Y-27632 Promotes the Repair Effect of Umbilical Cord Blood-Derived Endothelial Progenitor Cells on Corneal Endothelial Wound Healing

Purpose: 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. Methods: 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 mu L Opti-MEM I reduced serum medium (model group), 2 x 10(5) UCB EPCs diluted in 100 mu L Opti-MEM I reduced serum medium (EPC group), 100 mu M Y-27632 diluted in 100 mu L Opti-MEM I reduced serum medium (Y-27632 group), and 2 x 10(5) UCB EPCs supplemented with 100 mu M Y-27632 (final volume 100 mu L, 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. Results: Culturing UCB EPCs in CM supplemented with 10 mu M 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 alpha 1, Na+-K+-ATPase beta 1, 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. Conclusions: 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.

Automated summary

Y-27632 promotes the proliferation of UCB EPCs and their differentiation towards CECs, while intracameral injection of Y-27632 and UCB EPCs accelerates the healing of corneal endothelium injuries in rabbits.