Keratocytes produce thrombospondin .1. Evidence for cell phenotype-associated synthesis

Immunochemical techniques were used to determine whether cells of the avascular corneal stroma (keratocytes) have the ability to synthesize thrombospondin 1 (TSP1), a glycoprotein originally described in platelets and more recently implicated in regulating cell behavior (e.g., migration) during wound repair in vascular tissue. Immunoprecipitation experiments with metabolically labeled cells showed that bovine keratocytes in preconfluent cultures produced TSP1, but de novo TSP1 production could not be detected in confluent keratocyte cultures. Immunofluorescence studies of the preconfluent cells revealed that the keratocyte TSP1 was distributed in perinuclear grannies and peripheral foci. TSP1 expression also was observed in keratocytes cultured in a collagen matrix model of stromal wound healing and, in this model, immunogold labeling revealed TSP1 loci on keratocyte surfaces adjacent to collagen fibers in the matrices. TSP1 expression was not observed in the syncytial keratocytes of normal bovine eel-nea, The results indicate that keratocytes have the ability to synthesize TSP1, and do so in vitro under conditions which simulate corneal stroma repair, but suggest that keratocytes in a syncytial arrangement (as in the normal cornea) do not make TSP1. TSP1 may play a role in corneal pathologies which induce keratocytes to change from a syncytial to a wound repair phenotype, such as mechanical damage to the stroma, Local production of TSP1 might provide an alternative source to platelet-derived TSP1 during nonvascularized stromal tissue repair. (C) 1996 Academic Press, Inc.