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M. J. Maldonado, C. Gil-Puig, M. Arriero, J. Dotor, I. Ochoa, C. Díaz-Ruiz, J. Prieto, F. Borrás-Cuesta; In vitro Effect of Recently Identified Anti-TGF-β1 Peptides on the Rabbit Corneal Epithelial Cell Line, SIRC. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4293.
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The cytokine TGF-b is one of the chief mediators in the fibrosis induction process. In the eye, TGF-β activity has been implicated in causing corneal fibrosis and haze, stimulating the synthesis and secretion of extracellular matrix proteins. Among the major isoforms of TGF-β, TGF-β1 has been shown to be primarily involved in the process of fibrogenesis. The major signalling pathway of TGF-β is through its transmembrane receptor and activates the cytoplasmic Smad proteins, which are crucial mediating several TGF-β responses. The peptides P17 and P144 have been recently identified and shown to block TGF-β1 activity in liver and skin tissues. The aim of this investigation was to study the efficacy of the inhibitory effect of the peptides P17 and P144 on activation of Smad2 in SIRC (Statens Seruminstitut Rabbit Cornea) epithelial cells, which have fibroblast morphology.
SIRC cells were cultured to confluence. The cells were then exposed to serum-free media alone (control), or with different concentrations of TGF-β1 (3 and 5 ng/ml) with and without anti- TGF-β peptides P17 and P144 in different concentrations (50 and 200 µg/ml) for 1 hour. They were lysed then in phospho buffer for Western blot with phospho-specific antibody against Smad2. The p-Smad2 protein concentration was calculated as the p-Smad2/β-actin ratio from ten different experiments. Provided the parametric distribution of this variable, we performed two-way ANOVA and student t-tests.
Immunoblot analyses revealed that TGF-β1 induces activation of Smad2 (p-Smad2) in SIRC cell line (P = 0.0001). The p-Smad2 concentration diminished significantly with both 50 µg/ml and 200 µg/ml anti-TGF-β1 peptide P144 (P = 0.0001). However, the P17 peptide did not change significantly the TGF-β-induced activation of Smad2, neither at 50 nor at 200 µg/ml concentrations (P = 0.55). Also, we found no significant difference in effect between the two concentrations with either peptide (P = 0.25).
The p-Smad2 was shown to be activated by TGF-β1 in SIRC cells. This stimulation was abolished similarly by both concentrations of anti-TGF-β1 peptide P144, 50 and 200 µg/ml, while the P17 peptide did not inhibit the TGF-β-induced activation of Smad2, neither at 50 µg/ml nor at 200 µg/ml concentrations in vitro. The anti-TGF-β1 peptide P144 has therefore a potential for exhibiting a significant inhibitory effect in the corneal fibrosis process.
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