Identifying Vulnerable Plaque in Rheumatoid Arthritis Using Novel

JDMXXX10.1177/8756479320922512Journal of Diagnostic Medical SonographyWang et al. research-article9225122020

Original Research

Journal of Diagnostic Medical Sonography 2020, Vol. 36(4) 300–310 Identifying Vulnerable Plaque in © The Author(s) 2020 Article reuse guidelines: sagepub.com/journals-permissions Rheumatoid Arthritis Using Novel DOI:https://doi.org/10.1177/8756479320922512 10.1177/8756479320922512 Microbubble Contrast-Enhanced journals.sagepub.com/home/jdm Carotid Ultrasonography and Serum Biomarkers

Linda F. Wang, MD1 , Yaming Li, MD, MS2, Douglas P. Landsittel, PhD2, Steven E. Reis, MD3, Marc C. Levesque, MD, PhD4, Donald M. Jones, MS4, Rachel Gartland, MD4, Jennifer Avolio, MBA, RVT5, Ali Shoushtari, MD5, Zengbiao Qi, PhD4, Cameron Dezfulian, MD6, Larry W. Moreland, MD4, and Kimberly P. Liang, MD4

Abstract Objective: Rheumatoid arthritis (RA) is associated with increased risk of cardiovascular disease. Adventitial vasa vasorum density (aVVD), the vessel density of the vasa vasorum, is a surrogate measure for atherosclerotic plaque vulnerability. The purpose of this study was to compare the adventitial vasa vasorum density (aVVD) in RA and non-RA control participants using novel carotid artery contrast-enhanced ultrasound (CEUS). In addition, we investigate associations of aVVD with traditional cardiovascular (CV) risk factors, vascular and inflammatory biomarkers, and RA disease activity. Methods: The study was a cross-sectional analysis of patients with RA and control participants without RA or other autoimmune disease. CV disease risk, biomarkers, and CEUS images were collected on all patients. Results: aVVD was quantified in 86 patients with RA and 95 non-RA control participants. Nitrite, CD40L, E-selectin, matrix metalloproteinase 9, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, myeloperoxidase (MPO), high-sensitivity C-reactive protein (hsCRP), and erythrocyte sedimentation rate were measured. Median aVVD was higher in patients with RA (0.59 [0.47–0.69] vs 0.64 [0.54–0.62]; P = .02). In patients with RA, MPO was lower (253.5 [153.2–480] vs 470.8 [274.2–830.1] ng/mL; P = .0002) and ESR was higher (15.5 [11–25] vs 13 [9–20] mm/h; P = .02). aVVD was correlated with MPO (r = −0.33, P = .001) and hsCRP (r = 0.25, P = .02) in control participants only, associations that remained significant after adjusting for number of CV risk factors and age. No significant correlations were found between aVVD and RA disease activity measures. Conclusions: Using a novel application of CEUS, we found that aVVD, an early measure of plaque vulnerability, was significantly higher in RA than control subjects, even after adjusting for CV risk factors. Differences in correlation of aVVD with vascular biomarkers and CV risk factors suggest RA-related differences in atherosclerotic progression.

Keywords vulnerable plaque, rheumatoid arthritis, contrast-enhanced ultrasound, inflammation

1School of Medicine, University of Pittsburgh, Pittsburgh PA, USA Received August 31, 2019, and accepted for publication March 15, 2Department of Biomedical Informatics, University of Pittsburgh, 2020. Pittsburgh PA, USA 3Division of Cardiology, University of Pittsburgh, Pittsburgh PA, USA Corresponding Author: 4Division of Rheumatology and Clinical Immunology, University of Kimberly P. Liang, MD, Division of Rheumatology and Clinical Pittsburgh, Pittsburgh PA, USA Immunology, University of Pittsburgh, 3708 Fifth Ave., Suite #501, 5Clinical and Translational Science Institute, University of Pittsburgh, Pittsburgh, PA 15213, USA. Pittsburgh PA, USA Email: [email protected] 6Vascular Medicine Institute and Critical Care Medicine, University of Pittsburgh, Pittsburgh PA, USA Wang et al. 301

