Purpose: Previously reported from this laboratory are two distinct factors responsible for corneal endothelium modulation: basic fibroblast growth factor (bFGF) and the corneal endothelium modulation factor (CEMF) that is released by inflammatory cells. The altered phenotypes mediated by these two distinct factors--marked increase in cell proliferation, cell shape changes, and synthesis of fibrillar collagens--are identical. The current study sought to determine if bFGF is the direct mediator for corneal endothelium modulation and if CEMF plays a role in inducing bFGF production.
Methods: bFGF synthesis mediated by CEMF was analyzed by immunoblot assay; cycloheximide was used to block protein synthesis. bFGF-Specific antisense oligonucleotide primer was used to inhibit CEMF-mediated bFGF synthesis and to block further the autocrine activity of bFGF. Cell proliferation was measured by cell counting. The steady-state levels of RNA were determined by Northern blot analysis. CEMF was further purified to homogeneity by sequential electrophoresis, elution, and renaturation of protein.
Results: The synergistic effect of CEMF and bFGF on corneal endothelial cells was measured by their growth-promoting activity on quiescent corneal endothelial cells. There was a dose-dependent cell proliferation mediated by bFGF at any given CEMF concentration. Thus, bFGF at 10 ng/ml with CEMF at 2.5 micrograms/ml demonstrated saturable synergistic activity on endothelial cell proliferation. When the steady-state levels of collagen RNA were measured under these conditions, the untreated cells showed the doublets of 5.6 and 5.0 kb of alpha 2(I) collagen RNA. The cells treated simultaneously with bFGF and CEMF contained mostly lower transcript, compared to the significant level of upper transcript in control cells. However, there was no significant change in the level of 6.9-kb type IV collagen RNA qualitatively or quantitatively; nonetheless, the level of alpha 2(IV) collagen RNA was lowest in cells treated with bFGF plus CEMF. Neither exogenous bFGF nor CEMF caused induction of bFGF messenger RNA in corneal endothelial cells, whereas simultaneous treatment with bFGF and CEMF selectively enhanced the 4.9-kb transcript. When protein synthesis was inhibited by cycloheximide, bFGF synthesis was blocked in the presence of CEMF, leading to inhibition of corneal endothelium modulation. The effect on endothelial cell growth of bFGF antisense primer was analyzed. Antisense primer blocked by 50% the enhanced growth potential mediated by bFGF induced with CEMF. Finally, CEMF was purified to homogeneity: the purified protein is approximately 17 kD and assumes the modulating activities.
Conclusions: These findings suggest that de novo synthesis of bFGF induced by CEMF is required for corneal endothelium modulation.