Seventy-six consecutive patients who fulfilled the American College of Rheumatology criteria for RA 25 were invited to participate. Only two patients refused, and the remaining 74 were included. Patients receiving lipid-lowering and antidiabetic medications were excluded. The baseline clinical and routine laboratory characteristics of the included RA patients are reported elsewhere 26. Eighty-three individuals with no known diseases and who were not taking any medication agreed to act as controls. These were friends, patient friends and laboratory staff. Three of them were excluded because they were found to have impaired fasting glucose (plasma glucose >5.5 mmol/l); the remaining 80 were included in the study. The study was approved by the Ethics Committees for Research on Human Subjects (Medical) of the University of the Witwatersrand and the Milpark Hospital.

We recorded traditional cardiovascular risk factors in both RA patients and control individuals using previously reported methods (Table 1) 26. We also recorded the following nontraditional cardiovascular risk factors: circulating high-sensitivity C-reactive protein (hs-CRP), cytokines (IL-1, IL-6 and tumor necrosis factor [TNF]-α), cytokine suppressant therapy (disease-modifying antirheumatic drug [DMARD] and prednisone use) and biomarkers of endothelial dysfunction (Tables 1 and 2). Blood sampling was performed on the same day that clinical data were recorded. Fasting blood samples were taken between 08:00 and 10:00 hours. All laboratory analyses except for assessments of cytokines and biomarkers of endothelial dysfunction were performed within 2 hours of sampling. These comprised lipids, hs-CRP and other laboratory tests that were performed for the second component of the study, which was conducted in the RA patients only (see below). Blood samples drawn for determination of cytokines and biomarkers of endothelial dysfunction were stored at -70°C before laboratory testing. Cytokines and adhesion molecules were measured using enzyme-linked immunosorbent assays (Hiss Diagnostics GmbH, Freiburg, Switzerland). The intra-assay and inter-assay coefficients of variation (respectively) were 5.1% and 8.6% for IL-1, 3.4% and 5.2% for IL-6, 6.9% and 7.4% for TNF-α, 3.1% and 5.2% for VCAM-1, 4.1% and 7.7% for ICAM-1, and 5.4% and 6.0% for ELAM-1.

For the second component of the study, recorded variables other than cytokines and biomarkers of endothelial dysfunction are summarized in Tables 3 and 4. A descriptive analysis of these variables in the current cohort was previously reported 26. Although 10 patients were on thyroxine replacement therapy and eight had subclinical hypothyroidism (thyrotropin >4 μIU/ml and normal thyroxine levels) 27, none had clinical hypothyroidism (decreased thyroxine levels) at the time of the study. IgM rheumatoid factor was determined using an immunoturbidimetric test on the Olympus AU 600 analyzer. The intra-assay and inter-assay coefficients of variation for rheumatoid factor were 3.4% and 4.6%, respectively.

The CCAs were evaluated using high resolution B-mode ultrasound. Details of this investigation in the present cohort were previously reported 26.

The recorded baseline characteristics in the RA patients and control individuals comprised traditional cardiovascular risk factors, hs-CRP and cytokines (Table 1). RA patients were younger, exercised less, had a higher waist circumference, higher systolic and diastolic blood pressures, and higher triglyceride levels than did control individuals. Five RA patients had diabetes that was being treated with dietary intervention only. With regard to nontraditional cardiovascular risk factors, hs-CRP, IL-1, IL-6 and TNF-α concentrations were higher in patients than in control individuals, and DMARD and prednisone were used by 56 and 11 RA patients, respectively. No patient was being treated or had been treated with biological agents at the time of the study.

Results for biomarkers of endothelial dysfunction in patients and control individuals are shown in Table 2. In univariate analysis, all three biomarkers of endothelial dysfunction (VCAM-1, ICAM-1 and ECAM-1) were higher in patients than in control individuals. These differences remained significant after controlling for cytokine suppressant agent (DMARD and prednisone) use (model 1) or traditional cardiovascular risk factors (model 2). After controlling for nontraditional cardiovascular risk factors, the differences in ELAM-1 between patients and control individuals were no longer significant (model 3). When traditional and nontraditional cardiovascular risk factors were simultaneously controlled for, the differences in levels of all three biomarkers of endothelial dysfunction were no longer significant between patients and control individuals (model 4).

