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Soluble Vascular Cell Adhesion Molecules May be Protective of Future Cardiovascular Disease Risk: Findings from the PREVEND Prospective Cohort Study

Aim: Soluble cell adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1, E-selectin, and P-selectin, have been suggested to be associated with cardiovascular disease (CVD) risk; however, the nature and magnitude of the association between VCAM-1 and CVD risk is uncertain. We aimed to assess the association of VCAM-1 with CVD risk and determine its potential utility for CVD risk prediction. Methods: VCAM-1 concentrations were measured at baseline in the PREVEND prospective study of 2,638 participants. Hazard ratios (95% confidence intervals [CI]) and measures of risk discrimination for CVD (e.g., C-index) and reclassification (i.e., net reclassification improvement) of participants were assessed. Results: During a median follow-up of 9.9 years, 614 CVD events occurred. Plasma VCAM-1 was weakly associated with several cardiovascular risk markers. In analyses adjusted for established cardiovascular risk factors, the hazard ratio (95% CI) for CVD per 1 standard deviation increase in loge VCAM-1 was 0.91 (0.84–0.99; P = 0.020), which remained consistent after additional adjustment for body mass index, alcohol consumption, triglycerides, renal function, and C-reactive protein; hazard ratio (95% CI) 0.89 (0.82–0.97; P = 0.006). Comparing the top versus bottom quintiles of VCAM-1 levels, the corresponding adjusted hazard ratios were 0.74 (0.57–0.96; P = 0.023) and 0.70 (0.54–0.91; P = 0.007) respectively. Adding VCAM-1 to a CVD risk prediction model containing conventional risk factors did not improve the C-index or net reclassification. Conclusions: Plasma VCAM-1 is inversely and independently associated with CVD. However, VCAM-1 provides no significant improvement in CVD risk assessment beyond conventional CVD risk factors.

of type 2 diabetes 26) . Given the uncertainty in the existing evidence, our primary objective was to characterize the shape and assess the magnitude and independence of the prospective association between VCAM-1 concentrations and CVD risk using a population-based sample of 2,638 participants free from pre-existing CVD at baseline. A secondary objective was to investigate the extent to which VCAM-1 concentrations could improve the prediction of first-onset CVD when added to a conventional CVD risk prediction model.

Materials and Methods
This report was conducted according to STROBE (STrengthening the Reporting of OBservational studies in Epidemiology) guidelines for reporting observational studies in epidemiology (Appendix 1) 27) .

Study Design and Participants
The study population, consisting of a representative sample of inhabitants living in the city of Groningen in the Netherlands, were participants in the Prevention of Renal and Vascular End-stage Disease (PREVEND) study, a longitudinal general population-based study designed to investigate the natural course of urinary albumin excretion and its relationship to renal disease and CVD. The actual PREVEND cohort (N 8,592) was recruited from inhabitants aged 28 -75 years invited for pre-screening from the city of Groningen in the Netherlands. Baseline examinations and measurements were performed between 1997 and 1998. Participants with a prevalent history of CVD, liver disease, renal disease, or malignancy were excluded in the present analysis. Given that assays for VCAM-1 could only be measured in a random subset of PREVEND participants because of known to play a pivotal role in the development of atherosclerosis. Endothelial cells play an important role in the regulation of homeostasis, arterial wall structure and tension, and undisturbed blood flow 4) . Soluble cell adhesion molecules [such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin, and P-selectin], which are glycoproteins integral to the cell membrane, are responsible for the adhesion of different cells onto the endothelial surface 5) . Cell adhesion molecules facilitate the attachment of circulating leucocytes to the endothelium, to sites of inflammation, and their subsequent movement and accumulation in arterial walls 6-9) , all of which are processes pivotal in the development and progression of atherosclerosis 10) . E-selectin and P-selectin mediate transient rolling of leucocytes along the endothelium 11) , whiles VCAM-1 and ICAM-1 mediate stronger attachment of leucocytes to the endothelium 9) . Circulating levels of these cell adhesion molecules (including VCAM-1) are associated with several cardiovascular risk factors and markers 12) and higher levels have been detected in atherosclerotic lesions 13,14) and in patients with ischemic stroke, coronary artery disease, and CVD [15][16][17][18] .
Emerging evidence indicates that these soluble cell adhesion molecules may be linked to the future risk of CVD. A number of prospective studies, based in general population settings, have generally reported positive and independent associations between CVD outcomes and circulating levels of E-selectin, P-selectin, or ICAM-1 16, [19][20][21][22][23][24] ; however, the association of VCAM-1 with CVD is unclear as previous studies have mostly reported null associations between VCAM-1 and CVD outcomes 21,23,25) . A single study demonstrated an increased risk of cardiovascular mortality with increased levels of VCAM-1, but the association was particularly evident among subjects with a history

