Association Between C-Reactive Protein and Age-Related Macular Degeneration FREE

Age-related macular degeneration (AMD) is a burden to the elderly population, and its consequences are increasing because treatment options are limited. Prevention remains the best approach for decreasing the impact of this leading cause of blindness. Knowledge about modifiable factors related to AMD has increased considerably during the past decade, including most notably cigarette smoking,^1- 3 nutritional factors,^4- 7 obesity,^8- 9 and lipid levels.¹⁰

Many factors associated with AMD are also related to cardiovascular disease (CVD). We have hypothesized that cardiovascular disorders and AMD share common antecedents and proposed that novel biomarkers associated with CVD be evaluated for their potential relationship with AMD.¹¹ One of these factors is C-reactive protein (CRP), a marker of systemic inflammation, which has been shown to be an independent indicator of risk for cardiovascular and peripheral arterial disease.^12- 13 Given the similarity of the risk profile for the 2 diseases, we designed a study to explore the relationships between AMD and CVD biomarkers, including CRP.

The investigation of inflammatory biomarkers in AMD is rendered even more biologically plausible by the observation that inflammation is associated with angiogenesis and that neovascularization can occur in inflammatory eye diseases,¹⁴ similar to the most advanced and debilitating neovascular form of AMD. Furthermore, in addition to CVD, stroke and Alzheimer disease may also have an inflammatory component.^13,15 It is possible that AMD represents another chronic, age-related inflammatory disease that is manifested in the eye and other organs, including the heart and brain. Therefore, we examined the relationship between CRP levels and AMD in the multicenter Age-Related Eye Disease Study (AREDS).

This study is ancillary to the AREDS. Details of the AREDS design have been described elsewhere.¹⁶ AREDS is a prospective cohort study designed to assess the incidence, clinical course, prognosis, and risk factors for AMD and cataract. AREDS also includes a double-masked randomized clinical trial to assess the effects of high-dose antioxidants (vitamins C and E and beta carotene) on AMD and cataract and the effect of high-dose zinc on AMD.

Eleven AREDS clinical centers nationwide enrolled 4757 participants between February 1993 and January 1998. Participants include 1117 controls, 1063 with mild AMD, 1621 with intermediate AMD, and 956 with advanced AMD. All participants were randomized into 1 of 4 treatment groups. Persons with AMD were randomized to receive high-dose antioxidants, zinc, antioxidants and zinc, or a placebo. Persons without AMD were randomized to receive either high-dose antioxidant vitamins or a placebo.

Participants were aged 55 to 80 years at enrollment and were required to be in overall good health. Potential participants were excluded if they had diseases with a poor 7-year survival prognosis (eg, end-stage cancer, advanced heart disease), hemochromatosis or Wilson disease, or oxalate kidney stone, alcoholism, or drug abuse or were unwilling or unable to discontinue the use of nonstudy antioxidant vitamin or zinc supplementation. Persons were also excluded if they had visual acuity less than 20/32 in both eyes, advanced AMD or laser photocoagulation for AMD in both eyes, bilateral cataract extraction without signs of AMD, or other eye diseases that potentially compromised evaluation of study outcomes or if they used medications known to be toxic to the lens or retina.

Participants were followed up at 6-month intervals, when information was collected on changes in visual acuity, disease incidence and progression, and risk factors from a visual acuity test, dilated lens and fundus examination, and a clinical interview. In addition, at the annual visit (which occurred at 12-month intervals beginning 12 months after randomization), blood samples were drawn for specified AREDS tests, fundus and lens photographs were taken (except at the first annual visit), and a refraction was completed. Since the end of the clinical trial in April 2001, participants have been examined annually.

The AREDS Executive Committee, the AREDS Data and Safety Monitoring Committee, and the National Eye Institute approved this ancillary study in 1995. The 2 sites with the most participants were chosen to participate. The Massachusetts Eye and Ear Infirmary (Boston, Mass) and the Devers Eye Institute (Portland, Ore) enrolled 1026 participants (517 and 509, respectively) into the AREDS clinical trial. Between January 1996 and April 1997, 930 of the 1026 participants (91%) had blood specimens drawn after randomization for this ancillary study, 465 at each clinic. All but 1.3% of the specimens were from participants who had been fasting for at least 8 hours. Blood samples were processed immediately and then frozen in liquid nitrogen freezers at −140°C. The study was approved by the human subjects committees of the 2 clinical centers, and all participants signed an informed consent statement.

