Purpose: It is unknown why human corneal endothelium exhibits limited capacity to divide while the endothelia of other species, such as rabbit, divide in vivo at wounding and in culture. A potentially valuable source of information concerning why human endothelium has such a limited proliferative capacity lies in elucidating any differences in the molecular events governing the cell cycle of these two species. A recent study of the relative expression of cell cycle-associated proteins in donor corneas suggests that human corneal endothelial cells in vivo have not exited the cell cycle but are arrested in G1-phase. The purpose of the current study was to identify differences in cell cycle protein expression in human and rabbit endothelium that would explain the difference in their relative proliferative capacities. Specifically, the authors first ascertained the relative proliferative status of rabbit corneal endothelial cells in vivo. The expression and intracellular distribution of G1-phase regulatory proteins was then determined in both species, and the results were compared.
Methods: Corneas from New Zealand white rabbits (weight range, 2 to 3 kg) and from human donors (age range, 6 months to 67 years) were fresh frozen, cryostat sectioned, and prepared for indirect immunofluorescence microscopy using an established protocol. The following monoclonal antibodies were localized in rabbit corneal endothelium only: cyclins D, E, A, and B1; protein kinase p34cdc2; and Ki67, a marker of actively cycling cells. Localization patterns for the following G1-phase regulatory proteins were compared in both human and rabbit corneal endothelia: the tumor suppressors, pRb, p53, and p16INK4, and the transcription factor, E2F. Reverse transcription-polymerase chain reaction studies were conducted to detect mRNA for Ki67 in human and rabbit corneal cells.
Results: Cyclins D, E, and A were localized in the cytoplasm of rabbit corneal endothelium, whereas cyclins B1 and p34cdc2 were detected in the nucleus. No Ki67 protein or mRNA expression was detected in the endothelium of either species. In human and rabbit endothelia, p53 and p16INK4 were localized to the cytoplasm, whereas pRb was detected in the nucleus. E2F exhibited a nuclear and a cytoplasmic localization in each species.
Conclusions: The corneal endothelium of rabbits stained positively for cyclins D, E, and A and did not stain for Ki67, suggesting that, as in humans, rabbit corneal endothelium in vivo is arrested in G1-phase upstream from Ki67 synthesis. Cyclin E was located in the cytoplasm of rabbit cells, whereas it was found in the nucleus in human endothelium. The apparent difference in cellular distribution of cyclin E in these two species may be significant because this cyclin is active during the G1-/S-phase transition. It is possible that in situ human and rabbit corneal endothelial cells are arrested at different points within G1-phase and/or that the difference in relative proliferative capacity exhibited by the corneal endothelium in these two species may be caused by differences in their relative ability to overcome G1-phase arrest.