Peroxynitrite Causes Endoplasmic Reticulum Stress and Apoptosis in Human Vascular Endothelium

Abstract

Objective-Peroxynitrite, a potent oxidant generated by the reaction of NO with superoxide, has been implicated in the promotion of atherosclerosis. We designed this study to determine whether peroxynitrite induces its proatherogenic effects through induction of endoplasmic reticulum (ER) stress. Methods and Results-Human vascular endothelial cells treated with Sin-1, a peroxynitrite generator, induced the expression of the ER chaperones GRP78 and GRP94 and increased eIF2␣ phosphorylation. These effects were inhibited by the peroxynitrite scavenger uric acid. Sin-1 caused the depletion of ER-Ca 2ϩ , an effect known to induce ER stress, resulting in the elevation of cytosolic Ca 2ϩ and programmed cell death (PCD). Sin-1 treatment was also found, via 3-nitrotyrosine and GRP78 colocalization, to act directly on the ER. Adenoviral-mediated overexpression of GRP78 in endothelial cells prevented Sin-1-induced PCD. Consistent with these in vitro findings, 3-nitrotyrosine was observed and colocalized with GRP78 in endothelial cells of early atherosclerotic lesions from apolipoprotein E-deficient mice. Conclusions-Peroxynitrite is an ER stress-inducing agent. Its effects include the depletion of ER-Ca 2ϩ , a known mechanism of ER stress induction. The observation that 3-nitrotyrosine-containing proteins colocalize with markers of ER stress within early atherosclerotic lesions suggests that peroxynitrite contributes to atherogenesis through a mechanism involving ER stress. Key Words: endothelium Ⅲ nitric oxide Ⅲ endoplasmic reticulum Ⅲ atherosclerosis Ⅲ calcium E ndothelial dysfunction/injury represents a key early step in atherogenesis. The majority of risk factors for atherosclerosis, including, hyperlipidemia, hypertension, diabetes, and smoking, are associated with endothelial dysfunction. 1 A major function of the vascular endothelium is the regulation of vascular tone by NO through NO-induced smooth muscle relaxation. 2 Reduced NO bioavailability is a common feature in atherosclerosis and can result from oxidative stress. 3 In the apolipoprotein E-deficient (apoE Ϫ/Ϫ ) mouse model of atherosclerosis, endothelial dysfunction, as shown by decreased NO-mediated vasodilation to acetylcholine, correlates with increased atherosclerotic lesion size. 4 The availability of NO and superoxide (. O 2 -) within the atherosclerotic lesion creates the conditions for peroxynitrite formation because NO and . O 2 -react at physiological pH to form peroxynitrite. 5 A marker of peroxynitrite generation, 3-nitrotyrosine (3-NT) is elevated in human atherosclerotic lesions. 6,7 However, the mechanism by which reduced NO bioavailability and peroxynitrite formation contribute to atherosclerosis remains uncertain. See coverEndoplasmic reticulum (ER) stress, a cellular stress pathway induced by the accumulation of unfolded proteins in the ER, may offer an explanation for the contribution of peroxynitrite to atherosclerosis. ER stress results in an evolutionary conserved cellular response involving the upregulation of a set of genes, including ...

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