Patients with rheumatoid arthritis (RA) have an approxi- In addition to use of imaging markers to detect sub- mately twofold increased risk of developing coronary clinical atherosclerosis, vascular and inflammatory bio- artery disease1 and heart failure.2 However, traditional markers have been found to predict atherosclerosis and cardiac risk factors found in the general population, such CV events.12 It has been suggested that such biomarkers as dyslipidemia and elevated body mass index (BMI), do may have greater influence than traditional risk factors in not adequately account for the increase in cardiovascular the pathogenesis of CVD in RA.13 These biomarkers (CV) disease found in patients with RA.3,4 Chronic reflect various mechanisms involved in atherosclerosis, inflammation has been increasingly implicated in the including endothelial dysfunction, inflammation, plaque development of atherosclerotic disease in RA. The ele- destabilization, and plaque rupture.14,15 Biomarkers of vated risk of cardiovascular disease (CVD) in patients endothelial dysfunction include intercellular adhesion with RA correlates with elevation of RA disease-related molecules: intercellular adhesion molecule 1 (ICAM-1), markers of inflammation, including high-sensitivity vascular cell adhesion molecule 1 (VCAM-1), E-selectin, 16 C-reactive protein (hsCRP), anti–cyclic citrullinated pep- and whole blood nitrite (NO2). Included in this group of tide (CCP) antibodies, and increased RA disease activity.5 biomarkers are proinflammatory and autoimmunity cyto- Notably, in the general population, systemic markers of kines that include interleukin 6 (IL-6) and interleukin 17 inflammation such as hsCRP have been associated with (IL-17), respectively.17 Mediators of plaque instability atherosclerotic plaque instability.6 Methods to identify include CD40 ligand (CD40L), matrix metalloproteinase features of plaque vulnerability in patients with RA with 9 (MMP-9), and myeloperoxidase (MPO).18 subclinical CVD could facilitate improved CV risk strati- Prior studies have found associations between vascu- fication of patients with RA. lar biomarkers and common carotid intima-media thick- A key early event in the pathogenesis of vulnerable ness (cIMT), as a marker of subclinical atherosclerosis, in plaques is neovascularization of the adventitial vasa patients with RA. However, to date, there are no studies vasorum, the network of small vessels that nourish the reporting the use of the novel imaging technique of CEUS arterial wall. Histological studies of human carotid, iliac, to quantify aVVD, a surrogate measure of plaque vulner- and renal arteries have shown that the adventitial vasa ability, in the RA population. The purpose of this study vasorum density (aVVD) is increased in patients with was to compare aVVD in RA and non-RA control partici- symptomatic atherosclerosis, and this neovasculariza- pants using CEUS. In addition, the study investigated an tion develops prior to intimal thickening.7 Furthermore, association of aVVD with traditional CV risk factors, histological studies of human aortas have found that vascular and inflammatory serum biomarkers, and RA increased microvessel density correlates with pathologic disease activity. features of plaque instability and is significantly increased in ruptured plaques compared to nonruptured plaques.8 Methods Contrast-enhanced ultrasound (CEUS) is a novel Study Design method that has been used to visualize features of vulnerable atherosclerotic plaque, including neovascularization The study was a cross-sectional analysis of patients with of the vasa vasorum and intraplaque neovascularization. RA and control participants without RA or other autoim- Gas-filled microbubbles that resonate under the ultra- mune disease. The primary objective was to compare the sound beam are injected intravenously and act as intravas- levels of carotid artery aVVD between those with and cular contrast.9 A CEUS study performed on the carotid without RA. The secondary objective was to compare the arteries of 147 human participants demonstrated that the levels of vascular and inflammatory serum biomarker presence, degree of adventitial vasa vasorum, and plaque levels between RA and non-RA participants. In addition, neovascularization were significantly correlated with the association between aVVD and these serum biomark- CVD and prior adverse CV events.10 Histological valida- ers was assessed in both RA and non-RA participants tion of the quantification of vasa vasorum density by after adjusting for traditional CV risk factors. CEUS has been performed with an animal model. Among New Zealand white rabbits, the video intensity of the Study Population adventitia normalized to the video intensity of the arterial lumen was correlated with total cross-sectional area and Patients with RA (n = 90) were recruited from the number of microvessels in the adventitial vasa vasorum. Rheumatoid Arthritis Comparative Effectiveness Notably, serial CEUS studies demonstrated an increase in Research (RACER) study. RACER encompasses a regis- video intensity over time, suggesting the potential for try of longitudinal data collected from a cohort of over carotid vasa vasorum CEUS assessment as a method for 1000 patients with RA visiting the University of Pittsburgh monitoring plaque progression.11 Rheumatoid Arthritis center.19 Control participants (n = 302 Journal of Diagnostic Medical Sonography 36(4)

