Do biologic therapies reduce aortic inflammation in rheumatoid arthritis patients?

Objectives Rheumatoid arthritis (RA) patients have an increased risk of cardiovascular disease (CVD). In the present study, we evaluated the inflammatory activity of the ascending aorta in RA patients who received biological treatment. Methods We assessed the aortic wall inflammation of RA patients using 18F-fluorodeoxyglucose (FDG) positron emission tomography/computed tomography before and after 6 months of biologic therapies. We also compared the inflammatory activity at the aortic wall in RA patients with remission or low disease activity (RLDA) and those with moderate or high disease activity (MHDA). The aortic uptake was measured by the standardized uptake value (SUV) and the target-to-background ratio (TBR). Results A total of 64 patients were included in the analysis (mean age, 58.4 ± 13.8 years old; female, 77%). The Disease Activity Score for 28 joints (DAS28) erythrocyte sedimentation rate (ESR) had significantly decreased after 6 months: from 5.0 ± 1.2 to 3.3 ± 1.2 (p < 0.001). The FDG uptake in the ascending aorta changed from baseline to 6 months, showing a maximum SUV (SUVmax) of 1.83 ± 0.34 to 1.90 ± 0.34 (p = 0.059) and TBR of 1.71 ± 0.23 to 1.75 ± 0.24 (p = 0.222). The SUVmax and TBR after 6 months were significantly higher in the RLDA group than in the MHDA group (2.05 ± 0.32 vs. 1.79 ± 0.33 (p = 0.002) and 1.89 ± 0.33 vs. 1.65 ± 0.20 (p = 0.001), respectively). The percentage of monocytes also significantly increased from baseline to 6 months: from 5.9 ± 1.6 to 6.9 ± 2.6 (p = 0.032). Conclusion The inflammation activity at the ascending aorta in RA patients did not change significantly after 6 months of biological treatment. RA patients with a low disease activity or in clinical remission after 6 months of biological treatment still had an increased inflammatory activity at the aortic wall.

In the present study, we assessed the ascending aortic wall inflammation in RA patients who received biologic therapies using 18 F-FDG PET/CT. We also compared the inflammatory activity at the aortic wall in RA patients in remission or with a low disease activity (RLDA) and those with moderate or high disease activity (MHDA) after biologic treatment.

Study population
Sixty-four RA patients who underwent whole-body 18 F-FDG PET/CT at baseline and 6 months after the initiation of biologic therapies at Gunma University Hospital were enrolled in this study. All patients were diagnosed with RA according to the American College of Rheumatology criteria revised in 1987 and had a history of inadequate clinical response to previous treatments with nonbiological disease-modifying antirheumatic drugs (DMARDs), such as methotrexate (MTX). Therefore, these patients had been recommended for treatment with biologics.
The study protocol was approved by the Institutional Review Board of Gunma University Hospital. Written, informed consent was obtained from each patient before they participated in the study.

Imaging acquisition
Whole-body 18 F-FDG PET/CT was performed using a PET/CT scanner (Biograph 16; Siemens Medical Solutions Inc., Malvern, PA, USA) before and 6 months after the initiation of biologic therapies. In brief, 18 F-FDG (5 MBq/kg) was injected intravenously after at least 6 h of fasting. Patients were imaged approximately 60 min after FDG injection in the supine position. A non-contrastenhanced CT scan (140 kV, 120-240 mAs) for attenuation correction and anatomic co-registration was obtained before PET imaging. PET imaging was performed in the 3-dimensional mode with 3 min per bed position and slice thickness of 3.27 mm. The PET images were reconstructed using an ordered-subsets expectationmaximization algorithm into 128 × 128 matrices as previously described [11,13].

Imaging analyses
Image analyses were performed using the syngo.via software program (Siemens Healthcare, Erlangen, Germany). An independent radiologist blinded to the clinical data analyzed all of the PET/CT images. According to the recommendation of the Cardiovascular Committee of the European Association of Nuclear Medicine (EANM) [22], we used the maximum standardized uptake value (SUV max ) and the target-to-background ratio (TBR) as parameters for the FDG uptake in the ascending aorta. The SUV max and TBR correlate with histological markers of inflammation and have been used in several studies to measure the FDG uptake in the arterial wall [17,18,23,24].
Ascending aorta inflammation was quantified within each region of interest (ROI), containing the arterial wall and the lumen, along the length of the ascending aorta. The ROIs were manually drawn every 5 mm on axial images, starting 1 cm above the origin of the left main coronary artery and ending at the merging point with the aortic arch. The SUV is determined as the radioactivity concentration (kBq/ml) within an ROI divided by the decay-corrected amount of injected dose per patient's weight (kBq/g). The average of the SUV max (mean SUVmax ) for all ROIs of the entire ascending aorta was then calculated. Subsequently, the TBR was obtained as the mean SUV max divided by the background SUV max in the superior vena cava to correct for the blood activity.

