|Molecular Vision 2007;
Received 18 December 2006 | Accepted 30 October 2007 | Published 12 November 2007
Apolipoprotein E gene and retinal microvascular signs in older people: the Cardiovascular Health Study
Cong Sun,1 Gabriella Tikellis,1 Gerald Liew,2
Ronald Klein,3 Emily K. Marino Larsen,4
Tien Y. Wong1,5
1Centre for Eye Research Australia, University of Melbourne, VIC; 2Centre for Vision Research, University of Sydney, NSW, Australia; 3Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI; 4Department of Biostatistics, University of Washington, Seattle, WA; 5Singapore Eye Research Institute, National University of Singapore, Singapore
Correspondence to: Tien Yin Wong, Centre for Eye Research Australia, University of Melbourne, 32 Gisborne Street, East Melbourne 3002, Australia; Phone: +61 3 9929 8352; FAX: +61 3 9662 3859; email: firstname.lastname@example.org
Purpose: To examine the association between apolipoprotein E (APOE) gene polymorphism and retinal microvascular signs in an older population.
Methods: Retinal photographs were taken of 2,152 participants (1,831 whites, and 321 African-Americans), aged 69-96 years, who were participating in a population-based study of four United States communities. We used standardized protocols to assess photographs for the presence of retinal microvascular signs (retinopathy, arterio-venous nicking, and focal arteriolar narrowing) and a computer-assisted method to measure retinal vessel diameters. We analyzed DNA extracted from blood samples of participants for common allelic variants of the APOE gene.
Results: After adjusting for age, gender, systolic blood pressure, smoking, total serum cholesterol, and other risk factors, we found white participants carrying the ε2 and ε4 alleles were more likely to have arterio-venous nicking than the ε3/ε3 homozygotes, with odds ratio (OR) of 1.70 and confidence interval (CI) 95% (1.03-2.83) for the ε2 carriers and OR 1.74 (95% CI 1.06-2.84) for the ε4 carriers. Among white participants without hypertension, the associations remained significant for the ε4 carriers (OR 2.32, 95% CI 1.18-4.57). Whites, normotensive carriers of the ε2 allele had significantly narrower retinal arteriolar diameters (adjusted mean arteriolar diameter of 163.5 μm, 95% CI 160.1-167.0, p=0.03) compared to the ε3/ε3 homozygotes (167.8 μm, 95% CI 166.0-169.6). APOE gene polymorphism was not associated with retinopathy, focal narrowing, or retinal venular diameters in white participants. There were insufficient numbers of African-Americans for separate multivariate analysis.
Conclusions: This study provides little evidence that the APOE gene polymorphism plays a significant role in the pathogenesis of retinal microvascular changes in the general population. In the older white population, APOE ε2 and ε4 allele carriers were more likely to have arterio-venous nicking. Other retinal signs, however, were not related to APOE gene polymorphism.
The retinal microcirculation provides a unique opportunity to study in vivo changes of the human microvasculature . Recent population-based studies show that various retinal microvascular signs (e.g., retinopathy signs, arterio-venous nicking, and focal arteriolar narrowing) are associated with risk of systemic vascular diseases, including hypertension [2-4], stroke [5-7], and other cardiovascular diseases , independent of traditional risk factors. Despite these observations, the factors that influence the occurrence of these retinal signs are not fully understood. Genetic predisposition to retinal microvascular signs has been hypothesized , and supported by a recent genome-wide linkage analysis, demonstrating several potential genetic loci linked to retinal vessel diameters .
The apolipoprotein E (APOE) gene is an attractive candidate to investigate possible associations with retinal microvascular signs. The APOE gene (ε2, ε3, and ε4) is associated with various types of ocular, cardiovascular, and neurodegenerative disorders, including age-related macular degeneration [10,11], glaucoma [12,13], clinical stroke , coronary heart disease , and cerebral infarction . These associations are possibly be modulated by the effects of APOE on lipid metabolism and large vessel atherosclerosis [17-22]. It is not clear whether APOE gene influences the development of smaller vessel or microvascular disease. Previous studies have indicated that APOE polymorphism may be associated with magnetic resonance imaging (MRI)-defined cerebral white matter lesions, which are manifestations of cerebral microvascular disease [23,24]. However, a population-based study found APOE gene polymorphism was not associated with diabetic retinopathy .
