In this study we examined the effect of oxidized low density lipoprotein (ox-LDL) on the intracellular production of reactive oxygen species (ROS) in bovine aortic endothelial cells (BAECs) and whether this increase occurs through its binding to the endothelial receptor lectin-like ox-LDL receptor-1 (LOX-1). Furthermore, this study also aimed to ascertain whether the binding of ox-LDL to LOX-1 is associated with NF-B activation. ox-LDL induced a significant dose-dependent increase in ROS production after a 30-s incubation with BAECs (p < 0.01). ROS formation was markedly reduced in BAECs incubated with anti-LOX-1 monoclonal antibody (p < 0.001), while control nonimmune IgG produced no effect. ox-LDL induced a time-and dose-dependent significant increase in ROS formation only in CHO-K1 cells stably expressing bovine LOX-1 (p < 0.001), while no increase was present in CHO-K1 cells. The activation of the transcription factor NF-B in BAECs was evident after a 5-min incubation with ox-LDL and was attenuated by anti-LOX-1 monoclonal antibody. The conclusion is that one of the pathophysiological consequences of ox-LDL binding to LOX-1 may be the activation of NF-B through an increased ROS production.

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...

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Atherosclerosis is the major cause of death in patients with diabetes mellitus, accounting for more than 70 % of mortality in all forms of the disease [1,2]. There is plenty of evidence that monocytes are involved in the pathogenesis of atherosclerosis [3,4], the earliest morphological evidence of the disease being the binding of monocytes to the endothelium [5][6][7]. This adhesion of monocytes, a prerequisite for their recruitment and accumulation in the lesion, is probably due to the appearance of adhesion Diabetologia (1997) Summary Although elevated levels of soluble E-selectin and intercellular cell adhesion molecules-1 (ICAM-1) have been reported in non-insulin-dependent diabetes mellitus (NIDDM), it is not clear by what mechanism this elevation occurs and whether or not it is related to glycaemic control. In this study we analyse: 1) the relation of glycaemic control with the concentrations of E-selectin, vascular cell adhesion molecules-1 (VCAM-1) and ICAM-1 in NIDDM patients; 2) whether metabolic control can affect the oxidative stress (as measured by plasma hydroperoxide concentration and susceptibility of LDL to in vitro oxidation) and hence the adhesion molecule plasma concentrations. Thirty-four (19 males and 15 females) poorly controlled NIDDM patients were studied. All parameters were evaluated at the beginning of the study and after 90 days of dietary and pharmacological treatment. The treatment decreased HbA 1C (p < 0.001), E-selectin (p < 0.001), plasma hydroperoxides (p < 0.003) and the susceptibility of LDL to in vitro oxidation (lag phase) (p < 0.0001). Before treatment HbA 1C , lag phase and lipid hydroperoxides correlated with E-selectin plasma concentration (r = 0.51, -0.57 and 0.54, respectively, p < 0.01). There was also a correlation between HbA 1C and lag phase (p < 0.01) and between HbA 1C and lipid hydroperoxides (p < 0.01). In addition, the variations of HbA 1C , lag phase and lipid hydroperoxide values correlated with those for E-selectin concentration after 90 days' treatment (r = 0.54, -0.64 and 0.61, respectively, p < 0.01). In multiple linear correlation analysis, however, the partial correlation coefficients of HbA 1C (basal and variations) with Eselectin concentration (basal and variations) fell to non-significant values (r = 0.12 and 0.25, respectively) when LDL lag phase and plasma hydroperoxides were kept constant. The results indicate that the improvement of metabolic control in NIDDM patients is associated with a decrease of E-selectin plasma levels; they also suggest that glycaemic control per se is not directly implicated in determining E-selectin plasma concentration; glycaemic control could affect E-selectin concentration through its effect on oxidative stress. [Diabetologia (1997) 40: 584-589]

ox-LDL increased the intracellular formation of ROS through the ligation to a specific endothelial receptor. Preincubation of HUVECs with lacidipine, a calcium antagonist with antioxidant properties, significantly reduced the intracellular ROS formation induced by ox-LDL. We propose that the effect of lacidipine on adhesion molecule expression and on NF-kB activation can be explained by its effect on intracellular ROS formation.