High sensitivity C-reactive protein, other markers of inflammation, and the incidence of macular degeneration in women

Abstract

Background

Prospective information on whether high sensitivity C-reactive protein (hsCRP) or other biomarkers of inflammation predict age-related macular degeneration (AMD) remains limited.

Methods

We measured hsCRP, soluble intercellular adhesion molecule-1 (sICAM-1) and fibrinogen levels in baseline plasma samples from 27,687 participants with a mean age of 55 y and initially free of AMD in the Women’s Health Study. We prospectively ascertained 150 cases of AMD with vision loss of 20/30 or worse by self-report confirmed with review of medical records, during 275,852 person-years of follow-up (mean=10 y) and used proportional hazards models to examine the relationship of these biomarkers with AMD.

Results

After adjustment for multiple risk factors, the rate ratio (95% confidence interval) of AMD contrasting the highest versus lowest quintile of hsCRP was 3.09 (1.39-6.88), P(trend)=0.02. In similar models, the RR (CI) for sICAM-1 was 1.87 (0.97 to 3.58), P(trend)=0.07. The relationship of fibrinogen with AMD was J-shaped with a RR (CI) of 2.01 (1.07-3.75) for women in the highest fifth versus second fifth.

Conclusions

Elevated circulating levels of hsCRP, sICAM-1, and fibrinogen precede the development of visually significant AMD in women, providing further support for the hypothesis that inflammation may play a role in AMD.

Introduction

Recent evidence suggests that inflammation and abnormalities of innate immunity play a role in the pathogenesis of age-related macular degeneration (AMD)¹, the leading cause of blindness among older adults ². A strong association between a common variant of the gene for complement factor H (CFH) and AMD has recently imparted considerable weight to this hypothesis ^3-9. These data coincide with the view that low-grade inflammation plays a more general role in the aging process itself ¹⁰, as well as in other age-related disorders ¹¹. Patterns of AMD progression ^{12, 13} viewed in the context the development of an increasingly proinflammatory status with age ¹⁰ suggest the possibility that this disease may develop via at least two somewhat discrete steps. The first involves accumulation of extracellular debris beneath the retina at varying rates among individuals, which may plausibly be related to upregulation of immuno-inflammatory responses and the development of AMD ¹⁴. The second step, which only occurs in a subset, leads to neovascular lesions and/or geographic atrophy. At least in the case of neovascular AMD this process involves an overt inflammatory/neovascular response originating from the choroidal vasculature ^{12, 15}.

Circulating levels of high sensitivity C-reactive protein (hsCRP) have been intensively studied and a single measure reliably indicates the degree of underlying systemic inflammation in asymptomatic adults. Moreover, blood levels of hsCRP have gained recognition through prospective epidemiological studies as a useful clinical indicator of future cardiovascular risk. ^{16, 17} In light of the evidence linking inflammation and AMD it is of interest to determine whether hsCRP levels or other markers of inflammation are predictive of AMD, but thus far data are inconsistent and there are few prospective studies ^18-22. The present study was undertaken to investigate whether circulating levels of hsCRP, sICAM-1, and fibrinogen, are elevated prior to the development of clinically apparent AMD.

Methods

Study Population

The base population was the subset (N=28,345) of the 39,876 participants in the Women’s Health Study (WHS) who provided a baseline blood specimen ²³. From these, we excluded 302 subjects who had prevalent AMD at baseline and 356 women for whom hsCRP, sICAM-1, or fibrinogen measurements were not available. We followed the remaining 27,687 women from baseline until the date of diagnosis of AMD, death, or the last completed follow-up, whichever came first.

Between 1992 and 1995, the WHS enrolled 39,876 apparently healthy women in a randomized, double blind, placebo-controlled trial of alternate day low-dose aspirin (100 mg) and vitamin E (600 IU) in the primary prevention of cardiovascular disease and cancer. The study protocol was approved by the institutional review board of the Brigham and Women’s Hospital (Boston, MA), and written informed consent was obtained from all WHS participants. The recruitment, enrollment, and characteristics of the WHS population and primary trial results have been published ²⁴. Briefly, 95% of the WHS population is white and 75% are registered nurses while the remainder was recruited from among other health professions. The mean age at baseline was 54 y. Although WHS participants can be considered a select group, the reported prevalence of common medical conditions was comparable to the general population ²⁵.

