title: Reduction of endothelial cell related TGFbeta activity by thiols.
abstract: Transforming growth factor beta (TGFbeta) may play an important role in diseases characterized by pulmonary fibrosis. We have previously demonstrated that thiols inhibit the pro-oxidant effects of TGFbeta1 in bovine pulmonary artery endothelial cells (BPAEC). To help define the mechanism of this observation we have examined the effect of reduced (GSH) and oxidized (GSSG) glutathione, N-acetyl cysteine (NAC) and cysteine (CYS) on the biological activity of a) TGFbeta released by bovine pulmonary artery endothelial cells (BPAEC) into culture medium, and b) commercially available porcine platelet TGFbeta1. The biological activity of TGFbeta (following activation) released into the medium from cultured BPAEC was significantly reduced when the cells were cultured in the presence of 10 mM GSH or 10 mM NAC for 24 h (10 mM GSH: 85.7 +/- 50 pg/ml/10(6) cells and 10 mM NAC: 127.3 +/- 35 pg/ml/10(6) cells, compared with control: 541 +/- 8.9 pg/ml/10(6) cells; p < 0.05). Thiols (10 mM GSH, 10 mM NAC and 5 mM cysteine), added directly to cell-free conditioned medium or to a commercially available preparation of porcine platelet TGFbeta1 for 6-24 h had a similar inhibitory effect on the biological activity of TGFbeta and altered the structure of porcine platelet TGFbeta1 as determined by mass spectroscopy. These thiols failed to reduce the expression of TGFbeta mRNA in BPAEC as measured by a competitive polymerase chain reaction assay. Incubating endothelial cells or cell-free conditioned medium with GSSG did not alter the biological activity of TGFbeta. Lower doses of thiols (0.1-1 mM), that we have shown inhibit the antiproliferative and pro-oxidant effects of exogenous TGFbeta1 on BPAEC, had no direct effect on TGFbeta bioactivity. In summary, thiols are capable of reducing the effects of TGFbeta in biological systems through a direct effect on the TGFbeta molecule. However, this action appears to be dose-dependent, and at low doses (0.1-1 mM) thiols may also inhibit the actions of exogenous TGFbeta1 in cell culture through a mechanism involving the cellular redox status.
authors.affiliation: Department of Medicine/Tupper Research Institute, New England Medical Center/Tufts University School of Medicine, Boston, MA 02111, USA. alex.white@es.nemc.org
authors.forename: A C
authors.initials: AC
authors.lastname: White
authors.forename: E K
authors.initials: EK
authors.lastname: Maloney
authors.forename: S L
authors.initials: SL
authors.lastname: Lee
authors.forename: J J
authors.initials: JJ
authors.lastname: Lanzillo
authors.forename: B L
authors.initials: BL
authors.lastname: Fanburg
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D003545:Cysteine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism / Q000494:pharmacology; D019803:Glutathione Disulfide / Q000378:metabolism / Q000494:pharmacology; D010084:Oxidation-Reduction; D011651:Pulmonary Artery / Q000166:cytology; D013438:Sulfhydryl Compounds / Q000378:metabolism* / Q000494:pharmacology; D013552:Swine; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*
chemical_list: D017077:Culture Media, Conditioned; D013438:Sulfhydryl Compounds; D016212:Transforming Growth Factor beta; D005978:Glutathione; D003545:Cysteine; D019803:Glutathione Disulfide; D000111:Acetylcysteine