- Email: [email protected]
Analysis of ligand binding to the LDL receptor-related protein (LRP)
Tuesday 11 October: Workshop Abstracts WI 1 Lipoprotein receptors and reldted diseases
proteins (LP) subsequently added. The degree of inhibition was associated with the amount of ox-LDL internalized and with the degree of LDL oxidation. Aggregated forms of lipoproteins showed a greater reduction in &gradation than corresponding monomeric ones. Extracts of cells preincubated with ox-LDL demonstrated a reduction in lysosomal cathepsin B activity but not in cathepsin D activity. Extracts of untreated M$ or purified cathepsin B showed a reduction in cathepsin B activity following direct incubation with ox-LDL but not with LDL. Since cathepsin B is a thiol protease, it is likely that reactive groups on modified apo B-100 in ox-LDL interact with the thiol at the active site of the enzyme at the low lysosomal PH. To assess whether intracellular trafficking of ox-LDL was altered, we compare it to that of
17.5
acetyl LDL. We labeled each LP with colloidal gold of a different size, mixed them together, and incubated M$Jwith the two labeled LP. After 1 h about 30% of the endosomes contained gold of only one size or the other. Even when together in single endosomes, gold of two sizes were segregated to separate parts of the endosome. This was not due to a characteristic of the size of the colloidal gold, since mixtures of acetyl LDL labeled with both sizes of gold consistently co-localized in endosomes. Collectively, these results suggest that, in addition to the published evidence of apo B modification in ox-LDL playing a role in the poor processing of ox-LDL in M+, inactivation of cathepsin B by ox-LDL and possibly altered intracellular trafficking of ox-LDL might also be responsible.
Wll LIPOPROTEIN RECEPTORS AND RELATED DISEASES Molecular heterogeneity of familial hypercholesterolemia in the Arab communities in Israel &&&Q&J& Reshef A, Center for Research, Prevention and Treatment of Atherosclerosis, Hadassah Univ. Hosp., 91 I20 Jerusalem, Israel
The Arab population of Israel consists of several well-defined communities. Increased prevalence of familial hypercholesterolemia (FH) has been identified in Christian Arabs and Druze. These populations may represent only a minor fraction of the corresponding communities that reside in the region and it may be postulated that some of the mutations identified here originated in the neighboring countries. Convincing evidence for this phenomenon emerged through the identification of the Lebanese allele at the LDL receptor gene locus (stop 660) in homozygote FH cases from Lebanon and in Israeli Christian Arabs. Other mutations, including the J.D. mutation which is of Italian-Syrian origin (Y807C) and the Jerusalem mutation (El87K), have recently been identified in Israel. It is not yet known whether the Druze mutation which is most prevalent in Druze of the Golan Heights (stop 167) is also present in other countries. With the recent identification of a mutation in Moslem Arabs (delta G165) it is now possible to extend the search for FH cases in the region and determine the relative importance of each of these mutations in the local FH population. We have devised screening methods for the detection of each of these mutations. So far, the identification of heterozygote FH cases from these communities enabled the characterization of possible differences in plasma lipoprotein concentrations from those in other population groups in Israel. Although no differences in plasma LDL-C levels were demonstrated, plasma lipoprotein(a) levels differed significantly and were found to be lower in the Druze than in the Christian Lebanese. It remains to be shown whether these differences are reflected in the prevalence of cardiovasculardisease. Lipoprotein receptors: ancestors and relatives Bujo H, Novak S, Nimpf J, sr W.!, Dept. Mol. Genetics, Univ. and Biocenter Vienna, Dr. Bohr-Gasse 9/Z’,A-1030 Vienna, Austria
