Ultrasonography involvement of carotid, upper and lower limb arteries in a large cohort of systemic sclerosis patients

Data on macrovascular involvement in systemic sclerosis (SSc) are still debatable. The aim of this study was to estimate its prevalence and possible determinants in a large cohort.


| INTRODUC TI ON
Skin and visceral microvasculopathy is a typical characteristic of systemic sclerosis (SSc), together with abnormal widespread deposition of collagen and other proteins of extracellular matrix, as shown by some threatening and severe clinical manifestations such as digital ulcers, pulmonary artery hypertension (PAH) and scleroderma renal crisis. 1,2 Whether macrovasculopathy affects scleroderma patients has been the object of some studies leading to contrasting results. A possible reason is the great heterogeneity between studies, as underlined by a meta-analysis published in 2011. 3 The same paper showed that carotid intima-media thickness (cIMT) was found higher in SSc than controls in 6 out of 14 studies. 3 In those showing higher cIMT, differences with controls were found comparable to those shown in other diseases characterized by an increased cardiovascular risk such as rheumatoid arthritis (0.09 mm), 4 diabetes mellitus (0.13 mm) 5 and familial hypercholesterolemia (0.12 mm), 6  In previous studies, Doppler examination showed that the diameter of ulnar artery was narrower in 20 SSc patients compared to 20 controls 10 and an occlusion of ulnar artery was found in 17 out of 79 SSc patients. 11 The aim of the present study was to assess macrovascular involvement in a large and well-defined cohort of SSc patients by contemporaneously evaluating, by Doppler ultrasonography, 3 different arterial districts, that is carotid arteries and arteries of upper limbs (UL) and LL; in addition, we evaluated a great number of clinical features in order to study if any of them may be useful to select those SSc patients at increased atherosclerotic risk.

| PATIENTS AND ME THODS
For the present study we enrolled 155 consecutive outpatients affected by SSc followed at the Rheumatology Unit of Verona, Roma and Modena. All patients fulfilled the American College of Rheumatology/European League Against Rheumatism classification criteria for SSc. 12 The distinction between limited and diffuse cutaneous SSc was made according to LeRoy et al 13 criteria. Skin involvement was assessed by modified Rodnan skin score (mRSS). 14 Antinuclear antibodies (ANA) and anticentromere antibodies (ACA) were tested by indirect immunofluorescence on HEp-2 cells, and anti-Scl70 antibodies were searched by enzyme-linked immunosorbent assay method.
Laboratory evaluation also included erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), creatinine with estimated glomerular filtration rate (eGFR), total, high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol, triglycerides, glucose and homocysteine levels. Each patient underwent pulmonary function tests with diffusing capacity for carbon monoxide adjusted to hemoglobin (DLCO) and ratio of DLCO to alveolar volume (DLCO/ AV). At the same time body mass index (BMI) was calculated and disease severity score was assessed, as proposed by Medsger et al 15 The diagnosis of interstitial lung disease (ILD) and PAH was based upon lung high-resolution computed tomography and right heart catheterization, respectively. Digital ulcers were defined as ischemic ulcers located at the digit tip. Evaluation of the cardiovascular risk was made in agreement with the European Low Risk Chart proposed by the European Society of Cardiology (ESC), 16 which considers the following parameters: gender, age, systolic blood pressure, total cholesterol value and smoking habits.
All patients underwent Doppler ultrasonography (DUS) of the carotid arteries and of the UL and LL arteries. The following arteries were analyzed: common carotid, internal and external carotid, subclavian artery, humeral artery, ulnar artery, radial artery, common femoral artery, profunda femoral artery, superficial femoral artery, popliteal artery, anterior and posterior tibial artery. cIMT measurements were carried out after a 15-minutes resting interval and no intravenous vasodilators were given the day of the examination and during the 3 previous days. Patients underwent ultrasound measurement of cIMT at both common carotid arteries on the distal wall.
cIMT was examined by a skilled operator using a high-resolution linear probe (7.5 MHz) by means of automatic ultrasound detection of cIMT (Esaote MyLab 30 Gold-QIMT). Common carotids were examined at standard angles bilaterally: 1 cm proximally to the bulb, a segment of 2 cm was selected with the cursor of the system and IMT was automatically calculated. Median IMT for each common carotid artery was calculated and expressed in centimeters.
Plaques were defined as a localized increase of vessel wall profile

