Abstract 338: 20-HETE Contributes To Ischemia-induced Compensatory Neovascularization

Abstract

Objective: Compensatory neovascularizaiton is an important adaptation for recovery from critical ischemia. Recent studies identified the CYP4A-20-HETE system as a novel regulator of angiogenic processes. Here, we assessed the contribution of the 20-HETE system to ischemia-induced neovascularization and further explored its underlying molecular and cellular mechanisms. Methods: The mouse ischemia hindlimb angiogenesis assay was performed to evaluate the effect of systemic or local inhibition of the CYP4A-20-HETE system with the 20-HETE synthase inhibitor, DDMS or the 20-HETE antagonist, 20-HEDGE on the compensatory angiogenic responses. Laser Doppler Perfusion Imaging was conducted to assess hindlimb blood flow. Blood pressure was monitored with the non-invasive blood pressure monitoring system. 20-HETE production in ischemic muscles was also measured by LC-MS-MS analysis. IHC staining were carried out to quantify microvessel density and to assess the localization of 20-HETE system in hindlimb gracilis muscle. Finally, western blot was performed to explore the potential underlying signaling pathways. Results: Inhibition of 20-HETE synthesis or antagonizing its action either locally or systematically reduced the blood flow perfusion and microvessel formation in response to ischemia without affecting blood pressures. Importantly, ischemic hindlimb muscles showed markedly elevated 20-HETE synthesis compared to non-ischemic controls (96 ± 17 pg/mg of protein versus non-detectable amount). IHC staining showed that an upregulation of 20-HETE synthase ( cyp4a12a ) is co-localized with endothelial cells in the ischemic gracillis muscle, suggesting ischemia may induce the increased production of 20-HETE in the vasculature. Furthermore, the protein expression of HIF-1α, VEGF, VEGFR2, and p44/42 MAPK in ischemic hindlimb muscles were significantly induced. These increases were negated by DDMS or 20-HEDGE. Conclusions: Early increases in 20-HETE production in ischemic muscles may regulate ischemic angiogenesis via the induction of the HIF-1α/VEGF and MAPK pathways. These results strengthen the notion that 20-HETE may be a key regulator of ischemia-induced compensatory neovascularization processes.