Atherosclerosis. 92 ( 1992) 26 I-264 0 1992 Elsevier ScientificPublishersIreland, Ltd. All rights reserved0009-8981/92/$05.00

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Printed and Publishedin Ireland ATHERO04767

Symposium Report

High density lipoproteins: A. Corsini’,

physiopathology

F. Bernini’,

C. Vergani2

and clinical relevance

and A.L. Catapano’

‘Institute of Pharmacological Sciences and 21nstiture of Internal Medicine. University of Milan0 (Italy)

Summary

On September 13- 14 199 1 a Symposium on High Density Lipoproteins: Physiopathology and Clinical Relevance was held in Bellagio (Italy). This Symposium was aimed at discussing various aspects of HDL from epidemiology to the most recent advances in the understanding of HDL metabolism and factors (diets, drugs) affecting their levels.

Key words: Apolipoprotein A-I; LCAT; CETP; Triglycerides

Epidemiology

Dr. G. Assmann (Munster, Germany) opened the first session with a review of the epidemiological data on HDL obtained in a large prospective study in Germany (the PROCAM study). In agreement with other prospective studies, a very strong inverse relationship between HDL cholesterol and coronary artery disease (CAD) was found. Furthermore, an analysis of the prevalence of myocardial infarction according to total cholesterol, HDL cholesterol and plasma triglycerides indicated that the subgroup with low HDL cholesterol (c 35 mg/dl) and high triglyceride levels (> 200 mg/dl) was at the highest risk for CAD. In Dr. Assmann’s opinion the data cor-

Correspondence to: A. Corsini, Institute of Pharmacological Sciences, via Balzaretti 9, University of Milano, Italy.

roborate the findings of the Helsinki Heart Study in which the patients who most benefited from gemfibrozil treatment (reduced CAD) were those presenting with low HDL and high plasma triglycerides. Dr.. C. Vergani (Milano, Italy) described the results of an Italian prospective study in a working population aged 20-59 years (the Rivetti Heart Study). In this study hypoalphalipoproteinemia (defined as HDL chol ~40 mg/dl in women and < 35 mg/dl in men) was observed in 10% of women and 19.3% of men and represents the second most common form of dyslipidemia. Primaryhypoalphalipoproteinemia, defined as low levels of HDL with normal levels of total cholesterol and triglycerides, was detected in 1.2% of women and 4.3% of men. In Dr. Vergani’s opinion more attention should be paid to these forms of dyslipoproteinemia, which are relatively frequent in a free living population.

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Structure of HDL Dr. D. Small (Boston, MA, U.S.A.) discussed the structure of HDL, and the role of different apolipoproteins in determining size and distribution of HDL. In nascent discoidal HDL apo A-I is believed to stabilize the overall structure by surrounding the rim of these particles. As the particle becomes spherical, by the action of several enzymes, A-I changes conformation. The change in size of HDL (HDL3 - HDL3 is also related to the number of apo A-I per particle. Dr. Small also discussed the characteristics of apo A-I alpha helices. All positively charged amino acids are on one side of this structure and account for 60% or more of the structure. Depending upon the radius of the particle, apo A-I may be more deeply impinged into the surface phospholipids. In the author’s opinion these findings are in agreement with the conformational changes of apo A-I in HDL subfractions detected with several methods.

Physiopathology Dr. J. Shepherd (Glasgow, U.K.) reviewed the studies on HDL apoprotein metabolism performed in his laboratory. Two approaches were used, one employed intact radiolabelled HDL; the other isolated HDL apolipoprotein (A-I and A-II) labelled and re-incorporated into HDL. The data indicated that at least two metabolically distinct pools of apo A-I exist owing to the presence in the blood of two subfractions of HDL, one containing only apo A-I and another containing A-I and A-II. However, the interpretation of the data is further complicated by the presence of substantial but incomplete exchange of A-I between these particles. In Dr. Shepherd’s opinion further studies are required to understand these complex relationships. Dr. D. Ruder (Bethesda, MD, U.S.A.) discussed recent data obtained on the ‘in vivo’ catabolisms of the two major apo A-I containing HDL subfractions (Lp A-I and Lp A-I, A-II) obtained by immunoaffinity chromatography after injection of labelled apo A-I and apo A-II re-associated with autologous plasma. A-I catabolism in Lp A-I, A-II was slower than that of A-I in Lp A-I. In a second series of studies A-I labelled with ‘25I or 1311was

