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Abstract
Background Rheumatoid arthritis (RA) is associated with enhanced cardiovascular (CV) risk and subclinical vascular disease. The proinflammatory milieu has been linked to premature atherosclerosis and endothelial dysfunction in RA. While interleukin 17 (IL-17) is considered pathogenic in RA, its role in determining vascular dysfunction in this disease has not been systematically assessed.
Objectives To analyse candidate variables that might determine endothelial function in various vascular territories in a cohort of patients with RA receiving treatment with biological agents, with minimal traditional CV risk factors and low disease activity score.
Methods Patients with RA (n=50) receiving stable treatment with biological agents underwent measurement of conduit artery endothelial function by brachial artery flow-mediated dilatation; arterial compliance by pulse wave velocity (PWV) assessment; and endothelium-dependent microvascular testing with Endo-Pat2000 device to assess the reactive hyperaemia index (RHI). IL-17 was quantified by ELISA and disease activity was assessed by 28-joint count Disease Activity Score.
Results IL-17 was the main determinant of lower RHI in univariate and multivariate analysis. Traditional and non-traditional CV risk variables determined PWV, with a signifi cant positive association with IL-17 in univariate and multivariate analysis. In contrast, conduit endothelial function was mainly determined by rheumatoid factor titres in univariate and multivariate analysis. Anti-cyclic citrullinated peptide titres, specific disease-modifying antirheumatic drugs or biological agents and disease activity did not determine vascular function.
Conclusion In patients with RA treated with biological agents, IL-17 is a main predictor of microvascular function and arterial compliance. This study suggests that IL-17 may play a significant role in development of endothelial dysfunction and cardiovascular disease in RA.
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Introduction
The paradigm of chronic inflammation as a driver of atherosclerosis is widely accepted.1 Endothelial damage, vascular non-compliance and plaque development are observed in chronic inflammatory conditions, predisposing to accelerated vascular disease.2 Indeed, rheumatoid arthritis (RA) is associated with a 50% higher risk of death from cardiovascular disease (CVD) compared with healthy controls3; a risk comparable to that seen in type II diabetes mellitus.4 Furthermore, RA is considered an independent risk factor for multivessel coronary artery disease.5 Recently, evidence-based recommendations for CV risk management in RA have been proposed.6
In addition to overt CV events, patients with RA (particularly those with a positive rheumatoid factor (RF)) display increased prevalence of subclinical atherosclerosis. This has been reported in patients receiving long-term RA treatment7 and in those with low CV risk factors and disease activity.8 Furthermore, endothelial dysfunction, a phenomenon linked to CVD development,9 is seen early on during the course of the disease.10 11
The striking increases in CVD-associated morbidity and mortality in RA cannot be fully explained by Framingham risk factors. Rather, inflammation and metabolic disturbances present in RA probably contribute to accelerated atherosclerosis. However, it is unclear which factors play primary predisposing roles in RA-associated CVD, and whether the function of the various arterial territories in RA is modulated by the same predisposing factors. Indeed, variations among various vascular territories in their responsiveness to differing stimuli (eg, shear stress, acetylcholine) and their relative dependence on nitric oxide versus other endogenous vasodilators are well described. Therefore, it is conceivable that a conduit vessel such as the brachial artery may respond in a differing or discordant fashion to various inflammatory stimuli present in RA, in comparison with a resistance arteriole.12,–,14
Proinflammatory mediators, such as tumour necrosis factor (TNF) and interleukin 6 (IL-6), are increased in RA and previous evidence supports their potential role in CV damage, even after controlling for other CV risk factors.15 16 However, it is unclear if these cytokines represent the primary vascular dysfunction determinants in RA. Recent interest has focused on the role of interleukin 17 (IL-17) in RA pathogenesis,17 but little is known about its role in vascular disease development in this condition. IL-17 may accelerate myocardial fibrosis and promote atherosclerosis in non-RA animal models,18 19 and elevated circulating IL-17 is detected in patients with acute coronary syndromes.20 However, association studies between IL-17 levels and vascular function in RA are lacking.
