Reduced gene expression of Survivin in PBMCs from patients with limited systemic sclerosis

Abstract Background/Aims The major antinuclear autoantibodies of systemic sclerosis (SSc) associate with different skin score trajectories and risk of internal organ manifestations. To elucidate molecular differences between ANA-defined subgroups, we utilised the prospective BIOPSY cohort of well-characterised SSc patients. Methods The prospectively collected BIOPSY cohort recruited 52 SSc patients (21 early dcSSc, 15 established dcSSc, 16 lcSSc) and 16 healthy controls (HC). 36 (69%) of the SSc patients are female. Mean disease duration in the early dcSSc cohort was 24 months (sd 12 months), and in established dcSSc was 11.3 years. ANA frequency in BIOPSY reflected the overall dcSSc population: anti-topoisomerase-1 (ATA) n = 14 (27%), anti-RNA pol III (ARA) n = 12 (23%) and other n = 26 (50%). Mean baseline skin score (MRSS) for early dcSSc was 21 (sd 11.2). At a group level mRSS peak was 21.9 (11.8) at 3 months and fell to 19.1(10.5) at 12 months. Serum biomarkers of ECM turnover and fibrosis were measured three monthly and genome-wide transcriptomic profiling of whole skin and whole blood performed by RNA-Seq. Statistical analysis used RStudio with ANOVA, and Tukey post-hoc test. Differential gene expression used the Bioconductor limma software, with standard thresholds for significance. Results At baseline, there were differences in soluble markers between clinical SSc sugroups and HC but not for major ANA subgroups. However, we found clear differences in early dcSSc analysed by major ANA subset for longitudinal change in serum markers of fibrosis and in whole skin gene expression, suggesting a mechanistic basis for the distinct clinical phenotypes associated with hallmark ANAs. During follow-up, significant differences were observed in HA, TIMP1, and PIIINP at 6 and 12 months (p < 0.05), with stable levels in ATA+ patients compared to progressively increased levels in the other subgroups. There were 564 significantly differentially expressed genes in skin between early dcSSc and HC. Unsupervised clustering differentiated patients with ARA and ATA positivity with early dcSSc. 54 genes were significantly differentially expressed in skin between ATA and ARA patients. Whilst 179 genes were differentially expressed in whole blood between early dcSSc compared with HC, no genes could significantly differentiate ATA from ARA. Functional analysis using HALLMARK pathway analysis identified both shared pathways associated with SSc across ANA groups (e.g. TGF beta signaling, IL6 JAK STAT3 signalling, inflammatory response), and pathways only upregulated in patients with ATA (e.g. Wnt beta catenin signaling, Notch signaling), and ARA (e.g. interferon gamma response, adipogenesis). Conclusion We have found significant differences in skin gene expression and longitudinal change in serum markers by autoantibody specificity in dcSSc. Our findings have implications for SSc pathogenesis and support stratification by ANA subgroup in clinical studies. Disclosure K.E. Clark: None. C. Campochiaro: None. E. Csomor: Corporate appointments; employee of GSK. A. Taylor: Corporate appointments; employee of GSK. K. Nevin: Corporate appointments; employee of GSK. N. Galwey: Corporate appointments; employee of GSK. M.A. Morse: Corporate appointments; employee of GSK. V.H. Ong: None. E. Derrett-Smith: None. N. Wisniacki: Corporate appointments; employee of GSK. S. Flint: Corporate appointments; employee of GSK. C.P. Denton: Consultancies; Actelion, GlaxoSmithKline, Bayer, Sanofi, lnventiva, Boehringer Ingelheim, Roche, Bristol Myers Squibb, CSL Behring, UCB, Leadiant Biosciences, Corbus, Servier, Arxx Therapeutics.

Download Full-text

POS0330 NINTEDANIB (TYROSINE KINASE INHIBITOR) DOWNREGULATES THE TRANSITION OF CULTURED SYSTEMIC SCLEROSIS FIBROCYTES INTO MYOFIBROBLASTS AND THEIR PRO-FIBROTIC ACTIVITY

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2411 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 392.2-392

Author(s):

S. Soldano ◽

P. Montagna ◽

E. Gotelli ◽

S. Tardito ◽

S. Paolino ◽

...