| SSc cohort
The study population was composed of 155 subjects, 18 male and 137 female, of which 95 (69.3%) were in menopause status. Mean age was 57.9 ± 14.5 years; the disease duration was 11.4 ± 8.1 years and the time from onset of Raynaud's phenomena was 16.4 ± 11.7 years. The main clinical characteristics are reported in Table 1.

| Carotid atherosclerosis
Seventy-five patients (49%) had carotid plaques. The artery stenosis was <50% in the majority of subjects (51 cases); 4 and 2 patients showed a hemodynamically significant stenosis comprised between 50% and 60% and between 60% and 70%, respectively. The mean value of IMT was 0.09 mm.
In univariate analysis we found the following differences: scleroderma patients with plaques were older (P < .001), more frequently had a limited cutaneous pattern of disease (P = .030) and hypertension (P = .032), showed higher values of glucose (P = .005), disease severity score (P = .009), homocysteine (P = .014) and ESR (P = .001) and lower values of eGFR (P = .002). Current immunosuppressive therapy was negatively associated with the presence of carotid plaques (P = .017). In terms of autoantibodies, ACAs were more frequent in patients with plaques (58% vs 42%, P = .045). These data are shown in Table 2.  We performed 2 multivariate models ( Table 4). The first one considered all the variables with a P < .1 in univariate analysis (except ESC) and showed that older age (P = .001), higher disease severity scores (P = .034) and limited cutaneous pattern (P = .001) were significantly associated with carotid plaques. The second model considered the variables with a P < .1 in the first one with the addition of ESC score and diabetes mellitus and confirmed the significance of cutaneous pattern (P = .001) and ESR (P = .010) with only a trend for ESC (P = .074).

| LL artery involvement
The data were collected in 140 patients. Forty-nine patients (32.9%) had plaques at the LL arteries. The artery stenosis was <50% in the majority of subjects (32 cases), but 1 patient had a hemodynamically significant stenosis between 50% and 60% and 1 between 80% and 90%. Moreover, occlusion of the anterior tibial artery was found in three cases.
We divided LL involvement in proximal or distal accordingly to the localization of plaques, that is, proximal (43, 30.7%) or distal (14,9.3%) to the popliteal artery.
In multivariate analysis, predicted DLCO/AV (P = .004) was found lower in patients with plaques, while ESC score was higher (P = .005) ( Table 4).

| UL artery involvement
Only seven patients showed UL plaques. All had ulnar involvement and 2 had also radial and humeral plaques. No patients had subclavian involvement. In univariate analysis, patients with plaques had worse predicted FVC, DLCO and DLCO/VA (P = .021, .023 and .02, respectively), more frequent ulcers (P = .012 for active and P = .004 for previous), higher disease severity score (P = .043) and were more often on anti-endothelin treatment (P = .050) ( Table 2).
In multivariate analysis we performed 2 models as previously explained (Table 4). We preferred previous to active ulcers for the analysis given the bigger sample size in this group. Model 1 showed that only a trend for lower predicted FVC and DLCO/VA to be associated with UL plaques (P = .066 and P = .078, respectively). In model 2 predicted FVC was shown to be significantly associated with plaques (P = .023).

| Combined analysis
We then analyzed data according to the number of vascular sites involved, that is, carotid and/or proximal LLs and/or distal LLs. We did not consider UL involvement since no differences in its prevalence were found between patients with and without plaques in other sites.
Scleroderma patients with carotid plaques more frequently had also plaques at the LLs: 37 out of 73 cases (50.7%) vs 12 out of 67 cases (17.9%), P < .001. Table 5 summarizes univariate analysis that showed both traditional cardiovascular risk factors and disease characteristics to be associated with an increased number of sites involved.
When we performed the multivariate analysis only male gender was found to be a risk factor for multi-site involvement (data not shown).