101) were volunteers recruited from (1) participants of assessing smoking status, family history, diabetes, use of the Heart Strategies Concentrating on Risk Evaluation an antihypertensive, and lipid-lowering medications. (SCORE) study (n = 28) and (2) the general population Blood pressure and BMI were measured. Fasting blood- by flyers/advertisements (n = 73). Heart SCORE is a lon- work included blood glucose, insulin level, and a lipid gitudinal, descriptive community-based research study of panel for use in estimating insulin sensitivity. In addition, 2000 white and black participants in southwestern a beta-cell function according to the homeostatic model Pennsylvania. It was designed to evaluate risk stratifica- assessment for insulin resistance (HOMA-IR), which is a tion and population differences in CVD with prespecified clinical and epidemiological tool, was used, where the recruitment goals of recruiting participants with low range of 0.7 to 2 is considered the reference.22,23 Framingham risk (n = 800), intermediate to high Framingham risk (n = 1000), and those with established Assessment of RA Disease-Specific Risk CVD (n = 200).20 Inclusion criteria for patients with RA were age ≥18 Factors years, meeting 2010 American College of Rheumatology Patients with RA underwent additional disease-specific classification criteria for RA diagnosis,21 and partici- assessments. RA disease activity was measured using the pating in the longitudinal RACER study. Inclusion cri- Clinical Disease Activity Index (CDAI), which includes teria for control participants were age ≥18 years with tender joint count, patient global assessment, and asses- no diagnosis of RA or other autoimmune disease. sor global assessment, ranging from 0 to 76. The CDAI Exclusion criteria were the following: age <18 years, score is interpreted as follows: remission (0.0–2.8), low pregnancy, pulmonary hypertension, and contraindica- activity (2.9–10.0), moderate activity (10.1–22.0), and tions to use of the perflutren ultrasound contrast agent, high activity (22.1–76.0).24 Joint counts and assessor Definity (Lantheus Medical Imaging, North Bellerica, global assessment were recorded by trained staff mem- MA). At the time of the study, contraindications to bers. In addition, a physician-calculated disease activity Definity included presence of intracardiac and intrapul- measure, the Disease Activity Score (DAS-28), was monary shunts, unstable cardiopulmonary conditions, obtained. DAS-28 is a continuous index ranging from 0 and use of chronic oxygen therapy. Since then, contra- to 9.4, in which low disease activity is defined as ≤3.2, indications have been amended to include large cardiac moderate disease activity is defined as >3.2 to ≤5.1, and shunts (right to left, bidirection, or transient right to high disease activity is defined as >5.1.25 A commonly left), hypersensitivity to perflutren, and administration used cutoff point for remission in DAS-28 is <2.6.26 by intra-arterial injection. All participants provided In all participants, RA disease-related serum measures written informed consent. The study was approved by were obtained, including erythrocyte sedimentation rate the University of Pittsburgh Institutional Review Board (ESR), high-sensitivity C-reactive protein (hsCRP), rheu- and the Human Research Protection Office. matoid factor (RF), and anti-CCP antibody. CCP was In the final analysis, 97 control participants and 87 determined using a kit from Axis-Shield Diagnostic patients with RA were included. Two control participants Limited (Dundee, UK). RF and hsCRP were measured by and two patients with RA were not included due to a lack the University of Pittsburgh Medical Center (UPMC) of intravenous (IV) access, one control participant was clinical laboratory. excluded due to hypertension during the study visit, and Use of medications, including disease-modifying anti- one patient with RA withdrew from the study prior to data rheumatic drugs (DMARDs), biologic agents, nonsteroi- collection. The final CEUS image analysis included 95 dal anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 control participants and 86 patients with RA. Of the three (COX-2) inhibitors, and glucocorticoids, was recorded. excluded CEUS image sets, one control participant’s images were excluded for an incomplete contrast of the arterial lumens. One control participant and one RA par- Microbubble Contrast-Enhanced Carotid ticipant’s images had to be excluded due to poor quality. Sonography Participants were examined in a dimly lit, temperature- Assessment of Traditional Cardiovascular Risk controlled room on an examination table lying supine Factors with their heads turned slightly to the left. It was ensured that participants’ necks were relaxed, as contractions of Participants completed a study visit at the University of the sternocleidomastoid muscle (SCM) cause poor imag- Pittsburgh Vascular Clinical and Translational Research ing due to lack of ultrasound penetration due to difficult Center (VCTRC). All participants were assessed for tra- transducer placement. The common carotid artery (CCA), ditional cardiovascular risk factors using questionnaires carotid bulb, internal carotid artery (ICA), and external Wang et al. 303 carotid artery (ECA) were imaged for this study. Imaging repeated when necessary. The examiner ensured imaging was completed on both the right and left sides using an consistency with precontrast baseline imaging. After “ear-to-ear” approach. injection, the examiner waited for microbubbles to fully The CCA usually originates from the innominate artery fill the vessel before capturing any images; the fill time on the right side and the aortic arch on the left side. The was dependent on heart rate. During image capture, quiet CCA course runs anterolateral with the SCM muscle and breathing and breath suspension in neutral were preferred the internal jugular vein (IJV), as well as medial to the thy- over breath hold in full inspiration or expiration. First, the roid. The mid-to-distal area of the CCA runs parallel to the left CCA was evaluated, and then the right CCA was eval- skin surface. The IJV is distinguished from the CCA by uated in the same way. Cineclips were digitally stored as vein movements controlled mainly by respirations. The an Audio Video Interleave (AVI) file and reviewed offline. sonographic texture of the carotid wall consists of three The optimal time window for performing CEUS, after parallel layers. The internal and external layers are hyper- administration of the contrast agent, was approximately echoic, and the middle layer is hypoechoic. Most often, the one to two minutes. Once the contrast media were admin- CCA divides into the ICA and the ECA at the level of the istered, arterial lumen enhancement began approximately superior border of the thyroid. The point of this division is after 10 to 15 seconds and lasted for up to two to five min- a dilated bulbous area referred to as the carotid bulb. utes. The administration of the contrast media was repeated CEUS was performed along the CCAs bilaterally by a if deemed necessary.9,29 Injected contrast volume, lot num- trained sonographer with the Registered Vascular ber, and time of administration were documented. Technologist (RVT) credential. All examinations were Participants underwent electrocardiogram (EKG) mon- performed using a GE Vivid 7 (General Electric Company, itoring throughout the imaging procedure and were moni- Milwaukee, WI) equipped with a 9L linear multifre- tored for 30 minutes following completion. Following the quency transducer and a dedicated vascular scanning CEUS examination, the participant continued to lie in the package. The B-mode settings were adjusted to optimize supine position for 30 minutes to monitor their blood pres- the quality of the gray-scale images, and the pulse repeti- sure, heart rate, and oxygen saturation. During postproce- tion frequency (PRF) used with color Doppler flow imag- dure, the IV access was discontinued. ing was adjusted according to the flow velocity. Two suspected adverse reactions to the contrast media Carotid sonographic images were obtained using a were recorded. One control participant experienced onset of standardized protocol based upon the most current rec- diffuse hives, itching, and faintness that was treated with ommendations.27 In short, first the left CCA, carotid bulb, diphenhydramine 50 mg IV, methylprednisolone 125 mg IV, ICA, and ECA were evaluated using gray-scale sonogra- and famotidine 20 mg IV, and this person was transferred to phy, color Doppler, and pulsed-wave Doppler. The right the emergency department. With this treatment, symptoms CCA, carotid bulb, ICA, and ECA were evaluated in the resolved without complications. Another control participant same way. After acquisition of the standard carotid artery complained of mild shortness of breath and tongue tingling sonographic images, the contrast preset of the ultrasound that were not treated. These symptoms resolved in 16 min- system was selected. The preset uses an ultrasound pulse utes after injection without complications. sequence with a low mechanical index (0.25–0.35/7) to 28 avoid destruction of the microbubbles. Three sagittal Image Analysis images of the distal CCA were obtained in five-cycle cine-loops with the focal point placed at the (1) near wall, Analysis of aVVD was performed on stored CEUS images (2) vessel, and (3) far wall. and cine-loops. The analysis was performed on Myocardial An IV cannula was inserted in the participant’s left Contrast Echocardiography (MCE) 2.9 software (Yabko, arm, preferably the antecubital vein, to avoid interaction LLC, University of Virginia), which quantitatively mea- of the injector with the right-sided examiner. The vial of sures the video intensity of cine-loop images. In CEUS, the Definity was allowed to warm to room temperature adventitial layer is hyperechoic, and evidence of neovascu- before starting the activation procedure. Activation was larization is indicated by dynamic movement of echogenic performed by shaking the vial for 45 seconds using a bubbles, whereas fixed echogenic signals are treated as tis- Vialmix (Lantheus Medical Imaging). The designated sue acoustic reflectors.30 A region of interest (ROI) was registered nurse (RN) withdrew the material from the selected in the intimal-medial layer on the posterior (far) middle of the liquid in the inverted vial, while avoiding wall of the CCA, and video intensity was measured over six injecting any air, into a 10-cc saline syringe in which 1.5 diastolic frames, within the MCE software. The video mL of saline had been expressed prior. intensities were averaged using Microsoft Excel (Microsoft, The ultrasound contrast media were injected in boluses Redmond, WA) and normalized to the video intensity of an of 0.5 cc through the peripheral IV line, followed by a ROI within the artery lumen using Stata statistical software normal saline flush, and the bolus administration was (StataCorp LLP, College Station, TX). aVVD was 304 Journal of Diagnostic Medical Sonography 36(4)