Case-control definitions are adopted from a previous AREDS publication.⁹ According to reading center grading of fundus photographs at the visit most closely associated with the specimen draw, participants in this ancillary study were divided into 4 maculopathy groups by size and extent of drusen in each eye, presence of geographic atrophy, and neovascular disease. These groups, numbered serially and defined by increasing severity of drusen or type of AMD, were as follows.

Group 1 (No Drusen). In this group (n = 183), each eye had no drusen or nonextensive small drusen, no pigment abnormalities, no advanced AMD, and no disqualifying ocular conditions. Most participants had visual acuity of 20/32 or better in both eyes.

Group 2 (Intermediate Drusen). In this group (n = 200), at least 1 eye had 1 or more intermediate-sized drusen, extensive small drusen, or pigment abnormalities associated with AMD. Neither eye had large drusen, advanced AMD, or a disqualifying ocular condition. Most participants had visual acuity of 20/32 or better in both eyes.

Group 3 (Large Drusen or Intermediate AMD). In this group (n = 325), at least 1 eye had either 1 or more large drusen, approximately 20 intermediate-sized soft drusen, or approximately 65 intermediate-sized hard drusen. Neither eye had advanced AMD, a disqualifying ocular condition, or presence of geographic atrophy with diameter at least one eighth that of the average disc, and most participants had visual acuity of 20/32 or better in both eyes. Also included were persons in whom one eye met these criteria and the other eye had either a disqualifying ocular condition or visual acuity of 20/32 or less not caused by AMD.

Group 4 (Geographic Atrophy or Neovascular AMD–Advanced AMD). In this group (n = 222), at least 1 eye had geographic atrophy definitely present (with diameter at least one eighth that of the average disc; n = 58) or neovascular AMD (further defined below; n = 164). In most cases, the other eye had visual acuity of 20/32 or better, with no evidence of advanced AMD or a disqualifying ocular condition.

Neovascular AMD included choroidal neovascularization or retinal pigment epithelial (RPE) detachment in 1 eye (nondrusenoid RPE detachment, serous sensory, or hemorrhagic retinal detachment), subretinal hemorrhage, subretinal pigment epithelial hemorrhage, subretinal fibrosis, or evidence of confluent photocoagulation for neovascular AMD. The term neovascular is used as a summary term for this group of participants because most persons in this group have direct evidence of choroidal neovascularization, according to the assessment of fundus photographs. A few participants in this group had serous RPE detachments.

The AREDS clinical trial⁷ showed that rates of progression to advanced AMD in groups 1 and 2 were low (5-year rates of 0.5% and 1.3%, respectively), and they were therefore combined here into one larger control group. For regression analyses, to enhance statistical power, group 3 (5-year rate of progression of approximately 18%) was combined with group 4 (5-year rate of progression of approximately 43%) to form the case group.

Serum samples were thawed and assayed for CRP, which was measured with a high-sensitivity assay as in previous studies of CVD.^12- 13 The concentration of CRP was determined by using an immunoturbidimetric assay on the Hitachi 911 analyzer (Roche Diagnostics, Indianapolis, Ind), with reagents and calibrators from Denka Seiken (Niigata, Japan). In this assay, an antigen-antibody reaction occurs between CRP in the sample and an anti-CRP antibody that has been sensitized to latex particles and agglutination results. This antigen-antibody complex causes an increase in light scattering, which is detected spectrophotometrically, with the magnitude of the change proportional to the concentration of CRP in the sample. The coefficients of variation of the assay at concentrations of 0.91, 3.07, and 13.38 mg/L are 2.81%, 1.61%, and 1.1%, respectively.

General risk-factor and dietary interviews were conducted at baseline, and slit-lamp biomicroscopy and ophthalmoscopy were performed at the blood drawing. The baseline risk factor variables that were considered in the analyses can be divided into 5 classes: demographic, medical, dietary or supplementation, use of medication, and ocular factors. For analysis, continuous variables (body mass index, weight change from the age of 20 years, and sunlight exposure) were categorized by quartiles or tertiles, according to the group without drusen (group 1).

