The role of modified lipoproteins and antioxidants in atherosclerosis and cardiovascular disease

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Friday, 28 May 1999 Plenary lectures THE FORMATION, FATE, AND CLINICAL CONSEQUENCES OF ATHEROSCLEROTIC PLAQUE E. Falk. Dept. Cardiolog); Aurhus University Hospital (Skejhy). Denmark Coronary atherosclerosis is by far the most frequent cause of ischemic heart disease and plaque disruption with superimposed thrombosis, with or without concomitant vasospasm, is the main cause of the acute coronary syndromes of unstable angina, myocardial infarction, and sudden coronary death. Therefore, for event-free survival, the vital question is not why atherosclerosis develops but rather why atherosclerosis, after years of indolent growth, suddenly becomes complicated by life-threatening thrombosis. Therefore, we have to focus on plaque composition and vulnerability to rupture and plaque thrombogenicity rather than on plaque size and stenosis severity. Plaque Disruption: The risk of plaque disruption depends more on plaque vulnerability (plaque O,pe) than on degree of stenosis (plaque size): lipid-rich and soft plaques are more vulnerable and prone to rupture than collagen-rich and hard plaques. Furthermore, they are highly thrombogenic after disruption because of high content of tissue factor. There seem to be 3 major determinants of a plaque's vulnerability to rupture: I) size and consistency of the lipid-rich atheromatous core, 2)) thickness of the fibrous cap covering the core. and 3)) ongoing inflammation and repair processes within the fibrous cap. Lipid accumulation, cap thinning, lack of smooth muscle cells (smc), and macrophage-related inflammation destabilize plaques, making them vulnerable to rupture. In contrast, sine-related healing and repair processes stabilize plaques, protecting them against disruption. Plaque size or stenosis severity tell nothing about a plaque's vulnerability. Many vulnerable plaques are invisible angiographically due to their small size and compensatory vascular remodeling. Plaque Thrombosis: The most feared consequence of coronary plaque disruption is thrombotic occlusion of the artery. About 75% of thrombi responsible for acute coronary syndromes are precipitated by plaque disruption whereby the highly thrombogenic gruel is exposed to the flowing blood. In the remaining 25%, superficial plaque erosion without frank disruption (i.e., no deep injury) is usually present. Most disrupted plaques are resealed by a small mural thrombus, and only sometimes does a major luminal thrombus evolve. There are 3 major determinants of the thrombotic response to plaque disruption/erosion: I)) local thrombogenic substrate (tissue factor), 2) local flow disturbances (stenosis and high shear), and 3) systemic thrombotic propensity (platelets, coagulation, and fibrinolysis). The thrombotic response to plaque disruption is dynamic. Thrombosis/rethrombosis and thrombolysis/embolization occur simultaneously in many patients with acute coronary syndromes, with or without concomitant vasospasm, causing intermittent flow obstruction. The initial flow obstruction is usually due to platelet aggregation but fibrin is important for the subsequent stabilization of the early and fragile platelet thrombus. Therefore, both platelets and fibrin are involved in the evolution of a persisting coronary thrombus.

(TXA2) and prostaglandin H2 (PGI2) are endothelium-derived contracting factors. In contrast to TXA2 and PGH2 which activate platelets, endothelin-I has no platelet effects, but proliferative properties in vascular smooth muscle. Under physiological conditions, the endothelium plays a protective role as NO prevents adhesion of circulating blood cells, keeps the vasculature in a vasodilated state and inhibits vascular smooth muscle proliferation. In disease states, decreased NO release contributes to enhanced vasoconstriction, adhesion of platelets and monocytes and proliferation of vascular smooth muscle, all events which are involved in cardiovascular disease.

DYSLIPIDEMIA, INSULIN RESISTANCE AND APOB J. Scott, C. Shoulders, N. Navaratnam, T. Aitman. Imperial College School