quantified as the ratio of mean CCA adventitial to lumen factors (age, BMI, hypertension, diabetes, high choles- video intensity for each CCA (right and left) far wall. For terol, smoking, family or personal history of CVD) and each participant, the maximum aVVD of either the right or anti-inflammatory medications (Table 1). RA-related mea- left CCA was used in the statistical analyses. Blinded image sures included RF and CCP positivity, disease activity, and analyses were performed independently by two investiga- DMARD use (Table 2). Among patients with RA, median tors (K.P.L. and L.F.W.). A subset of 20 image analyses was (interquartile range [IQR]) disease duration was 9.9 (5.6– performed separately by the two investigators (K.P.L. and 17.1) years. Median (IQR) DAS-28 score was 0.927 (0.36– L.F.W.), with excellent interrater reliability; intraclass cor- 1.47) and CDAI score was 8.5 (3–16) (Table 2). Patients relations (ICCs) were >0.80 for all sets of ROIs. with RA were older, more hypertensive, and more hyper- cholesterolemic, and they had a higher number of CV risk Vascular and Inflammatory Biomarkers factors (40.2% vs 20.6% had three or more risk factors; P = .003) compared to control participants. Twelve patients CD40L, E-selectin, MMP-9, ICAM-1, VCAM-1, MPO, with RA and two control participants reported a personal hsCRP, and ESR were measured in serum. Whole blood history of CVD (P = .01). Other vascular comorbidities nitrite was measured by chemiluminescence following the did not differ significantly between groups, including his- protocol described by Piknova and Schechter.31 Blood sam- tory of stroke or cerebrovascular disease (RA, n = 3; con- ples were drawn during study visits on the same day as the trol, n = 1), atrial fibrillation (RA, n = 1; control, n = 1), CEUS studies were performed. Whole blood in a 10-mL and deep vein thrombosis or pulmonary embolism (RA, n serum separator tube was centrifuged at 800 g for 10 min- = 1; control, n = 1). Other comorbidities, including utes at room temperature. Serum was stored as 200-µL ali- chronic kidney disease, chronic lung disease, chronic liver quots at −80°C for laboratory testing. Enzyme-linked disease, peptic ulcer disease, sleep apnea, and hematologic immunosorbent assay (ELISA) kits from R&D Systems disorders (anemia, abnormal leukocyte, or platelet count), (Minneapolis, MN) were used to measure serum CD40L, did not differ significantly between groups (data not E-selectin, MMP-9, ICAM-1, VCAM-1, and MPO, accord- shown). Of these additional comorbidities, the most com- ing to the manufacturer’s protocols. Serum IL-6 and IL-17A mon were sleep apnea (RA, n = 8; control, n = 5) and concentrations were analyzed using kits from eBioscience hematologic disorder (RA, n = 16; control, n = 8). (San Diego, CA). The hsCRP and ESR were measured Regarding medication use, 36 patients with RA were according to UPMC clinical laboratory protocols. using biologics at the time of study, of which tumor necrosis factor (TNF) inhibitors were used most frequently (n = Statistical Analysis 35). Of the nonbiologics, most frequently reported DMARDs used, past or present, were methotrexate (MTX) Demographic data, CV risk factors, and biomarker levels (n = 70) and hydroxychloroquine (n = 37). One control were compared between RA and control participants using participant reported past DMARDs consisting of sulfasala- Wilcoxon rank-sum or χ2 tests. Association of aVVD with zine (n = 1). Corticosteroids, aspirin, and COX-2 inhibitor biomarkers and CV risk factors, stratified by case status, use was significantly higher among patients with RA, was examined using Pearson and Spearman correlations, whereas NSAID and statin use was similar between groups. respectively. The relationship between RA and aVVD was assessed using multiple linear regression modeling with Comparison of aVVD RA as the categorical exposure of interest and aVVD as the continuous outcome. aVVD was modeled as a function of An example of CEUS image interpretation, within MCE case status, age, sex, and CV risk factors. The relationship software, is depicted in Figure 1 with ROIs drawn in the between RA disease-related measures and aVVD was contrasted arterial lumen and in the adventitia (Figure 1). assessed in the same manner except only patients with RA aVVD was higher in patients with RA compared to were included. The relationship between medication use controls (P = .02) (Figure 2) with median (IQR) aVVD and aVVD was assessed using the rank-sum test or of 0.62 (0.54–0.77) vs 0.59 (0.47–0.69), respectively. In Kruskal-Wallis H test. All statistical analyses were per- linear regression models, RA case status was significantly formed using Stata (StataCorp LLP). associated with higher aVVD, even after adjusting for age, hypertension, hypercholesterolemia, number of CV risk factors, and personal history of CVD (P = .017). In Results multivariable models adjusting for age, BMI, hyperten- Demographic and Clinical Characteristics sion, diabetes, hypercholesterolemia, smoking, family or personal history of CVD, and number of CV risk factors, The recorded demographic and clinical characteristics of RA case status remained significantly associated with RA and control participants included traditional CV risk higher aVVD (P = .011). Wang et al. 305

Table 1. Demographic and Clinical Characteristics of RA and Control Participants.a

Variable Control (n = 97) RA (n = 87) P Value Age, median (IQR), y 56 (18) 61 (12) .01 Sex (female) 69 (72.6) 71 (81.6) .13 Race .56 Caucasian 76 (80) 72 (82.8) African American or Other 19 (20) 15 (17.2) BMI, median (IQR), kg/m2 27.15 (23.81–31.00)b 27.43 (24.73–31.24) .48 Obesity 28 (29.5) 32 (36.8) .4 Hypertension 18 (21.4) 39 (46.4) <.0001 Diabetes 7 (7.5) 7 (8.1) .85 High cholesterol 27 (28) 37 (43) .04 Smoking 41 (43.2) 49 (56.3) .06 Family history of CVD 27 (29) 26 (30.1) .71 Personal history of CVD 2 (2) 12 (14) .01 Number of CV risk factors .003 0 21 (22.1) 11 (12.6) 1 33 (34.7) 23 (26.4) 2 21 (22.1) 18 (20.7) ≥3 20 (21.1) 35 (40.2) HOMA-IR, median (IQR) 1.36 (0.96–2.58)c 2.53 (1.45–4.87)d .04 Corticosteroid use 34 (42)e 65 (74.7) <.001 NSAID use 75 (92.6)e 73 (83.9) .098 COX-2 inhibitor use 5 (6.2)e 31 (35.6) <.001 Aspirin use 51 (52.6) 38 (43.7) <.001 Statin use 24 (0.3)f 29 (33.3) .74

Abbreviations: BMI, body mass index; CV, cardiovascular; CVD, cardiovascular disease; HOMA-IR, homeostatic model assessment for insulin resistance; IQR, interquartile range; NSAID, nonsteroidal anti-inflammatory drug; RA, rheumatoid arthritis. aResults are expressed as number (%) unless indicated otherwise. Data were analyzed using Wilcoxon rank-sum or χ2 tests. bn = 96. cn = 30. dn = 45. en = 81. fn = 80.

