Activation of Rac1 and RhoA Preserve Corneal Endothelial Barrier Function

Invest Ophthalmol Vis Sci. 2016 Nov 1;57(14):6210-6222. doi: 10.1167/iovs.16-20031.

Abstract

Purpose: The corneal endothelium is responsible for the correct hydration of the corneal stroma. Corneal endothelial cells have a low proliferative capacity, so preserving their barrier function under suboptimal conditions that cause osmotic imbalance, such as those arising from corneal pathologies, age, cryopreservation, and transplantation, is essential for maintaining corneal transparency. We have investigated the signaling induced by hyperosmotic shock that reversibly disrupts corneal endothelial barriers in human endothelial cells and in murine corneas.

Methods: Endothelial barrier properties were analyzed in vitro by electric cell substrate impedance sensing (ECIS) and confocal microscopy of the human endothelial cell line B4G12-HCEC, and, ex vivo, by confocal microscopy and stimulated emission-depletion (STED) super-resolution microscopy of murine corneas. Cell signaling in response to hyperosmotic stress, induced with an excess of sodium chloride, was investigated in B4G12-HCECs. Rho GTPase activity was detected by pulldown assays with recombinant GST proteins fused to the Rho binding domains of Rho effectors.

Results: Hyperosmotic stress increased actin polymerization and activated the Rho GTPases Rac1 and RhoA, but not Cdc42. Rac1- and RhoA-mediated pathway inhibition had a minor effect on barrier disruption but partially delayed barrier reformation after stress withdrawal. In contrast, Rac1 and RhoA activation enhanced constitutive endothelial barrier function and accelerated barrier repair.

Conclusions: Our results indicate that Rac1 and RhoA activation do not mediate stress-induced cell contraction but are endothelial responses that act to restore and maintain barrier homeostasis. Therefore, pharmacological activation of these two GTPases could be a therapeutic strategy for preserving corneal endothelial barrier function.

MeSH terms

  • Animals
  • Capillary Permeability / physiology*
  • Cell Line
  • Endothelium, Corneal / cytology
  • Endothelium, Corneal / metabolism*
  • Female
  • Humans
  • Mice
  • Microscopy, Confocal
  • Signal Transduction
  • rac1 GTP-Binding Protein / metabolism*
  • rhoA GTP-Binding Protein / metabolism*

Substances

  • RAC1 protein, human
  • rac1 GTP-Binding Protein
  • rhoA GTP-Binding Protein