To investigate the proliferation of umbilical cord blood–derived endothelial progenitor cells (UCB EPCs) and the differentiation efficiency toward corneal endothelial cell (CEC)-like cells induced by rho-associated protein kinase (ROCK) inhibitor Y-27632 and to determine the most effective strategy for repairing corneal endothelium injuries in rabbits.
UCB EPCs were cultured in Endothelial Cell Growth Medium-2 (EGM-2) media or conditioned media (CM) from human CECs, with and without the addition of Y-27632. Bromo-deoxyuridine (BrdU) immunocytochemistry and cell counting kit-8 assays were used to examine the proliferation of the cells. Real-time polymerase chain reaction, western blot, and immunocytochemistry were used to detect the CEC markers. Nd:YAG laser was used to establish an appropriate endothelium injury model based on rabbit corneas. The following intracameral injections were then performed to repair the model: 100 μL Opti-MEM I reduced serum medium (model group), 2 × 105 UCB EPCs diluted in 100 μL Opti-MEM I reduced serum medium (EPC group), 100 μM Y-27632 diluted in 100 μL Opti-MEM I reduced serum medium (Y-27632 group), and 2 × 105 UCB EPCs supplemented with 100 μM Y-27632 (final volume 100 μL, EPC/Y-27632 group). The follow-up tests focused on corneal transparency, central corneal thickness, intraocular pressure, and in vivo confocal microscopy, which were performed to evaluate the healing of the wounds.
Culturing UCB EPCs in CM supplemented with 10 μM Y-27632 resulted in higher proliferation rates compared with EGM-2 media and CM. There were significantly improved protein levels of Zona Occludens 1, N-cadherin, Na+-K+-ATPase α1, Na+-K+-ATPase β1, and Pax6 and improved mRNA levels of collagen type IV and VIII and AQP1. The combined intracameral injection of Y-27632 and UCB EPCs accelerated the recovery of corneal transparency, regression of corneal edema, and healing of the corneal endothelium compared with the injections of Y-27632 and UCB EPCs on their own.
Y-27632 not only promotes the proliferation of UCB EPCs but also contributes to differentiation of UCB EPCs toward CECs in the presence of CM. The intracameral injection of Y-27632 itself promotes the healing of corneal endothelium wounds. On this basis, supplementing UCB EPCs with Y-27632 accelerates the healing of corneal endothelium wounds.