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(Stroke. 1999;30:1548-1553.)
© 1999 American Heart Association, Inc.

Original Contributions

Impact of Apolipoprotein E 4 and Vascular Disease on Brain Morphology in Men From the NHLBI Twin Study

C. DeCarli, MD; T. Reed, PhD; B. L. Miller, MD; P. A. Wolf, MD; G. E. Swan, PhD D. Carmelli, PhD

From the Department of Neurology, University of Kansas, Kansas City (C.D.); Department of Medical and Molecular Genetics, Indiana University, Indianapolis (T.R.); Department of Neurology, University of California at San Francisco–Mt Zion (B.L.M.); Department of Neurology, Boston University (Mass) (P.A.W.); and Health Sciences Division, SRI International, Menlo Park, Calif (G.E.S., D.C.).

Correspondence to Charles DeCarli, MD, Department of Neurology, Kansas University Medical Center, 3901 Rainbow Blvd, Kansas City, KS 66160-7314. E-mail cdecarli{at}kumc.edu

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Background and Purpose—Apolipoprotein E 4 genotype (ApoE4) has been associated with increased risk for cardiovascular disease morbidity or mortality. This appears to be mediated by an ApoE4-related increase in cardiovascular atherosclerosis. Given the similarities between risk factors for heart disease and risk factors for stroke, a positive association between ApoE4 and stroke would be expected. Since age-related brain atrophy and the extent of white matter hyperintensities (WMH) share similar risk factors, we examined the combined effect of ApoE4 and history of vascular disease on brain volume, WMH, and MRI evidence of stroke.

Methods—Subjects were the surviving members of the National Heart, Lung, and Blood Institute Twin Study. This is a longitudinal study of the effects of cardiovascular disease risk factors in community-dwelling male veterans. The fourth and final examination of this cohort included cerebral MRI and was completed in 1997. Apolipoprotein E (ApoE) genotype, quantitative measures of brain volume, WMH, and the presence of stroke on MRI were obtained from the 396 participants in the final examination. The presence or absence of a history of coronary heart disease, cerebrovascular disease, peripheral arterial disease, and ApoE genotype were determined for each subject.

Results—Of the 396 men, 88 (22%) had at least 1 ApoE4 allele. ApoE4 was not associated with differences in age or education. While the prevalence of vascular disease was generally greater in the ApoE4 group, this was only significant for coronary heart disease (29.8% in subjects without ApoE4 versus 40.7% in subject with ApoE4; P=0.03). ApoE4 subjects had significantly smaller brain volumes (942.4±34.5 versus 952.2±40.1 cm³; P=0.02). MRI evidence of stroke was detected in 88 (22%) of the subjects. The distribution of ApoE genotype was marginally different between subjects with MRI-detected stroke compared with those without. Further analysis revealed that the co-occurrence of cerebrovascular disease and ApoE4 was associated with significantly greater brain atrophy and WMH than either ApoE4 or cerebrovascular disease alone. Similar relations were seen for coronary heart disease and peripheral arterial disease.

Conclusions—We conclude that ApoE4 enhances the extent of brain abnormalities in the presence of various vascular diseases. We speculate that this effect may be mediated by an increased susceptibility to brain injury or impaired repair mechanisms associated with ApoE4.

Key Words: apolipoproteins • brain injuries • cerebrovascular disorders • epidemiology • magnetic resonance imaging

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Apolipoprotein E (ApoE) is a 299–amino acid protein important to lipid metabolism and other physiological processes.^{1 2 3} ApoE is polymorphic and encoded by alleles residing on chromosome 19q13.2 designated as 2 (ApoE2), 3 (ApoE3), and 4 (ApoE4). While these polymorphisms differ only by single cysteine-arginine amino acid substitutions at amino acids 112 and 158,¹ these substitutions result in important differences in physiological properties.^{3 4 5} For example, ApoE modulates the metabolism of the atherogenic apolipoprotein B–containing lipoproteins.⁴ Epidemiological evidence suggests that the ApoE4 genotype conveys a higher cardiovascular risk than does ApoE2 or ApoE3.^{3 4} Geographic variation in cardiovascular disease is associated with the prevalence of ApoE4 genotype⁴ among adults as well as the children of adults with cardiovascular morbidity or mortality.^{6 7 8} ApoE appears to modulate the impact of vascular disease by altering the level of serum cholesterol and LDL,⁴ and the ApoE4 genotype is associated with relative elevations in serum cholesterol.⁹ ApoE4 is also associated with a significantly greater surface area of atherosclerotic vascular disease in an unselected autopsy population¹⁰ as well as greater severity of angiographically defined coronary artery disease.¹¹ The cardiovascular effect of ApoE4 may not be fully described by estimates of symptomatic heart disease, since it also increases the risk of silent myocardial ischemia.¹² Therefore, while the risk for symptomatic atherosclerotic heart disease is multifactorial, the ApoE4 genotype appears to enhance the effect of these risk factors.⁴