Endothelial dysfunction is an early event in atherogenesis. In the presence of vascular risk factors, endothelial cells undergo phenotypic changes leading to decreased nitric oxide bioactivity, vasoconstriction, inflammation and thrombosis.2 Individuals with abnormal vasodilator function have increased CV event rates.3 CV risk can be assessed early on through functional measures of endothelial function, including arterial compliance, flow mediated dilatation (FMD) of large and medium vessels (conduit function) and microvascular endothelial function. These tests represent validated surrogate markers of vascular risk in diverse patient populations and are abnormal in RA.3 8 21 22
In this study, we assessed whether various traditional and non-traditional CV risk factors are associated with microvascular and conduit endothelial function and with arterial compliance in a cohort of patients with RA treated with biological agents, with low disease activity and low burden of traditional CV risk factors. We also examined if various vascular territories differ in the factors that determine their function in RA.
Subjects and methods
Subjects
The University of Michigan institutional review board approved this study, which complied with the Declaration of Helsinki. Patients with RA fulfilling the 1987 American College of Rheumatology diagnostic criteria23 were recruited from University of Michigan rheumatology clinic and by advertisement. For inclusion, patients had to be receiving stable doses of disease-modifying antirheumatic drugs (DMARDs) and/or biological agents for at least 3 months before enrolment. Patients were excluded if they were pregnant or breast feeding, smokers, had diabetes mellitus, congestive heart failure, acute infection or significant liver or renal disease. Patients receiving lipid-lowering drugs were required to be on stable doses of the drug for at least 6 months.
Vascular function measurements
Procedures were performed at the University of Michigan research vascular laboratory in a temperature controlled room, after patients fasted and held vasoactive drugs for at least 12 h.
Pulse wave analyses and arterial pulse wave velocity
After subjects had rested supine for 10 min, applanation tonometry was measured once at the right carotid and femoral arteries for 10 s each, following manufacturer's guidelines (Sphygmocor device; Atcor, Itasca, Illinois, USA). Three-lead ECG recordings were obtained simultaneously with the tonometry to calculate aortic pulse wave velocity (PWV) (a direct measure of arterial stiffness). The quality index was 80%.
Concomitant microvascular and conduit brachial endothelial function
Simultaneous measurement of conduit artery endothelial-dependent vasodilatation by brachial FMD and of microvascular endothelial-dependent vasodilatation by the Endo-Pat2000 device (Itamar, Caesarea, Israel) was performed on the dominant arm as described by us previously.24 Ten minutes after PWV determination, patients were connected to the device's finger probes and to a three-lead ECG system and ECG-triggered B-mode brachial artery measurements were performed by ultrasound.
Basal resting finger peripheral arterial tonometry (PAT) was recorded on one finger from each hand. During this period, basal brachial artery diameter (BAD) was recorded by dominant arm ultrasound for at least 10 s for FMD measurement. A dominant arm blood pressure (BP) cuff was inflated to 50 mm Hg above systolic BP for 5 min. Upon cuff deflation, reactive hyperaemia (RH) was created and RH-PAT recorded in the ipsilateral dominant hand finger for 5 min. The device's computer compared 120 s of baseline mean PAT with RH-PAT (mean PAT from 60 to 120 s after cuff release on dominant arm). Readings were standardised to contralateral hand PAT during same periods, providing the RH-PAT index.
Concomitantly, at 50–90 s after BP cuff release during RH, BAD was recorded to calculate FMD, following guidelines25 and as reported by us.26 27 A Terason 2000 ultrasound system (Terason, Burlington, Massachusetts, USA) with a 10.0 MHz linear array transducer was used. After cuff inflation, BAD was imaged longitudinally by B-mode imaging with the transducer 2–10 cm above the antecubital crease. Images were acquired at the end of each R wave on the ECG by a triggered event. Endothelial-dependent FMD was defined as the percentage change in BAD from baseline in response to RH (measured from the media-adventitia line (M-line) to the M-line). FMD = ((RH BAD – basal BAD)/basal BAD) × 100 (in %). Image analysis was performed with software from Medical Imaging Applications (Coralville, Iowa, USA).