We have previously reported the prevalence of retinal microvascular signs and its association with blood pressure in the Cardiovascular Health Study (CHS), a population-based study in older persons [26,27]. In the current study, we investigate its association with common allelic variants of APOE.
The CHS is a population-based cohort study of cardiovascular disease in adults aged 65 years and older . The study population and methodology are described in detail elsewhere . In brief, recruitment of the original cohort of 5,201 participants took place 1989-1990 at four field centers: Allegheny County, Pennsylvania; Forsyth County, North Carolina; Sacramento County, California; and Washington County, Maryland. An additional 687 eligible African-Americans were recruited from Forsyth County, Sacramento County, and Allegheny County. All participants completed a standard interview-administrated questionnaire relating to family history, medication use, and medical history including hospitalizations and prior diagnosis of cardiovascular diseases. Other examinations conducted included anthropometric measures, blood pressure, electrocardiography, carotid ultrasonography, echocardiography, and blood chemistry profiles [27,29]. Differences between those recruited and those not recruited have been presented elsewhere .
Retinal photography was offered to participants who returned for a follow up examination during 1997 to 1998, approximately 9 years after the baseline examination [26,27]. Of the 4,249 participants (95.5% of survivors) who were contacted at this examination, we initially excluded 29 (0.7%) participants whose race was neither white nor African-American, 491 (11.6%) without APOE gene data, and 1,577 (37.1%) people with missing data for retinal microvascular signs, leaving 2,152 (50.6% of the original 4,249) persons who were considered eligible for this analysis. The total number of cases with specific retinal microvascular signs varied in different analyses due to the different number of missing data for each retinal sign.
Retinal photography and grading
The retinal photography procedure and grading of retinal microvascular signs have been described in detail elsewhere [26,27]. In brief, during the 1997-1998 period, two 45-degree non-mydriatic retinal photographs of one randomly selected eye were obtained, one centered on the optic disc and another centered on the macula (Early Treatment for Diabetic Retinopathy Study, ETDRS standard fields 1 and 2). Photographs were evaluated at the Fundus Photograph Reading Center in Madison, Wisconsin, by two trained graders, masked to participant characteristics, according to a standardized protocol.
Retinal microvascular signs included the following: (1) retinopathy lesions (i.e., presence of microaneurysms, retinal hemorrhages, cotton wool spots, or hard exudates), (2) arterio-venous nicking, and (3) focal arteriolar narrowing. The grading and definition of these lesions were based on a standard protocol described in other reports . For retinopathy, the following lesions were evaluated in each of the four quadrants of the retina using a 'light-box': microaneurysms, blot hemorrhages, flame-shaped hemorrhages, soft exudates or cotton-wool spots, hard exudates, macular edema, intraretinal microvascular abnormalities, venous beading, new vessels at disc or elsewhere, and vitreous hemorrhage. Retinopathy was defined as being present if any of these lesions were definite or probable in any of the four quadrants. Focal narrowing was defined definite if an arteriole estimated to be 50 μm in diameter had a constricted area of 2/3 or less the width of proximal and distal vessel segments. Mild focal narrowing was considered present if the combined length of narrowing in the quadrant was less than 1/2 an optic disc diameter, moderate if between 1/2, and two optic disc in diameter, or severe if two optic disc diameter. Arteriovenous nicking was considered definite if the venous blood column was narrowed on both sides of its crossing under an arteriole. Definite arteriovenous nicking less than that in the ETDRS standard photograph 9 (with narrowing of the blood column by approximately 1/2) was considered mild/moderate, and nicking equalling or exceeding the standard was severe. For both focal narrowing and arteriovenous nicking, the grade "questionable" was used if the grader thought the lesion was present but had only 50% to 90% confidence to be sure . For our analysis of retinopathy, we only included participants without diabetes (n=1,521), as diabetic retinopathy has a different pathophysiology. We refer to these lesions as "non-diabetic retinopathy".
