H₂O₂-induced endothelial NO production contributes to vascular cell apoptosis and increased permeability in rat venules

Although elevated levels of H₂O₂ have been implicated to play important roles in the pathogenesis of various cardiovascular diseases, the underlying mechanisms remain unclear. This study aims to examine the effect of H₂O₂ on endothelial nitric oxide (NO) production in intact venules, and elucidate the role and mechanisms of NO in H₂O₂-induced increases in microvessel permeability. Experiments were conducted on individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp), and endothelial [Ca²⁺]i was measured on fura-2-loaded vessels. Perfusion of H₂O₂ (10 μM) caused a delayed and progressively increased endothelial [Ca²⁺]i and Lp, a pattern different from inflammatory mediator-induced immediate and transient response. Under the same experimental conditions, measuring endothelial NO via DAF-2 and the spatial detection of cell apoptosis by fluorescent markers revealed that H₂O₂ induced two phases of NO production followed by caspase activation, intracellular Ca²⁺ accumulation, and vascular cell apoptosis. The initial NO production was correlated with increased endothelial NO synthase (eNOS) Ser¹¹⁷⁷ phosphorylation in the absence of elevated endothelial [Ca²⁺]i, whereas the second phase of NO depended on increased [Ca²⁺]i and was associated with Thr⁴⁹⁵ dephosphorylation without increased Ser¹¹⁷⁷ phosphorylation. Inhibition of NOS prevented H₂O₂-induced caspase activation, cell apoptosis, and increases in endothelial [Ca²⁺]i and Lp. Our results indicate that H₂O₂ at micromolar concentration is able to induce a large magnitude of NO in intact venules, causing caspase activation-mediated endothelial Ca²⁺ accumulation, cell apoptosis, and increases in permeability. The mechanisms revealed from intact microvessels may contribute to the pathogenesis of oxidant-related cardiovascular diseases.