A.M. Pastorino

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

Oxidized low density lipoprotein (ox-LDL) has been suggested to affect endothelium-dependent vascular tone through a decreased biological activity of endothelium-derived nitric oxide (NO). Oxidative inactivation of NO is regarded as an important cause of its decreased biological activity, and in this context superoxide (O⨪2) is known to inactivate NO in a chemical reaction during which peroxynitrite is formed. In this study we examined the effect of ox-LDL on the intracellular NO concentration in bovine aortic endothelial cells and whether this effect is influenced by ox-LDL binding to the endothelial receptor lectin-like ox-LDL receptor-1 (LOX-1) through the formation of reactive oxygen species and in particular of O⨪2. ox-LDL induced a significant dose-dependent decrease in intracellular NO concentration both in basal and stimulated conditions after less than 1 min of incubation with bovine aortic endothelial cells (p < 0.01). In the same experimental conditions ox-LDL also induced O⨪2 generation (p < 0.001). In the presence of radical scavengers and anti-LOX-1 monoclonal antibody, O⨪2formation induced by ox-LDL was reduced (p < 0.001) with a contemporary rise in intracellular NO concentration (p < 0.001). ox-LDL did not significantly modify the ability of endothelial nitric oxide synthase to metabolizel-arginine to l-citrulline. The results of this study show that one of the pathophysiological consequences of ox-LDL binding to LOX-1 may be the inactivation of NO through an increased cellular production of O⨪2.

Predisposition to LDL oxidation in patients with and without angiographically established coronary artery disease

Oxidative modification of low density lipoprotein (LDL) may play an important role in the mechanism of atherosclerotic damage to blood vessels. In the present study the LDL isolated from the plasmas of 73 coronary artery disease (CAD) patients, 28 valvular heart disease (VHD) patients, 59 subjects affected by type IIa hyperlipoproteinemia and 71 controls was oxidatively modified by incubation with copper ions. In 15 CAD and 15 Type IIa patients and 15 controls the LDL chemical composition and polyunsaturated fatty acid (PUFA) content were also measured. Differences in the LDL susceptibilities to lipid peroxidation were studied by measuring the changes of fluorescence intensity. The lag phase in the CAD patients was found to be significantly lower than in the VHD and controls (P < 0.001). The lag phase in the type IIa patients was significantly higher than in the CAD patients (P < 0.01), and significantly lower than the VHD and controls (P < 0.01). The LDL isolated from the type IIa patients had an increase in the relative content of free and esterified cholesterol (P < 0.05), while the CAD patients had a decrease in the relative content of free cholesterol (P < 0.05), and an increase in the relative content of protein (P < 0.05). The lowest value of the LDL cholesterol to protein ratio and LDL size, was found in the CAD patients (P < 0.05). When expressed in micrograms/mg LDL cholesterol, the concentration of the LDL PUFAs was significantly higher in the CAD group than in the others (P < 0.05). The LDL alpha-tocopherol concentration was quite similar in the different groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidized low-density lipoprotein increases the production of intracellular reactive oxygen species in endothelial cells : inhibitory effect of lacidipine

Objective The mechanisms by which oxidized low-density lipoprotein (ox-LDL) induces the expression of adhesion molecules on endothelial cells (HUVECs) are still not clear. The signal transduction pathways for these binding molecules include the translocation of the transcription factor NF-kB and the intracellular reactive oxygen species (ROS) are said to play a key role in this process. Aim of this study was (1) to evaluate the effect of ox-LDL on intracellular production of ROS in culture of HUVECs; (2) to evaluate if the intracellular increase of ROS induced by ox-LDL is mediated by the binding to a specific endothelial receptor; (3) to ascertain if lacidipine can decrease ox-LDL-induced ROS production in HUVECs. Methods Five *MCu2 + ox-LDL were incubated with HUVECs for 5 min. 2′,7′-Dichlorofluorescein (DCF) as an expression of intracellular ROS production, was measured by flow cytometry. Results ox-LDL induced a significant dose-dependent increase in DCF production (P < 0.001) through the binding to a specific receptor. The preincubation of HUVECs with radical scavengers compounds and lacidipine significantly reduced (P < 0.001) the ox-LDL-induced DCF production. Conclusions 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.

