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A cell‐based screening assay to identify pharmaceutical compounds that enhance the regenerative quality of corneal repair
Author(s) -
Gordon Gabriel M.,
LaGier Adriana J.,
Ponchel Corinne,
Bauskar Aditi,
Itakura Tatsuo,
Jeong Shinwu,
Patel Nitin,
Fini M. Elizabeth
Publication year - 2016
Publication title -
wound repair and regeneration
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.847
H-Index - 109
eISSN - 1524-475X
pISSN - 1067-1927
DOI - 10.1111/wrr.12390
Subject(s) - luciferase , stromal cell , reporter gene , cell culture , transfection , microbiology and biotechnology , biology , viability assay , chemistry , gene , cancer research , gene expression , biochemistry , genetics
ABSTRACT The goal of this study was to develop and validate a simple but quantitative cell‐based assay to identify compounds that might be used pharmaceutically to give tissue repair a more regenerative character. The cornea was used as the model, and some specific aspects of repair in this organ were incorporated into assay design. A quantitative cell‐based assay was developed based on transcriptional promoter activity of fibrotic marker genes ACT2A and TGFB2 . Immortalized corneal stromal cells (HTK) or corneal epithelial cells (HCLE) were tested and compared to primary corneal stromal cells. Cells were transiently transfected with constructs containing the firefly luciferase reporter gene driven by transcriptional promoters for the selected fibrotic marker genes. A selected panel of seven chemical test compounds was used, containing three known fibrosis inhibitors: lovastatin (LOV), tyrphostin AG 1296 (6,7‐dimethoxy‐3‐phenylquinoxaline) and SB203580 (4‐(4‐fluorophenyl)‐2‐(4‐methylsulfinylphenyl)‐5‐(4‐pyridyl)1H‐imidazole), and four potential fibrosis inhibitors: 5‐iodotubercidin (4‐amino‐5‐iodo‐7‐(β‐D‐ribofuranosyl)‐pyrrolo(2,3‐d)pyrimidine), anisomycin, DRB (5,6‐dichloro‐1‐β‐D‐ribofuranosyl‐benzimidazole) and latrunculin B. Transfected cells were treated with TGFB2 in the presence or absence of one of the test compounds. To validate the assay, compounds were tested for their direct effects on gene expression in the immortalized cell lines and primary human corneal keratocytes using RT‐PCR and immunohistochemistry. Three “hits” were validated LOV, SB203580 and anisomycin. This assay, which can be applied in a high throughput format to screen large libraries of uncharacterized compounds, or known compounds that might be repurposed, offers a valuable tool for identifying new treatments to address a major unmet medical need. Anisomycin has not previously been characterized as antifibrotic, thus, this is a novel finding of the study.