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F. Wu, L. Roberts, K. Mussellman, B. Kane, N. Vij, J.R. Hassell, S. Chakravarti; Molecular Characterization of Murine Corneal Keratocytes, Fibroblasts and Myofibroblasts by Microarray Gene Expression Profiling . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4222.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The corneal keratocytes synthesize and maintain the corneal stromal matrix. Previous studies showed that cultured in the presence of serum, these cells assume a fibroblast phenotype, and exposed to TGF-ß the cell become myofibroblasts. To identify biological pathways, molecular events underlying these trans-differentiations and multiple markers for each cell type, we elucidated their gene expression patterns by microarray profiling. Methods: Keratocytes were isolated from collagenase-treated adult mouse corneas and plated in DMEM with1% platelet poor horse serum. After 36 h, the cells were switched to a) serum free-DMEM (SF), b) DMEM + 10% FCS (F) and c) DMEM + 10% FCS + 10 mg/ml TGF-ß (F+T) for 48 hours to establish keratocyte, fibroblast and myofibroblast morphologies, respectively. Total RNA was isolated from a) to c) and cornea, biotinylated, hybridized to MG-U74Av2 (Affymetrix) and analyzed as described previously [Lawrance, Hum Mol Gen 2001,10:445; Li, PNAS 2001,98:31]. Results: The SF, F, and F+T morphologies were as described previously for bovine and rabbit keratocytes, fibroblasts and myofibroblasts. Compared to the cornea, 270 genes were differentially expressed (>5 fold) in the cell cultures. Specific known corneal genes, namely aldehyde dehydrogenase, collagen type VI, and type VII, were up regulated in SF. Others up regulated in the cornea and SF included Bmp4, MAPK1, heat shock genes, dynein, HLA-2D. G1 to S phase transition 1 gene, cathepsins, were specifically elevated in SF. While phospholipase A2, IL-2 inducible T-cell kinase, MMP 14, procollagen types I, VIII and XI, laminin a2 and glypican were down regulated in the cornea and keratocyte, but elevated in the fibroblast and myofibroblast phenotypes. Genes down regulated in all cell types included discoidin domain receptors, cadherins, junction cell adhesion molecules, cell cycle regulators septin, spindlin, centrin, makorin. Collagen type IV, entactin, laminin g1, MMP3 and collagen XV were elevated primarily in SF and F. F+T showed up regulation of procollagen type XI, collagen type V, integrin b5, thrombospondin, and TNF receptor. Conclusion: The SF profile was more closely related to F than F+T. Growth arrest genes normally elevated in the cornea were down regulated in culture. Wound healing genes were up regulated in F+T. Genes differentially expressed in each cell type will be useful in developing biomarkers.
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