In univariate analysis, VCAM-1 was related to rheumatoid factor titres and low GFR, ICAM-1 to rheumatoid factor titres and IL-6, and ELAM-1 to IL-6 (Table 4). None of the other recorded baseline characteristics in the 74 RA patients were associated with biomarkers of endothelial dysfunction (Tables 4 and 5).

After controlling for traditional cardiovascular risk factors in multivariable regression models, IL-6 was predictive of all three biomarkers of endothelial dysfunction, and rheumatoid factor titre and low GFR were both predictive of VCAM-1 and ICAM-1 (Table 5). Additional controlling for thyrotropin levels did not materially alter these models (data not shown).

As previously reported, the median (range) CCA IMT was 0.654 mm (0.496–1.150 mm), and 23 (31%) patients had plaque 26. Twenty-one (28%) patients had a CCA IMT under 0.600 mm and no plaque. The role of clinical and routine laboratory characteristics as predictors of common carotid atherosclerosis in the present cohort was also previously reported 26. VCAM-1 concentrations correlated with the CCA IMT (r_s = 0.280; P = 0.016) and were higher in patients with plaque than in those without plaque (769 pg/ml [391–2073 pg/ml] versus 703 pg/ml [445–2001 pg/ml]; P = 0.043). The associations between CCA findings and other biomarkers did not achieve statistical significance.

We found that biomarkers of endothelial dysfunction were markedly higher in RA patients than in healthy control individuals. Increased circulating adhesion molecule concentrations have been reported in RA 23242829. Our patients also had higher hs-CRP and IL-1, IL-6 and TNF-α concentrations than did control individuals, but these nontraditional cardiovascular risk factors did not fully account for the differences in biomarkers of endothelial dysfunction between patients and control individuals. Indeed, the RA patients also had generally less favourable traditional cardiovascular risk factor profiles than healthy control individuals.

The younger age of the healthy control individuals included reflects the difficulties in recruiting healthy aged persons who are not taking any medication. Hence, we controlled for age as well as other traditional cardiovascular risk factors when assessing the differences in biomarkers of endothelial dysfunction between patients and control individuals. Of interest, the body mass indices were similar in both groups, but patients had higher waist circumference, blood pressure and triglyceride levels. The latter are features of the metabolic syndrome 530. Although differences in age and exercise habits might have contributed to these findings, features of the metabolic syndrome also cluster in RA because of inflammation-induced insulin resistance 530.

In multivariable models, traditional cardiovascular risk factors attenuated the differences in biomarkers of endothelial dysfunction between patients and control individuals to a lesser extent than did nontraditional cardiovascular risk factors. However, the differences in endothelial function between patients and control individuals were no longer significant only after both traditional and nontraditional cardiovascular risk factors had been controlled for. Our results also show the need for comprehensive assessment of cardiovascular risk factors in healthy individuals when comparing their endothelial function with that in RA patients.

We investigated the potential role of an extensive range of patient characteristics in endothelial dysfunction in RA. IL-6, rheumatoid factor titre and low GFR predicted endothelial dysfunction, as assessed using biomarkers.