Lacidipine inhibits the activation of the transcription factor NF-kappa B and the expression of adhesion molecules induced by pro-oxidant signals on endothelial cells

Objective The adhesion of monocytes to endothelium, an early event in atherosclerosis is mediated by cell adhesion molecules. Signal-transduction pathways for these binding molecules include the translocation of the transcription factor NF-κB; moreover, intracellularly generated oxygen-derived free radicals play a major role in this process. In this study we evaluated the extent to which lacidipine, a calcium antagonist with antioxidant properties, affects the expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin on human umbilical vein endothelial cells, induced by different pro-oxidant signals such as oxidized low density lipoprotein (LDL) and tumor necrosis factor-α (TNF-α). Methods We incubated 5 mmol/l Cu2+-oxidized LDL and TNF-α (2 ng/ml) with human umbilical vein endothelial cells for 48 and 6 h, respectively. ICAM-1, VCAM-1 and E-selectin were measured by flow cytometry. NF-κB was evaluated by electrophoretic mobility shift assay. Results The incubation of 5 μmol/l Cu2+-oxidized LDL not only caused a dose-dependent increase in ICAM-1, VCAM-1 and E-selectin (P <0.001), but also synergically increased their TNF-α–induced expression (P <0.001). The addition of lacidipine to human umbilical vein endothelial cells significantly reduced the expression of ICAM-1, VCAM-1 and E-selectin induced by TNF-α alone or with oxidized LDL (P <0.001). The reduction in adhesion molecule expression caused by lacidipine was paralleled by a significant fall in NF-κB translocation. Conclusions The results suggest that lacidipine may have prevented NF-κB-mediated adhesion molecule expression by exerting its effects on oxygen-derived free radicals. The results support previous observations that lacidipine may have therapeutic effects in atherosclerosis.

The platelet-endothelium interaction mediated by lectin-like oxidized low-density lipoprotein receptor-1 reduces the intracellular concentration of nitric oxide in endothelial cells

OBJECTIVES To address the potential role of the endothelial lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in the thrombotic system, in this study we first examined whether platelet interaction with LOX-1 generated reactive oxygen species (ROS) and superoxide (O2.-) and then investigated the relationship between the intracellular production of O2.- and the availability of nitric oxide (NO). BACKGROUND Oxidative inactivation of NO is regarded as an important cause of its decreased biologic activity which may favor platelet-dependent arterial thrombosis. METHODS Bovine aortic endothelial cells (BAECs) and Chinese hamster ovary-K1 cells stably expressing bovine LOX-1 (BLOX-1-CHO) were incubated at different times with human platelets. The ROS, O2.-, and NO were measured in cells by flow cytometry. RESULTS The incubation of BAECs and BLOX-1-CHO cells with human platelets induced a sharp and dose-dependent increase in intracellular concentration of ROS and O2.- (p from <0.01 to <0.001). The increase in intracellular concentration of O2.- was followed by a dose-dependent reduction in basal and bradykinin-induced intracellular NO concentration (p from <0.01 to <0.001). The increase in O2.- and the reduction of NO were inhibited by the presence of vitamin C and anti-LOX-1 monoclonal antibody (p < 0.001). CONCLUSIONS The results of this study show that one of the pathophysiologic consequences of platelet binding to LOX-1 may be the inactivation of NO through an increased cellular production of O2.-.

