenesis
Essential Role of the Vascular Erythropoietin Receptor
From the Chair of Experimental Cardiovascular Medicine (P.M., C.E.), Bristol Heart Institute, University of Bristol, United Kingdom.
Correspondence to Paolo Madeddu, Bristol Heart Institute, University of Bristol, Level 7, Bristol Royal Infirmary, Upper Maudlin Street, Bristol BS2 8HW, UK. E-mail madeddu{at}yahoo.com
See related article, pages 662–669
Key Words: limb ischemia • angiogenesis • vasculogenesis • endothelial progenitor cells • migration
Neovascularization and hematopoiesis are parts of an integrated and tightly regulated program aimed at creating the logistics for the delivery of cells, oxygen, and nutrients to the different organs of the body. This cooperation starts during the early stages of development, with endothelial and hematopoietic cells emerging in temporal and spatial proximity, in both the embryo and the extraembryonic yolk sac. During adulthood, hematopoiesis takes place mainly in the bone marrow (BM), where hematopoietic stem cells (HSC) reside in a specialized microenvironment, the "stem cell niche," which provides the optimal conditions for stem cell maintenance. Here too, HSC and endothelial progenitor cells (EPC) seem to be closely related, to such an extent that the distinction between these cells remains shadowy. For instance, the subset of CD34+ BM cells that coexpress the receptor for vascular endothelial growth factor, VEGFR2, is enriched for both HSC and endothelial precursors in mice and humans.1–3 Single CD34+VEGFR2+ cells from adult BM generate both hematopoietic and endothelial cells in vitro and have long-term proliferative capacity.4 When injected into the hindlimb muscles of immunodeficient SCIDbg mice, these hematopoietic CD34+VEGFR2+ cells accelerate the recovery from limb ischemia through stimulation of neovascularization and myogenesis.5
Additionally, integration of hematopoiesis and angiogenesis is supported by hormones, cytokines and growth factors. Erythropoietin (Epo) stimulates the proliferation of erythroid precursors and their differentiation into mature red cells,6 and also exerts in vivo proangiogenic and reendothelialization actions, through activation of PI3K/nitric oxide-dependent EPC mobilization.7,8 Accordingly, circulating Epo has been shown to be an independent predictor of EPC number and function in patients with coronary heart disease.7
Importantly, mature endothelial cells express Epo receptors (EpoR)9 and, following Epo stimulation, switch from quiescent state to an angiogenic phenotype.10–12 Thus, the promotion of neovascularization by Epo might be attributable to the direct stimulation of resident endothelial cells, the recruitment of BM stem cells that differentiate into endothelial cells in situ or the combination of both processes. How can we disentangle the truth from this complicated scenario?
Recently, the importance of the Epo-EpoR system in erythropoiesis and vasculogenesis was firmly established using lines of mutant mice lacking either the Epo or EpoR gene. EpoR deficient mice die of severe anemia between embryonic day 13 (E13) and E15. They also show a severely disorganized vascular network in the myocardium, leading to cardiac failure.13 Although these observations are consistent with participation of the EpoR in hematopoiesis and heart vasculogenesis, the precise contribution of EpoR to adult nonhematopoietic tissue remains obscure. To address this important question, Suzuki et al established transgene-rescued EpoR mice expressing EpoR exclusively in the hematopoietic lineage under the transcriptional control of the GATA-1 gene.14 These mice are viable and fertile, but, following exposure to a hypoxic environment, they develop accelerated pulmonary hypertension and impaired EPC recruitment to pulmonary endothelium.15
In the present issue of Circulation Research, Nakano et al report the results of further investigation into the consequences of EpoR gene disruption in nonhematopoietic tissues.16 Following induction of limb ischemia, transgene-rescued EpoR mice showed delayed healing, which was attributed to defective activation of the VEGF/VEGFR2 axis leading to impaired capillarization of limb skeletal muscles. The authors also observed that EPC mobilization is reduced in transgene-rescued EpoR mice; however, transplantation of wild-type BM could not correct the defect in capillarization, thus suggesting a preponderant role of resident endothelial cells in angiogenesis impairment.
A puzzling aspect of Nakano’s study is that neither Epo nor EpoR were modulated by ischemia in wild-type mice. We know from previous studies that hypoxia-induced expression of the Epo gene is under the control of a widespread oxygen-sensing mechanism that is not limited to Epo-producing cells (see Stockmann and Fandrey 2006, for review).17 Analysis of proteins that bound to the Epo enhancer under hypoxia was indeed instrumental to the identification of hypoxia-inducible factor-1 (HIF-1).18 Thus, the lack of Epo/EpoR upregulation in the ischemic muscle following femoral artery occlusion is at variance with the established concept that hypoxia increases Epo. Apparently, it also contradicts previous data from Nakano’s group showing upregulation of EpoR in hypoxia-induced pulmonary hypertension.16 How can we reconcile these discrepancies?
Recent studies have shown that VEGF-A is a previously unsuspected negative regulator of Epo synthesis.19 In Nakano’s study, ischemia induced VEGF-A upregulation in limb muscles of wild-type mice. VEGF-A might have suppressed Epo synthesis, thus contrasting the induction of Epo by hypoxia. However, Epo levels remained unchanged in transgene-rescued EpoR mice, which failed to show upregulation of VEGF-A following induction of ischemia. This discounts the possibility that VEGF-A plays a role in the control of Epo expression in this experimental setting.
Nakano et al limited their evaluation to the expression of EpoR at mRNA level. However, posttranscriptional modifications are also crucial for the functionality of this receptor.20 Epo induces EpoR dimerization, which is followed by Janus kinase (JAK2)-mediated EpoR phosphorylation. Phospho-EpoR creates docking sites for STAT5, Ras, and PI3K (Figure). It would therefore be of paramount importance to investigate the phosphorylation state of EpoR in endothelial cells exposed to hypoxia and ischemia.
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Constitutively active EpoR was previously linked to a point mutation, R129C, in the extracellular domain of EpoR, or to the formation of complexes with the envelope viral protein gp55 (see Constantinescu et al for review).20 Under normal conditions, EpoR is not constitutively active and Epo binding is required to induce signaling.21 However, a fraction of the receptor exists as preformed dimer, which seems to be rapidly activated at low Epo concentrations. We speculate that hypoxia might increase the affinity of EpoR for its natural ligand, possibly by influencing spontaneous dimerization. Alternatively, hypoxia may trans-activate EpoR through binding with (unknown) coreceptor or by influencing the phosphorylation state of the EpoR intracellular domain. EpoR contains an extended cytoplasmatic domain that acts as a docking site for the tyrosine kinase receptor KIT. Activation of KIT by its ligand causes tyrosine phosphorylation of EpoR and thereby promotes PI3K-dependent activation of VEGF-A.22 Thus, posttranscriptional modifications of EpoR may be required for hypoxia-induced activation of VEGF-A.
Another aspect of Nakano’s studies that deserves comment is the observed impairment of EPC mobilization in EpoR rescued mice, despite the fact that those mice have normal EpoR levels in the hematopoietic system. To reach the circulation, stem cells transmigrate from their niche to the central marrow region, which comprises the BM vasculature and is therefore referred to as the BM "vascular niche". BM microvasculature not only provides the logistics for nutrients and oxygen delivery to BM cells, but also acts as a gatekeeper controlling the exit of stem cells into the circulation. In EpoR rescued mice, endothelial cells of the BM vascular niche might be dysfunctional and thus disturb the transmigration of EPC and hematopoietic stem cells to the systemic circulation. If this possibility were proved true, then we should reformulate the concept of postnatal vasculogenesis as a unidirectional phenomenon in which EPC set the stage for neovascularization.
The recognition of a vascular EpoR system, which is endowed with significant pathophysiological functions, opens new exploitation horizons in cardiovascular medicine. In particular, targeting EpoR may prove useful for the treatment of ischemic cardiovascular disease, with the important caveat that an activated EpoR could increase the risk of cancerous angiogenesis.
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Acknowledgments |
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Sources of Funding
Dr Emanueli holds a British Heart Foundation (BHF) basic science lectureship. The Bristol Heart Institute is partner of the European Vascular Genomic Network of Excellence (EVGN).
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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Related Article:
Circ. Res. 2007 100: 662-669.
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