Risk Factor Assessment
During two outpatient visits by study participants, baseline data on sociodemographics, medical history, physical health, including anthropometric measurements, and risk markers for CVD were assessed. Blood biomarker analyses were performed on plasma and serum venous samples taken from participants after an overnight fast and 15 minutes of rest. Concentrations of VCAM-1 were determined in ethylenediaminetetraacetic acid (EDTA) plasma (that had been stored frozen at 80 for 15 years) using the sandwich enzyme-linked immunosorbent assay (Diaclone human sVCAM-1/CD106 ELISA, Diaclone SAS, France) with the minimal detection limit being 0.6 ng/ml. The inter-assay coefficients of variation were 5.94% and 1.44% in the lower normal and financial constraints, our final cohort for this analysis included 2,638 participants with complete information on serum VCAM-1, relevant covariates, and cardiovascular outcomes. Participants in the PREVEND study included men and women recruited from an ethnically homogeneous population, and therefore characteristics of excluded participants were generally not different from those included in the final analyses (Appendix 2). The derivation of the analytic sample is reported in Appendix 3. The medical ethics committee of the University Medical Center Groningen duly approved the PREVEND study, which was conducted in accordance with the Declaration of Helsinki. Each participant provided written informed consent for voluntary participation, which was documented in a consent form approved by the medical ethics committee. BMI, body mass index; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate (as calculated using the Chronic Kidney Disease Epidemiology Collaboration combined creatinine-cystatin C equation); HDL-C, high-density lipoprotein cholesterol; ; hsCRP, high sensitivity C-reactive protein; IQR, interquartile range; SBP, systolic blood pressure; UAE, urinary albumin excretion; VCAM-1, vascular cell adhesion molecule-1; WHR, waist-to-hip ratio tion for cardiovascular morbidity was retrieved from PRISMANT, the Dutch national registry of hospital discharge diagnoses 35) . All data were coded according to the International Classification of Diseases, Ninth Revision (ICD-9) until 01-01-2009. The data were coded according to ICD-10 codes after this date. First-onset CVD was defined as the combined endpoint of acute myocardial infarction, acute and subacute ischemic heart disease, coronary artery bypass grafting or percutaneous transluminal coronary angioplasty, subarachnoid hemorrhage, intracerebral hemorrhage, other intracranial hemorrhage, occlusion or stenosis of the precerebral or cerebral arteries, and other vascular interventions, such as percutaneous transluminal angioplasty or bypass grafting of peripheral vessels and aorta. Coronary heart disease events were defined as fatal or nonfatal myocardial infarction (MI), fatal or nonfatal ischemic heart disease (IHD), coronary artery bypass grafting (CABG), and percutaneous transluminal coronary angioplasty (PTCA). Stroke events were defined as subarachnoid higher normal range, respectively. Plasma glucose was measured by dry chemistry (Eastman Kodak, Rochester, New York). Total cholesterol, high-density lipoprotein cholesterol (HDL-C), high sensitivity C-reactive protein (hsCRP), triglycerides, serum creatinine, and serum cystatin C were measured using standard laboratory methods, which have been described previously [28][29][30][31][32] . Urinary albumin excretion (UAE) was calculated as the mean of two 24-hour urine collections. Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) combined creatinine-cystatin C equation 33) . Type 2 diabetes mellitus and hypertension were defined as previously reported 31,32,34) .