In multivariable analysis, IL-6 predicted endothelial dysfunction independent of traditional cardiovascular risk factors. Chronic cytokine release from inflamed joints was previously implicated in the increased production of adhesion molecules by endothelial cells in RA 431. Circulating cytokines could impair endothelial function directly 4 or through their effects on insulin sensitivity and on CRP and fibrinogen (a major determinant of erythrocyte sedimentation rate 32) production by the liver 4. In the present cohort of unselected RA patients, IL-6 was more strongly associated with endothelial dysfunction than were CRP, erythrocyte sedimentation rate and insulin resistance. In contrast to IL-6, circulating IL-1 and TNF-α concentrations were not associated with endothelial dysfunction. IL-1 and TNF-α are major proinflammatory cytokines in RA joints and stimulate IL-6 production by synovial fibroblasts 3334353637, whereas IL-6 is a major circulating cytokine in RA that induces the acute phase response, production of immunoglobulins by B cells and neuroendocrine alterations 33343738. IL-6 promotes adhesion molecule expression and stimulates macrophages to secrete monocyte chemotactic protein-1 39. Circulating IL-6 concentrations also predict cardiovascular disease in the general population, independent of hs-CRP levels 39.

Apart from IL-6, rheumatoid factor was also predictive of endothelial dysfunction independent of traditional cardiovascular risk factors in the present cohort. The mechanism underlying the strong association between rheumatoid factor and endothelial dysfunction in RA cannot be discerned from our data. However, rheumatoid factor may directly cause endothelial injury 31. Direct evidence for a role for humoral immunity in atherosclerosis was found by George and coworkers 40. In their study repeated intraperitoneal administration of IgG from serum of mice immunized with heat shock protein 65 enhanced fatty streak formation in mice in comparison with their control anti-bovine serum albumin injected littermates 40. Rheumatoid factor is produced by B cells that are highly effective at presenting antigens to T cells 41 and T-cell activation in rheumatoid synovium is B-cell dependent 42. The recently reported remarkable efficacy of B-cell depletion in rheumatoid factor positive RA with rituximab 43 indicates that B cells are key contributors to the immunopathogenesis of RA. In a recent autopsy report on two RA patients with coronary artery disease the coronary plaques and adventitia contained large numbers of B cells, whereas in coronary artery disease it is typical for lymphocytic infiltrates to consist almost exclusively of T cells 44. These reports and our findings support a role for humoral mechanisms in RA atherogenesis.

Finally, a low GFR also predicted endothelial dysfunction in our RA patients. Although only four (5%) patients had an elevated serum creatinine (>115 μmol/l), GFR estimation using the Cockcroft–Gault equation 45 revealed that 16 (22%) patients had a GFR under 60 ml/min 26, which is indicative of chronic kidney disease. The high prevalence of cardiovascular disease in individuals with chronic kidney disease has been amply reported 45. This is attributable to the high prevalence of traditional risk factors such as older age, high total cholesterol, low high-density lipoprotein cholesterol and diabetes, as well as to nontraditional risk factors such as oxidant stress, inflammation and, to a lesser extent, hyperhomocysteinaemia. In the present cohort, varimax rotated factor analysis confirmed strong relationships between low GFR, age and hyperhomocysteinaemia 26. Also, independent of age as well as other traditional cardiovascular risk factors, a low GFR remained independently predictive of endothelial dysfunction in our patients whereas hs-CRP was not associated with circulating adhesion molecules. In support of an important role of oxidant stress in cardiovascular disease complicating chronic kidney disease, both vitamin E 800 U daily and acetylcysteine 600 mg twice daily were shown to decrease cardiovascular events in randomized controlled trials in haemodialysis patients 45. Whether such interventions could decrease cardiovascular disease in RA may deserve further study.

In a previous study conducted by Wallberg-Jonsson and coworkes 23 in 39 RA patients, ICAM-1 and selectin concentrations were found to be related to ultrasonographically detected CCA and femoral artery plaque as well as to haemostatic factors of endothelial origin. We found that VCAM-1 was associated with ultrasonographically determined CCA IMT and plaque. Taken together, these data further support the contention that circulating adhesion molecules are linked to cardiovascular disease in RA.

We assessed cardiovascular risk comprehensively and our results are in keeping with previously reported paradigms of cardiovascular disease in RA 4531. However, our findings must be reproduced in a longitudinal study and investigations of a larger cohort is likely to reveal more independent associations between biomarkers of endothelial dysfunction and cardiovascular risk factors.