| ESC
We studied ESC score to predict cIMT as surrogate of developing atherosclerotic events. A significant correlation between cIMT and ESC was found (Spearman's correlation .300, P < .001) and patients with a cIMT > 0.09 cm had higher ESC scores (0 ± 2 vs 1 ± 2, P = .002). Indeed, ESC score was found to perform fairly in predicting a cIMT > 0.09 cm (area under the curve [AUC] 0.646, P = .003) and showed a very low sensitivity but a high specificity in identifying patients at high risk, that is with an ESC score ≥5% (49.2% and 97.4%, respectively). When considering an ESC score ≥1% as a marker of increased cIMT, it showed a specificity of 53.8% and a sensitivity of 70.5%.

| D ISCUSS I ON
In this study we have evaluated the macrovascular involvement in patients with SSc by performing a DUS of carotid, UL and LL and by collecting information on disease and cardiovascular risk factors (CRF). We have found that macrovascular involvement is quite common and that traditional CRF and some disease characteristics are associated with the development of plaques, not only in the univariate analysis that may be affected by age and disease duration, but also in multivariate models. In addition, we have confirmed that cIMT may be a useful red flag for macrovasculopathy also at LLs. Finally, ESC was found to perform fairly also in identifying SSc patients with subclinical atherosclerosis.  inflammation and may be affected by many factors, such as age and gender, our result was confirmed also after correcting for them, so its increase in vasculopathy patients may be actually related to the role of inflammation in atherosclerosis.

TA B L E 4 Multivariate analysis of possible determinants of plaques
Some disease characteristics have been found to increase the risk of macrovascular involvement independently from traditional CRF. Subjects with limited pattern were shown to have an increased risk of carotid plaques. Nordin et al 9 has previously reported a similar result for anti-centromere antibodies after correction for gender, age and disease duration. These antibodies have been found to be also associated with a lower ABPI. 23 Although in our cohort ACAs were showed to be more frequent in patients with carotid plaques only in univariate analysis, our results on limited pattern seems to support that those patients with a more pronounced microvascular than fibrotic process have an increased risk of carotid atherosclerosis, supporting a possible link between micro-and macrovascular involvement. On the other hand, one may argue that patients with a diffuse pattern or anti-Scl70 antibody positivity are more often on immunosuppressive drugs; it is worth noticing that, in our cohort, this treatment was found to be protective against carotid plaques in univariate analysis. Although the role of inflammation in atherosclerosis is well known, there is still a lack of data on the possible role of immunotherapy to prevent its progression. 24 We speculate that the between micro-and macrovasculopathy via a common pathologic pathway such as endothelial dysfunction. 25,26 Although these data support an intriguing link between micro-and macrovasculopathy, there is contrasting evidence on videocapillaroscopy. It was found to be related to ulnar involvement by Lescoat   Given that atherosclerosis is quite common also in SSc patients and that carotid DUS is a good screening tool, since cIMT correlates well with both ESC score and macrovasculopathy in other sites, our study further stresses that a thorough cardiovascular screening of SSc patients may be effective and very important in particular if we consider that peripheral atherosclerosis is a well-known risk factor for cardiovascular events in the general population and statins and aspirin are recommended in its treatment. 32 In addition, cardiovascular events accounted for about 29% of non-SSc-related deaths in the large EUSTAR cohort, 33 so its prevention is an issue also in SSc.
In conclusion, this study shows that macrovascular involvement is quite common in SSc patients and that, apart from a possible role of traditional risk factors, some disease characteristics are significantly associated with atherosclerotic plaques. In addition, we further underline the importance of screening for macrovascular involvement at LLs in those SSc patients with an increased cIMT. Finally, ESC score was found to have a fair performance in predicting subclinical atherosclerosis also in SSc patients. Our study suggests that a complete evaluation of patients is mandatory for rheumatologists for a comprehensive approach to this disease, that is still without a specific treatment, and a multidisciplinary and tailored therapy may allow longer survival.