Outcome Assessment for CVD
The primary endpoint in our study was firstonset CVD, with incident CHD and stroke as secondary endpoints. Ascertainment of dates and causes of death were by record linkage with the Dutch Central Bureau of Statistics. Information on hospitaliza- The cross-sectional associations of serum VCAM-1 with CVD risk markers were assessed by calculating Pearson's correlation coefficients adjusted for age and sex. Analyses of the associations between serum VCAM-1 concentrations and risk of cardiovascular outcomes involved time-to-event Cox proportional hazards models after confirmation of assumptions of proportionality of hazards 37) . Collinearity diagnostics using the variance inflation factor 38) showed no evidence of collinearity among covariates, including hemorrhage, intracerebral hemorrhage, other and unspecified intracranial hemorrhage, occlusion and stenosis of precerebral or cerebral arteries, and carotid obstruction. Further details on the definition of cardiovascular outcomes are provided in previous reports 34,36) .

Statistical Analyses
Skewed variables (e.g., VCAM-1, hsCRP, triglycerides, creatinine, and UAE) were natural logarithm (loge) transformed to achieve approximately normal distributions. Descriptive analyses were performed to summarize baseline characteristics of participants. The   model based on risk factors included in the Framingham CVD Risk Score (i.e., age, sex, smoking status, SBP, total cholesterol, and HDL-C) 44) . For participants with at least 10 years of follow-up, reclassification of participants was assessed using the net-reclassification-improvement (NRI) 45) across predicted 10-year cardiovascular risk categories of low ( 5%), intermediate (5 to 7.5%), and high ( ≥ 7.5%) risk 46) . Risk prediction analysis was restricted to participants without a known history of diabetes or CVD at baseline. All statistical analyses were conducted using Stata version 14 (Stata Corp, College Station, Texas).