Given the similarities between risk factors for heart disease and risk factors for stroke, a positive association between ApoE4 and stroke would be expected. Unfortunately, the relation among the various ApoE genotypes and stroke remains unclear,^{13 14 15 16 17} although recent evidence suggests that the ApoE4 allele status is overrepresented in Italian survivors of ischemic stroke.¹⁸ The reliance of epidemiological studies on clinically apparent stroke as the outcome may explain some of these differences. Elevated systolic blood pressure and other cerebrovascular risk factors are also associated with incidental (silent) cerebral infarction among community-dwelling older individuals.^{19 20 21} Similarly, cerebrovascular diseases (CVDs) increase the prevalence and severity of white matter hyperintensities (WMH) seen by MRI and increase the amount of age-related brain atrophy.^{22 23 24 25 26 27} If the ApoE4 genotype conveys an increased risk for CVD, then cerebrovascular-related brain differences may be enhanced. To test this hypothesis, we analyzed quantitative MRI results of 396 surviving members of the National Heart, Lung, and Blood Institute (NHLBI) Twin Study according to the extent of vascular disease and ApoE allele status.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Study Population
The NHLBI Twin Study is a longitudinal study of cardiovascular disease and associated cardiovascular disease risk factors in 514 pairs of male twins, 254 monozygotic and 260 dizygotic, born in 1917–1927 and aged 42 to 56 years when first examined in 1969–1972.^{28 29} Three follow-up examinations after 10, 16, and 25 years assessed cardiovascular disease status and collected repeated measurements of physiological, biochemical, and psychosocial risk factors.³⁰ In the most recent follow-up (1995–1997) of the NHLBI Twin Study, brain MRI was added to the sequence of tests previously given to these subjects. Analyses in the present study are limited to a subset of 396 individual twins, who participated in the fourth examination of this cohort and for whom MR volumetric data and ApoE genotyping were available. Included in this sample were 74 intact monozygotic pairs, 71 intact dizygotic pairs, and 106 singletons.

Cerebral MR Scans and Image Analysis
MR (1.5-T) scanning on GE scanners was performed at 4 study sites with the use of a conventional spin-echo, double-echo sequence in the axial orientation with repetition time of 2000 ms, echo time of 20/100 ms, 24-cm field of view, and 5-mm contiguous slices from the vertex to the foramen magnum imaged in a 256x192 matrix and interpolated to 256x256 with 1 excitation. Axial images were oriented parallel to the anterior commissure–posterior commissure line. After acquisition of the MR scans, the digital information was transferred to a central location for processing and analysis by one of the authors (C.D.), who is a neurologist with considerable neuroimaging experience and was blind to zygosity and medical history of subjects. Quantitative analysis of the MR scans was performed with the use of a custom written program operating on a SUN Microsystems Ultra 1 workstation. Image evaluation was based on a semiautomated segmentation analysis that involves operator-guided removal of nonbrain elements, as previously described.³¹ For segmentation of brain parenchyma from cerebrospinal fluid (CSF), a difference image was created by the subtraction of the second-echo image from the first-echo image. Image intensity nonuniformities were then removed from the difference image, and the resultant corrected image was modeled as a mixture of 2 gaussian probability functions.^{32 33} The segmentation threshold was determined at the minimum probability between the modeled CSF and brain matter intensity distribution.³¹ For segmentation of WMH from brain matter, the first and second echo images were summed, and after removal of CSF and correction of image intensity nonuniformities, a log-normal distribution was fitted to the summed image data. A segmentation threshold for WMH was a priori determined as 3.5 SDs in pixel intensity above the mean of the fitted distribution of brain parenchyma.

After image segmentation into CSF and brain matter was completed, the operator returned to the image to identify the presence or absence of cerebral infarction. The presence of cerebral infarction on MRI was determined from the size, location, and imaging characteristics of the lesion. The image analysis system allowed for superimposition of the subtraction image, the proton density image, and the T2-weighted image at x3 magnified view to assist in interpretation of lesion characteristics. Signal void, best seen on the T2-weighted image, was interpreted to indicate a vessel. Only lesions 3 mm qualified for consideration as cerebral infarcts. Other necessary imaging characteristics included the following: (1) CSF density on the subtraction image and (2) distinct separation from the circle of Willis vessels if the stroke was in the basal ganglia area.

