CD40 and CD40 ligand (CD40L) are costimulatory molecules of the tumor necrosis factor receptor superfamily and well known for their involvement in inflammatory diseases: atherosclerotic mouse models with disrupted CD40 signalling develop lesions of reduced size with a more stable plaque profile. This study investigated the potential of plasma and intraplaque levels of CD40 and CD40L as markers for cardiovascular disease (CVD) in humans and their association with plaque stability.
Soluble CD40 and CD40L (sCD40L) were measured in plasma in 1,437 subjects from The SUrrogate markers for Micro- and Macro-vascular hard endpoints for Innovative diabetes Tools (SUMMIT) cohort. Intra-plaque levels of sCD40 and sCD40L were measured in atherosclerotic plaque homogenates from 199 subjects of the Carotid Plaque Imaging Project (CPIP) cohort.
Both plasma sCD40 and sCD40L levels were elevated in individuals with prevalent stroke, while sCD40 levels also were higher in individuals with a prior acute myocardial infarction. Plasma levels of sCD40 correlated with carotid intima-media thickness and total carotid plaque area and were associated with risk of cardiovascular events over a 3-year follow-up period. Intra-plaque levels of sCD40 and sCD40L were associated with plaque components characteristic for plaque vulnerability and extracellular matrix remodelling.
Higher plasma sCD40 and sCD40L levels are associated with prevalent CVD. Plasma sCD40 levels also correlate with the severity of carotid atherosclerosis and predict future cardiovascular events, while intra-plaque levels correlate with a vulnerable plaque phenotype. Our findings thus demonstrate that elevated levels of sCD40 and sCD40L are markers of CVD.
Cardiovascular disease (CVD) is the leading cause of mortality worldwide, accounting for 17.9 million deaths each year [
CD40 and CD40 ligand (CD40L) are costimulatory molecules and members of the tumor necrosis factor (receptor) superfamily (TNF(R)SF) [
While not much is known about the specific actions of CD40 in human CVD, an association for CD40L in CVD has been suggested. Increased levels of platelet CD40L have been reported in patients with hypercholesterolemia [
To address the potential of sCD40 and sCD40L as markers of CVD, we have analysed plasma and carotid artery plaque levels of CD40/CD40L for associations with CV events, as well as carotid IMT and plaque vulnerability markers in two large human cohorts.
Extended methods are provided in the
Plasma samples were included from 1,438 subjects (
Carotid plaques (n=198) were collected from patients during carotid endarterectomies at the Vascular Department of Skane University Hospital (Malmö, Sweden) between 2005 and 2010 (The Carotid Plaque Imaging Project [CPIP]) [
Shapiro-Wilk and D’Agostino-Pearson omnibus K2 tests were used to assess Gaussian distribution. Variables found to be normally distributed are shown as mean with standard deviation while non-normally distributed variables are shown as median with interquartile range (IQR). For analysis of plasma, plaque histology sections and homogenates, the Mann-Whitney U test was used to compare groups and Spearman’s rank correlation was used for continuous variables. For comparisons between categorical variables, the chi-square test was used. A logistic regression model was used to test for associations with CV events and mortality in the SUMMIT cohort. The model was adjusted for age, diabetes and prevalent ischemic stroke, acute myocardial infarction (AMI), and hypertension as covariates. A
To assess associations between sCD40, sCD40L and CVD, we analysed the plasma from 1,438 subjects from the SUMMIT cohort using Proximity Extension Assay (PEA). Comparatively high sCD40 levels were associated with prevalent ischemic stroke (
