Triglycerides, hypertension, and smoking predict cardiovascular disease in dysbetalipoproteinemia.

BACKGROUND: Dysbetalipoproteinemia (DBL) is an autosomal recessive lipid disorder associated with a reduced clearance of remnant lipoproteins and is associated with an increased cardiovascular disease (CVD) risk. The genetic cause of DBL is apoE2 homozygosity in 90% of cases. However, a second metabolic hit must be present to precipitate the disease. However, no study has investigated the predictors of CVD, peripheral artery disease and coronary artery disease in a large cohort of patients with DBL. OBJECTIVE: The objectives of this study were to describe the clinical characteristics of a DBL cohort and to identify the predictors of CVD, peripheral artery disease, and coronary artery disease in this population. METHODS: The inclusion criteria included age ≥ 18 years, apoE2/E2, triglycerides (TG) > 135 mg/dL and VLDL-C/plasma TG ratio > 0.30. RESULTS: We studied 221 adult DBL patients, of which 51 (23%) had a history of CVD. We identified 3 independent predictors of CVD, namely hypertension (OR 5.68, 95% CI 2.13-15.16, P = .001), pack year of smoking (OR 1.03, 95% CI 1.01-1.05, P = .01) and TG tertile (OR 1.82, 95% CI 1.09-3.05, P = .02). The CVD prevalence was 51% in patients with hypertension and 18% in those without hypertension (P = .00001), and 30% in the highest TG tertile vs 15% in the lowest tertile (P = .04). Similarly, the CVD prevalence was higher in heavy smokers compared with nonsmokers (36% vs 13%, P = .006). CONCLUSION: Hypertension, smoking, and TG are independently associated with CVD risk in patients with DBL. Aggressive treatment should be initiated in patients with DBL because of the increased risk of CVD.

Compound heterozygotes for a novel mutation, apo E1 Nagoya (Arg142Ser) and Apo E2 (Arg158Cys), with severe type III hyperlipoproteinemia and familial hypercholesterolemia.

AIM: A patient with severe type III hyperlipoproteinemia and familial hypercholesterolemia (FH) was previously reported (Metabolism, 44,1995:460-465). In the current study, the patient's apolipoprotein (apo) E gene was analyzed. METHODS: An apo E isoform analysis was performed using isoelectric focusing and immunoblotting. In addition, after DNA preparation, a restriction fragment length polymorphism analysis and DNA sequence analysis were performed. RESULTS: The patient's apo E phenotype was E2/E1, and the genotype was ε2/ε2. The sequence analysis of the patient's DNA revealed a new variant of apo E, which involves a single substitution of one serine (AGC) for one arginine (CGC) at position 142, thereby adding one negatively charged unit to apo E2. Therefore, the patient was compound heterozygous for apo E1 (Arg142Ser) and apo E2 (Arg158Cys). CONCLUSIONS: A novel mutation, apo E1 Nagoya (Arg142Ser) in a patient with severe type III hyperlipoproteinemia with heterozygous FH was characterized. Since the presence of arginine at the amino acid residue 142 of apo E is considered to play an important role in binding to LDL receptors, the mutation apo E1 Nagoya (Arg142Ser) likely contributed to the expression of severe type III hyperlipoproteinemia in this patient.

Apolipoprotein E mutations: a comparison between lipoprotein glomerulopathy and type III hyperlipoproteinemia.

Apolipoprotein E (ApoE) serves as a ligand for the low-density lipoprotein (LDL) receptor and cell surface receptors of the LDL receptor gene family. More than 10 different causative apoE mutations associated with lipoprotein glomerulopathy (LPG) have been reported. ApoE polymorphisms including three common phenotypes (E2, E3, E4), and a variety of rare mutations can affect blood cholesterol and triglyceride levels. The N-terminal domain of apoE is folded into a four-helix bundle of amphipathic α-helices, and contains the receptor-binding domain in which most apoE mutations that cause LPG or dominant mode of type III hyperlipoproteinemia (HL) are located. No single apoE mutation has been reported that causes both LPG and the dominant mode of type III HL.

Resequencing the APOE gene reveals that rare mutations are not significant contributory factors in the development of type III hyperlipidemia.

