AB0168 NINTEDANIB (TYROSINE-KINASE INHIBITOR) INHIBITS THE TRANSITION OF CIRCULATING FIBROCYTES ISOLATED FROM SYSTEMIC SCLEROSIS PATIENTS INTO MYOFIBROBLASTS: AN IN VITROSTUDY

Background:Systemic sclerosis (SSc) is a chronic connective disease characterized by microvascular alterations, dysregulated immune response and fibrosis [1,2]. Myofibroblasts are alpha-smooth muscle actin (alphaSMA)+cells and play a crucial role in fibrosis, through the excessive synthesis and deposition of extracellular matrix (ECM) proteins, in particular fibronectin (FN) and type I collagen (COL1) [3]. Despite myofibroblasts primarily derive from resident fibroblasts transition and differentiation, another important source is represented by circulating fibrocytes [4]. 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 [5].Objectives:To investigate the possible effects of nintedanib in contrasting the ability of cultured mature fibrocytes from SSc patients to differentiate into profibrotic myofibroblasts.Methods:Circulating fibrocytes were obtained from peripheral blood mononuclear cells isolated from 5 limited cutaneous SSc patients (mean age 68 +/- 10 years) and then plated on FN-coated tissue culture dishes in growth medium (DMEM at 20% of fetal bovine serum, 1% of penicillin-streptomycin and 1% L-glutamine), to allow the adhesion of fibrocyte precursors. Adherent cells were maintained in growth medium for 8 days in order to allow their differentiation into fibrocytes. Differentiated fibrocytes were treated with nintedanib at the concentrations of 100nM and 1000nM for 3 and 24 hours (hrs) or maintained in growth medium without any treatment. The differentiation of fibrocytes into myofibroblasts was determined evaluating the gene expression of alphaSMA, fibroblast specific protein-1 (S100A4) COL1, FN and CXCR4 by quantitative real-time polymerase chain reaction, and the protein synthesis of alphaSMA, COL1 and FN by western blotting.Results:Nintedanib inhibited alphaSMA and S100A4 gene expression already at the concentration of 100nM in cultured fibrocytes and after 3 hrs of treatment, when compared with untreated cells. Furthermore, both concentrations of nintedanib (100nM and 1000nM) reduced the gene expression of COL1 and FN, whereas only 100nM downregulated the CXCR4 gene expression. At protein level, nintedanib 100nM and 1000nM reduced the synthesis of alphaSMA and COL1 after 24 hrs of treatment, whereas FN synthesis was reduced only by the nintedanib concentration of 1000nM.Conclusion:The preliminary results show that nintedanib may inhibit thein vitrotransition of SSc fibrocytes into myofibroblasts and their profibrotic activity, through the reduction of specific myofibroblast phenotype markers and ECM protein production. The results seem to suggest fibrocytes as further possible target of the antifibrotic action of nintedanib in SSc.References:[1]Cutolo M et al. Expert Rev Clin Immunol. 2019;15:753-64 2. Barsotti S et al. Clin Exp Rheumatol. 2016;34(Suppl.100):S3-S13 3. Wynn TA et al. Nat Med. 2012;18:1028-40. 4.Distler JHW et al. Arthritis Rheumatol. 2017;69:257-67 5.Hilberg F et al. Cancer Res. 2008;68:4774-82.Disclosure of Interests:Stefano Soldano: None declared, Giulia Martinelli: None declared, Samuele Tardito: None declared, Sabrina Paolino: None declared, Massimo Patanè: None declared, Emanuele Gotelli: None declared, Claudio Corallo: None declared, Carmen Pizzorni: None declared, Alberto Sulli Grant/research support from: Laboratori Baldacci, Carlotta Schenone: None declared, Vanessa Smith Grant/research support from: The affiliated company received grants from Research Foundation - Flanders (FWO), Belgian Fund for Scientific Research in Rheumatic diseases (FWRO), Boehringer Ingelheim Pharma GmbH & Co and Janssen-Cilag NV, Consultant of: Boehringer-Ingelheim Pharma GmbH & Co, Speakers bureau: Actelion Pharmaceuticals Ltd, Boehringer-Ingelheim Pharma GmbH & Co and UCB Biopharma Sprl, Maurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Actelion, Celgene, Consultant of: Bristol-Myers Squibb, Speakers bureau: Sigma-Alpha

