20-HETE Regulates Angiogenic Functions of Human Endothelial Progenitor Cells and Contributes to Ischemia-induced Compensatory Neovascularization

Objective: Circulating EPC contribute to postnatal neovascularization, which is an important adaptation for recovery from critical ischemia. It is thus essential to understand the factors that regulate EPC involvement in the neovascularization process. We have identified the CYP4A-20-HETE system as a novel regulator of EPC angiogenic functions in vitro . Here, we explored cellular mechanisms by which 20-HETE regulates EPC angiogenic functions and assessed its contribution to EPC-mediated angiogenesis and ischemia-induced compensatory neovascularization in vivo . Approach and Results: 20-HETE induced EPC adhesion to fibronectin and EC monolayer by 40 ± 5.6 and 67 ± 10%, respectively, which were accompanied by a rapid induction of VLA-4 and CXCR4 mRNA expression. Basal and 20-HETE-stimulated increases in adhesion were negated by inhibition of the CYP4A-20-HETE system. Real-time PCR analysis and EPC proliferation assay indicated that a positive feedback regulation exists between 20-HETE and HIF-1α/VEGF pathways. Furthermore, in vivo addition of EPC increased angiogenesis by 3.6±0.2 fold and these effects were markedly reduced by inhibition of 20-HETE system. Systemic inhibition of the CYP4A-20-HETE system reduced the compensatory neovascularization in response to ischemia in a mouse ischemic hindlimb model. Importantly, ischemic hindlimb muscles produce markedly elevated amounts of 20-HETE compared to non-ischemic controls (124 ± 27 versus 17 ± 17 pg/mg of protein). Conclusions: These results strengthen the notion that 20-HETE regulates the angiogenic functions of EPC in vitro and in vivo in autocrine and paracrine manner via up-regulation of key pro-angiogenic factors. Increases in 20-HETE at ischemic sites promote and contribute to compensatory neovascularization.