Figure 1. Smoking adjusted odds ratios (ORs) (vertical midline of each box) and 95% confidence intervals (horizontal bars) for the association between high-sensitivity C-reactive protein levels more than 3 mg/L (vs <1 mg/L) and risk of age-related macular degeneration in nested case-control samples from 5 prospective cohorts. The size of the squares is proportional to the inverse of the variance of the ORs (and reflects sample size). The diamond represents the summary OR estimate (center of diamond) and 95% confidence interval for the pooled estimate (horizontal points of diamond). HPFS indicates Health Professionals Follow-up Study; NHS, Nurses' Health Study; PHS, Physicians' Health Study; WAFACS, Women's Antioxidant and Folic Acid Cardiovascular Study; and WHS, Women's Health Study.
Figure 2. Smoking adjusted odds ratios (ORs) (vertical midline of each box) and 95% confidence intervals (horizontal bars) for the association between high-sensitivity C-reactive protein levels more than 3 mg/L (vs <1 mg/L) and risk of neovascular age-related macular degeneration in nested case-control samples from 4 prospective cohorts. The size of the squares is proportional to the inverse of the variance of the ORs (and reflects sample size). The diamond represents the summary OR estimate (center of diamond) and 95% confidence interval for the pooled estimate (horizontal points of diamond). HPFS indicates Health Professionals Follow-up Study; NHS, Nurses' Health Study; PHS, Physicians' Health Study; and WHS, Women's Health Study.
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Mitta VP, Christen WG, Glynn RJ, et al. C-Reactive Protein and the Incidence of Macular Degeneration: Pooled Analysis of 5 Cohorts. JAMA Ophthalmol. 2013;131(4):507–513. doi:10.1001/jamaophthalmol.2013.2303
SECTION EDITOR: LESLIE HYMAN, PhD
Author Affiliations: Department of Epidemiology, Harvard School of Public Health (Drs Mitta, Rimm, Hankinson, and Schaumberg), Division of Preventive Medicine (Drs Christen, Glynn, Ridker, and Schaumberg) and the Channing Laboratory (Dr Hankinson), Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, and the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School (Dr Schaumberg), Boston, Massachusetts; The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland (Dr Semba); and Department of Ophthalmology and Visual Sciences, University of Utah, Salt Lake City (Dr Schaumberg).
Importance This study adds to the evidence that elevated levels of high-sensitivity C-reactive protein (hsCRP) predict future risk of age-related macular degeneration (AMD). This information might shed light on underlying pathological mechanisms involving inflammation and could be of clinical utility in the identification of persons at high risk of AMD who may benefit from increased adherence to lifestyle recommendations, eye examination schedules, and therapeutic protocols.
Objective To investigate the relationship between hsCRP and future risk of AMD in US men and women.
Design Pooled analysis of prospective nested case-control data from the Women's Health Study and 4 other cohorts, the Physicians' Health Study, Women's Antioxidant and Folic Acid Cardiovascular Study, Nurses' Health Study, and Health Professionals Follow-up Study.
Setting A prospective nested case-control study within 5 large cohorts.
Participants Patients were initially free of AMD. We prospectively identified 647 incident cases of AMD and selected age- and sex-matched controls for each AMD case (2 controls for each case with dry AMD or 3 controls for each case of neovascular AMD).
Main Outcome Measures We measured hsCRP in baseline blood samples. We used conditional logistic regression models to examine the relationship between hsCRP and AMD and pooled findings using meta-analytic techniques.
Results After adjusting for cigarette smoking status, participants with high (>3 mg/L) compared with low (<1 mg/L) hsCRP levels had cohort-specific odds ratios (ORs) for incident AMD ranging from 0.94 (95% CI, 0.58-1.51) in the Physicians' Health Study to 2.59 (95% CI, 0.58-11.67) in the Women's Antioxidant and Folic Acid Cardiovascular Study. After testing for heterogeneity between studies (Q = 5.61; P = .23), we pooled findings across cohorts and observed a significantly increased risk of incident AMD for high vs low hsCRP levels (OR, 1.49; 95% CI, 1.06-2.08). Risk of neovascular AMD was also increased among those with high hsCRP levels (OR, 1.84; 95% CI, 1.14-2.98).
Conclusions and Relevance Overall, these pooled findings from 5 prospective cohorts add further evidence that elevated levels of hsCRP predict greater future risk of AMD. This information might shed light on underlying mechanisms and could be of clinical utility in the identification of persons at high risk of AMD who may benefit from increased adherence to lifestyle recommendations, eye examination schedules, and therapeutic protocols.
