L.M. Havekes

Macrophage scavenger receptor class A : A multifunctional receptor in atherosclerosis

In atherogenesis, elevated plasma levels of low density lipoprotein (LDL) lead to the chronic presence of LDL in the arterial wall. There, LDL is modified (eg, oxidized), and these modified lipoproteins activate endothelial cells, which attract circulating monocytes. These monocytes enter the vessel wall, differentiate into macrophages, and subject the modified lipoproteins to endocytosis through scavenger receptor pathways. This unrestricted uptake, which is not limited by intracellular cholesterol levels, eventually leads to the formation of lipid-filled foam cells, the initial step in atherosclerosis. Macrophage scavenger receptor class A (SRA) is thought to be one of the main receptors involved in foam cell formation, mediating the influx of lipids into the macrophages. In addition to this role in modified lipoprotein uptake by macrophages, the SRA has been shown to be important in the inflammatory response in host defense, cellular activation, adhesion, and cell-cell interaction. Given the importance of these processes in atherogenesis, these latter functions may prove to make the SRA a multifunctional player in the atherosclerotic process.

Apolipoprotein e4 allele and cognitive decline in elderly men

Abstract Abstract Objectives: To determine whether polymorphism of apolipoprotein E - notably, the e4 allele - predicts cognitive deterioration in the general population. Design: Population based cohort investigated in 1990 and in 1993. Setting: Zutphen, the Netherlands. Subjects: Representative cohort of 538 Dutch men aged 70-89 at baseline. Main outcome measures: Cognitive function assessed by mini mental state examination, change in cognitive function and incidence of impaired cognitive function at three years. Results: The baseline prevalence of impaired cognitive function (mini mental state examination score <=25) was higher among carriers of the e4 allele compared wth men without the allele (41.0% (55) v 31.1% (122) P=0.03), and this result was still valid after adjustment for age, occupation, smoking, alcohol use, and cardiovascular diseases. The decline in cognitive function at three years was largest in men homozygous for e4 (−2.4 points), intermediate in those heterozygous for e4 (−0.7 points), and lowest in men without e4 (−0.1 points), and it was independent of other risk factors (P=0.02). The risk of developing impaired cognitive function during follow up was significantly increased in allele carriers compared with non-carriers (27.6% (16/58) v 15.5% (32/207)). The adjusted odds ratio was 2.87 (95% confidence interval 1.29 to 6.42). Twenty two per cent of the risk of developing impaired cognitive function in this population may be attributable to the e4 allele. Conclusions: The apolipoprotein e4 allele predisposes to cognitive decline in a general population of elderly men.

Postprandial triglyceride response in young adult men and familial risk for coronary atherosclerosis.

Atherosclerosis starts early in life [1, 2]. Postprandial lipoprotein metabolism is proposed to be involved in this process [3]. Cholesteryl ester-rich remnants of triglyceride-rich lipoproteins may directly promote accumulation of cholesteryl esters in the arterial wall [4-6]. Further, it has been reported [7, 8] that the protective effect of increased levels of high-density lipoprotein (HDL) cholesterol on the risk for coronary artery disease may not only be explained by their role in reverse cholesterol transport but also by the relation between HDL and triglyceride metabolism [9]. Plasma triglycerides have an effect on HDL composition and HDL cholesterol levels [10]; an inverse relation between HDL2 levels and postprandial triglyceride levels has been shown [11]. Levels and composition of HDL could thus be a reflection of the effectiveness of triglyceride-rich lipoprotein catabolism. A deranged metabolism of triglyceride-rich lipoproteins in plasma has been reported in familial dysbetalipoproteinemia [12, 13], a disease associated with premature coronary atherosclerosis. Studies comparing patients with coronary artery disease and persons without the disease have shown that differences with respect to postprandial hypertriglyceridemia [14-16] and postprandial retinyl palmitate concentrations (a marker of chylomicrons and their remnants) are detectable after an oral lipid load [14, 15]. These results suggest a delayed clearance of these lipoproteins in patients with coronary artery disease. Thus, accumulating evidence indicates that postprandial lipemia plays an important role in causing coronary artery disease, and the implications with respect to treatment and primary prevention are increasingly being recognized [17]. Postprandial lipoprotein metabolism has not yet been studied in children and young adults with an increased risk for coronary atherosclerosis. In our study, male offspring of men with clinical manifestations of and angiographically proven coronary atherosclerosis were compared with male offspring of men who did not have coronary atherosclerosis (negative result after angiography). This approach enabled us to study a group of healthy young adults at high familial risk for developing clinical manifestations of coronary artery disease later in life. We assessed whether changes in the triglyceride response to a standardized oral lipid load, as reported in patients with coronary artery disease, can also be measured in healthy young male offspring of such patients. Methods Participants Men with very severe coronary artery disease (patients), defined as more than 70% occlusion in at least three major coronary vessels, were selected from coronary angiography databases of cardiology departments of the Zuiderziekenhuis Rotterdam (1988 to 1991), the University Hospital Rotterdam (1988 to 1989), the Refaja Ziekenhuis in Dordrecht (1990 to 1991), and the Antonius Ziekenhuis in Nieuwegein (1992), all hospitals situated in the Netherlands. Simultaneously, a reference group of men [controls] was selected who at coronary angiography had no or, at most, only minor lesions, defined as 20% stenosis or less in all coronary vessels. Further, participants were selected according to the following additional criteria: 1) age between 45 and 65 years; 2) blood pressure not exceeding 160/100 mm Hg; 3) absence of liver disease, diabetes mellitus, thyroid disease, and renal disease; 4) first coronary angiography within 2 years before examination for our study; and 5) first consultation of a physician for cardiac symptoms within 5 years before the examination for our study. Eligible participants were sent a letter asking whether they had a son 15 to 30 years of age and, if so, whether the son was willing to participate in the study. These sons (identified by their fathers) received a separate letter inviting them to have an oral lipid loading test. Participants also received a short questionnaire about smoking habits, alcohol intake, physical activity, and fat intake. We screened medical files of 629 patients whose coronary angiographic data met the criteria. Of these patients, 19 had died, 46 had diabetes mellitus, 6 had renal disease, 2 had thyroid disease, 183 had either no son or sons outside the required age range, 58 could not be contacted, 17 had no contact with their children, 78 had a cardiac history exceeding 5 years, and 63 could not be invited for other reasons (hospitalization, other serious diseases). Of 157 families (fathers and sons) who met all the criteria, 55 (either father or son) refused to participate in the study (response, 65%), leaving 102 fathers and 139 sons. The latter had oral lipid loading tests. The study protocol was approved by the medical ethics committees of the Zuiderziekenhuis Rotterdam and the University Hospital Rotterdam. Informed consent forms were obtained from all participants in the study. Baseline Measurements Fathers were asked to visit the hospital at 9:00 a.m. after fasting for at least 12 hours. Fathers responded to a questionnaire about the number of first-degree relatives who had had myocardial infarctions and about medication use at the time of the examination (medication was taken to the hospital where the examination took place). Systolic blood pressure and diastolic blood pressure were measured using a random zero sphygmomanometer (Hawksley, Lancing, United Kingdom). Fasting serum blood samples were drawn by antecubital venipuncture for measurement of levels of triglycerides, total cholesterol, low-density lipoprotein (LDL) cholesterol, and HDL cholesterol (and its subfractions HDL2 and HDL3). Height and weight were measured without shoes and without heavy clothing. The sons were invited to come to the hospital, after the same period of fasting, on a separate day to have an oral lipid loading test. For sons, questionnaires were used to obtain data about use of medication, fat intake, alcohol intake, and smoking habits, referring to a 1-month period before the examination for this study. Daily total fat intake was calculated from an 81-item semiquantitative food frequency questionnaire by using a computerized food-composition table [18]. In the sons, blood samples were taken by antecubital venipuncture for measurement of baseline levels of serum triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol (and its subfractions HDL2 and HDL3), apoprotein A-1, apoprotein A-2, apoprotein B, and retinyl palmitate concentrations. This baseline measurement of lipid levels was taken as the starting point (t0) for the oral lipid loading test. In all of the sons, apolipoprotein E was phenotyped. Oral Lipid Loading Test Sons of patients and sons of controls came to the hospital at 7:45 a.m. after an overnight fasting of 12 hours. Height and weight were measured first to calculate body surface area. Five minutes after the venipuncture for obtaining baseline lipid levels (at t0), all participants received a liquid lipid load, which consisted of a mixture of dairy cream (40% fat), egg yolk, milk powder, and retinyl palmitate (in aqueous solution) [15]. Participants received the lipid load in a dose based on their individual body surface area (77.5 g fat, 0.5 g cholesterol, and 27 000 IU of retinyl palmitate per square meter of body surface area). The mixture was consumed within 15 minutes. The participants received an antecubital venous catheter (Venflon Viggo AB, Helsingborg, Sweden), which was kept open during the test period by means of disposable obturators (Venflon). Through this catheter, blood samples were drawn at 2 (t2), 4 (t4), 5 (t5), 6 (t6), 7 (t7), 8 (t8), 10 (t10), and 12 (t12) hours after starting consumption of the oral lipid load. Total cholesterol, triglyceride, and retinyl palmitate concentrations were determined in serum isolated from these samples. During the 12-hour period, other sources of calories were withheld from participants. Because postprandial exercise has been reported to decrease postprandial lipemia [19], participants stayed in the hospital and were asked to refrain from heavy physical activity during the test period. Laboratory Analyses Serum total cholesterol levels were measured using an automated enzymatic method (Boehringer Mannheim, Mannheim, Germany) CHOD-PAP reagent kit [20]. Levels of HDL cholesterol and LDL cholesterol were measured by the same method after precipitation. For HDL cholesterol, the phosphotungstate method according to Burstein [21], with a minor modification as described by Grove [22], was used. For LDL cholesterol, precipitation was carried out with polyvinylsulfate (Boehringer Mannheim). Throughout the entire study period, results of total cholesterol and HDL cholesterol determinations were within limits of the quality control program of the World Health Organization Regional Lipid Reference Centre (Prague, Czechoslovakia). Levels of apoprotein A-1 and B were assayed using an automated immunoturbidimetric method (Kone Diagnostics, Espoo, Finland). Levels of apoprotein A-2 were determined by radial immunodiffusion against specific antiserum (Boehringer Mannheim, Germany) according to Cheung and Albers [23], with slight modifications. All automated analyses were carried out on the Kone Specific Analyzer (Kone Instruments) using frozen ( 20C) serum samples. High-density lipoprotein2 and HDL3 in serum were assayed as described by Gidez and colleagues [24] with slight modifications. High-density lipoprotein2 and HDL3 were separated using stepwise precipitation of apoprotein B containing lipoproteins with heparin/Mn2+ and HDL2 with dextran sulfate. Apolipoprotein E phenotyping was done by isoelectric focusing of delipidated serum followed by immunoblotting, using apolipoprotein E antiserum as first antibodies [25]. Retinyl palmitate analyses were done as described previously by Groot and colleagues [15]. Statistical Analysis Means SDs were calculated for baseline characteristics of all family members. The total cholestero

