Abstract
We have demonstrated previously that the Rho family GTPase Rac-1 is required for maintenance of endothelial barrier functions in mouse microvascular myocardial endothelial (MyEnd) cells in vitro as well as in rat mesenteric microvessels in vivo. In this study, we tested the hypothesis that specific activation of Rac-1 would stabilize microvascular endothelial barrier functions. For this purpose we used Escherichia coli Cytotoxic necrotizing factor (CNF-1) under conditions (300 ng/ml, 120 min) where it strongly activated Rac-1 and Cdc42 but not Rho A in MyEnd cells. Under these conditions, CNF-1 induced translocation of the actin-binding proteins cortactin and vasodilator-stimulated phosphoprotein (VASP) to cell junctions, increased the junction-associated actin filament belt, and reduced monolayer permeability. We also tested the effect of CNF-1 on endothelial barrier properties in vivo using single-perfused mesenteric microvessels. In contrast to cultured microvascular monolayers, CNF-1 did not reduce baseline barrier functions assayed as hydraulic conductivity (Lp). However, following 120 min pretreatment, CNF-1 significantly attenuated the peak Lp increase in response to platelet-activating factor (PAF, 10 nM) to 12.6±4×10−7 cm/(s cmH2O) compared to 46.2±10×10−7 cm/(s cmH2O) in experiments using PAF alone. These experiments indicate that activation of Rac-1 and Cdc42 stabilizes microvascular endothelial barrier functions in vitro and in vivo, likely by increasing the junction-associated actin cytoskeleton.
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Acknowledgements
We are grateful to Gabriele Königer and Joyce Lenz for skilful technical assistance. We thank U. Walter (Institute for Clinical Biochemistry and Pathobiochemistry, University of Würzburg) for supplying the VASP antiserum and G. Schmidt (Department of Pharmacology and Toxicology, University of Freiburg) for generously supplying CNF-1 toxin. These studies were supported in part by grants from the National Heart, Lung, and Blood Institute (HL44485 and HL28607), a grant from the Deutsche Forschungsgemeinschaft (SFB 487, TP B5) and the IZKF Würzburg (TP E23).
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Waschke, J., Burger, S., Curry, FR.E. et al. Activation of Rac-1 and Cdc42 stabilizes the microvascular endothelial barrier. Histochem Cell Biol 125, 397–406 (2006). https://doi.org/10.1007/s00418-005-0080-2
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DOI: https://doi.org/10.1007/s00418-005-0080-2