Atherosclerotic disease is increased in recent-onset rheumatoid arthritis: a critical role for inflammation

Rheumatoid arthritis patients

Patients (n = 40), including 13 men and 27 women with a mean age of 53 years (range 22–78 years), were recruited at first presentation between May 2004 and December 2005 when attending scheduled appointments at our specialized early RA clinic. All patients aged 18 years or older attending the early RA clinic during the study recruitment period were invited to take part in the study. All participants met the American College of Rheumatology 1987 revised criteria for diagnosis of RA. At presentation, five patients were taking low-dose prednisone, and three patients were using nonsteroidal anti-inflammatory drugs or cyclooxygenase-2 inhibitors. Unless contraindicated, all RA patients were started on triple therapy of methotrexate, sulphasalazine and hydroxychloroquine after RA diagnosis was confirmed, and the doses were titrated over time according to disease activity criteria with the aim of achieving clinical remission.

Nonrheumatoid arthritis patients

Control subjects (n = 40) were healthy volunteers recruited from the same geographical area, including 13 men and 27 women with a mean age of 53 years (range 21–80 years), matched to the RA patients' age, sex and CV risk factors. We matched against a database of >1,000 cIMTs recruited in a primary prevention setting. The primary match was for age and gender, after which we matched for number and type of risk factors on a categorical basis. While exact blood pressure or lipid levels were not matched, we were able to match for hypertension and hyperlipidemia, in addition to smoking status in almost all the cases.

Study procedure

The study was approved by the human research ethics committee at Princess Alexandra Hospital, and all subjects provided written informed consent. RA patients underwent a comprehensive clinical evaluation and measurements by a rheumatologist and a clinical metrologist, including the American College of Rheumatology core set of disease activity criteria, the Health Assessment Questionnaire and calculation of the disease activity score. Demographic variables were ascertained by questionnaire. Disease duration was determined by the length of RA symptoms. Disease activity in RA patients was measured using the disease activity score (DAS 4v), a validated composite score incorporating tender joint counts (out of 53) and swollen joint counts (out of 44), the erythrocyte sedimentation rate, and the patient global assessment of disease activity (100 mm visual analogue scale) [15].

Cardiovascular risk factor ascertainment

CV risk factors were ascertained among RA patients and controls at presentation. Cigarette smoking was assessed by questionnaire, which included details about past and present smoking habits, the number of cigarettes smoked per day and the smoking duration. Diabetes mellitus was classified as present if diagnosed by a physician or if patients were taking antidiabetic medications.

History of previous angina was classified as present if it had been reported by physician; and history of previous myocardial infarction was classified as present if development of either, first, a typical rise and gradual fall (troponin) or a more rapid rise and fall of biochemical markers of myocardial necrosis (with at least one of ischaemic symptoms, development of pathologic Q waves on ECG (electrocardiogram), or ECG changes indicative of ischaemia (ST-segment elevation or depression)) or, second, either new pathological Q waves on serial ECGs or pathological changes of healed or healing myocardial infarction was reported [16].

History of stroke or transient ischaemic attack was defined by admission to the hospital with computed tomography evidence of ischaemic occlusion or with carotid endarterectomy, or presentation with stroke/transient ischaemic attack symptoms with significant plaque on carotid ultrasound and neurological sequelae, with exclusion of subarachnoid haemorrhage and space-occupying lesions. Family history of CV attack or cerebrovascular attack before age 65 in first-degree relatives was determined by questionnaire.

The body mass index was calculated as weight (kilograms) divided by the square of the height (metres). History of hypercholesterolemia and hypertension were determined by diagnosis and recording as such in medical records by a physician, or use of lipid-lowering drugs or antihypertensive medication. Blood pressure was measured at first presentation.

