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I.M. Wormstone, J.A. Eldred, J.R. Reddan, G. Duncan; Identification of Signalling Pathways Involved in Tgfß2 Induced Matrix Contraction of Human Lens Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4640.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Matrix contraction is a major process in the light scattering formation of posterior capsule opacification (PCO) following cataract surgery. Previous work has shown that TGFß can induce contraction, however the downstream mechanisms giving rise to this event remain unresolved. Therefore, the aim of the present study was to determine the involvement of putative pathways in TGFß mediated matrix contraction. Methods: Cells from the human lens line FHL 124 were routinely cultured and seeded onto glass coverslips (immuno–detection of Smad2) or tissue culture dishes (Patch assay). Contraction was assessed using a patch growth assay, whereby all area covered by cells, was measured using imaging techniques following fixation in 4% formaldehyde and cell staining with Coomassie Blue. In addition, total protein content, determined by dye extractions was used to give an estimate of total cell population. To study Smad2 distribution immunocytochemistry was used. Following a 24hr periord of serum starvation, cells were maintained in the following conditions: Control medium +/– 10µM MEK inhibitor (U0126); 10ng/ml TGFß2 +/– U0126; Control medium +/– 10µM Rho kinase Inhibitor (Y–27632); 10ng/ml TGFß2 +/– Y–27632. The experimental duration for Smad2 staining and patch assays were 1 and 3 days respectively. Results: Addition of 10ng/ml TGFß2 to patch assays caused a significant reduction in detectable area, indicating contraction has occurred. Maintenance of cultures in the presence of either 10µM U0126 or 10µM Y–27632 had no significant effect on patch area when added to control medium. However, when either U0126 or Y–27632 was added in the presence of TGFß2, contraction was inhibited and the patch area was similar to control values. With respect to cell population all groups were similar. Immunocytochemistry for Smad2 showed clear nuclear translocation in response to 10ng/ml TGFß2. This signalling event was unaffected by either U0126 or Y–27632. Conclusions: The MAP kinase and Rho kinase signalling pathways play important roles in TGFß mediated matrix contraction of human lens cells. Induction of matrix contraction by TGFß2 does not appear to be a Smad dependent process as nuclear translocation was unaffected by MAPK and Rho kinase inhibition.
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