Inflammation plays a significant role in the incidence and progression of age-related macular degeneration (AMD),1-3 the leading cause of blindness among older adults in the United States.4 Drusen, subretinal deposits indicative of the onset of AMD, have been shown to contain fibrinogen, vitronectin, complement components, and C-reactive protein (CRP), proteins associated with generalized inflammation.5-7 Inflammatory cell debris has also been isolated from the outer surface of the Bruch membrane in eyes with AMD.8 The inflammatory hypothesis has been further strengthened by the discovery of a strong association between AMD and a common gene variant for complement factor H,9-12 as well as variants in other complement pathway genes.13-15
C-reactive protein activates the classic route of complement activation directly via cytokines through Fc receptor binding by antibodies, which enhances the inflammatory response.6 Circulating high-sensitivity CRP (hsCRP) levels have been widely studied as a nonspecific marker of systemic inflammation, and a single measure has been shown to reliably indicate the degree of underlying systemic inflammation in asymptomatic adults. Moreover, hsCRP blood levels have gained recognition through epidemiological studies as a useful clinical indicator of future cardiovascular risk.16,17 Given the evidence linking inflammation and AMD, it has been of natural interest to determine whether hsCRP levels also are predictive of AMD. Prior studies of hsCRP and AMD18-25 provide preliminary evidence of a relationship between hsCRP and AMD, but findings are mixed and there have only been 3 prospective studies. One of these prospective studies was of more than 27 000 women in the Women's Health Study (WHS) cohort.22 In this article, we have extended these findings by conducting a pooled analysis of prospective nested case-control data from the WHS and 4 other cohorts.
The study population consisted of prospective nested case-control samples of participants in 5 population-based cohort studies (WHS, Physicians' Health Study [PHS], Women's Antioxidant and Folic Acid Cardiovascular Study [WAFACS], Nurses' Health Study [NHS], and Health Professionals Follow-up Study [HPFS]). The recruitment, enrollment, and characteristics of the study populations have been published elsewhere.26-30 Briefly, the WHS, PHS, and WAFACS were all randomized, placebo-controlled trials initially designed to investigate the effect of aspirin or antioxidants on cardiovascular or cancer outcomes. The PHS consists of male physicians, while the WHS and WAFACS cohorts comprised female health professionals (primarily nurses). The HPFS and NHS were designed as observational cohort studies, consisting of male dentists, pharmacists, and other health professionals in the HPFS and solely female nurses in the NHS. Participants in the PHS and WHS were apparently healthy men and women, respectively, who were free of prior diagnoses of cancer or cardiovascular disease. Women in the WAFACS cohort were at high risk of cardiovascular disease, with a history of myocardial infarction or at least 3 major risk factors for cardiovascular disease. Participants in the NHS and HPFS cohorts were not restricted from participating based on initial health status. Together, these cohorts include a total of 33 142 men and 67 093 women with stored baseline blood samples. A description of the cohorts is provided in Table 1.
At baseline, participants provided a baseline blood sample and completed a mailed questionnaire on which they reported demographic information as well as a medical history and personal information on a number of lifestyle factors, including height, weight, and cigarette smoking history. Yearly follow-up questionnaires (every 2 years in the NHS and HPFS) provided reliable information on newly developed diseases and updated information on lifestyle factors. Subjects with prevalent AMD at baseline; subjects who did not provide a baseline blood specimen; or subjects for whom a hsCRP measurement was not available (because of various logistic reasons) were excluded from this analysis. The research protocol was approved by the institutional review boards at Brigham & Women's Hospital and the Harvard School of Public Health.
Procedures for our 2-stage documentation of incident AMD are nearly identical in each cohort and have been previously described and validated.22,31 On each study questionnaire, participants were asked to report any new diagnosis of AMD, including the month and year of diagnosis as well as the name and address of the diagnosing eye care professional, and for signed permission to review medical records. For each report of AMD, we sent a letter to the participant's ophthalmologist or optometrist to obtain information from the medical record on the date of diagnosis, best-corrected visual acuity at the time of diagnosis, date when visual acuity first reached 20/30 or worse in the affected eye, and the chorioretinal lesions that were present (drusen; retinal pigment epithelial changes including atrophy, hypertrophy, and retinal pigment epithelium detachment; geographic atrophy; subretinal neovascular membrane; or disciform scar). We confirmed a diagnosis of AMD for purposes of this study if 1 or more typical lesions were documented and associated with a visual acuity loss of 20/30 or worse. In those cases in which other ocular anomalies were also present, we asked the eye care professional to judge whether the visual acuity would be expected to be 20/30 or worse as a result of AMD alone. We defined neovascular AMD as the documented presence of a retinal pigment epithelium detachment, subretinal neovascular membrane, or disciform scar that was not due to other causes (eg, histoplasmosis or choroidal rupture). Dry AMD included cases with the documented presence of drusen and/or retinal pigment epithelial changes but with no signs of neovascular AMD. We classified participants based on the most severely affected eye.
