Abstract
Objectives: Rheumatoid arthritis (RA) is a chronic, inflammatory disease associated with increased risk of cardiovascular (CV) disease. Arterial stiffness (AS) is an independent predictor of CV events. This study aimed to analyse local carotid AS parameters in seronegative and seropositive RA patients. Design: Of 347 consecutive RA patients, we selected specifically those who were free of established CV diseases and risk factors. As a result, 140 patients (126 women, 52.2 ± 10 years) and 140 healthy controls (122 women, 52.7 ± 8.0 years) were enrolled into this study. The common carotid AS was evaluated using radio frequency echo-tracking system to determine the local carotid pulse wave velocity (cPWV) and carotid intima-media thickness (cIMT). Based on rheumatoid factor (RF) and/or anti-citrullinated protein antibody (ACPA) positivity, RA patients were categorized into seronegative and seropositive subgroups. Results: Carotid PWV was determined to be significantly higher in all patients and subgroups than controls (p < .001 for all). Although cIMT was similar between the patients, controls and seropositive subgroup, seronegative patients had significantly higher cIMT compared to controls (p = .035) and seropositive group (p = .010). Moreover, a significant positive correlation was found between cPWV and age (r: 0.603, p < .001), ESR (r: 0.297, p = .004), ACPA (r: 0.346, p = .001) and cIMT (r: 0.290, p = .005) in seropositive patients. Conclusions: RA per se is sufficient to cause arteriosclerosis in the absence of classical CV risk factors. However, arterial hypertrophy is only increased in seronegative patients but not in seropositive group.
Introduction
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease associated with increased cardiovascular (CV) morbidity and mortality [Citation1]. The increased CV morbidity and mortality in patients with RA can not be entirely explained by traditional CV risk factors and chronic inflammation seems to act as an independent CV risk factor [Citation2,Citation3]. Chronic inflammation plays an important role in the pathogenesis of both atherosclerosis and RA and accentuates established CV risk factors [Citation3]. It contributes to the increased CV risk by promoting vascular changes and increasing arterial stiffness (AS) [Citation4]. AS is a powerful predictor of all-cause mortality in addition to CV outcomes and it can predict CV events in RA patients [Citation5,Citation6]. Therefore, it is increasingly recognized as a surrogate end point for CV disease. Numerous invasive and non-invasive methods measuring AS have been described. The most widely used and validated techniques involve the assessment of pulse waves as they travel over a significant portion of the arterial tree; regional (over shorter arterial segments) and local (carotid or femoral) AS are emerging as promising biomarkers [Citation7].
Carotid intima-media thickness (cIMT) of the common carotid artery (CCA) is a useful noninvasive anatomical structural measure of subclinical CV disease and the most popular technique used to study early structural changes in the arterial wall [Citation8]. Enhanced AS and cIMT are both associated with systemic rheumatoid inflammation [Citation4,Citation9,Citation10], all of which add to the cardiovascular phenomenon in RA.
After the identification of rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPAs), two subgroups of RA have been recognized as seronegative and seropositive [Citation11]. Previous studies have shown that seropositive patients have more severe inflammatory activity than patients with seronegative RA [Citation12,Citation13]. However, in a recent study, seronegative patients were detected to have more inflammatory activity compared to those with seropositive RA [Citation14]. Because, there is an association between inflammation and atherosclerosis, we aimed to investigate AS and cIMT, measured locally in the CCA by high-resolution echotracking system, in seronegative and seropositive RA patients.
Materials and methods
Study design and population
This was an observational (case-control) study performed in our hospital between November 2013 and March 2016. Consecutive patients with RA who met the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) classification criteria and did not have malignancy, chronic renal failure or other concomitant acute or chronic inflammatory disease were comprehensively studied for AS and arterial hypertrophy. Of all 347 patients examined, we identified those who did not have a history of atherosclerotic CV disease (coronary artery disease, peripheral artery disease, ischemic stroke/transient ischemic attack), were never smokers and between 18–60 years of age, had no hypertension, diabetes mellitus, dyslipidemia. Patients using biologic agents and on regular drug therapies for concomitant diseases were also excluded. As a result, 140 patients fulfilled these criteria and were eligible for analysis. Besides, 140 age and gender matched healthy control subjects, also free of CV risk factors and established CV disease were included into this study. These healthy subjects were recruited from a pool of individuals who were admitted to our polyclinic for a variety of complaints and to whom a through examination for subclinical atherosclerosis had also been performed. Demographic characteristics, biochemical data were obtained for all study population. Duration of disease and current medications (disease-modifying anti-rheumatic drugs, steroids, methotrexate and non-steroidal anti-inflammatory drugs) were determined. Informed consent was obtained from each subject and the study protocol was approved by the local ethical committee of our hospital.
