Abstract 5442: Inhibition Of Prolyl Hydroxylase Domain Protein Promotes Therapeutic Neovascularization
Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that regulates expression of genes involved in O2 homeostasis, including potent stimulators of angiogenesis. Prolyl Hydroxylases Domains proteins (PHDs) mediate O2-dependent hydroxylation of two conserved proline residues of HIF-1α subunit, and subsequently trigger HIF-1α ubiquitylation and proteasome targeting. Inhibition of PHD may therefore upregulates HIF-1-dependent signaling and promotes post-ischemic neovascularization. Mice (n=8 per group) with right femoral artery ligation were treated, by in vivo electrotransfer, with plasmids encoding for an irrelevant shRNA (shCON) or shRNA directed against HIF-1α (shHIF), PHD1 (shPHD1), PHD2 (shPHD2) and PHD3 (shPHD3). PHDs silencing induced a specific transient downregulation of their respective mRNA and protein levels at day 2 after ischemia. Inhibition of PHDs upregulated HIF-1α and carbonic anhydrase IV, a paradigmatic marker of HIF-1 activity, mRNA and protein levels. This was associated with an increase in VEGF-A and eNOS mRNA and protein levels, two key hypoxia-inducible pro-angiogenic genes. In addition, MCP-1 mRNA levels and the number of MAC-3-positive macrophages were also enhanced in ischemic leg of mice treated with shPHD2 and 3. Activation of HIF-1α related pathways was associated with changes in post-ischemic neovascularization. At day 14, silencing of PHD2 and 3 increased vessel density by 2.2- and 2.5-fold, capillary density by 1.8- and 2.1-fold and foot perfusion by 1.2- and 1.4-fold, respectively when compared to shCON (p<0.001). shPHD1 displayed a lower pro-angiogenic effect. Of interest, co-administration of shHIF-1α with shPHD3 abrogated shPHD3-related effects suggesting that activation of HIF-1α-dependent pathways mediated the pro-angiogenic effects of PHD silencing. Therefore, direct inhibition of PHDs and more particularly PHD3 promoted therapeutic revascularization via specific activation of HIF-1 target genes.