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G. A. Secker, Q. P. Schwarz, A. J. Shortt, P. T. Khaw, R. R. Ali, J. T. Daniels; Pax6 Deficient Fibroblasts Display an Exaggerated Wound Healing Response. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3502.
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The PAX6/Pax6 gene is a hierarchical transcription factor regulating a multitude of genes involved in eye development and adult homeostasis. Heterozygous Pax6+/- mice are small eyed and provide an excellent model for aniridia and the progressive nature of associated corneal abnormalities. The underlying process of these abnormalities is poorly understood and is thought to be due to stem cell failure however, it has been recently proposed that it may be due to a deficiency in the stem cell niche and adjacent corneal stroma. Therefore, this study has begun to evaluate the significance of the corneal stroma, by comparing corneal fibroblasts from Pax6+/- and Pax6+/+ mice. Furthermore human corneal fibroblasts were manipulated with siRNA targeting PAX6.
Corneal fibroblasts were isolated using explant culture and maintained in DMEM with 10% serum. Immunohistochemistry was used to locate Pax6 expression in tissue sections and cultured cells. Free floating fibroblast populated collagen gels were cultured in serum containing media and percentage contraction was measured at days 1, 3, 5 and 7 using Image Tool software. Fibroblast proliferation was analysed using Alamar Blue reagent with fluorescence readings being recorded up to 48 hours. Migration in response to serum was determined using a transwell chemotaxis assay. Cells were fixed, stained and counted following a 16 hour incubation with results being expressed as percentage migration.
Fibroblast Pax6 expression was predominantly localised to the limbal stroma, with decreased expression observed in Pax6+/- mice. Importantly, corresponding levels of Pax6 expression was maintained in cultured murine cells. Furthermore, cells from Pax6+/- mice and PAX6 silenced human cells displayed increased collagen gel contraction, increased cell proliferation and decreased cell migration.
Recent studies have indicated the involvement of the corneal stroma as a factor in aniridia disease progression. Our data suggests abnormal PAX6/Pax6 expression by stromal fibroblasts leads to an exaggerated wound healing response, including increased matrix contraction and cell proliferation together with impaired cellular migration. These results suggest a role for corneal fibroblasts in the progression of aniridia and suggest that therapeutic strategies under development may need to target both epithelial and fibroblast cells of the cornea in order to achieve maximal patient benefit.
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