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4.P.314 Nidogen is a physiological ligand for the scavenger receptor in liver endothelial cells
362 4.P.312
Thursday 9 October 1997 : Posters Lipoprotein receptors Identification and molecular characterization of LR11 from man and chicken
Kouichi Seimiva, Sonja Moerwald', Hideaki Bujo, Hiroyuki Yamazaki, Jun Kusunoki, Tatsuro Kanaki, Nobuhiro Morisaki, Johannes Nimpf 1 , Wolfgang J . Schneider', Yasushi Saito. Second Department of Internal Medicine, Chiba University School of Medicine, Chiba, Japan; 1 Department of Molecular Genetics, Biocenter and University of Vienna, A-1030, Vienna, Austria Receptors belonging to the LDL receptor gene family play key roles in cellular endocytosis of a broad spectrum of ligands including spent, biologically inactive and/or unwanted plasma carrier complexes, certain toxins, yolk precursors, as well as circulating plasma lipoproteins . We have recently discovered and molecularly characterized a novel and unusually complex member of the LDL receptor gene family from the rabbit, termed LR11 (LDL receptor relative with 11 binding repeats) . LR11 is a complex seven-domain mosaic protein containing, among other structural elements, a cluster of 11 LDLR ligand binding repeats and a domain with homology to VPS 10, a yeast receptor for vacuolar protein sorting . Here we present the molecular structures, sites of expression, and chromosomal localization of human and chicken LRl ls. The most striking properties of LR11s are their (i) amazingly high degree of structural conservation (over 80% identity among mammals and birds) with 100% identity in the membrane spanning and cytoplasmic domains of rabbit and man ; (ii) lack of regulation by cholesterol and estrogen ; and (iii) high levels of expression in brain. The conservation of the features of LRlis from two such diverse species as man and chicken suggest possible brain-specific functions, in concert with another recently identified brain-specific LDL receptor family member, i .e ., LR8B/apoE receptor 2 .
cialized extracellular matrices which modulate cellular activities and consist mainly of glycoproteins including type IV collagen, laminin, nidogen and proteoglycans . To study endocytosis, 125 I-labelled proteins were added to monolayer cultures of liver endothelial cells (LEC) . Nidogen was effectively endocytosed by LEC, but globular fragments of type IV collagen (7-S, NC1) or laminin (fragments of laminin) were not . To study the receptor specificity the 125 1-nidogen was incubated together with an excess of various unlabelled substances known to bind different receptors in LEC . The results indicated that only ligands for scavenger receptors (poly I, formaldehyde-treated albumin) were able to compete with nidogen for uptake . To study the elimination of nidogen in vivo ' 25 1-nidogen was injected intravenously into rats . Nidogen was eliminated from the circulation, by a biphasic kinetics : 42% was removed by tt/ 2 (a) = 0 .7 min, and 58% by a slow t,12 (,B) = 12 min . 45% of the 125 I-nidogen was found in the liver 60 minutes after injection, 10% in the kidneys and 35% in the blood . Analysis of the isolated liver cells showed that most of the label was located in the LEC . Coinjection with formaldehyde-treated albumin significantly increased the circulatory survival of 125 I-nidogen . Previously, it has been shown that scavenger receptors ligands include chemically modified proteins, such as oxidized LDL and maleylated albumin, but not their unmodified counterparts . We have shown that globular fragments of procollagen are cleared from the blood circulation by scavenger receptors (Melkko et al. 1994 J . Exp. Med . 179 : 405-412). This together with the finding that nidogen is a ligand for scavenger receptors suggest that one of the scavenger receptors physiological functions is to take part in the catabolism of unmodified proteins .