Baseline blood specimens were collected and stored in liquid nitrogen freezers until the time of analysis, when samples were thawed and the levels of the inflammatory markers were measured in a core laboratory. Levels of hsCRP were analyzed using a validated immunoturbidimetric assay on the Hitachi 911 analyzer (Roche Diagnostics) by using reagents and calibrators from Denka Seiken.32 A single technician, masked to case-control status, performed all assays. Levels were similar to expected values for hsCRP in a population of healthy middle-aged men and women.33
We used prospective nested case-control designs within each of the 5 cohort studies in which cases of incident AMD were confirmed (as defined earlier). Controls were randomly selected from among those participants in the cohort who had not been diagnosed with AMD and who had also provided a baseline blood specimen. Controls were matched to cases by age (±1 year), with up to 3 controls per case. We identified a total of 647 case-control sets who met our eligibility criteria.
We used conditional logistic regression to examine the relation of hsCRP levels with subsequent development of AMD. The hsCRP levels were categorized using cutoff points of less than 1 mg/L, 1 to 3 mg/L, and greater than 3 mg/L, defined a priori based on the joint recommendation of the American Heart Association and the Centers for Disease Control and Prevention for clinical assessment of cardiovascular risk34 and for consistency with other recent studies of hsCRP and AMD, as well as for comparison with the literature on associations of hsCRP with cardiovascular disease.22 In initial analyses, we obtained cohort-specific, smoking-adjusted odds ratios (ORs) and 95% confidence intervals of AMD for moderate (1-3 mg/L) and high levels (>3 mg/L) of CRP compared with low levels (<1 mg/L) as a referent. All models included terms for the respective randomized treatment assignments depending on cohort (aspirin, β-carotene, vitamin E, folic acid/vitamin B6/vitamin B12, or vitamin C). We tested for linear trend across categories of the markers by entering a single ordinal score variable (0, 1, or 2) in the regression model. We then extended these models to adjust for other potential confounders including body mass index, use of antihypertensive and cholesterol-lowering drugs, and dietary intake of ω-3 fatty acids, lutein/zeaxanthin, and zinc. Models were based on case-control sets for whom complete data were available on all covariates of interest. A 2-tailed P value of .05 was considered a statistically significant result. Pooled ORs were calculated with the random-effects estimator and heterogeneity assessed with the Cochran Q test using Stata 10.1 (StataCorp). All other analyses were carried out using SAS version 9.2 (SAS Institute Inc).
Compared with control subjects, cases had significantly higher levels of hsCRP at baseline in each cohort except for the PHS, as well as higher body mass index and a larger proportion of current cigarette smokers. The baseline characteristics of cases and controls from each cohort are shown in Table 2.
After adjusting for current smoking status, participants with high hsCRP levels had cohort-specific ORs of incident AMD ranging from 0.94 (95% CI, 0.58-1.51) in the PHS to 2.59 (95% CI, 0.58-11.67) in WAFACS, whereas participants with moderate hsCRP levels had cohort-specific ORs of developing AMD ranging from 0.95 (95% CI, 0.67-1.35) in the PHS to 1.54 (95% CI, 0.80-2.94) in the HPFS, compared with participants with low hsCRP levels (Table 3). Further adjustment for other risk factors, including body mass index and dietary intake of ω-3 fatty acids, lutein/zeaxanthin, and zinc, resulted in similar cohort-specific ORs for hsCRP and AMD (Table 3). No significant interactions (P values >.15) were noted between hsCRP and randomized treatment assignment (aspirin, β-carotene, vitamin E, vitamin C, or folic acid/vitamin B6/vitamin B12) in the case-control populations derived from randomized trials (PHS, WHS, and WAFACS). Adjustment for pack-years rather than current cigarette smoking did not impact the overall study findings (data not shown).
A formal test for heterogeneity between studies showed no statistically significant heterogeneity among the cohorts (Q = 5.61; P = .23). We therefore pooled results from all 5 cohort studies to obtain an overall estimate of the association between hsCRP and incident AMD (total n = 647 incident cases of AMD and n = 1480 controls). The combined ORs for AMD for increasing tertiles of hsCRP were 1.17 (95% CI, 0.92-1.47) and 1.48 (95% CI, 1.06-2.08) compared with participants with low hsCRP levels (Figure 1).
