The Binding of Oxidized Low Density Lipoprotein (ox-LDL) to ox-LDL Receptor-1 Reduces the Intracellular Concentration of Nitric Oxide in Endothelial Cells through an Increased Production of Superoxide

Abstract

Endothelium-dependent relaxation is impaired in animals with atherosclerosis (1-3), which has been linked to a decreased production and/or biological activity of endotheliumderived nitric oxide (NO) 1 (4, 5). Oxidative inactivation of NO is regarded as an important cause of its decreased biological activity (6). The vascular release of superoxide (O 2 . ) radicals is sharply increased in atherosclerotic arteries (7,8), and O 2 . is known to inactivate NO in a chemical reaction during which the cytotoxic radical peroxynitrite is formed (9, 10). The presence of peroxynitrite-derived nitrotyrosines has recently been demonstrated in human atherosclerotic lesions (11). Oxidized low density lipoprotein (ox-LDL) has been observed to induce abnormalities in endothelial function, which may be relevant for the progression of atherosclerotic lesions (12). In particular functional alterations of the endothelial cells may be involved in the reduction of vasodilation, in response to stimuli that induce NO release, in isolated arteries exposed to ox-LDL (13).Recently, an endothelial receptor for ox-LDL, called lectinlike ox-LDL receptor-1 (LOX-1) was cloned from cultured bovine aortic endothelial cells (BAECs) (14). It has been suggested that ox-LDL uptake through this receptor may be involved in endothelial activation or dysfunction in atherogenesis (14). In this context we recently reported that ox-LDL binding to LOX-1 determined a significant increase in the generation of reactive oxygen species (ROS) in endothelial cells (15). In this report we investigated the relationship between the intracellular production of ROS and in particular of O 2. and the intracellular concentration of NO in cultures of BAECs exposed to ox-LDL. MATERIALS AND METHODSLDL Isolation-Whole blood, obtained by venipuncture from healthy volunteers after 12 h of fasting, was collected into Vacutainer tubes (Becton Dickinson, Meylan, France) containing EDTA (1 mg/ml) and processed for LDL separation within 1 day by sequential flotation in NaBr solution (16) containing 1 mg/ml EDTA.LDL Oxidation and Modification-Cu 2ϩ -modified LDL (1.7 mg of protein/ml) was prepared by exposure of LDL to 5 M CuS0 4 for 18 h at 37°C as described previously (17,18). The extent of LDL oxidation was determined by thiobarbituric acid-reactive substances as reported (18). Protein was measured by the Pierce BCA protein assay reagent (19). Malondialdehyde-modified LDL (MDA-LDL) was prepared according to a previously described method (20, 21). Acetylation of LDL was achieved by repeated additions of acetic anhydride (22).Cell Cultures-BAECs were isolated and cultured as described previously (23). Cells used for experiments were at passage levels between * This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Ministry of Health, Labour and Welfare of Japan; the Organization for Pharmaceutical Safety and Research; Takeda Science Foundation; and DNO Medical Research Foundation. The costs of publication of this...

Lectin‐like Oxidized Low‐density Lipoprotein Receptor‐1, a New Promising Target for the Therapy of Atherosclerosis?

“…Incubation with ox-LDL also induced the increased production of intracellular superoxide anion and hydrogen peroxide, which was also mediated by LOX-1 (Cominacini et al 2000(Cominacini et al , 2001. It has also been shown that the binding of ox-LDL to LOX-1 decreases intracellular nitric oxide levels through an increased production of superoxide anion, which could inactivate nitric oxide (Cominacini et al 2001). Both superoxide anion and hydrogen peroxide are products of LOX-1 activation and inducers of LOX-1 expression (Nagase et al 2001).…”

“…Most of the information about the signal transduction pathways of LOX-1 came from the activation or inhibition of LOX-1 by ox-LDL and LOX-1 antibody. Using bovine aortic endothelial cells and CHO-K1 cells transfected with the LOX-1 gene (LOX-1-CHO-K1), Cominacini et al (2000Cominacini et al ( , 2001 revealed that the binding of ox-LDL to LOX-1 induces a sharp increase of intracellular reactive oxygen species (ROS) production determined by 2 ,7 -dichlorofluorescein diacetate (DCFH 2 -DA) assay as early as 5 minutes after the start of incubation with ox-LDL. Native LDL (nLDL), acetylated LDL (Ac-LDL) and malondialdehyde-modified LDL (MDA-LDL) show no effect.…”

Mechanisms of H2O2-induced oxidative stress in endothelial cells

“…Endothelial cell dependent relaxation of vascular smooth muscle cells (SMC) is impaired in atherosclerosis (Cominacini et al 2001) due to decreased availability of nitric oxide (NO). Reduction in NO availability may result in an additional down-regulation of NO formation, though this is still controversial (Ignarro 1990 ).…”

“…NADPH oxidase, NOS to a lesser extent, and xanthine oxidase are implicated in increased ROS in endothelial cells upon exposure to minimally oxidized LDL (oxLDL, (Heinloth et al 2000;Cominacini et al 2001;Rueckschloss et al 2001;Stepp et al 2002). NADPH oxidase is also an important contributor to O 2 ·-levels in smooth muscle cells due to H 2 O 2 exposure ).…”

Lectin-like oxidized LDL receptor-1 is a cell-adhesion molecule involved in endotoxin-induced inflammation

“…The expression of LOX-1 is induced by stimuli as rapidly as other kinds of cell-adhesion molecules and selectins, suggesting that LOX-1 is in the so-called class of immediate early genes. LOX-1 is a potent mediator of ''endothelial dysfunction'': binding of endothelial LOX-1 by ligands induces superoxide generation, inhibits nitric oxide production, enhances endothelial adhesiveness for leukocytes, and induces expression of chemokines (14)(15)(16). The versatile activities of LOX-1 include the ability to bind not only oxidized LDL, but also aged red blood cells, apoptotic cells, and activated platelets (17,18).…”