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OBJECTIVES: 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-ß, 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-ß-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-ß-stimulated gel contraction similar to other antioxidants DPI and NAC, but not SOD or catalase. EGCG suppressed TGF-ß-induced ROS production in all fibroblasts. Furthermore, EGCG inhibited TGF-ß or PDGF-BB-induced phospho-extracellular signal-regulated kinase (ERK)1/2 MAP kinase and NF-κB activity in SSc fibroblasts. CONCLUSION: 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.

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BACKGROUND: Arterial mechanical parameters are modified in women with polycystic ovary syndrome (PCOS), before and during pregnancy. This study tested the hypothesis that aortic mechanics and endothelial function are modified in the mifepristone-treated rat model of PCOS. METHODS: Female rats injected daily with mifepristone or vehicle for 7-9 days were assessed by ultrasound to allow estimation of aortic stiffness index and compliance. The influence of acetylcholine (ACh) and sodium nitroprusside (SNP) on dissected phenylephrine-contracted aortic rings was assessed. RESULTS: Aortic compliance was reduced by 67% in mifepristone-treated rats versus controls (P<0.05), while stiffness index was increased 2.3-fold (P<0.02). ACh-induced dilation was less in aortic rings from mifepristone-treated rats (P=0.022) and was less sensitive to the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (P<0.001), while SNP-induced dilation was greater (P=0.001). CONCLUSIONS: Aortic mechanics in vivo and endothelial function in vitro were consistently perturbed in mifepristone-treated rats. Aortic ring behaviour suggested that NO release was depressed or degradation elevated, with a compensatory increase in NO sensitivity and/or activation of a non-NO-mediated relaxation mechanism. The mifepristone-treated rat is a valid model for investigation of the vascular deficits seen in PCOS.

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Nitric oxide is a gas and a free radical which is now recognised to have very important physiological roles. It is synthesised enzymatically from the amino acid L-arginine in a number of tissues using the three isoforms of nitric oxide synthase, one of which is inducible and can form much large amounts of NO. NO is important in the endothelium-dependent regulation of blood flow and pressure as well as inhibiting the activation of blood platelets. NO is recognised as a neurotransmitter at least in certain types of nerves. Along with other free radicals, NO is also important in the primary defence mechanisms against attack by micro-organisms. NO has a close interaction with iron-containing proteins and binds to haem. By this process NO activates a haem-containing enzyme called soluble guanylyl cyclase which is activated a thousand fold to produce the signalling molecule cyclic GMP. This has many effects at the molecular level to set in train the pathways which propagate the diverse physiological actions of NO. Although this pathway through cyclic GMP is important, this is by no means the only mechanism by which NO influences the activities of the cell. These alternative pathways depend on modification of the structure of enzymes and structural proteins in several different ways. Most of these modifications result from the actions of NO with other free radicals such as oxygen and superoxide anions to produce reactive oxidants. The oxidants modify the proteins by, among others, nitrosation and nitration of proteins of thiol groups and aromatic amino acids respectively. These changes introduce potential new subtleties to the effects on NO on cellular function which are only now being explored. Protein modifications by NO are even more evident in many inflammatory disorders and may account, at least to some extent, to the pathology seen in these conditions.

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