Effects of troglitazone on in vitro oxidation of LDL and HDL induced by copper ions and endothelial cells

Summary Troglitazone is a new oral antidiabetic agent able to reduce lipid peroxidation. In this study we evaluated its effect on the susceptibility of LDL and HDL to in vitro oxidation induced by copper ions and endothelial cells. In Cu ++ -induced LDL modification, different amounts of troglitazone were added to aliquots of the same pool of plasma with subsequent ultracentrifuge separation of LDL and HDL. Differences in LDL and HDL susceptibility to in vitro oxidation with Cu ++ were studied by measuring the changes in fluorescence intensity (expressed as lag phase). LDL derived from plasma incubated with different amounts of troglitazone were also incubated with umbilical vein endothelial cells (HUVEC), the modification being monitored by LDL relative electrophoretic mobility and fluorescence. During Cu ++ - and HUVEC-induced LDL oxidation, the decay rate of vitamin E, and the potency of troglitazone as a radical scavenger in comparison with vitamin E were also studied. Troglitazone determined a significant, dose-dependent decrease in Cu ++ -induced LDL and HDL oxidation. Incubation with HUVEC was also followed by a progressive, significant decrease of LDL relative electrophoretic mobility and fluorescence intensity. During Cu ++ - and HUVEC-induced-LDL modification, troglitazone significantly reduced the rate of vitamin E decay. In this study we also demonstrated that under the same oxidative stress, troglitazone was much more potent as a radical scavenger than vitamin E. In conclusion, the results demonstrate that troglitazone can reduce LDL and HDL in vitro oxidation and that, during this process, it can protect vitamin E. In addition to ensuring blood glucose control, the drug may therefore be useful in inhibiting lipoprotein peroxidation. [Diabetologia (1997) 40: 165–172]

Predisposition to LDL oxidation during copper-catalyzed oxidative modification and its relation to α-tocopherol content in humans

The predisposition to LDL oxidation during copper-catalyzed oxidative modification and its relationship with LDL alpha-tocopherol concentration was studied in 41 control subjects. The results show that the predisposition of LDL to oxidation expressed as duration of the inhibition period and rate of the propagation period varied greatly in the controls, but did not correlate with the values of LDL alpha-tocopherol. On the contrary the experiments with alpha-tocopherol incorporated in LDL demonstrate that even small increases of incorporated alpha-tocopherol, under circumstances where other variables were probably largely unaffected, increased proportionally the length of the inhibition period and reduced the rate of the propagation period. The values of LDL alpha-tocopherol achieved after the enrichment turned out to be positively correlated with the duration of the inhibition period and negatively with the rate of the propagation period. Finally the results of this study also show that there was a variability in the LDL alpha-tocopherol decay of different subjects under the same oxidative stress. In our conditions however, the time in which alpha-tocopherol contributed to the LDL protection was much shorter than the mean length of the inhibition period. The results demonstrate that the variability in the predisposition to LDL oxidation during copper-catalyzed oxidative modification is not determined only by the concentration of alpha-tocopherol in LDL and that therefore its value as a sole indicator of antioxidant status is probably inadequate.

The calcium channel blocker lacidipine reduces the development of atherosclerotic lesions in the apoE-deficient mouse.

Background Lacidipine is a widely used calcium-channel blocker, which has both long-lasting antihypertensive activity and also antioxidant properties. Previous studies have demonstrated the ability of lacidipine to reduce the development of atherosclerotic lesions in several animal models. Objective The present study investigated the anti-atherosclerotic potential of lacidipine in the apoE-deficient mouse, an experimental model of atherosclerosis showing progressively complex and widespread lesions which closely resemble the inflammatory-fibrous plaques seen in humans. Methods Lacidipine was administered daily by gavage for 10 weeks at dose levels of 0 (control), 0.3, 1.0 and 3.0 mg/kg. Results Lacidipine administration reduces the extension of atherosclerotic lesions in the aorta of the apoE-deficient mouse without affecting plasma lipid levels. We also show that apoE-deficient mice have four-fold higher values of the proatherogenic peptide, endothelin, compared with the wild-type C57BL/6 mouse and that lacidipine administration reduced, in a dose-dependent manner, the concentrations of plasma endothelin. Conclusion Lacidipine has anti-atherogenic effects in the apoE-deficient mouse, and reduces plasma endothelin concentrations.