Keyword(s):

Gene Expression ◽

Systemic Sclerosis ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Kinase Inhibitor ◽

Adherent Cells ◽

Research Support ◽

Fibrotic Process ◽

Circulating Fibrocytes

Background:Fibroblast-to-myofibroblast transition is one of the fundamental steps involved in the fibrotic process that characterise systemic sclerosis (SSc) [1]. Myofibroblasts are α-smooth muscle actin (αSMA) positive cells that contribute to fibrosis through the excessive synthesis and deposition of extracellular matrix (ECM) proteins, primarily fibronectin (FN) and type I collagen (COL1) [2].Among the cells involved in the fibrotic process of SSc, circulating fibrocytes seem to have an emerging role as an important source of fibroblasts and myofibroblasts [3].Nintedanib is a tyrosine kinase inhibitor approved for the treatment of idiopathic pulmonary fibrosis that interferes with the signalling pathways involved in the pathogenesis of fibrosis (4). Nintedanib was recently demonstrated to have a beneficial effect in patients with interstitial lung disease (ILD) associated with SSc (5).Objectives:To investigate nintedanib effect in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity.Methods:Circulating fibrocytes were obtained from 14 SSc patients (mean age 64±14 years), who fulfilled the 2013 ACR/EULAR criteria for SSc and that underwent complete disease staging in a day-hospital setting at the Rheumatology Division of Genoa University. Five age-matched healthy subjects (HSs) were also analysed. All SSc patients and HSs signed the informed consent and the local EC approved the study. Peripheral blood mononuclear cells were isolated by density gradient centrifugation and plated on FN-coated dishes. After overnight culture, non-adherent cells were removed, and adherent cells were maintained in growth medium for 8 days (T8) to obtain fibrocytes [6]. T8-cultured SSc fibrocytes were maintained in growth medium (untreated cells) or treated with nintedanib 0.1μM and 1μM for 3 and 24 hours. Fibroblast specific protein-1 (S100A4) and αSMA, as markers of fibroblast/myofibroblast phenotype, together with COL1 and FN, were investigated by qRT-PCR and Western blotting. Non-parametric Mann-Whitney and Wilcoxon tests were used for the statistical analysis.Results:Significantly elevated gene and protein expressions of αSMA, S100A4, COL1 and FN were observed in SSc fibrocytes compared to HS fibrocytes (gene: αSMA p<0.001; others p<0.0001; protein: all p<0.05). In accordance with the antibody positivity for Scl70 and the presence or absence of ILD at CT scan, SSc patients were grouped as either Scl70 positive patients with ILD (Scl70+ILD+) or Scl70 negative patients without ILD (Scl70-ILD-). Significant αSMA, S100A4, COL1 and FN gene expressions were found in fibrocytes from Scl70+ILD+ compared to HS fibrocytes (αSMA p<0.001; others p<0.0001). Moreover, fibrocytes from Scl70+ILD+patients showed a more significant gene expression of fibroblasts/myofibroblasts markers compared to Scl70-ILD-patients (p<0.01 for S100A4), whereas no differences were observed for ECM gene expression.Nintedanib reduced the gene and protein expression of αSMA, COL1 and FN in SSc fibrocytes compared to untreated ones with different statistical significance.Noteworthy, nintedanib significantly downregulated αSMA, S100A4, COL1 and FN gene expression (all p<0.05) in Scl70+ILD+fibrocytes, whereas only that of S100A4 and FN was significantly downregulated (p<0.05) in Scl70-ILD- fibrocytes compared to untreated cells.Conclusion:Nintedanib seems to downregulate in vitro the transition of fibrocytes into myofibroblasts and their pro-fibrotic activity, particularly in cells isolated from Scl70+ILD+SSc patients.References:[1]Cutolo M et al. Exp Rev Clin Immunol. 2019;15:753-64.[2]Van Caam A et al. Front. Immunol. 2018;9:2452.doi:10.3389/fimmu.2018.02452.[3]Distler JH et al. Arthritis Rheumatol. 2017;69:257-67.[4]Distler O et al. New Eng J Med. 2019; 380:2518-28.[5]Maher TB et al. Arthritis Rheumatol.2020.doi:10.1002/art.41576.[6]Cutolo M et al. Arthritis Res Ther. 2018;20:157.doi:10.1186/s13075-018-1652-6.Acknowledgements:We thank Stefano-Lutz Willing for the scientific support through the study.Disclosure of Interests:Stefano Soldano: None declared, Paola Montagna: None declared, Emanuele Gotelli: None declared, Samuele Tardito: None declared, Sabrina Paolino: None declared, Claudio Corallo: None declared, Carmen Pizzorni: None declared, Alberto Sulli: None declared, Carlotta Schenone: None declared, Greta Pacini: None declared, Vanessa Smith: None declared, Maurizio Cutolo Grant/research support from: I received grant/research support from Bristol-Myers Squibb, Boehringer, Celgene

Download Full-text

Chronic progressive sensory ataxic neuropathy associated with limited systemic sclerosis

Journal of the Neurological Sciences ◽

10.1016/j.jns.2005.10.010 ◽

2006 ◽

Vol 241 (1-2) ◽

pp. 103-106 ◽

Cited By ~ 6

Author(s):

Yasuyuki Nobuhara ◽

Mineki Saito ◽

Rina Goto ◽

Yoshihito Yoshidome ◽

Miwako Kawamura ◽

...

Keyword(s):

Systemic Sclerosis ◽

Ataxic Neuropathy ◽

Limited Systemic Sclerosis

Download Full-text

Patients with systemic sclerosis-associated pulmonary arterial hypertension express a genomic signature distinct from patients with interstitial lung disease

Journal of Scleroderma and Related Disorders ◽

10.1177/2397198318764780 ◽

2018 ◽

Vol 3 (3) ◽

pp. 242-248 ◽

Cited By ~ 6

Author(s):

Matthew Moll ◽

Romy B Christmann ◽

Yuqing Zhang ◽

Michael L Whitfield ◽

Yu Mei Wang ◽

...

Keyword(s):

Gene Expression ◽

Systemic Sclerosis ◽

Pulmonary Arterial Hypertension ◽

Interstitial Lung Disease ◽

Arterial Hypertension ◽

Lung Disease ◽

Expression Profiles ◽

Area Under The Curve ◽

Gene Expression Profiles ◽

Pulmonary Arterial

Objective: Pulmonary arterial hypertension and interstitial lung disease are major causes of mortality in systemic sclerosis. We used a previously identified microarray biomarker to determine whether systemic sclerosis-pulmonary arterial hypertension and systemic sclerosis-interstitial lung disease patients demonstrate distinct gene expression profiles. Methods: Peripheral blood mononuclear cells were collected from healthy controls ( n = 10), systemic sclerosis patients without pulmonary hypertension (systemic sclerosis-no pulmonary arterial hypertension, n = 39), and systemic sclerosis-pulmonary arterial hypertension patients ( n = 21; mean pulmonary arterial pressure ≥25, pulmonary capillary wedge pressure ≤15, and pulmonary vascular resistance ≥3 Wood units) diagnosed by right heart catheterization. Systemic sclerosis-interstitial lung disease patients were defined as those with evidence of fibrosis on chest computed tomography and significant restriction (forced vital capacity <70% predicted, n = 11). Systemic sclerosis-pulmonary arterial hypertension biomarker included 69 genes selected by unbiased statistical screening of three publicly available microarray studies. RNA levels were measured by NanoString Technologies. Gene expression levels that were significantly correlated with pulmonary arterial hypertension (multiple statistical measures) were chosen as inputs into a forward selection logistic regression model. Results: When interstitial lung disease patients were included ( n = 64), four genes (S100P, CD8B1, CCL2, and TIMP1) and male sex predicted pulmonary arterial hypertension with a high level of accuracy (area under the curve = 0.83). Without interstitial lung disease patients ( n = 53), two genes (THBS1 and CD8B1) and male sex predicted pulmonary arterial hypertension with a high level of accuracy (area under the curve = 0.80). When examining systemic sclerosis patients with borderline elevated pulmonary pressures (mean pulmonary arterial pressure = 21–24 mmHg), gene expression changes closely resembled the systemic sclerosis-pulmonary arterial hypertension group, except for THBS1. Conclusion: Systemic sclerosis-pulmonary arterial hypertension and systemic sclerosis-interstitial lung disease have similar but distinct gene expression profiles. Many gene expression changes occur early in the disease course, potentially allowing early detection. THBS1 appears to be an important mediator in the development of pulmonary arterial hypertension-predominant phenotype. Further prospective investigation is warranted.

