Biomarkers in Systemic Sclerosis: An Overview

Systemic sclerosis (SSc) is a complex autoimmune disease characterized by significant fibrosis of the skin and internal organs, with the main involvement of the lungs, kidneys, heart, esophagus, and intestines. SSc is also characterized by macro- and microvascular damage with reduced peripheral blood perfusion. Several studies have reported more than 240 pathways and numerous dysregulation proteins, giving insight into how the field of biomarkers in SSc is still extremely complex and evolving. Antinuclear antibodies (ANA) are present in more than 90% of SSc patients, and anti-centromere and anti-topoisomerase I antibodies are considered classic biomarkers with precise clinical features. Recent studies have reported that trans-forming growth factor β (TGF-β) plays a central role in the fibrotic process. In addition, interferon regulatory factor 5 (IRF5), interleukin receptor-associated kinase-1 (IRAK-1), connective tissue growth factor (CTGF), transducer and activator of transcription signal 4 (STAT4), pyrin-containing domain 1 (NLRP1), as well as genetic factors, including DRB1 alleles, are implicated in SSc damage. Several interleukins (e.g., IL-1, IL-6, IL-10, IL-17, IL-22, and IL-35) and chemokines (e.g., CCL 2, 5, 23, and CXC 9, 10, 16) are elevated in SSc. While adiponectin and maresin 1 are reduced in patients with SSc, biomarkers are important in research but will be increasingly so in the diagnosis and therapeutic approach to SSc. This review aims to present and highlight the various biomarker molecules, pathways, and receptors involved in the pathology of SSc.

The purpose of this review is to present an overview the various molecules, pathways, and biomarker receptors involved in SSc pathology.
Histopathologically, SSc-ILD is characterized by an early pulmonary infiltration of inflammatory cells into the lung parenchyma and can be classified into specific disease patterns, including non-specific interstitial pneumonia (NSIP), habitual interstitial pneumonia (UIP), organized pneumonia and lymphoid pneumonia [83][84][85][86][87][88][89][90].Among the different patterns of ILD, the most common pattern in SSc is NSIP (50-77% of SSc patients with ILD) with bilateral involvement typically starting at the bases.The UIP pattern occurs more rarely during SSc and is characterized by shorter survival than NSIP.SSc-ILD is detected after diagnosis by high resolution CT (HR-CT) and progression by both HR-CT and pulmonary function tests (PFT) (Figure 1) [95][96][97][98].Studies have shown how quantitative CT scanning can be used to detect early SSc-ILD and differentiate it from interstitial pneumonia and to follow the progression of SSc-ILD more precisely [95][96][97][98][99].
Note that these changes can also be detected in other connective tissue diseases, a they are more frequent in SSc.RP is followed by telangiectasias, ischemic DU, scars, periungual microvascular abnormalities, pulmonary arterial hypertension and cardiac disease affecting function and exercise tolerance [6,88,89,[95][96][97][98]110-1 are considered outcomes of vascular damage in SSc.Pulmonary hypertension (PH) is hemodynamic condition, defined by a mean pulmonary arterial pressure (mPAP) > 20 mmHg at rest.PAH (pulmonary arterial hypertension) is characterized by a mean pulmonary arterial pressure (mPAP) greater than 20 mmHg and pulmonary arterial wedge pressure (PAWP) less than 15 mmHg on right heart catheterization (RHC) (Figure 4) with a PVR > 3 WU in the absence of significant interstitial lung disease (ILD) [116][117][118][119]. PAH is due to vasculopathy of the small and medium calibre pulmonary arteries (pulmonary arterial hypertension) which leads to vascular remodelling and to increase in pulmonary vascular resistance (PVR).PAH occurs in 7% to 19% of SSc patients depending on the population and duration of the disease [116][117][118][119][120][121].