Determination of serum IL-17
High binding enzyme immunoassay/radioimmunoassay 96-well plates were coated with anti-human IL-17 in carbonate buffer, and incubated at 4°C overnight. Serum samples and standards (prepared from recombinant human IL-17 (eBioscience, San Diego, California, USA)) were added to wells, incubated for 2 h at room temperature, followed by addition of biotinylated anti-IL-17 (eBioscience, San Diego, California, USA) and streptavidin horseradish peroxidase (Biolegend, San Diego, California, USA) for 2 h. After sample colour change with tetramethylbenzidine reagent (BD Biosciences, San Jose, California, USA) addition, absorbance was quantified on a microplate reader at 450 nm wavelength.
Other laboratory measurements
Lipid profile, high-sensitivity C-reactive protein (hsCRP), insulin and glucose were measured in the Michigan Diabetes Research and Training Center. RF, anti-cyclic citrullinated peptide (anti-CCP) antibodies, erythrocyte sedimentation rate (ESR) and complete blood count were measured at the University of Michigan Central Laboratories.
Insulin resistance assessment
Insulin resistance was assessed with the Homoeostatic Model Assessment (HOMA1-IR)28 using the formula: HOMA1-IR = (fasting plasma insulin (µU/ml) × fasting plasma glucose mg/dl))/405.
Statistical analysis
Measures of vascular endothelial function were analysed as dependent variables. Continuous variables were summarised using means and SDs, and categorical variables by counts and percentages. Log transformation was applied to variables with highly skewed values (hsCRP, ESR, anti-CCP, IL-17 and RF). For univariate analyses, one-way analysis of variance or linear regression methods were used to evaluate the effect of each predictor on vascular function. Forward stepwise multivariate regression analyses were performed to identify the effect of predictors in the presence of other factors associated with vascular dysfunction. Variables with p<0.15 in the univariate analyses were included in the multivariate analyses, to examine their true association with outcome measures in the presence of other factors. The above analyses were repeated for the 33 subjects with detectable levels of IL-17. A p value ≤0.05 was considered significant. Procedures were done in SAS 9.2.
Results
Demographic and clinical characteristics
Fifty patients treated with stable doses of biological agents, with or without concomitant DMARD therapy, were analysed. Of these, 3 (6%) were treated with abatacept, 15 (30%) with adalimumab, 24 (48%) with etanercept, 2 (4%) with infliximab and 6 (12%) with rituximab. Thirty-one of these patients (62%) were also receiving DMARDs: 4 (13%) leflunomide, 25 (81%) methotrexate, 2 (6%) sulfasalazine, while 27 (54%) of all patients were on prednisone (mean daily dose±SD=5.8 ± 2.9 mg).
Demographics and information on disease duration and activity and autoantibodies are included in table 1. Clinical characteristics, vascular function and inflammatory markers are shown in table 2.
Demographic and clinical features of study participants
Vascular function and laboratory markers of CV risk and inflammation
Factors associated with vascular function in RA
Table 3 shows the association between vascular measurements and clinical and laboratory features. Patients who were anti-CCP positive versus negative had higher IL-17 levels (3128.9 pg/ml vs 1829.2 pg/ml, p=0.01). Men had higher BAD (p=0.004), haematocrit (p=0.003) and diastolic BP (p=0.047) than women.
Univariate analysis of determinants of vascular function in RA
Microvascular endothelial function (RH index (RHI)) was significantly negatively associated with IL-17 and hsCRP in univariate and multivariate analysis (tables 3 and 4). The negative association between IL-17 and RHI persisted when only subjects with detectable IL-17 levels were included in the analysis (n=33, p=0.03). Other variables that were negatively associated with RHI were also linked to inflammation and included ESR, triglycerides, white cell count and RF levels, but these associations did not persist in multivariate analysis. There were no significant associations between traditional CV risk factors and RHI. Patients with undetectable IL-17 levels had a significantly higher RHI than those with measurable IL-17 levels (2.4% vs 2.0%, p=0.03). RA disease duration was significantly lower among patients with undetectable IL-17 (10.7 years vs 20.1 years, p=0.01).