Two parallel pathways of lipoprotein transport can be studied in the laying hen, as this animal manages to maintain systemic homeostasis while transporting large amounts of lipoproteins and other yolk precursors into the growing oocytes. The largest oocyte ovulates after having accumulated in only 7 days as much as 2.5 g protein, 230 mg cholesterol and 5 g triglycerides! The source for yolk are lipoproteins and other macromolecules synthesized under the control of estrogen in the liver, from where they are secreted into the plasma compartment and subsequently transported into the oocytes. Simultaneously, somatic cells are supplied in regulated and regulatory fashion with these very components. Our
studies on lipoprotein receptor-mediated regulation of oocyte growth in the chicken have revealed that in order to master the task of dual lipoprotein transport systems, the laying hen expresses several highly related but distinct genes related to that of the LDL receptor. For oocyte growth via yolk precursor uptake, the receptors are (i) a 95-kDa protein which possibly is the ancestor of the mammalian VLDL receptors, and (ii) a large LDLreceptor related protein (LRP), an old relative of the LRP family. Somatic cells express an LDL receptor (130 kDa) and a second LRP (600kDa; Nimpf et al. J Biol Chem 1994; 269: 212-219). The pairwise expression of these cell-type specific genes is indicative of specific functions with regard to ligand recognition and transport, corroborated by distinct differences in regulation of the respective genes. For instance, the LDL receptor of somatic tissues preferentially transports LDL and is sterol-regulated, whereas the 95kDa oocyte receptor binds VLDL and vitellogenin (a lipophosphoglycoprotein found only in oviparous animals) and is refractory to feedback regulation. Further insights into the structure/function/regulation relationships of the growing LDL receptor gene family are gained from delineating the subtle ligand-binding differences among the avian receptors on one hand, and between avian and mammalian gene products on the other hand. The laying hen has provided better understanding of the biological basis for simultaneous expression of highly related genes in one and the same organism. The VLDL receptor and related molecules: role and function m, Tohoku University Gene Research Center, l-l Tsutsumidori-Amamiya, Aoba, Sendai 981, Japan
Lipoprotein receptors mediate the uptake of plasma lipid-canying lipoproteins and thereby play a key role both in intracellular lipid metabolism and in the clearance of lipoproteins from the plasma. The LDL receptor, consisting of five functional domains, binds lipoproteins that contain apo B 100 and/or apo E, and carries them into cells. Recently we have isolated a cDNA highly similar to the LDL receptor. Cells expressing this cDNA bound only ape Econtaining lipoproteins and carried them into cells via receptormediated endocytosis. This newly discovered receptor, designated ‘VLDL receptor’, consists of five functionaI domains that closely resemble those of the LDL receptor. Although the structure and ligand binding of the VLDL receptor are closely related to those of the LDL receptor, the chromosomal location, tissue distribution and regulation of the two receptors are different. Analysis of ligand binding to the LDL receptor-related protein (LW Willnow Orth K, Herz J, Dept. of Mol. Genetics, Univ. of Texas, SouthLestern Medical Center, Dallas, TX 7523.5, USA
LRP, a member of the LDL receptor gene family, is a large multi-
Atherosclerosis X, Montreal, October 1994
176
Tuesday 1 I October: Workshop Abstracts Wl2 HDL-cell interaction
