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1 From the Institute for Wound Research and Department of Ophthalmology, University of Florida, Gainesville, Florida; 2 Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida; and 3 University of Florida Genetics Institute, Gainesville, Florida.
PURPOSE. To characterize changes over time in the genomic expression profile of rat corneas after excimer laser photorefractive keratectomy (PRK), in an effort to better understand the cellular response to injury and the dynamic changes that occur in gene expression patterns as a wound heals.
METHODS. The corneal gene expression profile of 1176 genes at 3 and 7 days after PRK was determined and compared with untreated corneal gene expression patterns by interrogating commercially available cDNA arrays with labeled target cDNA prepared from pooled total RNA harvested from the respective treatment group of adult male rats. The gene expression patterns were inferred based on the hybridization intensities of the probes on the cDNA arrays. The hybridization signals were globally normalized and filtered. The data were analyzed by using hierarchical and k-means clustering algorithms before and after normalization of variances.
RESULTS. Of the 1176 cDNA elements on the array, 588 consistently produced similar results in replicate experiments and comprised the data set analyzed in this work. In total, 73 genes were identified, with expression levels that differed by at least threefold at either 3 or 7 days after PRK. At 3 days after PRK, 70 genes were identified with expression levels that differed by more than threefold, compared with the expression level in untreated animals. The expression of 42 genes increased by threefold or more, whereas expression of 28 genes decreased by threefold or more. By day 7 after PRK, the number of genes displaying more than a threefold difference in expression pattern was reduced to 27 genes, 20 of which showed elevated levels, whereas 7 exhibited decreased levels. Hierarchical clustering of the 588 studied genes produced 10 clusters with correlation coefficients of 0.9 or greater. To determine whether any of the clusters were overrepresented by genes with related functions, the cumulative hypergeometric probability was calculated by obtaining the observed number of functionally related genes within each of the 10 clusters. Seven of the clusters were statistically overrepresented by one or more categories of functionally related genes, such as cell cycle regulators, transcription factors, and metabolic pathway genes. Clustering analysis of 56 genes generally considered to influence corneal wound healing produced 10 gene clusters with correlation coefficients of at least 0.9. Expression of 23 of these 56 genes increased at day 3, then decreased at day 7 to levels similar to those on day 0. These included several growth factors (VEGF, FGF, IGF-I), proteases (PAI-1, PAI-2A) and protease inhibitors (TIMP-2 and TIMP-3). Expression of nine genes increased on both days 3 and 7 compared with expression on day 0 (e.g., TGFB1, TGFBIIR, M6P/IGFR-2), and no genes decreased on both days 3 and 7, compared with day 0.
CONCLUSIONS. Microarray analysis of 1176 identified 588 genes with reproducible patterns of expression in rat corneas on days 3 and 7 after PRK and 73 genes with a threefold change in expression compared with untreated corneas. Hierarchical clustering of these 588 genes identified 10 clusters of genes with very similar patterns of expression. Clustering of genes with similar patterns of expression implies a common regulatory pathway for the genes within a cluster, and identifies potential new targets for regulating corneal wound healing.
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