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(Circulation. 2009;120:50-59.)
© 2009 American Heart Association, Inc.

Molecular Cardiology

Inhibition of Prolyl Hydroxylase Domain Proteins Promotes Therapeutic Revascularization

Céline Loinard, MSc; Amandine Ginouvès, PhD; José Vilar, PhD; Clément Cochain, MSc; Yasmine Zouggari, MSc; Alice Recalde, MSc; Micheline Duriez, BS; Bernard I. Lévy, MD, PhD; Jacques Pouysségur, PhD; Edurne Berra, PhD^*; Jean-Sébastien Silvestre, PhD^*

From the Paris Cardiovascular Research Center, INSERM U970, Hôpital Européen Georges Pompidou, Université Paris 5, Paris, France (C.L., J.V., C.C., Y.Z., A.R., M.D., B.I.L., J.S.); and Institute of Developmental Biology and Cancer, CNRS-UMR6543, University of Nice, Nice, France (A.G., J.P., E.B.). The present address for E.B. is Cell Biology and Stem Cells Unit, CICbioGUNE, Derio, Spain.

Correspondence to Jean-Sebastien Silvestre, PhD, Paris Cardiovascular Research Center, INSERM U970, Hôpital Européen Georges Pompidou, Université Paris 5, 56 rue Leblanc, 75015 Paris, France. E-mail Jean-Sebastien.Silvestre{at}inserm.fr

Received August 7, 2008; accepted May 8, 2009.

Background— The hypoxia-inducible transcription factor (HIF) subunits are destabilized via the O₂-dependent prolyl hydroxylase domain proteins (PHD1, PHD2, and PHD3). We investigated whether inhibition of PHDs via upregulating HIF might promote postischemic neovascularization.

Methods and Results— Mice with right femoral artery ligation were treated, by in vivo electrotransfer, with plasmids encoding for an irrelevant short hairpin RNA (shRNA) (shCON [control]) or specific shRNAs directed against HIF-1 (shHIF-1), PHD1 (shPHD1), PHD2 (shPHD2), and PHD3 (shPHD3). The silencing of PHDs induced a specific and transient downregulation of their respective mRNA and protein levels at day 2 after ischemia and, as expected, upregulated HIF-1. As a consequence, 2 key hypoxia-inducible proangiogenic actors, vascular endothelial growth factor-A and endothelial nitric oxide synthase, were upregulated at the mRNA and protein levels. In addition, monocyte chemotactic protein-1 mRNA levels and infiltration of Mac-3-positive macrophages were enhanced in ischemic leg of mice treated with shPHD2 and shPHD3. Furthermore, activation of HIF-1-related pathways was associated with changes in postischemic neovascularization. At day 14, silencing of PHD2 and PHD3 increased vessel density by 2.2- and 2.6-fold, capillary density by 1.8- and 2.1-fold, and foot perfusion by 1.2- and 1.4-fold, respectively, compared with shCON (P<0.001). shPHD1 displayed a lower proangiogenic effect. Of interest, coadministration of shHIF-1 with shPHD3 abrogated shPHD3-related effects, suggesting that activation of endogenous HIF-1-dependent pathways mediated the proangiogenic effects of PHD silencing.

Conclusions— We demonstrated that a direct inhibition of PHDs, and more particularly PHD3, promoted therapeutic revascularization. Furthermore, we showed that activation of the HIF-1 signaling pathway is required to promote this revascularization.

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CLINICAL PERSPECTIVE

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