Demographic. The demographic variables included age, sex, race, education, and sunlight exposure (adult lifetime average annual ocular UV-B exposure, adapted from McCarty et al).¹⁷

Medical. Medical variables included history of smoking, body mass index, weight change (increase or decrease) since the age of 20 years, hypertension (systolic blood pressure >160 mm Hg, diastolic blood pressure >90 mm Hg, or current use of antihypertensive medication), history of CVD (at least 1 of the following: newly developed heart disease after enrollment but before blood draw, occurrence of a stroke or myocardial infarction after enrollment but before blood draw, history of angina and taking an angina medication [dipyridamole, propranolol, β-blocker, calcium channel blocker, nitroglycerin, or isobide dinitrate], taking a CVD medication [furosemide, angiotensin-converting enzyme inhibitor, digoxin, blood-thinning medication, cholesterol-lowering medication]), diabetes (under treatment for diabetes), and arthritis.

Dietary or Supplementation. The dietary or supplement variables included an antioxidant index and use of study treatment containing antioxidants. The antioxidant index was based on dietary results from a modified Block Food Frequency questionnaire (AREDS Manual of Operations, 1992) completed at the participant's baseline visit. Three measures were assessed: carotenoid intake (alpha carotene, beta carotene, lutein, lycopene, and beta-cryptoxanthin), vitamin C intake, and vitamin E intake. Participants were grouped as having high antioxidant intake (above the highest quartile of intake for 2 of the 3 measurements), low antioxidant intake (below the lowest quartile of intake for 2 of the 3 measurements), or mixed antioxidant intake.

Participants randomized to receive the study supplements containing high-dose antioxidants or high-dose antioxidants and zinc comprised the antioxidant treatment group. Participants randomized to receive the study supplements containing zinc or placebo comprised those not in the antioxidant treatment group.

Use of Medication. Use of medication was defined as current use with 5 or more lifetime years of regular use. These medications included hydrochlorothiazide, diuretics (other than hydrochlorothiazide), aspirin, antacids, nonsteroidal anti-inflammatory drugs, thyroid hormones, β-blockers, and, for women, estrogen and progesterone.

Ocular. Ocular variables included iris color and refractive error. Iris color was graded at the reading center by comparing photographs of each eye with standards on a scale from 1 (light or blue) to 4 (dark or brown); a person's eyes were considered light if both eyes were code 1, dark if both eyes were code 4, and mixed if at least 1 eye was code 2 or code 3 or eyes were not of the same code. A person was considered myopic if both eyes were myopic by −1.0 diopters (D) spherical equivalent refractive error or more, hyperopic if both eyes had +1.0 D spherical equivalent refractive error or more, or other, which includes emmetropes and mixed cases.

The median values and interquartile ranges of CRP were calculated for each maculopathy group, and the most advanced AMD grade was compared with group 1 by using a nonparametric test of all P values. Conditional logistic regression analysis (SAS procedure LOGISTIC; version 8.02, SAS Institute Inc, Cary, NC) was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) after the population was divided into quartile groups according to the quartile cut points of CRP values for the disease-free group 1, as done in previous studies of CRP and CVD.^12- 13 Prevalence ORs, which describe the association between disease and CRP (comparing cases in groups 3 and 4 with controls in groups 1 and 2), were computed for each CRP quartile group relative to the lowest quartile group. A test for linear trend was calculated according to the median levels of CRP within each quartile group.

Multivariate ORs were estimated from conditional logistic regression models and were adjusted for age (57-65, 66-70, and 71-83 years), sex, race (white vs other), smoking (ever smoked vs never smoked), education (never completed high school, high school graduate, some college, or college graduate), body mass index (measured as weight in kilograms divided by the height in meters squared; <23.9, 23.9-29.9, >29.9), antioxidant index (low, mixed, high), diabetes, history of CVD, hypertension, and antioxidant treatment (taking study supplement containing antioxidants vs taking study supplement containing no antioxidants). History of CVD and hypertension were not correlated. Other risk factors described previously were evaluated as potential covariates but did not reach statistical significance in this ancillary study population (including weight change from the age of 20 years, sunlight exposure, arthritis, anti-inflammatory drugs, thyroid hormones, β-blocker use, hormone use [women], other medications, iris color, and refractive error).

To evaluate a possible threshold effect, additional analyses were performed comparing cases with controls according to levels of CRP for group 1 above and below the 90th percentile and above and below the mean CRP plus 2 SDs. Finally, to evaluate effect modification by cigarette smoking, additional logistic regression analyses were conducted to determine the ORs for AMD in 6 subgroups defined by never and ever smoking and low, intermediate, and high tertiles of CRP.