Biomarkers in Systemic Sclerosis Skin Involvement
As previously reported, among the hallmarks of SSc, skin involvement is one of the pivotal symptoms and is used to classify SSc patients in routine clinical practice into three different subgroups, limited skin involvement (lcSSc), diffuse skin involvement (dcSSc), and limited SSc (lSSc) [1][2][3][4][5].Although in different sizes and except for patients with SSc sine Scleroderma (ssSSc), the skin is always involved in SSc.SsSSc is a very rare subgroup characterized by the complete or partial absence of skin involvement but with the presence of internal organ involvement and typical serologic abnormalities.
The mRss has some disadvantages, for example, it cannot detect small but clinically relevant changes in skin thickness over time.Recently, several studies have reported the usefulness of high-frequency skin ultrasound (US) for early identification of skin involvement in patients with SSc [6][7][8][9].Most of the authors used a US equipped with a probe at a frequency of 10-30 MHz, as high frequencies are needed to study skin thickness to achieve good resolution, even if penetration is poor.This allows good visualization that can distinguish the epidermis, dermis, and subcutaneous fat, providing a thickness determination and qualitative assessment of the skin; the authors made a comparison with mRss, the current gold standard for the study of skin compromise in patients with SSc [17] (Figure 5).
The mRss has some disadvantages, for example, it cannot detect small but clinically relevant changes in skin thickness over time.Recently, several studies have reported the usefulness of high-frequency skin ultrasound (US) for early identification of skin involvement in patients with SSc [6][7][8][9].Most of the authors used a US equipped with a probe at a frequency of 10-30 MHz, as high frequencies are needed to study skin thickness to achieve good resolution, even if penetration is poor.This allows good visualization that can distinguish the epidermis, dermis, and subcutaneous fat, providing a thickness determination and qualitative assessment of the skin; the authors made a comparison with mRss, the current gold standard for the study of skin compromise in patients with SSc [17] (Figure 5).Skin involvement in SSc patients has a great impact on patients' quality of life as it can cause pruritus, depigmentation, edema, traction ulcers, and movement difficulties; however, it is not associated with increased mortality.Skin involvement is initially due to edema caused by microvascular lesions and inflammation, subsequently to increased Skin involvement in SSc patients has a great impact on patients' quality of life as it can cause pruritus, depigmentation, edema, traction ulcers, and movement difficulties; however, it is not associated with increased mortality.Skin involvement is initially due to edema caused by microvascular lesions and inflammation, subsequently to increased collagen deposition.For these reasons, the skin thickens and it is impossible to pinch it in a normal skin fold.In SSc patients, skin biopsies reveal increased thickness of the dermis and increased amount of collagen deposition, and the disease usually begins with finger involvement in a centripetal pattern.Skin thickening is universal in SSc and generally required for a definitive diagnosis with some exceptions [116][117][118][119].There are many candidate biomarkers for skin disease in SSc.In skin biopsies, the expression levels of TGF-β1, TGF-βR1 and TGF-βR2 are higher in SSc patients than in healthy subjects [134][135][136][137][138][139][140].Cutaneous gene expression of macrophage-associated biomarkers (CD14, IL-13RA1) and TGF-β-associated biomarkers (OSMR SERPINE1, CTGF) is associated with cutaneous disease progression in SSc [134][135][136][137][138][139][140].Marginal zone protein B1 (MZB1) appears to be a good biomarker for cutaneous fibrosis [116][117][118][119]. Circulating levels of periostin are elevated in SSc with extensive cutaneous fibrosis [116][117][118][119]. Sirtuins are NAD-dependent protein deacetylases that regulate angiogenesis; SIRT1 and SIRT3 correlate with the degree of cutaneous fibrosis of the SSc [116][117][118][119]. Adiponectin is reduced in skin affected by SSc [134][135][136][137][138][139][140].The fibrillar collagen molecule COL4A1, the matricellular protein COMP, the gene encoding spondin-SPON1, and TNC, another ECM protein, have recently been upregulated in the skin of SSc individuals, and all these molecules completely distinguish the normal-skinned SSc [116][117][118][119].Other investigators found that the upregulated genes IL-13RA1, OSMR and SERPINE 1 were the most predictive of progressive skin disease [134][135][136][137][138][139][140].

