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Lovastatin upregulates receptor-mediated uptake of acetylated LDL in the human Mono Mac 6 cell line
Monday IO October 1994: PosterAbstracts Modified lipoproteins
11761 Antiatherosclerotic
effects of the new antioxidant Mrz 3/124 in different animal models of hyperiipidemia wuelfroth, Bartmann A, Engel L, Heinle H*, Hopstock CC, Wolfe-Coote S**, Dept. Pharm&ology, Mere dr Co: GmbH & Co., PO Box 111353, 60048 Frankfurt, Germany; *Inst. Physiology, Univ. Tiibingen. Germany; **Medical Research Council, Tygerberg, South Aftica
A drug, Mrz 3/124, with antioxidant and lipid-lowering activity was investigated in different animal models. Ma 3/124 lowered cholesterol in rabbits and monkeys fed an atherogenic diet over a period of 4 and 8 weeks respectively. As a model for type IIa familial hypercholesterolemia, the Watanabe rabbit was used. The effect on triglycerides was determined in normolipemic rata and in hypertriglyceridemic streptozotocin-induced diabetic rats. Cholesterol and triglyceride could be reduced in the different models by 20-502 and up to 75% respectively. Antioxidant capacity was evaluated in the lipoprotein fractions by measuring TBARs and conjugated dienes. Half-maximal inhibition was observed between 1 and 5 PM in vitro and ex vivo after feeding animals the atherogenic diet plus drug. To evaluate the drug effect on the formation of an atherosclerotic plaque, a rabbit model was used in which endothelial dysfunction, induced by electric stimulation of the carotid arteries led to a profound atherosclerotic plaque after 4 weeks. Treatment with Ma 3/124 reduced the number of newly formed cell layers. The inhibition of SMC proliferation and the antioxidant and antilipemic properties suggest that the drug opposes atberogenesis on several levels. Lovastatin upregulates receptor-medlated uptake of acetvlated LDL in the human Mono Mac 6 cell line &&&&& I?rboticky N, Weber PC, Inst. fiir Prophylare und
pij
Epidemiologie der Kreislauflrankheiten, Univ. Miinchen, Germany
Ludwig-Maximilians-
(1781 * The primary site of apolipoprotein-B modification by advanced glycosylation endproducts (AGE-LDL) lies within a 28.kDa region located N-terminal to the LDL-receptor binding domain wR, Mitchell R, Vlassara H, Cerami A, Picower Inst., Ma&asset, NY 11030, USA
Advanced glycosylation endproducts (AGES) arise from, glucosederived Amadori products and have been implicated in diabetic atherogenesis. We recently reported the presence of an AGE modified form of LDL (AGELDL) which circulates in high amounts in individuals with diabetes (Proc Nat1 Acad Sci 1993; 90: 6434-6438). LDL modified bv advanced elvcosvlation exhibits delayed &sma clearance k&tics when &cte~into tmnsgenie mice expressing the human LDL receptor. Furthermore, diabetic patients treated with the advanced glycosylation inhibitor aminoguanidine show a decrease in circulating LDL levels, which suggests that the AGE modification of LDL contributes to high LDL levels in vivo. Chemical modification of the basic residues of apo B has been shown to interfere with the ability of LDL to undergo mceptormediated uptake. Because AGBs form selectively on lysine and arginine residues, we considered that advanced glycosylation might produce adducts near the LDL-receptor binding domain and interfere with LDL uptake. We used AGE-specific antibodies to perform Western biotting analysis of apo B peptides obtained by V8 protease digestion. Although apo B has a high content of basic residues, AGE-immunoreactivity was localized predominantly to a single peptide (MW = 28 kDa). AGE formation within this peptide could also be shown to be blocked specifically by coincubation with aminoguanidine. N-terminal microsequencing identified the 28 kDa peptide to begin at position 1332 of the apo B sequence. The carboxyl terminus was calculated to be at amino acid 1564, i.e. 1788 residues N-terminal to the putative LDLreceptor binding domain. These data point to the high reactivity and specificity of this site for AGE formation and provide further evidence for long-range interactions between the LDLreceptor binding domain and remote regions of the apo B pplypeptide. 11791 m,
Acetylated LDL (ace@-LDL) receptors, also known as scavenger receptors, mediate the uptake of modified e.g. oxidized lipoproteins by macrophages in the aaerial wall and thus play a crucial role in the generation of foam cells. The HMG-CoA reductase inhibitor lovastatin is known to upregulate LDL receptors by inhibiting cellular cholesterol biosynthesis. We studied the influence of lovastatin on the scavenger receptor activity in the human monocytic Mono-Mac-6 (MM6) cell line. Lovastatin caused a significant dose-dependent increase in ‘251-labeled ace@-LDL degradation (+22%; +54%; +80%; with 0.4; 4; 10,uM lovastatin, respectively). Receptor-specific binding of ace@-LDL was also enhanced (+115%) by lovastatin (10pM). Scatchard analysis indicated a substantial increase in binding sites (&ax 55 vs. 95 ng ace@-LDUmg cell protein, control vs. lovastatin). This reflects an increase in the number of scavenger receptors from approximately 17 000 to 30 OOO/cell.Receptor affinity was only slightly increased. The effect of lovastatin could be reversed by simultaneous co-incubation with mevalonic acid (500,~M), indicating a direct relationship to the inhibition of the cholesterol synthesis pathway. The results demonstrate an upregulation of scavenger receptor activity in monocytic cells by lovastatin and thereby suggest a possible involvement of HMG-CoA reductase inhibitors in foam cell formation.
