Abstract
Objective: Compensatory neovascularization is an important adaptation for recovery from critical ischemia. The CYP4A-derived 20-HETE were recently identified as a novel regulator of angiogenesis. Here, we assessed the contribution of 20-HETE to ischemia-induced neovascularization and its underlying molecular and cellular mechanisms. Methods: The mouse ischemia hindlimb angiogenesis assay was established to study the role of CYP4A-20-HETE in compensatory angiogenesis using the 20-HETE synthase inhibitor (DDMS, 10mg/kg/d) and the 20-HETE antagonist (6, 15-20-HEDGE, 10mg/kg/d). Hindlimb blood flow and microvessels density in ischemic hindlimb muscle were measured by Laser Doppler Perfusion Imaging and Immunofluorescent staining, respectively. 20-HETE production in ischemic muscles was measured by LC-MS-MS. Western blot was performed to explore the underlying signaling pathways. Flow cytometry analysis was used to assess the contribution of 20-HETE to mobilization and homing of bone marrow (BM)-derived endothelial progenitor cells (EPC) to ischemic neovascularization. Results: 20-HETE inhibition by DDMS and 6, 15-20-HEDGE reduced the blood flow perfusion and microvessel formation in response to ischemia by 46±2.3% and 34±1.2%, respectively. Importantly, ischemic hindlimb muscles showed markedly elevated 20-HETE synthesis compared to non-ischemic controls (96 ± 17 pg/mg protein vs. non-detectable). HIF-1α, VEGF, and VEGFR2 expression were markedly induced in ischemic hindlimb muscles, but were negated by DDMS and 6, 15-20-HEDGE. Furthermore, ischemia induced BM-derived EPC mobilization and subsequent EPC homing to the ischemic hindlimb muscle by 5.5±0.7 and 20±2.8 folds, respectively, whereas DDMS and 6, 15-20-HEDGE significantly attenuated these processes. Conclusions: An increase in 20-HETE production in ischemic muscles regulates ischemic compensatory neovascularization via the induction of the HIF-1α/VEGF pathways that support the mobilization and homing of BM-derived EPC. These results strengthen the notion that 20-HETE is a key regulator of ischemic compensatory angiogenesis via regulation of EPC function and may represent a novel therapeutic target.
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