Abstract 15873: Novel Contributions of Neutrophil-derived Myeloperoxidase and Hypochlorous Acid to 20-hydroxyeicosatetraenoic Acid Production That Drives Post-ischemic Angiogenesis

Abstract

Introduction: Compensatory angiogenic response to ischemia is often insufficient in maintaining adequate tissue perfusion resulting in critical limb ischemia and amputation. Identifying a novel mechanism by which angiogenesis occurs in these conditions is clinically relevant. We recently uncovered that an increase in 20-HETE, an arachidonic acid metabolite of CYP4A/F ω-hydroxylases, regulates post-ischemic angiogenesis. However, the underlying mechanism resulting in this increase is unknown. Hypothesis: Neutrophil-derived myeloperoxidase (MPO) and hypochlorous acid (HOCl) critically contribute to post-ischemic 20-HETE increases that drive angiogenesis. Methods: Hindlimb ischemia was established in mice depleted of neutrophils, macrophages, and MPO (MPO -/- ). Angiogenesis was assessed by laser doppler perfusion imaging and micro-vessel density quantitation in the hindlimb gracilis muscles. MPO and HOCl were detected in these tissues using immunohistochemistry and a HOCl-specific fluorophore. We also determined the effects of MPO and HOCl on 20-HETE production, the expression of 20-HETE synthase CYP4A11, and hypoxia inducible factor-1α in cultured endothelial cells (EC) using LC/MS/MS, real time-PCR and western blot analysis, respectively. Results: We found that ischemia failed to increase 20-HETE production in mice depleted of neutrophils and MPO (13 ± 1.5 vs 35 ± 5 and ~2 ± .25 vs 35 ± 5 pg/mg of protein, respectively), accompanied with a decreased post-ischemic angiogenic phenotype. We also detected the formation of MPO and HOCl in post-ischemic gracilis muscles. MPO and HOCl also significantly stimulate CYP4A11 expression and 20-HETE production (40±12 vs 8±5 pg/mg of protein) in EC. Furthermore, HOCl quickly induces CYP4A11 mRNA/protein expression (2-fold,) and the protein expression of HIF-1α (2-fold) in as little as 15 min. Conclusion: Our studies establish for the first time that neutrophil-derived MPO and HOCl are responsible for promoting 20-HETE increases that critically drive angiogenesis post ischemia. Thus, identifying these novel mediators can further future therapeutic strategies to balance angiogenic responses during ischemia as well as treating diseases that are associated with abnormal angiogenesis.