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Autoantibodies against oxidized LDL and lipoprotein-antibody (Lp-Ab) autoimmune complexes in human atherosclerosis
38
Monday 10 October 1994: Poster Abstracts Modtfied lipoproteins
protective effect against LDL peroxidation in a whole organism where other antioxidant systems am known to come into play. The present study consists of two parts: experimental and clinical. The experiments showed that a single intravenous injection of a large dose of human HDLj (200 mg protein) into rabbits with induced hypercholesterolemia (plasma cholesterol 500700 mg/dl) was accompanied by a significant elevation of plasma HDL and led to a decrease (P < 0.05) of conjugated dienes and trienes by 20-30% after 6 h. A decreased level of conjugated dienes (determined using both spectrophotometty and HPLC) remained stable for 24 h after HDL administration. In the clinical investigations we found weak but statistically significant negative correlation between HDL-C and plasma conjugated dienes (r = -0.262; P < 0.01; n = 11l), and between HDLC and hydroperoxides of apo B-containing lipoproteins (r = -0.420; P < 0.01; n = 116). in the combined male groups of healthy subjects and CHD patients. We conclude that HDL also does take part in the protection of plasma lipids from peroxidation in vivo. Autoantibodies against oxidized LDL and lipoprotein-antibody (Lp-Ab) autoimmune complexes in buman atherosclerosis Denisenko AD, Makovejchuk EG, Vinogradov AG, Kuznetzov AS, mAN, Dept. of Biochem., Inst. of Exp. Med., 12 Acad.
11411
Pavlov Str., St. Petersburg 197376, Russia
Lp-Ab autoimmune complexes were isolated from human plasma and saline extracts of aortic intima using ultracentrifugation and affinity chromatography. The concentration of Lp-IgG autoimmune complex in plasma of IHD patients was 12 times that of healthy persons. The content of fluorescent products reflecting the extent of lipid peroxidation in the plasma and aortic autoimmune complexes was three times that in free LDL. The antibodies isolated from Lp-Ab complexes of plasma or aorta reacted with apo B in an immunoblot test. This finding suggests that the autoantigenicity of lipoprotein is caused by peroxidative modification of apo B. The ability of peroxidized lipoproteins to induce an immune response was confumed in the experiments in which immunization of rabbits with autologous LDL subjected to peroxidation in vitro led to the appearance of specific antibodies in the same animals, as evidenced by ELISA and double immunodiffusion tests. Application of immunomodulator TactivinR in the treatment of IHD patients lowered the concentration of plasma Lp-Ab immune complexes and improved the clinical course of IHD. A similar effect was observed after elimination of immune complexes from blood using the procedure known as extracorporeal cryoprecipitation of heparinized plasma. Desialylated, small/dense, electronegative, oxidized low density lipoproteins constitute an atherogenic multiply modified lipoprotein subfraction Qrekhov AN, Institute of Experimental Cardiology, 3rd
1
Cherepkovskaya Street 15A, 121552 Moscow, Russia
Blood plasma of patients with angiographically assessed coronary atherosclerosis contains atherogenic modified LDLs with low sialic acid content. The desialylated LDL subfraction is chsracterized by altered lipid composition, smaller sire, higher density, negative charge and tendency to aggregate. There is evidence that multiply modified desialylated LDL is oxidatively modified lipoprotein. Desialylated LDL is more susceptible to oxidation than native LDL, and its content of lipid-soluble vitamin A and E is lower. In desialylated LDL there is twice to four times as much oxysterols as in native sialylated LDL, and an elevated lysophosphatidylcholine level
We consider these data as a direct indication of oxidative modification of naturally occurring, multiply modified LDL circulating in the blood of patients. We believe that this LDL subfraction is identical to the electronegative, small dense, oxidized lipoprotein which is being studied by other investigators. 1
Metabolism of native and naturally occurring moditied low density lipoprotein in human aortic intbual
cells Tertov v\l, Inst. Exp. Cardiol.,
3rd Cherepkovskaya St. I5A,
121552 Moscow, Russia