Biomarkers in the Gastrointestinal Systemic Sclerosis Impairment
Gastrointestinal (GI) involvement can be found in approximately 80% of SSc patients (in both the lcSS subgroup and the dcSSc subgroup) and represents a severe manifestation of the disease.All tracts of the gastrointestinal system can be affected, from microstomia to gastroesophageal reflux and gastrointestinal dysmotility to intestinal pseudoobstruction and fecal incontinence [43,[195][196][197][198].Gastrointestinal involvement may be present from the early stage of the disease and may be asymptomatic making its diagnosis difficult [43,[195][196][197][198]. Gastrointestinal system involvement in SSc is profound and includes intestinal and esophageal dysmotility and fibrosis, intestinal ischemia, primary sclerosing cholangitis, primary biliary cirrhosis, bacterial overgrowth, increase in intesti-nal malignancies, and intestinal inflammation, among other complications [43,[195][196][197][198].Approximately 50% of SSc patients complain of symptoms due to gastric involvement with early satiety, postprandial fullness, bloating, nausea, and epigastric pain.Another complication is represented by the so-called "watermelon stomach" due to microvascular ectasias in the stomach which lead to microhemorrhages and chronic anemia.Patients with SSc may also present with intestinal malabsorption due to mucosal surface reduction and bacterial overgrowth, and small bowel involvement may also manifest as intestinal pseudoobstruction [43,[195][196][197][198].Furthermore, fecal incontinence is frequent among SSc patients.Treatment of gastrointestinal complications typically focuses on individual problems of gastroesophageal reflux (proton pump inhibitors, H2-blockers, sucralfate), stenosis (dilatation), dysmotility, and bacterial overgrowth (erythromycin, azithromycin, metoclopramide, domperidone, cisapride) [43,[195][196][197][198].In the line of biomarkers, there are elevated fecal levels of the inflammatory biomarker calprotectin in SSc, suggesting that fecal calprotectin could be an effective biomarker for intestinal disease [43,[195][196][197][198].

Conclusions
The field of biomarkers in SSc continues to expand in scope and complexity.The sheer number of molecules, pathways, and receptors involved in the pathology of SSc reflect the many complexities of the disease.Several biomarkers are intimately involved in both the pathogenesis and characteristics of the disorder.A biomarker, as broadly defined by the National Institutes of Health Working Group on Biomarkers, is a measure that can be applied for purposes as varied as disease diagnosis, staging, prognostication, measuring or predicting treatment response, and even defining surrogate outcomes [199].Biomarkers are likely to be of increasing importance for early diagnosis, assessment of disease course and activity monitoring, as well as therapeutic responses, in SSc patients.Furthermore, the introduction of genomics, proteomics, and metabolomics is deepening our understand-ing of the pathophysiology and architecture of SSc.With these developments, a breadth of candidate biomarkers are being studied but the challenge lies in finding readily measurable biomarker which offer specific diagnostic and prognostic value above that of the actually imaging and functional techniques.The numerous environmental triggers and epigenetic mechanisms involved in the pathogenesis of SSc make finding a single biomarker which can accurately represent SSc a further challenge.Biomarkers in scleroderma are being developed to inform overall prognosis, predict treatment response, and quantify outcomes which had been previously defined only clinically.Individual biomarkers such as CXCL4, adiponectin, and CCL18 are demonstrating prognostic utility for fibrotic and vascular complications to an extent they may be clinically useful.Furthermore, N-terminal-pro-BNP and BNP have a well-described association with PAH and systolic pulmonary arterial pressure in SSc, thought to reflect the increasing stress on the right ventricle with increasing pulmonary pressures, and subsequent studies have demonstrated a prospective utility of pro-BNP levels for later development of PAH.Overexpression of type I IFN, TGF-β, PPAR-γ, PI3K-Akt, as well as serum levels of adiponectin, MMP-9, MMP-12, LOX, ADAM12, THBS1, COMP could be used as potential biomarkers of SSc-related skin fibrosis [40][41][42][43][44][45][46][47][76][77][78][79][80][81][82][83][84][85][86][87].Moreover, recent studies stressed importance of genetic pathway dependent on TGF-β and serum levels of IL-6, KL-6, SP-D, and CCL18 as prominent biomarkers for assessing the severity of fibrosis in SSc-related ILD.In the line of biomarkers, there are elevated fecal levels of the inflammatory biomarker calprotectin in SSc, suggesting that fecal calprotectin could be an effective biomarker for intestinal disease.The most important biomarkers of scleroderma renal crisis include increased levels of serum soluble CD147 and CD163, renin, mannose-binding lectin, endothelin-1, soluble vascular adhesion molecules, E-selectin, lipocalin-2, angiogenin, apelin, chemerin, complement components and NT-proBNP levels.However, with the exception of autoantibodies, today there are no routinely measured biomarkers in SSc and reliable validation of the many potential biomarkers is lacking [40][41][42][43][44][45][46][47][76][77][78][79][80][81][82][83][84][85][86][87][102][103][104][105][106][107][108][109][110].Several studies shown that specific autoantibodies are important serologic markers for determination subclasses and clinical features of SSc.ANA might not only represent biomarkers of disease but also play a pathogenic role through immune-mediated mechanisms and molecular mimicry.Moreover, the presence of ANA, NVC abnormalities and recurrent Raynaud's phenomenon are predictive factors for progression to definitive SSc.In particular, anti-topoisomerase I antibodies are predictive, in the first 3 years of disease of the development of diffused skin involvement and digital ulcers (DU), as well as severe interstitial lung disease (ILD) [65][66][67][68][69][70][71][72][73][74][76][77][78][79][80][81][82][83][84][85][86][87][119][120][121][122][123][124][125][126][127].The anti-centromere autoantibodies (ACA) are associated with PAH and anti-topoisomerase I autoantibodies with ILD.Finally, anti-RNA polymerase III autoantibodies may be biomarkers for rapid progression of skin thickening, gastric antral vascular ectasia, SSc-associated tumors and scleroderma renal crisis.In summary, identifying biomarkers, which can offer diagnostic and prognostic certainty, may help SSc patients to receive preventative treatment as part of a personalised medicine approach.Finally, large randomised controlled trials, which have facilitated new licensed treatments in SSc, have also offered valuable insight into the response of candidate biomarkers but further large scale studies focussing on biomarkers are needed to validate these in order to incorporate them into routine disease stratification.

