Can Modulators of Inflammation Serve as Biomarkers for Subclinical Atherosclerosis in Rheumatoid Arthritis? Kimberly P. Liang1, Douglas P. Landsittel2, Suresh R. Mulukutla3, Steven E. Reis3, Marc C. Levesque1, Donald M. Jones1, Rachel Gartland1, Ali Hakim Shoushtari4, Flordeliza S. Villanueva3, Hunter C. Champion5, Larry W. Moreland1 1Division of Rheumatology; 2Center for Research on Health Care Data Center; 3Division of Cardiology; 4Clinical and Translational Science Institute; 5Division of Pulmonary, Allergy and Critical Care Medicine; University of Pittsburgh. ABSTRACT INTRODUCTION RESULTS RESULTS, cont. DISCUSSION Mean age, BMI, and gender frequency were similar (p≥0.19) between RA (59.3 years, 28.9, 72% female) and controls (54.7 years, 28.7, 67% female). (Table 1) Race was significantly different (4.4% African-American or other in RA vs. 28.6% in controls; p=0.001). (Table 1) RA subjects (n=44) had mean +/- SD CDAI of 8.55 +/- 8.05 (median 6.75, IQR 2.75-14.75), and mean +/- SD disease duration of 13.06 +/- 9.58 years (median 9.39, IQR 5.47-17.89). Mean cIMT was higher in RA (0.88) than controls (0.79); p=0.02. (Table 2 and Figure 1) Detectable IL-6 presence was higher in RA (31.8%) than controls (14.3%); p=0.03. (Table 2) Mean (+/-SD) MPO was lower in RA (594.9 +/- 670.9) than controls (727.4 +/- 464.6) (p=0.01). (Table 2 and Figure 2) Plaque presence was found in 47.8% of RA cases vs. 37.7% of controls (p=0.28). None of the other serum biomarkers were significantly different between RA vs. controls. ICAM-1 (r=0.36, p<0.001), VCAM-1 (r=0.28, p=0.002), and E-selectin (r=0.32, p<0.001) were positively associated with cIMT in the overall group. (Figure 3) Mean cIMT was higher in those with IL-6 above detection limit (0.91 vs. 0.80; p=0.05). Though not significant, detectable IL-6 was higher in those with plaque (28.3%) than in those without plaque (14.9%); p=0.08. Mean (+/-SD) CD40L and MPO were lower in those with plaque presence (8385 +/- 3803 for CD40L and 554 +/- 454 for MPO) compared to plaque absence (9433 +/- 3535 for CD40L and 754 +/- 605 for MPO). (Figure 4) Results were similar when examined in the RA and control groups separately, though the magnitude of correlations with cIMT was slightly higher in the control group for ICAM-1 and E-selectin (r=0.4). In the RA group only, mean ICAM-1 was higher (292) in those with plaque than in those without plaque (234) (p=0.045). Previous studies have shown a higher rate of subclinical atherosclerosis, as measured by cIMT, in RA than non-RA subjects.1-2 However, in Carotti et al’s study,1 no significant associations were found between common carotid artery (CCA) cIMT and clinical or laboratory assessments of disease activity. Our findings confirm a higher mean cIMT in RA than control subjects. Our study assessed the CCA cIMT. Kobayashi et al’s study2 suggested higher severity of carotid bulb (but not CCA) atherosclerosis in RA than controls. Further studies are warranted of both bulb and CCA cIMT in RA. In RA, immune complexes that fix C1q may be a source of arterial injury initiating atherogenesis. These complexes bind to C1q receptors on the endothelium, triggering an upregulation of adhesion molecules such as E-selectin, ICAM-1 and VCAM-1 on the endothelial surface.3 One hypothesis of atherogenesis is that this intense immune/inflammatory reaction may precipitate plaque ulceration, rupture and thrombosis,4 a process that may be accentuated in RA. Recent studies have shown that serum concentrations of ICAM-1, VCAM-1, and E-selectin are higher in RA patients than controls, independent of traditional CV risk factors,5-6 and VCAM-1 was associated with increased cIMT and plaque in RA.6 Our study showed correlations of all 3 adhesion molecules with cIMT in the overall group, with slightly larger magnitudes of correlation in the control group separately. In our study, IL-6, a marker of systemic inflammation, was positively correlated with cIMT and had a trend for correlation in those with plaque vs. those without plaque. This corroborates the importance of inflammation in RA and atherosclerosis. Unexpectedly, our study showed an inverse correlation of CD40L and MPO with cIMT and plaque presence; these biomarkers have been associated with high-risk atherosclerotic lesions and CVD in clinical studies in the general