Y-27632, a ROCK Inhibitor, Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing

Abstract

Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.

The core planar cell polarity gene,Vangl2, directs adult corneal epithelial cell alignment and migration

“…Consistent with previous studies [25–27], it was found that inhibition of ROCK activity using Y-27632 increased the rate of healing of mouse corneal epithelial cells, whereas inhibition of JNK using SP600125 (as described in the Materials and methods section) decreased the rate of wound healing (mean ± s.e.m. ; SP600125 7.99 µm h −1  ± 0.95; DMSO 14.13 µm h −1  ± 1.14; t -test: p  = 0.001; n  = 7; 8: Y-27632 26.06 ± 1.53; control 16.50 ± 2.02; t -test: p  = 0.001; n  = 11; 9; figure 3 c,d ).…”

“…We also pharmacologically inhibited two downstream mediators of PCP signalling, ROCK and JNK, and found that neither prevented wound healing. Consistent with previous data [27], ROCK inhibition increased the rate of wound healing, associated with disruption of the cytoskeleton leading to apparent stretching of cells behind the wound edge. Together with the observation that the actin cytoskeleton was not grossly defective in Vangl2-deleted corneal epithelial cells, these data suggest that reorganization of the actin cytoskeleton (the primary role of ROCK in this system) is not the main mechanism through which PCP acts during the wound-healing migration response.…”

“…; SP600125 7.99 µm h −1  ± 0.95; DMSO 14.13 µm h −1  ± 1.14; t -test: p  = 0.001; n  = 7; 8: Y-27632 26.06 ± 1.53; control 16.50 ± 2.02; t -test: p  = 0.001; n  = 11; 9; figure 3 c,d ). Previous data have suggested that increased wound-healing rate of epithelia upon inhibition of ROCK activity is substantially owing to cytoskeletal disruption causing cells to spread into the wound [26,27]. Labelling the actin cytoskeleton of corneal epithelial cells with Alexa488-conjugated phalloidin after Vangl2 -deletion, inhibition of JNK and inhibition of ROCK activity was consistent with this possibility (figure 3 e–h ).…”

CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability

“…To investigate the transcriptional profile of CD109 + and CD200 + cells, expression of putative LSC markers ΔNp63, ABCB5, C/EBPδ, BMI1, AXIN2, FZD7, CHD3, WNT7A, CK14, and CK15 [12,16,[39][40][41][42][43], corneal epithelial differentiation marker CK3 [44] and marker of proliferative cells Ki67 [45] was assessed by qRT-PCR.…”

New Human Organotypic Corneal Tissue Model for Ophthalmic Drug Delivery Studies

“…34 Recent success in the isolation and expansion of primary human corneal epithelial cells has allowed for a reliable and reproducible source of primary human corneal epithelial cells for the development of novel organotypic corneal tissue models. 35,36 Primary cultured human corneal epithelial cells grown on permeable supports can address questions concerning drug transport mechanisms by avoiding species extrapolation and the influence of modified gene expression due to cell immortalization. 27,37 These model systems can be valuable for corneal penetration studies since they allow high sample throughput, access to both the apical and basolateral sides, and A to B and also B to A transport studies.…”