Uncoupling keratocyte loss of corneal crystallin from markers of fibrotic repair

PURPOSE: Corneal crystallins are lost from resident cells of the corneal stroma during wound repair, and this is associated with a loss of cell transparency. The goal of this study was to identify factors inducing loss of the corneal crystallin transketolase (TKT). METHODS: A cell culture model of freshly isolated rabbit corneal keratocytes was used. Fibrotic markers included cell proliferation, adoption of a "fibroblastic" spindle-shaped morphology associated with cytoskeletal rearrangement, loss of TKT, and expression of alpha-smooth muscle actin (alpha-sm actin), a marker for the myofibroblast. RESULTS: When freshly isolated keratocytes were cultured in the continuous presence of 10% calf serum, the high level of intracellular TKT protein was reduced dramatically within 24 to 48 hours. In contrast, TKT protein was retained in cells maintained in the absence of serum. When cells were prevented from proliferating by exposure to serum for <24 hours or by continuously exposing to serum at a contact-inhibiting plating density, TKT loss was inhibited. TKT loss was induced by treatment of serum-free cultures with the serum cytokines platelet-derived growth factor or basic fibroblast growth factor, both of which also stimulated keratocyte proliferation, although not other changes associated with fibrosis. However, TKT loss was not induced by treatment of serum-free cultures with a third serum cytokine, transforming growth factor- (TGF)-beta, even though TGF-beta stimulated cell proliferation at low doses and induced the fibroblastic spindle-shape and express alpha-sm actin at high doses. CONCLUSIONS: TKT loss in corneal keratocytes can be induced by PDGF or bFGF and this loss can be uncoupled from other fibrotic markers. Targeting these cytokines or the signaling pathways that they activate could enable retention of corneal crystallin in stromal cells during repair, a more regenerative outcome. The result would be enhanced clarity of the cornea.