title: Activation of Rac1 and RhoA Preserve Corneal Endothelial Barrier Function.
abstract: OBJECTIVE
  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.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
authors.forename: María C
authors.initials: MC
authors.lastname: Ortega
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain 2Department of Ophthalmology, Fundación Jiménez Díaz, Madrid, Spain 3Instituto de Investigación Sanitaria, Fundación Jiménez Díaz, Madrid, Spain 4Department of Ophthalmology, Hospital Universitario Rey Juan Carlos, Móstoles, Madrid, Spain.
authors.forename: Diana
authors.initials: D
authors.lastname: Santander-García
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Marcos-Ramiro
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Barroso
authors.affiliation: King's College London, Randall Division of Cell and Molecular Biophysics, New Hunt's House, Guy's Campus, London, United Kingdom.
authors.forename: Susan
authors.initials: S
authors.lastname: Cox
authors.affiliation: Department of Ophthalmology, Fundación Jiménez Díaz, Madrid, Spain 3Instituto de Investigación Sanitaria, Fundación Jiménez Díaz, Madrid, Spain.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Jiménez-Alfaro
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
authors.forename: Jaime
authors.initials: J
authors.lastname: Millán
mesh_terms: D000818:Animals; D002199:Capillary Permeability / Q000502:physiology*; D002460:Cell Line; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism*; D005260:Female; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D015398:Signal Transduction; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: C107953:RAC1 protein, human; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein