H2O2‐induced Microvessel Barrier Dysfunction: the Interplay Between Reactive Oxygen Species, Nitric Oxide, and Peroxynitrite

Elevated H2O2 is implicated in many cardiovascular diseases. We previously demonstrated that H2O2-induced endothelial nitric oxide synthase (eNOS) activation and excessive NO production contribute to vascular cell injury and increases in microvessel permeability. However, the mechanisms of excessive NO-mediated vascular injury and hyperpermeability remain unknown. This study aims to examine the functional role of NO-derived peroxynitrite (ONOO-) in H2O2-induced vascular barrier dysfunction by elucidating the interrelationships between H2O2-induced NO, superoxide, ONOO-, and changes in endothelial [Ca2+](i) and microvessel permeability. Experiments were conducted on intact rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca2+](i), NO, and O-2(-) were assessed with fluorescence imaging. Perfusion of vessels with H2O2 (10 mu mol/L) induced marked productions of NO and O-2(-), resulting in extensive protein tyrosine nitration, a biomarker of ONOO-. The formation of ONOO- was abolished by inhibition of NOS with N-G-Methyl-L-arginine. Blocking NO production or scavenging ONOO- by uric acid prevented H2O2-induced increases in endothelial [Ca2+](i) and Lp. Additionally, the application of exogenous ONOO- to microvessels induced delayed and progressive increases in endothelial [Ca2+](i) and microvessel Lp, a pattern similar to that observed in H2O2-perfused vessels. Importantly, ONOO- caused further activation of eNOS with amplified NO production. We conclude that the augmentation of NO-derived ONOO- is essential for H2O2-induced endothelial Ca2+ overload and progressively increased microvessel permeability, which is achieved by self-promoted amplifications of NO-dependent signaling cascades. This novel mechanism provides new insight into the reactive oxygen and/or reactive nitrogen species-mediated vascular dysfunction in cardiovascular diseases.