Optimal injection sites for therapeutic angiogenesis: HGF-mediated regulation of HIF-1α via MAPK/PI3K pathways in hypoxic endothelial cells

Therapeutic angiogenesis offers a promising strategy for patients with critical limb-threatening ischemia (CLTI) who are unsuitable candidates for revascularization. However, the optimal administration sites for gene therapy agents, such as pCK-HGF-X7, remains undefined. Clinical trials commonly employ multiple intramuscular injections at sites of arterial occlusion; yet the necessity and efficacy of such extensive and repetitive protocols remains unclear. Targeted injections into ischemic tissues or their margins may improve therapeutic outcomes. Moreover, the molecular mechanisms by which hepatocyte growth factor (HGF)/c-Met signaling regulates hypoxia-inducible factor-1 alpha (HIF-1 alpha) expression under hypoxic conditions are not fully understood. This study aims to elucidate these molecular mechanisms in endothelial cells under hypoxic conditions and to identify the most effective injection sites for therapeutic angiogenesis agents. The effects of various HGF isoforms/complexes on human aortic endothelial cells (HAECs) were evaluated under normoxic and hypoxic conditions, focusing on proliferation, migration, and tube formation. Pathway inhibitors were used to explore the underlying mechanisms in hypoxic HAECs, and the findings were validated in a rat hindlimb ischemia model. Results demonstrated that HGF723 and HGF728 activated the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways, regulating HIF expression and significantly enhanced endothelial cell proliferation, migration, and tube formation, particularly under hypoxia. Despite these cellular effects, HGF treatment did not significantly improve tissue perfusion or neovascularization in normal rat hindlimbs. However, in ischemic rat hindlimbs, it markedly promoted angiogenesis and improved tissue perfusion in the gastrocnemius muscle. These findings indicate that therapeutic angiogenesis agents should primarily target hypoxic tissues, extending to the interface between normoxic and hypoxic regions, to optimize treatment efficacy.