functional receptor implicated in the hepatic uptake of chylomicron remnants. In addition to apo E-rich lipoproteins and lipoprotein lipase, LRP also endocytoses aa-macroglobulin, plasminogen activators and complexes of these proteases with their specific inhibitor, plasminogen activator inhibitor-l (PAI-1). Thus, LRP not only functions in lipoprotein metabolism but is also involved in the regulation of fibrinolysis. Disturbances in either of these diverse biological systems contribute significantly to the development of atherosclerotic lesions. Ligand binding and competition experiments suggests that several of the ligands bind to independent sites on LRP. To identify specific binding regions on the receptor we have used recombinant DNA techniques to dissect LRP into chimetic minireceptors each of which expresses a different region of the extracellular domain of LRP. Like the wildtype receptor, these minireceptors actively endocytose ligands from the extracellular environment of transfected cells, but their ligand-binding profile is restricted. Using this approach we have identified binding regions on LRP for receptor-associated protein (RAP), tissue-type plasminogen activator @PA) and tPA/PAI-1 and urokinase/PAI-1 complexes. Characterization and purification of the tipolysis-stimulated receptor &ihain BE, Yen FT, Mann CJ, Delplanque B, Guermani LM, Troussard AA, Khahou J, INSERM Unit 391, Universite’de Rennes I, France
The lipolysis-stimulated receptor (LSR) mediates binding, internalization and lysosomal degradation of apo B-containing lipoproteins in human fibroblasts lacking the LDL-receptor and in primary hepatocytes of rata and homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits. LSR binds both apo B and apo E, and has highest affinity for TG-rich lipoproteins. Lipoprotein binding to LSR occurs only after the receptor has been activated by free fatty acid, a process that is reversible. Characterization of the mechanism of activation indicates that the interaction of LSR protein with a specific free fatty acid causes conformational changes of the protein that reveal a lipoprotein-binding domain. Ligand blot analysis of rat liver membrane proteins separated by SDS-PAGE and transferred to nitrocellulose, revealed a single band corresponding to a protein of 180 kDa. Using gel filtration, anion-exchange chromatography and gel-band elution techniques, a protein of similar molecular weight and demonstrating LSR activity was purified to homogeneity. LSR activation by lipolytic products, high affinity for TG-rich lipoproteins and inhibition by lactoferrin am consistent with this receptor acting primarily for the clearance. of chylomicron remnants. In addition LSR can bind LDL, and is present in subjects lacking the LDL receptor. Screening for pharmacological agents able to activate LSR revealed that rifampin derivatives were potent in vitro stimulators of LSR activity (>lO-fold that of free fatty acid). Administration of these compounds to homozygous WHHL rabbits (50 mg/kg) caused strong (>50%) reduction of plasma cholesterol that persisted over the 3 weeks of treatment without noticeable side effects. Our current hypotbesis is that LSR contributes significantly to the clearance of chylomicron remnants, and that it serves as an alternative pathway for the removal of LDL. LSR therefore provides a new target for pharmacological management of familial hypercholesterolemia.
Structure and function of macrophage scavenger receptors mT, Suzuki S, Kurihara Y, Kurihara H, Doi T, Jishage K, Kamada N, Ueda 0, Matsumoto A, Honda M, Wada Y, Yazaki Y, Itakura H, Univ of Tokyo, Tokyo; CSK Research Park; and Univ. of Osaka, Osaka, Japan
We have established scavenger receptor-deficient mice by gene targeting. In addition to foam cells in the atherosclerotic lesion, several types of perivascular cells, including Mato’s perivascular fluorescent cells of brain microarteries, express scavenger receptors. Scavenger receptors mediate the uptake of various negatively charged macromolecules and also mediate the cation-independent adhesion of macrophages. These functions play critical roles in atherogenesis. Scavenger receptors consist of six domains. Domain 1 mediates endocytosis. Domain 3 (coiled-coil) mediates pH-dependent ligand association. Domain 4 (collagen) contains a ligand-binding ‘charged collagen’ structure. Two synthetic apo BlOO peptides can inhibit acetyl-LDL binding to the scavenger receptor without modification. One peptide can inhibit only after acetylation. The ligand/receptor interaction was analyzed. We have synthesized and tested over 600 compounds or drugs inhibiting the modification of lipoproteins by affecting the scavenger pathway. Among them several compounds were effective in the prevention of atherosclerosis in both WHHL rabbits and in animal models in which a high-cholesterol diet is fed. Characterization of abnormal macrophage metabolism in a normocholesterolemic kindred with xanthoma
