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In vivo regulation of VLDL receptor mRNA in rabbit
Tuesday I1 October 1994: Poster Abstracts Receptors
115
1
content of VLDL receptor mRNA in heart was 3-6 times higher than the control value. The content of VLDL receptor mRNA in gastrocnemius was doubled. We could not detect VLDL receptor mRNA in liver. Very weak expression of the LDL receptor was detected in rabbit heart ventricle. Enhanced expression of VLDL receptor mRNA in the ventricle and atrium from rabbits treated with thyroxine was confirmed. These results suggest that estradiol exerts its effect transcriptionally in the heart, which requires a constant energy supply. Although it cannot be ruled out that the plasma level of VLDL is lowered by the enhanced expression of liver LDL receptor, it is also possible that the estrogen receptor interacts directly with the VLDL receptor gene.
Centro de Investigacicin, Hosp. La Fe, 46009-Valencia, Spain
1
The LDL-receptor fragment responsible for binding LDL (amino acids 187-2 12) has been analyzed by Pin technology to determine the minimal size of amino acid sequence binding of ape B. It was shown that Cys-201 and three negatively charged amino acid residues in its microenvironment play an important role in the binding of LDL by the synthetic octa-peptide. If this peptide is made hydrophdbic it should be possible to incorporate it into the cell membrane to simulate the LDL receptor. Study of the LDL-binding capacity of such peptide-enriched cells is under investigation. Effect of scavenger receptor-interacting ligands on leukocyte adhesion to the endothelium of rat carotid artery &&Q&A, Zhniga A, Gabald6n M, Unidad de Histoquimia, Ligand binding to the scavenger receptors is competitively inhibited by dextran sulfate, fucoidan, albumin modified by formaldehyde treatment (Form-albumin) and polyinosinic acid (Poly I), but not by polycytidylic acid (Poly C).We have studied the effect of scavenger receptor-interacting ligands on leukocyte adhesion to the endothelium of rat carotid artery both under basal conditions and in the presence of albumin, and the effect of inhibitors of the synthesis of endotbelial receptors involved in leukocyte binding (actinomycin D, cycloheximide and dexamethasone) on leukocyte adhesion produced by albumin. The study was performed in Wistar rats and the i.v. doses per 100 g administered to groups of six rats were: bovine albumin A7906 (Sigma Chem.) and Form-albumin, 25 mg; Poly I and Poly C, 800 g; fucoidan, 1 mg; dextran sulfate 500 000, 1.25 mg; actinomycin D, 10 g; dexamethasone, 50 g; cycloheximide, 40 g. Leukocyte adhesion was determined 18 h after stimulus in surface preparations of carotid artery by the non-specific esterase reaction. Results expressed as esterase (+) leukocytes/mm2 were (mean f SD): control 7 k 6; Poly I 308 & 132; fucoidan, dextran sulfate and Form-albumin did not differ significantly from controls; albumin 62 + 12; albumin plus the following were: fucoidan 17 + 7; dextran sulfate 8 & 4; dexamethasone 28 f 15; actinomytin D 10 It 8 and cycloheximide 26 & 6. The massive adhesion found with Poly I can be explained by the increase in ICAM- receptors it produces in endothelial cells in vitro. The inhibitory effect found with dextran sulfate and fucoidan, together with the rapid clearance of albumin from the circulatibn, suggest that like other modified albumins, bovine albumin A-7906 which has been prepared by heat shock may be altered in such a way as to be recognized by scavenger receptors. In vivo regulation of VLDL receptor mRNA in rabbit 1 Jineami, Masuzaki H, Mizuno M, Matsuoka N, Yamamoto T*, Nakao K, 2nd Div., Dept. qf Med., Kyoto Univ. Fat. of Med., Sakyo-Ku, Kyoto 606; *Tohoku Univ. Gene Res. Center, Aoba Ku, Sendai 981, Japan
The VLDL receptor, a newly identified lipoprotein receptor, has homologous domain structure with the LDL receptor but has one additional cysteine-rich repeat in the ligand-binding domain. This receptor mRNA is expressed in the heart, muscle and adipose tissue, all of which are active in fatty acid metabolism. This ligand specificity and tissue distribution characterize the VLDL receptor. So far, the regulation of the VLDL receptor expression has not been studied. In order to elucidate the role of this receptor in vivo lipid metabolism, we administered pharmacological doses of 17a-ethinyl estradiol into rabbits and analyzed by RNA blot and RNase protection assay the expression of VLDL receptor in the rabbits’ heart and liver in comparison with LDL receptor expression. The
+ Expression of q-macroglobulin receptor/ LDL recep tar-related protein and scavenger receptor in human atherosclerotic lesions Luoma 1, Hiltunen T, S%rkioja T+, Moe&up SK*, Gliemann J*, Kodama T#, Nikkari T, YUHerttuala S, Univ. of Tampere, Box
607, FIN-33101 Tampere, Finland;+Univ. Oulu, Oulu, Finland; *Univ. of Aarhus, Aarhus, Denmark; #Univ. of Tokyo, Tokyo, Japan