Baseline Characteristics and Correlates of VCAM-1
Data were available for 2,638 participants without a known history of CVD at baseline. The mean age of overall participants at baseline was 51 (SD 13) years, and 49% were males ( Table 1). The mean (SD) loge VCAM-1 was 6.79 (0.45) ng/ml. Except for VCAM-1 levels, there were significant differences in clinically relevant subgroups and levels of risk markers between participants who did and did not develop CVD. Loge VCAM-1 levels were weakly correlated with risk markers for CVD. Baseline VCAM-1 concentrations were higher by 7% in men compared with women, by 17% in people with diabetes compared with people without diabetes, and by 6% in people with a history of hypertension compared with those without a history. The levels were lower by 6% in cur-between UAE and eGFR. To characterize the shape of the association, hazard ratios (HRs) estimated within quintiles of baseline VCAM-1 concentrations relative to the bottom quintile were plotted against mean loge VCAM-1 levels in each quintile using floating absolute risks (FARs) 39) as described in detail previously 40,41) . Vascular cell adhesion molecule-1 was modeled as both continuous (per 1 SD) higher loge serum VCAM-1 levels) and categorical (quintiles defined according to the baseline distribution of serum VCAM-1 level) variables. To assess the independence of the association, HRs were calculated with progressive adjustment for age and sex, other established CVD risk factors (smoking status, history of diabetes, SBP, total cholesterol, and HDL-C), potential confounders [body mass index (BMI), alcohol consumption, fasting glucose, triglycerides, eGFR, and hsCRP], and UAE. Effect modification by individual characteristics, such as age, sex, and other cardiovascular risk markers, were assessed using interaction tests. To limit potential biases due to reverse causation, we carried out subsidiary analyses that excluded the first two years of follow-up, participants on regular antihypertensive medication, and participants on regular lipid-lowering medication (statins).
To assess whether adding information on VCAM-1 to a model that included conventional cardiovascular risk factors 42) is associated with improvement in CVD risk prediction, we calculated measures of discrimination for censored time-to-event data (Harrell's C-index 43) ) and reclassification. To investigate the change in C-index, we added VCAM-1 to a     Table 2).
A near log-linear inverse relationship was observed between VCAM-1 levels and CVD risk in analyses adjusted for established CVD risk factors (Fig. 1). CI, confidence interval; HR, hazard ratio; Q, quintile; SD, standard deviation; VCAM-1, vascular cell adhesion molecule-1 Model 1: Age and sex Model 2: Model 1 plus smoking status, history of diabetes, systolic blood pressure, total cholesterol, and high-density lipoprotein-cholesterol Model 3: Model 2 plus body mass index, alcohol consumption, glucose, loge triglycerides, estimated glomerular filtration rate (as calculated using the Chronic Kidney Disease Epidemiology Collaboration combined creatinine-cystatin C equation), and loge high sensitivity C-reactive protein Model 4: Model 3 plus loge urinary albumin excretion cardiovascular risk markers. In analyses adjusted for established CVD risk factors, we observed an inverse near linear association of VCAM-1 with risk of CVD. The association remained consistent on further adjustment for several potential confounders, including UAE. The findings were consistent across several clinically relevant subgroups and levels of cardiovascular risk markers. The associations were similar in several sensitivity analyses. Similar inverse associations were observed for CHD risk. There was no statistically significant evidence of an association with stroke in analyses adjusted for established risk factors and several potential confounders; however, further adjustment for UAE strengthened the association due to a negative confounding effect of UAE on the association. This was unexpected given the positive correlation of VCAM-1 with UAE and the knowledge that albuminuria is a putative marker for endothelial dysfunction and is independently associated with increased vascular risk 47) . Indeed, further exploration of the current data showed a positive and independent association between UAE and stroke risk. Findings from the assessments of improvements in measures of risk discrimination and reclassification indicate that additional information on VCAM-1 measurements provides no significant improvement in CVD risk prediction.

Comparison with Previous Work
To our knowledge, this is the first study to assess the association between VCAM-1 and the composite outcome of CVD and to report inverse associations first two years of follow-up, participants on regular antihypertensive medication, and participants on regular lipid-lowering medication ( Table 4). The associations did not vary significantly by levels or categories of several clinically relevant individual characteristics (Fig. 2). In separate analyses for other cardiovascular outcomes, an inverse association was also demonstrated for CHD in analyses adjusted for several established risk factors and potential confounders; however, for stroke, the initial null association observed in analyses adjusted for established risk factors and several potential confounders became statistically significant with additional adjustment for loge UAE (Table 3).

VCAM-1 and CVD Risk Prediction
A CVD risk prediction model containing risk factors based on the Framingham CVD Risk Score yielded a C-index of 0.7590 (95% CI: 0.7404 -0.7776). After addition of information on VCAM-1 levels, the C-index was 0.7596 (0.7410 to 0.7783), representing a non-significant increase of 0.0006 ( 0.0014 to 0.0026; P 0.543). There was no improvement in the classification of participants into predicted 10-year CVD risk categories (NRI: 0.00%, 0.41% to 1.42%; P 0.996).

