title: Transforming growth factor-beta mRNA and protein in hypertrophic scar tissues and fibroblasts: antagonism by IFN-alpha and IFN-gamma in vitro and in vivo.
abstract: Hypertrophic scarring (HSc) following burn injury is a common, disfiguring, and functionally limiting form of dermal fibrosis, compromising recovery. Previously, elevated levels of transforming growth factor-beta1 (TGF-beta1), a fibrogenic cytokine, were found in wounds and serum of severely injured patients, antagonized in part by treatment with systemic interferon-alpha2b (IFN-alpha2b) both in vitro and in vivo. It is hypothesized that in wound healing after injury, platelets are an initial source of TGF-beta, but wound fibroblasts may be capable, after activation, of autoamplification of the initial response to injury by increasing TGF-beta mRNA and protein that may subsequently be responsive to IFN therapy with IFN-alpha or IFN-gamma or both. Using three pairs of site-matched HSc and normal fibroblasts from the same individuals, nonconfluent and near confluent fibroblasts were treated with TGF-beta, and cell proliferation and collagen production were assayed using cell counting and 18O2 isotopic uptake into hydroxyproline before analysis by gas chromatography-mass spectrometry (GC-MS). HSc and normal fibroblasts were assayed for the production of TGF-beta protein secretion using ELISA for TGF-beta1, TGF-beta2, and TGF-beta3 after acidification of medium samples from 96-h cultures. HSc and normal fibroblasts were treated with IFN-alpha2b or IFN-gamma or both for 96 h. Quantitative RT-PCR and Northern analysis were performed using newly synthesized internal standards for human TGF-beta1. TGF-beta stimulates both HSc and normal fibroblast proliferation. Collagen synthesis is greater in HSc than in normal fibroblasts and is maximally stimulated at 75 pM TGF-beta. TGF-beta stimulated collagen metabolism is antagonized by IFN-alpha or IFN-gamma or both in an additive fashion. HSc and normal fibroblasts not only possess the mRNA for TGF-beta1 but also secrete mature TGF-beta protein. Treatment of HSc and normal fibroblasts with IFN-alpha2b or IFN-gamma antagonizes TGF-beta protein production, and additive effects occur. RT-PCR demonstrates that after IFN treatment, downregulation of TGF-beta1 mRNA accounts in part for the reduction in protein secretion in HSc fibroblasts. Elevations of systemic TGF-beta may be due to wound fibroblasts. TGF-beta synthesis and antagonism of fibroblast TGF-beta protein secretion occurs with either IFN-alpha or IFN-gamma, in part by downregulation of TGF-beta1 mRNA levels.
authors.affiliation: Department of Surgery, University of Alberta, Edmonton, Canada. etredget@gpu.srv.ualberta.ca
authors.forename: E E
authors.initials: EE
authors.lastname: Tredget
authors.forename: R
authors.initials: R
authors.lastname: Wang
authors.forename: Q
authors.initials: Q
authors.lastname: Shen
authors.forename: P G
authors.initials: PG
authors.lastname: Scott
authors.forename: A
authors.initials: A
authors.lastname: Ghahary
mesh_terms: D017439:Cicatrix, Hypertrophic / Q000097:blood / Q000235:genetics* / Q000378:metabolism*; D003094:Collagen / Q000096:biosynthesis; D015536:Down-Regulation / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012867:Skin / Q000187:drug effects / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000097:blood / Q000235:genetics* / Q000378:metabolism*; D014945:Wound Healing / Q000187:drug effects / Q000235:genetics / Q000502:physiology
chemical_list: D000077190:Interferon alpha-2; D016898:Interferon-alpha; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D007371:Interferon-gamma; D003094:Collagen