Abstract
Abstract: :
Purpose: Bovine corneal endothelial cells (BCEC) have been shown previously in our lab to synthesize and secrete endothelin–1 (ET–1), bind ET–1 specifically to receptors of the ETA subclass and subsequently elevate intracellular calcium. This investigation explores ET–1's potential as an autocrine mitogenic factor in corneal endothelial cells and whether its mitogenic activity is mediated through the cyclin–dependent kinase inhibitor, p27. Methods: BCEC were isolated from bovine eyes and grown in DMEM–10% serum. Tritiated thymidine incorporation was determined in quiescent cultures at approximately 60% confluence in 12–well plates. Agonists were added and after a 16–h incubation, the cells were pulsed with 0.5 µCi/ml [methyl–3H]–thymidine for 6 h. Labeled DNA was extracted and solubilized in 1N NaOH and 1% SDS and counted by LSC. Immunofluorescent localization of p27 was performed using an anti–p27 polyclonal antibody in BCEC grown on glass coverslips after fixation in cold methanol. Healing was examined in confluent BCEC grown in 12–well plates following a 1mm wide wound that was scribed using a silicon rubber tipped spatula. Results: ET–1 stimulated a dose–dependent increase in DNA synthesis. Known mitogens, EGF (10 ng/ml) and bFGF (2 ng/ml) induced large increases in DNA synthesis (83% and 151%, respectively). When ET–1 (100nM) and EGF were present together, the stimulation was 18% greater than the sum of their individual responses. P27 demonstrated a punctate nuclear localization that was far more intense in confluent (contact inhibited) cells than in sparse cultures. Treatment of cells with 100 nM ET–1 led to a decrease in the intensity of p27 staining. In addition, ET–1 accelerated the rate of wound closure in BCEC. Conclusions: ET–1 appears to have mitogenic properties in BCEC that also potentiate the effects of EGF. Stimulation of cell proliferation leads to enhanced wound healing that appears to be mediated by suppressing the expression of p27.
Keywords: cornea: endothelium • proliferation • wound healing