Summary of Main Findings
In this population-based study of individuals without a history of CVD at baseline, baseline VCAM-1 levels were weakly correlated with several (PRIME), which enrolled apparently healthy men aged 50 -59 years at baseline 21) . Similarly, no statistically significant evidence of an association was found between VCAM-1 and CHD risk in the Health Professional Follow-up Study 48) . On the contrary, Jager and colleagues, in a 7.4 year follow-up of the Hoorn study of 631 participants, demonstrated elevated VCAM-1 levels to be independently associated with cardiovascular mortality in subjects with type 2 diabetes 26) . However, a subset of study participants (24%) had a history of CVD at baseline, and though the study authors accounted for this in their models, stratified analysis by history of CVD was not conducted.
between VCAM-1 and the outcomes of CVD, CHD, and stroke. Previous studies conducted on the topic have mostly reported no evidence of any associations of VCAM-1 with cardiovascular outcomes. In analysis of the British Regional Heart Study cohort, Malik and colleagues demonstrated no statistically significant evidence of an association of VCAM-1 with CHD 25) . In addition, they conducted a pooled analysis of four studies (including their study), but this did not change the null findings. Luc and colleagues found no evidence of an association between VCAM-1 and the risk of acute coronary events in their analysis of the Epidemiological Study of Myocardial Infarction Study Hazard ratios were adjusted for age, sex, smoking status, history of diabetes, systolic blood pressure, total cholesterol, and high-density lipoprotein cholesterol; CI, confidence interval (bars); CVD, cardiovascular disease; Estimated GFR, glomerular filtration rate (as calculated using the Chronic Kidney Disease Epidemiology Collaboration combined creatinine-cystatin C equation); HDL-C, high-density lipoprotein cholesterol; HR, hazard ratio; hsCRP, high sensitivity C-reactive protein; SD, standard deviation; UAE, urinary albumin excretion; , P -value for interaction; cut-offs used for fasting glucose, body mass index, systolic blood pressure, total cholesterol, HDL-C, triglycerides, estimated GFR, and hsCRP are median values.  14,52) . A previous study has also suggested that ICAM-1 and VCAM-1 function at opposite ends of the atherosclerosis spectrum and that their interaction with CVD involves a more complex process 48) . There is also a possibility that the null associations demonstrated in previous studies may be attributable to study design and participant characteristics such as sample size, duration of follow-up, and age and sex of participants. Compared to some previous studies 21,48,53) , our study had a longer follow-up and a higher event rate. Given the postulated role of VCAM-1 in the early atherosclerotic process 54,55) , longer follow-up durations may be needed to demonstrate any association that we demonstrated in our study. In addition, the majority of previous studies have demonstrated these null associations in cohorts only limited to men, whereas the current study involved both men and women 21,48,56) . Indeed, our subgroup analyses showed no statistically significant associations in both genders, though there were trends towards protective associations. Finally, while our cohort involved relatively younger to older participants, previous studies employed middle-aged to elderly participants in their samples 21,48,56) . These explanations and mechanisms are however speculative and further work is warranted to identify key underlying biological mechanisms through which VCAM-1 may play a protective role in the development of CVD.

Implications of Findings
The findings of our study may have clinical implications. Irrespective of the irrelevance of the utility of VCAM-1 in CVD risk prediction, its use for CVD risk prevention and treatment may still have relevance. The overall evidence supports the possibility that elevated VCAM-1 levels might protect from CVD events in the general population. It highlights the vascular endothelium as a potential therapeutic target for the prevention and/or treatment of CVD. Circulating VCAM-1 levels may be a potential biomarker for monitoring the effects of treatment on the vascular endothelium. Nonetheless, given that this is the first study to demonstrate these findings; further studies are needed to replicate these findings.

Strengths and Limitations
This is the first simultaneous and comprehensive assessment of the association of VCAM-1 with the risk of incident composite CVD, CHD, and stroke. Other strengths include the nationally representative sample, the large endpoint numbers by most standards, the high response and long-term follow-up, and measurements on a comprehensive panel of lifestyle In our study, stratified analysis by history of diabetes showed no statistically significant evidence of effect modification. A protective effect of VCAM-1 on CVD risk was observed for participants without a history of diabetes, but the results were not statistically significant for participants with a history of diabetes. Given the small sample and low event rate for this subgroup, the null results could be attributed to low power; however, a trend towards a protective effect was observed.