. . WA,
Clin. Res. Inst. of Montreal, 110 Pine Ave West, Montreal, H2W lR7, Canada
It has been hypothesized that foam cells in cutaneous xanthomas result from the uptake by the macrophage scavenger receptor (MSR) of plasma LDL which may be modified due to prolonged circulation in hyperlipidemia. We have observed in two siblings (FC, LC) extensive xanthelasma and planar xantbomas in the absence of hyperlipidemia. Blood monocytes from FC showed a 60-fold higher rate of de radation of oxidized LDL than those from a normal subject. Id I-AcLDL degradation by blood monocytes from LC was 1523 ng/mg cell protein/5 h (n = 2) vs. 464 f 97 (mean f SEM) for four control subjects. Accumulation of esterified cholesterol was 3-9 times higher in macrophages from LC than controls. Monocyte SR mRNA, measured with fluorescence detection of PCR-amplified revere transcribed RNA, was 0.62 f 0.05 in LC (ratio SRI/?-actin) (n = 3 measurements) vs. 0.095 f 0.05 (n = 4 controls) and rose during 7 days in culture to 64.8 ?r 2.1 and 28.4 zb11 in macrophages, for LC and controls, respectively. Among all monocytes studied, variation in AcLDL degradation rate was completely accounted for by SR mRNA levels (r = 0.99, n = 5 subjects). Mean MSR mRNA in monocytes from 2 of 3 brothers and the son of LC was about 20 times higher than the average in controls. In the LC kindred, markedly high monocyte SR mRNA segregated with abnormal cellular phenotype. characterized by over accumulation of lipid. Thus, the presence of xanthoma in the absence of hyperlipidemia was associated with precocious expression of the MSR mRNA in monocytes. These studies help to clarify the regulation of expression of the scavenger receptor and the role of this receptor in foam cell formation and atberogenesis.
W12 HDL-CELL INTERACTION Cholesterol efflux from cells in culture w, Davidson WS, Yancey P, Kilsdonk EPC, de la Llera Moya M, Dept. of Biochem., Med. Coil. of Pennsylvania, Philadelphia, PA, USA
We have studied cellular cholesterol efflux and have focused on the contribution of both cell and acceptor properties. At the cellular level we have observed that treatment of cells with 25hydroxycholesterol reduces cholesterol efflux to phospholipid-
Atherosclerosis X, Montreal, October 1994
proteins (LP) subsequently added. The degree of inhibition was associated with the amount of ox-LDL internalized and with the degree of LDL oxidation. Aggregated forms of lipoproteins showed a greater reduction in &gradation than corresponding monomeric ones. Extracts of cells preincubated with ox-LDL demonstrated a reduction in lysosomal cathepsin B activity but not in cathepsin D activity. Extracts of untreated M$ or purified cathepsin B showed a reduction in cathepsin B activity following direct incubation with ox-LDL but not with LDL. Since cathepsin B is a thiol protease, it is likely that reactive groups on modified apo B-100 in ox-LDL interact with the thiol at the active site of the enzyme at the low lysosomal PH. To assess whether intracellular trafficking of ox-LDL was altered, we compare it to that of
17.5
acetyl LDL. We labeled each LP with colloidal gold of a different size, mixed them together, and incubated M$Jwith the two labeled LP. After 1 h about 30% of the endosomes contained gold of only one size or the other. Even when together in single endosomes, gold of two sizes were segregated to separate parts of the endosome. This was not due to a characteristic of the size of the colloidal gold, since mixtures of acetyl LDL labeled with both sizes of gold consistently co-localized in endosomes. Collectively, these results suggest that, in addition to the published evidence of apo B modification in ox-LDL playing a role in the poor processing of ox-LDL in M+, inactivation of cathepsin B by ox-LDL and possibly altered intracellular trafficking of ox-LDL might also be responsible.