Clinical and laboratory evaluations
RA disease activity was assessed using the Disease Activity Score for 28 joints (DAS28) based on the ESR at baseline and 6 months after the initiation of biologic therapies. We also measured the white blood cell (WBC) count, serum levels of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), matrix metalloproteinase-3 (MMP-3), anti-cyclic citrullinated peptide antibodies (ACPA), and rheumatoid factor antibodies (RF).
The patients were divided into 2 groups according to the DAS28-ESR at 6 months: RLDA in those with a DAS28-ESR < 3.2 and MHDA in those with a DAS28-ESR ≥ 3.2. The clinical parameters and FDG uptake for the ascending aorta at baseline were compared between these two groups.

Statistical analyses
Data analyses were performed using the IBM SPSS Statistics 25 software program (IBM Corp., Armonk, NY, USA). Data were expressed as the mean ± standard deviation for parametric variables, as the median and interquartile range for nonparametric variables, and as numbers and percentages for categorical variables. For the comparison of continuous data between two clinical response groups, an unpaired Student's t-test was used. For the comparison of continuous data before and after treatment, paired Student's t-test or Wilcoxon's signedrank test was used. For the comparison of categorical data, the chi-square test or Fisher's exact test was used. A value of p < 0.05 was considered statistically significant.

Results
A total of 64 patients were included in the analysis (mean age, 58.4 ± 13.8 years old; females, 77%) ( Table 1). The mean disease duration was 13.1 ± 11.8 years. A total of 70% of the patients received MTX, and 48% received prednisolone (PSL). Figure 1 shows the 18 F-FDG PET/ CT findings for a case at baseline and 6 months after the initiation of biologic therapy. The mean FDG uptake values for the ascending aorta and the clinical parameters at baseline and 6 months after the initiation of biologic treatments are shown in Table 2. Although all clinical parameters except for the ACPA were significantly decreased at 6 months compared with the baseline, the mean SUV max and TBR were not significantly changed after 6 months ( Table 2). We compared the FDG uptake at the ascending aorta between the two groups (RLDA group vs. MHDA group) at baseline and after 6 months of treatment. Figure 2 shows the increases in the mean SUV max and TBR values for the ascending aorta at 6 months after the initiation of treatment, especially in the RLDA group. The mean SUV max value at 6 months was significantly higher in the RLDA group than in the MHDA group (2.05 ± 0.32 vs. 1.79 ± 0.33; p = 0.002). The TBR value at baseline was significantly higher in the RLDA group than in the MHDA group (1.79 ± 0.26 vs. 1.65 ± 0.19; p = 0.018). After 6 months, the TBR value was also significantly higher in the RLDA group than in the MHDA group (1.89 ± 0.33 vs. 1.65 ± 0.20; p = 0.001).
At baseline, the WBC count and percentage of monocytes were significantly higher in the RLDA group than in the MHDA group (p = 0.005 and p = 0.035) ( Table 3). Regarding the WBC subtypes at 6 months, there were significantly lower percentages of neutrophils (p < 0.001) and significantly higher percentages of lymphocytes (p < 0.001) in the RLDA group than in the MHDA group ( Table 4). The percentage of monocytes significantly increased from baseline to 6 months (5.9 ± 1.6 to 6.9 ± 2.6; p = 0.032).
We compared the FDG uptake and clinical parameters between the groups with and without anti-tumor necrosis factor (TNF) drug treatment, but there were no significant differences between these two groups (data not shown). The data are expressed as the mean ± standard deviation, with the exception of skewed variables ( a ), which are represented as medians (interquartile range) SUV max maximum standardized uptake value; TBR tissue-to-background ratio; ESR erythrocyte sedimentation rate; CRP C-reactive protein; WBC white blood cell count; MMP-3 matrix metalloproteinase-3; ACPA anti-cyclic citrullinated peptide antibody; RF rheumatoid factor; DAS28 Disease Activity Score in 28 joints   uptake parameters of the ascending aorta for the two clinical response groups. The mean SUV max and TBR values of the ascending aorta at 6 months after biologic treatment were significantly higher in the group with a DAS28-ESR < 3.2 (indicating remission or low disease activity) than in the group with a DAS28-ESR ≥ 3.2 (indicating moderate or high disease activity). SUV max , maximum standardized uptake value; TBR, target-to-background ratio; DAS28, Disease Activity Score in 28 joints; RLDA, remission or low disease activity; MHDA, moderate or high disease activity