The aim of this study was to investigate binding, uptake and intracellular degradation of native and naturally occurring modified (desialylated) low density lipoprotein (nom-LDL) by smooth muscle cells cultured from uninvolved and atherosclerotic intima of human aorta. Earlier we have demonstrated that nom-LDL, but not native LDL, causes lipid accumulation in normal intimal cells. Nom-LDL was separated from native LDL by lectin chromatography on RCA12o-agarose. Binding and uptake of native *251LDL by cells cultured from normal intima was 2- to 4-fold lower than that of cells isolated from atherosclerotic lesions. Binding and uptake of 1251-nom-LDLby both types of cells was 1.5- to 4fold higher than that of native LDL Related rate of degradation of nom-LDL was 1.5 and 5fold lower than degradation of native LDL by normal and atherosclerotic cells, respectively. The low rate of nom-LDL degradation was also demonstrated in homogenates of intimal cells. Activities of lysosomal proteinases of atherosclerotic cells were decreased lower than in normal cells. Pepstatin A, a cathepsin D inhibitor, completely inhibited lipoprotein degradation, while serine, thiol or metallo-proteinase inhibitors had no particular effect. This fact reveals that cathepsin D is involved in the initial stage of apo B degradation by intimal smooth muscle cells. These data show that increased uptake and decreased lysosoma1 degradation of modified (desialylated) LDL may be the main cause of lipid accumulation in human aortic smooth muscle cells. Aggregation and increased macrophage uptake of 11441 LDLs modified by cellular acidic proteases Morgan, Hart D, Leake DS, Biochem. and Physiol., Univ. of Reading, Berkshire, UK
LDL incubated with sonicated macrophages or arterial endothelial or smooth muscle cells at pH 4.5 (proteolysed-LDL) showed partial proteolysis of its protein moiety, apolipoprotein B-100. Following re-isolation by ultracentrifugation, the proteolysed LDL was taken up and degraded much faster by intact macrophages than control LDL. This enhanced uptake could be inhibited almost entirely by the addition of cathepsin inhibitors during the incubation of LDL with the macrophage sonicate. The extent of the increased cellular uptake by macrophages of proteolysed LDL was dependent on the time of incubation with the macrophage sonicate and the concentration of the sonicate. The LDL articles underwent aggregation during their proteolysis. Thus Y251-labeled LDL that had been incubated with a high concentration of macrophage sonicate (1OOpg protein/ml) did not migrate from the well during agarose gel electrophoresis. In addition, electron microscopy of proteolysed LDL showed large-scale aggregation of the LDL particles. The aggregation of LDL modified by acidic proteases may help to explain why it is taken up faster by macroph;gs. I-labeled LDL with purified cathepsins B and Incubation of D alone or in combination produced a complete loss of intact apo B-100 and a number of fragmentation products. Pre-incubation with these purified cathepsins, however, did not consistently lead to increased uptake of the LDL by intact macrophages. which suggests that, although the cathepsins are necessary, other factors
Atherosclerosis X, Montreal, October 1994
Monday 10 October 1994: Poster Abstracts Modtfied lipoproteins
protective effect against LDL peroxidation in a whole organism where other antioxidant systems am known to come into play. The present study consists of two parts: experimental and clinical. The experiments showed that a single intravenous injection of a large dose of human HDLj (200 mg protein) into rabbits with induced hypercholesterolemia (plasma cholesterol 500700 mg/dl) was accompanied by a significant elevation of plasma HDL and led to a decrease (P < 0.05) of conjugated dienes and trienes by 20-30% after 6 h. A decreased level of conjugated dienes (determined using both spectrophotometty and HPLC) remained stable for 24 h after HDL administration. In the clinical investigations we found weak but statistically significant negative correlation between HDL-C and plasma conjugated dienes (r = -0.262; P < 0.01; n = 11l), and between HDLC and hydroperoxides of apo B-containing lipoproteins (r = -0.420; P < 0.01; n = 116). in the combined male groups of healthy subjects and CHD patients. We conclude that HDL also does take part in the protection of plasma lipids from peroxidation in vivo. Autoantibodies against oxidized LDL and lipoprotein-antibody (Lp-Ab) autoimmune complexes in buman atherosclerosis Denisenko AD, Makovejchuk EG, Vinogradov AG, Kuznetzov AS, mAN, Dept. of Biochem., Inst. of Exp. Med., 12 Acad.
