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S.A. K. Harvey, Y. Du, J.L. Funderburgh, E. DeGarmo, N. SundarRaj; Human Corneal Keratocytes: Microarray Analysis of the Phenotypic Shift to Fibroblasts . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1207.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Wounding activates the quiescent dendritic keratocytes of the corneal stroma, which undergo a phenotypic shift to proliferative fibroblasts. Similarly, isolated keratocytes cultured in defined serum–free medium are activated to fibroblasts on exposure to serum. We used this serum–exposure model to compare human keratocyte gene expression with that of matched fibroblasts. Methods: Keratocytes isolated from four human corneas were cultured with or without serum. Total RNA was extracted from these 8 independent samples, and processed using appropriate products (Affymetrix: Santa Clara, CA). First, 35 – 40 ng was used as a source of mRNA for two–cycle cDNA amplification. The resultant cDNA was subjected to in vitro transcription, yielding 75 – 100 µg of biotinylated cRNA. Analysis of cRNA on HG–U133 Plus 2.0 GeneChips characterized 54,675 probe sets, representing 47,000 transcripts and variants. Signal prior to normalization was 304 ± 63 (mean ± SD, n=8) with 33 ± 2 % of probe sets called present. Results: We found 810 panels which were consistently changed in the same direction in all four paired comparisons. Of these the strongest candidates had > 2–fold change for every paired comparison: 120 unique, characterized transcripts were relatively increased in fibroblasts and 45 were relatively increased in keratocytes. Fibroblast increases included transcripts (HGNC designation) associated with cell cycle control or mitosis (ASPM, CCNA2, CCNB1, CCNB2, CDC2, CDC20, CDCA8, CDKN2C, CENPF, also BUB1B, CUL4B, KIF23, MCM5, NEDD9, SKP2, SMC4L1, STK6); altered extracellular matrix synthesis (COL1A1, COL3A1, COL5A1, COL5A2, COL8A1, EFEMP1, LOX, LOXL2, TNC); and altered cytoskeletal function (COTL1, FSCN1, MYH10, PFN1, TAGLN, TPM1, TPM2). Keratocyte increases included presumptive corneal crystallins (ADH1B, ALDH3A1) and metallothioneins (MT1A, MT1F, MT1G, MT1H, MT1X) which can protect against oxidative stress. Conclusions: The subset of strong candidates described above includes known or plausible changes associated with the keratocyte shift to fibroblast; i.e., decreases in crystallins and in an oxidative stress protection system and increases in systems supporting proliferation and remodeling of the cytoskeleton and extracellular matrix. A closer review of other candidates, and comparison of the present study with equivalent published murine data, will further our understanding of corneal responses to wounding.
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