Intrarater and interrater reliabilities for this method have been published.^{31 32}

ApoE Genotyping
For ApoE structural locus genotyping, the polymerase chain reaction was used to amplify 244–base pair fragments that contain variant amino acid residues 112 (cystinearginine=4 allele) and 158 (argininecystine=2 allele). Polymerase chain reaction products were then digested with the restriction enzyme HhaI and electrophoresed on an 8% polyacrylamide nondenaturing gel.³⁴ In this procedure, the most common 3 allele is cut by HhaI at position 158; the 4 allele is cut twice by the addition of a second restriction site at position 112; and the less frequent 2 allele lacks either recognition site. Genotyping by using this technique was completed on 589 individuals, showing allele frequencies of 2=0.09, 3=0.76, and 4=0.15, consistent with expected frequencies in other white populations.³⁵

Statistical Analysis
Differences in demographics, health histories, and brain morphology were evaluated between ApoE4 carriers, defined as either homozygote or heterozygote for the ApoE4 allele, and noncarriers. We used t tests for continuous variables and the ² test for categorical variables. The relationship of brain MR volumes to cardiovascular disease was examined in the total sample and separately in ApoE4 carriers and noncarriers. A regression model was used to adjust volumes for age, education, and head size.

To investigate the combined effect of the ApoE4 allele and cardiovascular disease, we divided subjects into 4 groups: subjects without the ApoE4 allele and without disease (reference group); subjects without ApoE4 and with disease; subjects with ApoE4 and without disease; and subjects in whom both ApoE4 and cardiovascular disease were present. These groups were entered into an ANCOVA model that adjusted for age, education, and head size.

Thirteen pairs of dizygotic twins were discordant for the presence of the ApoE4 allele. All ApoE4 twins had the 4/3 genotype. Of the non-ApoE4 cotwins, 9 were 3/3 genotype, 2 were 2/3 genotype, and 1 was 2/2 genotype. One discordant pair was excluded from the analysis because the ApoE4 carrier twin had the 4/2 genotype. This subject pair was excluded from the analysis because of the rare occurrence of this genotype and the potential for the offsetting effects of the 4/2 combination.

For this report we used all the available MR data, including data from both members of a twin pair as well as singletons. Because a potential bias may exist in estimating the standard error of a regression coefficient calculated from a sample of nonindependent observations (ie, twin pairs), we used bootstrap methods³⁶ (ie, resampling the data with replacement) to calculate empirical estimates of the standard errors of our estimates. In critical cases, in which the significance level was marginal, we created 1000 bootstrap data sets using the twin pair as the unit for resampling. All analyses were conducted with the SAS statistical package (version 6.09).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The mean age of twin subjects when they underwent MR scanning was 72.3±2.9 years. Table 1 summarizes the demographics, health histories, age, and adjusted MR volumes by presence or absence of the ApoE4 allele.

View this table: [in this window] [in a new window]   Table 1. Subject Characteristics by Presence or Absence of the Apo-4 Allele

The incidence of vascular disease was generally higher among ApoE4 carriers than noncarriers (Table 1) but reached statistical significance only for coronary heart disease (CHD) (40.7% versus 29.8%; P=0.03) and was marginally significant for peripheral arterial disease (PAD) (12.7% versus 7.4%; P=0.07). After adjustment for age and head size, average brain volumes were significantly smaller among ApoE4 carriers than noncarriers (942.4±34.5 versus 952.2±40.1 cm³; P=0.02). WMH volumes, however, were not significantly greater in ApoE4 carriers than noncarriers (4.4±5.7 versus 4.1±6.1 cm³; P>0.05). Similar group differences in brain measures according to ApoE4 status were present for the discordant dizygotic twins, although these differences did not reach statistical significance. ApoE4 noncarriers had a mean brain parenchyma volume of 951.8±35.3 cm³ and a mean WMH volume of 4.2±4.7 cm³, whereas ApoE4 carriers had a mean brain parenchyma volume of 942.1±35.2 cm³ (P=0.45) and mean WMH volume of 6.4±10.2 (P=0.53).

In addition, 88 subjects had stroke identified on MRI. The majority of these strokes were asymptomatic (60% without reported history); 72% were small (<1 cm in maximum diameter) and were located in the area of the lenticulostriate distribution (ie, 87% of small strokes were located in the lenticulostriate distribution; the remainder were in subcortical white matter). We compared the distribution of ApoE4 between subjects with MR evidence of stroke (n=88) and those without MR evidence of stroke (n=308). The difference in the distribution of ApoE alleles for subjects with MR evidence of stroke (ApoE2=5%, ApoE3=67%, and ApoE4=28%) compared with those subjects without MR evidence of stroke (ApoE2=12%, ApoE3=68%, and ApoE4=20%) did not reach statistical significance (P=0.08).