Biochemical examination
After at least 8-hour fasting, plasma glucose, blood urea nitrogen, creatinine, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), C-reactive protein (CRP), RF, and ACPA levels and erythrocyte sedimentation rate (ESR) were measured and recorded. RF was measured by nephelometry; a level of 20 U/ml or more was considered positive. ACPAs were measured by enzyme linked immunosorbent assay (ELISA; Axis Shields Diagnostics, Dundee, UK) and considered positive above a cut off value of 17 U/mL as suggested by the manufacturer.
Carotid arterial stiffness assessment
Subjects were studied after 15 min of supine rest, in a temperature controlled environment. Studies were performed using a color Doppler ultrasound machine (MyLab25; ESAOTE) with a 7.5-MHz linear array probe, implemented with a high-resolution echo-tracking system allowing accurate measurements of carotid diameter changes [Citation15]. Pressure waveforms were noninvasively obtained using arterial diameter change waveforms calibrated based on systolic and diastolic blood pressure (BP) values measured with a cuff-type manometer applied to the upper arm. The main indices of carotid AS β index were automatically calculated according to established formulas [Citation16]. Common cIMT was automatically and simultaneously measured as the mean of the relative values of the last 8 s on the same image sequence. cIMT was measured in the far wall of the CCA, 1 cm proximal to the carotid bulb.
Statistical analysis
Continuous variables with normal distribution are presented as mean ± SD, otherwise as median with interquartile range. The assumption of normality of distribution was evaluated through the Shapiro-Wilk test. Normal distributions of the values were assessed by using the Kolmogorov-Smirnov test. The Mann Whitney U and Student t test were used for analysis of continuous variables when appropriate. Categorical variables were expressed as absolute (n) and relative (%) frequencies. They were analyzed by using chi-squared or Fischer’s exact test. The Spearman or Pearson correlation analysis was used to assess the relationship between various variables and carotid pulse wave velocity (cPWV). Furthermore, difference of cPWV values between RA patients and controls after controlling for possible confounding factors was examined through an appropriate multivariate model. The same statistical model was used to check for the effect of confounding factors on the established correlations among patients with RA. The presented p-values in parentheses were referred to comparisons of each subjects in the patient, seronegative and seropositive groups with the controls whereas p values at right column are derived for difference between the group of seronegative vs. group of seropositive in and . A p value < .05 was considered statistically significant. All statistical analysis was carried out using SPSS version 20 (Released 2011. IBM Corp., Armonk, NY).
Table 1. Demographic and clinical features of the study population.
Table 2. Local carotid stiffness parameters of the study population.
Results
A total of 140 patients with mean age of 52.2 ± 10.7 years and 140 healthy controls with a mean age of 52.7 ± 8.0 were included into this study. The median disease duration of patients was 8.9 ± 8.2 (range: 1–38) years. Overall, 126 patients (90%) were women and 14 patients (10%) were men. Among them, 48 patients (34.3%) were seronegative and 92 patients (65.7%) were seropositive.
The demographic and clinical features of the study population are shown in . Accordingly, there were not significant differences in terms of age, gender, BMI, systolic blood pressure (SBP), diastolic blood pressure (DBP), mean BP, pulse pressure (PP), TC and LDL-C levels between both groups. However, HDL-C, CRP, RF, ACPA levels and ESR were significantly higher among patients compared to controls (p < .001 for all). On the contrary, TG level was found to be significantly higher in control subjects than patients (1.97 ± 1.0 vs 1.25 ± 0.60 mmol/L, respectively, p < .001). When RA subgroups were compared with controls, it was detected that only seronegative group but not seropositive ones had significantly higher SBP (p = .049) and PP (p = .047) levels. In addition, seronegative patients were significantly more overweight than seropositive patients (30.0 ± 5.7 vs 27.7 ± 5.7 kg/m2, respectively, p = .044). Although, the levels of both inflammatory biomarkers, ESR and CRP level were higher in seronegative patients compared with seropositives, only the elevation in CRP level reached the statistical significance (23.6 ± 3.0 vs 14.4 ± 1.9 nmol/L, respectively, p = .033).