Download Full-text

Primary Biliary Cirrhosis Associated with Systemic Sclerosis: Diagnostic and Clinical Challenges

International Journal of Rheumatology ◽

10.1155/2011/976427 ◽

2011 ◽

Vol 2011 ◽

pp. 1-12 ◽

Cited By ~ 20

Author(s):

Cristina Rigamonti ◽

Dimitrios P. Bogdanos ◽

Maria G. Mytilinaiou ◽

Daniel S. Smyk ◽

Eirini I. Rigopoulou ◽

...

Keyword(s):

Liver Disease ◽

Systemic Sclerosis ◽

General Population ◽

Primary Biliary Cirrhosis ◽

Early Identification ◽

Case Reports ◽

Biliary Cirrhosis ◽

Antimitochondrial Antibodies ◽

Favorable Prognosis ◽

Limited Systemic Sclerosis

Patients with primary biliary cirrhosis (PBC) often have concurrent limited systemic sclerosis (SSc). Conversely, up to one-fourth of SSc patients are positive for PBC-specific antimitochondrial antibodies (AMA). The mechanisms responsible for the co-occurrence of these diseases are largely unknown. Genetic, epigenetic, environmental, and infectious factors appear to be important for the pathogenesis of the disease, but the hierarchy of events are not well defined. Patients with SSc and PBC have an increased morbidity and mortality compared with the general population, but whether the presence of both diseases in an affected individual worsens the prognosis and/or outcome of either disease is not clear. Some case reports suggested that the presence of SSc in PBC patents is associated with a more favorable prognosis of the liver disease, whereas others report an increased mortality in patients with PBC and SSc compared to patients with PBC alone. This paper discusses the features of patients with PBC-associated SSc. Our aims are to clarify some of the pathogenetic, diagnostic, and clinical challenges that are currently faced in the routine management of these patients. We also intend to provide some practical hints for practitioners that will assist in the early identification of patients with PBC-associated SSc.

Download Full-text

Positive Antinucleolar Antibodies on Direct Immunofluorescence of Lesional Skin of a Patient With Limited Systemic Sclerosis

American Journal of Dermatopathology ◽

10.1097/00000372-900000000-98247 ◽

2018 ◽

pp. 1

Author(s):

Cristian A. Valenzuela-Oñate ◽

Jorge Magdaleno-Tapial ◽

Violeta Zaragoza-Ninet ◽

Victor Alegre de Miquel

Keyword(s):

Systemic Sclerosis ◽

Direct Immunofluorescence ◽

Lesional Skin ◽

Limited Systemic Sclerosis

Download Full-text

Association between gene expression profiling of skin lesion and autoantibody in patients with systemic sclerosis

10.1101/2020.04.12.20063131 ◽

2020 ◽

Author(s):

Jun Inamo

Keyword(s):

Gene Expression ◽

Systemic Sclerosis ◽

Skin Lesion ◽

Topoisomerase I ◽

Cellular Response ◽

Rna Polymerase Iii ◽

Functional Enrichment ◽

Keratinocyte Differentiation ◽

Study Cohort ◽

Specific Autoantibody

AbstractObjectiveThe aim of this study was to investigate relevance between type of autoantibody and gene expression profile in skin lesion of systemic sclerosis (SSc), and identify specifically dysregulated pathways.MethodsSixty-one patients with SSc from the Genetics versus Environment in Scleroderma Outcome Study cohort and thirty-six healthy controls (HC) are included. Differentially expressed genes (DEGs) were extracted and functional enrichment and pathways analysis were conducted.ResultsCompared with HC, lists consisting of 2, 71, 10, 144 and 78 DEGs were created for patients without specific autoantibody, anti-centromere (ACA), anti-U1 RNP (RNP), anti-RNA polymerase III (RNAP) and anti-topoisomerase I (ATA) antibody, respectively. While part of enriched pathways overlapped, distinct pathways were identified except those without specific autoantibody: keratinocyte differentiation in ACA, NF-kB signaling and cellular response to transforming growth factor beta stimulus in RNAP, interferon alpha/beta signaling of RNP and cellular response to stress in ATA.ConclusionPathogenic pathways were identified according to type of autoantibodies by leveraging gene expression data of patients and controls from multi-center cohort. The current study will promote to explore new therapeutic target for SSc.Key messageDistinct pathways are associated with type of autoantibody in skin lesion of systemic sclerosis.

