Abstract
Purpose: :
To determine the efficacy of ROCK inhibitors (Y-27632 or H-1152) and statin drugs in stimulating corneal endothelial cell proliferation and migration, and to stratify which agents are most effective as potential pharmacologic treatments for corneal endothelial disease, to be included in future animal and human experiments.
Methods: :
Corneal endothelial cells (CECs) were isolated from freshly enucleated adult porcine eyes using trypsin/EDTA digestion and were further characterized with known markers. The CECs (derived from passage 2 to 4) were grown to confluence on Collagen IV-coated culture dishes with complete growth media and corneal endothelial cell growth supplements. The CECs treated with different pharmacological agents were examined for changes in the distribution profile of ZO-1, paxillin and actin cytoskeleton by immunofluorescence staining and images were captured using confocal microscopy. Proliferation of CECs treated with ROCK inhibitors (Y-27632 and H-1152) and statins (lovastatin) was quantified using Click-iT EdU HCS Assay (Invitrogen), and migration of treated CECs was quantified using Cell Migration Assay system (Platypus Technologies Oris).
Results: :
Porcine corneal endothelial cells (CECs) were successfully cultured and passaged from the endothelial surface area of the cornea utilizing the trypsin/EDTA digestion protocol. Monolayers of confluent CECs treated with 10 µM Y-27632 showed a complete collapse of actin cytoskeletal architecture with markedly reduced actin stress fibers, tight junctions, and cell adhesions within two hours of drug administration, as assessed by phalloidin, ZO-1, and paxillin staining, respectively. Cell morphology was largely restored within 48 hours of drug treatment with Y-27632. Proliferation of CECs was enhanced by administration of 10 µM Y-27632, 2 µM H-1152, and 10 µM lovastatin compared to control. H-1152 demonstrated the greatest stimulatory effect on proliferation of CECs with a 1.4-fold increase in proliferation compared to control, followed by lovastatin and Y-27632. Migration of CECs was enhanced by administration of 10 µM Y-27632, 2 µM H-1152, and 10 µM lovastatin compared to control. Similarly, H-1152 demonstrated the most profound stimulatory effect on CEC migration over 74 hours, followed by Y-27632 and lovastatin.
Conclusions: :
This study demonstrated the enhancing effects of lovastatin and ROCK inhibitors (Y-27632 and H-1152) on CEC migration and proliferation in vitro in association with changes induced in cell morphology, cell adhesions and cell-cell junctions. These ongoing studies demonstrate that H-1152, of those agents tested, is most effective in achieving increased proliferation and migration of CECs.
Keywords: cornea: endothelium • proliferation • wound healing