Table 2 summarizes brain measures according to the presence or absence of vascular disease as well as the joint occurrence of vascular disease and ApoE4 allele. The second column of Table 2 summarizes brain measures according to the presence or absence of a history of CVD, CHD, and PAD in brain parenchyma, intracranial fluid, and WMH. After adjustment for age and head size, subjects with CVD, CHD, or PAD had significantly smaller brain volumes and larger CSF volumes than those without disease. By contrast, only those with CVD had a significantly larger volume of WMH than those without disease.

View this table: [in this window] [in a new window]   Table 2. Mean MR Volumes According to ApoE Genotype and Cardiovascular Disease History

When subjects were further stratified according to ApoE4 status (Table 2), the following pattern of associations emerged. In the absence of cardiovascular disease (CVD, CHD, PAD), the presence or absence of the ApoE4 genotype showed no effect on brain parenchyma, intracranial fluid, or WMH volumes. For example, in subjects without CVD, parenchyma volume was 948 cm³ in those with the ApoE4 genotype and 954 cm³ in those without (mean difference=6 cm³; P>0.10). Similarly, in subjects without CVD, there was no significant effect of ApoE4 on WMH volume (mean volumes of 3.3 and 3.5 cm³, respectively). Subjects with both cardiovascular disease and the ApoE4 genotype, however, showed the most reduction in brain volume and the largest increase in WMH volume. Thus, because the effect of ApoE4 on brain volume was restricted only to subjects with cardiovascular disease, we conclude that the ApoE4 genotype enhances the effect of cardiovascular diseases on brain volumes. The presence of ApoE4 genotype alone is not, however, a risk factor for brain atrophy or WMH.

The Figure is a graphic display of the joint effect of ApoE4 alleles and presence or absence of CVD on WMH. The presence of CVD significantly increased WMH volumes in ApoE4 noncarriers, but in those subjects with both CVD and the ApoE4 allele, the amount of WMH was substantially larger than the amount that would be expected from the separate effects of CVD and ApoE4 combined (Table 2). Thus, strong evidence for the presence of a synergistic effect is suggested (F_1,384=4.75, P<0.03).

View larger version (34K): [in this window] [in a new window]   Figure 1. Relation between CVD, ApoE4 status, and WMH volume adjusted for age, education, and head size. + indicates presence; -, absence. The combined effect of ApoE4+ and CVD+ is associated with >6 cm3 increase in WMH volume compared with the absence of both factors.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
This is the first report, to our knowledge, to examine the relation between the presence or absence of ApoE4 genotype and cardiovascular disease in terms of brain morphology. We found that subjects with both cardiovascular disease and ApoE4 genotype showed the greatest reduction in brain volumes and the largest increases in WMH volumes. Because the effect of ApoE genotype was restricted to subjects with cardiovascular disease, we conclude that ApoE4 enhances this effect. In addition, we noted a reduced frequency of the ApoE2 genotype and an increased frequency of the ApoE4 genotype in subjects with MRI evidence of stroke.

These findings are consistent with similar studies of cardiovascular disease in which disease severity is enhanced for individuals with both cardiovascular disease and the ApoE4 genotype.^{3 4 6 7 8 10 11} While this study did not assess the mechanisms that might explain these findings, it is plausible that the ApoE genotype may induce the observed differences through increased atherogenesis or enhanced susceptibility to CVD-associated ischemic processes. Both age-related brain atrophy and increased WMH share cardiovascular risk factors similar to stroke,^{24 25 37 38} and an ischemic mechanism has been postulated as the pathogenesis for both of these processes.^{39 40 41} WMH can be induced in chronic ischemic animal models,³⁹ and moderately severe brain atrophy is present in spontaneously hypertensive rats.⁴¹ Thickening of the vascular intima as well as atherogenesis occurs in the small and medium-sized arteries of hypertensive brains, which are the terminal vascular supply for the white matter areas susceptible to WMH formation.⁴² Increased atherogenesis in these vessels might reduce blood flow and induce ischemic damage to the white matter and could explain the effect of ApoE4 on enhanced WMH.⁴³ A similar analogy is less apparent as an explanation for the enhanced effect of ApoE4 on brain atrophy. Deficiencies in apolipoprotein E, however, are associated with increased neuronal susceptibility to injury.⁴⁴ ApoE4 appears less effective in sustaining lipoprotein-related neuronal growth and repair mechanisms.^{1 2 3} Since cardiovascular disease is known to lead to brain atrophy and increased WMH volumes, any further susceptibility to injury conveyed by ApoE4 could explain the combined effects of ApoE4 and cardiovascular risk factors seen here. As in previous reports,^{45 46} we did not see a primary effect of ApoE genotype on brain or WMH volumes in our nondemented older subjects, further supporting the notion that another injurious process must accompany the presence of the ApoE4 genotype to induce the effects noted. Similarly, it is unlikely that the presence of incipient Alzheimer's disease (AD) in our ApoE4 subjects could explain these results. While the AD process is associated with brain atrophy, this appears independent of the ApoE genotype,⁴⁵ and careful studies have shown that the AD process is not associated with increased amounts of WMH.⁴⁷ The absence of significant differences in brain volumes or WMH in the ApoE4 subjects without vascular disease again supports the notion that an increased prevalence of incipient AD is an unlikely explanation for the observed effects.

