Abstract
Purpose :
Promoting cell migration is pivotal to corneal endothelial wound healing and can facilitate regeneration of functional corneal endothelium after descematorhexis. The aim of this study is to provide functional and mechanistic insights of targeting non-muscle myosin II (NMII) to promote corneal endothelial wound healing in vitro and in vivo.
Methods :
Bovine corneal endothelial cells (BCECs) exposed to Y27632, a Rho kinase inhibitor, and blebbistatin, a selective NMII inhibitor, were analyzed for the migration speed, directionality, and in vitro wound healing by live-cell imaging. Changes in lamellipodial dynamics and actin retrograde flow rates were revealed by kymography analysis. The differential effects of Y27632 and blebbistatin on cell junctional integrity were analyzed by Western blotting analysis, immunofluorescence staining and paracellular permeability assay. The overall effects of Y27632 and blebbistatin were verified in rabbit corneal endothelial wounding model.
Results :
Y27632 and blebbistatin treatments suppressed NMII activity, leading to reduced cell contraction during migration. Both agents promoted directional migration of BCECs, reflected by increased directionality ratio and mean square displacements, and accelerated in vitro wound healing. The lamellipodial protrusion persistence was increased, and actin retrograde flow was decreased after NMII inhibition. Counteracting lamellipodial protrusion by ARP2/3 inhibitor abolished this migration-promoting effect. Although both Y27632 and blebbistatin accelerated wound healing, cell junctional integrity and barrier function were better preserved after blebbistatin treatment, leading to more rapid corneal deturgescence in rabbit corneal endothelial wounding model.
Conclusions :
NMII inhibition promotes CEC migration and wound healing in vitro and in vivo, which may facilitate restoring functional corneal endothelium by descemetorhexis approach.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.