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Serum resistin is predictive marker of development of new digital ulcers in systemic sclerosis

Systemic sclerosis (SSc) is autoimmune disease characterized by endothelial dysfunction and microvascular damage. Resistin has been implied in microvascular dysfunction. Objective of this study is to evaluate the association between baseline resistin and development of new digital ulcers (DUs) in SSc patients. At baseline, serum resistin has been assessed in 70 female SSc patients and 26 healthy controls (HC). In SSc patients, clinical assessment was performed at baseline and after a 52-weeks follow-up. Serum resistin level was increased in SSc patients compared to HC [5.89 ng/ml (2.5 ng/ml–8.1 ng/ml) vs 2.3 ng/ml (0.4 ng/ml–2.4 ng/ml), p = 0.0004)]. Resistin was lower (p = 0.005) in SSc patients with early capillaroscopic pattern than patients with active or late capillaroscopic pattern [2.49 ng/ml (0.89 ng/ml–5.81 ng/ml) vs 7.11 ng/ml (3.48 ng/ml–11.35 ng/ml) and 6.49 ng/ml (3.35 ng/ml–8.87 ng/ml), respectively]. After a 52-weeks follow-up, 34 (48.6%) patients developed new DUs. Median serum resistin was significantly higher in patients with new DUs than in patients without new DUs [6.54 ng/ml (3.35 ng/ml–11.02 ng/ml) vs 4.78 ng/ml (1.06 ng/ml–7.6 ng/ml), p = 0.019]. Kaplan–Meier curves show a significantly reduced free survival from DUs in patients with increased resistin (p = 0.002). In multivariate analysis, resistin is associated with the development of new DUs. Increased serum resistin level is a predictive marker of new DUs in SSc.


Introduction
Systemic sclerosis (SSc) is an autoimmune disease characterized by microvascular damage, autoimmunity-mediated inflammation and fibroblast activation. In SSc, the endothelial dysfunction is one of the most important features that involves both the macro-and microvascular network [1]. Digital ulcers (DUs) are the main vascular complication of chronic hypoxia consequent to endothelial dysfunction [2]. High modified Rodnan skin score (mRSS) and diffuse cutaneous (dcSSc) subset, anti-topoisomerase I antibody, higher value of systolic pulmonary arterial pressure (sPAP), increased intrarenal arterial stiffness, late capillaroscopic pattern and increased percentage of CD21 low B cells are known risk factors for development of new DUs in SSc patients [3][4][5][6][7][8].
Adipose tissue is involved in many inflammatory processes. Various soluble factors produced by adipose tissue, known as adipocytokines or adipokines, have been characterized, such as resistin, adiponectin, leptin [9,10].
Human resistin is a 12.5 kDa polypeptide, which belongs to a family of cysteine-rich proteins, called resistin-like molecules (RELMs), implicated in the regulation of inflammatory processes [11]. Resistin has a specific role in the development of microvascular damage and endothelial dysfunction [12,13]. When incubated with resistin, endothelial cells (EC) respond by a greater production of endothelin-1 (ET-1) and augment ET-1 mRNA expression [14]. Moreover, although resistin did not affect the basal release of endothelium-derived nitric oxide (NO), it significantly augmented pathological inflammation increasing the expression of the vascular cell adhesion molecule 1 (VCAM-1) and the chemoattractant chemokine MCP-1, involved in early atherosclerotic lesion formation [15].

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Several studies indicate that resistin is associated with specific organ manifestations in SSc, especially the vascular ones [16][17][18][19][20]. Sawicka et al. [18] found an association between serum resistin and interstitial lung disease (ILD), arthralgia, esophageal involvement and inflammatory parameters in SSc patients. Masui et al. [19] hypothesized a possible role of serum resistin level as marker for pulmonary vascular involvement in SSc, suggesting a possible contribution of resistin to the pathogenesis of SSc pulmonary arterial hypertension (PAH). Moreover, serum resistin level is elevated in SSc patients with DUs compared to SSc patients without DUs [20].
Aim of this study is to evaluate the role of serum resistin level in development of new DUs in SSc patients.

Subjects
Seventy consecutive SSc females patients [median age 55 years (IQR 43 years-62 years)], fulfilling the American College of Rheumatology/European League Against Rheumatism Collaborative Criteria for SSc, were enrolled in this study [21]. Forty-one (58.6%) had dcSSc and twenty-nine (41.4%) had limited cutaneous SSc (lcSSc) according to Le Roy et al. [22]. Demographic and clinical features of SSc patients are shown in Table 1.
Exclusion criteria were active malignancies, acute and chronic kidney disease, recent surgery (< 1 year), recent cardiovascular or cerebrovascular events (< 1 year), recent exposition to chemotherapy or implantation of autologous adipose tissue-derived cells for the treatment of DUs, peripheral arterial disease, coagulopathies, diabetes. Smokers, pregnancy and breastfeeding were excluded.
Twenty-six healthy controls (HC) matched for sex and age [median age 50 years (IQR 37 years-64 years)] were also enrolled in this study.
The subjects' written consent was obtained according to the Declaration of Helsinki, and the study was approved by the ethics committee of Sapienza University (IRB approval 377).

