Inhibition of Prolyl Hydroxylase Domain Proteins Promotes Therapeutic Revascularization

Background-The hypoxia-inducible transcription factor (HIF) subunits are destabilized via the O-2-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 alpha (shHIF-1 alpha), 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 alpha. 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 alpha-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 alpha 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. (Circulation. 2009; 120: 50-59.)