The trial demonstrated a significant 24% reduction in risk of ischemic stroke in the aspirin group, but no significant effect on the risk of myocardial infarction, hemorrhagic stroke, or death from cardiovascular causes. Similarly, there was no overall effect of low-dose aspirin for the prevention of total, breast, colorectal or other site-specific cancers, but a protective effect for lung cancer could not be ruled out. Final results for the vitamin E arm of the WHS trial showed no overall benefit for major cardiovascular events or cancer, no effect on total mortality, and decreased cardiovascular mortality.

Assessment of AMD

Procedures for our two-stage documentation of incident AMD are identical to those used in the Physicians’ Health Study, which have been previously described and validated ^{26, 27}. On each study questionnaire we asked participants about the diagnosis of AMD, including the month and year of diagnosis, the name and address of the diagnosing eye doctor, and for signed permission to review medical records. For each report of a diagnosis of AMD, we sent a letter to the participant’s eye doctor to obtain information from the medical record on the date of diagnosis, best-corrected visual acuity at the time of diagnosis, the date when visual acuity first reached 20/30 or worse, and the chorio-retinal lesions that were present (drusen; RPE changes including atrophy, hypertrophy and RPE detachment; geographic atrophy; subretinal neovascular membrane; disciform scar). We confirmed a diagnosis of AMD for purposes of this study if one 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 doctor to judge whether the visual acuity would be expected to be 20/30 or worse due to AMD alone. The visual acuity criterion was included to reduce the possibility of surveillance bias, and because we were interested in determinants of visually significant disease. We defined neovascular AMD as the documented presence of an RPE detachment, subretinal neovascular membrane, or disciform scar that was not due to other causes (e.g. histoplasmosis, choroidal rupture). Dry AMD included those 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. Follow-up in the WHS trial was 97% percent complete for morbidity outcomes including AMD.

Measurement of Biomarkers

Baseline blood specimens were collected in EDTA and stored in liquid nitrogen freezers until the time of analysis when samples were thawed and levels of the inflammatory markers were measured in a core laboratory certified by the National Heart Lung and Blood Institute/Centers for Disease Control and Prevention Lipid Standardization program. Levels of hsCRP were analyzed using a validated immunoturbidometric method as previously described (Denka Seiken, Tokyo, Japan) ²⁸. Levels were similar to expected values for hsCRP in a population of healthy middle-aged women ²³. Levels of sICAM-1 and fibrinogen were determined by a commercially available ELISA method (R&D Systems).

Assessment of other Risk Factors

At entry into the WHS, participants completed a mailed questionnaire on which they reported information on demographic information including their age, race/ethnicity, highest educational level, and household income level as well as a detailed medical history and personal information on a large number of lifestyle factors including height and weight from which we calculated the body mass index, cigarette smoking history, medication use (including postmenopausal hormones, antihypertensives, and cholesterol lowering medications) and diet assessed via a validated semi-quantitative food frequency questionnaire. We followed the cohort for an average of 10 y with annual questionnaires to update risk factor and health status information.

Statistical Analysis

We divided levels of hsCRP, sICAM-1 and fibrinogen into fifths based on the distribution in the study population. We also examined relationships for categories of hsCRP formed using cut points of <1 mg/L, 1 to 3 mg/L and >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 risk.²⁹ In initial analyses, we obtained age- and smoking-adjusted rate ratios (RR) and 95% confidence intervals (CI) of AMD for each of the upper four versus the lowest fifth of hsCRP and the other markers in proportional hazards regression models. All models included terms for randomized treatment assignments to aspirin and vitamin E. We tested for linear trend across categories of the markers by entering a single ordinal score variable in the regression model, and alternatively by including the natural log transformed levels of each biomarker as a continuous variable. We then extended these models to adjust for other potential confounders including use of hormone therapy, antihypertensive medications, and cholesterol lowering drugs; body mass index; and dietary intake of omega-3 fatty acids, lutein/zeaxanthin, and zinc. We further examined whether cigarette smoking, aspirin assignment, or use of hormone therapy might modify the association between each marker and AMD by including interaction terms in the age and smoking-adjusted regression models. We were interested in potential effect modification by these variables based on evidence linking these common exposures with both levels of inflammatory markers as well as with AMD. Finally, We also obtained estimates for the age and smoking-adjusted relationships of each marker with neovascular AMD, but due to the relatively small number of these cases, we did not extend the models to control for other potential confounders.