Comparison of Vascular and Inflammatory lipidemia, hypertension, diabetes, and family history of Biomarkers CVD) was performed in control participants and patients with RA separately. These variables did not significantly Results for vascular and inflammatory biomarkers in RA predict aVVD with F(8, 72) = 0.50, P = .85, R2 = 0.05 in and control participants are shown in Table 3. Patients with control participants and F(8, 70) = 0.85, P = .56, R2 = RA had lower MPO (P = .0002) and higher ESR levels (P 0.09 in patients with RA. When stratified by case status, the = .01) than control participants. aVVD was correlated with correlation of aVVD with MPO in controls remained sig- MPO (r = −0.33, P = .001) and hsCRP (r = 0.25, P = .02) nificant in linear regression models adjusting for age, sex, by Pearson correlation in control participants (Table 4). No and CV risk factors, F(8, 72) = 1.84, P = .001, R2 = 0.09. significant correlations were found between aVVD and bio- The correlation of aVVD with hsCRP in controls became markers in patients with RA (Table 4). Number of CV risk nonsignificant in this adjusted linear regression analysis, factors and personal history of CVD were not significantly F(9, 71) = 0.53, P = .402, R2 = 0.06. No substantial correlated with aVVD in both RA and controls (data not changes in the other associations between aVVD and bio- shown). Among patients with RA, disease duration, CDAI, markers were found with linear regression (data not shown). DAS-28, and medication use were not significantly corre- In multivariable linear regression models adjusting only for lated with aVVD (data not shown). those variables that were significantly different between RA and controls (age, hypertension, hypercholesterolemia, Association of aVVD With Biomarkers number of CV risk factors, and personal history of CVD), nitrite was significantly associated with higher aVVD (P = Multiple linear regression modeling of aVVD adjusting for .044), and MPO was significantly associated with lower age, sex, and CV risk factors (obesity, smoking, hyper- aVVD (P = .002) in controls only. No significant 306 Journal of Diagnostic Medical Sonography 36(4)

Table 2. RA-Related Biomarkers, Disease Activity Scores, and DMARD Use.a

Variable Control (n = 97) RA (n = 87) P Value RF positive 7 (7) 50 (57) <.001 CCP positive 22 (23) 61 (70) <.001 Disease duration, y — 9.9 (5.6–17.1)b — DAS-28 — 0.93 (0.36–1.47) — CDAI — 8.5 (3–16) — DMARD usec 1 (1) 80 (92) <.001 Biologics current use 0 (0) 36 (41) <.001 TNF inhibitor current use 0 (0) 35 (40) <.001 Other biologics current use 0 (0) 9 (10) <.001 MTX <.001 Current use 0 (0) 52 (60) Past use 0 (0) 18 (21) Hydroxychloroquine <.001 Current use 0 (0) 21 (24) Past use 0 (0) 16 (18) Sulfasalazine <.001 Current use 0 (0) 7 (8) Past use 1 (1) 9 (10) Leflunomide <.001 Current use 0 (0) 7 (8) Past use 0 (0) 9 (10) Other DMARD .006 Current use 0 (0) 2 (2) Past use 1 (1) 8 (9)

Abbreviations: CCP, cyclic citrullinated peptide; CDAI, Clinical Disease Activity Index; DAS-28, Disease Activity Score; DMARD, disease- modifying antirheumatic drug; MTX, methotrexate; TNF, tumor necrosis factor; RA, rheumatoid arthritis; RF, rheumatoid factor. aResults are expressed as number (%) or median (interquartile range). bn = 73. cIn addition to “current use” and “past use,” participants could also report “unknown” for each type of medication. No participants reported past or unknown use for biologics overall and for TNF inhibitors. “Other biologics” comprised tocilizumab, rituximab, and tofacitnib. “Other DMARD” comprised azathioprine, auranofin/gold, gold sodium thiomalate, minocycline, cyclosporine, and penicillin.

Discussion It is believed that this cross-sectional study is the first to compare aVVD in patients with RA with controls using the novel CEUS. The median (IQR) aVVD of 0.59 (0.47– 0.69) in control participants is similar to the ratio (mean ± SD) of 0.577 ± 0.119 reported by Arcidiacono et al.32 In that study, the average of right and left common carotid aVVD, using CEUS, was used to measure aVVD in healthy participants without classical CV risk factors.32 An early measure of plaque vulnerability, common carotid artery aVVD is significantly higher in RA compared to control participants, suggesting increased vasa vasorum neovascularization in patients with RA. This study also sought to explore the idea that inflammatory pathways in RA may promote vasa vasorum neovascularization, result- Figure 1. Measurement of adventitial vasa vasorum density. (A) Lumen region of interest (ROI) intensity and (B) ing in the creation of more unstable plaques. adventitial ROI intensity. Prior attempts to identify subclinical CVD in patients with RA have predominately used traditional gray-scale associations were seen between these biomarkers and carotid sonography to assess common cIMT. It has also aVVD in the RA group in these models. been used to detect the presence of carotid plaques, Wang et al. 307

Figure 2. Adventitial vasa vasorum density (aVVD) between rheumatoid arthritis (RA) and control participants. Median aVVD (interquartile range) was 0.62 (0.54–0.77) in patients with RA vs 0.59 (0.47–0.69) in control participants (P = .02). (A) Box plot. (B) Dot plot.