No Effect of C-Reactive Protein on Early Atherosclerosis Development in Apolipoprotein E*3-Leiden/Human C-Reactive Protein Transgenic Mice

Objective—C-reactive protein (CRP) has been associated with risk of cardiovascular disease. It is not clear whether CRP is causally involved in the development of atherosclerosis. Mouse CRP is not expressed at high levels under normal conditions and increases in concentration only several-fold during an acute phase response. Because the dynamic range of human CRP is much larger, apolipoprotein E*3-Leiden (E3L) transgenic mice carrying the human CRP gene offer a unique model to study the role(s) of CRP in atherosclerosis development. Methods and Results—Atherosclerosis development was studied in 15 male and 15 female E3L/CRP mice; E3L transgenic littermates were used as controls. The mice were fed a hypercholesterolemic diet to induce atherosclerosis development. Cholesterol exposure did not differ between E3L/CRP and E3L mice. Plasma CRP levels were on average 10.2±6.5 mg/L in male E3L/CRP mice, 0.2±0.1 mg/L in female E3L/CRP mice, and undetectable in E3L mice. Quantification of atherosclerosis showed that lesion area in E3L/CRP mice was not different from that in E3L mice. Conclusion—This study demonstrates that mildly elevated levels of CRP in plasma do not contribute to the development of early atherosclerosis in hypercholesterolemic E3L/CRP mice.

In the absence of the low density lipoprotein receptor, human apolipoprotein C1 overexpression in transgenic mice inhibits the hepatic uptake of very low density lipoproteins via a receptor-associated protein-sensitive pathway.

