Proliferative capacity of corneal endothelial cells

Abstract

The corneal endothelial monolayer helps maintain corneal transparency through its barrier and ionic "pump" functions. This transparency function can become compromised, resulting in a critical loss in endothelial cell density (ECD), corneal edema, bullous keratopathy, and loss of visual acuity. Although penetrating keratoplasty and various forms of endothelial keratoplasty are capable of restoring corneal clarity, they can also have complications requiring re-grafting or other treatments. With the increasing worldwide shortage of donor corneas to be used for keratoplasty, there is a greater need to find new therapies to restore corneal clarity that is lost due to endothelial dysfunction. As a result, researchers have been exploring alternative approaches that could result in the in vivo induction of transient corneal endothelial cell division or the in vitro expansion of healthy endothelial cells for corneal bioengineering as treatments to increase ECD and restore visual acuity. This review presents current information regarding the ability of human corneal endothelial cells (HCEC) to divide as a basis for the development of new therapies. Information will be presented on the positive and negative regulation of the cell cycle as background for the studies to be discussed. Results of studies exploring the proliferative capacity of HCEC will be presented and specific conditions that affect the ability of HCEC to divide will be discussed. Methods that have been tested to induce transient proliferation of HCEC will also be presented. This review will discuss the effect of donor age and endothelial topography on relative proliferative capacity of HCEC, as well as explore the role of nuclear oxidative DNA damage in decreasing the relative proliferative capacity of HCEC. Finally, potential new research directions will be discussed that could take advantage of and/or improve the proliferative capacity of these physiologically important cells in order to develop new treatments to restore corneal clarity.

Figure 1

Diagrams illustrating the positive (A) and negative (B) regulation of the cell cycle. Springer-Verlag is the original copyright holder.

Figure 2

Evidence that HCEC in the wound area of ex vivo corneas (A) and in culture (B) can actively proliferate. Endothelium in the donor cornea in (A) was scrape-wounded, incubated in culture medium, immunostained for Ki67 (green) and counterstained with DAPI (red) to detect nuclei. Nuclei within the wound area are positively stained for Ki67, whereas nuclei in cells in the unwounded part of the endothelial monolayer are not stained for Ki67. HCEC cultured from a 67-year old donor show positive Ki67 staining. Orig. mag.= 16X (A) and 20X (B). Lippincott Williams & Wilkins is the original copyright holder of the image in (B).

Figure 3

Growth curves demonstrating age-related differences in relative proliferative capacity in HCEC in an ex vivo cornea scrape wound (A) and in culture (B). Graph in (A) presents the average percent of actively cycling cells in the wound area from at least 3 corneas per age-group. Graph in (B) presents direct cell counts at various times after initiation of the culture. Adapted from Joyce. 2005. Exp. Eye Res. 81: 629-638. Springer-Verlag is the original copyright holder of this image.

Figure 4

Effect of mild oxidative stress on the proliferative response of HCEC. Subconfluent HCECs cultured from a 26-year old donor were incubated in the presence of 0, 25, 50, or 100 mM H₂O₂ as the oxidative stressor. Cell numbers were determined using a WST-8 spectrophotometric assay. Results are presented as absorbance at 450 nm. The Association for Research in Vision and Ophthalmology is the original copyright holder.