Discussion
In the present study, we found that the inflammation activity of the ascending aorta in RA patients did not significantly change after 6 months of biologic treatment. RA patients with RLDA after 6 months had a significantly higher FDG uptake at the aortic wall than those with MDLA.
Biological agents showed beneficial effects on reducing RA symptoms and disease activity by suppressing inflammation. Since atherosclerosis is a chronic inflammation process, these drugs may be also effective in reducing CVD risk in RA. Previous reports have shown that 8-week anti-TNF-α therapy reduced aortic inflammation in RA patients [25]. However, the FDG uptake of the arterial wall after treatment was still significantly higher in RA patients than in the control group.
In our study, although the disease activity and serum markers of RA patients significantly decreased after 6 months of biologic treatment, the inflammatory activity of the ascending aorta still existed. This result indicated that the cardio-protective effects of biologic therapy might not apply to all RA patients at six months.
A cross-sectional study in the USA demonstrated that RA patients who needed anti-TNF therapy to continue their clinical remission had a greater FDG uptake at the ascending aorta than those receiving non-biological DMARDs [26]. In addition to the high disease activity before the biological treatment, our patients had a long disease duration after the onset of RA. These patients' condition might have led to the remnant aortic inflammation after 6 months of biological treatment, even if their joints had a good clinical response to biological drugs. Our results indicated that if we were intending to use biological drugs to achieve a cardio-protective effect, we should ensure RA patients maintain a low disease activity for a long time.
The FDG uptake in the aortic wall was significantly higher in RA patients with RLDA than in those with MHDA at 6 months. This was a controversial result. However, as mentioned above, our follow-up period was only 6 months and thought to be insufficient to reduce systemic inflammation by RA. It is thus possible that a longer treatment period led to the reduced aortic inflammation in the RLDA group.
In addition, a 3-year, prospective, observational study in an Italian cohort clearly showed that the maintenance of remission was associated with a reduced risk of atherosclerosis [27]. Our results showed that while the use of biologic therapies had reduced the clinical symptoms at 6 months, the aortic wall inflammation in these patients persisted despite 6 months of biologic treatment. Therefore, tight control of the RA activity in these patients should be maintained, and their CVD risk should be carefully estimated.
In this study, we also noted an elevation of monocytes at baseline and at 6 months in the RLDA group. An increased activation of circulating monocytes has been reported in patients receiving anti-TNF therapy [26] and the elevations of monocyte subpopulation can be seen in RA patients with coronary artery atherosclerosis and are associated with an increased CVD risk [28,29]. Furthermore, a previous in vivo imaging study reported that the peripheral blood mononuclear cell accumulation was  The data are expressed as the mean ± standard deviation RLDA remission and low disease activity; MHDA moderate and high disease activity; WBC white blood cell correlated with the arterial wall inflammation assessed on hybrid single-photon emission computed tomography/CT [30]. These previous findings suggest that the increased percentage of monocytes might be involved in arterial wall inflammation in RA patients, even after biologic therapy. Several limitations associated with the present study warrant mention. First, traditional cardiovascular risk factors could not be fully investigated because of the lack of such information. Second, the selection of biologic agents was left to each physician. Since different drugs have different mechanisms through which they act on RA pathogenesis, treatment bias may exist. Third, since FDG-PET/CT is a nuclear imaging modality, repeated examinations might lead to increased radiation exposure. Finally, this was a short-term study. To determine whether or not biological therapies are useful for reducing aortic inflammation and CVD risk in RA patients, further longterm follow-up research should be conducted.

Conclusions
We demonstrated that the inflammation activity of the ascending aorta in RA patients was not significantly altered after 6 months of biologic treatment. RA patients with a low disease activity or in clinical remission after 6 months of such treatment still had an increased inflammatory activity at the aortic wall.