To quantify retinal arteriolar and venular diameters, the fundus photographs were digitized with a high-resolution scanner. The diameters of all arterioles and venules coursing through a specified area half (1/2) to one (1) disk diameter from the optic disk were measured by a computer-assisted program . The individual arteriolar and venular diameters were combined into summary measures, referred to as central arteriolar equivalent (CRAE) and central retinal venular equivalent (CRVE) using formulas by Parr and Hubbard . CRAE and CRVE have approximately normal distribution, with smaller equivalents representing narrower average arteriolar and venular diameters in that eye, respectively .
Quality control procedures were implemented during the retinal grading. In the CHS, there were 71 intra-grader and 69 inter-grader re-readings. The intra-grader kappa statistics ranged from 0.90 for retinopathy, 0.49 for arterio-venous nicking and 0.57 for focal arteriolar narrowing, while the inter-grader kappa statistics were 0.88, 0.43, and 0.31, respectively. For arteriolar and venular equivalents, the intra- and inter-grader correlation coefficients ranged 0.67-0.91.
Apolipoprotein E genotyping
Genotyping of APOE in the CHS has been previously described [31,32]. For analysis, we analyzed separately the three common alleles of APOE (ε2, ε3, and ε4) and its six common genotypes. The three major allelic forms of the APOE gene were determined in the Core Molecular Genetics facility at the University of Vermont College of Medicine by the method of Hixson and Vernier . The two primers used for polymerase chain reaction amplification, done in 96-well microtiter plates, were 5'-GGC ACG GCT GTC CAA GGA-3' and 5'-ACA GAA TTC GCC CCG GCC TGG TAC AC-3'. Amplitaq T4 DNA polymerase was obtained from Perkin-Elmer, Wellesley, Mass; the restriction enzyme HhaI was obtained from New England BioLabs, Ipswich, Mass. DNA samples known to be ε4/ε2 and ε2/ε3 were analyzed with each batch as positive controls. The restriction patterns were determined with the use of agarose electrophoresis .
Definition of other variables
Participants underwent cardiovascular disease risk factor assessment and clinical and laboratory evaluation during the course of the study [34-36]. Relevant portions are highlighted in this paper. Blood pressure levels were taken according to a standardized protocol . Hypertension was defined as systolic blood pressure 140 mmHg, diastolic blood pressure 90 mmHg, or the combination of self-reported high blood pressure diagnosis and use of anti-hypertensive medications. Medical history, medication use, cigarette smoking and alcohol consumption status were ascertained from questionnaires. Diabetes was defined according to the American Diabetes Association criteria : treatment with either oral hypoglycemic agents or insulin in the year before the examination; or having fasting blood glucose of 126 mg/dl for those not using any hypoglycemic agents in the past year. Technicians assessed body mass index (BMI; kg/m2) and the waist-hip ratio. Blood collection, processing, and definitions for fasting plasma glucose, total cholesterol, LDL and HDL cholesterol, and triglycerides are described elsewhere [29,38]. All variables defined were based on the 1997-1998 clinic examination, concurrent with retinal photography, except data on most blood chemistries (e.g., HDL cholesterol and triglyceride used in this analysis), which was taken from the 1992-1993 examination.
Using the chi square test, we examined whether the distribution of APOE genotypes in whites and African-Americans was in Hardy-Weinberg equilibrium. Where the reliability of p-values based on chi square approximations was questionable, we report p-values for Fisher's exact test. We also examined the distribution of APOE allele carriers (ε2, ε3, and ε4) and specific genotypes by presence or absence of any retinal microvascular signs using the chi square test or Fisher's exact test. We analyzed the associations of polymorphisms of the three APOE allele (ε2, ε3, ε4) carriers and six genotypes (ε2/ε2, ε2/ε3, ε2/ε4, ε3/ε3, ε3/ε4, and ε4/ε4) with different retinal microvascular signs including retinal vessel diameters. Only the results of the allele analyses are presented. We defined carriers of the ε2 allele as persons with genotypes ε2/ε2, ε2/ε3, and ε2/ε4, and carriers of the ε4 allele as persons with genotypes ε3/ε4 and ε4/ε4. The method of analysis using allele carriers status rather than the allele itself was based on previous reports, with most studies defining carriers of the ε2 allele as those having the ε2/ε2 or ε2/ε3 genotype, and carriers of the ε4 allele as those having the ε3/ε4 or ε4/ε4 genotype, with the ε3/ε3 genotype acting as a reference group . We also performed an additional analysis in which persons with the ε2/ε4 genotype were grouped as ε4 allele carriers.