of Medicine. Hammersmith Hospital, Du-Cane Road, London WI2 0NN, UK The metabolic overlap syndromes hyperlipidemia, diabetes mellitus, insulin resistance, obesity and hypertension, together constitute the major risk factors for coronary heart disease. The genetic predisposition to these conditions is strong, but complex and many o f the genes involved have not been identified. The identification or the genes or metabolic pathways involved in these disorders, would lead to new diagnostic and therapeutic targets with wide applicability. The spontaneously hypertensive rat (SHR) is a model of essential hypertension which also displays abnormalities of lipid metabolism and insulin resistance similar to those found in the human metabolic syndromes. These include raised blood triglycerides and fauy acids, defective catecholamine mediated lipolysis and excessive growth of intra-abdominal adipocytes. Similar abnormalities have been identified in combined hyperlipidemia, maturity onset diabetes mellitus and obesity in humans. We are using the SHR as a model to identify genes and pathways which are likely to be in common with those causing the human diseases. We have recently identified loci on chromosomes 4 and 12 in SHR that confer insulin resistance. The chromosome 4 locus also confers abnormalities of catecholamine mediated lipolysis. The chromosome 4 locus has been mapped, using radiation hybrids and the lipid and insulin resistance abnormalities isolated in a congenic rat strain. Using cDNA microarrays and radiation hybrid mapping, we have now identified a defective SHR gene that resides at the peak of linkage to these SHR QTLs. The SHR coding sequence for this gene contains multiple sequence variants caused by unequal recombination of a duplicated ancestral gene. The gene encodes a plasma membrane protein on the PPARX pathway. The encoded protein product is undetectable in SHR adipocyte plasma membrane. The known biological functions of this protein suggest that this gene underlies some, if not all, of these SHR QTL phenotypes. CD36 is an important candidate for susceptibility to the human insulin resistance syndromes. The human gene is being examined for linkage and association in haman subjects with insulin resistance from different ethnic backgrounds and subjects with combined hyperlipidemia. The results of these studies, together with the results of recent linkage studies in familial combined hyperlipidemia, will be presented.

NITRIC OXIDE AND ENDOTHELIN AS MEDIATORS OF ATHEROSCLEROSIS

THE ROLE OF MODIFIED LIPOPROTEINS AND ANTIOXIDANTS IN ATHEROSCLEROSIS AND CARDIOVASCULAR DISEASE

T.E Liischer. Division of CardioloKl; UniuersiO, Hospital Ziirich.

B. Frei. L. Pauling Institute. Oregon State University, USA

Switzerland The circulation is controlled by the central nervous system, hormones and local vascular mechanism. The endothelium is in a strategic anatomical position in the blood vessel wall between the blood (and platelets and monocytes) and vascular smooth muscle. Endothelial cells are stimulated by mechanical and hormonal signals and it release mediators modulating contraction and proliferation of vascular smooth muscle, platelet function, coagulation and monocyte adhesion. Nitric oxide (NO) and prostacyclin (PGI~) and a putative hyperpolarizing factor (EDHF) mediate relaxation. NO inhibits smooth muscle proliferation and (with PGI2) platelet function. Bradykinin induced NO production is regulated by angiotensin converting enzyme on the endothelium; this enzyme converts angiotensin 1 into angiotensin 11, and inactivates bradykinin. Endothelin-l, thromboxane A2

It is well established that oxidatively modified LDL (ox-LDL), but not native LDL, is recognized by scavenger receptors on macrophages, resulting in the conversion of resident macrophages in the vascular wall into lipid-laden foam cells. In addition, ox-LDL is chemotactic for monocytes and chemostatic for macrophages, facilitating monocyte recruitment to, and preventing macrophage egress from, the vascular wall. Furthermore, minimally modified (oxidized) LDL stimulates expression of monocyte chemotactic proteinI (MCP-l) and adhesion molecules by endothelial cells, leading to marked increase in monocyte-endothelial interactions and monocyte migration into the subendothelial space. Ox-LDL also inhibits the biological action of endothelium-derived relaxing factor (EDRE nitric oxide), stimulates smooth muscle cell proliferation, is cytotoxic, and promotes a prothrombotic milieu, among numerous other atherogenic effects.

71st EAS Congress and Satellite Symposia

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Friday 28 May 1999 Workshop: Apolipoproteins stratcture, fitnction and genetics

