H₂ O₂ -induced microvessel barrier dysfunction: the interplay between reactive oxygen species, nitric oxide, and...

Elevated H₂ O₂ is implicated in many cardiovascular diseases. We previously demonstrated that H₂ O₂ -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 H₂ O₂ -induced vascular barrier dysfunction by elucidating the interrelationships between H₂ O₂ -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 O₂^- were assessed with fluorescence imaging. Perfusion of vessels with H₂ O₂ (10 µmol/L) induced marked productions of NO and O₂^- , 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 H₂ O₂ -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 H₂ O₂ -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 H₂ O₂ -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.