Similar to sCD40, higher plasma sCD40L levels were associated with prevalent stroke (
Plaque burden in the carotid artery was assessed via ultrasound imaging at study inclusion and we searched for possible associations between sCD40 and sCD40L and plaque burden. A positive Spearman’s rank correlation was found between both plasma sCD40 and sCD40L levels and total plaque area (r=0.355,
Arterial stiffness—a well-known factor associated with atherosclerosis progression [
To elucidate whether plasma sCD40 and sCD40L levels predicted carotid atherosclerosis progress, in addition to correlating with plaque burden at study inclusion, we compared the initial IMT measurements calculated via ultrasound imaging with IMT measurements taken after 3 years. Plasma sCD40L, but not sCD40, levels were associated with change—specifically plaque growth—in maximum IMT in the CCA and mean and maximum IMT in the bulb region of the plaques (
As sCD40L levels correlated with carotid plaque progression, we then investigated the potential for plasma sCD40 and sCD40L levels to predict future CV events using logistic regression. Interestingly, the level of sCD40, but not sCD40L, in plasma was associated with the occurrence of fatal or non-fatal CV events during the 3 years follow-up period (OR, 1.3; 95% CI, 1.042 to 1.625) after adjusting for age, diabetes, hypertension as well as prevalent AMI and stroke at baseline (
To further explore the presence of CD40 and CD40L in atherosclerosis we examined their expression pattern in carotid endarterectomy plaques from the CPIP cohort (
To further explore the presence of CD40 and CD40L in atherosclerosis we examined their expression in homogenates from 198 carotid endarterectomy plaques of the CPIP cohort (here comprised of both the soluble and cell associated isoforms). Disrupted CD40 signalling in a murine atherosclerosis model results in increased stabilisation characterised by reductions in lipid core size and inflammatory cell presence accompanied by increased collagen content [
CD40 ligation has been previously well-described to induce pro-inflammatory cytokine and chemokines, including interleukin (IL)-1β, IL-6, IL-8, IL-12, TNF-α, and chemokine (C-C motif) ligand 5 (CCL5)
The reported increase in fibrous cap thickness and collagen content in plaques from CD40-deficient mice has been proposed to result from the accompanying decreased plaque expression and activity of matrix metalloproteinase (MMP)-2 and -9 together with increased tissue inhibitor of metalloproteinase (TIMP)-1 expression, and has thus prompted the suggestion that CD40 signalling destabilizes plaques by promoting extracellular matrix (ECM) degradation and, as a result, fibrous cap thinning [
As sCD40 and sCD40L plasma levels were higher in subjects who had suffered a stroke in the SUMMIT cohort, we next analysed whether sCD40 and/or sCD40L levels are similarly elevated in the plaques from individuals after a cerebrovascular event (median time between symptom and surgery was 14 days [IQR, 7 to 22]; the CPIP cohort). Levels of CD40 and CD40L measured in plaque strongly correlate with each other (r=0.613,
With this study, we set out to assess the value of sCD40 as a possible marker of CVD and investigate the biological associations that support a role for CD40 in atherogenesis. Our findings that sCD40 levels in plasma are associated with carotid plaque burden, while levels in carotid plaques are associated with a vulnerable plaque phenotype, strengthens the potential for sCD40 as marker for and mediator of CVD.