BACKGROUND: APOE (apolipoprotein E gene) 2/2 genotype and an apolipoprotein B/total cholesterol (ApoB/TC) ratio <0.15 are diagnostic for type III hyperlipidemia. We hypothesized that patients with APOE genotype 2/3 or 2/4 and an ApoB/TC ratio <0.15 may have a mutation in their epsilon 3 or 4 allele, resulting in a type III hyperlipidemia phenotype. OBJECTIVE: We tested this hypothesis. METHODS: The DNA sequence of all 4 exons and exon/intron boundaries of the APOE (plus 600 bp upstream of exon 1) of 47 patients with APOE 2/3 and 18 patients with APOE 2/4 genotype and an ApoB/TC ratio <0.15 was determined. As controls the APOE sequence of 53 APOE genotype 2/3 and 20 APOE genotype 2/4 probands with ApoB/TC ratio >0.15 was determined. The sequence analysis was extended to include 47 patients with APOE genotype 3/3, 14 with APOE genotype 3/4, and 3 with APOE genotype 4/4 and an ApoB/TC ratio <0.15. Finally, we determined the sequence of the APOE gene in 145 patients with an ApoB/TC ratio >0.15 and who had triglycerides above the 90th percentile for age and sex. RESULTS: No deleterious variants in the APOE gene were observed in patients with APOE genotype other than 2/2 and an ApoB/TC ratio <0.15. Only a single probably deleterious variant, K72E, was observed in patients with triglycerides above the 90th percentile. CONCLUSIONS: Patients with an ApoB/TC ratio <0.15 do not have an increased likelihood of mutation in the APOE gene, and rare variants in the APOE gene are not important in the development of hypertriglyceridemia.

Cardiac and vascular phenotypes in the apolipoprotein E-deficient mouse.

Cardiovascular death is frequently associated with atherosclerosis, a chronic multifactorial disease and a leading cause of death worldwide. Genetically engineered mouse models have proven useful for the study of the mechanisms underlying cardiovascular diseases. The apolipoprotein E-deficient mouse has been the most widely used animal model of atherosclerosis because it rapidly develops severe hypercholesterolemia and spontaneous atherosclerotic lesions similar to those observed in humans. In this review, we provide an overview of the cardiac and vascular phenotypes and discuss the interplay among nitric oxide, reactive oxygen species, aging and diet in the impairment of cardiovascular function in this mouse model.

A dynamic model of calcific nodule destabilization in response to monocyte- and oxidized lipid-induced matrix metalloproteinases.

Vulnerable plaque remains clinically undetectable, and there is no accepted in vitro model. We characterize the calcific nodules produced by calcifying vascular cells (CVC) in ApoE-null mice, demonstrating increased destabilization of cultured nodules in the presence of oxidized low-density lipoprotein (oxLDL) and monocytes under pulsatile shear stress. CVC implanted in the subcutaneous space of hyperlipidemic mice produced nodules revealing features of calcific atherosclerotic plaque including a fibrous cap, cholesterol clefts, thin shoulder, lipids, and calcium mineral deposits. CVC nodules seeded in the pulsatile flow channel (τ(avg) = 23 dyn/cm(2), ∂τ/∂t = 71 dyn·cm(-2)·s(-1)) underwent deformation and destabilization. Computational fluid dynamics revealed distinct shear force profiles on the nodules. Presence of oxLDL or monocytic THP-1 cells significantly increased the numbers of nodules destabilized from the substrate. Both oxLDL and THP-1 increased matrix metalloproteinase (MMP) activity in CVC. The MMP inhibitor GM6001 significantly reversed oxLDL- and THP-1-induced nodule destabilization, whereas overexpression of MMP-9 increased destabilization. These findings demonstrate that CVC-derived nodules resembled calcific atherosclerotic plaque and were destabilized in the presence of active lipids and monocytes via induction of MMPs.

Biophysical analysis of apolipoprotein E3 variants linked with development of type III hyperlipoproteinemia.

BACKGROUND: Apolipoprotein E (apoE) is a major protein of the lipoprotein transport system that plays important roles in lipid homeostasis and protection from atherosclerosis. ApoE is characterized by structural plasticity and thermodynamic instability and can undergo significant structural rearrangements as part of its biological function. Mutations in the 136-150 region of the N-terminal domain of apoE, reduce its low density lipoprotein (LDL) receptor binding capacity and have been linked with lipoprotein disorders, such as type III hyperlipoproteinemia (HLP) in humans. However, the LDL-receptor binding defects for these apoE variants do not correlate well with the severity of dyslipidemia, indicating that these variants may carry additional properties that contribute to their pathogenic potential. METHODOLOGY/PRINCIPAL FINDINGS: In this study we examined whether three type III HLP predisposing apoE3 variants, namely R136S, R145C and K146E affect the biophysical properties of the protein. Circular dichroism (CD) spectroscopy revealed that these mutations do not significantly alter the secondary structure of the protein. Thermal and chemical unfolding analysis revealed small thermodynamic alterations in each variant compared to wild-type apoE3, as well as effects in the reversibility of the unfolding transition. All variants were able to remodel multillamelar 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) vesicles, but R136S and R145C had reduced kinetics. Dynamic light scattering analysis indicated that the variant R136S exists in a higher-order oligomerization state in solution. Finally, 1-anilinonaphthalene-8-sulfonic acid (ANS) binding suggested that the variant R145C exposes a larger amount of hydrophobic surface to the solvent. CONCLUSIONS/SIGNIFICANCE: Overall, our findings suggest that single amino acid changes in the functionally important region 136-150 of apoE3 can affect the molecule's stability and conformation in solution and may underlie functional consequences. However, the magnitude and the non-concerted nature of these changes, make it unlikely that they constitute a distinct unifying mechanism leading to type III HLP pathogenesis.

Effect of zeaxanthin and antioxidant supplementation on vascular endothelial growth factor (VEGF) expression in apolipoprotein-E deficient mice.

PURPOSE: Apolipoprotein E(-/-) deficient (apoE(-/-)) mice develop hypercholesterolemia, atherosclerosis, and retinal alterations. We studied the oxidative status and vascular endothelial growth factor (VEGF) expression in murine retinal pigment epithelium-choroid (RPE) and Bruch's membrane (BM) ultrastructure and the effect of zeaxanthin. METHODS: Ten 6-month-old C57BL/6 and 40 apoE(-/-) mice were divided into four groups (n = 10 each) and fed different diets for 12 weeks based on body weight: wild type (WT) and apoE(-/-) (AE-Con) mice standard rodent chow; apoE(-/-) mice (AES) standard rodent chow with ascorbate (800 mg/kg), tocopherol (1053 mg/kg), and zinc (135 mg/kg); and apoE(-/-) mice the last diet plus zeaxanthin with either 0.4 g/kg (AES-Z04) or 4 g/kg feed (AES-Z4). RESULTS: Plasma total cholesterol (TC) and triglycerides (TG) and urine lipid peroxidation (isoprostanes) were measured. VEGF expression was determined in RPE-choroid homogenates. Zeaxanthin uptake was assessed in liver and retina by high-performance liquid chromatography; the retinal ultrastructure was analyzed by electron microscopy. AE-Con mice had higher plasma TC (p < 0.001) and TG (p < 0.001) values than WT mice. AE-Con mice had higher RPE-choroid-VEGF levels than WT mice (p < 0.05), BM thickness (p < 0.001) and presence of basal laminar deposits (BLamD). AES-Z4 resulted in lower urinary isoprostanes (p = 0.054) and lower VEGF expression in the RPE-choroid (p < 0.01). BM in the AES-Z4 animals had less confluent BLamD than AE-Con, AES, or AES-Z04 animals. CONCLUSIONS: We have reported that supplementation with zeaxanthin and antioxidants may delay or reverse alterations in the RPE and deposits in BM, and reduced VEGF expression observed in apoE(-/-) mice.

Lipoprotein glomerulopathy induced by ApoE-Sendai is different from glomerular lesions in aged apoE-deficient mice.