Clinical assessment
The main clinical indexes were assessed for all SSc patients at baseline and every six months for a follow-up period of 52 weeks. Skin involvement was assessed by mRSS [23]. The activity and severity of disease were assessed by disease activity index (DAI) [24] and disease severity scale (DSS) [25], respectively. Nailfold videocapillaroscopy (NVC) was performed with a videocapillaroscope (Pinnacle Studio Version 8) equipped with a 500 × optical probe. The nailfold of the second, third and fourth finger was examined in each patient. According to Cutolo et al., patterns identified include early, active and late [26]. DUs were defined according to Amanzi et al. [27].    (Table 3).

Discussion
In this study, we found increased serum resistin level in SSc patients compared to HC. We found that serum resistin is increased in SSc patients with early NVC pattern and in patients with new DUs.
The association of serum resistin with DUs in SSc was described in cross-sectional studies [19,20]. It is well known that ET-1 is strongly upregulated in SSc patients and it plays a key role in the pathogenesis of major vascular complications of SSc such as DUs and PAH [28,29]. In RAPIDS 1-2, authors demonstrated that ET-1 is increased in SSc patients with new DUs and bosentan, a dual endothelin receptor antagonist, treatment was associated with a 30% reduction in the number of new DUs. The authors conclude that treatment with bosentan may be effective in preventing new DUs [30,31]. Olewicz-Gawlik et al. [20] demonstrated that SSc patients with DUs have higher serum resistin level compared to SSc patients without DUs. Few studies demonstrate that resistin is increased in PAH. Masui et al. [19] found serum resistin levels significantly increased in patients with elevated right ventricular systolic pressure (RVSP) than in those with normal RVSP; the authors concluded that elevation of resistin levels is associated with proliferative obliterative vasculopathy, especially pulmonary arterial involvement leading to PAH. In the same study, the authors found an higher prevalence of DUs in SSc patients with elevated serum resistin levels supporting the previous data regarding the pro-angiogenic property of resistin [14,15]. A higher serum resistin level seems to be associated with ILD, arthralgia, esophageal involvement and inflammatory parameters in SSc patients [18]. We can suppose that through the increased production of ET-1, resistin may determine a proliferative vascular disease, characterized by a mio-intimal proliferation, leading to the typical blood flow alterations in SSc [16,32,33]. We hypothesized that resistin may play a role in the pathogenesis of new DUs by up-regulation of endothelial ET-1 synthesis. In SSc patients, high serum resistin level was associated with non-vascular SSc complications.
In this study, after a 52-weeks follow-up, 48.6% SSc patients developed new DUs and median serum resistin level was significantly higher in patients with new DUs than in patients without new DUs. In univariate analysis, we demonstrated that resistin, early capillaroscopic pattern, mRSS, DAI and DSS are predictive markers of new DUs, conversely, in multivariate analysis, only resistin is a predictive marker of new DUs.
In literature, markers of development of new DUs are widely reported: mRSS, dcSSc, anti-topoisomerase I antibody, late capillaroscopic pattern, PAH, increased intrarenal arterial stiffness and increased percentage of CD21 low B cells [3][4][5][6][7][8]. Hachulla et al. [3] found that development of new DUs is associated with high mRSS and development of DUs typically occurred within 5 years of the first non-Raynaud clinical symptom of SSc in the majority of patients. In addition, Tiev et al. [4] demonstrated that early onset of SSc, increased duration of SSc, high mRSS, and presence of anti-topoisomerase I antibodies were associated with prior or current DUs. DUs development correlates with capillaroscopic damage and Sebastiani et al. [5] proposed CSURI as a novel tool with the ability to predict the development of DUs in SSc patients. In a recent EUS-TAR observational cohort study of patients with newly diagnosed DUs, dcSSc, anti-topoisomerase I antibody and PAH were associated with presence of any DU at the prospective visit [6]. Rosato [7]. Moreover, Visentini et al. [8] found that the median percentage of CD21 low B cells was significantly higher in patients with new DUs than in patients without new DUs and showed a significantly reduced free survival from new DUs in SSc patients with CD21 low B cells > 10%. In multivariate analysis, CD21 low B cells were associated with the development of new DUs and the authors concluded that CD21 low B cells > 10% are a predictive marker of onset of new DUs [8].
For the first time, we demonstrated that that serum resistin level is a predictive marker of development of new DUs in SSc patients.
We can conclude that serum resistin level may be a predictive marker of new DUs development in SSc patients. Larger prospective studies are needed to evaluate the potential role of serum resistin as a biomarker predictive of new DUs.
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