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Gene expression profile of ARPE-19 during repair of the monolayer
S Singh1, J J Zheng, S C Peiper
1Department of Ophthalmology & Visual Sciences, Kentucky Lions Eye Center, University of Louisville School of Medicine, KY-40202, USA.
Background:
The purpose of this study was to identify the profile of gene expression during retinal pigment epithelial (RPE) wound repair.
Methods:
ARPE-19 cells derived from a human RPE cell line were grown for 4 weeks and injured by creating multiple concentric wounds. Unwounded cultures served as controls. During the proliferative phase of wound repair, total RNA was extracted from control and wounded cultures, and a [32P]dATP-labeled cDNA probe was synthesized and hybridized to Atlas Arrays (Clontech, Palo Alto, Calif.) containing 588 cDNAs. The autoradiograms obtained were then analyzed using the Molecular Dynamics software program. Semiquantitative PCR was carried out to confirm up-regulation of four genes associated with wound repair. ELISA was performed to quantitate the secreted MCP-1.
Results:
In wounded cultures prominent up-regulation (greater than fivefold) was seen for genes encoding DNA synthesis and DNA repair proteins. A greater than threefold increase was seen for genes encoding mitogen-activated protein kinase, CD44, MCP-1 (monocyte chemotactic protein), thymosin beta-10, and HDGF (hepatoma-derived growth factor), among others. Genes encoding tumor suppressors were downregulated three- to five-fold in the wounded compared with the unwounded cultures. Semiquantitative PCR confirmed up-regulation of transcripts for thymosin beta-10, HDGF, CD44, and MCP-1. ELISA showed a 20% increase in secreted MCP-1.
Conclusions:
Gene array analysis revealed a differentiation program that included increased expression of genes involved in wound repair (adhesion molecules, cytokines, signal transducers), along with increased MCP-1 secretion. The RPE may be an early participant in the inflammatory response that occurs with proliferative vitreoretinopathy.