Prolyl-Hydroxylases Inhibition Enhances the Efficiency of Cell and Non-Cell Based Strategies for Treatment of Critical Limb Ischemia

Therapeutic neovascularization represents an alternative treatment modality for patients with advanced ischemic coronary disease or critical limb ischemia (CLI). Upregulation of HIF-1α, through specific PHDs inhibition, can be thought of as a master switch that coordinates the expression of a wide repertoire of genes involved in regulating vascular growth and remodeling. The aim of this study was to unravel the efficiency of specific prolyl-hydroxylase domain protein (PHDs) silencing in both cell and non cell-based therapeutic strategies in CLI. We first showed that PHD2 and PHD3 mRNA levels were up-regulated whereas that of HIF-1α were down-regulated in mononuclear circulating cells and in ischemic legs of diabetic and non diabetic patients with CLI. We then used short hairpin RNAs (shRNAs) to downregulate murine PHD1, PHD2 or PHD3 levels and analyzed their efficiency as a therapeutic strategy in diabetic mice with hindlimb ischemia. PHDs silencing increased post-ischemic neovascularization. This increase was associated with an up-regulation of two key pro-angiogenic factors, VEGF-A and endothelial NOS and with that of pro-arteriogenic factors including fractalkine, MCP-1, and SDF-1 leading to activation of postischemic immunoinflammatory response. In addition, concomitant silencing of PHD2 and HIF-1α abrogated the shPHD2-related effects, demonstrating that activation of HIF-1α signaling mediated the pro-angiogenic and pro-arteriogenic effects of shPHD2. Finally, we assessed the putative beneficial effects of PHDs silencing on human endothelial progenitor cells (hEPC) or bone-marrow derived mesenchymal stem cells (hMSC)-based therapies. Cells were transfected with siRNA directed against PHDs and injected in Nude mice with hind-limb ischemia. We showed that PHDs silencing enhanced human cells therapeutic effect in the ischemic leg of Nude mice compared to animals receiving untreated cells. In conclusion, our study reveals, for the first time, that silencing of PHDs by the transient and local upregulation of endogenous HIF-1α improves therapeutic angiogenesis. This study also paves the way for future strategy based on administration of siRNAs directed against PHDs to enhance the efficiency of cell-based strategy.