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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

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SAT0301 CIRCULATING FIBROCYTES FROM SYSTEMIC SCLEROSIS PATIENTS AS POSSIBLE TARGET OF CTLA4-IG TREATMENT: AN IN VITRO STUDY

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.4626 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 1096.1-1096

Author(s):

S. Soldano ◽

P. Montagna ◽

S. Paolino ◽

E. Alessandri ◽

C. Pizzorni ◽

...

Keyword(s):

Gene Expression ◽

Systemic Sclerosis ◽

Costimulatory Molecule ◽

Skin Fibroblasts ◽

Type I ◽

Research Support ◽

Hla Dr ◽

Antigen Presenting ◽

Circulating Fibrocytes

Background:muscle actin (aSMA)+cells involved in the overproduction of extracellular matrix proteins, primarily fibronectin (FN) and type I collagen (COL1) at the level of damaged tissues (1). These cells may originate from different cell types including fibroblasts, endothelial and epithelial cells, and fibrocytes (1). Circulating fibrocytes are bone marrow progenitor cells expressing specific markers of hematopoietic (CD34, CD45, and MHC class II) and stromal cells (COL1 and COL3), chemokine receptors (CCR2, CCR7), and CXCR4 (2). CXCR4 regulates fibrocyte migration into injured tissues allowing their differentiation into fibroblasts/myofibroblasts (2).In vitro, fibrocytes differentiate from circulating CD14+monocytes showing an antigen-presenting capability through the expression of HLA-DR and costimulatory molecule CD86 (2). CTLA4-Ig fusion protein (abatacept) interacts with CD86 on cell surface of antigen presenting cells (APCs), such as macrophages and endothelial cells (3,4).Objectives:To investigate the possible effect of CTLA4-Ig treatment on cultured human fibrocytes and skin fibroblasts isolated from the same systemic sclerosis patients (SSc pts).Methods:Fibrocytes isolated from the peripheral blood mononuclear cells of SSc pts and healthy subjects (HSs) were cultured on fibronectin-coated plates in DMEM at 20% of FBS; for further 8 days (T8) to allow their complete differentiation. Differentiated fibrocytes were maintained in growth medium or treated with CTLA4-Ig at different concentrations (10, 50, 100, and 500μg/ml) for 3 hours. Fibroblasts were isolated from the skin biopsies of the same patients and HSs, cultured until the 3rdpassage in RPMI at 10% FBS and then treated with CTLA4-Ig for 24 and 48 hours. Fibrocytes were characterized as CD45+CXCR4+COL1+cells and the expression of CD86 and HLA-DR was also evaluated. The gene expression of aSMA, COL1, CXCR4, TGFb1 and CD86 was investigated by quantitative real-time polymerase chain reaction in cultured fibrocytes and skin fibroblasts. In cultured skin fibroblasts, COL1 and fibronectin synthesis was evaluated by Western blotting.Results:Treatment with CTLA4-Ig for 3 hours significantly downregulated aSMA and COL1 gene expression in cultured SSc fibrocytes at T8 (p<0.01, p<0.05 vs. untreated fibrocytes), whereas no modulatory effect was observed on the TGFbeta1 and CXCR4 gene expression. In cultured SSc skin fibroblasts, CTLA4-Ig did not induce any significant effect on CD68, TGFb1, COL1 and FN gene expression as well as COL1 and FN protein synthesis, both after 24 and 48 hours. Of note, these cultured SSc skin fibroblasts showed a low expression of CD86.Conclusion:Due to their high expression of CD86, circulating fibrocytes seem to be more responsive to CTLA4-Ig treatment than the skin fibroblasts isolated from the same SSc patient.References:[1]Cutolo M et al. Expert Rev Clin Immunol. 2019;15:753-64.[2]Bucala R. Mol Med.2015;2:S3-5.[3]Cutolo M et al. Clin Exp Rheumatol. 2015;33:250-4.[4]Brizzolara R et al. J Rheumatol. 2013;40:738-40.Disclosure of Interests:Stefano Soldano: None declared, Paola Montagna: None declared, Sabrina Paolino: None declared, Elisa Alessandri: None declared, Carmen Pizzorni: None declared, Greta Pacini: None declared, Federica Goegan: None declared, Alberto Sulli Grant/research support from: Laboratori Baldacci, Carlotta Schenone: None declared, Vanessa Smith Grant/research support from: The affiliated company received grants from Research Foundation - Flanders (FWO), Belgian Fund for Scientific Research in Rheumatic diseases (FWRO), Boehringer Ingelheim Pharma GmbH & Co and Janssen-Cilag NV, Consultant of: Boehringer-Ingelheim Pharma GmbH & Co, Speakers bureau: Actelion Pharmaceuticals Ltd, Boehringer-Ingelheim Pharma GmbH & Co and UCB Biopharma Sprl, Maurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Actelion, Celgene, Consultant of: Bristol-Myers Squibb, Speakers bureau: Sigma-Alpha