Our results, however, differ from previous reports. Three previous MRI studies have assessed the relation between brain morphology, cardiovascular diseases, and ApoE genotype in large groups of nondemented older individuals.^{48 49 50} In one of these studies, no significant effect of ApoE4 was seen on measures of brain atrophy or WMH⁴⁸ ; however, no attempt was made to assess the effect of the co-occurrence of ApoE4 and vascular disease. A second study found a significant increase in WMH with the co-occurrence of ApoE3 genotype and vascular disease.⁴⁹ While each of these studies was population based, similar to our study, some important differences could explain the different results. First, previous studies were cross-sectional and relied on the subject's history and screening laboratories to identify the presence or absence of vascular disease. Our longitudinal study was designed to evaluate repeatedly the various forms of vascular disease by confirming the subject's history through extensive review of the medical records as well as multiple tests for presymptomatic vascular disease. Second, we used quantitative brain image analysis. Previous studies that relied on categorical ratings of MRI may be less sensitive to the subtle but continuous effects revealed by our quantitative measures. Third, our cohort was considerably older than the other cohorts.^{48 49} CVD risk increases with age, and the duration of various risk factors plays an important role in the occurrence of brain atrophy and WMH.³⁷ Therefore, if ApoE4 acts to enhance the susceptibility to brain injury from these processes, these effects are more likely to be seen in an older population. Fourth, our population consisted exclusively of men. These relations may differ for women. Further study of these effects in women would address this issue.

Finally, we found a decreased prevalence of ApoE2 genotype and increased prevalence of ApoE4 genotype in our subjects with MRI evidence of stroke. While still controversial, epidemiological studies have shown both a reduced prevalence of ApoE2⁵¹ and an increased prevalence of ApoE4¹⁸ in individuals with symptomatic stroke. Our population was relatively small with 396 subjects, but we found that 88 subjects had MRI evidence of stroke. This prevalence is consistent with prior population-based MRI studies.^{24 25 38} Since the ApoE4 genotype is associated with an increased prevalence of silent myocardial infarction,¹² it is possible that we are observing a similar phenomenon with MRI strokes, 60% of which were clinically asymptomatic. The enhanced ability of MRI to detect clinically silent stroke, therefore, may explain our ability to allude to differences in ApoE genotype distributions between individuals with and without MRI evidence of stroke. Further work is necessary to confirm or refute this observed trend.

Conclusions
We observed a significant enhancement of the effect of various vascular diseases and ApoE4 genotype on brain atrophy and WMH volume in the men of the NHLBI Twin Study. Importantly, no significant effect on these measures was seen with ApoE4 alone, and only minimal effects were seen in non-ApoE4 individuals with a coincident history of CVD or cardiovascular disease. In addition, there was a trend toward a difference in the frequency of ApoE2 and ApoE4 genotypes between subjects with MRI evidence of stroke compared with subjects without MRI evidence of stroke. We speculate that ApoE4 enhances brain injury in the presence of various vascular diseases in men. Further work with women would clarify this relation for the general population.

	Acknowledgments

 
This study was supported by grant HL51429 from the NHLBI. We would also like to acknowledge the assistance of Lisa Jack and James Garner in the processing of the MR images.

	Footnotes

 
Reviews of this article were directed by Dr Hermes Kontos. Because of possible conflict of interest, Dr Mark Dyken was not involved in the review process.

Received October 15, 1998; revision received May 13, 1999; accepted May 13, 1999.

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