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Antioxidants, such as vitamins C and E, may inhibit LDL oxidation, thereby inhibiting the above mentioned atherogenic mechanisms of oxLDL. Vitamin C forms the first line of antioxidant defense in human plasma, and vitamin C supplementation in humans increases the resistance of plasma to lipid peroxidation. Similarly, vitamin E supplementation increases the resistance of plasma-derived LDL to oxidative modification. These observations are supported by studies showing that vitamin C and/or vitamin E supplementation in healthy subjects, smokers and various patient groups, such as hypercholesterolemics and diabetics, significantly reduces in t~it~o levels of F2-isoprostanes, prostagtandin-like endproducts of lipid peroxidation. In addition, vitamins C and E may exert beneficial effects on cardiovascular disease (CVD) by improving cellular antioxidant status. In monocytes, vitamins C and/or E decrease cytokine production and adhesiveness to endothelial cells. Vitamin E inhibits smooth muscle cell proliferation and platelet aggregation, probably by inhibiting protein kinase C activation. In endothelial cells, vitamins C and/or E prevent dysfunction and maintain production o f nitric oxide (NO). The biologic activity of NO is impaired in CVD patients, as measured by flow-mediated vasodilation, but restored by acute or chronic vitamin C supplementation. Numerous studies have demonstrated significant improvement by vitamin C supplementation or infusion of vasodilation in smokers, following a high-fat meal, and in patients with diabetes, hypercholesterolemia, hypertension, chronic heart failure or angina. The inhibitory effects on LDL oxidation and the improvement of cellular antioxidant status may explain, in part, the inverse associations observed in numerous epidemiological studies between lowered CVD risk and vitamin C and/or E intake. This evidence and the limited trial data will be reviewed.

using a primed, constant, infusion of I-[13C]-Ieucine. In seven men on the reduction diet, IDL and LDL apoB kinetics were also determined. ApoB isotopic enrichment was measured using gas-chromatography mass spectrometry and SAAM-II was used to estimate the fractional turnover rates. Subcutaneous and visceral adipose tissue at the L3 vertebra was quantified by magnetic resonance imaging. With weight reduction there was a significant decrease (p < 0.05) in BMI, waist circumference and visceral adipose tissue. The plasma concentrations of total cholesterol, triglyceride, insulin and lathosterol also significantly decreased (p < 0.05). Compared with weight maintenance, weight reduction significantly decreased VLDL apoB concentration, pool size, and hepatic secretion of VLDL apoB (A +2.5±4.6 vs A -14.7+4.0 mg/kg fat free mass/day, p = 0.010), but did not significantly alter its fractional catabolism. Weight reduction was also associated with an increased fractional catabolic rate of LDL apoB (0.24+0.07 vs 0.54+0.10 pools/day, p = 0.002) and conversion of VLDL to LDL apoB (11.7±2.5 vs 56.3±11.4%, p = 0.008). Change in hepatic VLDL apoB secretion was significantly correlated with the change in visceral adipose tissue area Ir = 0.59, p = 0.043) but not plasma concentrations o f insulin, free fatty acids or lathosterol. The data support the hypothesis that reduction in visceral adipose tissue is associated with a decrease in the hepatic secretion of VLDL apoB and this may be due to a decrease in portal lipid substrate supply. Weight reduction may also increase the fractional catabolism of LDL apoB, but this requires further evaluation. LOW DENSITY LIPOPROTEIN LIGAND FUNCTION IN PATIENTS WITH MUTANT APOLIPOPROTEIN B-100 ASSESSED BY TWO-COLOR FLUORESCENCE FLOW CYTOMETRY B. Raungaard, E Heath, J.U. Brorholt-Petersen, H.K. Jensen, O. Faergeman.

Workshop:

Apolipoproteins

structure, function and genetics GENETIC ARCHITECTURE OF LIPOPROTEIN(a) G. Utermann, M. Ogorelkova, H.G. Kraft. Institute for Medical Biology and Human Genetics/Uru~ersity lnnsbruck. 6020 lnnsbruck. Austria The apolipoprotein(a) gene is a remarkable example for a quantitative trait locus (QTL) in humans. In Caucasians and Asians variationat this locus is the major determinant of the concentrations in plasma of the atherogenic lipoprotein(a). Lp(a) is associated with CHD and stroke possibly because of it's funtion as an "interlooper" into the fibrinolytic system. Concentrations of Lp(a) differ over thousandfold between individuals and severalfold among ethnic groups. Genetic epidemiology, twin-, family- and sib-pair linkage studies have resulted in the dissection of this quantitative character and demonstrated that the relation of the apo(a) locus to Lp(a) concentration is complex and that the genetic architecture of the Lp(a) trait differs among major human groups. Three types of variation have been identified in the apo(a) gene. A size polymorphism in the coding region (K IV type 2 repeats), a pentanucleotide repeat polymorphism in the promoter (5'PNRP) and sequence variation in coding and non-coding regions of the gene including a C/T polymorphism at +93 which creates an additional ATG start codon but also affects transcription. The causal +93 C/T effect is masked by linkage disequilibrium in Caucasians. Analysis of apo(a) K IV 6-10 exons revealed the existance of population specific spectra of polymorphism in this domain. Sib-pair and allele sharing approaches have demonstrated that mutations in apo B and in the LDL-R which abolish binding of LDL-R and result in familial hypercholesterolemia raize Lp(a) concentrations and increase the variability of the trait. The mechanism for this is presently unclear. APOLIPOPROTEIN B-100 KINETICS IN VISCERAL OBESITY AND THE EFFECTS OF WEIGHT REDUCTION EM. Riches, G.E Watts, J. Hua 1, G.R. Stewart2, R.P. Naoumova 3, P.H.R. Barrett 4. University Department of Medicine and Western Australia