The 10-year coronary risk was calculated using the Framingham equation. Laboratory data included fasting total cholesterol levels, low-density lipoprotein levels, high-density lipoprotein levels, very-low-density lipoprotein levels, triglyceride levels, glucose levels, C-reactive protein (CRP) levels, the erythrocyte sedimentation rate, haemoglobin levels, anticitrullinated peptide levels, rheumatoid factor levels, antinuclear antibodies, and high-resolution HLA-DRB genotyping.

Measurement of intima-media thickness and plaque

Analyses were carried out on RA patients within 1–4 weeks after RA diagnosis, and generally before commencement of antirheumatic therapy. Ultrasound images were frozen by ECG triggering (top of R-wave) to minimize variability depending on changes in the cIMT and lumen diameter occurring during the cardiac cycle [7, 17]. Offline analysis of the images was performed on a workstation using standard software for automated measurement of the intima-media thickness (IMT) (QLab 4.2.1 with IMT plugin, version 1.1; Philips Ultrasound, Bothell, WA, USA). The algorithm uses a maximum-slope edge-detection technique, beginning in the vessel lumen and detecting the first maximum slope of the change in signal for the near and far walls, and repeating the analysis to identify the media–adventitia interface. The software calculates the mean and standard deviation for the IMT thickness in a region-of-interest box, placed by the observer perpendicular to the vessel walls, with the measurements performed on the R wave of the ECG.

Care was taken not to include any plaque detected in the carotid artery in the IMT measurement. Images were obtained of the anterior, lateral and posterior views from both the right and left carotid arteries; because of extraneous noise and gain dependency in the near field, however, only the measurements of far wall views were recorded. These measurements were averaged to provide a mean IMT. The intra-observer variation and the coefficient of variance of the IMT from this institution are 0.01 ± 0.04 mm (5%). For each subject the average IMT of six frozen images was calculated, and the mean cIMT ((left + right)/2) was taken as a measure of the wall thickness of the distal common carotid artery.

Both extracranial carotid arterial systems were extensively scanned to identify plaque, which was defined as a focal widening relative to adjacent segments, with protrusion into the lumen composed of calcified deposits (hard plaque), noncalcified deposits (soft plaque) or a combination of calcification and noncalcified material. Plaque was considered present if visualized in the full diameter of the vessel; that is, both the proximal and distal parts of the plaque were attached to the typical double-lined intima-media structure and the double lines were also visible on the opposite side of the lumen [18]. Examination and analysis of the cIMT and plaque were performed by the same investigator (SH).

Statistical analysis

Continuous variables are described as the mean ± standard deviation, and categorical variables presented as the percentage. Log transformations were applied to non-normally distributed data. The chi-squared test was used to analyse categorical variables. cIMT differences between RA patients and controls were tested with a two-sample t test. The association of the cIMT with age, continuous CV variables and disease activity variables was evaluated using linear regression analysis.

In the absence of previous cIMT data in early RA, an a priori power calculation was not possible concerning the study sample size. Based on an expected IMT of 0.58 ± 0.09 (the mean in middle-aged asymptomatic primary prevention patients in our practice), however, we anticipated that a group of 40 early RA patients and 40 matched control individuals would have a 90% power to permit the detection of the expectation of a 10% greater IMT in RA patients, and a significant difference (P < 0.05) in the IMT between RA patients and control subjects. In view of the sample size of 40, the four variables that were significant at P ≤ 0.01 on univariate linear regression analysis were analysed in a multivariate backwards stepwise linear regression model.

Logistic regression was used to look for correlations between CRP levels <5 and ≥ 5 with traditional CV risk factors. A CRP value of 5 was chosen as a clinically useful discriminator of values above and below the normal reference range.

Mediation analysis was carried out as previously described, where indicated [19]. All analyses used Stata 9/SE statistical software (Stata Corp, College Station, Texas, USA).

