Purchase this article with an account.
M. Fini, A. J. LaGier, C. Ponchel, G. Gordon; A Novel Screen to Identify Inhibitors of Corneal Fibrosis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):393.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Corneal scarring is a significant clinical problem throughout the world. IN this study we used a novel high throughput screening assay to identify novel inhibitors of TGF-beta2, a key mediator of corneal fibrosis in order to reduce corneal scarring.
A construct was created containing the the firefly luciferase gene driven by the TGF-beta2 promoter. This construct was co-transfected with a renilla luciferase gene under the control of a CMV promoter into a human corneal limbal epithelial cell line (HCLE), generously provided by Dr. Ilene Gipson. HCLEs were treated with 10 ng/ml of TGF-beta2 (to induce an autocrine feedback loop) with or without various compounds for 24 hours. The firefly to renilla activity ratio was then quantified to determine the relative TGF-beta2 promoter activity to see which compounds could block transcription of the TGF-beta2 gene. Statistical significance of observations was determined using a student’s t-test.
We performed a pilot experiment screening 7 synthetic chemical compounds that we hypothesized may inhibit TGF-beta2 expression. Of the seven compounds studied, compounds A (an actin polymerization inhibitor) and C (a JNK/SAPK signaling pathway activator) reduced TGF-beta2 induced promoter activity though the effects were not quite statistically significant. Only compound G (a p38 MAPK pathway inhibitor) significantly reduced TGF-beta2 promoter expression. Compound G also had no apparent affect on cell morphology or cell survival.
Though only in the initial pilot phase of testing, we have developed a high throughput screening process for identifying TGF-beta2 inhibitors, and even identified at least one possible compound. Applying this screening model to drug libraries with thousands of compounds will uncover more TGF-beta2 inhibitors, some of which may be used in the clinic to reduce corneal complications and scarring in the near future.
This PDF is available to Subscribers Only