Figure 1 .
Figure 1.A 63-year-old female with the diagnosis of SSc.(A) Axial High-resolution CT scan shows traction bronchiectasis (white arrows) in a back-ground of diffuse ground-glass opacities, related to a fibrotic nonspecific pneumonia (NSIP) patter.(B) Using the mediastinal window setting the dilatation of the main pulmonary artery (32 mm) can also be recognized as sign of pulmonary hypertension, a common complication of the disease.Moreover a dilatation of the esophagus (*) can be seen (operator E.B., Radiology Unit, University of Trieste, University Hospital of Cattinara).

Figure 1 .
Figure 1.A 63-year-old female with the diagnosis of SSc.(A) Axial High-resolution CT scan shows traction bronchiectasis (white arrows) in a back-ground of diffuse ground-glass opacities, related to a fibrotic nonspecific pneumonia (NSIP) patter.(B) Using the mediastinal window setting the dilatation of the main pulmonary artery (32 mm) can also be recognized as sign of pulmonary hypertension, a common complication of the disease.Moreover a dilatation of the esophagus (*) can be seen (operator E.B., Radiology Unit, University of Trieste, University Hospital of Cattinara).

Figure 3 .
Figure 3. Nailfold capillaroscopy images (×200).Figure (A) shows the capillaroscopic find healthy subject with capillaries of normal number and shape, i.e., the so-called U-sha hairpin-like morphology; (B-D) show the capillaroscopic changes in the scleroderma patte early (B), active (C) and late (D) phases, respectively and associated with a progressive dist the architecture of normal capillaries, which gradually deviate from the A pattern.In part Figure 3. Nailfold capillaroscopy images (×200).Figure (A) shows the capillaroscopic find healthy subject with capillaries of normal number and shape, i.e., the so-called U-sha hairpin-like morphology; (B-D) show the capillaroscopic changes in the scleroderma patte early (B), active (C) and late (D) phases, respectively and associated with a progressive dist the architecture of normal capillaries, which gradually deviate from the A pattern.In part

