April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
A Chemical Screen to Identify Pharmacological Inhibitors of Corneal Fibrosis
Author Affiliations & Notes
  • Gabriel M. Gordon
    USC Institute for Genetic Medicine, Keck School of Medicine of USC, Los Angeles, California
  • M. Elizabeth Fini
    USC Institute for Genetic Medicine, Keck School of Medicine of USC, Los Angeles, California
  • Footnotes
    Commercial Relationships  Gabriel M. Gordon, None; M. Elizabeth Fini, None
  • Footnotes
    Support  NIH Grant EY09828, NIH grant P30-EY003040
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 2028. doi:
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      Gabriel M. Gordon, M. Elizabeth Fini; A Chemical Screen to Identify Pharmacological Inhibitors of Corneal Fibrosis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2028.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Corneal repair occurs on a spectrum from regenerative to fibrotic, the former restoring corneal clarity, the latter causing scarring and obstructed vision. We are developing a method to efficiently screen chemical libraries, using transcriptional activity of surrogate marker genes for fibrosis as the read-out in a cell-based assay performed in high content format. This study examined use of the α-smooth muscle actin (SMA) gene.

Methods: : A surrogate reporter construct was created utilizing 1 kb of the SMA gene promoter to drive expression of the firefly luciferase gene (a gift of Dr. Michael Keogh). Test cells were co-transfected with this construct along with a renilla luciferase gene driven by the constitutively active CMV promoter, which serves as a readout for cell viability. We used primary rabbit keratocytes (PRK) or an immortalized human keratocyte cell line (HTK; a gift of Dr. Jamie Jester). Transfected cells were treated for 24 hours with TGF-β2 (T2) to activate the program of fibrotic gene expression. A subset of cells were co-treated with 7 different test compounds. The firefly to renilla expression ratio (F/R ratio) was calculated to identify compounds that block SMA gene activity, while also maintaining cell viability. SMA gene and protein expression were also assessed by RT-PCR and immunocytochemistry (ICC).

Results: : T2 treatment increased the F/R ratio by 2.7 fold in HTKs. Co-treatment with anisomycin (ANI), lovastatin (LOV), iodotubercidin (IODO), and SB203580 (SB) significantly reduced the F/R ratio by 0.1, 1.4, 0.6, and 1.1 fold respectively. Similarly, T2 treatment of PRKs resulted in a 4.5 fold increase in the F/R ratio and treatment with ANI, LOV, IODO, and SB reduced the ratio to 1.7, 1, 0.9, and 1.1 fold respectively. RT-PCR analysis showed T2 treatment increased SMA mRNA by 10 fold in HTKs and 69 fold in PRKs. ANI treatment reduced this to 1.3 fold in HTKs and 3.2 fold in PRKs while SB treatment reduced expression to 2.6 fold in HTKs and 17.7 fold in PRKs. ICC staining of both HTKs and PRKs showed that ANI and SB both significantly reduced SMA protein levels. We will show how combining this assay with a second assay utilizing the T2 gene as surrogate (reported last year), provides a more comprehensive picture of a compound’s anti-fibrotic effect.

Conclusions: : We have developed a high throughput screening method for identifying inhibitors of corneal fibrosis, and we have identified two lead compounds. Applying this screening model to chemical libraries with thousands of compounds will uncover more potential inhibitors of corneal scarring, which can then be further assessed in a clinical setting.

Keywords: cornea: stroma and keratocytes • wound healing • drug toxicity/drug effects 

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