41
LDL oxidation and intermittent claudiition: assw ciation of lag phase to plasma HDL concentration Mijlgaard J, Olsson AG, Clinical Research Center and
Dept. of Internal Medicine, Faculty of Health Science, Hospital, 581 85 Linkiiping, Sweden
Univ.
It has been shown that the susceptibility of LDL to oxidation is related to severity of coronary artery disease among patients with myocardial infarct. The aim of this study was to investigate if LDL from patients with peripheral atherosclerosis (n = 29) was more susceptible to copper-induced oxidation than LDL from age-, sex-, and smoking-matched controls (n = 29). Associations between plasma lipoprotein concentrations and LDL oxidation were also investigated. LDL was prepared by ultracentrifugation and dialyzed. Oxidation was initiated with CuSO4 (1.67,~M). The production of conjugated dienes was continuously monitored at 234 nm. Several oxidation indices, including lag phase, were calculated from the oxidation curve. There were no significant differences in oxidation indices or plasma lipoprotein concentrations between patients and controls. Mean lag phase + SD were (patients/controls) 99.7 + 14.8/ 104.6f 12.9 min (P= 0.19), respectively. There was an overrepresentation of patients with very short lag phase. Correlation analysis (n = 58) between lag phase and plasma lipoprotein concentrations showed a positive association to HDb-cholesterol (r = 0.41, P = 0.0014) and a negative relation to the logarithm of VLDL-TG concentration (r = -0 3 1, P = 0.016). We found no significant difference in the susceptibility of LDL to copper-induced oxidation between patients with peripheral atherosclerosis and controls. HDb-cholesterol may have a protective effect on LDL oxidation. 1 w,
Malondialdehyde-modified HDL impairs hepatic sterol metabolism Brunet S, Gavin0 V, Tuchwebcr B, Levy E, Centre de
Atherosclerosis X, Montreal, October 1994
11761 Antiatherosclerotic
effects of the new antioxidant Mrz 3/124 in different animal models of hyperiipidemia wuelfroth, Bartmann A, Engel L, Heinle H*, Hopstock CC, Wolfe-Coote S**, Dept. Pharm&ology, Mere dr Co: GmbH & Co., PO Box 111353, 60048 Frankfurt, Germany; *Inst. Physiology, Univ. Tiibingen. Germany; **Medical Research Council, Tygerberg, South Aftica
A drug, Mrz 3/124, with antioxidant and lipid-lowering activity was investigated in different animal models. Ma 3/124 lowered cholesterol in rabbits and monkeys fed an atherogenic diet over a period of 4 and 8 weeks respectively. As a model for type IIa familial hypercholesterolemia, the Watanabe rabbit was used. The effect on triglycerides was determined in normolipemic rata and in hypertriglyceridemic streptozotocin-induced diabetic rats. Cholesterol and triglyceride could be reduced in the different models by 20-502 and up to 75% respectively. Antioxidant capacity was evaluated in the lipoprotein fractions by measuring TBARs and conjugated dienes. Half-maximal inhibition was observed between 1 and 5 PM in vitro and ex vivo after feeding animals the atherogenic diet plus drug. To evaluate the drug effect on the formation of an atherosclerotic plaque, a rabbit model was used in which endothelial dysfunction, induced by electric stimulation of the carotid arteries led to a profound atherosclerotic plaque after 4 weeks. Treatment with Ma 3/124 reduced the number of newly formed cell layers. The inhibition of SMC proliferation and the antioxidant and antilipemic properties suggest that the drug opposes atberogenesis on several levels. Lovastatin upregulates receptor-medlated uptake of acetvlated LDL in the human Mono Mac 6 cell line &&&&& I?rboticky N, Weber PC, Inst. fiir Prophylare und
pij
Epidemiologie der Kreislauflrankheiten, Univ. Miinchen, Germany
Ludwig-Maximilians-
(1781 * The primary site of apolipoprotein-B modification by advanced glycosylation endproducts (AGE-LDL) lies within a 28.kDa region located N-terminal to the LDL-receptor binding domain wR, Mitchell R, Vlassara H, Cerami A, Picower Inst., Ma&asset, NY 11030, USA
