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Thrombin-induced endothelial barrier disruption in intact microvessels: role of RhoA/Rho kinase-myosin phosphatase

G P van Nieuw Amerongen1, R J P Musters, E C Eringa

  • 1Laboratory for Physiology, Institute for Cardiovascular Research, Vrije Universiteit University Medical Center, Amsterdam, The Netherlands. nieuwamerongen@vumc.nl

American Journal of Physiology. Cell Physiology
|March 21, 2008
PubMed

Insights

Thrombin increases endothelial permeability via Rho kinase-mediated myosin light chain-2 (MLC2) phosphorylation, disrupting endothelial barrier integrity in rat kidney arterioles. This mechanism underlies prolonged, but not transient, endothelial barrier dysfunction.

Area of Science:

  • Vascular Biology
  • Cellular Physiology
  • Microcirculation Research

Background:

  • Endothelial hyperpermeability is critical for regulating blood flow and tissue fluid balance.
  • Myosin light chain-2 (MLC2) phosphorylation drives endothelial cell contraction and barrier function.
  • Thrombin is a known inducer of endothelial hyperpermeability, primarily through Rho kinase (ROCK)-mediated pathways.

Purpose of the Study:

  • To investigate the in situ effects of thrombin on endothelial cell morphology and barrier integrity in intact microvessels.
  • To elucidate the role of Rho kinase (ROCK) and MLC2 phosphorylation in thrombin-induced endothelial barrier dysfunction.
  • To differentiate the mechanisms of prolonged versus transient endothelial barrier dysfunction.

Main Methods:

  • Digital three-dimensional imaging microscopy of pressurized rat kidney arterioles.
  • Visualization of cytoskeletal dynamics (F-actin), paracellular flux, and MLC2 phosphorylation in endothelial cells (ECs).
  • Measurement of Rho kinase (ROCK) activity using MYPT1-phosphorylation as a surrogate marker.

Main Results:

  • Thrombin induced F-actin formation and EC rounding in situ, without apparent gaps, but decreased barrier integrity.
  • Increased MLC2 phosphorylation correlated with thrombin-induced barrier dysfunction, mediated by Rho kinase (ROCK).
  • Histamine-induced hyperpermeability showed no detectable Rho kinase (ROCK) activity on F-actin filaments.

Conclusions:

  • Thrombin directly alters endothelial cell morphology and barrier integrity in intact microvessels.
  • Enhanced Rho kinase (ROCK) activity is a key contributor to prolonged endothelial barrier dysfunction induced by thrombin.
  • The findings distinguish mechanisms underlying transient versus sustained endothelial barrier compromise.

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