A Factor in the Corneal Stroma that Stimulates Keratocyte Proliferation without Loss of Keratocyte Morphology

Abstract

Abstract: : Purpose: To determine the function of a factor isolated from corneal stroma that activates keratocytes to proliferate without loss of morphology Methods: Fresh corneal stromas were homogenized using a Polytron in 5 ml DMEM/F12 per gram of stroma. This homogenate was extracted overnight at 4 °C and insoluble material removed by centrifugation. Collagenase isolated keratocytes from bovine corneas were plated in DMEM/F12 containing attachment factors and then switched to DMEM/F12, DMEM/F12 containing extract or 10% fetal bovine serum on day 1. Keratocytes were plated at a low density (110 cells/mm2) and treated with the extract or fetal bovine serum for 48 hours starting on day 1. The cells were radiolabeled with 3H-thymidine (10 uCi/ml DMEM/F12) for 48 hours. Incorporation was measured by liquid scintillation and DNA content by DNA fluorometry. Results: Keratocytes cultured in the stromal extract and in serum proliferate but only those in extract retained their dendritic morphology. Keratocytes cultured in 20% extract proliferated at the same level as keratocytes cultured in serum for the first 48 hours. In long-term culture, however, the keratocytes in the stromal extract grew at a slower rate and to a lower final density than keratocytes in serum, while keratocytes cultured in DMEM/F12 did not grow. Keratocytes plated out at higher initial densities show a decreased response to the extract compared to serum, as determined by thymidine incorporation. Conclusions: These results suggest that a readily extractable factor that causes keratocytes to proliferate but retain their morphology is present in the corneal stroma. The growth of keratocytes in extract is contact inhibited at a lower cell density than growth in serum. Keratocytes in the corneal stroma are normally contact inhibited and do not proliferate. Once the cornea is wounded, keratocytes close to the wound area undergo apoptosis. This creates a cell void area resulting in the loss of contact inhibition by adjacent keratocytes. They become activated and grow into the void until they are in contact with each other again. We propose that the factor in the extract may be responsible for activating keratocytes following corneal wounding in vivo.

Keywords: cornea: stroma and keratocytes • wound healing • cell death/apoptosis