title: A human cellular system for analyzing signaling during corneal endothelial barrier dysfunction.
abstract: Correct corneal endothelial barrier function is essential for maintaining corneal transparency. However, research on cell signaling pathways mediating corneal endothelial barrier dysfunction has progressed more slowly than that involving other cellular barriers because of the lack of human corneal endothelial cell models. Here we have optimized the culture of the human corneal endothelial cell (HCEC) line B4G12 as a model for studying paracellular permeability. We show that B4G12-HCECs form confluent monolayers with stable cell-cell junctions when cultured on plastic, but not glass, surfaces precoated with various extracellular matrix components. Cell morphometry and measuring intercellular spaces and transendothelial electric resistance indicate that B4G12-HCECs form optimal monolayers on collagen and fibronectin. Based on the use of specific inhibitors, it has been proposed that the Rho-regulated kinases, ROCK-I and ROCK-II, mediate actomyosin-induced contraction in corneal endothelial cell barriers. ROCKs are effectors of RhoA, RhoB and RhoC. We show that the GTPase RhoA and its effector ROCK-II are predominantly expressed in B4G12-HCECs and primary human corneal endothelial cells. The activation of Rho GTPases during acute barrier disruption has not been investigated in corneal endothelial cells. RhoA, but not other related GTPases that are highly expressed in B4G12-HCECs, such as Rac1 and Cdc42, is transiently activated during barrier disruption in response to the inflammatory mediator thrombin. Pharmacological inhibition of RhoA and ROCK reduces B4G12-HCEC acute contraction. We propose that exploiting B4G12-HCECs is a useful experimental strategy for gaining further insight into the signaling pathways involved in human corneal endothelial barrier function.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain; Department of Ophthalmology, Hospital Universitario Rey Juan Carlos, Mostoles, Spain; Department of Ophthalmology, Fundación Jiménez Díaz, Madrid, Spain; Instituto de Investigación Sanitaria, Fundación Jiménez Díaz, 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: María Cristina
authors.initials: MC
authors.lastname: Ortega
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Benito-Martínez
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Barroso
authors.affiliation: Department of Ophthalmology, Fundación Jiménez Díaz, Madrid, Spain; Instituto 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. Electronic address: jmillan@cbm.csic.es.
authors.forename: Jaime
authors.initials: J
authors.lastname: Millán
mesh_terms: D002199:Capillary Permeability / Q000502:physiology*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism*; D015398:Signal Transduction
keywords: Actomyosin; Barrier function; Corneal endothelium; Rho GTPase; Rho kinase; RhoA; Thrombin