Possible Explanations for Findings
Our findings would suggest that higher circulating concentrations of VCAM-1 are protective of cardiovascular outcomes. There was however no statistically significant evidence of an association of VCAM-1 with the specific outcome of stroke, which could be due to the low event rate. Though previous studies conducted in general population settings have failed to demonstrate any statistically significant association of VCAM-1 with CVD risk, an increased risk of CVD with elevated levels of VCAM-1 has been suggested, given that circulating levels of other soluble adhesion molecules (such as E-selectin, P-selectin, or ICAM-1) have been found to be positively and independently associated with CVD outcomes 16, [19][20][21][22][23][24] . Indeed, the therapeutic effect of leech enzyme extract on atherosclerosis (a traditional Chinese medicine commonly used for treating CVD) has been shown to be manifested via inhibiting the expression of adhesion molecules such as ICAM-1 and VCAM-1 49) . The mechanistic pathways for the link between elevated VCAM-1 levels and its protective effect on the development of CVD are unclear, as increased expression of soluble adhesion molecules have been suggested to play a direct role in the pathogenesis of atherosclerosis 50) via endothelial dysfunction and inflammation. These observations may reflect the differences in the functions of these adhesion molecules and suggest that there may be important pathophysiologic differences between these molecules and the genesis of atherosclerosis. E-selectin is expressed exclusively by endothelial cells and therefore may be a better marker of endothelial dysfunction, whereas ICAM-1 and VCAM-1 are expressed on a number of cells, including endothelial cells and leukocytes 51) . Whiles E-selectin and P-selectin mediate transient rolling of leucocytes along the endothelium 11) , ICAM-1 and VCAM-1 allow stronger leukocyte rolling and attachment to the endothelium 9) . Intercellular adhesion molecule-1, which has been consistently demonstrated to be associated with incident vascular events, is highly expressed by endothelial cells in human atheroma, and low levels have been expressed on normal endothelium. In contrast, VCAM-1 is not expressed at all on normal endothe-and biological markers that permitted adequate adjustment for potential confounders. Reverse-causation bias was minimized as individuals with prevalent CVD at baseline were excluded from the analyses at the onset. The analyses were detailed, which included comparison of the associations at different levels or categories of vascular risk markers, several sensitivity analyses to supplement and contextualize the principal findings, and risk prediction analyses. Limitations include the potential for residual confounding due to errors in measurements of risk marker and other unmeasured confounders; absence of repeat measurements of VCAM-1 which precluded the ability to correct for regression dilution bias, which could have underestimated the association between long-term VCAM-1 levels and CVD; and analyses which were based on a predominantly white-European population, therefore the findings may not be generalizable to individuals of different ethnicities. In addition, blood samples for VCAM-1 measurements involved long-term storage which could have resulted in under-estimation of the associations due to sample degradation. However, it has been reported that VCAM-1 is not known to be influenced by repeated freeze-thaw cycles after storage 57) .
In conclusion, available evidence for the first time shows that VCAM-1 is protective of incident CVD. Addition of information on VCAM-1 to conventional risk factors provides no significant improvement in CVD risk prediction in the general population. Further studies are needed to replicate this association and the etiological mechanisms underlying these data require investigation.

Funding Sources
The Dutch Kidney Foundation supported the infrastructure of the PREVEND program from 1997 to 2003 (Grant E.033). The University Medical Center Groningen supported the infrastructure from 2003 to 2006. Dade Behring, Ausam, Roche, and Abbott financed laboratory equipment and reagents by which various laboratory determinations could be performed. The Dutch Heart Foundation supported studies on lipid metabolism (Grant 2001-005). The Dutch Top Institute Food and Nutrition supported the measurements of sVCAM-1 (Grants within the Cardiovascular Health program, CH-001 and CH-003). The contents of this paper are solely the responsibility of the authors and do not represent the views of the Sponsors. The sponsors did not participate in the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation of the manuscript.