4.P.315 4 .P.313
Parallel regulation of cholesterol 7a-hydroxylase and high density lipoprotein binding proteins in the hamster
C. S€rougne', J.L. Smith 2 , M . Souidi l , N . Fidge 3 , R . Davis 4 , C . Lutton' . 'Orsay, France ; 2 Brisbane, Australia; 3 Melbourne, Australia; 4San Diego, USA The present study was performed to examine whether the expression of 2 HDL binding proteins, HB1 and HB2, vary under different dietary conditions in liver and whether their levels are associated with cholesterol 7a hydroxylase, the rate-limiting enzyme for the conversion of cholesterol to bile acids, the major removal pathway for cholesterol from the body . Five groups of 4 week old hamsters were fed for 1 week either a commercial diet or a semi-synthetic lithogenic diet or this diet supplemented with inhibitors of HMG-CoA reductase, glutathione synthesis or ACAT . A sixth group of 9 week old hamsters were fed the commercial diet for 1 week . Liver total membranes were prepared for the measurements of HB1 and HB2 mass and liver microsomes were used for the determination of cholesterol 7a hydroxylase mass and activity. Immunodetection of these 3 proteins was performed by dot blots using specific antibodies . Cholesterol 7a hydroxylase activity was measured using the method of Martin et al . (J . Lipid Res ., 1993, 34, 581-588), recently improved in our laboratory . The mean cholesterol 7a hydroxylase activity in all the lithogenic groups was lower than in the control group whereas it was higher for the 9 week old hamsters . Similar changes were observed for the mass of cholesterol 7a hydroxylase, HB 1 and HB2, except for the simvastatin-treated group, which was not different from the control group, . On combining the results from all groups, a strong positive correlation was found between cholesterol 7a hydroxylase and both HB1 and HB2 mass (n = 60, r = 0 .672 and 0.626, respectively, P = 0.0001) . Expression of HB1 and HB2 varied with the different diets . The strong positive relationship between HB1 and HB2 and cholesterol 7a hydroxylase suggests that these proteins have an important function in cholesterol homeostasis, possibly regulating the supply of HDL-cholesterol for conversion into bile acids .
4 .P.314
Nidogen is a physiological ligand for the scavenger receptor in liver endothelial cells
B . Smedsrod t , J . Melkko l , U . Mayer2 , S . Johansson' . 'Department of Experimental Pathology, University of Tromso, N-9037 Tromso, Norway ; 2 Max-Planck-Institut fur Biochemie, D-82152 Martinsried, Germany; 3 Department of Medical Chemistry, University of Uppsala, S-75123, Sweden The purpose of this study was to determine if globular proteins in basement membrane are ligands for scavenger receptors . Basement membranes are spe-
Stimulation of LDL receptor expression by LDL-immuno-apheresis in patients with heterozygous familial hypercholesterolemia
J. Streicher, P. Valent, H . Schmidtl, G. Sengolge, S . Banyai, W.H. Horl, K . Derfler. University of Vienna ; 'University of Graz, Austria The aim of the present study was to investigate the effect of low density lipoprotein (LDL)-immunoapheresis on residual LDL receptor expression in patients with heterozygous familial hypercholesterolemia (FH) and insufficient response to lipid lowering agents . Using a monoclonal antibody LDL receptor expression was determined by flow cytometry of peripheral blood monocytes (PBMC) in 7 heterozygous FH patients prior to, after, 24 and 48 hours after LDL-apheresis, as well as in 10 healthy volunteers . In addition in vitro stimulation experiments by culturing PBMC for 48 hours under LDL deficient conditions were performed . LDL receptor expression was calculated by transforming the mean fluorescence intensity into the number of antibody binding sites per cell (S/C) . Prior to LDL-apheresis, LDL receptor density was comparable in patients (2014 ± 359 S/C) and controls (1782 ± 252 S/C), whereas under in vitro conditions LDL receptor expression of controls exceeded that of FH patients by .16 times . However, immediately after apheresis LDL receptor expression significantly increased to a maximum of 3640 f 423 S/C, i .e . almost the same level as obtained by in vitro stimulation (3632 f 572 S/C) . This results demonstrate, that despite drug therapy LDL-apheresis significantly further stimulates the residual LDL receptor expression in FH patients . This effect may be partially responsible for the delayed reappearance of hypercholesterolemia in between of two treatments.
