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(Stroke. 1996;27:1755-1759.)
© 1996 American Heart Association, Inc.

Articles

Apolipoprotein E Polymorphism Is Associated With Segment-Specific Extracranial Carotid Artery Intima-Media Thickening

James G. Terry, MS; George Howard, DrPH; Michele Mercuri, MD, PhD; M. Gene Bond, PhD John R. Crouse, III, MD

the Departments of Internal Medicine (J.R.C., J.G.T.), Public Health Sciences (G.H.), and Neurobiology and Anatomy (M.M., M.G.B.), Bowman Gray School of Medicine, Winston-Salem, NC.

Correspondence to John R. Crouse III, MD, Department of Internal Medicine/Section on Endocrinology, Medical Center Blvd, Winston-Salem, NC 27157.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Background and Purpose Apolipoprotein E (apoE) polymorphism affects plasma cholesterol and may influence risk of atherosclerosis. We investigated the association of apoE with carotid artery wall thickening (an index of atherosclerosis) in individuals with and without coronary artery disease (CAD).

Methods ApoE phenotypes were resolved in 260 individuals equally represented by angiographically determined CAD case subjects and disease-free control subjects. Carotid artery intima-media thickening (IMT) was evaluated by B-mode ultrasound. Associations of apoE (E2, E3, or E4) with risk factors and IMT were evaluated in general linear models adjusted for age, sex, and CAD status with and without other traditional risk factors.

Results Total cholesterol (TC) and LDL cholesterol were associated with apoE isoforms. Mean TC and LDL cholesterol were lower in E2 (n=33) carriers than E3 (n=155) and E4 (n=66) carriers (each P<.001). IMT also varied by apoE. E2 carriers had less common carotid IMT than E3 and E4 carriers (P<.01), while internal carotid IMT was less in E2 and E3 carriers than in E4 carriers (P<.02). Bifurcation IMT was not associated with apoE (P=.24). ApoE polymorphism remained associated with common (P<.01) and internal (P<.04) IMT, and the association of apoE with mean IMT of all sites reached significance (P<.04) after adjustment for age, sex, CAD status, TC, LDL cholesterol, HDL cholesterol, triglycerides, diabetes, hypertension, and smoking.

Conclusions ApoE polymorphism is associated with segment-specific carotid IMT. The association of apoE with carotid IMT was statistically independent of apoE-associated variation in LDL cholesterol levels.

Key Words: apolipoproteins • atherosclerosis • carotid arteries • coronary artery disease • ultrasonics

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Apolipoprotein E plays a key role in dietary and hepatic cholesterol trafficking.^{1 2} ApoE mediates receptor uptake of TG-rich lipoproteins and may also participate in reverse cholesterol transport.^{1 2} In humans, apoE polymorphism contributes to variation in plasma cholesterol levels.¹ Accordingly, variant forms of apoE affecting plasma cholesterol concentration could predispose individuals to (or protect them from) atherosclerosis.

The apoE gene codes for three protein isoforms designated E2, E3, and E4.^{1 2} E3 is most common and is thought to be the parent form of apoE from which the E2 and E4 variants arose by single amino acid substitutions.² The E2 variant lacks a critical positive charge near its receptor binding region, making it defective as a ligand relative to E3 and E4.² The E4 variant has a positive charge substitution that may alter its lipoprotein affinity relative to E2 and E3.^{3 4} These critical charge differences are thought to affect lipoprotein metabolism in individuals with variant apoE isoforms.

