title: Modulation of collagen type I, fibronectin and dermal fibroblast function and activity, in systemic sclerosis by the antioxidant epigallocatechin-3-gallate.
abstract: OBJECTIVE
  SSc is characterized by the overproduction of extracellular matrix (ECM) proteins, such as collagen and fibronectin, by activated fibroblasts, as well as oxidative stress. This study investigates the anti-fibrotic potential of the antioxidant epigallocatechin-3-gallate (EGCG) on activated dermal fibroblasts from SSc patients.
  
  
  METHODS
  Dermal fibroblasts from a cell line (AG), healthy individuals (CON) and SSc patients were treated with EGCG, TGF-β, PDGF-BB or other antioxidants [antioxidants superoxide dismutase (SOD), catalase, N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI)]. Collagen type I, fibronectin, connective tissue growth factor (CTGF), α-smooth muscle actin and mitogen-activated protein (MAP) kinases were measured by ELISA and western blot. Fibroblast contractile forces were measured by collagen gel contraction. Reactive oxygen species (ROS) were assessed by dichlorofluorescein assay and nuclear factor κ beta (NF-κB) activity by DNA binding assay.
  
  
  RESULTS
  EGCG (1-100 µM) dose-dependently decreased collagen type I secretion in culture medium after 24 h in AG fibroblasts. Collagen type I protein expression in cell lysates was also significantly reduced by 40% in EGCG-treated cells (40 µM). Furthermore, EGCG also down-regulated TGF-β-induced collagen type I, fibronectin and CTGF. Similarly, in CON fibroblasts EGCG decreased basal and stimulated collagen type I, fibronectin and CTGF after 24 h, while in SSc the effects of the antioxidant were apparent after 48 h. Fibroblast-mediated contraction of collagen gels was inhibited by EGCG as early as 1 h in AG fibroblasts, and in the CON and SSc fibroblasts. Additionally, EGCG also inhibited TGF-β-stimulated gel contraction similar to other antioxidants DPI and NAC, but not SOD or catalase. EGCG suppressed TGF-β-induced ROS production in all fibroblasts. Furthermore, EGCG inhibited TGF-β or PDGF-BB-induced phospho-extracellular signal-regulated kinase (ERK)1/2 MAP kinase and NF-κB activity in SSc fibroblasts.
  
  
  CONCLUSIONS
  The results suggest that the antioxidant, EGCG, can reduce ECM production, the fibrotic marker CTGF and inhibit contraction of dermal fibroblasts from SSc patients. Furthermore, EGCG was able to suppress intracellular ROS, ERK1/2 kinase signalling and NF-κB activity. Taken together, EGCG may be a possible candidate for therapeutic treatment aimed at reducing both oxidant stress and the fibrotic effects associated with SSc.
authors.affiliation: Centre for Rheumatology and Connective Tissue Disease, University College London Medical School, Royal Free Campus, London, UK. a.dooley@medsch.ucl.ac.uk
authors.forename: Audrey
authors.initials: A
authors.lastname: Dooley
authors.forename: Xu
authors.initials: X
authors.lastname: Shi-Wen
authors.forename: Nima
authors.initials: N
authors.lastname: Aden
authors.forename: Thomas
authors.initials: T
authors.lastname: Tranah
authors.forename: Nirupa
authors.initials: N
authors.lastname: Desai
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Denton
authors.forename: David J
authors.initials: DJ
authors.lastname: Abraham
authors.forename: Richard
authors.initials: R
authors.lastname: Bruckdorfer
mesh_terms: D000704:Analysis of Variance; D000975:Antioxidants / Q000378:metabolism*; D001706:Biopsy; D016022:Case-Control Studies; D002392:Catechin / Q000031:analogs & derivatives* / Q000378:metabolism; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism*; D006801:Humans; D017382:Reactive Oxygen Species / Q000378:metabolism; D012595:Scleroderma, Systemic / Q000378:metabolism*
chemical_list: D000975:Antioxidants; D024042:Collagen Type I; D005353:Fibronectins; D017382:Reactive Oxygen Species; D002392:Catechin; C045651:epigallocatechin gallate