Table 3. Vascular and Inflammatory Biomarkers in RA and Control Participants.a

Biomarker Control RA P Value Nitrite,b µM 0.15 (0.04–0.21) 0.17 (0.03–0.24) .11 CD40L, pg/mL 8361 (6682–10,490) 8070 (6300–10,189) .44 E-selectin, ng/mL 29.2 (20.8–42.9) 33 (23.2–43.1) .19 MMP-9, ng/mL 372.3 (335.9–618.2) 372.3 (265.7–644.9) .13 MPO, ng/mL 470.8 (274.2–830.1) 253.5 (153.2–480) .0002 ICAM-1, ng/mL 234.3 (189.5–271.9) 234.3 (190.1–285.8) .57 VCAM-1, ng/mL 760.2 (653.8–986.4) 760.2 (631–964.4) .31 IL-6 detected,c pg/mL 22 (23.2) 31 (35.6) .08 IL-17 detected,c pg/mL 6 (6.3) 2 (2.3) .20 hsCRP, mg/dL 0.14 (0.07–0.32) 0.19 (0.09–0.8) .07 ESR,d mm/h 13 (9–20) 15.5 (11–25) .01

Abbreviations: CD40L, CD40 ligand; ESR, erythrocyte sedimentation rate; hsCRP, high sensitivity C-reactive protein; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; MMP-9, matrix metalloproteinase 9; MPO, myeloperoxidase; RA, rheumatoid arthritis; VCAM-1, vascular cell adhesion molecule 1. aResults are expressed as median (interquartile range) unless indicated otherwise. Data were analyzed using Wilcoxon rank-sum or χ2 tests. bTotal, n = 110; control, n = 56; RA, n = 54. cResults expressed as number (%). dTotal, n = 176; control, n = 94; RA, n = 82. measures that are predictors of CV events in the general adventitial VV has been shown to increase prior to intimal population. In a meta-analysis examining the relationship thickening in humans and animals,7,34,35 thus suggesting between RA and imaging markers of CV risk, presence of that measuring aVVD could identify earlier stages of neo- carotid plaques and cIMT was significantly higher in vascularization in the development of atherosclerosis pre- patients with RA compared to controls, suggesting that ceding the increase in cIMT. In addition, when directly RA is significantly associated with subclinical athero- compared with gray-scale carotid sonography, CEUS has sclerosis and CV risk.33 This study’s results using a novel been found to be superior for visualization of the intima- application of CEUS align with these findings but offer media complex in performing cIMT measurements and several advantages. improves detection of carotid plaques.36 CEUS offers the additional capability of quantifying The lack of correlation between aVVD and traditional adventitial neovascularization in a noninvasive manner, CV risk factors, in both RA and control participants, may whereas gray-scale carotid sonography cannot distinguish indicate that certain risk factors contribute more to CV, vessel density in the vasa vasorum. Of note, increased warranting further study. Staub et al.10 also report a lack cIMT reflects intimal thickening thought to result from of association between VV enhancement on CEUS and adventitial vasa vasorum (VV) neoangiogenesis, yet CV risk factors but found that participants with more 308 Journal of Diagnostic Medical Sonography 36(4)