ApoE is codominantly inherited, resulting in three homozygous and three heterozygous phenotypes.^{1 2} Plasma cholesterol levels are thought to vary among individuals with differing apoE phenotypes.¹ E2 carriers have been shown to have lower mean TC and LDL-C levels than either E3 or E4 carriers.¹ E4 carriers generally have higher TC and LDL-C levels than either of the other forms.¹ Differential receptor and lipoprotein affinity among apoE forms may be largely responsible for apoE-associated variation in plasma lipid levels.^{2 3 4}

Most cross-sectional studies have shown association of E4 with increased prevalence of premature CAD defined by angiography^{5 6 7 8} or of symptomatic CHD.^{9 10} Two prospective studies have also demonstrated associations of E4 with incident CHD.^{11 12} Population studies indicate that E4 allele frequencies are highest in countries where CHD mortality is highest¹ and frequencies are reduced in the elderly.¹³ However, deaths from Alzheimer's disease may contribute to age-associated decreases in E4 frequency.¹⁴ In fact, a recent report suggests no important effect of apoE polymorphism on risk of incident CHD in the elderly.¹⁵

The association of variant forms of apoE with extracranial carotid atherosclerosis has not been well studied. A single study in participants free from clinically manifest CHD found that the E3/2 genotype was associated with preclinical carotid atherosclerosis.¹⁶ Conflicting associations of apoE with ischemic stroke that may relate to carotid atherosclerosis have also been reported.^{17 18} E2 was associated with stroke in one cross-sectional study,¹⁷ but allele frequencies within the control group that were not consistent with previous reports may have affected the results.¹ A second, similarly designed study found E4 more prevalent in those with stroke.¹⁸ However, E4 was not associated with incident stroke in the elderly.¹⁵

To study the association of apoE polymorphism with atherosclerosis of the coronary and carotid arterial beds, we phenotyped apoE in 260 individuals who underwent coronary angiography and carotid ultrasonography.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Participants
CAFUS is an ongoing epidemiological investigation of risk factors for coronary and cerebrovascular disease.¹⁹ The study participants were selected from symptomatic North Carolina Baptist Hospital/Bowman Gray School of Medicine patients who had been referred for coronary angiography.^{19 20}

Exclusion criteria for CAFUS have been described previously^{19 20} and include MI within the previous 6 weeks, previous coronary artery bypass surgery or angioplasty, use of certain medications, or presence of clinical conditions thought to alter plasma lipids. Age younger than 45 years was also an exclusion criterion for CAFUS because the study was intended to assess risk factors and relationships between coronary and carotid artery disease, and those associations are weaker in younger individuals.²⁰

The sampling goal of the study was to assess 70 each of male and female CAD case and control subjects. Because they are relatively "rare" in the angiography population, all eligible disease-free men and women and all women with CAD were assessed. Men with CAD were more frequent, and therefore <20% of those eligible were selected at random to participate in the study.¹⁹ A total of 280 participants (69 male control subjects, 71 male case subjects, 70 female control subjects, and 70 female case subjects) entered the study. The selection strategy and study population have been described in more detail previously.^{19 20} Of the 280 CAFUS participants, apoE phenotype and Lp(a) levels were determined in 260 participants (130 control subjects and 130 CAD case subjects) based on the availability of plasma frozen at -80°C. The study was approved by the institutional review board, and all participants provided informed consent to participate in CAFUS.

Methods
Study participants were classified as CAD case or control subjects on the basis of coronary angiography. Individuals with 50% stenosis in one or more coronary arteries were classified as CAD case subjects, while control subjects had no detectable coronary stenosis.^{19 20} Patients with detectable stenosis <50% were excluded from the CAFUS.

Extracranial carotid artery IMT was quantified by B-mode ultrasound as previously described.^{19 20 21} Examinations were performed with the aid of a Biosound 2000 II s.a. high-resolution ultrasound unit equipped with an 8-MHz transducer. Images were transcribed to videotape. Examination procedures and quality control for the instrument and for certified sonographers and readers were strictly maintained as described.^{20 21} Both sonographers and readers were blind to participant CAD case-control status.

Carotid artery segments from which IMT measurements were made were identified relative to the bifurcation: the internal carotid (1 cm distal to the flow divider), the area of the bifurcation or bulb (the segment proximal to the flow divider and distal to the origin of the bulb), and the common carotid (1 cm proximal to the origin of the bulb) were examined. A pair of ultrasonic interfaces was used to measure IMT of each segment: the adventitia-media and the intima-lumen interfaces on the near wall and a mirror pair on the far wall. Each segment was examined from circumferential angles to identify the thickest intima-media site, and the entire examination was recorded by the sonographer. Maximum IMT was determined at each site by certified readers as described.^{20 21} IMT was measured in the right and left carotid arteries on both far and near walls of the common, bifurcation, and internal arterial segments (four measures per segment, 12 measures total).

Reproducibility of this B-mode ultrasound protocol has been carefully examined.²¹ Repeated baseline scans in 878 MIDAS participants were in excellent agreement (ß-coefficient=.99 for overall mean maximum IMT).²¹ Intrascan and interscan differences for overall mean maximum IMT among sonographers and readers ranged from -0.007±0.154 to 0.004±0.160 mm in the MIDAS evaluation.²¹

Blood for lipid analyses was obtained from fasting participants 6 to 8 weeks after catheterization, as described.¹⁹ Plasma was immediately isolated by low-speed centrifugation and divided into aliquots for storage at -80°C. Traditional lipid risk factors were measured in the Bowman Gray Lipid Analytic Laboratory by standardized methods of the Centers for Disease Control and Prevention. Cholesterol and TG analyses were performed on whole plasma with and without heparin-MnCl₂ precipitation with the use of a Technicon RA 1000 Auto-analyzer.²² LDL-C was determined after ultracentrifugation at d=1.006 within the d=1.006 infranate²² or estimated by the Friedewald formula²³ when plasma quantity was insufficient for ultracentrifugation (n=4). Lp(a) was measured with the use of the Strategic Diagnostics Inc enzyme-linked immunoassay (interassay coefficient of variation <8.2%).

ApoE phenotypes were determined by the method of Kataoka et al.²⁴ This modification of a previously published method²⁵ allows rapid and accurate determination of apoE phenotypes. Briefly, 10 µL of plasma is diluted with a reducing buffer consisting of 5 mmol/L dithiothreitol (Sigma) in 0.25% Tween 20, and samples are then incubated overnight at 4°C.²⁴ Treated plasma is subjected to isoelectric focusing followed by Western blotting, as described.²⁵ ApoE phenotypes are identified by a primary polyclonal antibody to human apoE (International Immunology Corp) followed by an alkaline phosphatase–labeled secondary antibody (INCSTAR). Controls that had been verified by restriction enzyme genotyping²⁶ and isoelectric focusing phenotyping^{24 25} in two separate laboratories were included on each gel.

Study participants were interviewed by the study coordinator using standardized questionnaires to obtain demographic information. Hypertension was coded positive if prior diagnosis was indicated or if systolic blood pressure was >150 mm Hg and/or diastolic blood pressure was >90 mm Hg. The presence of diabetes mellitus was defined by history of the disease or fasting glucose >140 mg/dL.

Statistical Analysis
Segment-specific mean maximum IMT of both near and far walls in right and left carotids was evaluated in each of the three arterial segments. The mean maximum score of all 12 arterial measurements was also evaluated. There was little missing IMT information (93% of sites were successfully interrogated); hence, the simple average was used as the overall mean maximum score for all sites. Carotid IMT least-squares mean±SE values are presented.

A general linear model (SAS procedure GLM) was used to evaluate associations of apoE with participant characteristics such as lipid parameters and carotid IMT.²⁷ In models with lipid outcomes, apoE was covaried with age, sex, and CAD status. Lp(a) and TG were analyzed by nonparametric tests or were log-transformed for multivariate analyses because of their departure from the normal distribution. The association of apoE with carotid IMT was evaluated by the general linear model with adjustment for age, sex, and CAD status in the presence or absence of other risk factors.²⁷ Categorical variables and apoE frequencies were tested by ² (SAS procedure FREQ). Because of the relative infrequency of some apoE phenotypes, apoE was designated as E2 (E2/2 or 3/2), E3 (E3/3), or E4 (E4/4 or 4/3) before analysis. Six participants with the E4/2 phenotype were omitted from analysis because of classification problems. Analyses were performed on SAS.²⁸

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
ApoE isoform frequencies among the 260 participants in the present study were 0.08 for E2, 0.77 for E3, and 0.15 for E4. These apoE frequencies agree well with those previously reported for North American populations.¹ Approximately 60% of participants were E3 homozygotes, 23% were E4/3 heterozygotes, and 12% were E3/2 heterozygotes; the other phenotypes (E2/2, E4/4, and E4/2) were relatively infrequent. The frequency of E2, E3, and E4 carriers was not significantly different between CAD patients and control subjects or between men and women (P=.871 and .392, respectively).

Table 1 presents participant characteristics according to their apoE isoform. TC and LDL-C were significantly associated with apoE (both P<.001, general linear model adjusted for age, sex, and CAD status). No other significant associations of apoE with risk factors were apparent.

View this table: [in this window] [in a new window]   Table 1. Participant Characteristics by ApoE Isoform

Segment-specific and overall comparisons of carotid artery IMT according to apoE isoform are presented in Table 2. Common carotid IMT adjusted for age, sex, and CAD status (model 1) varied significantly by apoE isoform. IMT in E2 carriers was less than that of either E3 or E4 carriers (P<.005, E2 versus E3 or E4). The association of apoE with common carotid IMT was further assessed by the general linear model adjusted for age, sex, CAD status, TC, LDL-C, HDL-C, TG, smoking status, diabetes mellitus, and hypertension (model 2). Risk factor–adjusted IMT in the common carotid remained significantly associated with apoE. IMT of E2 carriers was less than either E3 or E4 carriers (P<.016 for each comparison).

View this table: [in this window] [in a new window]   Table 2. Carotid Artery IMT by Arterial Segment and ApoE Isoform

No significant associations among apoE isoforms and carotid bifurcation IMT were found for either multivariable model. However, internal carotid IMT adjusted for age, sex, and CAD status varied significantly with apoE. Internal carotid IMT of E3 carriers was less than that of E4 carriers (P<.016), and IMT of participants with E2 tended to be less than that of E4 carriers as well (P=.06). Internal carotid IMT remained associated with apoE after adjustment for potential confounding risk factors. E4 carriers had greater internal carotid IMT than E3 carriers in model 2 (P<.015).

Overall mean carotid IMT (mean maximum of all measured sites) was marginally associated with apoE (P=.052) in model 1. However, the association of overall mean carotid IMT with apoE reached significance after other potential risk factors were included in the model (P=.032). The overall mean carotid IMT was significantly greater in those with E4 than those with E3 (P<.015). While E2 and E3 carriers had similar overall mean IMT, E2 versus E4 comparisons did not reach significance (P=.07) for model 2.

Restricting these analyses to those with the E3/2, E3/3, and E4/3 phenotypes (ie, dropping from analysis those few participants homozygous for E2 [n=2] and E4 [n=7]) did not appreciably alter the association of apoE with carotid IMT. After adjustment for age, sex, CAD status, and risk factors (model 2), apoE phenotype was associated with common (P=.006), internal (P=.021), and overall carotid (P=.017) IMT. Common carotid IMT was lower in those with the E3/2 phenotype than in those with either E3/3 or E4/3 (P<.008 for each comparison). Internal carotid and overall IMT values were greater in E4/3 heterozygotes than in E3 homozygotes (P<.008 for each comparison).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
ApoE polymorphism contributes to variation in plasma cholesterol levels among individuals and populations, thus potentially affecting the risk of CHD.¹ However, the association of apoE with CHD may only partially relate to apoE-associated variation in TC and LDL-C.^{1 7 10 11 29}

ApoE facilitates cellular uptake of dietary and hepatic cholesterol transported within remnant lipoproteins, but functional differences in apoE isoforms may affect lipoprotein metabolism.^{1 2} Variant apoE forms have been shown by Mahley² to have differential affinities for the LDL receptor and the lipoprotein receptor–related protein.³⁰ These in vitro studies have shown that E2 binds poorly to both LDL receptor (<2% of E3 or E4 binding)² and lipoprotein receptor–related protein (<40% of E3 or E4 binding).³⁰ Other studies have shown preferential association of E4 with large, TG-rich lipoproteins relative to E2 or E3.^{3 4}

Differences in lipoprotein metabolism help to explain associations of E2 with lower and E4 with higher plasma cholesterol. E2 carriers exhibit delayed postprandial remnant clearance³¹ partially due to defective lipoprotein binding by hepatic receptors.^{2 30} Consequently, hepatic LDL receptor may be upregulated and plasma cholesterol lowered in E2 carriers.^{1 2 31} The E4 isoform associates well with TG-rich chylomicrons^{3 4 31} and promotes efficient clearance by high-affinity binding to remnant receptors.³⁰ Rapid hepatic clearance of chylomicron and VLDL remnants may downregulate LDL receptor and raise plasma cholesterol in E4 carriers.^{1 2 31}

In the present study, analyses adjusted for age, sex, and CAD status confirmed previous reports that TC and LDL-C varied according to apoE isoform. E2 carriers had lower mean TC and LDL-C than either E3 or E4 carriers (P<.001). Although most studies have previously shown higher plasma cholesterol in those with E4 relative to E3, the association of lower cholesterol with E2 is stronger.^{1 32} No other lipids were significantly associated with apoE in the present study. Lp(a) levels were previously reported to be higher in E4 carriers,³³ but this association has not been confirmed.³⁴

ApoE-associated variation in cholesterol levels has created interest in apoE variants as heritable risk factors for atherosclerosis. In the present study, no significant difference in apoE isoform frequency was apparent between CAD patients and disease-free control subjects (P=.871). Despite association of apoE variants with CHD,^{5 6 7 8 9 10 11 12} the association of E4 with higher LDL-C is attenuated with age.³⁵ Also, previously reported associations of apoE polymorphism with CHD and atherosclerosis were often in participants younger than those in the present study.^{5 6 8 10 11 29} A study of 91 men younger than 50 years referred for angioplasty found the E4 allele in 42% of participants younger than 40 years but in only 21% of those aged 40 to 50 years.⁸ Exclusion of individuals younger than 45 years in the present study may have masked associations of apoE frequency with CAD.

ApoE polymorphism was strongly associated with carotid IMT in the present study. Analyses adjusted for age, sex, CAD status, and lipid risk factors indicated that apoE isoforms were associated with IMT in the common (P<.01) and internal (P<.04) carotids. Overall mean IMT was significantly associated with apoE after adjustment for other risk factors (P<.04). No significant interactions were found between apoE and sex or CAD status. Therefore, associations of apoE with IMT were similar in men and women and in those with and without prevalent CAD.

Significantly thinner common carotid walls were present in those with E2 versus those with either E3 or E4 after adjustment for other risk factors. In the internal segment and for overall mean score, carotid IMT was significantly less in E3 carriers than in E4 carriers. Carotid IMT was similar in E2 and E3 carriers in the internal segment and for overall mean score, but univariate comparisons of E2 and E4 IMT were of borderline significance, possibly because of lower E2 frequency. These same segment-specific associations of apoE polymorphism with carotid IMT were also present when the data set was restricted to only those with the E3/2, E3/3, and E4/3 phenotypes. Thus, heterozygous inheritance of variant alleles was sufficient for these associations.