In contrast to the paucity of data in the literature for sCD40 as a marker for CVD, sCD40L has been previously widely linked to CVD via increased levels in patients with hypercholesterolemia [
Much less is known about the possible role of sCD40 as a marker for CVD. Though sCD40 appears to be actively produced under physiological conditions (as suggested by comparatively large amounts of sCD40 found in urine of healthy individuals) [
Efficient targeting of inflammatory processes can successfully stabilize vulnerable plaques in mice [
Neither CD40 nor CD40L was associated with time between cerebrovascular symptoms and endarterectomy, suggesting they are not merely upregulated as part of the general healing response that occurs after plaque rupture. However, though sCD40 levels were associated with a less stable carotid plaque phenotype, plaque levels were nonetheless similar in plaques from symptomatic and asymptomatic patients. This inconsistency may, at least in part, be explained by the accompanying association with components of the ECM turnover machinery, leading to a net effect in CD40-rich plaques, where enough ECM synthesis of stable plaque elements balances out the more vulnerable elements, resulting in a plaque phenotype still stable enough not to rupture/erode and cause cerebrovascular symptoms. This in turn may indicate a local protective element in CD40-CD40L signalling, in addition to its well-described actions promoting the inflammatory process. We hypothesize that this may demonstrate the contrasting results of CD40 and CD40L interactions with (and in) different cell types of varying functions, such as macrophages, T cells, and SMCs in a model where CD40-signalling simultaneously for example promotes inflammation via macrophages and ECM synthesis via SMCs, even if the phenotypes of these cells have also been widely discussed [
Also interesting from a therapeutic standpoint is the study by Fernandez et al. [
As limitations, it must be noted that correlations do not indicate causality, and we cannot rule out that the reported associations may also result from an upregulation of sCD40 and sCD40L stimulated by an upstream signalling cascade or events. It is also important to bear in mind that in the plaque homogenates derived from the CPIP cohort it is not possible to distinguish between soluble and cell-bound CD40 and CD40L. The analysis thus shows the net effect of both isoforms, even if the effects of the individual isoforms may differ. Moreover, though it is impossible to be certain of the exact type of ischemic stroke, as subjects with atrial fibrillation were excluded in both cohorts, and—in the CPIP cohort—no signs of lacunar infarctions were found (assessed by brain computed tomography), the probability of the underlying mechanisms to be atrial fibrillation and small vessel disease rather than atherosclerosis can be considered low. Unfortunately, the presence of cardiac valvular disease is impossible to exclude as not all patients underwent echocardiography. Finally, as we in both cohorts measured sCD40L in plasma to evaluate its potential as a non-cell associated marker, platelet expression of CD40L—previously reported e.g., by Cha et al. [
In the present study we report for the first time elevated plasma sCD40 levels in individuals with a prior ischemic stroke or MI, as well as an association with arterial stiffness, carotid plaque burden, and IMT, and, finally, an association with increased risk of future CV events (
Supplementary materials related to this article can be found online at
Patient demographics in the CPIP and SUMMIT study cohorts
Spearman correlations between sCD40 and sCD40L in plasma (arbitrary unit) and the subjects’ general clinical characteristics (SUMMIT cohort)
Median with IQR values of sCD40 and sCD40L in plasma (A.U., SUMMIT cohort) when comparing between two groups (MannWhitney U test)
Spearman correlations between the circulating levels of sCD40 and sCD40L (A.U., SUMMIT cohort) and change in IMT in the CCA and the carotid bulb region assessed by vascular ultrasound (at the 3-year follow-up point compared to study inclusion)
Sensitivities, specificities and their 95% CI at various cut-offs of predicted values from LR in predicting atherosclerotic vascular events
Spearman correlations of CD40 and CD40L (A.U., CPIP cohort) measured in plaque homogenate versus plaque elements
The authors have no financial conflicts of interest.
This work was supported by funding from the Innovative Medicines Initiative (the SUMMIT consortium), IMI-2008/115006, Swedish Research Council, Swedish Heart and Lung Foundation, Swedish Society for Medical Research, Swedish Society of Medicine, Emil and Wera Cornell foundation, Hjelt foundation, ALF Grants Region Skane, Bundy Academy, Diabetes foundation, Diabetes Research & Wellness Foundation, SUS foundations and funds, Stroke foundation, the Royal Physiographic Society in Lund, The Swedish Heart and Lung Association, The Swedish Stroke Association, Albert Påhlsson’s foundation and Swedish Foundation for Strategic Research (Dnr IRC15-0067). Angela C. Shore is supported by the UK National Institute of Health Research (NIHR) Exeter Clinical Research Facility. The views in this manuscript are those of the authors and not necessarily those of the NIHR or the UK Department of Health.
The authors wish to thank Mihaela Nitulescu, Ana Persson, Lena Sundius, and Fong To for expert technical assistance.