High-Density Lipoprotein Cholesterol Concentrations and Postheparin Hepatic and Lipoprotein Lipases in Obesity: Relationships with Plasma Insulin Levels

The concentration of high-density lipoprotein (HDL) cholesterol in patients with severe obesity is generally subnormal. The exact mechanism linking obesity with reduced levels of HDL cholesterol remains unclear. In this study we evaluated the postheparin plasma lipolytic enzymes lipoprotein lipoprotein lipase (LPL) and hepatic lipase (HL) in a group of 24 obese women compared with controls and analyzed the interrelationships between insulin, postheparin lipolytic enzymes and HDL subfractions. Total HDL cholesterol was significantly lower in the obese subjects than in the controls, and the difference was mainly due to HDL2 cholesterol concentrations. Mean fasting glucose, insulin and the summated means of glucose (sigma glucose) after the oral glucose tolerance test (OGTT) were not significantly different in the two groups. The summated means of insulin (sigma IRI) after the OGTT were significantly higher in the obese women than in the controls. LPL, HL and the HL-to-LPL ratio were significantly higher in the obese women than in the controls. HL and LPL correlated positively with sigma glucose, sigma IRI and body mass index (BMI) and negatively with plasma triglycerides. Partial correlation analysis demonstrated that, when exposed to similar sigma IRI values, HL and LPL were no longer correlated with sigma glucose, plasma triglycerides and BMI. HDL2 cholesterol levels were negatively correlated with HL, posthepatic plasma lipolytic activity, sigma glucose, plasma triglycerides and BMI. HDL2 cholesterol concentrations were directly correlated with LPL. Partial correlation analysis showed that when exposed to similar HL and LPL values, HDL2 cholesterol values were no longer correlated with sigma glucose, sigma IRI, plasma triglycerides and BMI. Therefore, our results demonstrate that the low HDL2 cholesterol levels found in obese women may be related to the high levels of HL and to the high HL-to-LPL ratio which in turn could be determined by the peripheral insulin resistance.

Determination of lipid hydroperoxides in native low-density lipoprotein by a chemiluminescent flow-injection assay

Lipid hydroperoxides have been implicated in the pathogenesis of atherosclerosis. This work was therefore set up to obtain a fast and specific chemiluminescent assay for measuring hydroperoxides in native low-density lipoprotein (LDL). The apparatus was a complete HPLC system including two pumps, an autosampler, a computer and a chemiluminescent detector with a T-mixing coil in the place of the column. Samples were injected from the autosampler and mixed with luminescent reagent (3 microM luminol and 1 microM microperoxidase in 0.1 M carbonate buffer (pH 10)) in the T-piece. To generate a calibration curve, linoleic acid hydroperoxide was obtained by incubating soybean lipoxygenase with linoleic acid. The calculated conjugated diene concentration was in good agreement with the nominal linoleic acid hydroperoxide concentration. The chemiluminescence was linear with the amount of linoleic acid hydroperoxide injected and the detection limit was about 3 pmol linoleic acid hydroperoxide. The chemiluminescence induced by copper-oxidized LDL was linear with concentration; the detection limit, when compared with linoleic acid hydroperoxide, was similar. The reproducibility of the linoleic acid hydroperoxide and of oxidized LDL hydroperoxide was examined in single pools. The coefficient of variation on the triplicates of each pool was about 3%. The titre of the linoleic acid hydroperoxide and oxidized LDL peroxides was quite stable for at least 10 days when stored under argon at 4 degrees C in the presence of EDTA. The mean value of the LDL hydroperoxides in 16 control subjects was 145.20 +/- 98.81 pmol/mg LDL protein. In conclusion, the microperoxidase-luminol-dependent chemiluminescence flow-injection assay is a rapid, sensitive and selective method for measuring lipid hydroperoxides in native LDL.