Results

At baseline, the mean (SD) age of women followed in the study was 54.6 (7.03) years. Additional baseline characteristics of the study population are presented in Table 1. The median levels of the inflammatory markers were: 2.01 mg/L for hsCRP, 343 ng/ml for sICAM-1, and 351 mg/L for fibrinogen. During an average follow-up of 10 years for a total of 275,852 person years of follow-up, we confirmed 150 cases of AMD associated with vision loss of 20/30 or worse, including 32 cases of neovascular AMD.

Table 1.

Baseline characteristics of the study population.

	Mean (SD) or Percent
	Women who remained free of AMD with vision loss (N=27,537)	Women who developed AMD with vision loss (N=150)
Age (years)	54.6 (6.99)	65.7 (7.17)
Body mass index (kg/m2)	25.9 (4.97)	26.5 (5.27)
Dietary intake
Omega 3 fatty acids (g)	1.39 (0.39)	1.47 (0.47)
Lutein/zeaxanthin (mcg)	3690 (2838)	3732 (2498)
Zinc (mg)	14.4 (9.79)	15.2 (8.66)
Current cigarette smoking, %	11.7	16.8
Hormone therapy, %	43.6	35.3
Treated hypertension, %	13.2	27.3
Use of cholesterol lowering drugs, %	3.2	8.7
Randomized treatments
Aspirin, %	50.1	46.0
Vitamin E, %	50.0	50.0

After adjusting for age and cigarette smoking, we observed an increased incidence of AMD among women across fifths of hsCRP (P for trend=0.006) and sICAM-1 (P for trend=0.03). In contrast, the association with fibrinogen appeared to be J-shaped, with elevated RR among those in both the highest and lowest fifths compared with women in the second fifth of the distribution (Table 3). Using women in the lowest fifth of hsCRP as the reference, women in the highest fifth of hsCRP levels had a RR (CI) of 3.22 (1.50 to 6.91). Compared to women with hsCRP<1mg/L, the RR (CI) for AMD was 1.54 (0.94 to 2.52) for hsCRP between 1 and 3 mg/L, and 1.89 (1.18 to 3.03) for hsCRP>3 mg/L (P for trend=0.003). The RR (CI) contrasting the highest versus lowest fifth of sICAM-1 was 1.88 (1.03 to 3.43). Women with fibrinogen levels in the highest versus second fifth had a RR (CI) for AMD of 2.29 (1.27 to 4.12), and the trend across categories was not significant (P for trend=0.08).

Further adjustment for additional risk factors including use of hormone therapy, antihypertensive medications, and cholesterol lowering drugs; body mass index; and dietary intake of omega-3 fatty acids, lutein/zeaxanthin, and zinc resulted in RR (CI) estimates of 3.09 (1.39 to 6.88) for the contrast of extreme fifths (P for trend=0.02). In the models using recommended clinical cut-points for hsCRP, the RR (CI) were 1.52 (0.91 to 2.53) for hsCRP between 1 and 3 mg/L, and 1.83 (1.09 to 3.08) for hsCRP>3 mg/L versus hsCRP<1 mg/L (P for trend=0.02). In multivariable-adjusted models for sICAM-1 and fibrinogen, the trend across fifths of sICAM-1 was no longer significant (P for trend=0.08), although the RR contrasting the highest versus lowest fifth was unchanged. The J-shaped association with fibrinogen persisted with a RR (CI) of 2.01 (1.07 to 3.75) for women in the highest versus second fifth. In similar multivariable models, we also tested the linear association between the natural log of each marker and incidence of AMD, and observed a corresponding multivariable-adjusted RR (CI) of 1.25 (1.06 to 1.47), P=0.009 for hsCRP, 3.54 (1.55 to 8.06), P=0.003 for sICAM-1, and 1.20 (0.52 to 2.78), P=0.67 for fibrinogen.

We found no significant interactions between hsCRP levels and randomized aspirin assignment (P=0.39), cigarette smoking (P=0.27), or use of hormone therapy (P=0.70). Interactions of these factors with sICAM-1 levels were also not significant (P=0.37 for aspirin, P=0.95 for smoking, P=0.83 for hormone therapy). With regard to fibrinogen, there were no interactions with aspirin (P=0.38) or cigarette smoking (P=0.29). However, there was modest evidence for interaction with hormone therapy (P=0.04). In separate models for women using hormone therapy, there was a significant linear trend across fifths of fibrinogen (P for trend=0.01); whereas among women who did not use hormone therapy the trend was not significant (P for trend=0.94) because the greatest risk of AMD was observed among women in the lowest fifth of fibrinogen levels among whom the RR (CI) was 2.88 (1.21 to 6.88) as compared to women in the second fifth of fibrinogen levels (Figure).