11411
Pavlov Str., St. Petersburg 197376, Russia
Lp-Ab autoimmune complexes were isolated from human plasma and saline extracts of aortic intima using ultracentrifugation and affinity chromatography. The concentration of Lp-IgG autoimmune complex in plasma of IHD patients was 12 times that of healthy persons. The content of fluorescent products reflecting the extent of lipid peroxidation in the plasma and aortic autoimmune complexes was three times that in free LDL. The antibodies isolated from Lp-Ab complexes of plasma or aorta reacted with apo B in an immunoblot test. This finding suggests that the autoantigenicity of lipoprotein is caused by peroxidative modification of apo B. The ability of peroxidized lipoproteins to induce an immune response was confumed in the experiments in which immunization of rabbits with autologous LDL subjected to peroxidation in vitro led to the appearance of specific antibodies in the same animals, as evidenced by ELISA and double immunodiffusion tests. Application of immunomodulator TactivinR in the treatment of IHD patients lowered the concentration of plasma Lp-Ab immune complexes and improved the clinical course of IHD. A similar effect was observed after elimination of immune complexes from blood using the procedure known as extracorporeal cryoprecipitation of heparinized plasma. Desialylated, small/dense, electronegative, oxidized low density lipoproteins constitute an atherogenic multiply modified lipoprotein subfraction Qrekhov AN, Institute of Experimental Cardiology, 3rd
1
Cherepkovskaya Street 15A, 121552 Moscow, Russia
Blood plasma of patients with angiographically assessed coronary atherosclerosis contains atherogenic modified LDLs with low sialic acid content. The desialylated LDL subfraction is chsracterized by altered lipid composition, smaller sire, higher density, negative charge and tendency to aggregate. There is evidence that multiply modified desialylated LDL is oxidatively modified lipoprotein. Desialylated LDL is more susceptible to oxidation than native LDL, and its content of lipid-soluble vitamin A and E is lower. In desialylated LDL there is twice to four times as much oxysterols as in native sialylated LDL, and an elevated lysophosphatidylcholine level
We consider these data as a direct indication of oxidative modification of naturally occurring, multiply modified LDL circulating in the blood of patients. We believe that this LDL subfraction is identical to the electronegative, small dense, oxidized lipoprotein which is being studied by other investigators. 1
Metabolism of native and naturally occurring moditied low density lipoprotein in human aortic intbual
cells Tertov v\l, Inst. Exp. Cardiol.,
3rd Cherepkovskaya St. I5A,
121552 Moscow, Russia
The aim of this study was to investigate binding, uptake and intracellular degradation of native and naturally occurring modified (desialylated) low density lipoprotein (nom-LDL) by smooth muscle cells cultured from uninvolved and atherosclerotic intima of human aorta. Earlier we have demonstrated that nom-LDL, but not native LDL, causes lipid accumulation in normal intimal cells. Nom-LDL was separated from native LDL by lectin chromatography on RCA12o-agarose. Binding and uptake of native *251LDL by cells cultured from normal intima was 2- to 4-fold lower than that of cells isolated from atherosclerotic lesions. Binding and uptake of 1251-nom-LDLby both types of cells was 1.5- to 4fold higher than that of native LDL Related rate of degradation of nom-LDL was 1.5 and 5fold lower than degradation of native LDL by normal and atherosclerotic cells, respectively. The low rate of nom-LDL degradation was also demonstrated in homogenates of intimal cells. Activities of lysosomal proteinases of atherosclerotic cells were decreased lower than in normal cells. Pepstatin A, a cathepsin D inhibitor, completely inhibited lipoprotein degradation, while serine, thiol or metallo-proteinase inhibitors had no particular effect. This fact reveals that cathepsin D is involved in the initial stage of apo B degradation by intimal smooth muscle cells. These data show that increased uptake and decreased lysosoma1 degradation of modified (desialylated) LDL may be the main cause of lipid accumulation in human aortic smooth muscle cells. Aggregation and increased macrophage uptake of 11441 LDLs modified by cellular acidic proteases Morgan, Hart D, Leake DS, Biochem. and Physiol., Univ. of Reading, Berkshire, UK
LDL incubated with sonicated macrophages or arterial endothelial or smooth muscle cells at pH 4.5 (proteolysed-LDL) showed partial proteolysis of its protein moiety, apolipoprotein B-100. Following re-isolation by ultracentrifugation, the proteolysed LDL was taken up and degraded much faster by intact macrophages than control LDL. This enhanced uptake could be inhibited almost entirely by the addition of cathepsin inhibitors during the incubation of LDL with the macrophage sonicate. The extent of the increased cellular uptake by macrophages of proteolysed LDL was dependent on the time of incubation with the macrophage sonicate and the concentration of the sonicate. The LDL articles underwent aggregation during their proteolysis. Thus Y251-labeled LDL that had been incubated with a high concentration of macrophage sonicate (1OOpg protein/ml) did not migrate from the well during agarose gel electrophoresis. In addition, electron microscopy of proteolysed LDL showed large-scale aggregation of the LDL particles. The aggregation of LDL modified by acidic proteases may help to explain why it is taken up faster by macroph;gs. I-labeled LDL with purified cathepsins B and Incubation of D alone or in combination produced a complete loss of intact apo B-100 and a number of fragmentation products. Pre-incubation with these purified cathepsins, however, did not consistently lead to increased uptake of the LDL by intact macrophages. which suggests that, although the cathepsins are necessary, other factors
Atherosclerosis X, Montreal, October 1994