We used logistic regression models to estimate the odds ratios (OR) and 95% confidence intervals (CI) for retinal microvascular signs by APOE alleles using the ancestral ε3/ε3 genotype as the reference group, adjusted for age, gender, race, fasting glucose, systolic blood pressure, cigarette smoking status, BMI, total plasma cholesterol, and HDL cholesterol. We constructed models with and without adjustment for total cholesterol and HDL cholesterol, since these factors may be involved in the causal pathway. We used general linear models to obtain adjusted mean retinal vessel diameters.
We analyzed data in the total cohort and for whites and African-Americans separately, since APOE genotype frequencies differ between races. We performed multivariate analysis for the whites group only, as multivariate analysis for African-Americans was limited by small numbers. We also conducted multivariate analysis stratified by hypertension status, as hypertension is strongly related to retinal microvascular signs. Finally, we examined for potential interaction with age, gender, race, diabetes status, triglyceride, HDL cholesterol, and cigarette smoking status by including appropriate cross-product terms in the logistic regression models. All p-values are two sided and all statistical analysis was carried out using STATA 9.0 (Stata Corp., College Station, Texas).
Table 1 presents the comparison of participants included (n=2,152) and not included (n=2,097) from the analysis. Participants excluded were more likely white (p<0.001, data not shown), female, and on average 2.1 years older than those included in this analysis. After controlling for age, gender, and race, those excluded were less likely to be high school graduates, more likely to have diabetes, hypertension, and to have elevated diastolic blood pressure and higher fasting glucose. Table 1 also shows the baseline characteristics of participants, stratified by race. Whites carrying ε2 allele were significantly older, and had lower total plasma cholesterol than carriers of the ε3 and ε4 alleles. African-American, carriers of the ε2 allele had significantly higher BMI, were more likely to have hypertension, but had lower total plasma cholesterol compared to the ε3 and ε4 carriers. In both whites and African-Americans, mean systolic and diastolic blood pressure did not differ significantly between the three APOE allele groups.
Table 2 presents the distribution of APOE genotype and allele frequencies in whites and African-Americans. Allele frequencies were 8.9% (ε2), 78.9% (ε3), and 12.2% (ε4) in whites, and 14.6% (ε2), 69.0% (ε3), and 16.4% (ε4) in African-Americans. There was no evidence of departure from Hardy-Weinberg equilibrium of APOE genotypes in whites (p=0.22) or African-Americans (p=0.63).
The distribution of the APOE alleles and genotype by specific retinal microvascular signs stratified by race (whites, African-Americans) is presented in Table 3. Overall, ε3/ε3 was the dominant genotype and ε3 the dominant allele in both whites and African-Americans. There was no significant difference in the distribution of APOE allele or genotype in subjects with retinal microvascular signs compared to those with no retinal microvascular signs in either whites or African-Americans. There was a borderline significant association for arterio-venous nicking in terms of distribution of the APOE genotype (p=0.06).
Table 4 shows results of the associations between APOE polymorphism and retinal microvascular signs in whites only. After adjusting for age, gender, systolic blood pressure, cigarette smoking status, total serum cholesterol and other risk factors, white participants carrying the ε2 and ε4 alleles were more likely to have arterio-venous nicking than the ε3/ε3 homozygotes, with (OR 1.70, 95% CI 1.03-2.83 for the ε2 carriers and OR 1.74, 95% CI 1.06-2.84 for the ε4 carriers). Among white participants, the associations remained significant for the ε4 carriers (OR 2.32, CI 1.18-4.57; data not shown). Multivariate analysis without adjustment for total and HDL cholesterol did not alter these associations (data not shown). In a separate analysis, when the ε4 allele carriers were defined as those who had ε2/ε4, ε3/ε4, and ε4/ε4 genotype, the associations were similar, with slightly weaker associations for arterio-venous nicking (OR 1.61, 95% CI 1.00-2.59; data not shown). There was similarly no significant association of the carriers of the ε4 allele with focal narrowing (OR 0.90; 95%CI 0.58-1.38) or retinopathy (OR 1.05, 95% CI 0.67-1.64; data not shown).
