In order to determine the effect of antibodies against electronegative low-density lipoprotein LDL(-) on atherogenesis, five groups of LDL low receptor-deficient (LDLr-/-) mice (6 per group) were immunized with the following antibodies (100 µg each): mouse anti-LDL(-) monoclonal IgG2b, rabbit anti-LDL(-) polyclonal IgG or its Fab fragments and mouse irrelevant monoclonal IgG and non-immunized controls. Antibodies were administered intravenously one week before starting the hypercholesterolemic diet (1.25% cholesterol) and then every week for 21 days. The passive immunization with anti-LDL(-) monoclonal IgG2b, polyclonal antibody and its derived Fab significantly reduced the cross-sectional area of atherosclerotic lesions at the aortic root of LDLr-/- mice (28.8 ± 9.7, 67.3 ± 17.02, 56.9 ± 8.02 µm² (mean ± SD), respectively) compared to control (124.9 ± 13.2 µm²). Vascular cell adhesion molecule-1 protein expression, quantified by the KS300 image-analyzing software, on endothelium and the number of macrophages in the intima was also decreased in aortas of mice treated with anti-LDL(-) monoclonal antibody (3.5 ± 0.70 per field x 10) compared to controls (21.5 ± 3.5 per field x 10). Furthermore, immunization with the monoclonal antibody decreased the concentration of LDL(-) in blood plasma (immunized: 1.0 ± 1.4; control: 20.5 ± 3.5 RLU), the amount of cholesterol oxides in plasma (immunized: 4.7 ± 2.7; control: 15.0 ± 2.0 pg COx/mg cholesterol) and liver (immunized: 2.3 ± 1.5; control: 30.0 ± 26.0 pg COx/mg cholesterol), and the hepatic content of lipid hydroperoxides (immunized: 0.30 ± 0.020; control: 0.38 ± 0.15 ng/mg protein). In conclusion, antibodies against electronegative LDL administered intravenously may play a protective role in atherosclerosis.

The objective of the present study was to identify disturbances of nitric oxide radical (·NO) metabolism and the formation of cholesterol oxidation products in human essential hypertension. The concentrations of·NO derivatives (nitrite, nitrate, S-nitrosothiols and nitrotyrosine), water and lipid-soluble antioxidants and cholesterol oxides were measured in plasma of 11 patients with mild essential hypertension (H: 57.8 ± 9.7 years; blood pressure, 148.3 ± 24.8/90.8 ± 10.2 mmHg) and in 11 healthy subjects (N: 48.4 ± 7.0 years; blood pressure, 119.4 ± 9.4/75.0 ± 8.0 mmHg).Nitrite, nitrate and S-nitrosothiols were measured by chemiluminescence and nitrotyrosine was determined by ELISA. Antioxidants were determined by reverse-phase HPLC and cholesterol oxides by gas chromatography. Hypertensive patients had reduced endothelium-dependent vasodilation in response to reactive hyperemia (H: 9.3 and N: 15.1% increase of diameter 90 s after hyperemia), and lower levels of ascorbate (H: 29.2 ± 26.0, N: 54.2 ± 24.9 µM), urate (H: 108.5 ± 18.9, N: 156.4 ± 26.3 µM), ß-carotene (H: 1.1 ± 0.8, N: 2.5 ± 1.2 nmol/mg cholesterol), and lycopene (H: 0.4 ± 0.2, N: 0.7 ± 0.2 nmol/mg cholesterol), in plasma, compared to normotensive subjects. The content of 7-ketocholesterol, 5alpha-cholestane-3ß,5,6ß-triol and 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol in LDL, and the concentration of endothelin-1 (H: 0.9 ± 0.2, N: 0.7 ± 0.1 ng/ml) in plasma were increased in hypertensive patients. No differences were found for ·NO derivatives between groups. These data suggest that an increase in cholesterol oxidation is associated with endothelium dysfunction in essential hypertension and oxidative stress, although ·NO metabolite levels in plasma are not modified in the presence of elevated cholesterol oxides.

The biodistribution and removal from plasma (measured as fractional clearance rate, FCR, per hour) of native and oxidatively modified 99mtechnetium-labeled ß-very low density lipoprotein (99mTc-ß-VLDL) were investigated in hypercholesterolemic (HC) and control (C) three-month old New Zealand rabbits. The intracellular accumulation of ß-VLDL labeled with 99mTc was studied in vitro in THP-1 cells and monocyte-derived macrophages isolated from rabbits. After intravenous injection into C rabbits, copper-oxidized ß-VLDL (99mTc-ox-ß-VLDL) was cleared from the circulation faster (0.362 ± 0.070/h) than native ß-VLDL (99mTc-nat-ß-VLDL, 0.241 ± 0.070/h). In contrast, the FCR of 99mTc-ox-ß-VLDL in HC rabbits was lower (0.100 ± 0.048/h) than that of 99mTc-nat-ß-VLDL (0.163 ± 0.043/h). The hepatic uptake of radiolabeled lipoproteins was lower in HC rabbits (0.114 ± 0.071% injected dose/g tissue for 99mTc-nat-ß-VLDL and 0.116 ± 0.057% injected dose/g tissue for 99mTc-ox-ß-VLDL) than in C rabbits (0.301 ± 0.113% injected dose/g tissue for 99mTc-nat-ß-VLDL and 0.305 ± 0.149% injected dose/g tissue for 99mTc-ox-ß-VLDL). The uptake of 99mTc-nat-ß-VLDL and 99mTc-ox-ß-VLDL by atherosclerotic aorta lesions isolated from HC rabbits (99mTc-nat-ß-VLDL: 0.033 ± 0.012% injected dose/g tissue and 99mTc-ox-ß-VLDL: 0.039 ± 0.017% injected dose/g tissue) was higher in comparison to that of non-atherosclerotic aortas from C rabbits (99mTc-nat-ß-VLDL: 0.023 ± 0.010% injected dose/g tissue and 99mTc-ox-ß-VLDL: 0.019 ± 0.010% injected dose/g tissue). However, 99mTc-nat-ß-VLDL and 99mTc-ox-ß-VLDL were taken up by atherosclerotic lesions at similar rates. In vitro studies showed that both monocyte-derived macrophages isolated from rabbits and THP-1 macrophages significantly internalized more 99mTc-ox-ß-VLDL than 99mTc-nat-ß-VLDL. These results indicate that in cholesterol-fed rabbits 99mTc-ox-ß-VLDL is slowly cleared from plasma and accumulates in atherosclerotic lesions. However, although the extent of in vitro uptake of 99mTc-ox-ß-VLDL by macrophages was high, the in vivo accumulation of this radiolabeled lipoprotein by atherosclerotic lesions did not differ from that of 99mTc-nat-ß-VLDL.