Of the 930 participants in this ancillary study, 61% were women and 39% were men, and the mean age was 69 years. Most participants (71%) had some college or higher education. Forty-one percent of participants had never smoked, 51% were former smokers, and 8% were current smokers. The median CRP value for all participants was 2.7 mg/L, with an overall range of 0.2 to 117 mg/L, and the 90th percentile range was 0.2 to 10.6 mg/L. The CRP values did not differ according to age groups (55-65, 66-70, and ≥71 years).

Table 1 displays the relationships between baseline characteristics and maculopathy groups, unadjusted for other variables. Significant differences (P<.05) between individuals in maculopathy groups 3 and 4 and individuals in groups 1 and 2 included sex (lower proportion of women), smoking status (lower proportion of never smokers), and education (lower proportion with a college degree).

Median baseline serum levels of CRP were higher among participants who had more severe maculopathy (Table 2). The difference between the median value for the most advanced maculopathy group 4 (3.4 mg/L) and the median for maculopathy group 1 (2.7 mg/L) was statistically significant (P = .02).

Table 3 displays the ORs for risk of AMD according to the quartile of CRP, for maculopathy case groups 3 and 4 compared with groups 1 and 2, after adjustment for various other known and potential factors associated with AMD. In an age- and sex-adjusted model, persons above the highest quartile of CRP had higher risk of AMD (OR, 1.53; 95% CI, 1.03-2.28). The trend for an increase in risk of maculopathy with increase in CRP was statistically significant (P = .02). After adjustment for additional covariates, the trend for an increase in risk remained significant for the highest quartile of CRP (OR, 1.65; 95% CI, 1.07-2.55; P for trend = .02). In a separate analysis (data not shown) that compared group 4 maculopathy with that of group 1, the effect estimate was similar for the highest level of CRP (OR, 1.72; 95% CI, 0.88-3.38) but was not significant, probably because of the reduced sample size (P for trend = .09).

Table 4 displays the association between CRP and maculopathy, with different cut points for values of CRP. Persons with CRP levels above the 90th percentile had a significantly increased risk, with an OR of 1.75 (95% CI, 1.12-2.75) for the age- and sex-adjusted model and an OR of 1.92 (95% CI, 1.20-3.06) for the full multivariate model. Persons with CRP values more than 2 SDs above mean levels for the study cohort were also at increased risk, with an OR of 1.89 (95% CI, 0.98-3.66) for the age- and sex-adjusted model and an OR of 2.03 (95% CI, 1.03-4.00) for the full multivariate model.

To explore whether the effect of CRP was modified by cigarette smoking, a consistently strong risk factor for AMD, we computed ORs for AMD in analyses in which participants were stratified into 6 groups according to smoking (never, ever) and tertile of CRP, as shown in Table 5. For smokers and never smokers, higher levels of CRP were associated with higher risk of AMD. Persons in the high-risk group (current and past smokers with the highest level of CRP) had a statistically significant, 2.16-fold higher risk (95% CI, 1.33-3.49) of maculopathy compared with the low-risk group (those who never smoked and had the lowest CRP level), after adjustment for other factors. Among never smokers, the odds of developing AMD were 2.03 in the highest tertile of CRP (95% CI, 1.19-3.46) compared with individuals in the lowest quartile of CRP, after adjustment for other factors. To evaluate this relationship further, we analyzed the effect of smoking (ever, never) stratified by tertile of CRP (data not shown). Cigarette smoking increased risk of AMD more than 1.7-fold in the lower 2 tertiles of CRP—ORs, 1.79 (95% CI, 1.06-3.00) and 1.90 (95% CI, 1.12-3.22)—but there was no association between smoking and AMD in the highest level of CRP (OR, 1.01; 95% CI, 0.61-1.69). The highest levels of CRP appear to increase risk of AMD independent of smoking.

In this study, CRP levels were associated with AMD. After adjustment for age, sex, and other variables, including smoking and body mass index, CRP levels were significantly higher among individuals with intermediate and advanced stages of AMD compared with controls. The magnitude of the association ranged from an OR of 1.65 to 2.16 for the highest levels of CRP. Risk of AMD was lowest among individuals who had low CRP values and never smoked. In contrast, risk tended to be highest among smokers who also had higher levels of CRP. Even among individuals who never smoked, the risk of AMD was increased 2-fold among those with the highest category of CRP compared with the lowest level of CRP as the referent category.

