Elsevier

Matrix Biology

Volume 26, Issue 8, October 2007, Pages 625-632
Matrix Biology

Endogenous endothelin-1 signaling contributes to type I collagen and CCN2 overexpression in fibrotic fibroblasts

https://doi.org/10.1016/j.matbio.2007.06.003Get rights and content

Abstract

Fibrosis is excessive scarring caused by the accumulation of extracellular matrix proteins and is a common end pathway in many chronic diseases. Endothelin-1 is a possible contributor to the persistent fibrotic phenotype of fibroblasts isolated from fibrotic lesions. In this report we used a specific dual endothelin A/B receptor antagonist, bosentan, to determine the role of endogenous endothelin signaling in maintaining the profibrotic phenotype of lung fibroblasts from scleroderma patients. Bosentan treatment of lung fibroblasts cultured from normal individuals and individuals with scleroderma was assessed using Affymetrix genome-wide expression profiling, real-time polymerase chain reaction and Western blot analysis and revealed that approximately one-third of the transcripts elevated greater than two-fold in fibrotic fibroblasts were reduced by Bosentan treatment. Genes whose overexpression in fibrotic fibroblasts that were dependent on endogenous endothelin signaling included the matrix or matrix-associated genes type I collagen, fibronectin and CCN2. The elevated adhesive property of fibrotic fibroblasts was also reduced by endothelin receptor antagonism. Basal expression of collagen, fibronectin and CCN2 and adhesion to matrix was not affected. Thus endogenous endothelin signaling contributes to the fibrotic phenotype of fibrotic fibroblasts, suggesting that antagonizing endothelin receptors may be of benefit in combating fibrotic disease.

Introduction

The normal tissue repair program requires the de novo production of connective tissue, which is comprised of mesenchymal cells and extracellular matrix (ECM). Should the repair program be appropriately terminated, normal tissue function is essentially restored. However, if the tissue repair program continues unabated, excessive production and contraction of can ECM occurs, resulting in pathological scarring. Scars are characterized by the presence of a specialized form of fibroblast, termed the myofibroblast (Desmouliere, 1995, Harrison et al., 1991), which is responsible both for normal tissue repair and also the excessive production and contraction of extracellular matrix (ECM) within fibrotic lesions (Desmouliere, 1995, Harrison et al., 1991). Excessive scarring can, in turn, result in pathological fibrotic diseases, which can culminate in organ failure and death.

The vasoconstrictive peptide endothelin-1 (ET-1) appears to be a key contributor to fibrosis (Shi-Wen et al., 2004a). Elevated levels of circulating ET-1, and increased ET-1 production, exist in patients with pulmonary fibrosis that correlate with the severity of the fibrotic phenotype (Miyauchi et al., 1992, Kawaguchi et al., 1994, Abraham et al., 1997, Teder and Noble, 2000). In cell culture, ET-1 promotes ECM production and contraction by fibroblasts (Shi-Wen et al., 2004a, Shi-Wen et al., 2004b). The scarring disease scleroderma (systemic sclerosis, SSc) is a disease of unknown etiology characterized by microvascular injury, autoimmune inflammatory responses, and severe and often progressive fibrosis of the lung (Denton and Black, 2003). In vitro studies have consistently shown that fibroblasts isolated from scars of scleroderma patients retain their pro-fibrotic phenotype for several passages in cell culture (Ivarsson et al., 1993, Shi-Wen et al., 1997, Chen et al., 2005). Thus we have used scleroderma fibroblasts as a model system for understanding the nature of persistent fibrosis. ET-1 is overproduced by fibrotic fibroblasts isolated from scleroderma patients (Shi-Wen et al., 2006). Endogenous ET-1 activity in these fibrotic fibroblasts directly contributes to the contractile phenotype of the this cell type as blocking ET-1 signaling by the specific ET-1 dual receptor antagonist bosentan greatly reduced the ability of scleroderma fibroblasts to contract a collagen gel matrix and the overexpression of the pro-adhesive and pro-contractile proteins α-smooth muscle actin (α-SMA), ezrin, moesin and paxillin (Shi-Wen et al., 2004a). These results suggest that enhanced ET-1 signaling contributes to the persistent fibrotic phenotype. However, whether bosentan might also alleviate the excessive production of matrix and matrix-associated proteins associated with scleroderma such as type I collagen, CCN2 (formerly called connective tissue growth factor, CTGF) and fibronectin is not known. Indeed the mechanism underlying the overexpression of these latter proteins by fibrotic fibroblasts is poorly understood.

In this report, we investigate the extent to which endogenous ET-1 signaling contributes to normal tissue repair and fibrotic responses by assessing whether the dual ETA/ETB receptor antagonist bosentan affects the fibrotic phenotype of fibroblasts isolated from lungs of fibrosing alveolitis associated with systemic sclerosis (FASSc) patients. Our studies provide evidence that endothelin-1 receptor antagonism might be beneficial in reversing chronic fibrosis.

Section snippets

Affymetrix gene of normal and FASSc lung fibroblasts reveals that Bosentan antagonizes 56/136 genes overexpressed by FASSc fibroblasts

To evaluate to what extent endogenous endothelin signaling contributes to the phenotype of fibrotic fibroblasts, we cultured normal and scleroderma lung fibroblasts until confluence. Cells were serum-starved for 18 h, and incubated for an additional 24 h in the presence or absence of the dual ETA/ETB receptor antagonist bosentan (10 μM). Total RNA was prepared from these cells, reverse transcribed, and applied to Affymetrix U133A arrays. A total of six patients and normal individuals were

Discussion

Fibrotic disease is an often fatal and has no therapy. Recently, however, several candidate mediators of the fibrotic phenotype have emerged, including ET-1 (Shi-Wen et al., 2004a, additional refs). In this report, we investigate the ability of bosentan, an ET/A receptor antagonist (Clozel and Salloukh, 2005), to prevent the overexpression of pro-fibrotic genes by FASSc fibroblasts, and the abilities of FASSc fibroblasts to excessively adhere to ECM. These data extend our previous findings that

Patients and cell culture

Fibroblasts were grown by explant culture from open lung biopsy specimens from SSc patients taken for histological staging of lung fibrosis, and control samples were taken from normal lungs not used for transplant. The group of 6 SSc patients fulfilled the criteria of the American College of Rheumatology for the diagnosis of SSc with lung involvement. Patients were female. Fibroblasts were used at passage 3 (Shi-Wen et al., 1997, Shi-Wen et al., 2001). Informed consent and ethical approval were

Acknowledgements

This work was supported by the Raynaud's and Scleroderma Association Trust, the Scleroderma Foundation, the Arthritis Research Campaign, the Nightingale Trust, the Welton Foundation, the Ontario Thoracic Society, the Canadian Foundation for Innovation and the Canadian Institutes of Health Research. We thank Martine Clozel (Actelion) for providing bosentan. A.L. is a New Investigator of the Arthritis Society (Scleroderma Society of Ontario) and a recipient of an Early Researcher Award.

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