OBJECTIVE: A mutant of apolipoproteinE (apoE), ApoE-Sendai (Arg145Pro), is one of the major causative factors of human lipoprotein glomerulopathy (LPG). An apoE-deficient mouse with introduced ApoE-Sendai gene (ApoE-Sendai mouse) developed a murine counterpart of LPG, whereas it was also reported that apoE-deficient mouse (apoE KO mouse) spontaneously developed LPG-like lesion regardless of introduction of ApoE-Sendai gene. In the present study, we differentiated renal lesions between these two models by detailed analyses of histology and lipoprotein profile, and clarified the role of apoE variants. METHOD: ApoE-Sendai mice were induced by injection of adenovirus vectors. The kidneys showing LPG-like lesions in apoE-Sendai and apoE KO mice were histopathologically evaluated. Plasma lipids and lipoproteins of both mice were also examined. RESULTS: Histological alteration of the kidney in ApoE-Sendai mice was observed with light microscopy (in 40 out of 50 mice; mild 24, moderate 13, severe 3). Characteristic lesions were dilated vascular lumens mimicking lipoprotein thrombi in human LPG. Similar changes were found in hematoxylin-eosin stained sections of aged apoE KO mice. Meanwhile, periodic acid-Schiff, Azan Mallory, and Oil red O/Sudan III stained sections revealed that the dilated lumens of ApoE-Sendai mice mainly contained lipids and lipoproteins but those of aged apoE KO mice contained much other materials, e.g., proteins and fibrils. These findings were supported by electron micrographs, in which round-shaped droplets indicating lipoproteins were observed in ApoE-Sendai mice but not in aged apoE KO mice. In the kidney of apoE KO mice many anti-mouse CD68 Ab positive cells were detected. This contrasts with the result seen in ApoE-Sendai mice. The plasma lipoprotein compositions of the two types of mice were totally different. CONCLUSION: It was certain that the kidneys of aged apoE KO mice showed morphological alteration, but the histological findings of glomerular lesions were different from those seen in the kidneys of ApoE-Sendai mice. According to the histological findings and plasma lipoprotein profile, ApoE-Sendai mice, not apoE KO mice, is a murine model for human LPG. This means that apoE variants are essential to LPG.

The expression of type III hyperlipoproteinemia: involvement of lipolysis genes.

Type III hyperlipoproteinemia (HLP) is mainly found in homozygous apolipoprotein (APO) E2 (R158C) carriers. Genetic factors contributing to the expression of type III HLP were investigated in 113 hyper- and 52 normolipidemic E2/2 subjects, by testing for polymorphisms in APOC3, APOA5, HL (hepatic lipase) and LPL (lipoprotein lipase) genes. In addition, 188 normolipidemic Dutch control panels (NDCP) and 141 hypertriglyceridemic (HTG) patients were genotyped as well. No associations were found for four HL gene polymorphisms and two LPL gene polymorphisms and type III HLP. The frequency of the rare allele of APOC3 3238 G>C and APOA5 -1131 T>C (in linkage disequilibrium) was significantly higher in type III HLP patients when compared with normolipidemic E2/2 subjects, 15.6 vs 6.9% and 15.1 vs 5.8%, respectively, (P<0.05). Furthermore, the frequencies of the APOA5 c.56 G>C polymorphism and LPL c.27 G>A mutation were higher in type III HLP patients, though not significant. Some 58% of the type III HLP patients carried either the APOA5 -1131 T>C, c.56 G>C and/or LPL c.27 G>A mutation as compared to 27% of the normolipidemic APOE2/2 subjects (odds ratio 3.7, 95% confidence interval=1.8-7.5, P<0.0001). The HTG patients showed similar allele frequencies of the APOA5, APOC3 and LPL polymorphisms, whereas the NDCP showed similar allele frequencies as the normolipidemic APOE2/2. Patients with the APOC3 3238 G>C/APOA5 -1131 T>C polymorphism showed a more severe hyperlipidemia than patients without this polymorphism. Polymorphisms in lipolysis genes associate with the expression and severity of type III HLP in APOE2/2.

Antioxidant effects of vitamins C and E, multivitamin-mineral complex and flavonoids in a model of retinal oxidative stress: the ApoE-deficient mouse.

The aim of the current study was to investigate the biochemical changes in the plasma and retina of apolipoprotein E deficient (apoE-/-) mice supplemented with various antioxidants. Ten wild type (WT-Con, C57BL/6) and 10 apoE-/- (AE-Con) mice received drinking water. Another 40 apoE-/- animals were divided into four groups of 10 mice each and received either chromocarbe diethylamine (AE-CD, 50mg/kg), cyaninosides chloride (AE-CC, 50mg/kg), multivitamin complex (AE-MC, 50mg/kg), or vitamins C and E (AE-CE, 100mg/kg and 200IU/kg). Cholesterol, triglycerides, and lipid peroxidation (thiobarbituric acid reactive substances [TBARS]) were measured in plasma, and TBARS and nitric oxide metabolites (NOx) concentration were determined in retinal homogenates. Transmission electron microscopy was performed to examine the retinal ultrastructure. AE-Con mice had significantly (P<0.05) increased oxidative stress in the plasma and retina with augmented production of retinal NOx compared with WT-Con mice. Retinal TBARS decreased in the AE-MC and AE-CE animals compared with the AE-Con group (P<0.05 and P<0.01, respectively). Only AE-CE treatment significantly (P<0.01) lowered retinal NOx. Morphologic retinal changes in the AE-Con group decreased in the AE-CE and AE-MC groups. There were no significant changes in the biochemical and structural parameters in the AE-CD and AE-CC groups. AE-Con mice had increased systemic and retinal oxidative stress compared with WT-Con animals. Vitamins C and E and the multivitamin-mineral complex reduced oxidative stress and ultrastructural retinal changes in this murine model of hypercholesterolemia.