A typical feature of human atherosclerotic lesions is the presence of macrophage- and smooth muscle cell (SMC)-derived foam cells. Three receptor systems may be involved in the development of these foam cells: a2-macroglobulin receptor/LDL receptor related protein (a2-MlULRP), scavenger receptor and LDL receptor. We have studied the expression of these receptors in human atherosclerotic lesions by in situ hybridization and immunocytochemistry. SMCs expressed a~-MR/LRP mRNA and protein in normal human aorta and in both early and advanced human lesions No scavenger receptor expression was found in lesion SMCs. Macrophages expressed a2-MRLRP as well as scavenger receptor mRNAs and proteins in early and late lesions, whereas LDL receptor expression was not found in human lesions. There are several findings suggesting that a~-MIULRP may have a role in the formation of foam cells: (a) our results show that lesion macrophages and SMCs expressed a2-MR/LRP, (b) it has been shown by others that apo E-enriched remnant lipoproteins and LPL-triglyceride-rich lipoprotein complexes can be bound and internalized by q-MR/LRP Also, (c) q-MIULRP is not down-regulated by increased cellular cholesterol content. Scavenger receptors, on the other hand, are likely to be involved in the uptake of modified forms of LDL by lesion macrophages. Our results demonstrate that, so far, the only lipoprotein receptor expressed in lesion SMCs in vivo is q-MR/LRP. Scavenger receptors arc expressed only in lesion macrophages. Both recep tars may contribute to the development of human atherosclerotic lesions. 1 mL,
Relationship of LDL receptor gene defects to familial hypercholesterolemia in childhood Levy E, Feoli-Fonseca JC, Lambert M, Depts. of
Nutrition and Pediatrics, H6pital Ste-Justine and Univ. qf Montreal, Quebec, Canada H3T 1 CS
Quantitative abnormalities in LDL metabolism are the most striking features of familial hypercholesterolemia (FH), a common autosomal codominant disorder. The naturally occurring mutations within the LDL receptor gene are responsible for the aberrant phenotype. Since few studies have defined FH in the pediatric population by molecular analysis, the present investigation aimed to characterize the relationship of genotype to biochemical phenotype in a French Canadian pediatric population. A group of 88 unrelated French Canadian children with a persistent baseline increase in LDL-C levels (~3.3 mM) and a posi-
Atherosclerosis X, Montreal, October 1994
115
1
content of VLDL receptor mRNA in heart was 3-6 times higher than the control value. The content of VLDL receptor mRNA in gastrocnemius was doubled. We could not detect VLDL receptor mRNA in liver. Very weak expression of the LDL receptor was detected in rabbit heart ventricle. Enhanced expression of VLDL receptor mRNA in the ventricle and atrium from rabbits treated with thyroxine was confirmed. These results suggest that estradiol exerts its effect transcriptionally in the heart, which requires a constant energy supply. Although it cannot be ruled out that the plasma level of VLDL is lowered by the enhanced expression of liver LDL receptor, it is also possible that the estrogen receptor interacts directly with the VLDL receptor gene.
Centro de Investigacicin, Hosp. La Fe, 46009-Valencia, Spain
1
The LDL-receptor fragment responsible for binding LDL (amino acids 187-2 12) has been analyzed by Pin technology to determine the minimal size of amino acid sequence binding of ape B. It was shown that Cys-201 and three negatively charged amino acid residues in its microenvironment play an important role in the binding of LDL by the synthetic octa-peptide. If this peptide is made hydrophdbic it should be possible to incorporate it into the cell membrane to simulate the LDL receptor. Study of the LDL-binding capacity of such peptide-enriched cells is under investigation. Effect of scavenger receptor-interacting ligands on leukocyte adhesion to the endothelium of rat carotid artery &&Q&A, Zhniga A, Gabald6n M, Unidad de Histoquimia, Ligand binding to the scavenger receptors is competitively inhibited by dextran sulfate, fucoidan, albumin modified by formaldehyde treatment (Form-albumin) and polyinosinic acid (Poly I), but not by polycytidylic acid (Poly C).We have studied the effect of scavenger receptor-interacting ligands on leukocyte adhesion to the endothelium of rat carotid artery both under basal conditions and in the presence of albumin, and the effect of inhibitors of the synthesis of endotbelial receptors involved in leukocyte binding (actinomycin D, cycloheximide and dexamethasone) on leukocyte adhesion produced by albumin. The study was performed in Wistar rats and the i.v. doses per 100 g administered to groups of six rats were: bovine albumin A7906 (Sigma Chem.) and Form-albumin, 25 mg; Poly I and Poly C, 800 g; fucoidan, 1 