Cell Cycle Control and Replication in Corneal Endothelium

Excessive loss of endothelial cells causes loss of the barrier function of the corneal endothelium, resulting in bullous keratopathy, permanent corneal clouding, and loss of visual acuity. In vivo repair of the endothelium following cell loss occurs by cell enlargement and migration, rather than by cell division. Human corneal endothelial cells (HCEC) do not divide in vivo, because they are inhibited in G1-phase of the cell cycle; however, they retain proliferative capacity. The cell cycle is divided into multiple phases. After mitogenic stimulation, cells enter G1-phase to prepare cells for DNA duplication, which occurs in S-phase. Cells then move into G2-phase to prepare cells for division, which occurs in M-phase. Movement of cells from G1- to S-phase can be prevented by the activity of the cyclin-dependent kinase inhibitors, p27Kip1, p21Cip1, and p16INK4a. These inhibitors prevent activation of the transcription factor, E2F, which is required for S-phase entry. Several factors contribute to inhibition of the proliferation of HCEC in vivo, including formation of strong cell-cell contacts (contact inhibition), lack of autocrine or paracrine growth factor stimulation, and the suppressive effect of transforming growth factor-beta2. This inhibition appears to be mediated, in large part, by p27Kip1. Although HCEC retain the ability to divide, their capacity to proliferate decreases with increasing age. This decrease is characterized by an age-related reduction in the rate of cell cycle entry and in the relative number of dividing cells. Evidence strongly suggests that this age-related decrease is the result of an up-regulation of the expression and activity of p21Cip1 and p16INK4a, but not of p27Kip1. HCEC can be induced to divide by overcoming or bypassing G1-phase inhibition using molecular biological approaches. The most promising approach so far is ectopic expression of the transcription factor, E2F2, which increases endothelial cell proliferation in ex vivo corneas from both young ( 50 years old). Proliferative capacity and the expression of senescence characteristics are also affected by endothelial topography. Cells within the central 6.0 mm diameter of the endothelium in corneas from older donors exhibit the lowest proliferative capacity and contain the highest percentage of senescent cells. The age- and topographically related decrease in proliferative capacity observed in HCEC is not due to the presence of critically short telomeres, but appears to result from sub-lethal oxidative nuclear DNA damage. Research has led to a new hypothesis regarding the molecular basis for the age- and topographically related decrease in proliferative capacity. This hypothesis states that, with increasing age, oxidative stress increases in HCEC due to their high metabolic activity and due to chronic light exposure. This results in a gradual increase in oxidative nuclear DNA damage, which leads to a decreased ability to divide, mediated by the G1-phase inhibitors, p21Cip1, and p16INK4a. This new hypothesis provides the basis for further exploration of the molecular mechanisms under-lying the age- and topographically related decrease in proliferative capacity of HCEC. This exploration could lead to the development of methods to prevent or reverse the effects of oxidative stress on these cells, thereby increasing their ability to divide in order to repair the endothelial monolayer and prevent the devastating effect on vision of the loss of endothelial barrier function.