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Related Experiment Videos

ROCK mediates thrombin's endothelial barrier dysfunction.

J M Carbajal1, M L Gratrix, C H Yu

  • 1Department of Physiology, The University of Arizona, and The Benjamin W. Zweifach Microcirculation Laboratories, Department of Veteran Affairs Medical Center, Tucson, Arizona 85723, USA.

American Journal of Physiology. Cell Physiology
|July 18, 2000
PubMed
Summary
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Thrombin increases endothelial permeability via Rho-associated protein kinase (ROCK) dependent stress fibers. However, large hole formation is ROCK-independent, suggesting partial ROCK involvement in overall hyperpermeability.

Area of Science:

  • Cell biology
  • Vascular biology
  • Biochemistry

Background:

  • Endothelial hyperpermeability, a hallmark of inflammation, is regulated by Rho GTPase signaling.
  • Thrombin stimulation of endothelial cells leads to increased F-actin stress fibers and contractile tension.
  • The role of Rho-associated protein kinase (ROCK) in thrombin-induced endothelial barrier dysfunction requires further elucidation.

Purpose of the Study:

  • To investigate the dependency of thrombin-induced endothelial hyperpermeability on ROCK signaling.
  • To determine the specific mechanisms by which ROCK inhibition affects endothelial barrier function.
  • To differentiate the roles of ROCK in stress fiber formation versus paracellular hole formation.

Main Methods:

  • Utilized Y-27632, a specific ROCK inhibitor, in bovine pulmonary artery endothelial cell (EC) monolayers.

Related Experiment Videos

  • Assessed myosin light chain phosphorylation (MLCP) and tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) and paxillin via Western blotting.
  • Analyzed F-actin organization and content using digital imaging and measured monolayer permeability with a size-selective assay.

    • Main Results:

    • Y-27632 treatment enhanced EC monolayer barrier function by reducing small-pore number, decreasing F-actin content, and reorganizing F-actin to cell-cell junctions.
    • ROCK inhibition prevented thrombin-induced MLCP, stress fiber formation, and increased phosphotyrosine levels of paxillin and p125(FAK).
    • While Y-27632 attenuated, it did not abolish thrombin-induced large paracellular hole formation, indicating a partially ROCK-dependent process.

    Conclusions:

    • Thrombin-induced stress fiber formation in endothelial cells is dependent on ROCK signaling.
    • Thrombin-induced formation of large paracellular holes is largely independent of ROCK.
    • Overall thrombin-induced endothelial hyperpermeability is partially regulated by ROCK, highlighting distinct pathways involved in barrier disruption.