Integrin-Linked Kinase Regulates Vasomotor Function by Preventing Endothelial Nitric Oxide Synthase Uncoupling

Abstract

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...

iNOS-Derived Nitric Oxide Induces Integrin-Linked Kinase Endocytic Lysosome-Mediated Degradation in the Vascular Endothelium

“…Confirming our earlier observations, 6 Western blot analysis revealed a significant decrease in total ILK expression in atherosclerotic carotid arteries versus healthy vessels ( Figure 1A). However, iNOS levels were increased in atherosclerotic vessels compared with healthy arteries, suggesting a link between ILK downregulation and iNOS expression.…”

“…6,7,20 Thus, we tested superoxide levels in MAEC isolated from WT and iNOS knockout mice, finding an increase in superoxide production in TNF-α-treated WT MAEC, which was not observed in iNOS knockout mice ( Figure 2B). This increase could also be detected in WT MAEC treated with 2 different NO donors ( Figure 2C), which differ in the timing of NO release.…”

“…5 Integrin-linked kinase (ILK), a canonical component of the integrin cell adhesion machinery, binds to eNOS, protects eNOS from uncoupling, and prevents vascular oxidative stress by preventing reactive oxygen species production by uncoupled eNOS. 6 In addition, ILK can regulate eNOS expression. 7 ILK upregulation promotes a broadly cardioprotective role.…”

“…7 ILK upregulation promotes a broadly cardioprotective role. 8 In the vasculature, ILK regulates angiogenesis, 9 endothelial survival, 10 apoptosis, 11 inflammation, 12 vasomotor tone, 6 vascular remodeling, 13 and cardiac remodeling. 14 In addition, ILK plays an important role during heart and vascular wall development.…”

“…16 We had previously reported the strike correlation between attenuation of ILK and atherosclerosis progression. 6 We, therefore, questioned whether iNOS-derived NO could be involved in ILK-decreased expression during atherosclerosis and vascular remodeling. In the current work, we show that NO released by iNOS decreases ILK levels and induces ILK endocytosis and degradation in the lysosomal compartment ILK.…”