Extracellular vesicle-mediated HIF-1α delivery promotes durable vascular remodeling via endothelial Dll1-dependent CD163+ macrophage differentiation.

Critical limb ischemia (CLI) is a severe vascular disease with limited pharmacologic treatments and a high risk of amputation. Conventional therapeutic angiogenesis, mainly based on single growth factors, has failed to achieve stable and functional vessel formation. Recent insights indicate that interactions between endothelial cells and immune cells, particularly perivascular macrophages, are critical for durable vascular remodeling. Here, we engineered a pH-responsive intein-based platform to load extracellular vesicles (EVs) with a stabilized, constitutively active form of hypoxia-inducible factor-1α (HIF-1α). In this study, we demonstrate that EV-mediated HIF-1α delivery markedly improves perfusion and reduces necrosis in a murine hindlimb ischemia model. HIF-1α-EVs activated a regenerative vascular program that coupled new vessel growth with structural maturation, resulting in enlarged collateral arteries and an expansion of type H and CD34+ endothelial populations. Notably, HIF-1α-EV treatment also increased CD163+ perivascular macrophages, which are known to support arteriogenesis by vessel stabilization. Mechanistically, EV-mediated delivery of HIF-1α upregulated endothelial Delta-like ligand 1 (Dll1), which directed macrophage differentiation toward a CD163+ reparative phenotype, thereby reinforcing vascular maturation and restoring blood flow. Collectively, this transcription factor delivery strategy offers a promising therapeutic avenue for durable revascularization in CLI and potentially other ischemic diseases.