Figure 3 .
Figure 3. Nailfold capillaroscopy images (×200).Figure (A) shows the capillaroscopic findings in a healthy subject with capillaries of normal number and shape, i.e., the so-called U-shaped and hairpin-like morphology; (B-D) show the capillaroscopic changes in the scleroderma pattern in the early (B), active (C) and late (D) phases, respectively and associated with a progressive distortion of the architecture of normal capillaries, which gradually deviate from the A pattern.In particular in figures (B,D), megacapillaries (defined as capillaries >50 µm) and an increase (B) and a subsequent decrease (C) in capillary density, respectively, are observed.The later picture (D) is characterised by a further accentuation of these anomalies, with a greater decrease in capillary density and an additional number of giant capillaries.In (B), there are also areas of microhaemorrhages, observable in the early phase but also present in the active phase (operators L.M. and B.R, Pulmonology Unit, University of Trieste, University Hospital of Cattinara).
Figure 3. Nailfold capillaroscopy images (×200).Figure (A) shows the capillaroscopic findings in a healthy subject with capillaries of normal number and shape, i.e., the so-called U-shaped and hairpin-like morphology; (B-D) show the capillaroscopic changes in the scleroderma pattern in the early (B), active (C) and late (D) phases, respectively and associated with a progressive distortion of the architecture of normal capillaries, which gradually deviate from the A pattern.In particular in figures (B,D), megacapillaries (defined as capillaries >50 µm) and an increase (B) and a subsequent decrease (C) in capillary density, respectively, are observed.The later picture (D) is characterised by a further accentuation of these anomalies, with a greater decrease in capillary density and an additional number of giant capillaries.In (B), there are also areas of microhaemorrhages, observable in the early phase but also present in the active phase (operators L.M. and B.R, Pulmonology Unit, University of Trieste, University Hospital of Cattinara).

Figure 4 .
Figure 4. Graph obtained from a right heart catheterisation procedure performed at our centre in a 68-year-old patient with Systemic Sclerosis with pulmonary involvement and interstitial disease.Pulmonary hypertension is in fact one of the complications of pulmonary involvement by systemic sclerosis and affects about 12% of patients with this rheumatological disease.This graph shows on the y-axis respectively the ECG monitoring (derivation VII) which is performed throughout the procedure (a), in the picture (b) it is also possible to observe the measurement of the pulmonary arterial pressure (PAP) and the end-expiratory pulmonary artery wedge pressure (PAWP) in the picture (c), (operator P.G., Pulmonology Unit, University of Trieste, University Hospital of Cattinara).

Figure 4 .
Figure 4. Graph obtained from a right heart catheterisation procedure performed at our centre in a 68-year-old patient with Systemic Sclerosis with pulmonary involvement and interstitial disease.Pulmonary hypertension is in fact one of the complications of pulmonary involvement by systemic sclerosis and affects about 12% of patients with this rheumatological disease.This graph shows on the y-axis respectively the ECG monitoring (derivation VII) which is performed throughout the procedure (a), in the picture (b) it is also possible to observe the measurement of the pulmonary arterial pressure (PAP) and the end-expiratory pulmonary artery wedge pressure (PAWP) in the picture (c), (operator P.G., Pulmonology Unit, University of Trieste, University Hospital of Cattinara).

Figure 5 .
Figure 5. Example of measurement of dermal thickness (yellow arrows) by skin high-frequency US (18 MHz probe) in a healthy subject (A) and in an SSc patient (B) at the level of arm (operator L.R, Pulmonology Unit, University of Trieste, University Hospital of Cattinara).

Figure 5 .
Figure 5. Example of measurement of dermal thickness (yellow arrows) by skin high-frequency US (18 MHz probe) in a healthy subject (A) and in an SSc patient (B) at the level of arm (operator L.R, Pulmonology Unit, University of Trieste, University Hospital of Cattinara).

Table 1 .
Correlation between autoantibodies, clinical manifestations and skin subset.