Figure.

Rate ratios and 95% confidence intervals for the association between plasma fibrinogen levels and risk of age-related macular degeneration among subgroups of women who were either non-users (panel A) or users (panel B) of post-menopausal hormone therapy.

In models for the subset of 32 cases of neovascular AMD, the age and smoking-adjusted RR (CI) was 3.22 (0.92 to 11.23) for hsCRP between 1 and 3 mg/L, and 2.97 (0.86 to 10.30) for hsCRP>3 mg/L. Confidence intervals were wide reflecting the uncertainty of the estimates given the relatively small number of cases, and the trend was not statistically significant, P for trend=0.14. There was no significant trend across fifths of sICAM-1 (P for trend=0.91) or fibrinogen (P for trend =0.06). However, if we collapsed the lower three fifths of fibrinogen to form a more robust reference group the RR (CI) was 3.41 (1.46 to 7.95) among women in the upper two fifths.

Discussion

CRP is a major acute-phase reactant principally regulated by proinflammatory cytokines that is elevated in response to infection, injury, and other insults. At the same time, CRP levels are quite stable among healthy individuals ^{30, 31} and reflect the degree of underlying systemic inflammation. The measurement of CRP by highly sensitive techniques has gained clinical acceptance as an adjunct in the assessment of cardiovascular risk. ^{32, 33} Results of previous cross-sectional studies of the association between hsCRP and AMD have been inconsistent ^{18, 20-22} but are more subject to possible bias as compared with a prospective study. Of prior prospective studies, one had limited power and failed to find an association,²² while the other demonstrated a faster rate of progression to later stages of AMD among patients with higher levels of hsCRP ¹⁹. In the present study, the first large prospective cohort study of initially healthy individuals, hsCRP levels predict incident AMD, the leading cause of vision impairment in older adults ². Women with hsCRP levels in the highest versus lowest fifth had a >3-fold higher incidence of AMD. The incidence of AMD was also increased approximately 2-fold among women with the highest levels of sICAM-1 and fibrinogen.

hsCRP is the most consistent of several biomarkers of inflammation in predicting cardiovascular disease ³³. In addition to being a sensitive biomarker of inflammation, CRP may have direct pathophysiological significance, for instance, through its ability to induce complement activation and thereby contribute to tissue damage ³⁴. Both CRP and complement proteins have been identified in and adjacent to ocular drusen and Bruch’s membrane ^{35, 36}. There is speculation that repeated cycles of complement attack on retinal pigment epithelial (RPE) cells could lead to AMD ^{1, 35}. This idea is further supported by the recent findings of a strong association between a common genetic variant of CFH and risk of AMD ^{3-7, 26}. Of particular interest in light of the present findings is that the variant form of CFH is predicted to bind CRP less effectively and thereby deter its ability to reduce deposition of the terminal attack complex ³⁷, the result being an overall increase in inflammatory activity. There is emerging evidence that associations between CRP and AMD may be interrelated with the CFH variant ³⁸. Though we were not able to assess this possibility in the present study, further study of this hypothesis would be of interest.

Elevated levels of both sICAM-1 and fibrinogen have also been linked with cardiovascular disease. Widely expressed in the vasculature but also in RPE ³⁹, sICAM-1 is involved in the adhesion and transendothelial migration of leukocytes ⁴⁰. Raised circulating levels sICAM-1 are indicative of a state of endothelial dysfunction and increased interaction with leukocytes, with consequent activation of target cells and induction of inflammatory activity ^{41, 42}. Since the shedding of ICAM-1 is not restricted to endothelial cells, its presence in the circulation may relate to nonendothelial tissue injury and leukocyte activation ⁴⁰. We observed a linear relationship between baseline sICAM-1 levels and risk of AMD such that women in the highest fifth have a nearly 2-fold increased risk of AMD. No association between sICAM-1 and AMD was shown in two prior reports ^{19, 21}. However, one study had low power ²¹ and, in the other ¹⁹, sICAM-1 levels were lower than in ours and the study endpoint was progression of preexisting AMD lesions rather than the incident cases we studied.