Wll LIPOPROTEIN RECEPTORS AND RELATED DISEASES Molecular heterogeneity of familial hypercholesterolemia in the Arab communities in Israel &&&Q&J& Reshef A, Center for Research, Prevention and Treatment of Atherosclerosis, Hadassah Univ. Hosp., 91 I20 Jerusalem, Israel
The Arab population of Israel consists of several well-defined communities. Increased prevalence of familial hypercholesterolemia (FH) has been identified in Christian Arabs and Druze. These populations may represent only a minor fraction of the corresponding communities that reside in the region and it may be postulated that some of the mutations identified here originated in the neighboring countries. Convincing evidence for this phenomenon emerged through the identification of the Lebanese allele at the LDL receptor gene locus (stop 660) in homozygote FH cases from Lebanon and in Israeli Christian Arabs. Other mutations, including the J.D. mutation which is of Italian-Syrian origin (Y807C) and the Jerusalem mutation (El87K), have recently been identified in Israel. It is not yet known whether the Druze mutation which is most prevalent in Druze of the Golan Heights (stop 167) is also present in other countries. With the recent identification of a mutation in Moslem Arabs (delta G165) it is now possible to extend the search for FH cases in the region and determine the relative importance of each of these mutations in the local FH population. We have devised screening methods for the detection of each of these mutations. So far, the identification of heterozygote FH cases from these communities enabled the characterization of possible differences in plasma lipoprotein concentrations from those in other population groups in Israel. Although no differences in plasma LDL-C levels were demonstrated, plasma lipoprotein(a) levels differed significantly and were found to be lower in the Druze than in the Christian Lebanese. It remains to be shown whether these differences are reflected in the prevalence of cardiovasculardisease. Lipoprotein receptors: ancestors and relatives Bujo H, Novak S, Nimpf J, sr W.!, Dept. Mol. Genetics, Univ. and Biocenter Vienna, Dr. Bohr-Gasse 9/Z’,A-1030 Vienna, Austria
Two parallel pathways of lipoprotein transport can be studied in the laying hen, as this animal manages to maintain systemic homeostasis while transporting large amounts of lipoproteins and other yolk precursors into the growing oocytes. The largest oocyte ovulates after having accumulated in only 7 days as much as 2.5 g protein, 230 mg cholesterol and 5 g triglycerides! The source for yolk are lipoproteins and other macromolecules synthesized under the control of estrogen in the liver, from where they are secreted into the plasma compartment and subsequently transported into the oocytes. Simultaneously, somatic cells are supplied in regulated and regulatory fashion with these very components. Our
studies on lipoprotein receptor-mediated regulation of oocyte growth in the chicken have revealed that in order to master the task of dual lipoprotein transport systems, the laying hen expresses several highly related but distinct genes related to that of the LDL receptor. For oocyte growth via yolk precursor uptake, the receptors are (i) a 95-kDa protein which possibly is the ancestor of the mammalian VLDL receptors, and (ii) a large LDLreceptor related protein (LRP), an old relative of the LRP family. Somatic cells express an LDL receptor (130 kDa) and a second LRP (600kDa; Nimpf et al. J Biol Chem 1994; 269: 212-219). The pairwise expression of these cell-type specific genes is indicative of specific functions with regard to ligand recognition and transport, corroborated by distinct differences in regulation of the respective genes. For instance, the LDL receptor of somatic tissues preferentially transports LDL and is sterol-regulated, whereas the 95kDa oocyte receptor binds VLDL and vitellogenin (a lipophosphoglycoprotein found only in oviparous animals) and is refractory to feedback regulation. Further insights into the structure/function/regulation relationships of the growing LDL receptor gene family are gained from delineating the subtle ligand-binding differences among the avian receptors on one hand, and between avian and mammalian gene products on the other hand. The laying hen has provided better understanding of the biological basis for simultaneous expression of highly related genes in one and the same organism. The VLDL receptor and related molecules: role and function m, Tohoku University Gene Research Center, l-l Tsutsumidori-Amamiya, Aoba, Sendai 981, Japan