Flow chart showing the study population of the The SUrrogate markers for Micro- and Macro-vascular hard endpoints for Innovative diabetes Tools (SUMMIT) cohort, including excluded subjects and subjects with/without cardiovascular disease (CVD).
CD40 and CD40 ligand (CD40L) localization in endarterectomy plaques. CD40 and CD40L immunoreactivity was found in particular in the shoulder (A, B) and cap (C, D) regions. Squares in insets mark enlarged plaque regions. Scale bars are 50 μm (far left and right image columns), 20 μm (middle column), and 1 mm (insets, far left column) (n=22 plaques).
Graphic representation of plasma soluble CD40 (sCD40) in cardiovascular disease. Higher plasma sCD40 levels are associated with prevalent cardiovascular disease, severity of carotid atherosclerosis and predict future cardiovascular events (CV events). MI, myocardial infarction.
Spearman correlations between the circulating levels of sCD40 and sCD40L (arbitrary unit, SUMMIT cohort) and carotid plaque burden
Vascular ultrasound |
CD40 (n=1,438 at inclusion; n=798 at follow-up) |
CD40L (n=1,438 at inclusion; n=798 at follow-up) |
||||
---|---|---|---|---|---|---|
r | r | |||||
Total plaque area |
0.355 | <1×10–16 | <1×10–16 | 0.409 | <1×10–16 | <1×10–16 |
Maximum plaque area (mm2) | 0.322 | <1×10–16 | <1×10–16 | 0.340 | <1×10–16 | <1×10–16 |
No. of plaques, right carotid artery | 0.354 | <1×10–16 | <1×10–16 | 0.152 | 2.0×10–8 | 1.0×10–7 |
No. of plaques, left carotid artery | 0.398 | <1×10–16 | <1×10–16 | 0.142 | 1.9×10–7 | 7.6×10–7 |
Plaque length (mm) | 0.316 | <1×10–16 | <1×10–16 | 0.286 | 1.3×10–15 | 9.3×10–15 |
Plaque height (mm) | 0.223 | 5.9×10–10 | 1.2×10–9 | 0.164 | 0.000006 | 0.000018 |
Lumen diameter reduction (%) | 0.129 | 0.00041 | 0.00041 | 0.224 | 5.2×10–10 | 3.1×10–9 |
Pulse wave velocity | 0.247 | 8.0×10–8 | <1×10–16 | 0.057 | 0.052 | 0.052 |
sCD40, soluble CD40; sCD40L, soluble CD40 ligand; SUMMIT, The SUrrogate markers for Micro- and Macro-vascular hard endpoints for Innovative diabetes Tools.
Measured at study inclusion;
Sum of all plaque areas/patient.
Spearman correlations between the circulating levels of sCD40 and sCD40L (arbitrary unit, SUMMIT cohort) and IMT in the CCA and the carotid bulb region assessed by vascular ultrasound (measured at study inclusion)
IMT measurements | CD40 (n=1,438 at inclusion; n=798 at follow-up) |
CD40L (n=1,438 at inclusion; n=798 at follow-up) |
||||
---|---|---|---|---|---|---|
r | r | |||||
Right | ||||||
Mean IMT in CCA | 0.139 | 2.7×10–7 | 1.1×10–6 | 0.053 | 0.048 | 0.256 |
Max IMT in CCA | 0.122 | 0.000006 | 0.000018 | 0.012 | 0.65 | 0.903 |
Mean IMT in bulb | 0.234 | 1.4×10–16 | 6.6×10–16 | 0.081 | 0.005 | 0.039 |
Max IMT in bulb | 0.268 | <1×10–16 | <1×10–16 | 0.077 | 0.008 | 0.055 |
Left | ||||||
Mean IMT in CCA | 0.088 | 0.001 | 0.0020 | 0.042 | 0.12 | 0.472 |
Max IMT in CCA | 0.073 | 0.007 | 0.0070 | 0.000 | 0.99 | 0.990 |
Mean IMT in bulb | 0.203 | 9.1×10–13 | 4.5×10–12 | 0.017 | 0.54 | 0.903 |
Max IMT in bulb | 0.240 | <1×10–16 | <1×10–16 | 0.038 | 0.19 | 0.570 |
sCD40, soluble CD40; sCD40L, soluble CD40 ligand; SUMMIT, The SUrrogate markers for Micro- and Macro-vascular hard endpoints for Innovative diabetes Tools; IMT, intima-media thickness; CCA, common carotid artery.