Although the test for heterogeneity among cohorts was not significant, the observed findings from the PHS appeared qualitatively disparate compared with those of the other cohorts. We therefore probed the PHS data in an attempt to identify any factors that might help explain this observation. In particular, based on differences between the PHS and the other cohorts, we estimated the association of hsCRP with AMD in the PHS across: (1) quartiles of age at baseline, (2) randomized assignment to aspirin vs placebo, and (3) quartiles of the date of diagnosis of AMD during follow-up. These analyses failed to identify any important differences in the association between hsCRP and AMD by these variables. We also tested for heterogeneity by sex, calculating sex-specific ORs for AMD by pooling results from the 3 women-only cohorts and separately for the 2 men-only cohorts. The ORs comparing individuals with high vs low levels of baseline hsCRP levels were 1.64 (95% CI, 1.15-2.34) among women and 1.39 (95% CI, 0.59-3.29) in men (Table 4); however, there was no evidence of heterogeneity in these findings (Q = 0.12; P = .73).
Finally, we performed a separate analysis of the subset of cases with the neovascular form of AMD. There were an insufficient number of cases of neovascular AMD (n = 7) in the WAFACS cohort to allow model convergence, so this cohort was not included in this analysis. In the 4 cohorts with sufficient numbers of cases, ORs comparing individuals with high vs low baseline hsCRP levels ranged from 1.64 (95% CI, 0.75-3.58) in the NHS cohort to 2.32 (95% CI, 0.76-7.10) in the HPFS (Table 5), with no evidence of heterogeneity (Q = 0.284; P = .96). In pooled results from the 4 cohorts with sufficient numbers of cases of incident neovascular AMD (n = 183 cases, n = 546 controls), the combined ORs for neovascular AMD were 1.84 (95% CI, 1.14-2.98) for participants with high hsCRP levels and 1.04 (95% CI, 0.67-1.64) for participants with moderate hsCRP levels, compared with participants with low hsCRP levels (Figure 2).
This analysis of 5 prospective case-control studies provides further evidence that a single measurement of hsCRP more than 3 mg/L predicts an increased risk of developing AMD over many years. After matching for age and controlling for cigarette smoking, individuals with baseline hsCRP levels more than 3 mg/L had a 50% increased risk of incident AMD and a nearly 2-fold increased risk of neovascular AMD. In conjunction with other lines of evidence, these findings support the theory that low-grade systemic inflammation contributes to AMD development in the general population.35
To our knowledge, the current study, pooling 647 incident cases of AMD and 1480 controls across 5 cohorts, comprises the largest group of prospectively ascertained cases to date. Although the specificity of AMD diagnosis is high in these cohorts,36 ascertainment of AMD cases may have been incomplete. However, the impact of any missed cases would be minimal in this prospective nested case-control study. For most cases included in the present study, the type or size of drusen was not collected when confirming cases, which might lead to some misclassification of AMD, though other analyses based on these same cases and controls have shown strong and consistent associations with known genetic and nongenetic AMD risk factors, suggesting any such bias is likely to be small.37,38 Inclusion of AMD cases and controls from large prospective cohorts of US men and women improves the generalizability of conclusions, but the inclusion of only health professionals may limit the generalizability, particularly if the distribution of hsCRP levels is shifted toward lower levels as might be expected if these populations of health professionals are more healthy than the general population.
There was some variability in estimates of association among the 5 cohorts, but this heterogeneity was not statistically significant and was not apparent in subgroup analyses of the more severe neovascular AMD cases. Investigation of possible sources of heterogeneity identified a stronger association in the 3 cohorts with more recent blood collections as compared with the 2 cohorts (PHS and NHS) with blood collections in the 1980s. Although the stability of hsCRP in frozen blood samples has been previously demonstrated,39 there is always a possibility that some degradation of hsCRP occurred in the stored samples. The design of the study precludes any direct analysis of this potential source of bias, but the most likely impact of degradation over time would be a null-ward bias in associations.