Advanced glycosylation endproducts (AGES) arise from, glucosederived Amadori products and have been implicated in diabetic atherogenesis. We recently reported the presence of an AGE modified form of LDL (AGELDL) which circulates in high amounts in individuals with diabetes (Proc Nat1 Acad Sci 1993; 90: 6434-6438). LDL modified bv advanced elvcosvlation exhibits delayed &sma clearance k&tics when &cte~into tmnsgenie mice expressing the human LDL receptor. Furthermore, diabetic patients treated with the advanced glycosylation inhibitor aminoguanidine show a decrease in circulating LDL levels, which suggests that the AGE modification of LDL contributes to high LDL levels in vivo. Chemical modification of the basic residues of apo B has been shown to interfere with the ability of LDL to undergo mceptormediated uptake. Because AGBs form selectively on lysine and arginine residues, we considered that advanced glycosylation might produce adducts near the LDL-receptor binding domain and interfere with LDL uptake. We used AGE-specific antibodies to perform Western biotting analysis of apo B peptides obtained by V8 protease digestion. Although apo B has a high content of basic residues, AGE-immunoreactivity was localized predominantly to a single peptide (MW = 28 kDa). AGE formation within this peptide could also be shown to be blocked specifically by coincubation with aminoguanidine. N-terminal microsequencing identified the 28 kDa peptide to begin at position 1332 of the apo B sequence. The carboxyl terminus was calculated to be at amino acid 1564, i.e. 1788 residues N-terminal to the putative LDLreceptor binding domain. These data point to the high reactivity and specificity of this site for AGE formation and provide further evidence for long-range interactions between the LDLreceptor binding domain and remote regions of the apo B pplypeptide. 11791 m,
Acetylated LDL (ace@-LDL) receptors, also known as scavenger receptors, mediate the uptake of modified e.g. oxidized lipoproteins by macrophages in the aaerial wall and thus play a crucial role in the generation of foam cells. The HMG-CoA reductase inhibitor lovastatin is known to upregulate LDL receptors by inhibiting cellular cholesterol biosynthesis. We studied the influence of lovastatin on the scavenger receptor activity in the human monocytic Mono-Mac-6 (MM6) cell line. Lovastatin caused a significant dose-dependent increase in ‘251-labeled ace@-LDL degradation (+22%; +54%; +80%; with 0.4; 4; 10,uM lovastatin, respectively). Receptor-specific binding of ace@-LDL was also enhanced (+115%) by lovastatin (10pM). Scatchard analysis indicated a substantial increase in binding sites (&ax 55 vs. 95 ng ace@-LDUmg cell protein, control vs. lovastatin). This reflects an increase in the number of scavenger receptors from approximately 17 000 to 30 OOO/cell.Receptor affinity was only slightly increased. The effect of lovastatin could be reversed by simultaneous co-incubation with mevalonic acid (500,~M), indicating a direct relationship to the inhibition of the cholesterol synthesis pathway. The results demonstrate an upregulation of scavenger receptor activity in monocytic cells by lovastatin and thereby suggest a possible involvement of HMG-CoA reductase inhibitors in foam cell formation.
41
LDL oxidation and intermittent claudiition: assw ciation of lag phase to plasma HDL concentration Mijlgaard J, Olsson AG, Clinical Research Center and
Dept. of Internal Medicine, Faculty of Health Science, Hospital, 581 85 Linkiiping, Sweden
Univ.
It has been shown that the susceptibility of LDL to oxidation is related to severity of coronary artery disease among patients with myocardial infarct. The aim of this study was to investigate if LDL from patients with peripheral atherosclerosis (n = 29) was more susceptible to copper-induced oxidation than LDL from age-, sex-, and smoking-matched controls (n = 29). Associations between plasma lipoprotein concentrations and LDL oxidation were also investigated. LDL was prepared by ultracentrifugation and dialyzed. Oxidation was initiated with CuSO4 (1.67,~M). The production of conjugated dienes was continuously monitored at 234 nm. Several oxidation indices, including lag phase, were calculated from the oxidation curve. There were no significant differences in oxidation indices or plasma lipoprotein concentrations between patients and controls. Mean lag phase + SD were (patients/controls) 99.7 + 14.8/ 104.6f 12.9 min (P= 0.19), respectively. There was an overrepresentation of patients with very short lag phase. Correlation analysis (n = 58) between lag phase and plasma lipoprotein concentrations showed a positive association to HDb-cholesterol (r = 0.41, P = 0.0014) and a negative relation to the logarithm of VLDL-TG concentration (r = -0 3 1, P = 0.016). We found no significant difference in the susceptibility of LDL to copper-induced oxidation between patients with peripheral atherosclerosis and controls. HDb-cholesterol may have a protective effect on LDL oxidation. 1 w,
Malondialdehyde-modified HDL impairs hepatic sterol metabolism Brunet S, Gavin0 V, Tuchwebcr B, Levy E, Centre de
Atherosclerosis X, Montreal, October 1994