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A. R. Djalilian, M. A. Shafiq, A. Namavari; mTOR Pathway in Keratocyte-To-Myofibroblast Transformation in the Cornea. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6213.
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Previous studies have looked at the mTOR pathway in fibrotic processes including those in the skin and the liver. This study was performed to investigate the role of mTOR pathway in keratocyte-to-myofibroblast transformation in the cornea.
Pig corneal keratocytes were isolated and cultured in DMEM/F12. To investigate the effect of TGF-beta on mTOR pathway, cells were treated with TGF-beta and were harvested at different timepoints (0.5, 1, 2, 4, 8, 12, 24, 48 hrs). To investigate the role of mTOR pathway in keratocyte-to-myofibroblast differentiation, isolated pig corneal cells or whole pig corneas were cultured in the same medium and were incubated with or without (control group) TGF-beta or Rapamycin for 5-7 days in the presence or absence of 5% fetal bovine albumin. Cultured cells or tissues were analyzed by Western blot and immunostaining for mTOR and its downstreams as well as keratocyte and myofibroblast markers and extracellular matrix components.
mTOR pathway was activated in 2-4 hours after stimulation with TGF-beta as detected by higher level of p-mTOR (23%), p-S6 Kinase (63%), p-Ribosomal S6 Protein (303%), and p-eIFB4 (102%) in Western blot. Inhibition of mTOR pathway by Rapamycin resulted in lower levels of alpha-smooth muscle actin (55%) and fibronectin (81%) and higher level of aldehyde dehydrogenase 1 (52%) after stimulation of cells with TGF-Beta for 5-7 days as detected by western blot and immunostaining.
mTOR pathway plays a role in keratocyte-to-myofibroblast transformation and Rapamycin can negatively regulate this transformation by inhibiting mTOR pathway.
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