4 .P.316
Generation of very low density lipoprotein receptor overexpressing mice
Paul J . Tacken, Andre van der Zee, Ko Willems van Dijk, Rune R . Frants, Louis M . Havekes, Marten H . Hofker. Department of Human Genetics, Leiden University, TNO-PG Gaubius Laboratory, Leiden, The Netherlands One of the first steps in atherosclerosis is considered to be the formation of fatty streaks . The very low density (VLDL) receptor is a good candidate gene for this form of lipid accumulation, because it is expressed in endothelial cells, smooth muscle cells and macrophages, it recognizes chylomicrons, VLDL and ,B-VLDL, and its expression is not downregulated by intracellular cholesterol . To study the VLDL receptor and its possible role in atherosclerosis, we created mice that overexpressed the human VLDL receptor . To obtain a native expression pattern, the entire gene including 15 kb of 5' and 20 kb of 3' sequences was used . Two large genomic fragments spanning this entire region were isolated from a cosmid library and co-injected into mouse oocytes . The three transgenic founders that were generated all carried multiple copies of the intact gene, showing that homologous recombination between the two
11th International Symposium on Atherosclerosis, Paris, October 1997
Thursday 9 October 1997 : Posters Lipoprotein receptors Identification and molecular characterization of LR11 from man and chicken
Kouichi Seimiva, Sonja Moerwald', Hideaki Bujo, Hiroyuki Yamazaki, Jun Kusunoki, Tatsuro Kanaki, Nobuhiro Morisaki, Johannes Nimpf 1 , Wolfgang J . Schneider', Yasushi Saito. Second Department of Internal Medicine, Chiba University School of Medicine, Chiba, Japan; 1 Department of Molecular Genetics, Biocenter and University of Vienna, A-1030, Vienna, Austria Receptors belonging to the LDL receptor gene family play key roles in cellular endocytosis of a broad spectrum of ligands including spent, biologically inactive and/or unwanted plasma carrier complexes, certain toxins, yolk precursors, as well as circulating plasma lipoproteins . We have recently discovered and molecularly characterized a novel and unusually complex member of the LDL receptor gene family from the rabbit, termed LR11 (LDL receptor relative with 11 binding repeats) . LR11 is a complex seven-domain mosaic protein containing, among other structural elements, a cluster of 11 LDLR ligand binding repeats and a domain with homology to VPS 10, a yeast receptor for vacuolar protein sorting . Here we present the molecular structures, sites of expression, and chromosomal localization of human and chicken LRl ls. The most striking properties of LR11s are their (i) amazingly high degree of structural conservation (over 80% identity among mammals and birds) with 100% identity in the membrane spanning and cytoplasmic domains of rabbit and man ; (ii) lack of regulation by cholesterol and estrogen ; and (iii) high levels of expression in brain. The conservation of the features of LRlis from two such diverse species as man and chicken suggest possible brain-specific functions, in concert with another recently identified brain-specific LDL receptor family member, i .e ., LR8B/apoE receptor 2 .
cialized extracellular matrices which modulate cellular activities and consist mainly of glycoproteins including type IV collagen, laminin, nidogen and proteoglycans . To study endocytosis, 125 I-labelled proteins were added to monolayer cultures of liver endothelial cells (LEC) . Nidogen was effectively endocytosed by LEC, but globular fragments of type IV collagen (7-S, NC1) or laminin (fragments of laminin) were not . To study the receptor specificity the 125 1-nidogen was incubated together with an excess of various unlabelled substances known to bind different receptors in LEC . The results indicated that only ligands for scavenger receptors (poly I, formaldehyde-treated albumin) were able to compete with nidogen for uptake . To study the elimination of nidogen in vivo ' 25 1-nidogen was injected intravenously into rats . Nidogen was eliminated from the circulation, by a biphasic kinetics : 42% was removed by tt/ 2 (a) = 0 .7 min, and 58% by a slow t,12 (,B) = 12 min . 45% of the 125 I-nidogen was found in the liver 60 minutes after injection, 10% in the kidneys and 35% in the blood . Analysis of the isolated liver cells showed that most of the label was located in the LEC . Coinjection with formaldehyde-treated albumin significantly increased the circulatory survival of 125 I-nidogen . Previously, it has been shown that scavenger receptors ligands include chemically modified proteins, such as oxidized LDL and maleylated albumin, but not their unmodified counterparts . We have shown that globular fragments of procollagen are cleared from the blood circulation by scavenger receptors (Melkko et al. 1994 J . Exp. Med . 179 : 405-412). This together with the finding that nidogen is a ligand for scavenger receptors suggest that one of the scavenger receptors physiological functions is to take part in the catabolism of unmodified proteins .
