Soluble CD40L and Cardiovascular Risk in Women
Background The immune-signaling dyad CD40/CD40L promotes atherogenesis, and patients with unstable angina have elevated plasma levels of soluble CD40L (sCD40L) and membrane-bound CD40L. It is unknown, however, whether elevations of circulating sCD40L precede the onset of acute cardiovascular symptoms.
Methods and Results In a prospective, nested case-control evaluation of healthy middle-aged women, mean concentrations of sCD40L at baseline were significantly higher among 130 participants who subsequently developed myocardial infarction, stroke, or cardiovascular death (cases), compared with 130 age- and smoking-matched women who remained free of cardiovascular disease (controls) during a 4-year follow-up (2.86 ng/mL for cases versus 2.09 ng/mL for controls; P=0.02). Women with concentrations above the 95th percentile of the control distribution (>3.71 ng/mL) had a significantly increased relative risk (RR) of developing future cardiovascular events (RR, 3.3; 95% CI, 1.2 to 8.6; P=0.01) that remained after adjustment for usual cardiovascular risk factors (multivariate RR, 2.8; 95% CI, 0.9 to 8.0; P=0.05).
Conclusions High plasma concentrations of sCD40L may be associated with increased vascular risk in apparently healthy women.
Received July 18, 2001; revision received September 13, 2001; accepted September 14, 2001.
Previously, we and others have demonstrated that the multipotent immunomodulator CD40L and its receptor CD40, expressed on vascular endothelial cells, smooth muscle cells, mononuclear phagocytes, and platelets, promote a wide array of pro-atherogenic functions in vitro.1–5 Ligation of CD40 on endothelial cells, smooth muscle cells, or mononuclear phagocytes triggers the expression of various proinflammatory mediators, such as the cytokines interleukin (IL)-1, IL-6, IL-12, tumor necrosis factor-α, and interferon-γ; the chemokines IL-8, monocyte chemoattractant protein-1, and RANTES (regulated upon activation, normal T cell expressed and secreted); intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1; the matrix metalloproteinases-1, -2, -3, -7, -8, -9, -10, -11, -12, and -13 as well as the procoagulant tissue factor.5 These observations implicate CD40L in the various stages of atherogenesis, including the initiation and progression of atherosclerotic lesions as well as acute complications. In accord with the predicted role the CD40/CD40L dyad in atherogenesis, disruption of CD40 signaling significantly diminished lesion formation and progression in hypercholesterolemic mice.6 Interruption of CD40/CD40L interactions also prevented the progression of established atheroma and promoted processes associated with plaque stabilization in humans.7,8 These in vitro and in vivo studies identified CD40L as a potential therapeutic target for treatment of atherosclerosis, although the underlying mechanisms by which interruption of CD40 signaling affects atherogenesis require further investigation.
In addition to the 39-kDa, cell-associated form, CD40L also occurs in a soluble, biologically fully active form (sCD40L).9 Interestingly, patients with unstable angina have higher concentrations of functional sCD40L than do patients with stable angina or healthy volunteers, possibly as a result of release from activated platelets or T lymphocytes (U. Schönbeck, PhD, et al, unpublished observations, 2001).10 It remains unknown, however, whether plasma concentrations of sCD40L also have diagnostic or prognostic value among apparently healthy individuals before the onset of acute cardiovascular events.
We performed a prospective, nested case-control analysis among participants in the Women’s Health Study (WHS), an ongoing primary prevention trial evaluating the efficiency of vitamin E and low-dose aspirin in 28 263 middle-aged American women with no history of cardiovascular disease or cancer.11 Blood samples were collected in EDTA and stored in liquid nitrogen until analysis. For this study, 130 women who subsequently developed either nonfatal myocardial infarction or stroke or died from acute cardiovascular events during the initial 4-year follow-up period were selected as case subjects. A committee of physicians using standardized procedures classified end points. For each confirmed case, a control participant of the same age (±2 years) and similar smoking status (former, current, or never) who remained free of reported cardiovascular disease was selected.
Baseline plasma sCD40L concentrations were measured by ELISA (Bender MedSystems). Briefly, diluted (1:5) plasma samples were applied in triplicate to 96-well plates precoated with mouse anti-human CD40L antibody and mixed (1:2) with a horseradish-peroxidase–labeled secondary mouse anti-human CD40L antibody (2 hours). Subsequently, plates were washed and antibody binding determined by colorimetry using 3,3′-5,5′-tetramethylbenzidine substrate. Absorbance was read at 650 nm and plasma concentrations of sCD40L were determined by comparison with serial dilutions of recombinant human CD40L. Analysis was performed in a blinded fashion. Intra-assay variation among the triplicates for all samples was less than 15%. Lipid levels were measured in a laboratory that participates in the Centers for Disease Control standardization.
