Recombinant TIMP-1 and -2 enhance the proliferation of rabbit corneal epithelial cells in vitro and the spreading of rabbit corneal epithelium in situ.

PURPOSE: The repair of the corneal epithelium is modulated by matrix metalloproteinases. The present study examined the effects of recombinant (r-) tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and -2) on the proliferation of cultured epithelial cells from rabbit cornea, and on the spreading of a sheet of squamous epithelium of rabbit cornea placed in an organ culture system. METHODS: DNA synthesis of the cells, with or without r-TIMPs, was determined by an immunoassay for BrdU incorporation. Cell proliferation was assayed by measuring MTT mitochondrial activity. Epithelial spreading was evaluated by culturing small corneal blocks for 24 h in the presence or absence of each agent. Cryosections were prepared and the epithelial growth on the cut stromal surface was measured. RESULTS: Each agent, r-TIMP-1 (at 50 and 100 ng/ml) and r-TIMP-2 (at 50 ng/ml) significantly enhanced the DNA synthesis and MTT activity of the corneal epithelial cells in vitro. Relative to the untreated control cells, DNA synthesis was increased 2.4-fold by r-TIMP-1 and 2.3-fold by r-TIMP-2. r-TIMP-1 (at 100 and 200 ng/ml) and r-TIMP-2 (at 10 and 50 ng/ml) each significantly enhanced the spreading of the corneal epithelium. Relative to the untreated control tissue, spreading of the epithelial sheet was increased 1.7-fold by r-TIMP-1 and 1.4-fold by r-TIMP-2. Higher concentrations of r-TIMP-1 and r-TIMP-2 did not further enhance either the DNA synthesis of the cultured cells or the spreading of the epithelium. CONCLUSIONS: Exogenous TIMPs enhanced the spreading of the corneal epithelium and proliferation of cultured corneal epithelial cells. Findings suggest that endogenous TIMPs may influence the healing of corneal epithelium in vivo.