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Tyrosine Kinase Inhibitor Sunitinib Delays Platelet-Induced Coagulation: Additive Effects of Aspirin

Thrombosis and Haemostasis ◽

10.1055/s-0041-1730312 ◽

2021 ◽

Author(s):

Delia I. Fernández ◽

Alicia Veninga ◽

Bibian M. E. Tullemans ◽

Constance C. F. M. J. Baaten ◽

Linsey J. F. Peters ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Cancer Patients ◽

Kinase Inhibitor ◽

Synergistic Effects ◽

Thrombus Formation ◽

Type I ◽

Glycoprotein Vi ◽

Fibrin Formation ◽

Increased Risk

Abstract Background Sunitinib is a multitarget tyrosine kinase inhibitor (TKI) used for cancer treatment. In platelets, sunitinib affects collagen-induced activation under noncoagulating conditions. We investigated (1) the effects of sunitinib on thrombus formation induced by other TK-dependent receptors, and (2) the effects under coagulating conditions. Cardiovascular disease is a comorbidity in cancer patients, resulting in possible aspirin treatment. Sunitinib and aspirin are associated with increased bleeding risk, and therefore we also investigated (3) the synergistic effects of these compounds on thrombus and fibrin formation. Methods Blood or isolated platelets from healthy volunteers or cancer patients were incubated with sunitinib and/or aspirin or vehicle. Platelet activation was determined by TK phosphorylation, flow cytometry, changes in [Ca2+]i, aggregometry, and whole blood perfusion over multiple surfaces, including collagen with(out) tissue factor (TF) was performed. Results Sunitinib reduced thrombus formation and phosphatidylserine (PS) exposure under flow on collagen type I and III. Also, sunitinib inhibited glycoprotein VI-induced TK phosphorylation and Ca2+ elevation. Upon TF-triggered coagulation, sunitinib decreased PS exposure and fibrin formation. In blood from cancer patients more pronounced effects of sunitinib were observed in lung and pancreatic as compared to neuroglioblastoma and other cancer types. Compared to sunitinib alone, sunitinib plus aspirin further reduced platelet aggregation, thrombus formation, and PS exposure on collagen under flow with(out) coagulation. Conclusion Sunitinib suppresses collagen-induced procoagulant activity and delays fibrin formation, which was aggravated by aspirin. Therefore, we urge for awareness of the combined antiplatelet effects of TKIs with aspirin, as this may result in increased risk of bleeding.

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