Table 4. Pearson Correlations of Adventitial Vasa Vasorum feature of early vulnerable plaque, and most participants Density With Vascular and Inflammatory Biomarkers in RA had no personal history of CVD. This was compared to an and Control Participants. elevated MPO, found in patients presenting to the emer- Control RA gency department with acute coronary syndromes. This may support the idea that MPO elevation is a later finding Biomarker r P Value r P Value in the progression of CVD.40 The biomarker levels in Nitrite 0.265 .05 0.042 .76 patients with RA were also likely affected by DMARD CD40 0.091 .38 −0.129 .24 use, as a wide variety of cytokines, including IL-6, decrease 41,42 E-selectin −0.045 .67 −0.090 .41 in response to DMARDs. MMP-9 0.030 .77 0.142 .19 This study did not demonstrate an association between MPO −0.332 .001 −0.047 .66 aVVD and RA disease duration or disease activity, as ICAM-1 0.060 .57 0.076 .49 measured by CDAI and DAS-28. Similarly, studies of VCAM-1 0.095 .36 −0.093 .40 cIMT have not consistently correlated with RA disease hsCRP 0.250 .02 −0.057 .61 activity.43 Such results are inconsistent with the known ESR 0.198 .06 −0.082 .47 association between CVD and increased RA disease- related markers of inflammation.5 This study’s results Abbreviations: ESR, erythrocyte sedimentation rate; hsCRP, high sensitivity C-reactive protein; ICAM-1, intercellular may have been influenced by the low disease activity adhesion molecule 1; MMP-9, matrix metalloproteinase 9; MPO, scores of the patients with RA, of whom 96% were in myeloperoxidase; RA, rheumatoid arthritis; VCAM-1, vascular cell clinical remission or with low disease activity as catego- adhesion molecule 1. rized by the DAS-28. In a study of patients with RA in whom 70% had a DAS-28 score in clinical remission or pronounced VV were more likely to have CVD. This with low disease activity, cIMT was not associated with study did not find an association between aVVD and per- presence of RA, DAS-28, RF level, or anti-CCP level.44 sonal history of CVD, but it was not powered to detect such an association. It only had two control participants Study Strengths and 12 patients with RA reporting a positive CV history.10 Strengths of this study include large sample size and use To assess the possible contribution of traditional CV of novel quantitative CEUS technique in a new popula- risk factors to the elevated aVVD in patients with RA, tion. In addition, the concomitant systematic collection of multiple linear regression analysis was performed with traditional CV risk factors, vascular and inflammatory case status as the primary predictor and aVVD as the out- biomarkers, and RA disease activity measures allowed come, adjusting for age, sex, and traditional CV risk fac- for examination of potential relationships between aVVD tors. In the multiple linear regression models, RA case and the pathophysiology of CVD in RA. status was significantly associated with higher aVVD even in the adjusted models, suggesting that the increased Study Limitations presence of traditional CV risk factors in the patients with RA does not completely account for their elevated aVVD This is a cross-sectional study with an inability to assess compared to controls. The measurement of aVVD in aVVD longitudinally. Larger prospective studies are patients with RA in this study highlights a potential appli- needed to determine the relationship between aVVD and cation of CEUS to detect subclinical vulnerable athero- CVD risk in patients with RA. While quantitative, the sclerosis where assessment of traditional CV risk factors method used for measuring aVVD involved user selection may underestimate atherosclerosis. of ROI, and future studies could likely benefit from more The lack of significant associations of aVVD with bio- standardized and automated methods of measuring aVVD, markers in patients with RA could reflect differential such as the software under development for measuring effects of these biomarker pathways in subclinical athero- intraplaque neovascularization with CEUS.45,46 Ideally, to sclerosis compared to controls. Of note, carotid intraplaque better distinguish the effect of traditional CV risk factors neovascularization assessed using CEUS and histology has on aVVD, the study groups should have similar baseline not consistently been associated with inflammation.37 This traditional CV risk factors. may suggest a need for further study of temporal relationships between inflammation and neovascularization. The Conclusion finding that aVVD inversely correlated with MPO in control participants was unexpected given prior studies show- The novel CEUS technique may be used to noninvasively ing higher levels of MPO in individuals with CVD.38,39 quantify aVVD, an early measure of plaque vulnerability. This finding could be from variability in measured MPO aVVD is significantly higher in patients with RA com- levels within this study. However, increased aVVD is a pared to control participants, even after adjusting for Wang et al. 309 traditional CV risk factors. Differences in association of 4. Gonzalez A, Maradit Kremers H, Crowson CS, et al: Do aVVD with traditional CV risk factors and inflammatory cardiovascular risk factors confer the same risk for car- and vascular biomarkers in patients with RA compared to diovascular outcomes in rheumatoid arthritis patients control participants suggest there may be RA-related dif- as in non-rheumatoid arthritis patients? Ann Rheum Dis ferences in atherosclerotic progression that require further 2008;67(1):64–69. 5. Maradit-Kremers H, Nicola PJ, Crowson CS, Ballman KV, study. Gabriel SE: Cardiovascular death in rheumatoid arthritis: a population-based study. Arthritis Rheum 2005;52(3): Acknowledgments 722–732. We thank the study coordinators Louise DeRiso and Maria 6. Lombardo A, Biasucci LM, Lanza GA, et al: Inflammation Jaksec; Dr. Flordeliza Villanueva and Dr. Suresh Mulukutla for as a possible link between coronary and carotid plaque guidance on aVVD analysis techniques; Bernadette Sendon for instability. Circulation 2004;109(25):3158–3163. technical assistance; and the University of Pittsburgh Vascular 7. Fleiner M, Kummer M, Mirlacher M, et al: Arterial neovas- Medicine Institute. cularization and inflammation in vulnerable patients: early and late signs of symptomatic atherosclerosis. Circulation Declaration of Conflicting Interests 2004;110(18):2843–2850. The authors declared no potential conflicts of interest with respect 8. Moreno PR, Purushothaman KR, Fuster V, et al: Plaque to the research, authorship, and/or publication of this article. neovascularization is increased in ruptured atherosclerotic lesions of human aorta: implications for plaque vulnerability. Circulation 2004;110(14):2032–2038. Funding 9. Schinkel AFL, Kaspar M, Staub D: Contrast-enhanced The authors disclosed receipt of the following financial support ultrasound: clinical applications in patients with atheroscle- for the research, authorship, and/or publication of this article: rosis. Int J Cardiovasc Imaging 2016;32(1):35–48. This research study was funded by the National Institute of 10. Staub D, Patel MB, Tibrewala A, et al: Vasa vasorum and Arthritis and Musculoskeletal and Skin Diseases of the National plaque neovascularization on contrast-enhanced carotid Institutes of Health (NIH) award number 5K23AR061407-02 and ultrasound imaging correlates with cardiovascular disease the National Center for Research Resources (NCRR) grant 2UL1 and past cardiovascular events. Stroke 2010;41(1):41–47. RR024153-06. The Clinical and Translational Science Institute at 11. Moguillansky D, Leng X, Carson A, et al: Quantification the University of Pittsburgh is supported by the NIH Clinical and of plaque neovascularization using contrast ultrasound: a Translational Science Award program, grant UL1 TR001857. histologic validation. Eur Heart J 2011;32(5):646–653. 12. Malik I, Danesh J, Whincup P, et al: Soluble adhesion mole- ORCID iD cules and prediction of coronary heart disease: a prospective Linda F. Wang https://orcid.org/0000-0001-9036-5561 study and meta-analysis. Lancet 2001;358(9286):971–976. 13. Solomon DH, Curhan GC, Rimm EB, Cannuscio CC, Supplemental Material Karlson EW: Cardiovascular risk factors in women with and without rheumatoid arthritis. Arthritis Rheum Scan the QR code with your smartphone to view the online 2004;50(11):3444–3449. supplemental videos available at https://journals.sagepub.com/ 14. Libby P: Inflammation in atherosclerosis. Arterioscler doi/suppl/10.1177/8756479320922512. Thromb Vasc Biol 2012;32(9):2045–2051. 15. Raggi P, Genest J, Giles JT, et al: Role of inflammation in the pathogenesis of atherosclerosis and therapeutic inter- ventions. Atherosclerosis 2018;276:98–108. 16. Galkina E, Ley K: Vascular adhesion molecules in atherosclerosis. Arterioscler Thromb Vasc Biol 2007;27(11): 2292–2301. 17. Ait-Oufella H, Taleb S, Mallat Z, Tedgui A: Recent References advances on the role of cytokines in atherosclerosis. 1. Maradit-Kremers H, Crowson CS, Nicola PJ, et al: Arterioscler Thromb Vasc Biol 2011;31(5):969–979. Increased unrecognized coronary heart disease and sudden 18. Silvestre-Roig C, de Winther MP, Weber C, Daemen MJ, deaths in rheumatoid arthritis: a population-based cohort Lutgens E, Soehnlein O: Atherosclerotic plaque destabi- study. Arthritis Rheum 2005;52(2):402–411. lization: mechanisms, models, and therapeutic strategies. 2. Nicola PJ, Maradit-Kremers H, Roger VL, et al: The Circ Res 2014;114(1):214–226. risk of congestive heart failure in rheumatoid arthritis: a 19. Shaw Y, Chang C-CH, Levesque MC, Donohue JM, population-based study over 46 years. Arthritis Rheum Michaud K, Roberts MS: Timing and impact of decisions 2005;52(2):412–420. to adjust disease-modifying antirheumatic drug therapy for 3. del Rincon ID, Williams K, Stern MP, Freeman GL, rheumatoid arthritis patients with active disease. Arthritis Escalante A: High incidence of cardiovascular events in a Care Res 2018;70(6):834–841. rheumatoid arthritis cohort not explained by traditional car- 20. Bambs C, Kip KE, Dinga A, Mulukutla SR, Aiyer AN, diac risk factors. Arthritis Rheum 2001;44(12):2737–2745. Reis SE: Low prevalence of “ideal cardiovascular health” 310 Journal of Diagnostic Medical Sonography 36(4)

