130 - Scavenging of Reactive Isolevuglandins in Mitochondria Reduces Vascular Oxidative Stress and Attenuates Hypertension

Abstract

Free radical oxidation of arachidonic acid produces highly reactive isolevuglandins (isoLG) which causes mitochondrial dysfunction by opening of the mitochondrial permeability transition pore (mPTP). Inhibition of the mPTP regulatory subunit cyclophilin D reduces isoLG-induced mitochondrial dysfunction (Free Radic Biol Med 2010;49:567-79). We found that depletion of cyclophilin D diminishes mitochondrial O 2 ●– , improves vascular relaxation and reduces hypertension (Hypertension 2016;67:1218-27). We hypothesized that hypertension is associated with accumulation of mitochondrial isoLG and mitochondria-targeted isoLG scavenger reduces vascular oxidative stress and attenuates hypertension. To test this hypothesis we developed mitochondria-targeted isoLG scavenger 4-(aminomethyl)-3-hydroxyphenoxy) butyl)triphenyl-phosphonium (mito2HOBA) by conjugation of lipophilic triphenylmethylphosphonium cation to 2-hydroxybenzylamine (2HOBA). Mito2HOBA is a water soluble compound which was well tolerated by cultured human aortic endothelial cells (HAECs) and by mice receiving it in drinking water. It was found that mito2HOBA (50 nM) inhibited mitochondrial O 2 ●– production (MitoSOX/HPLC) and prevented cardiolipin oxidation (LS-MS) in HEACs incubated with TNFα+Angiotensin II while untargeted analog 2HOBA (50 nM) was not effective. The functional role of mitochondrial isoLG was tested in vivo using angiotensin II model of hypertension. C57Bl/6J mice were infused with angiotensin II (0.7 mg/kg/day) or saline (Sham) and received mito2HOBA in the drinking water (0.1 g/L). It was found that mito2HOBA diminished angiotensin II-induced hypertension. Hypertension was associated with increased formation of mitochondrial isoLG measured by Western blot analysis of heart mitochondria using the anti-isoLG D11 scfv antibody and 2-fold decrease in kidney ATP. mito2HOBA reduced accumulation of isoLG adducts in the heart mitochondria and prevented drop in kidney ATP in angiotensin II-infused mice. Decrease of NO is a hallmark of endothelial dysfunction in hypertension due to vascular oxidative stress. We examined vascular oxidative stress by measurements of aortic O 2 ●– using fluorescent O 2 ●– probe DHE and HPLC, and analysis of endothelial NO using Electron Spin Resonance and specific NO spin trap Fe(DETC) 2 . It was found that mito2HOBA reduced vascular O 2 ●– in angiotensin II-infused mice and preserved endothelial NO. These data support the role of mitochondrial oxidative stress and mitochondrial isoLG in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isoLG may have therapeutic potential in cardiovascular disease.