Download Full-text

Systematic analysis of the literature in search of defining systemic sclerosis subsets

The Journal of Rheumatology ◽

10.3899/jrheum.201594 ◽

2021 ◽

pp. jrheum.201594

Author(s):

Tatiana Nevskaya ◽

Janet E. Pope ◽

Matthew A. Turk ◽

Jenny Shu ◽

April Marquardt ◽

...

Keyword(s):

Gene Expression ◽

Systemic Sclerosis ◽

Original Data ◽

Response To Therapy ◽

Classification Systems ◽

Skin Involvement ◽

Multisystem Disease ◽

Systematic Analysis ◽

Tissue Samples ◽

Gene Expression Signatures

Objective Systemic sclerosis (SSc) is a multisystem disease with heterogeneity in presentation and prognosis. An international collaboration to develop new SSc subset criteria is underway. Our objectives were to identify systems of SSc subset classification and synthesize novel concepts to inform development of new criteria. Methods Medline, Cochrane MEDLINE, CINAHL, EMBASE and Web of Science were searched from their inceptions to December 2019 for studies related to SSc sub-classification, limited to humans without language or sample size restrictions. Results Of 5686 citations, 102 articles reported original data on SSc subsets. Subset classification systems relied on extent of skin involvement and/or scleroderma-specific autoantibodies (n=61), nailfold capillary patterns (n=29), molecular, genomic and cellular patterns (n=12). While some systems of subset classification confer prognostic value for clinical phenotype, severity, and mortality; only subsetting by gene expression signatures in tissue samples has been associated with response to therapy. Conclusion Subsetting on extent of skin involvement remains important. Novel disease attributes including SSc-specific autoantibodies, nailfold capillary patterns and tissue gene expression signatures have been proposed as innovative means of SSc subsetting.

Download Full-text

Large-scale analysis of longitudinal skin gene expression in systemic sclerosis reveals relationships of immune cell and fibroblast activity with skin thickness and a trend towards normalisation over time

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-221352 ◽

2021 ◽

pp. annrheumdis-2021-221352

Author(s):

Brian Skaug ◽

Marka A Lyons ◽

William R Swindell ◽

Gloria A Salazar ◽

Minghua Wu ◽

...

Keyword(s):

Gene Expression ◽

Systemic Sclerosis ◽

Immunohistochemical Staining ◽

Large Scale ◽

Immune Cell ◽

Treatment Strategies ◽

Principal Component ◽

Skin Thickness ◽

High Baseline ◽

Over Time

ObjectivesDetermine relationships between skin gene expression and systemic sclerosis (SSc) clinical disease features, and changes in skin gene expression over time.MethodsA total of 339 forearm skin biopsies were obtained from 113 SSc patients and 44 matched healthy controls. 105 SSc patients had a second biopsy, and 76 had a third biopsy. Global gene expression profiling was performed, and differentially expressed genes and cell type-specific signatures in SSc were evaluated for relationships to modified Rodnan Skin Score (mRSS) and other clinical variables. Changes in skin gene expression over time were analysed by mixed effects models and principal component analysis. Immunohistochemical staining was performed to validate conclusions.ResultsGene expression dysregulation was greater in SSc patients with affected skin than in those with unaffected skin. Immune cell and fibroblast signatures positively correlated with mRSS. High baseline immune cell and fibroblast signatures predicted higher mRSS over time, but were not independently predictive of longitudinal mRSS after adjustment for baseline mRSS. In early diffuse cutaneous SSc, immune cell and fibroblast signatures declined over time, and overall skin gene expression trended towards normalisation. On immunohistochemical staining, most early diffuse cutaneous SSc patients with high baseline T cell and macrophage numbers had declines in these numbers at follow-up.ConclusionsSkin thickness in SSc is related to dysregulated immune cell and fibroblast gene expression. Skin gene expression changes over time in early diffuse SSc, with a tendency towards normalisation. These observations are relevant for understanding SSc pathogenesis and could inform treatment strategies and clinical trial design.

Download Full-text