Lipoprotein receptors mediate the uptake of plasma lipid-canying lipoproteins and thereby play a key role both in intracellular lipid metabolism and in the clearance of lipoproteins from the plasma. The LDL receptor, consisting of five functional domains, binds lipoproteins that contain apo B 100 and/or apo E, and carries them into cells. Recently we have isolated a cDNA highly similar to the LDL receptor. Cells expressing this cDNA bound only ape Econtaining lipoproteins and carried them into cells via receptormediated endocytosis. This newly discovered receptor, designated ‘VLDL receptor’, consists of five functionaI domains that closely resemble those of the LDL receptor. Although the structure and ligand binding of the VLDL receptor are closely related to those of the LDL receptor, the chromosomal location, tissue distribution and regulation of the two receptors are different. Analysis of ligand binding to the LDL receptor-related protein (LW Willnow Orth K, Herz J, Dept. of Mol. Genetics, Univ. of Texas, SouthLestern Medical Center, Dallas, TX 7523.5, USA
LRP, a member of the LDL receptor gene family, is a large multi-
Atherosclerosis X, Montreal, October 1994
176
Tuesday 1 I October: Workshop Abstracts Wl2 HDL-cell interaction
functional receptor implicated in the hepatic uptake of chylomicron remnants. In addition to apo E-rich lipoproteins and lipoprotein lipase, LRP also endocytoses aa-macroglobulin, plasminogen activators and complexes of these proteases with their specific inhibitor, plasminogen activator inhibitor-l (PAI-1). Thus, LRP not only functions in lipoprotein metabolism but is also involved in the regulation of fibrinolysis. Disturbances in either of these diverse biological systems contribute significantly to the development of atherosclerotic lesions. Ligand binding and competition experiments suggests that several of the ligands bind to independent sites on LRP. To identify specific binding regions on the receptor we have used recombinant DNA techniques to dissect LRP into chimetic minireceptors each of which expresses a different region of the extracellular domain of LRP. Like the wildtype receptor, these minireceptors actively endocytose ligands from the extracellular environment of transfected cells, but their ligand-binding profile is restricted. Using this approach we have identified binding regions on LRP for receptor-associated protein (RAP), tissue-type plasminogen activator @PA) and tPA/PAI-1 and urokinase/PAI-1 complexes. Characterization and purification of the tipolysis-stimulated receptor &ihain BE, Yen FT, Mann CJ, Delplanque B, Guermani LM, Troussard AA, Khahou J, INSERM Unit 391, Universite’de Rennes I, France
The lipolysis-stimulated receptor (LSR) mediates binding, internalization and lysosomal degradation of apo B-containing lipoproteins in human fibroblasts lacking the LDL-receptor and in primary hepatocytes of rata and homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits. LSR binds both apo B and apo E, and has highest affinity for TG-rich lipoproteins. Lipoprotein binding to LSR occurs only after the receptor has been activated by free fatty acid, a process that is reversible. Characterization of the mechanism of activation indicates that the interaction of LSR protein with a specific free fatty acid causes conformational changes of the protein that reveal a lipoprotein-binding domain. Ligand blot analysis of rat liver membrane proteins separated by SDS-PAGE and transferred to nitrocellulose, revealed a single band corresponding to a protein of 180 kDa. Using gel filtration, anion-exchange chromatography and gel-band elution techniques, a protein of similar molecular weight and demonstrating LSR activity was purified to homogeneity. LSR activation by lipolytic products, high affinity for TG-rich lipoproteins and inhibition by lactoferrin am consistent with this receptor acting primarily for the clearance. of chylomicron remnants. In addition LSR can bind LDL, and is present in subjects lacking the LDL receptor. Screening for pharmacological agents able to activate LSR revealed that rifampin derivatives were potent in vitro stimulators of LSR activity (>lO-fold that of free fatty acid). Administration of these compounds to homozygous WHHL rabbits (50 mg/kg) caused strong (>50%) reduction of plasma cholesterol that persisted over the 3 weeks of treatment without noticeable side effects. Our current hypotbesis is that LSR contributes significantly to the clearance of chylomicron remnants, and that it serves as an alternative pathway for the removal of LDL. LSR therefore provides a new target for pharmacological management of familial hypercholesterolemia.