Logistic regression analysis of association between the circulating levels of sCD40 (sCD40, SUMMIT cohort) and cardiovascular events (adjusted for confounding factors
Variable | Confidence interval | Odds ratio | |
---|---|---|---|
sCD40 | 1.042–1.625 | 1.3 | 0.020 |
Age | 0.996–1.042 | 1.0 | 0.109 |
Diabetes | 0.759–1.681 | 1.1 | 0.549 |
Hypertension | 0.596–1.319 | 0.9 | 0.553 |
Acute myocardial infarction | 1.178–2.467 | 1.7 | 0.005 |
Stroke | 1.058–3.507 | 1.9 | 0.032 |
sCD40, soluble CD40; SUMMIT, The SUrrogate markers for Micro- and Macro-vascular hard endpoints for Innovative diabetes Tools.
Odds ratios were adjusted for age (years) and prevalent diabetes, hypertension, prior acute myocardial infarction, and stroke (yes vs. no).
Spearman correlations of CD40 and CD40L (A.U., CPIP cohort) measured in plaque homogenate versus plaque elements
Variable | CD40 (n=198) |
CD40L (n=198) |
||||
---|---|---|---|---|---|---|
r | r | |||||
Histology (area %) | ||||||
Smooth muscle α-actin | –0.070 | 0.33 | 0.33 | –0.142 | 0.048 | 0.094 |
Calcium (van Kossa) | –0.208 | 0.004 | 0.016 | –0.268 | 0.00017 | 0.00068 |
Oil Red O | 0.199 | 0.005 | 0.016 | 0.043 | 0.55 | 0.55 |
Vulnerability index |
0.178 | 0.014 | 0.029 | 0.180 | 0.014 | 0.041 |
Cytokines (pg/g) | ||||||
CXCL1 | 0.228 | 0.002 | 0.012 | 0.268 | 0.002 | 0.008 |
IL-6 | 0.305 | 0.00032 | 0.0022 | 0.235 | 0.001 | 0.007 |
IL-12p70 | 0.211 | 0.004 | 0.020 | 0.230 | 0.002 | 0.008 |
Tumor necrosis factor α | 0.391 | 3.4×10–8 | 3.1×10–7 | 0.290 | 6.9×10–5 | 0.0006 |
CCL5 | 0.021 | 0.772 | 0.772 | 0.200 | 0.007 | 0.014 |
CCL2 | 0.175 | 0.017 | 0.034 | 0.186 | 0.001 | 0.007 |
CCL4 | 0.202 | 0.006 | 0.020 | 0.186 | 0.011 | 0.014 |
PDGF-AA/AB/BB | 0.268 | 0.0002 | 0.0018 | 0.243 | 0.001 | 0.007 |
Plaque components (mg/g) | ||||||
oxLDL (μU/g) | 0.236 | 0.001 | 0.004 | 0.259 | 0.00041 | 0.0037 |
CD40L, CD40 ligand; A.U., arbitrary unit; CPIP, Carotid Plaque Imaging Project; CXCL1, C-X-C motif chemokine ligand 1; IL, interleukin; CCL, chemokine (C-C motif) ligand; PDGF, platelet-derived growth factor; oxLDL, oxidized low density lipoprotein.
Calculated as (CD68++glycophorin A++Oil Red O+ area%) / (smooth muscle α-actin++collagen++von Kossa+area%).