Nintedanib downregulates the transition of cultured systemic sclerosis fibrocytes into myofibroblasts and their pro-fibrotic activity

Maurizio Cutolo,Emanuele Gotelli,Paola Montagna,Samuele Tardito,Sabrina Paolino,Carmen Pizzorni,Alberto Sulli,Vanessa Smith,Stefano Soldano

doi:10.1186/s13075-021-02555-2

Abstract

BackgroundCirculating fibrocytes are an important source of fibroblasts and myofibroblasts, which are involved in fibrotic processes, including systemic sclerosis (SSc).The study aimed to investigate the effect of nintedanib (a tyrosine kinase inhibitor) in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity.MethodsCirculating fibrocytes were obtained from 18 SSc patients and 5 healthy subjects (HSs). Cultured SSc fibrocytes were maintained in growth medium (untreated cells) or treated with nintedanib 0.1 and 1 μM for 3 and 24 h. Fibroblast-specific protein-1 (S100A4) and α-smooth muscle actin (αSMA), as markers of fibroblast/myofibroblast phenotype, together with type I collagen (COL1) and fibronectin (FN), were investigated by qRT-PCR and Western blotting. Non-parametric tests were used for statistical analysis.ResultsSignificantly 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). Interestingly, an increased gene and protein expression of αSMA and S100A4 was found in fibrocytes from SSc patients positive for anti-Scl70 and with interstitial lung disease (ILD) (Scl70+ILD+) compared to Scl70−ILD− patients (S100A4: gene: p < 0.01; protein: p < 0.05), whereas no differences were observed for COL1 and FN.Nintedanib reduced gene and protein expression of αSMA, S100A4, COL1, and FN in SSc fibrocytes compared to untreated ones with different statistical significance.Noteworthy, nintedanib significantly downregulated gene and protein expression of αSMA, S100A4, COL1, and FN in Scl70+ILD+ fibrocytes (all p < 0.05), whereas only that of S100A4 and FN was significantly downregulated (p < 0.05) in Scl70−ILD− fibrocytes compared to the related untreated cells.ConclusionsNintedanib 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.

Highlights

Systemic sclerosis (SSc) is a complex chronic and progressive autoimmune connective tissue disease whose pathophysiology is characterized by microvascular damage, immune-inflammatory response, and diffuse fibrosis at the level of the skin and internal organs, including lung and heart [1]
Fibrosis is primarily determined by the persistent activation and transition of fibroblasts into α-smooth muscle actin positive myofibroblasts, which are considered the key effector cells of the excessive and progressive deposition of extracellular matrix (ECM) macromolecules, such as type I collagen (COL1) and fibronectin (FN) [2,3,4]
Fibrocytes are thought to be circulating fibroblast precursors expressing both hematopoietic and stromal cell markers (CD34, CD45, Major histocompatibility complex (MHC) class II), as well as Type I collagen (COL1), which are used as the minimum criteria for identifying these cells in in vitro cultures, together with the surface expression of C-X-C chemokine receptor type 4 (CXCR4) [4, 9, 10]

Summary

Introduction

Systemic sclerosis (SSc) is a complex chronic and progressive autoimmune connective tissue disease whose pathophysiology is characterized by microvascular damage, immune-inflammatory response, and diffuse fibrosis at the level of the skin and internal organs, including lung and heart [1]. Fibrosis is primarily determined by the persistent activation and transition of fibroblasts into α-smooth muscle actin (αSMA) positive myofibroblasts, which are considered the key effector cells of the excessive and progressive deposition of extracellular matrix (ECM) macromolecules, such as type I collagen (COL1) and fibronectin (FN) [2,3,4]. Inhibition of accumulation, activation, and transition of fibroblasts into myofibroblasts might contribute to reduce the fibrotic process and potentially to alter the natural development of many important fibrotic diseases, including SSc [5, 6]. Circulating fibrocytes are an important source of fibroblasts and myofibroblasts, which are involved in fibrotic processes, including systemic sclerosis (SSc). The study aimed to investigate the effect of nintedanib (a tyrosine kinase inhibitor) in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity

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