There were no significant associations between APOE polymorphism with retinopathy, focal arteriolar narrowing, or retinal venular diameter. Among normotensive white participants, ε2 allele carriers had significantly narrower retinal arteriolar diameters (adjusted mean arteriolar diameter of 163.5 μm, 95% CI 160.1-167.0, p=0.03) compared to the ε3/ε3 homozygotes (167.8 μm, 95% CI 166.0-169.6; data not shown).
Finally, we found no significant interactions between APOE gene polymorphisms with age, gender, race, hypertension, diabetes status, triglyceride, HDL, or cigarette smoking status (p>0.10 for all cross-product terms; data not shown). Although the association between APOE and arterio-venous nicking was slightly stronger in normotensive white participants than hypertensive white participants, the OR in the two strata (normotensive and hypertensive) were similar. Additionally, formal test of interaction by including cross-product terms in the models showed no evidence of statistically significant interaction of the association of APOE and retinal microvascular signs with hypertension.
Retinal microvascular signs have been suggested to be markers of systemic vascular diseases, and have been shown to predict a variety of cardiovascular disorders . While these retinal microvascular signs are related to traditional vascular risk factors such as hypertension, hyperglycemia, and cigarette smoking, it is still unclear whether retinal microvascular signs are also influenced by genetic factors. Such information may provide insights into the pathogenesis of ocular and systemic vascular diseases.
In the CHS, we report largely weak and inconsistent associations between APOE polymorphism and retinal microvascular signs. Among white participants, carriers of both the ε2 and ε4 alleles were more likely to have arterio-venous nicking, after controlling for other risk factors such as age, elevated blood pressure, cholesterol, and triglyceride levels. These associations strengthened in normotensive whites, with ε2 allele carriers having significantly narrow retinal arterioles than ε3/ε3 genotype carriers. We found no associations of APOE polymorphism with retinopathy, focal arteriolar narrowing, or retinal venular diameter.
There are few studies available for direct comparison with the current study. In the Atherosclerosis Risk in Communities (ARIC) study, a population-based study in middle-aged people, narrower arteriolar diameters was also associated with white, normotensive ε2 allele carriers. However, white ε4 allele carriers were less likely to have arterio-venous nicking (OR 0.8, 95% CI 0.7-1.0) whereas African-American ε2 allele carriers were more likely to have arterio-venous nicking (OR 1.3, 95% CI 1.0-1.8) . The contrasting findings between the two studies may be related to the differences in age between the two populations (average age 78 years in CHS as compared to 60 years in the ARIC study) and in the frequency of different vascular risk factors such as hypertension, hyperglycemia and other risk factors, which have strong effects on the retinal microvasculature. Such differences may mask more subtle differences in genetic susceptibility to retinal microvascular signs related to APOE polymorphism.
The association of APOE polymorphism with arterio-venous nicking is difficult to explain. APOE has been associated with hypertension  and is thought to contribute to arteriolar smooth muscle proliferation and differentiation , as well as vascular endothelial function . These processes have also been linked to the pathogenesis of arterio-venous nicking . Thus, associations between APOE polymorphism and arterio-venous nicking may partially reflect underlying genetic susceptibility to elevated blood pressure, endothelial dysfunction, and other systemic processes. However, given the moderate number of analyses we conducted, it is possible that the association of arterio-venous nicking and the APOE gene may be a chance finding. In the literature, the genetic contribution of the ε2 allele is typically opposite to that of the ε4 allele. However, in our study, the associations of arterio-venous nicking were similar for the ε2 and ε4 allele, which we were unable to explain.