population.7-9 This may be due to limitation of our small sample size and wide variability in these biomarkers’ levels. Alternatively, this may indicate differential effects of these biomarker pathways in subclinical atherosclerosis in RA patients; future larger studies are required. BACKGROUND: Rheumatoid arthritis (RA) is independently associated with a higher risk of cardiovascular disease (CVD) and premature atherosclerosis. Mechanisms of atherosclerosis include (1) Endothelial dysfunction/activation mediated by intercellular adhesion molecules (e.g., ICAM-1, VCAM-1, E-selectin) and oxidative stress (e.g., through MPO activity); (2) Inflammation mediated by cytokines (e.g., IL-6); (3) Plaque stability mediated by CD40-CD40L interactions; and (4) Proteolysis/Plaque rupture mediated by proteolytic enzymes (e.g. MMP-9). This study evaluates whether these serum biomarkers are higher in RA subjects, and/or associated with subclinical carotid atherosclerosis. METHODS: Carotid ultrasounds were performed with measurement of intima-media thickness (cIMT, using maximum of both sides) and plaque presence (of either side), and serum biomarkers (ICAM-1, VCAM-1, E-selectin, MPO, IL-6, CD40L, and MMP-9) were measured by ELISA in all subjects (46 RA cases and 70 controls). Each of the biomarkers, cIMT, plaque presence, and demographic data, were tested for differences between cases and controls, using the Wilcoxon rank-sum test (for continuous data) or chi-square test. The relationship between each serum biomarker and cIMT or plaque presence was tested by Spearman correlations or rank-sum test (for IL-6 only, which was categorized as above detection limit [for 21%] or not). RESULTS: Mean age, BMI, and gender frequency were similar (p≥0.19) between RA (59.3 years, 28.9, 72% female) and controls (54.7 years, 28.7, 67% female); race was significantly different (4.4% African-American or other in RA vs. 28.6% in controls; p=0.001). Mean cIMT and detectable IL-6 presence were higher in RA (0.88  and 31.8%) than controls (0.79 and 14.3%; p=0.02 and 0.03). Mean (+/-SD) MPO was lower in RA (594.9+/-670.9) than controls (727.4+/-464.6) (p=0.01). Plaque presence was found in 47.8% of RA cases vs. 37.7% of controls (p=0.28). None of the other serum biomarkers were significantly different between RA vs. controls. ICAM-1 (r=0.36, p<0.001), VCAM-1 (r=0.28, p=0.002), and E-selectin (r=0.32, p<0.001) were positively associated with cIMT in the overall group . Mean cIMT was higher in those with IL-6 above detection limit (0.91 vs. 0.80; p=0.05). Mean CD40L and MPO were lower in those with plaque presence (8385 for CD40L and 554 for MPO) compared to plaque absence (9433 for CD40L and 754 for MPO).  Results were similar when examined in the RA and control groups separately, though the magnitude of correlations with cIMT was slightly higher in the control group for ICAM-1, VCAM-1, and E-selectin. In the RA group only, mean ICAM-1 was higher (292) in those with plaque than in those without plaque (234) (p=0.05). CONCLUSIONS: This study confirms previous observations that cIMT is higher in RA than non-RA subjects. Markers of endothelial dysfunction (ICAM-1, VCAM-1, E-selectin) are significantly associated with higher cIMT in both RA and non-RA subjects. This suggests possible common pathways of atherosclerotic progression as evidenced by cIMT and biomarker elevation. Further studies are needed to determine the predictive ability of these and other serum biomarkers in CVD risk stratification algorithms in RA patients. Rheumatoid arthritis (RA) is independently associated with a higher risk of cardiovascular disease (CVD) and premature atherosclerosis. Mechanisms of atherosclerosis include: Endothelial dysfunction/activation mediated by intercellular adhesion molecules (e.g., ICAM-1, VCAM-1, E-selectin) and oxidative stress (e.g., through MPO activity) Inflammation mediated by cytokines (e.g., IL-6) Plaque stability mediated by CD40-CD40L interactions Proteolysis/Plaque rupture mediated by proteolytic enzymes (e.g. MMP-9). The objective of this study was to evaluate whether these serum biomarkers are higher in RA subjects, and/or associated with subclinical carotid atherosclerosis. METHODS AND MATERIALS Figure 3. Correlations of ICAM-1, VCAM-1, and E-selectin with cIMT 46 RA cases and 70 controls were