mg; dextran sulfate 500 000, 1.25 mg; actinomycin D, 10 g; dexamethasone, 50 g; cycloheximide, 40 g. Leukocyte adhesion was determined 18 h after stimulus in surface preparations of carotid artery by the non-specific esterase reaction. Results expressed as esterase (+) leukocytes/mm2 were (mean f SD): control 7 k 6; Poly I 308 & 132; fucoidan, dextran sulfate and Form-albumin did not differ significantly from controls; albumin 62 + 12; albumin plus the following were: fucoidan 17 + 7; dextran sulfate 8 & 4; dexamethasone 28 f 15; actinomytin D 10 It 8 and cycloheximide 26 & 6. The massive adhesion found with Poly I can be explained by the increase in ICAM- receptors it produces in endothelial cells in vitro. The inhibitory effect found with dextran sulfate and fucoidan, together with the rapid clearance of albumin from the circulatibn, suggest that like other modified albumins, bovine albumin A-7906 which has been prepared by heat shock may be altered in such a way as to be recognized by scavenger receptors. In vivo regulation of VLDL receptor mRNA in rabbit 1 Jineami, Masuzaki H, Mizuno M, Matsuoka N, Yamamoto T*, Nakao K, 2nd Div., Dept. qf Med., Kyoto Univ. Fat. of Med., Sakyo-Ku, Kyoto 606; *Tohoku Univ. Gene Res. Center, Aoba Ku, Sendai 981, Japan
The VLDL receptor, a newly identified lipoprotein receptor, has homologous domain structure with the LDL receptor but has one additional cysteine-rich repeat in the ligand-binding domain. This receptor mRNA is expressed in the heart, muscle and adipose tissue, all of which are active in fatty acid metabolism. This ligand specificity and tissue distribution characterize the VLDL receptor. So far, the regulation of the VLDL receptor expression has not been studied. In order to elucidate the role of this receptor in vivo lipid metabolism, we administered pharmacological doses of 17a-ethinyl estradiol into rabbits and analyzed by RNA blot and RNase protection assay the expression of VLDL receptor in the rabbits’ heart and liver in comparison with LDL receptor expression. The
+ Expression of q-macroglobulin receptor/ LDL recep tar-related protein and scavenger receptor in human atherosclerotic lesions Luoma 1, Hiltunen T, S%rkioja T+, Moe&up SK*, Gliemann J*, Kodama T#, Nikkari T, YUHerttuala S, Univ. of Tampere, Box
607, FIN-33101 Tampere, Finland;+Univ. Oulu, Oulu, Finland; *Univ. of Aarhus, Aarhus, Denmark; #Univ. of Tokyo, Tokyo, Japan
A typical feature of human atherosclerotic lesions is the presence of macrophage- and smooth muscle cell (SMC)-derived foam cells. Three receptor systems may be involved in the development of these foam cells: a2-macroglobulin receptor/LDL receptor related protein (a2-MlULRP), scavenger receptor and LDL receptor. We have studied the expression of these receptors in human atherosclerotic lesions by in situ hybridization and immunocytochemistry. SMCs expressed a~-MR/LRP mRNA and protein in normal human aorta and in both early and advanced human lesions No scavenger receptor expression was found in lesion SMCs. Macrophages expressed a2-MRLRP as well as scavenger receptor mRNAs and proteins in early and late lesions, whereas LDL receptor expression was not found in human lesions. There are several findings suggesting that a~-MIULRP may have a role in the formation of foam cells: (a) our results show that lesion macrophages and SMCs expressed a2-MR/LRP, (b) it has been shown by others that apo E-enriched remnant lipoproteins and LPL-triglyceride-rich lipoprotein complexes can be bound and internalized by q-MR/LRP Also, (c) q-MIULRP is not down-regulated by increased cellular cholesterol content. Scavenger receptors, on the other hand, are likely to be involved in the uptake of modified forms of LDL by lesion macrophages. Our results demonstrate that, so far, the only lipoprotein receptor expressed in lesion SMCs in vivo is q-MR/LRP. Scavenger receptors arc expressed only in lesion macrophages. Both recep tars may contribute to the development of human atherosclerotic lesions. 1 mL,
Relationship of LDL receptor gene defects to familial hypercholesterolemia in childhood Levy E, Feoli-Fonseca JC, Lambert M, Depts. of
Nutrition and Pediatrics, H6pital Ste-Justine and Univ. qf Montreal, Quebec, Canada H3T 1 CS
Quantitative abnormalities in LDL metabolism are the most striking features of familial hypercholesterolemia (FH), a common autosomal codominant disorder. The naturally occurring mutations within the LDL receptor gene are responsible for the aberrant phenotype. Since few studies have defined FH in the pediatric population by molecular analysis, the present investigation aimed to characterize the relationship of genotype to biochemical phenotype in a French Canadian pediatric population. A group of 88 unrelated French Canadian children with a persistent baseline increase in LDL-C levels (~3.3 mM) and a posi-
Atherosclerosis X, Montreal, October 1994