Molecular mechanisms of type III hyperlipoproteinemia: The contribution of the carboxy-terminal domain of ApoE can account for the dyslipidemia that is associated with the E2/E2 phenotype.

Apolipoprotein E2, which has an R158 for C substitution, has reduced affinity for the LDL receptor and is associated with type III hyperlipoproteinemia in humans. Consistent with these observations, we have found that following adenovirus-mediated gene transfer, full-length apoE2 aggravates the hypercholesterolemia and induces hypertriglyceridemia in E-deficient mice and induces combined hyperlipidemia in C57BL/6 mice. Unexpectedly, the truncated apoE2-202 form that has an R158 for C substitution when expressed at levels similar to those of the full-length apoE2 normalized the cholesterol levels of E-deficient mice without induction of hypertriglyceridemia. The apoE2 truncation increased the affinity of POPC-apoE particles for the LDL receptor, and the full-length apoE2 had a dominant effect in VLDL triglyceride secretion. Hyperlipidemia in normal C57BL/6 mice was prevented by coinfection with equal doses of each, the apoE2 and the apoE2-202-expressing adenoviruses, indicating that truncated apoE forms have a dominant effect in remnant clearance. Hypertriglyceridemia was completely corrected by coinfection of mice with an adenovirus-expressing wild-type lipoprotein lipase, whereas an inactive lipoprotein lipase had a smaller effect. The findings suggest that the apoE2-induced dyslipidemia is not merely the result of substitution of R158 for C but results from increased secretion of a triglyceride-enriched VLDL that cannot undergo lipolysis, inhibition of LpL activity, and impaired clearance of chylomicron remnants. Infection of E(-)(/)(-)xLDLr(-)(/)(-) double-deficient mice with apoE2-202 did not affect the plasma cholesterol levels, and also did not induce hypertriglyceridemia. In contrast, apoE2 exacerbated the hypercholesterolemia and induced hypertriglyceridemia, suggesting that the LDL receptor is the predominant receptor in remnant clearance.

Clinical features of type III hyperlipoproteinemia: analysis of 64 patients.

The clinical and biochemical characteristics of type III hyperlipoproteinemia are described in 64 patients (35 males and 29 females). Homozygosity for apolipoprotein E2, the presence of an abnormally cholesterol-rich very low density lipoprotein fraction (beta-VLDL) and an elevated ratio of very low density lipoprotein cholesterol to plasma triglycerides (> 0.3; normal ratio about 0.2) were the basis for the diagnosis. Mean serum cholesterol and triglyceride concentrations at the first visit in the clinic were 426 +/- 221 and 719 +/- 996 mg/dl, respectively. The mean age at diagnosis of the disorder was 49 years in males and 53 years in females. There was a high prevalence of obesity (72%), xanthomas (42%), and atherosclerosis (39%), especially peripheral vascular disease (31%). Early and correct diagnosis of this familial lipoprotein disorder seems necessary because of the prompt and beneficial response to therapeutic interventions.

[Apolipoprotein E phenotyping--useful in the study of hyperlipoproteinemia type III]. / Apolipoprotein E-fenotyping--nyttig ved utredning av hyperlipoproteinemi type III.

Apolipoprotein E (apo E) is a protein constituent of several plasma lipoproteins. Its major physiological role is to mediate the interaction of these lipoproteins and receptors. Three common apo E alleles and six phenotypes are known. The phenotype E2/E2 is associated with type III hyperlipoproteinemia. As a consequence of the increasing clinical interest in apo E phenotypes, especially in relation to the diagnosis of type III hyperlipoproteinemia, we have established a method for apo E phenotyping. Allele frequencies from a population of 211 healthy individuals are presented. The results show that, like as in Finland and Sweden the e4 allele is more frequent than in non-Nordic countries.