Role of TNF-α-induced reactive oxygen species in endothelial dysfunction during reperfusion injury

We hypothesized that neutralization of TNF-α at the time of reperfusion exerts a salubrious role on endothelial function and reduces the production of reactive oxygen species. We employed a mouse model of myocardial ischemia-reperfusion (I/R, 30 min/90 min) and administered TNF-α neutralizing antibodies at the time of reperfusion. I/R elevated TNF-α expression (mRNA and protein), whereas administration of anti-TNF-α before reperfusion attenuated TNF-α expression. We detected TNF-α expression in vascular smooth muscle cells, mast cells, and macrophages, but not in the endothelial cells. I/R induced endothelial dysfunction and superoxide production. Administration of anti-TNF-α at the onset of reperfusion partially restored nitric oxide-mediated coronary arteriolar dilation and reduced superoxide production. I/R increased the activity of NAD(P)H oxidase and of xanthine oxidase and enhanced the formation of nitrotyrosine residues in untreated mice compared with shams. Administration of anti-TNF-α before reperfusion blocked the increase in activity of these enzymes. Inhibition of xanthine oxidase (allopurinol) or NAD(P)H oxidase (apocynin) improved endothelium-dependent dilation and reduced superoxide production in isolated coronary arterioles following I/R. Interestingly, I/R enhanced superoxide generation and reduced endothelial function in neutropenic animals and in mice treated with a neutrophil NAD(P)H oxidase inhibitor, indicating that the effects of TNF-α are not through neutrophil activation. We conclude that myocardial ischemia initiates TNF-α expression, which induces vascular oxidative stress, independent of neutrophil activation, and leads to coronary endothelial dysfunction.