The lack of significant associations between apoE and bifurcation IMT may relate to anatomic and structural differences between the bifurcation and other carotid segments. The geometry of the bifurcation and perhaps the artery wall itself may affect the association between apoE and IMT. Alternatively, IMT measurement error may be greater in the bifurcation where the arterial walls are not parallel, thus obscuring associations and increasing the likelihood of a type II statistical error.

To our knowledge, this study represents one of only two reports of an association of apoE polymorphism with carotid IMT measured by B-mode ultrasound. A case-control substudy in the ARIC cohort found that the E3/2 genotype was more frequent in participants with preclinical atherosclerosis (n=145) than in control subjects (n=224) after adjustment for other risk factors.¹⁶ In contrast to the ARIC substudy, E2 was associated with lower IMT (ie, less atherosclerosis) in the present study. Differences in study design may explain the discrepant findings; those with clinically manifest CHD were excluded from ARIC, whereas half of the participants in our own study had documented CAD. However, the trend toward lower mean IMT in E2 versus E4 carriers in the present study was consistent in both disease-free control subjects and CAD patients.

In the present study, the association of apoE with carotid IMT persisted after adjustment for TC and LDL-C. Others have suggested that the association of apoE with atherosclerosis cannot be totally attributed to variation in TC and LDL-C.^{7 10 11 29} Hixson et al²⁹ found that after adjustment for age, TC, and smoking, apoE genotype still accounted for 3% to 11% of the variance in atherosclerosis of the aorta in young men. The mechanism by which apoE isoforms could be associated with atherosclerosis independent of TC and LDL-C is not known. However, metabolism of LDL-precursor or remnant lipoproteins may differ in those with variant apoE isoforms.^{1 31} Alternatively, apoE variants may differ in their ability to effect cholesterol efflux from peripheral vascular cells.^{36 37}

The present study may be restricted by case-control participant selection. Participants were symptomatic angiography patients and, although half were free of disease, generalizability of the results to "healthier" populations may be limited. These hospital patients likely had greater carotid IMT than asymptomatic individuals, which may have affected the associations. Also, risk factors that were not measured in the present study (such as other apolipoprotein polymorphisms) may have contributed to these associations. Finally, these associations should be confirmed in a larger study because the number of E2 carriers was small and some data were missing in the present study.

In summary, significant associations of apoE polymorphism with carotid IMT were found in the present study even though no associations of apoE frequency with CAD were detected. IMT of E4 and E3 carriers exceeded that of E2 carriers in the common carotid. IMT of E4 carriers exceeded that of E2 and E3 carriers in the internal carotid and for overall mean carotid IMT. The association of apoE with carotid IMT persisted after adjustment for TC and LDL-C. These data suggest that E2 is associated with less and E4 with more carotid atherosclerosis independent of apoE-associated variation in plasma TC and LDL-C.

	Selected Abbreviations and Acronyms

 

apoE = apolipoprotein E ARIC = Atherosclerosis Risk in Communities CAD = coronary artery disease CAFUS = Carotid Artery Follow-up Study CHD = coronary heart disease HDL-C = HDL cholesterol IMT = intima-media thickening LDL-C = LDL cholesterol Lp(a) = lipoprotein(a) MI = myocardial infarction MIDAS = Multicenter Isradipine Diuretic Atherosclerosis Study TC = total cholesterol TG = triglycerides

	Acknowledgments

 
This study was supported by grant R01-HL-35333 from the National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md, and the General Clinical Research Center of the Bowman Gray School of Medicine, grant M01 RR-07122. The authors wish to acknowledge the assistance of Julia Robertson, Programmer, General Clinical Research Center. We are indebted to Drs Michael Paidi, Shinkuro Kataoka, and Barbara Howard at the Medlantic Research Institute and Lori Kelly and Dr Robert Ferrell at the University of Pittsburgh for their assistance in establishing the apoE phenotyping method.

Received March 1, 1996; revision received May 30, 1996; accepted June 18, 1996.

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