In contrast to the linear relations of hsCRP and sICAM with AMD, the association with fibrinogen levels appears J-shaped. More detailed investigation suggested that the increased risk of AMD among women in the lowest fifth of the distribution is limited to those women who were not using hormone therapy. This finding may have biological relevance in light of data showing a reduction in fibrinogen levels among women using hormones ⁴³ and other data suggesting that hormone therapy may reduce the risk of AMD ⁴⁴. Further study would be needed, however, before firm conclusions can be reached.

Fibrinogen has been identified in drusen ³⁶ and Bruch’s membrane ⁴⁷; and prior epidemiological evidence from cross-sectional studies supports a relationship of fibrinogen with AMD ^50-52.Fibrinogen is a hemostatic factor and acute phase proinflammatory protein ^{45, 53} that could conceivably affect risk of AMD through reductions in choroidal blood flow ⁴⁶, direct effects on the vascular wall ⁴⁸, or other effects of chronic inflammation; though it remains possible that it is merely a marker of risk.

The prospective methodology and large sample-size of the present study lessens the likelihood of bias. Nevertheless, the study was limited by a single measurement of each biomarker; increasing the chance that intra-individual variation may have affected the findings. However, the study benefited from state-of-the-art assays and is consequently unlikely to have been impacted by laboratory issues. Moreover, misclassification of exposure levels is expected to bias estimates toward the null and is thus an unlikely explanation for the positive findings. Although we controlled for known AMD risk factors, residual confounding remains a concern as in any epidemiological study. Ascertainment of AMD may have been incomplete as it relied on self-reports from participants that were later confirmed by review of medical record data. However, the prospective cohort approach minimizes any impact of missed cases so long as the specificity of diagnosis is high ⁵⁴. Generalizability of these findings from female health professionals to the larger population of women and men is also uncertain.

Together with the discovery that a common variant of CFH strongly increases the risk of AMD ^{3-9, 26}, these data further substantiate an important role for the closely connected processes of inflammation and innate immunity in the pathogenesis of this sight threatening and incurable disease. Specifically, the current data demonstrate that although AMD is not associated with any overt clinical signs of ocular inflammation, higher circulating levels of hsCRP, sICAM-1, and fibrinogen predict future risk of AMD. hsCRP in particular has already been adopted as a useful adjunct in cardiovascular risk prediction, and may also aid in motivating patients to alter risky lifestyle behaviors. Further study of the interrelationships of genetic predisposition with these factors, and the possible clinical utility of the measurement of inflammatory biomarkers such as hsCRP in the setting of AMD should be considered.

Table 2.

Incidence rate ratios and 95% confidence intervals for age-related macular degeneration among initially healthy women according to fifths of high-sensitivity C-reactive protein (hsCRP), soluble intercellular adhesion molecule-1 (sICAM-1), and fibrinogen.

	Model 1*		P-value	Model 2†		P-value
	RR	CI		RR	CI
hsCRP (mg/L)
<0.63	1.00			1.00
0.63-1.44	2.72	1.25-5.96		2.67	1.22-5.87
1.45-2.72	2.92	1.35-6.31		2.71	1.24-5.94
2.73-5.16	3.35	1.56-7.16		3.13	1.43-6.86
≥5.17	3.22	1.50-6.91	0.006	3.09	1.39-6.88	0.02
sICAM-1 (ng/mL)
<291	1.00			1.00
291-325	1.20	0.61-2.37		1.33	0.65-2.71
326-359	1.28	0.67-2.43		1.48	0.75-2.92
360-408	1.25	0.67-2.36		1.35	0.69-2.66
≥409	1.88	1.03-3.43	0.03	1.91	0.97-3.58	0.07
Fibrinogen (mg/L)
<297	1.93	0.98-3.80		1.92	0.95-3.90
297-332	1.00			1.00
333-368	1.44	0.75-2.76		1.52	0.78-2.97
369-416	1.49	0.80-2.80		1.47	0.77-2.83
≥417	2.29	1.27-4.12	0.08	2.01	1.07-3.75	0.29

^*Model 1: Adjusted for randomized treatment assignments, age, and current cigarette smoking.

^†Model 2: Adjusted for factors in model 1 and additionally for use of hormone therapy, antihypertensive medications, and cholesterol lowering drugs; body mass index; and dietary intake of omega-3 fatty acids, lutein/zeaxanthin, and zinc.