To study the role of apoC1 in lipoprotein metabolism, we have generated transgenic mice expressing the human APOC1 gene. On a sucrose-rich diet, male transgenic mice with high APOC1 expression in the liver showed elevated levels of serum cholesterol and triglyceride compared with control mice (5.7+/-0.7 and 3.3+/-2.1 vs. 2.7+/-0.1 and 0.4+/-0.1 mmol/liter, respectively). These elevated levels were mainly confined to the VLDL fraction. Female APOC1 transgenic mice showed less pronounced elevated serum lipid levels. In vivo VLDL turnover studies revealed that, in hyperlipidemic APOC1 transgenic mice, VLDL particles are cleared less efficiently from the circulation as compared with control mice. No differences were observed in the hepatic production and extrahepatic lipolysis of VLDL-triglyceride. Also, VLDL isolated from control and APOC1 transgenic mice were found to be equally good substrates for bovine lipoprotein lipase in vitro. These data indicate that the hyperlipidemia in APOC1 transgenic mice results primarily from impaired hepatic VLDL particle clearance, rather than a defect in the hydrolysis of VLDL-triglyceride. To investigate which hepatic receptor is involved in the apoC1-mediated inhibition of VLDL clearance, APOC1 transgenic mice were bred with an LDL receptor-deficient (LDLR(-/-)) background. In addition, control, LDLR(-/-), and LDLR(-/-)/APOC1 mice were transfected with adenovirus carrying the gene for the receptor-associated protein (Ad-RAP). Both serum cholesterol and triglyceride levels were strongly elevated in LDLR(-/-)/APOC1 mice compared with LDLR(-/-) mice (52+/-19 and 36+/-19 vs. 8.4+/-0.9 and 0.5+/-0.2 mmol/liter, respectively), indicating that apoC1 inhibits the alternative VLDL clearance pathway via the remnant receptor. Transfection of LDLR(-/-) mice with Ad-RAP strongly increased serum cholesterol and triglyceride levels, but to a lesser extent than those found in LDLR(-/-)/APOC1 mice (39+/-8 and 17+/-8 vs. 52+/-19 and 36+/-19 mmol/liter, respectively). However, in LDLR(-/-)/APOC1 mice the transfection with Ad-RAP did not further increase serum cholesterol and triglyceride levels (52+/-19 and 36+/-19 vs. 60+/-10 and 38+/-7 mmol/liter, respectively). From these studies we conclude that, in the absence of the LDLR, apoC1 inhibits the hepatic uptake of VLDL via a RAP-sensitive pathway.

The lipoprotein lipase (Asn291 → Ser) mutation is associated with elevated lipid levels in families with familial combined hyperlipidaemia

Familial combined hyperlipidaemia (FCHL) is one of the major genetic causes of coronary heart disease (CHD) and is characterised by elevated levels of plasma cholesterol and/or triglycerides in individuals within a single family. Decreased lipoprotein lipase (LPL) activity has been found in some cases of FCHL. A recent study revealed a common mutation in the LPL gene, LPL(Asn291-->Ser), with a frequency of 9.3% in Dutch FCHL patients (Reymer et al,. Circulation, 90 (1994) I-998). This mutation was found in 3 out of 17 FCHL families. Extensive family studies were subsequently performed to determine the effect of this mutation on the phenotypic expression of FCHL. Using a pedigree-based maximum likelihood estimate, we demonstrated that the LPL(Asn291-->Ser) mutation significantly affects the levels of plasma and very low density lipoprotein (VLDL) triglycerides (2.03 +/- 0.21 vs. 1.14 +/- 0.13 and 1.21 +/- 0.16 vs. 0.62 +/- 0.09 mmol/l, carriers and non-carriers, respectively) and VLDL- and high density lipoprotein (HDL) cholesterol (0.83 +/- 0.10 vs. 0.38 +/- 0.06 and 1.02 +/- 0.08 vs. 1.29 +/- 0.05 mmol l, carriers and non-carriers, respectively), but not those of plasma and low density lipoprotein (LDL) cholesterol. These findings indicate that the LPL(Asn291-->Ser) mutation is associated with elevated lipid levels, indicating it may be one of the genetic factors predisposing to FCHL in the families studied.

Apolipoprotein E polymorphism affects plasma levels of lipoprotein(a)

In a group of 303 healthy Caucasian adults of both sexes we studied the influence of the apolipoprotein E (apo E) polymorphism on plasma levels of Lipoprotein(a) (Lp(a). The APOE*2 allele was found to decrease the mean plasma Lp(a) level by 24.8%, whereas the APOE*4 allele increased the mean Lp(a) level by 25.7%. These effects were parallel to the effect of apo E polymorphism on plasma cholesterol and low density lipoprotein (LDL)-cholesterol. For the Lp(a) levels, the genetic variance associated with the APOE locus contributed about 4% to the total phenotypic variance. For plasma cholesterol and LDL-cholesterol this contribution was 4.5 and 6.3%, respectively. We also found a significant positive correlation between LDL-cholesterol and Lp(a) levels. Since the apo E polymorphism effects LDL-receptor activity, we conclude that, at least in healthy normolipidemic individuals, plasma levels of Lp(a) are modulated by the LDL-receptor activity.