Multivariate analysis of determinants of vascular function in RA
For PWV, univariate analysis showed a positive association with both traditional (age, heart rate, systolic BP (SBP), Framingham risk score) and non-traditional risk factors (IL-17, ESR and white cell count) (table 3). In multivariate analysis, age, heart rate and IL-17 continued to significantly predict PWV (p=0.01, p=0.02, p=0.02, respectively) (table 4). The positive association between IL-17 and PWV persisted when only subjects with detectable IL-17 were included in the analysis (n=33, p=0.004).
In analysis of conduit FMD, RF levels negatively correlated with FMD in univariate and multivariate analysis (tables 3 and 4). No other traditional or non-traditional CV risk factors predicted conduit FMD.
No significant associations were seen between the various vascular function tests and 28-joint count Disease Activity Score, HOMA, anti-CCP, insulin, RA disease duration, serum lipids, specific DMARDs or biological agents, vasoactive drugs or statin therapy (data not shown).
Discussion
Increased CVD causes significant excess mortality in RA, and exists independently of traditional Framingham risk factors.29,–,31 Increased subclinical atherosclerosis prevalence and severity are found in patients with poorly controlled RA5 32 33 but also in those receiving long-term DMARD treatment.7 Despite widespread use of biological agents alone and in combination with DMARDs, there is no evidence that CV-related morbidity and mortality in RA in the USA is decreasing. Conversely, the mortality gap between RA and the general population appears to be widening.34,–,36 While improvements in endothelial function and PWV and decreases in inflammatory markers and insulin resistance have been reported in patients with RA treated with TNF inhibitors, it is not clear that these benefits are sustained or contribute to significant decreases in CV events and mortality.21 37,–,40 Furthermore, while a link between proinflammatory responses and vascular damage has been proposed, various conflicting results exist on the association between specific inflammatory markers and endothelial dysfunction at various vascular territories, as well as during various stages of the natural history of plaque development and acute coronary syndrome.
In this study of patients with well-controlled RA and low Framingham CV risk factors receiving biological agents we investigated the association of inflammatory markers and traditional CV risk factors with FMD, a measure of conduit artery vasodilator function; with RH, a measure of microvascular vasodilator function; and with PWV, a measure of arterial stiffness. Abnormalities in these measures have been linked to enhanced CV risk in various patient populations.41 42
Our results show that IL-17 is independently negatively associated with microvascular function and positively associated with large vessel arterial compliance in patients with RA treated with biological agents, primarily anti-TNF agents. These observations may have clinical relevance because endothelial dysfunction is strongly linked to pathogenesis and clinical expression of future vascular damage. Furthermore, patients with non-detectable IL-17 had significantly higher microvascular function. Also, we have reproduced results similar to those found by other groups in other patient populations, with variations in determinants of vascular function in different vascular territories.43 Indeed, the only determinants of conduit FMD were RF levels, while IL-17 and inflammatory markers associated with lower RHI and higher PWV.
These findings support the hypothesis that systemic inflammation represents a mechanistic link between risk factors and vascular dysfunction in both the microvasculature and large vessel territories. RH is a complex response44 and the association observed between IL-17 and other inflammatory markers with lower RHI may reflect endothelial dysfunction in the microvasculature. Furthermore, reports that changes in the microcirculation may be important predictors of CVD in women44 can be extrapolated to a disease with female predominance like RA. Thus, our results indicate a potentially important role for IL-17 and the inflammatory burden in CV prognosis. RA is associated with impaired large muscular artery function, resulting in arterial stiffness.45 The latter correlates with a risk of morbidity and mortality due to CVD.46 47 Although arterial stiffness has been linked to IL-6 and TNF,48 our study is, to our knowledge, the first report of associations between IL-17 and higher PWV.