Previous cross-sectional studies of the relationship between hsCRP and AMD have provided mixed results. Two clinic-based studies18,19 noted an association between hsCRP level and AMD, while 1 cross-sectional population-based study did not.23 The increased likelihood of selection bias among cross-sectional and clinic-based studies lends greater weight to the conclusions of more recent prospective studies, which minimize selection and surveillance bias, and also provide information on hsCRP levels prior to the onset of AMD. Of 3 recent population-based prospective studies, 1 noted no association,24 whereas 2 others21,22 reported an increased incidence of AMD among individuals with higher baseline levels of hsCRP. One of these studies22 was a full cohort analysis of WHS and therefore included the same set of incident AMD cases that are included herein in the nested case-control sample from that population. That study found a 90% increased incidence of AMD among women with an hsCRP level more than 3 mg/L. The results from the WHS case-control analysis in this study were consistent with the full WHS cohort analysis. In the other prospective study in which a significant association was observed, Boekhoorn et al21 noted a 40% increased incidence of early AMD among persons with an hsCRP level more than 3.26 mg/L and an 80% increased incidence of late AMD cases (neovascular AMD and central geographic atrophy) among persons with an hsCRP level more than 3.23 mg/L each compared with persons with an hsCRP level less than 0.83 mg/L. In a study of 254 individuals with early AMD at baseline, Robman et al40 recently observed an 80% increased risk of AMD progression over 7 years. Our results are also consistent with a recent meta-analysis41 of 11 studies (9 cross-sectional and 2 prospective), in which there was a 2-fold increase in late AMD (primarily neovascular cases) and a 31% increase in overall AMD in subjects with an elevated hsCRP level more than 3 mg/L.
C-reactive protein has emerged over the past decade as an important risk marker for cardiovascular and other age-related diseases. Data from the WHS, 1 of the 5 cohorts included herein, for example, showed a 66% increased risk of coronary heart disease among women with a baseline hsCRP level more than 3 mg/L,42 a magnitude similar to the increased risk of AMD we previously observed in the WHS, as well as in pooled findings from the 5 cohorts in this report. Although a role of inflammation and innate immunity/complement dysregulation in AMD is now established,13-15,43 a direct role for CRP in AMD causation remains a topic of research and debate.
The ability of CRP to induce complement activation, coupled with the presence of complement components in subretinal drusen, suggests a possible etiological role of CRP in the pathogenesis of AMD. Supporting this hypothesis, prior laboratory work has shown that the common AMD-associated Y402H variant of complement factor H attenuates its binding affinity for CRP, particularly at higher concentrations of CRP,44 and thus reduces deactivation of the complement cascade. This may result, at least in theory, in alterations in the retinal pigment epithelium, damage to the underlying Bruch membrane, and deposition of drusen and progression to AMD.7,45,46 Such findings concur with evidence from a recent epidemiological study that suggests that risks of AMD associated with higher hsCRP levels are greater among individuals with the Y402H genotype.47 We think it is important to continue to study whether CRP interacts with complement factor H or other AMD-associated genes and plays a direct role in any of these pathways, since pharmacologic modification of CRP levels is a possibility. However, the use of statin drugs to lower CRP levels, as is being tested for prevention of cardiovascular outcomes, does not appear promising for AMD in light of good evidence that such drugs have no effect on AMD progression.48
In conclusion, our pooled data from 5 prospective studies demonstrate that persons with an elevated hsCRP level more than 3 mg/L (a level used to indicate increased risk of cardiovascular disease) have a higher incidence of AMD. Given these findings, and the similar results of a recent meta-analysis,13 studies might be considered, for example, to determine whether measurement of hsCRP could be useful to motivate individuals with higher risk levels to make lifestyle changes (eg, smoking cessation, dietary modification, and weight loss) or have regular eye examinations so that any interventions to prevent vision loss from AMD could be initiated in a timely fashion. These data also support continued investigation of the possibility that hsCRP may contribute in some way to the pathogenesis of AMD. If shown, such a finding would further support interventions to lower systemic CRP levels to prevent AMD onset and progression.
Correspondence: Debra A. Schaumberg, ScD, OD, MPH, Harvard Medical School, Division of Preventive Medicine, Brigham & Women's Hospital, 900 Commonwealth Ave E, 3rd Floor, Boston, MA 02215 (email@example.com).
Submitted for Publication: May 13, 2012; final revision received October 16, 2012; accepted November 13, 2012.
Published Online: February 7, 2013. 10.1001/jamaophthalmol.2013.2303. Corrected February 14, 2013.
Conflict of Interest Disclosures: Dr Ridker is listed as a coinventor on patents held by the Brigham & Women's Hospital that relate to the use of inflammatory biomarkers in cardiovascular disease and diabetes that have been licensed to AstraZeneca and Siemens.
Funding/Support: This work was supported by National Institutes of Health grants EY017362, EY013834, EY06633, EY009611, CA047988, HL043851, CA87969, CA49449, HL35464, CA34944, CA40360, HL26490, HL34595, and HL046959.
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