associated with Lp A-I and Lp A-I, A-II respectively. Again the residence time of A-I in Lp A-I was shorter than that of apo A-I in Lp A-I, A-II. In the author’s opinion these data indicate that these particles have different metabolic pathways. Dr. Rader also presented data on familial hyperalphalipoproteinemia and cholesteryl ester transfer protein (CETP) deficiency. In the former case the high levels of plasma apo A-I were due to an increase in the apo A-I production rate, while in CETP deficiency they were due to a lower fractional catabolic rate. Both conditions presented with high levels of Lp A-I. Dr. C. Fielding (San Francisco, CA, U.S.A.) presented data on the role of HDL subfractions, separated by two dimensional electrophoresis, in the removal of cholesterol from cultured cells. Apo A-I-containing HDL are heterogeneous and the smallest particles (with pre /3 electrophoretic mobility) are most effective in promoting ‘reverse cholesterol transport’, even though they account for only l-2% of total plasma apo A-I. These lipoproteins contain a single A-I molecule and are not a direct substrate for LCAT. Dr. Fielding presented data supporting the concept that small pre fi HDL are precursors for large discoidal HDL which are the most effective substrate for LCAT. The action of this enzyme upon cholesterol may provide lipids which contribute to the formation of mature HDL particles. Dr. J. Oram (Seattle, WA, U.S.A.) reviewed the most recent data on the role of the HDL ‘receptor’ pathway in the ‘reverse cholesterol transport’. Dr. Oram presented data indicating that the interaction of HDL with their receptor triggers the translocation of cholesterol from intracellular compartments to the plasma membrane where the sterol can be removed. Apo A-I in HDL and to a lesser extent A-II and A-IV interact with the putative receptor and activate protein kinase C. Additional studies on the characterization of the putative HDL receptor were presented. A protein of 110 kDa possessing many features expected for the HDL receptor (i.e. up-regulation by cholesterol loading of cells and specificity for HDL apolipoproteins) was isolated. The protein sequence (derived from cDNA) comprises of 14 imperfect tandem repeats each containing two amphiphatic helices, but no membrane spanning do-

263 main. Transfection of cells with this cDNA leads to increased cell surface binding of HDL. Dr. F. Bernini (Milano, Italy) presented results of studies on the properties of the HDL binding site in EAhy 926 cells, a permanent human cell line possessing several differentiated functions of vascular endothelium. The HDL binding site in EAhy 926 is similar to that present in fibroblasts, smooth muscle cells, and endothelial cells. Upregulation of HDL binding by cholesterol was npt prevented by blockage of protein synthesis, and also occurred in formaldehyde-fixed, non-living cells. In Dr. Bernini’s opinion these data indicate that EAhy 926 cells are a good model for studying the HDL interaction with endothelial cells and a mechanism independent of cellular metabolism is involved in the cholesterol-mediated up-regulation of HDL binding sites. Dr. H. Funke (Miinster, Germany) reviewed the most recent data on LCAT deficiency from his laboratory. He presented results on the sequence analysis of the LCAT gene in 5 families with classical LCAT deficiency and in two families with fish eye disease (FED). In both families with FED a missense mutation on codon 123 (Ile - Thr) was identified. In the 5 families with LCAT deficiency 6 different defective alleles were identified. In a Canadian family the homozygous patients were compound heterozygous at codon 93 (ala - Thr) and 158 (Arg - Cys). In the other 4 families the homozygous were true genetic homozygous. Most of the identified defects were missense mutations, but a non-sense mutation giving rise to a stop codon was identified also. In Dr. Funke’s opinion since the observed defects do not cluster in a specific region it can be hypothesized that LCAT consist of several distinct structurally important domains. Dr. L. Agellon (New York, NY, U.S.A.) reviewed the most recent data on the effect of CETP on lipoprotein metabolism in mice, a species which normally lacks CETP activity. High expression of human CETP in transgenic mice results in a lowering of plasma cholesterol due to a decrease of HDL cholesterol without effect on VLDL, a finding that is at variance with the well known relation between VLDL and HDL levels. To refine this model further a strain of mice that produces both human A-I and CETP was created. In these

mice the reduction of HDL was much more impressive, suggesting that the speciation of HDL induced by the expression of human apo A-I in mice promotes the activity of CETP. In the author’s opinion the transgenic mice model can be useful in providing further insight into the role of CETP in lipid metabolism. Dr. G. Assmann (Miinster, Germany) discussed the identification of several probands from unrelated families with genetically determined structural variants of apo A-I. Although several of these mutations had no impact on HDL cholesterol, some mutations did, as for instance in a family with carriers of the 165 Pro - Arg, 173 Arg - Cys and a 202 frameshift giving rise to a protein of 229 aa. However, CAD is not a hallmark in these patients, even though in some families corneal opacity was detected. Of interest also was the deletion 107 (Lys - 0): a mutation which impairs the ability of apo A-I to activate LCAT (40% of normal). Dr. Assmann also discussed the recent finding of a patient with no detectable apo A-I in her plasma owing to a mutation for which she was homozygous. In Dr. Assmann’s opinion the search for naturally occurring mutants of apo A-I may contribute to a better understanding of the structure-function relationship in apo A-I. Dr. F. Pugani (Milano, Italy) discussed the identification of a polymorphic site in the promoter region of apolipoprotein A-I, due to an adenine to guanine transition 78 base pairs upstream from the transcription start of the apo A-l gene. The allelic frequency of this polymorphism increased with increasing levels of plasma HDL cholesterol. DNA sensitivity to DNase cleavage indicated different conformation in the regions surrounding the polymorphic area. In the author’s opinion these data support the concept that this polymorphism is located in an area of the apo A-I promoter that modulates A-I gene expression. Dr. P. Roma (Milano, Italy) reported kinetic studies on the catabolism of HDL in man with hypoalphalipoproteinemia. The reduced apo A-l levels in 3 probands with familial hypoalpha was due to an increased fractional catabolic rate not related to an abnormal behaviour of apo A-I. Kinetics were also studied in carriers of the A-I Milan0 mutation (Arg 173 - Cys). A-I Milan0 had a faster catabolism as compared to normal A-I