While IL-17 may play crucial in RA pathogenesis, its potential role in endothelial dysfunction and increased CVD in this disease remains unclear. Evidence from atherosclerosis animal models supports a role for this cytokine in plaque formation. IL-17A is proatherogenic by promoting monocyte/macrophage recruitment into the aortic wall.49 There is constitutive expression of IL-17E by resident plaque cells, and IL-17-expressing B cells and neutrophils in advanced and complicated plaques, indicating a complex contribution of IL-17 family cytokines to atherosclerosis.50 Elevated plasma IL-17 levels in patients with acute myocardial infarction have been observed51 and IL-17 induces an inflammatory phenotype on vascular smooth muscle cells.52 IL-17 is also critical for the maintenance of angiotensin II-induced hypertension and vascular dysfunction, adding another link between the inflammatory milieu in RA and traditional CV risk factors.53 Until more evidence supports a mechanistic link between IL-17 and CVD in RA, however, we cannot exclude the possibility that this cytokine might be merely a marker for the presence of other factors that account mechanistically for the observed variation in vascular function. How TNF, IL-6 and IL-17 interact to enhance CV risk in RA also deserves further investigation. Further, as there is evidence that vascular dysfunction in RA may not be improved by short-term treatment with conventional DMARDs,54 it will be interesting to assess how targeted anti-IL-17 treatments modify vascular function.
A proportion of patients with RA in this study had undetectable IL-17 levels. This supports previous reports that show undetectable serum IL-17 in a subset of patients with RA and healthy controls.55 56 Undetectable IL-17 in our population could reflect various factors, including downregulation secondary to biological agents and/or DMARDs or genetic polymorphisms.57 Whether this subset of patients is relatively protected from vascular dysfunction warrants future investigation.
Importantly, in this RA cohort with well-controlled disease, there were no associations between vascular abnormalities and RA disease activity scores, supporting previous findings that effective RA treatment may lead to arterial stiffness improvement.21
This study has some limitations. While the association between RA-induced systemic inflammation and impaired vascular function remains significant when adjusted for other factors, only prospective studies have the potential to discern a causal relationship. The studied cohort was predominantly white, and findings may not apply to other ethnic groups. Several factors showing correlation may be intercorrelated, and finding independent effects can be difficult even after multivariate adjustment. No a priori power calculations were obtained. Furthermore, the cohort examined in this study represents a subgroup of patients treated with biological agents and is therefore not representative of patients with RA as a whole, or other RA subgroups such as TNF-naive patients. Euthyroid status was not assessed before inclusion and it was not possible to reliably calculate cumulative prednisone dose. Future studies should assess if IL-17 continues to be an important determinant of vascular function in other RA groups including patients with more active disease or those with recent diagnosis.
Finally, we did not observe associations between levels of anti-CCP antibodies and the vascular markers studied, while there was significant negative association between RF and conduit vascular function. This supports evidence from a large population-based study demonstrating association between CVD and the presence of RF but not anti-CCP,58 but does not support the findings of extensive subclinical atherosclerosis observed in patients with RA who are anti-CCP-positive versus negative,59 or the observed independent association of anti-CCP antibodies with the development of ischaemic heart disease.60 These conflicting results from various studies of autoantibodies and CVD in RA61 62 indicate that the exact role played by autoantibodies in accelerated atherosclerosis in RA is still undefined.
Overall, the results of this study suggest that factors predicting conduit vascular function in patients with RA differ from those associated with large-vessel arterial compliance and microvascular function. Our findings support IL-17 as a potential marker of vascular risk in RA in small and large vascular territories and a causative association between this cytokine and CVD. Furthermore, our results indicate that, even in patients with well-controlled RA receiving stable doses of biological agents, factors inherent to the disease, including RF, IL-17 and inflammatory markers, continue to be the main determinants of vascular function. Indeed, this study supports growing evidence that, although RA treatment has improved over time, the inflammatory milieu may continue to determine vascular function even in patients with well-controlled disease and low traditional CV risk factors. Whether IL-17 represents an attractive therapeutic target for both RA disease manifestations and for possible amelioration of CV risk warrants further examination.
References
Footnotes
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Funding This work was supported by the National Institutes of Health through PHS grant RO1 HL086553 (to MJK) and UL1RR024986 (University of Michigan CTSA). WM was supported by K12HD001438 from the National Institutes of Health. This work was also supported in part by the Michigan Diabetes Research and Training Center, University of Michigan.
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Competing interests None.
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Ethics approval This study was conducted with the approval of the University of Michigan institutional review board.
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Provenance and peer review Not commissioned; externally peer reviewed.