Figure 6. Intracellular H₂O₂ in endothelial cells regulate endothelial nitric oxide synthase activation in vivo.

A, intracellular redox status was measured by 2′,7′-dichlorfluorescein-diacetate (DCF-DA) staining in gated CD31⁺/CD45⁻ population of collagenase-digested ischemic muscles at day 3. The dotted lines indicate the background signals without DCF-DA. B, ischemic muscles from Wild-type (WT) and Tie2-driven catalase transgenic (Cat-Tg) mice at day 3 were isolated and incubated. Their H₂O₂ production was measured by Amplex Ultra Red assay. C, harvested ischemic and non-ischemic muscles at day 3 were analyzed for protein expression of phosphorylated and total form of endothelial nitric oxide synthase (eNOS), Akt and ERK1/2 (as control) by Western analysis. Densitometry analysis in activation (phosphorylation) of each protein is shown. All data shown are mean+SE (n = 3 mice per group, *p<0.05, **p<0.01 and ***p<0.001).