4.P.315 4 .P.313
Parallel regulation of cholesterol 7a-hydroxylase and high density lipoprotein binding proteins in the hamster
C. S€rougne', J.L. Smith 2 , M . Souidi l , N . Fidge 3 , R . Davis 4 , C . Lutton' . 'Orsay, France ; 2 Brisbane, Australia; 3 Melbourne, Australia; 4San Diego, USA The present study was performed to examine whether the expression of 2 HDL binding proteins, HB1 and HB2, vary under different dietary conditions in liver and whether their levels are associated with cholesterol 7a hydroxylase, the rate-limiting enzyme for the conversion of cholesterol to bile acids, the major removal pathway for cholesterol from the body . Five groups of 4 week old hamsters were fed for 1 week either a commercial diet or a semi-synthetic lithogenic diet or this diet supplemented with inhibitors of HMG-CoA reductase, glutathione synthesis or ACAT . A sixth group of 9 week old hamsters were fed the commercial diet for 1 week . Liver total membranes were prepared for the measurements of HB1 and HB2 mass and liver microsomes were used for the determination of cholesterol 7a hydroxylase mass and activity. Immunodetection of these 3 proteins was performed by dot blots using specific antibodies . Cholesterol 7a hydroxylase activity was measured using the method of Martin et al . (J . Lipid Res ., 1993, 34, 581-588), recently improved in our laboratory . The mean cholesterol 7a hydroxylase activity in all the lithogenic groups was lower than in the control group whereas it was higher for the 9 week old hamsters . Similar changes were observed for the mass of cholesterol 7a hydroxylase, HB 1 and HB2, except for the simvastatin-treated group, which was not different from the control group, . On combining the results from all groups, a strong positive correlation was found between cholesterol 7a hydroxylase and both HB1 and HB2 mass (n = 60, r = 0 .672 and 0.626, respectively, P = 0.0001) . Expression of HB1 and HB2 varied with the different diets . The strong positive relationship between HB1 and HB2 and cholesterol 7a hydroxylase suggests that these proteins have an important function in cholesterol homeostasis, possibly regulating the supply of HDL-cholesterol for conversion into bile acids .
4 .P.314
Nidogen is a physiological ligand for the scavenger receptor in liver endothelial cells
B . Smedsrod t , J . Melkko l , U . Mayer2 , S . Johansson' . 'Department of Experimental Pathology, University of Tromso, N-9037 Tromso, Norway ; 2 Max-Planck-Institut fur Biochemie, D-82152 Martinsried, Germany; 3 Department of Medical Chemistry, University of Uppsala, S-75123, Sweden The purpose of this study was to determine if globular proteins in basement membrane are ligands for scavenger receptors . Basement membranes are spe-
Stimulation of LDL receptor expression by LDL-immuno-apheresis in patients with heterozygous familial hypercholesterolemia
J. Streicher, P. Valent, H . Schmidtl, G. Sengolge, S . Banyai, W.H. Horl, K . Derfler. University of Vienna ; 'University of Graz, Austria The aim of the present study was to investigate the effect of low density lipoprotein (LDL)-immunoapheresis on residual LDL receptor expression in patients with heterozygous familial hypercholesterolemia (FH) and insufficient response to lipid lowering agents . Using a monoclonal antibody LDL receptor expression was determined by flow cytometry of peripheral blood monocytes (PBMC) in 7 heterozygous FH patients prior to, after, 24 and 48 hours after LDL-apheresis, as well as in 10 healthy volunteers . In addition in vitro stimulation experiments by culturing PBMC for 48 hours under LDL deficient conditions were performed . LDL receptor expression was calculated by transforming the mean fluorescence intensity into the number of antibody binding sites per cell (S/C) . Prior to LDL-apheresis, LDL receptor density was comparable in patients (2014 ± 359 S/C) and controls (1782 ± 252 S/C), whereas under in vitro conditions LDL receptor expression of controls exceeded that of FH patients by .16 times . However, immediately after apheresis LDL receptor expression significantly increased to a maximum of 3640 f 423 S/C, i .e . almost the same level as obtained by in vitro stimulation (3632 f 572 S/C) . This results demonstrate, that despite drug therapy LDL-apheresis significantly further stimulates the residual LDL receptor expression in FH patients . This effect may be partially responsible for the delayed reappearance of hypercholesterolemia in between of two treatments.
4 .P.316
Generation of very low density lipoprotein receptor overexpressing mice
Paul J . Tacken, Andre van der Zee, Ko Willems van Dijk, Rune R . Frants, Louis M . Havekes, Marten H . Hofker. Department of Human Genetics, Leiden University, TNO-PG Gaubius Laboratory, Leiden, The Netherlands One of the first steps in atherosclerosis is considered to be the formation of fatty streaks . The very low density (VLDL) receptor is a good candidate gene for this form of lipid accumulation, because it is expressed in endothelial cells, smooth muscle cells and macrophages, it recognizes chylomicrons, VLDL and ,B-VLDL, and its expression is not downregulated by intracellular cholesterol . To study the VLDL receptor and its possible role in atherosclerosis, we created mice that overexpressed the human VLDL receptor . To obtain a native expression pattern, the entire gene including 15 kb of 5' and 20 kb of 3' sequences was used . Two large genomic fragments spanning this entire region were isolated from a cosmid library and co-injected into mouse oocytes . The three transgenic founders that were generated all carried multiple copies of the intact gene, showing that homologous recombination between the two
11th International Symposium on Atherosclerosis, Paris, October 1997