Clinical features

Carotid intima-media thickness

When compared with healthy matched control subjects, RA patients exhibited a greater average cIMT than control subjects (0.64 ± 0.13 mm versus 0.58 ± 0.09 mm, P = 0.03) (Figure 1) and an increased prevalence of carotid atherosclerotic plaque (14 (35%) versus 3 patients (7.5%), P = 0.01). We repeated the analysis after removing patients with previous CV events and their controls, and the results did not differ (0.63 ± 0.02 mm versus 0.57 ± 0.01 mm, P = 0.03). These data indicate that patients presenting with early RA have increased atherosclerotic burden as evidenced by increased cIMT and carotid arterial plaque, compared with matched control subjects.

Determinants of carotid intima-media thickness

Univariate and multivariate analyses were carried out to determine CV risk factors or inflammatory factors that might contribute to atherosclerosis in early RA. A positive association was shown between the cIMT and the CRP level, the disease activity score (DAS 4v), age, male sex, the pack-year history of smoking, history of smoking and systolic blood pressure at the time of presentation with RA (Table 3 and Figure 2). The slope of the regression line with age was steeper in RA patients (slope = 0.011, R² = 0.58) than in control individuals (slope = 0.009, R² = 0.66), suggesting a more rapid rate of thickening of cIMT in RA patients than controls with increasing age (Figure 2). RA patients with previous CV events and their matched control subjects were removed for this analysis. After addition of RA as an interactive term to the regression analysis, the presence of RA did not affect the relationship between age and cIMT. The role of inflammatory activity became clearer upon multivariate analysis, which revealed a strong association between the cIMT, age and CRP level at presentation (Table 3, adjusted R² = 0.64).

Variable	R2 value	Standardized β coefficient	Standard error	t value	P value	Confidence interval
Univariate model
Number of swollen joints	0.12	0.391	0.041	2.25	0.032	0.008–0.174
Disease activity score (DAS4v)a	0.16	0.431	0.079	2.74	0.01	0.056–0.377
C-reactive protein level (mg/l)a	0.14	0.407	0.025	2.71	0.01	0.018–0.121
Rheumatoid factor presenceb			0.047		0.54	0.072–0.117
Age at onset of rheumatoid arthritisa	0.56	0.759	0.001	7.19	<0.001	0.008–0.014
Male sexb			0.066		0.018	-0.298 to -0.030
Pack-year history	0.10	0.320	0.001	2.09	0.044	0.000–0.005
History of smoking, everb			0.062		0.002	-0.329 to -0.079
Systolic blood pressurea	0.27	0.540	0.184	3.63	<0.001	0.292–1.040
Cholesterol level	0.01	0.146	0.197	0.82	0.417	-0.564 to -0.239
Multivariate model
Age at onset of rheumatoid arthritis	0.64		0.002	6.11	<0.001	0.007–0.012
C-reactive protein level (mg/l)			0.001	2.18	0.038	0.000–0.004

Adjusted R² of the multivariate linear regression model = 0.69. ^aVariables entered into the multivariate model. Parameters determined by univariate linear regression analysis or t test^b.

Determinants of C-reactive protein

We hypothesized that CRP and other markers of inflammation were major contributors to the cIMT in RA, and this was indeed the case. To further understand contributors to the cIMT in early RA, we determined the relationship between the CRP level at presentation and traditional CV risk factors. The CRP level at disease onset was related to age (P = 0.016; Figure 2), and with each yearly increase in age there was a corresponding 5% increase in probability of CRP > 5 (P = 0.035). The low-density lipoprotein cholesterol level and triglycerides showed a positive linear relationship to CRP, and the high-density lipoprotein cholesterol level showed a negative linear relationship to CRP level at disease onset (P < 0.05). Presence of CRP > 5 was significantly associated with a history of smoking (P = 0.015). Those who had ever smoked were at greater risk of CRP > 5, with an odds ratio of 5.6 (P = 0.019). History of smoking was the only variable to maintain a significant association with CRP level at presentation in a multivariate model (P = 0.02). Further analysis demonstrates no mediation of CRP on the relationship of cIMT with traditional CV risk factors, except for body mass index.