Means and proportions for baseline clinical characteristics of the study participants were computed and compared using either Student’s t test or the χ2 statistic. Relative risk (RR) of developing future cardiovascular events associated with increasing levels of sCD40L at baseline were then computed in a series of logistic regression analyses that divided the study sample according to the 50th, 75th, 90th, 95th, and 99th percentile cutpoints of the control distribution for sCD40L. All P values are 2-tailed and all confidence intervals are computed at the 95% level.
Table 1 shows the baseline clinical characteristics of the study participants. As expected, women who developed cardiovascular disease during follow-up were more likely at study entry to be obese, hypertensive, or diabetic, or to have a family history of premature atherosclerosis, compared with women who remained free of disease. LDL cholesterol and triglyceride levels were higher at baseline among cases, whereas HDL cholesterol levels were lower (all P≤0.01). Use of hormone replacement therapy did not differ significantly between the 2 groups.
Overall, plasma levels of sCD40L at baseline among cases exceeded that in controls (2.86±0.35 versus 2.09±0.19 ng/mL; P≤0.02) (Table 1). This difference was almost entirely the result of an excess of particularly high values among the case subjects. The great majority of cases and control subjects had similar levels of sCD40L at study entry (Figure). However, 11 cases had baseline levels of sCD40L in excess of the 99th percentile cutpoint for the control distribution, compared with only 1 control subject (P≤0.01).
RRs for developing future cardiovascular events, according to the prespecified cutpoints defined by the distribution of the study controls, rose with increasing concentrations of sCD40L and became statistically significant with levels of sCD40L in excess of the 95th and 99th percentile cutpoints (RR: 3.29 [P≤0.02] and 11.83 [P≤0.01], respectively) (Table 2).
An additional post-hoc analysis was performed, comparing clinical characteristics among the 12 participants with levels of sCD40L in excess of the 99th percentile cutpoint with the 248 participants with lower levels. Age, smoking, body mass index, LDL and HDL cholesterol levels, and hormone replacement therapy were similar between these 2 study groups (Table 3). Study participants with particularly elevated levels of sCD40L had somewhat higher rates of hypertension and a family history of premature coronary artery disease, but neither of these differences achieved statistical significance. None of the 12 women with markedly elevated baseline levels of sCD40L had diabetes. Moreover, there were no significant differences in the time from randomization to the cardiovascular event between the 11 cases with extreme sCD40L levels and the remaining cases with lower sCD40L (15.9 versus 19.5 months; P=0.3). Assignment to aspirin versus placebo presumably did not affect our observation because within the group of 12 subjects showing the highest sCD40L concentrations, 6 were randomly assigned to aspirin and 6 to placebo. In addition, the blood samples assayed for sCD40L were drawn before randomization.
Previous reports on this cohort have documented an association of plasma levels of C-reactive protein, IL-6, serum amyloid A, and ICAM-1 with increased cardiovascular risk.12 However, we observed no significant correlation between these parameters and sCD40L.
In this prospective, nested case-control study of apparently healthy middle-aged women, markedly elevated plasma concentrations of sCD40L at baseline (>5.54 ng/mL) foretold a significantly increased risk of future cardiovascular events. Previous studies demonstrated that patients with unstable angina had significantly higher serum levels of sCD40L when compared with patients with stable angina and controls.10 In this circumstance, activated platelets or T lymphocytes may release sCD40L secondarily. The present study, however, demonstrates elevation of sCD40L concentrations in some women before events that may result from acute thrombosis.
Little is known regarding the mechanisms yielding release of soluble forms of CD40L. Potential sources for sCD40L in plasma include platelets and T lymphocytes, as well as mononuclear phagocytes and endothelial cells.1–5 The tendency of family history of cardiovascular disease to correlate with enhanced sCD40L plasma levels suggests that genetic factors might contribute to our observation. Although we found no association between sCD40L and C-reactive protein, IL-6, or ICAM-1 levels, the absolute number of subjects with markedly elevated sCD40L was small, and CD40/CD40L-independent mechanisms may well pertain to women who develop cardiovascular complications despite low sCD40L levels. However, our finding suggests that high plasma concentrations of sCD40L reflect aspects of risk distinct from those gauged by other inflammatory markers. Administration of aspirin after the measurement of sCD40L does not appear to attenuate subsequent risk of a cardiovascular event, unlike C-reactive protein.13
In conclusion, these limited prospective data provide evidence that elevated plasma concentrations of sCD40L at baseline may identify certain apparently healthy women at high risk for cardiovascular events, an observation that we believe should stimulate further evaluation of sCD40L as a biomarker for cardiovascular disease.
This study was supported in part by grants from the National Heart, Lung and Blood Institute (HL-34636, HL-56985, HL-58755, and HL-63293). Dr Ridker was further supported by an Established Investigator Award from the American Heart Association and by a Distinguished Clinical Scientist Award from the Doris Duke Charitable Foundation. The authors also acknowledge Karen Williams for editorial assistance.
↵*The first 2 authors contributed equally to this work.
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