Novel Contributions of Neutrophil‐derived Myeloperoxidase and Hypochlorous Acid to 20‐HETE Production that drives Post‐Ischemic Angiogenesis

Abstract

Objective20‐hydroxy‐eicosatetraenoic acid (20‐HETE), an arachidonic acid metabolite produced by cytochrome P450 (CYP) 4A/F ω‐hydroxylases, was recently uncovered as a novel contributor to ischemia‐induced angiogenesis. The current study aims to determine whether neutrophil‐derived myeloperoxidase (MPO) and hypochlorous acid (HOCl) mediate post‐ischemic 20‐HETE increases that drive angiogenesis and its underlying mechanism.MethodsMice were subjected to a femoral artery ligation with the contralateral hindlimb acting as the non‐ischemic control. Hindlimb angiogenesis was assessed by laser doppler blood perfusion imaging (LDPI) as well as quantifying the microvascular density in the gracilis muscles where angiogenesis is taking place. Endothelial cells (EC) in culture were subjected to physiological concentrations of MPO and HOCl. Gracilis muscles and EC were analyzed for 20‐HETE production by LC/MS/MS. The mRNA expression of the 20‐HETE synthesizing enzyme, CYP4A11 was measured by real‐time PCR. Protein expression of CYP4A11 and HIF1a were analyzed using western blotting. The rate and mechanism of HOCl entry into EC was evaluated using live‐cell fluorescence microscopy with a HOCl‐specific indicator dye (FDOCl‐1) in the presence/absence of chloride channel blocker 9‐anthracenecarboxylic acid (9‐Ac) and/or gap junction inhibitor 18‐beta‐glycyrrhetinic acid (18β‐GA).ResultsIn animals that were selectively depleted of neutrophils, ischemia failed to induce 20‐HETE increases in the gracilis muscle compared to the controls (13 ± 1.5 vs 35 ± 5 pg/mg of protein). Additionally, MPO‐null mice failed to show increased post‐ischemic 20‐HETE (~2 ± .25 vs 35 ± 5 pg/mg of protein) as well. Both animals display a decreased post‐ischemic angiogenenic phenotype. In EC, the addition of MPO or HOCl markedly stimulated the expression of CYP4A11 and production of 20‐HETE (40 ± 12 vs 8 ± 5 pg/mg of protein). Upregulation of CYP4A11 transcript/protein (>2.5‐fold) and HIF1a protein expression (2‐fold) was observed as early as 15 minutes post‐HOCl exposure. The mechanism of HOCl entry was also exmained by FDOCl‐1 fluorescence quantification, which was markedly decreased in EC pre‐treated with 9‐Ac or 18β‐GA by approximately 40% and 60%, respectively.ConclusionOur study demonstrates for the first time the contribution of MPO and HOCl in promoting 20‐HETE production that drives post‐ischemic angiogenesis. Moreover, this study aims to further expand on the known mechanisms involved in regulating ischemia‐induced angiogenesis. These new findings have high clinical relevancy in studying vascular pathologies by identifing novel mechanisms that contribute to pathological angiogenesis.Support or Funding InformationThis study was supported by grants from AHA[11SDG6870004 (AMG) and 17GRNT33430003 (AMG)], the NIH [HL34300 (M.L.S.) and grant DK38226 (J.R.F.)]; & the R.A. Welch Foundation [GL625910 (J.R.F.)].