The local carotid AS parameters of the study population are presented in . RA patients had a significantly elevated cPWV in comparison with control subjects (7.37 ± 1.70 vs 6.49 ± 0.92 m/s, respectively, p < .001). However, the other parameters of local carotid AS, namely carotid distension, distensibility, diameter, SBP, DBP, PP and IMT were similar between patients and controls. When the subgroup analysis was performed, seronegative patients had significantly higher cIMT than controls (6.4 ± 0.12 vs 5.9 ± 0.12 mm, respectively, p = .035) and seropositive patients (6.4 ± 0.12 vs 5.8 ± 0.13 mm, respectively, p = .010). Further, seronegative patients was determined to have significantly elevated cPWV than seropositives (7.68 ± 2.24 vs 7.12 ± 1.34 m/s, respectively, p = .031). Moreover, we found significantly higher carotid distensibility (p = .022), carotid diameter (p < .001), local carotid SBP (p = .017) and carotid PP (p = .001) levels in seronegative group compared to patients with seropositive RA.
The correlation between cPWV and clinical and other laboratory parameters are shown in . According to these results, we determined a significant positive correlation with cPWV and SBP (r: 0.465, p = .001), mean BP (r: 0.343, p = .020), brachial PP (r: 0.481, p = .001), carotid PP (r: 0.687, p < < .001) but a significant negative correlation with carotid distensibility (r: −0.735, p < .001) in RF/ACPA negative patients. In fact, we found a significant positive correlation between cPWV and age (r: 0.603, p < .001), BMI (r: 0.274, p = .022), urea (r: 0.257, p = .013), creatinine (r: 0.242, p = .020), LDL-C (r: 0.229, p = .030), TG (r: 0.277, p = .008), ESR (r: 0.277, p = .004) and ACPA (r: 0.346, p = .001) levels, disease duration (r: 0.292, p = .013), SBP (r: 0.519, p < .001), DBP (r: 0.244, p = .019), mean BP (r: 0.364, p < .001), brachial PP (r: 0.538, p < .001) and carotid PP (r: 0.552, p < .001) in seropositive patients. There were also a significant positive correlation between cPWV, IMT (r: 0.290, p = .005) and diameter (r: 0.229, p = .028) whereas a significant negative correlation between cPWV, distensibility (r: −0.542, p < 0.001) and HDL-C (r: −0.390, p < .001) in RF/ACPA positive patients.
Table 3. Correlation between carotid stiffness and other clinical and laboratory parameters in rheumatoid arthritis subgroups.
However, after adjusting for possible confounder (obesity), the association between cPWV and LDL-C and carotid diameter did not remain statistically significant in seropositive patients (p = .20 and 0.11, respectively). On the other hand, a statistically significant negative correlation between cPWV and eGFR was detected (r: −0.549, p < .001) in seropositive group. In addition, the association between mean BP and cPWV did not remain statistically significant in seronegative group as well (p = .07).
Discussion
In this study, RA patients was found to have increased carotid AS than controls. In contrast, cIMT was similar between the patient and control groups. When subgroup analysis was performed, we demonstrated for the first time that patients with seronegative RA have a significant increase in cPWV than seropositive RA patients despite similar disease duration. We have also showed significantly higher cIMT in seronegative RA patients compared to those with seropositive RA. Furthermore, a significant positive correlation was observed between cPWV, cIMT, CRP and disease duration in seropositive RA group. The determined signs of increased carotid AS and cIMT in seronegative patients may be an evidence of the more severe inflammatory activity and atherosclerotic process due to the already known relationship between inflammation and atherosclerosis. Atherosclerosis is a major factor contributing to mortality in RA.