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in control and A-I Milan0 patients. The faster catabolism was related to the structural abnormality of this apoprotein. In Dr. Roma’s opinion these studies suggest that multiple metabolic defects may underlie hypoalphalipoproteinemia in man. Diets, drugs and HDL Dr. J. Patsch (Innsbruck, Austria) discussed post prandial lipemia, and the relationship between TG and HDL levels. The strong inverse relationship between TG and HDL supports the concept that TG may play a major role in the onset of CAD. Furthermore, studies on the post prandial lipemia in subjects with angiographically demonstrated CAD showed an increased response to a fatty meal. In the author’s opinion post prandial lipemia could be a test for detecting subjects at risk for CAD. Dr. S. Eisenberg (Jerusalem, Israel) discussed the effects of diets and drugs which reduce plasma triglycerides on HDL. Diets rich in polyunsaturated fatty acids lower HDL by reducing the A-I production rate. Drugs which reduce plasma TG (bezatibrate) cause a measurable increase in plasma HDL cholesterol, which, however, is weakly related to the changes of TG, lipoprotein lipase, and cholesterol ester/protein ratio suggesting a direct effect of the drug on the HDL system. In the author’s opinion this finding indicates that regulation of the HDL system by drugs or diets is mediated by several distinct mechanisms. Dr. J.C. Fruchart (Lille, France) discussed the effect of hypolipidaemic drugs on HDL subfractions, containing A-I (Lp A-I) and those containing A-I plus A-II (Lp A-I, A-II). The physiological function of Lp A-I appears to be related to cholesterol eMux from cells while Lp A-I, A-II is essentially inactive. Furthermore Lp A-I is decreased in patients with CAD. Cholestyramine and simvastatin treatment increased Lp A-I, while pravastatin and fenotibrate increased Lp A-I, AII. The effect of the drugs was related to the baseline level of Lp A-I; simvastatin increased Lp A-I levels in patients with Lp A-I < 35 mg/dl; fenofibrate induced a decrease of Lp A-I only in patients with high levels of Lp A-I. In the author’s opinion these findings may be important for pharmacological and epidemiological studies. Dr. C.R. Sirtori (Milano, Italy) discussed the

role of phospholipids (PL) in atherosclerosis. Evidence from several ‘in vitro’ and ‘in vivo’ studies suggest that unsaturated PL may improve the clearance of atherogenic lipoproteins and mitigate the effect of atherosclerotic diets. Dr. Sirtori described a recent pharmacodynamic study performed in normolipidemic volunteers with double labelled phosphatidylcholine (PC). The choline moiety (3H) showed an early incorporation into HDL and, subsequently, into LDL. The 14C radioactivity (linoleic acid) was rapidly incorporated into VLDL triglyceride and later transferred to HDL, compatible with a loss of acyl groups. The polyunsaturated PC remained in plasma longer than previously recognized. In the author’s opinion this study provides clues to the possible therapeutic role of essential phospholipids. Dr. G. Salvioli (Modena, Italy) presented data on phospholipids and bile composition. He reviewed the role of phospholipids in determining bile properties and discussed the role of polyunsaturated PC on these properties. In rats, when linoleic acid was perfused, dilinoleic PC became the major PL in bile. Similar studies performed in man indicate that the dilinoleic PC content in bile was increased. However, no long-term data are available and, in the author’s opinion, the effect on bile litogenecity still remains to be addressed. In summary, the current knowledge of factors regulating high density lipoprotein levels was presented. Several issues were raised which will require further study in order to be fully understood, yet a significant amount of information is being gathered on HDL. No simple answer can explain how HDL relates to CAD. It is likely that several factors (diets, hormones, drugs) interfere with HDL, but only now are we beginning to understand the complexity of the metabolic pathways regulating the plasma levels of this fascinating lipoprotein class. Acknowledgements

The authors’ work described in this summary was supported by MPI and CNR (Progetto Finalizzato Invecchiamento) Publication 923090. We wish also to thank Dr. C. Lanzoni (Rhbne Poulenc-Rorer) for the generous support that made the meeting possible and Miss Maddalena Marazzini for typing the manuscript.

High density lipoproteins: physiopathology and clinical relevance.

On September 13-14 1991 a Symposium on High Density Lipoproteins: Physiopathology and Clinical Relevance was held in Bellagio (Italy). This Symposium ...
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