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J. G. Browne, R. Kane, N. Oliver, J. K. Crean, C. J. O' Brien; Induction of EMT in ARPE-19 Cells by Recombinant Human CTGF. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5722.
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Connective Tissue Growth Factor (CTGF) is a recognised downstream mediator of transforming growth factor-beta (TGF-ß) in many fibrotic diseases. Recent studies have shown that CTGF levels are increased in the vitreous humour in patients with proliferative vitreoretinopathy (PVR). PVR can be defined by the growth and contraction of membranes within the retina. Intraocular diseases resulting from various stimuli have PVR as there endpoint. CTGF has been found in PVR associated membranes. ARPE-19 is a spontaneously arising human RPE cell line with normal karyology which forms normal polarised epithelial monolayers. In this study we observed epithelial mesenchymal transition like changes of ARPE-19 cells following treatment with CTGF.
ARPE-19 cells were cultured in dulbecco's modified eagle's medium nutrient mixture F-12 ham containing 10% FCS, 2mM L-Glutamine, 50 U/ml penicillin and 50 µg/ml streptomyocin. On becoming confluent these cells were cultured in KI media for 24 hours. Serum restricted cells were then cultured treated with 25 ng/ml recombinant CTGF. Morphologcal changes were observed using a light microscope. Immuofluoresence and Western blotting was performed to investigate changes in EMT markers including ZO-1 and vimentin. Signalling pathways affected by CTGF were also investigated including the p38 MAPK and the p42/44 MAPK pathways.
Changes from an epithelial to mesenchyal like phenotype of ARPE-19 cells were observed upon treatment with CTGF. Immunoflouresence staining showed reduction in levels of ZO-1, while vimentin levels increased. Western blotting also showed changes in the levels of EMT markers including α smooth muscle actin. Signalling events following treatment of ARPE-19 cells were also observed. CTGF treatment was seen to increase phosphorylation of both p38 and p42/44.
TGF-ß is a well documented inducer of EMT. In this study we investigated a downstream mediator of TGF-ß, CTGF for its potential to inducer EMT. Changes in both gross morphology and subcellular structure indicate that CTGF is capable of inducing EMT in ARPE-19 cells. Elevated levels of CTGF in the vitreous may thus be contribute to the morphological changes seen in PVR.
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