Results: ILK expression was detected in the endothelial cell layer of nonatherosclerotic vessels but was absent from the endothelium of atherosclerotic arteries. Live ultrasound imaging revealed that acetylcholine-mediated vasodilatation was impaired in cKO mice. These mice exhibited lowered agonist-induced nitric oxide synthase (NOS) activity and decreased cyclic guanosine monophosphate and nitrite production. ILK deletion caused endothelial NOS (eNOS) uncoupling, reflected in reduced tetrahydrobiopterin (BH4) levels, increased BH2 levels, decreased dihydrofolate reductase expression, and increased eNOS-dependent generation of superoxide accompanied by extensive vascular protein nitration. ILK reexpression prevented eNOS uncoupling in cKO cells, whereas superoxide formation was unaffected by ILK depletion in eNOS-KO cells, indicating eNOS as a primary source of superoxide anion. eNOS and ILK coimmunoprecipitated in aortic lysates from control animals, and eNOS-ILK-shock protein 90 interaction was detected in human normal mammary arteries but was absent from human atherosclerotic carotid arteries. eNOS-ILK interaction in endothelial cells was prevented by geldanamycin, suggesting heat shock protein 90 as a binding partner. Key Words: atherosclerosis Ⅲ oxidative stress Ⅲ uncoupling protein E ndothelial dysfunction is defined as impaired endothelium-dependent relaxation of blood vessels in response to the endogenous vasodilator nitric oxide (NO). Endothelial dysfunction is concomitant with changes in vascular structure associated with many forms of vascular disease, such as hypertension and atherosclerosis. 1 Atherosclerotic lesions develop mostly in areas exposed to disturbed blood flow, whereas endothelial cells exposed to laminar flow are protected against inflammatory activation and show higher relative expression of endothelial NO synthase (eNOS) and superoxide dismutase. 2,3 Most NO in the vasculature is produced by eNOS, with a minor contribution from neuronal-type nNOS expressed in vascular smooth muscle cells. Under inflammatory conditions, vascular cells can express iNOS, which produces large amounts of NO and contributes further to vascular damage. 4 eNOS can be activated by hemodynamic forces, autacoids, hormones, and growth factors. NO relaxes vessels via activation of soluble guanylyl cyclase (sGC). The resulting elevated levels of cyclic guanosine monophosphate (cGMP) activate cGMP-dependent protein kinase type I (cGKI), which phosphorylates downstream targets that regulate the Original received July 29, 2011; revision received December 13, 2011; accepted December 14, 2011. In November 2011 actin-myosin cytoskeleton and the calcium clearing mechanism, leading to vasorelaxation. 5 Many endothelial cell molecules, including integrins, sense shear stress. 6,7 Integrin-linked kinase (ILK), a key regulator of blood vessel integrity, is a phosphoinositide 3-kinasedependent serine/threonine kinase that binds to the cytoplasmic domain of ß-integrin and lies upstream of many intracellular signaling pa...

Objective-Hydrogen peroxide (H 2 O 2 ) is an important mediator in the vasculature, but its role in the regulation of soluble guanylate cyclase (sGC) activity and expression is not completely understood. The aim of this study was to test the effect of H 2 O 2 on sGC expression and function and to explore the molecular mechanism involved. Methods and Results-H 2 O 2 increased sGC␤1 protein steady-state levels in rat aorta and aortic smooth muscle cells (RASMCs) in a time-and dose-dependent manner, and this effect was blocked by catalase. sGC␣2 expression increased along with ␤1 subunit, whereas ␣1 subunit remained unchanged. Vascular relaxation to an NO donor (sodium nitroprusside) was enhanced by H 2 O 2 , and it was prevented by ODQ (sGC inhibitor). cGMP production in both freshly isolated vessels and RASMCs exposed to H 2 O 2 was greatly increased after sodium nitroprusside treatment. The H 2 O 2 -dependent sGC␤1 upregulation was attributable to sGC␤1 mRNA stabilization, conditioned by the translocation of the mRNA-binding protein HuR from the nucleus to the cytosol, and the increased mRNA binding of HuR to the sGC␤1 3Ј untranslated region. HuR silencing reversed the effects of H 2 O 2 on sGC␤1 levels and cGMP synthesis. has been proposed to be an endothelium-derived hyperpolarizing factor in peripheral arteries. 12,13 H 2 O 2 may also dilate arteries by alternative mechanisms, such as the stimulation of the production of prostanoids 14 or the activation of soluble guanylate cyclase (sGC). 15 Moreover, under some circumstances, H 2 O 2 may increase the vascular content of endothelial nitric oxide synthase and to stimulate its activity, with subsequently increased synthesis of nitric oxide (NO). 16 -18 Most of cellular effects of NO depend on the interaction with its main receptor, sGC, and ROS could interact with this receptor, thus modifying the cellular response to NO. In fact, several studies have shown that cGMP levels increase during ischemia, a pathological condition characterized by increased ROS synthesis. 19,20 In addition, sGC can be modulated by ROS, modifying its activity after brief exposition to oxidants, [21][22][23] and recent work suggests that sGC expression and activity may be attenuated by ROS in rat aortic smooth muscle cells (RASMCs) 24 and ovine pulmonary artery smooth muscle cells. 25 Moreover, Courtois et al 26 proposed that a superoxide aniondependent sGC downregulation may be involved in the genesis of the hypertension induced by chronic lead administration. Conclusion-OurThe present study was devoted to analyzing the ability of ROS, particularly H 2 O 2 , to modulate the cellular content of MethodsDetails regarding materials, animal studies, RASMC isolation, and immunohistochemical studies; measurement of cGMP, RNA isolation, and RNA interference; RNA-electrophoretic mobility shift assay (RNA-EMSA); and statistics are provided in an expanded Methods section in the online Data Supplement, available at http://atvb.ahajournals.org. Vascular ReactivityThe preparation of the aortic ri...