studied. Carotid ultrasounds were performed with measurement of intima-media thickness (cIMT, using maximum of both sides) and plaque presence (of either side). Serum biomarkers were measured by ELISA in all subjects: ICAM-1, VCAM-1, E-selectin (adhesion molecules) Myeloperoxidase (MPO) Interleukin-6 (IL-6) CD40L Matrix metalloproteinase-9 (MMP-9) Each of the biomarkers, cIMT, plaque presence, and demographic data, were tested for differences between cases and controls, using: Wilcoxon rank-sum test (for continuous data) or Chi-square test (for categorical data) The relationship between each serum biomarker and cIMT or plaque presence was tested by Spearman correlations or rank-sum test (for IL-6 only, which was categorized as above detection limit or not). Only 21% of subjects had detectable IL-6 levels. Table 2. cIMT and Serum Biomarkers of Atherosclerosis in RA and Controls RA (n=46) Controls (n=70) p-value cIMT (mm) 0.88 ± 0.23 0.79 ± 0.19 0.021 ICAM-1 (ng/mL) 260.4 ± 78.7 239.4 ± 79.8 0.18 VCAM-1 (ng/mL) 881.4 ± 238.5 902.2 ± 312.0 0.75 E-selectin (ng/mL) 34.0 ± 11.0 34.2 ± 13.2 0.83 MPO (ng/mL) 594.9 ± 670.9 727.4 ± 464.6 0.011 IL-6 (pg/mL) above detection limit (%) 31.8 14.3 0.025 CD40L (pg/mL) 9034.2 ± 4762.6 9086.7 ± 2890.6 0.25 MMP-9 (ng/mL) 446.7 ± 318.3 480.8 ± 213.4 0.085 ICAM-1: r = 0.361, p<0.001 VCAM-1: r= 0.283, p=0.002 CONCLUSIONS This study confirms previous observations that cIMT is higher in RA than non-RA subjects. Markers of endothelial dysfunction (ICAM-1, VCAM-1, E-selectin) are significantly associated with higher cIMT in both RA and non-RA subjects. This suggests possible common pathways of atherosclerotic progression as evidenced by cIMT and biomarker elevation. Further studies are needed to determine the predictive ability of these and other serum biomarkers in CVD risk stratification algorithms in RA patients. All values are mean ± SD unless indicated otherwise. cIMT is the maxiumum of both sides. Research reported in this poster was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (NIH) under Award Number 5K23AR061407-02 and was made possible by Grant Number 2UL1 RR024153-06 from the National Center for Research Resources (NCRR), a component of the NIH, and NIH Roadmap for Medical Research (support for CTSI). This work was also made possible by Genentech, NIH / NIAMS Award Number 1RC2-AR-058989 (support for RACER study); the Margaret J Miller endowed chair for Arthritis Research (Dr. Moreland); and institutional funds from the University of Pittsburgh Division of Rheumatology and Clinical Immunology and the Department of Medicine. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NCRR or NIH, Genentech, or the University of Pittsburgh. The authors also acknowledge Louise DeRiso, Dawn McBride, Min Shi, and Maria Jaksec (research coordinators). Table 1. Demographic Characteristics of RA and Controls eSelectin: r = 0.318, p<0.001 Figure 1. cIMT in RA vs. Controls Figure 2. MPO in RA vs. Controls RA (n=46) Controls (n=70) p-value Age (yrs), mean ± SD 59.3 ± 12.3 54.7 ± 16.3 0.19 BMI (kg/m2), mean ± SD 28.9 ± 6.3 28.7 ± 7.1 0.74 Female gender (%) 71.7 67.1 0.60 Caucasian race (%) 93.5 72.9 0.001 African-American or other race (%) 6.5 27.1 Figure 4. Box Plots of CD40L and MPO by Plaque Presence REFERENCES Carotti M, Salaffi F, Mangiacotti M, Cerioni A, Giuseppetti GM, Grassi W. Atherosclerosis in rheumatoid arthritis: the role of high-resolution B mode ultrasound in the measurement of the arterial intima-media thickness]. Reumatismo 2007;59:38-49. 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Soluble CD40 ligand levels indicate lipid accumulation in carotid atheroma: an in vivo study with high-resolution MRI. Arterioscler Thromb Vasc Biol 2003;23:e11-4. Schonbeck U, Varo N, Libby P, Buring J, Ridker PM. Soluble CD40L and cardiovascular risk in women. Circulation 2001;104:2266-8. Wong ND, Gransar H, Narula J, Shaw L, Moon JH, Miranda-Peats R, Rozanski A, Hayes SW, Thomson LE, Friedman JD, Berman DS. Myeloperoxidase, subclinical atherosclerosis, and cardiovascular disease events. JACC Cardiovasc Imaging. 2009 Sep;2(9):1093-9. CONTACT Kimberly P. Liang, MD University of Pittsburgh Division of Rheumatology and Clinical Immunology Email: liangkp@upmc.edu Phone: (412) 383-8846 Wilcoxon rank-sum test, p=0.0235 Wilcoxon rank-sum test, p=0.0462