The identification of RF and ACPAs has led to the division of RA into two subgroups as seronegative and seropositive [Citation14]. Thereafter, serological status has become an important factor in both the diagnosis and prognostification of the disease. Seropositive RA patients have been shown to have a more severe disease course [Citation17,Citation18]. However, the clinical presentation and disease course of seronegative patients are generally reported as less severe, although studies are conflicting [Citation19]. Although previous studies [Citation12,Citation13] have determined more inflammatory activity in seropositive RA patients, a recent study demonstrated contradictory findings [Citation14]. Chronic inflammation observed in RA is very important for early atherosclerosis since inflammation plays a major role in initiation and progression of atherosclerosis [Citation20]. AS is one of the earliest indicator of structural and functional changes within the arterial wall [Citation21]. It is also an early manifestation of atherosclerotic CV diseases [Citation22]. There are multitude of techniques available to evaluate and quantify AS by measuring several parameters such as PWV or augmentation index [Citation21]. PWV is a measure of AS which can be assessed using different techniques [Citation22]. We used a radiofrequency-based wall-tracking system implemented in a standard ultrasound equipment to assess local CS during routine measurement of cIMT [Citation15]. Such a combined evaluation of local arterial structure and function may further improve the evaluation of vascular aging and early atherosclerotic injury.
Carotid IMT is a non-invasive ultrasound biomarker of early, preclinical atherosclerosis and a predictor of CV events [Citation23]. Gonzalez-Juanatey et al. [Citation24] showed greater cIMT in RA patients who experienced CV events compared with the remaining RA patients who did not have CV complications. Moreover, cIMT demonstrated significant correlations with disease severity [Citation25], CRP [Citation26] and ESR [Citation27] values in RA patients. We excluded RA patients with traditional CV risk factors to eliminate possible confounders. We found that patients with seronegative RA have significantly higher cIMT than those with seropositive RA. Ambrosino et al. [Citation4] determined a more severe inflammatory status significantly impact on cIMT. From this point of view, our results support the hypothesis that, patients with seronegative RA have more inflammatory activity compared with seropositive RA. We have also found significant correlation between cPWV and cIMT, CRP and disease duration in seropositive RA patients. This result may also in line with the fact that, long-standing chronic inflammation is associated with more severe atherosclerosis. Kim et al. [Citation28] investigated the major determinants of AS in 262 RA patients and determined that age, BMI, BP, lipid profile and glucose but not disease-related factors such as disease duration, ESR, CRP and seropositivity were significantly correlated with brachial-ankle PWV. Although, our results were mostly consistent with these findings, differently, we also found a significantly positive correlation between cPWV and disease duration, ESR and ACPA levels. This could be due to the enrollment of patients with longer disease duration (mean disease duration 9 years compared with 6.5 years) in the present study. In another study evaluating the effect of inflammation on AS in 77 RA patients, aortic PWV was found to have significant correlation with CRP, a marker of inflammation, and mean BP but not with disease duration [Citation29]. Contrary to these findings, we did not find a significant correlation between cPWV and CRP level but we found a significant correlation of cPWV with disease duration. This could be due to the fact that the population in this study was relatively younger and not treated with biologic agents.
Traditional CV risk factors in the general population include cigarette smoking, insulin resistance, diabetes, hypertension, dyslipidemia, physical inactivity and obesity [Citation30]. One of the challenges of studying the contributions of these risk factors in RA is that the factors themselves can be simultaneously affected by inflammation and treatments for RA. Although evidence is conflicting, some studies suggest an increase of these risk factors in RA. Whether or not traditional CV risk factors are increased in the RA population, they do not fully explain the elevated CV risk in RA patients. Therefore, our patient population was chosen from those without established CV disease and these risk factors to eliminate the influence of possible confounders on local CS parameters and cIMT and to be able to demonstrate the pure effect of chronic inflammation on these surrogate CV markers.
2010 ACR/EULAR criteria for RA that were applied to select the patients in this study are at least 6 points based on the following: (1) up to 5 points for joint affection, (2) up to 3 points for serology, (3) 1 point for symptom duration and (4) 1 point for acute phase reactants. Accordingly, since the seronegative patients don’t receive points for serology, they necessarily have to get more points based on joint affection, disease duration and acute phase reactants. Because disease duration was equal in the two subgroups in our study, the seronegative patients probably have more severe diseases in terms of acute phase reactants and joint affection. Thus, it is probably the selection of patients who obtain 6 points, rather than the absence of seropositivity, that plays a role. This may explain our results.
In conclusion, we have shown that patients with seronegative RA had significantly increased cPWV and cIMT in comparison with seropositive patients and controls. In addition, a significant positive correlation was determined between cPWV and cIMT, CRP and disease duration in seropositive RA patients. As a result we believe that, patients with seronegative RA may have more inflammatory activity than those with seropositive RA and they should be followed more carefully in terms of CV events.
Acknowledgements
No financial support for this paper is present.
Disclosure statement
The authors report no conflicts of interest.
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