Strengths of this study include a study population drawn from the community, standardized measurement of retinal microvascular signs from photographs, and detailed information on a variety of risk factors. Important limitations of our study should also be mentioned. First, there was insufficient statistical power of performing meaningful multivariate analyses in African-Americans to detect significant associations, if they existed. Second, because retinal photographs were only taken after nine years of follow-up and retinal microvascular signs have been demonstrated to be associated with cardiovascular mortality [44,45], it is possible that selective mortality might have obscured any finding of an association between retinal microvascular changes and APOE polymorphism (e.g., those with certain APOE alleles and retinal microvascular abnormalities may have died prior to retinal photography). However, the age-gender-race-adjusted APOE allele frequencies in the 2,152 participants included in our analysis and the allele frequencies observed in 5,222 participants at baseline were not significantly different (p=0.86; data not shown). Third, the CHS used a 45 ° non-stereoscopic retinal photograph of only one randomly selected eye to determine the presence of retinal microvascular abnormalities. As a result, there may be more grading variability with this method, compared to the grading of stereoscopic retinal photographs of two eyes taken through dilated pupils in the Beaver Dam Eye Study . As discussed in a previous publication , in our elderly study population, the prevalence of age-related cataract and other ocular pathologies was high, and thus, a significant proportion of photographs were ungradeable. Moreover, the reproducibility of the grading of some retinal signs (e.g., focal arteriolar narrowing) was low. Therefore, non-differential misclassification of retinal microvascular abnormalities may have weakened any associations we observed. Another limitation of our study relates to the low prevalence of retinal microvascular signs in the population. Our study has an 80% power to detect an association of the ε2 (or ε4) allele with focal narrowing with a minimum OR of 2.1.
In summary, in the CHS study population, we found few consistent associations of APOE gene polymorphism with retinal microvascular signs. Among white persons, carriers of the ε2 and ε4 alleles were more likely to have arterio-venous nicking than the ε3/ε3 homozygotes, while controlling for age, gender, diabetes and hypertension status, and other risk factors. However, we found no associations of APOE gene polymorphism with retinopathy, focal arteriolar narrowing, or retinal venular diameter. These data provide little evidence that the APOE gene polymorphism plays a significant role in the pathogenesis of retinal microvascular changes. Further research in this area is required to seek possible genetic determinants of retinal microvascular signs in the population.
The research reported in this article was supported by contracts N01-HC-85079 through N01-HC-85086, N01-HC-35129, N01 HC-15103, N01 HC-55222, and U01 HL080295 from the National Heart, Lung, and Blood Institute, with additional contribution from the National Institute of Neurological Disorders and Stroke. A full list of participating CHS investigators and institutions can be found at CHS. Additional support was provided by grant R21-HL077166 from NHLBI, NIH and the Sylvia and Charles Viertel Clinical Investigator Award (T.Y.W.).
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40. Liew G, Shankar A, Wang JJ, Klein R, Bray MS, Couper DJ, Sharrett AR, Wong TY. Apolipoprotein E gene polymorphisms and retinal vascular signs: the atherosclerosis risk in communities (ARIC) study. Arch Ophthalmol 2007; 125:813-8.
41. Bhavani AB, Sastry KB, Reddy NK, Padma T. Lipid profile and apolipoprotein E polymorphism in essential hypertension. Indian Heart J 2005; 57:151-7.
42. Hui DY. Apolipoprotein E-induced cell signaling in the vessel wall. Rev Endocr Metab Disord 2004; 5:335-41.
43. Klein R, Sharrett AR, Klein BE, Chambless LE, Cooper LS, Hubbard LD, Evans G. Are retinal arteriolar abnormalities related to atherosclerosis?: The Atherosclerosis Risk in Communities Study. Arterioscler Thromb Vasc Biol 2000; 20:1644-50.
44. Wong TY, Klein R, Klein BE, Tielsch JM, Hubbard L, Nieto FJ. Retinal microvascular abnormalities and their relationship with hypertension, cardiovascular disease, and mortality. Surv Ophthalmol 2001; 46:59-80.
45. Wong TY, Klein R, Nieto FJ, Klein BE, Sharrett AR, Meuer SM, Hubbard LD, Tielsch JM. Retinal microvascular abnormalities and 10-year cardiovascular mortality: a population-based case-control study. Ophthalmology 2003; 110:933-40.
46. Klein R, Klein BE, Moss SE, Wang Q. Hypertension and retinopathy, arteriolar narrowing, and arteriovenous nicking in a population. Arch Ophthalmol 1994; 112:92-8.