Apolipoprotein E3-Leiden. A new variant of human apolipoprotein E associated with familial type III hyperlipoproteinemia.

SummaryA variant of apolipoprotein E, denoted apo E3-Leiden, has been identified in a 41-year-old male suffering from type III hyperlipoproteinemia with xanthomatosis. Apo E3-Leiden focus in the E3 position. In contrast with normal apo E3, apo E3-Leiden is defective in binding to the low density lipoprotein (LDL) receptor and does not contain cysteine as evaluated by cysteamine treatment of very low density lipoprotein followed by isoelectric focusing and conventional protein staining and by amino acid analysis. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, apo E3-Leiden displays an electrophoretic mobility intermediate to that of normal apo E3 and apo E2 (Arg158→Cys). The mother and four siblings of the proband also have apo E3-Leiden and hyperlipoproteinemia type III; three of them with xanthomatosis. Two siblings do not show apo E3-Leiden in their VLDL fraction and do not have hyperlipoproteinemia type III. In the VLDL fractions of all affected family members only the presence of apo E3-Leiden could be detected after cysteamine treatment and isoelectric focusing followed by conventional protein staining. However, isoelectric focusing of cysteaminetreated sera followed by immunoblotting, using anti-apo E antiserum as first antiserum, demonstrates the presence of low amounts of normal apo E3 in addition to apo E3-Leiden in serum of the affected family members. These results indicate that all affected family members are heterozygotes E3/E3-Leiden and suggest that in this family type III hyperlipoproteinemia is transmitted as a dominant trait.

Apolipoprotein E genotype and association between smoking and early onset Alzheimer's disease.

Abstract Objective: To investigate the hypothesis that differential survival between smokers and nonsmokers leading to a decrease in the frequency of the e4 allele of the apolipoprotein E gene may explain the inverse relation between smoking history and early onset Alzheimers disease. Design: A population based case-control study. Setting: The four northern provinces of the Netherlands and metropolitan Rotterdam. Subjects: 175 patients with early onset Alzheimers disease and two independent control groups of 159 and 457 subjects. Main outcome measures: Frequencies of the apolipoprotein e4 allele and relative risk of early onset Alzheimers disease. Results: The inverse association between smoking history and early onset Alzheimers disease could not be explained by a decrease in the frequency of the apolipoprotein e4 allele. Among carriers of this allele with a family history of dementia subjects with a history of smoking had a strongly reduced risk of early onset Alzheimers disease (odds ratio 0.10 (95% confidence interval 0.01 to 0.87)). Conclusions: The results suggest that the inverse relation between smoking history and early onset Alzheimers disease cannot be explained by an increased mortality in carriers of the apolipoprotein e4 allele who smoke. The association is strongly modified by the presence of the apolipoprotein e4 allele as well as by a family history of dementia. Key messages Key messages This study shows that the inverse association between smoking history and early onset Alzheimers disease cannot be explained by a shift in frequency of the apolipoprotein e4 allele The inverse relation was significant only in subjects with a family history of dementia who carry the e4 allele Our study suggests that clinical trials with nicotine or nicotine derivatives have the greatest chance of success in patients with familial Alzheimers disease who carry the apolipoprotein e4 allele

In vivo catabolism of biologically modified LDL.

Incubation of human low density lipoprotein (LDL) at 37 degrees C in the presence of human umbilical vein endothelial cells (EC) caused a time-dependent shift in the charge and density of LDL. The physical changes of the human LDL occurred parallel with an increase in its clearance from the serum and uptake in the liver when injected into rats. The serum decay of the EC-modified LDL (44 hours incubation) was 20 times faster than for control LDL. EC-modified LDL, cleared from the blood, was quantitatively recovered in the liver. Isolation of the different liver cell types (parenchymal, Kupffer, and endothelial cells) after in vivo injection of 125I-EC-modified LDL showed that approximately 30 times more radioactivity was associated with the endothelial cells than with the parenchymal cells (per milligram of cell protein). In vitro experiments indicated that EC-modified-LDL was processed by the rat liver endothelial cells via a high affinity, saturable pathway related to the pathway by which these cells processed acetyl-LDL. We concluded that, if EC-modified LDL is generated in vivo, the liver, and in particular the endothelial cells, forms the major protection system against the occurrence of atherogenic particles in the blood.