To our knowledge, this is the first report of an association between CRP values and AMD in a large cohort of cases and controls. We found only one other study¹⁸ of early age-related maculopathy that showed no association with CRP. However, in that study, cigarette smoking was also not associated with maculopathy among the 29 variables assessed, and the authors suggested that this might be due to a "substantial rate of non-participation."

Our findings have important implications and lend support to an evolving hypothesis that inflammation is associated with the pathogenesis of AMD.^11,19- 20 Several mechanisms are potentially involved that could lead to inflammatory responses, including oxidative stress caused by known risk factors for AMD, such as smoking,^1- 2 insufficient antioxidants in the diet,^3,5 dietary fat,^4- 5 and obesity.⁸ Smoking is one of the most consistent risk factors for AMD, yet many individuals who have never smoked develop AMD. In fact, we found that higher CRP values increased risk of AMD among smokers and individuals who never smoked, independent of the other risk factors in the model. Therefore, factors other than smoking in these individuals might create an adverse milieu or damage the RPE-retina-choroidal complex in some way, which in turn could lead to an inflammatory stimulus and increase CRP values. Elevated CRP levels have been found in other chronic diseases associated with aging, including coronary heart disease, stroke, and Alzheimer disease,^12- 13,15 so AMD may have similar pathogenetic processes. CRP is a measure of systemic inflammation but may also be associated with local (intraocular) inflammatory factors or immune function. These results provide insight into potentially important mechanisms and will stimulate additional epidemiologic and basic science research to sort out primary (causal) and secondary events.

Unique features of this study include the evaluation of a systemic biomarker for inflammation in a large and well-characterized population of patients with and without maculopathy from 2 geographic areas in the United States. Further strengths of this study include the standardized collection of risk factor information, including direct measurements of blood pressure and body mass index, and classification of maculopathy by means of standardized ophthalmologic examinations and fundus photography. Misclassification was unlikely because CRP values were quantified by using objective laboratory methods without knowledge of the participants' maculopathy status, and AMD grade was assigned without knowledge of CRP status.

Residual confounding is a concern in many epidemiologic studies. We controlled for known AMD risk factors and those associated with AMD in this study cohort. For example, obesity and cigarette smoking are related to AMD and are also related to increased levels of CRP and other systemic inflammatory markers.²¹ CRP was significantly and independently related to AMD in this study after adjustment for these confounding factors. Although some unmeasured and therefore uncontrolled factors might still be confounding this relationship, they would have to be highly associated with CRP and be a strong risk factor for AMD to explain these results.

Our study population consists of patients with a range of maculopathy and some individuals without AMD who participated in a randomized trial of nutritional supplements. Results were not altered after adjustment for assignment to antioxidants within the randomized trial. Controls were more likely to be women, to be nonsmokers, and to have more education, so selection bias should be considered. However, these analyses adjusted statistically for these differences, and previous case-control analyses of the entire AREDS cohort, as well as this subset at 2 centers, demonstrate an association with known risk factors for AMD similar to that of other study populations. Although this is a selected population, the cases likely represent the typical patient with AMD, and the overall population is comparable to others in this age range in terms of smoking status and prevalence of obesity. The large sample size and well-characterized study population provided a unique opportunity to evaluate our hypotheses. Moreover, the biological effects of CRP are not likely to differ in major ways among various populations of patients with AMD.

Measures of CRP were taken from single fasting blood specimens that were stored in a repository at −140°C until analyzed. These are standard methods that are in use in several large-scale epidemiologic studies throughout the country. In fact, these are the same methods used in the studies^12- 13 that established CRP as a marker for CVDs. The medians and ranges of CRP in the various quartiles in our study are similar to those of other published studies^12- 13 of CRP and CVDs. Because a single measurement was taken, we cannot evaluate the effects of changes in the levels of this biomarker over time. Follow-up studies are needed.

In summary, to the best of our knowledge, this is the first study to implicate CRP as a systemic inflammatory marker for the development of AMD. Higher CRP values were found to be significantly related to AMD independent of established risk factors, including smoking and obesity. Among smokers and nonsmokers, higher baseline CRP levels were associated with an increased risk of AMD. These results may shed light on the mechanisms and pathogenesis of AMD development and prognosis. Moreover, CRP levels may add clinically relevant predictive information about risk of AMD in addition to known risk factors. Anti-inflammatory agents might have a role in preventing AMD, and inflammatory biomarkers such as CRP may provide a method of identifying individuals for whom these agents and other therapies would be more or less effective. These results and hypotheses should be evaluated further with prospective studies and possibly randomized trials.