in a community-based population: the heart strategies 34. Kwon HM, Sangiorgi G, Ritman EL. Adventitial vasa vaso- concentrating on risk evaluation (Heart SCORE) study. rum in balloon-injured coronary arteries: visualization and Circulation 2011;123(8):850–857. quantitation by a microscopic three-dimensional computed 21. Aletaha D, Neogi T, Silman AJ, et al: 2010 Rheumatoid tomography technique. J Am Coll Cardiol 1998;32(7): arthritis classification criteria: an American College of 2072–2079. Rheumatology/European League Against Rheumatism col- 35. Sedding DG, Boyle EC, Demandt JAF. Vasa vasorum laborative initiative. Ann Rheum Dis 2010;69(9):1580–1588. angiogenesis: key player in the initiation and progression 22. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, of atherosclerosis and potential target for the treatment of Treacher DF, Turner RC: Homeostasis model assessment: cardiovascular disease. Front Immunol 2018;9:706. insulin resistance and beta-cell function from fasting plasma 36. Shah BN, Chahal NS, Kooner JS, Senior R: Contrast- glucose and insulin concentrations in man. Diabetologia enhanced ultrasonography vs B-mode ultrasound for visu- 1985;28(7):412–419. alization of intima-media thickness and detection of plaques 23. Cacho J, Sevillano J, de Castro J, Herrera E, Ramos MP: in human carotid arteries. Echocardiography 2017;34(5): Validation of simple indexes to assess insulin sensitivity 723–730. during pregnancy in Wistar and Sprague-Dawley rats. Am J 37. Demeure F, Bouzin C, Roelants V. Head-to-head com- Physiol Endocrinol Metab 2008;295(5):E1269–E1276. parison of inflammation and neovascularization in human 24. Aletaha D, Nell VP, Stamm T, et al: Acute phase reactants carotid plaques: implications for the imaging of vulnerable add little to composite disease activity indices for rheuma- plaques. Circ Cardiovasc Imaging 2017;10(5):e005846. toid arthritis: validation of a clinical activity score. Arthritis 38. Wong ND, Gransar H, Narula J. Myeloperoxidase, sub- Res Ther 2005;7(4):R796–806. clinical atherosclerosis, and cardiovascular disease events. 25. van der Heijde DM, van ’t Hof M, van Riel PL, van de Putte JACC Cardiovasc Imaging 2009;2(9):1093–1099. LB: Development of a disease activity score based on judg- 39. Nussbaum C, Klinke A, Adam M, Baldus S, Sperandio ment in clinical practice by rheumatologists. J Rheumatol M: Myeloperoxidase: a leukocyte-derived protagonist of 1993;20(3):579–581. inflammation and cardiovascular disease. Antioxid Redox 26. Fransen J, van Riel PL: DAS remission cut points. Clin Exp Signal 2013;18(6):692–713. Rheumatol 2006;24(6, suppl 43):S29–S32. 40. Ferrante G, Nakano M, Prati F. High levels of systemic 27. Stein JH, Korcarz CE, Hurst RT, et al: Use of carotid ultra- myeloperoxidase are associated with coronary plaque ero- sound to identify subclinical vascular disease and evaluate sion in patients with acute coronary syndromes. Circulation cardiovascular disease risk: a consensus statement from the 2010;122(24):2505–2513. American Society of Echocardiography Carotid Intima- 41. Kremer JM, Lawrence DA, Hamilton R, McInnes IB: Media Thickness Task Force. Endorsed by the Society for Long-term study of the impact of methotrexate on serum Vascular Medicine. J Am Soc Echocardiogr 2008;21(2):93– cytokines and lymphocyte subsets in patients with active 111; quiz 189–190. rheumatoid arthritis: correlation with pharmacokinetic 28. Porter TR, Abdelmoneim S, Belcik JT, et al: Guidelines measures. RMD Open 2016;2(1):e000287. for the cardiac sonographer in the performance of contrast 42. Ally MM, Hodkinson B, Meyer PW. Circulating anti- echocardiography: a focused update from the American citrullinated peptide antibodies, cytokines and genotype Society of Echocardiography. J Am Soc Echocardiogr as biomarkers of response to disease-modifying antirheu- 2014;27(8):797–810. matic drug therapy in early rheumatoid arthritis. BMC 29. Ten Kate GL, van den Oord SC, Sijbrands EJ. Current status Musculoskelet Disord 2015;16:130. and future developments of contrast-enhanced ultrasound of 43. Gauri LA, Fatima Q, Diggi S, Khan A, Liyakat A, Nagar K: carotid atherosclerosis. J Vasc Surg 2013;57(2):539–546. Study of carotid artery intimomedial thickness in patients 30. Coli S, Magnoni M, Sangiorgi G. Contrast-enhanced ultra- with rheumatoid arthritis and its co-relation with sever- sound imaging of intraplaque neovascularization in carotid ity of the disease. J Assoc Physicians India 2017;65(7): arteries: correlation with histology and plaque echo- 37–40. genicity. J Am Coll Cardiol 2008;52(3):223–230. 44. van Breukelen-van der Stoep DF, van Zeben D, Klop B. 31. Piknova B, Schechter AN: Measurement of nitrite in blood Association of cardiovascular risk factors with carotid samples using the ferricyanide-based hemoglobin oxida- intima media thickness in patients with rheumatoid arthri- tion assay. Methods Mol Biol 2011;704:39–56. tis with low disease activity compared to controls: a cross- 32. Arcidiacono MV, Rubinat E, Borras M, et al: Left carotid sectional study. PLoS One 2015;10(10):e0140844. adventitial vasa vasorum signal correlates directly with age 45. Akkus Z, Hoogi A, Renaud G. New quantification meth- and with left carotid intima-media thickness in individuals ods for carotid intraplaque neovascularization using without atheromatous risk factors. Cardiovasc Ultrasound contrast-enhanced ultrasound. Ultrasound Med Biol 2015;13:20. 2014;40(1):25–36. 33. Ambrosino P, Lupoli R, Di Minno A, Tasso M, Peluso R, 46. Zhang Q, Li C, Han H. Spatio-temporal quantification of Di Minno MN: Subclinical atherosclerosis in patients with carotid plaque neovascularization on contrast enhanced rheumatoid arthritis: A meta-analysis of literature studies. ultrasound: correlation with visual grading and histopathol- Thromb Haemost 2015;113(5):916–930. ogy. Eur J Vasc Endovasc Surg 2015;50(3):289–296.