Structure and function of macrophage scavenger receptors mT, Suzuki S, Kurihara Y, Kurihara H, Doi T, Jishage K, Kamada N, Ueda 0, Matsumoto A, Honda M, Wada Y, Yazaki Y, Itakura H, Univ of Tokyo, Tokyo; CSK Research Park; and Univ. of Osaka, Osaka, Japan
We have established scavenger receptor-deficient mice by gene targeting. In addition to foam cells in the atherosclerotic lesion, several types of perivascular cells, including Mato’s perivascular fluorescent cells of brain microarteries, express scavenger receptors. Scavenger receptors mediate the uptake of various negatively charged macromolecules and also mediate the cation-independent adhesion of macrophages. These functions play critical roles in atherogenesis. Scavenger receptors consist of six domains. Domain 1 mediates endocytosis. Domain 3 (coiled-coil) mediates pH-dependent ligand association. Domain 4 (collagen) contains a ligand-binding ‘charged collagen’ structure. Two synthetic apo BlOO peptides can inhibit acetyl-LDL binding to the scavenger receptor without modification. One peptide can inhibit only after acetylation. The ligand/receptor interaction was analyzed. We have synthesized and tested over 600 compounds or drugs inhibiting the modification of lipoproteins by affecting the scavenger pathway. Among them several compounds were effective in the prevention of atherosclerosis in both WHHL rabbits and in animal models in which a high-cholesterol diet is fed. Characterization of abnormal macrophage metabolism in a normocholesterolemic kindred with xanthoma
. . WA,
Clin. Res. Inst. of Montreal, 110 Pine Ave West, Montreal, H2W lR7, Canada
It has been hypothesized that foam cells in cutaneous xanthomas result from the uptake by the macrophage scavenger receptor (MSR) of plasma LDL which may be modified due to prolonged circulation in hyperlipidemia. We have observed in two siblings (FC, LC) extensive xanthelasma and planar xantbomas in the absence of hyperlipidemia. Blood monocytes from FC showed a 60-fold higher rate of de radation of oxidized LDL than those from a normal subject. Id I-AcLDL degradation by blood monocytes from LC was 1523 ng/mg cell protein/5 h (n = 2) vs. 464 f 97 (mean f SEM) for four control subjects. Accumulation of esterified cholesterol was 3-9 times higher in macrophages from LC than controls. Monocyte SR mRNA, measured with fluorescence detection of PCR-amplified revere transcribed RNA, was 0.62 f 0.05 in LC (ratio SRI/?-actin) (n = 3 measurements) vs. 0.095 f 0.05 (n = 4 controls) and rose during 7 days in culture to 64.8 ?r 2.1 and 28.4 zb11 in macrophages, for LC and controls, respectively. Among all monocytes studied, variation in AcLDL degradation rate was completely accounted for by SR mRNA levels (r = 0.99, n = 5 subjects). Mean MSR mRNA in monocytes from 2 of 3 brothers and the son of LC was about 20 times higher than the average in controls. In the LC kindred, markedly high monocyte SR mRNA segregated with abnormal cellular phenotype. characterized by over accumulation of lipid. Thus, the presence of xanthoma in the absence of hyperlipidemia was associated with precocious expression of the MSR mRNA in monocytes. These studies help to clarify the regulation of expression of the scavenger receptor and the role of this receptor in foam cell formation and atberogenesis.
W12 HDL-CELL INTERACTION Cholesterol efflux from cells in culture w, Davidson WS, Yancey P, Kilsdonk EPC, de la Llera Moya M, Dept. of Biochem., Med. Coil. of Pennsylvania, Philadelphia, PA, USA
We have studied cellular cholesterol efflux and have focused on the contribution of both cell and acceptor properties. At the cellular level we have observed that treatment of cells with 25hydroxycholesterol reduces cholesterol efflux to phospholipid-
Atherosclerosis X, Montreal, October 1994