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 O₂ ⁻ were assessed with fluorescence imaging. Perfusion of vessels with H₂O₂ (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 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.