Heart Research Institute, University of Western Australia, Royal Perth Hospital, Perth; I Department of Radiology, Royal Perth Hospital, Perth; 2Faculty of Science, University of Western Australia, Nedlands, Western Australia," JLipoprotein Team, MRC Clinical Sciences Centre. Imperial College of Medicine, Hammersmith Hospital, London, UK; 4Department of Bioengineering, University of Washington, Seattle, WA, USA

Department of Medicine and Cardiology Aarhus Amtssygehus UniverstO" Hospital. Aarhus. Denmark Defects in the structure of apolipoprotein (apo) B-100 reduce binding of low density lipoprotein (LDL) to LDL receptors. This condition, designated familial defective apoB-100 (FDB), is caused by mutations in the apoB100 gene. To test a fluorescence flow cytometric assessment of LDL ligand function we examined LDL prepared from patients with molecularly verified heterozygosity for the Arg3s0o-GIn mutation (n = 9). We compared the results to measurements of LDL ligand function in healthy individuals (n = 11) and in relatives to the FDB patients without known apoB-100 gcne mutations (n = 9). LDL ligand function was determined by a competition assay: Ebstein Barr virus transformed lymphoblasts from a normolipidemic individual were incubated at 37"C either with pure LDL prepared from one of the study subjects, pure fluorescently conjugated LDL (DiI-LDL), or a 3:1 mixture of LDL and DiI-LDL. The ability of LDL to compete with DilLDL in binding to LDL receptors was expressed as the ratio of median fluorescence of cells incubated with a mixture of LDL and DiI-LDL to median fluorescence of cells incubated with pure LDL (Dil-ratio). Dilratios were divided by median fluorescence of cells incubated with pure DiI-LDL to take into account variations in LDL receptor number on cell surfaces. A comparable subpopulation of lymphoblasts was identified using a fluorescently conjugated B-cell specific monoclonal antibody. Knowing the apoB-100 genotype of the FDB patients and relatives allowed us to compare the diagnostic capability of our functional assay with that of DNA diagnosis. Normal LDL ligand function was defined as the mean adjusted Dil-ratio for healthy individuals. The adjusted Dil-ratios ranged for healthy individuals from 82 to 132% of normal, for relatives from 81 to 152% of normal, and for patients from 54 to 78% of normal. Thus, adjusted Dil-ratios for individuals with heterozygous FDB were completely separated from ratios for healthy individuals and relatives. Our data show that LDL ligand function is significantly reduced in patients heterozygous for the Arg350o-GIn mutation compared to healthy individuals and relatives. We suggest that the two-color fluorescence flow cytometric assay described here can be used to test patients for a major reduction in LDL ligand function.

(This work was in part supported by the Danish Heart Foundation). CONTRIBUTION OF APOLIPOPROTEIN A-I CENTRAL DOMAIN TO THE FORMATION OF LIPOPROTEINS. AN IN VIVO ANALYSIS BY ADENOVIRUS-MEDIATED GENE TRANSFER Y.L. Marcel, D. McManus. Lipoprotein & Atherosclerosis Group.

University of Ottawa Heart Institute, Ottawa, Ontario, Canada We investigated the effect of reduction in visceral obesity on the kinetics of apolipoprotein B-100 (apoB) metabolism in a controlled dietary intervention study in 26 obese men. Hepatic secretion of VLDL apoB was measured

To analyze structure-function relationships in the central domain of apoA-I we deleted sequences predicted to form pairs of antiparallel amphipathic a-

71st EAS Congress and Satellite Symposia