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Omer Iqbal, Wells Brambl, Charles Bouchard; The role of Transforming growth factor beta 2 (TGF-beta 2) in the pathogenesis of keratoconus.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2914.
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© ARVO (1962-2015); The Authors (2016-present)
We sought out to test the hypothesis that TNF-alpha and/or gentle mechanical stimulation of Human Corneal Endothelial Cells (HCEC) induces the expression of activated TGF-beta2.
HCEC’s were obtained courtesy of Dr. Monika Valtink and cultured in Basal F99 medium as per Dr. Valtink’s protocol. Cells were trypsinized and plated at a concentration of a 12k cells/mL in a 24 well plate and allowed to grow to 100% confluency. Cells were washed and media replaced with DMEM and 10% FBS. Cells were exposed to sham, 1 ng/mL, 10 ng/mL, or 100 ng/mL of TNF-alpha. Half of those cells were exposed to gentle mechanical stimulation. After 16 hours of incubation supernatants were collected and stored at -80C. TGF-beta2 concentration in supernatants was assayed using eBioscience TGF-beta2 platinum ELISA, which were then subjected to one-way ANOVA compared to control sample.
When compared to control samples, HCEC cells exposed to 10 ng/mL TNF-alpha expressed more TGF beta 2, 346.7 vs. 512.2 ng/mL respectively (p=0.0096). When compared to control samples, HCEC cells exposed to 100 ng/mL TNF-alpha expressed increased TGF beta 2, measuring 346.7 vs 505.1 ng/mL respectively (p=0.0435). However there was no significant effect of gentle mechanical stimulation of cells in the presence or absence of TNF-alpha.
TNF-alpha exposed cells at concentrations greater than or equal to 10 ng/mL had increased secretion of TGF-beta2. These data suggest that an initial inflammatory response may trigger increased secretion of TGF-beta 2 by HCEC's, therefore possibly increasing fibrosis of the stroma as seen in Keratoconus. Interestingly, this effect was not enhanced by gentle mechanical stimulation. However, this study is limited by the short time course of the experiment, which obviates the lack of applicability to chronic eye rubbing. Future testing will evaluate Human Corneal Epithelial Cells and their response to TNF-alpha and whether inhibition of Smad3 rescues increased TGF-beta 2 secretion in response to TNF-alpha. Clinical studies are warranted to validate these results.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
HCEC secretion of TGF-Beta 2 in sham, mechanical stimulation alone, 1 ng/mL TNF-alpha, 1 ng/mL TNF-alpha + mechanical stimulation, 10 ng/mL TNF-alpha, 10 ng/mL TNF-alpha + mechanical stimulation, 100 ng/mL TNF-alpha, and 100 ng/mL TNF-alpha + mechanical stimulation.
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