The 55 kDa keratin K12 and the 59 kDa keratin K4 were used as biochemical markers of differentiated corneal and conjunctival epithelium, respectively, to follow the temporal and spatial appearance of these cell types during embryonic development of the mouse eye. K12 was first detected in corneal epithelial cells of day 15 mouse embryos in a small subpopulation of superficial cells. At later developmental stages only suprabasal corneal epithelium expressed K12, however, in post-natal and adult cornea all cell layers were K12-positive. K4 was first observed, in 14 and 15 day embryos, in a subpopulation of epithelial cells which had invaginated from surface ectoderm to form the lid buds. From embryonic day 16 on K4 was detected in all areas of developing conjunctival epithelium. Some superficial corneal epithelial cells also expressed K4 during embryonic development, but by immunofluorescence microscopic criteria, this keratin was localized exclusively to the conjunctiva in post-natal and adult eye. Expression of the 50 kDa 'basal-type' keratin K14 was also examined in this study. Similarly to K4, K14 was first noted in epithelium comprising the lid bud at embryonic day 14. Between 14 and 17 days of development some epithelial cells in the putative fornix of the conjunctiva did not express K14. Although corneal epithelial cells expressed K14 during development, in adult cornea only certain basal cells did so. These results suggest that the invagination of surface ectoderm to form the presumptive eyelid may be coupled to the initiation of differentiation of ocular surface epithelium.

TGF beta, but not TGF alpha, may play an important role in corneal epithelial cell wound healing by accelerating the reformation of the adhesion complex and subsequent epithelial cell-extracellular matrix adhesion.

Laminin 5 functions to promote cell-matrix adhesion and therefore is hypothesized to abrogate apoptosis initiated through the loss of epithelial cell contact with extracellular matrix. Laminin 5 levels are decreased in epithelial cells cultured in a hypoxic environment. Exposure of epithelial cells to hypoxia may induce apoptotic pathways transmitted through changes in mitochondrial membrane potential. Using an apoptosis assay based on mitochondrial membrane integrity, the effect of hypoxia (2% oxygen) on human corneal epithelial cell viability was determined. Both a virally transformed corneal epithelial cell line and third passage corneal epithelial cells were resistant to hypoxia-mediated apoptosis for up to 5 days in culture. However, at 7 days in culture, a statistically significant increase in apoptosis was noted in hypoxic corneal epithelial cells compared to normoxic (20% oxygen) controls. Increased apoptosis in hypoxic epithelium at 7 days in culture correlated with decreased deposition of laminin 5 into the extracellular matrix, as determined by western blot analysis and immunofluorescence microscopy. Additionally, the extracellular processing of the α3 and γ2 chains of laminin 5 was negatively impacted by corneal epithelial cell exposure to hypoxia for 7 days. Treatment of human corneal epithelial cells cultured in 20% oxygen with function-inhibiting antibodies to laminin 5 for 2 or 3 days resulted in a statistically significant decrease in proliferation, and concomitant increase in apoptosis, compared with untreated normoxic controls. Based on these results, it appears that mechanisms of hypoxia-mediated apoptosis in human corneal epithelial cells may be initiated by the loss of processed laminin 5 in the extracellular matrix or by the loss of laminin 5-epithelial cell communication and transmitted through mitochondria.