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cAMP induced Rac 1-mediated cytoskeletal reorganization in microvascular endothelium

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Abstract

It is well established that cAMP stabilizes endothelial barrier functions, in part by regulation of VE-cadherin via EPAC/Rap 1. The aim of the present study was to investigate whether cAMP activates Rac 1 in microvascular endothelium. In human dermal microvascular endothelial cells (HDMEC), treatment with forskolin/rolipram (F/R) to increase cAMP by as well as the Epac/Rap 1-stimulating cAMP analogue 8-pCPT-2′-O-methyl-cAMP (O-Me-cAMP) stabilized endothelial barrier properties as revealed by raised transendothelial electrical resistance (TER). Under these conditions, immunostaining of VE-cadherin and claudin 5 were increased and linearized. This was paralleled by activation of Rac 1 by 153 ± 16% (F/R) or 281 ± 65% (O-Me-cAMP) whereas activity of Rho A was unchanged. F/R and O-Me-cAMP increased the peripheral actin belt and recruited the Rac 1 effector cortactin to cell junctions, similar to direct activation of Rac 1 by CNF-1. Thrombin was used to further test the physiologic relevance of cAMP-mediated Rac 1 activation. Thrombin-induced drop of TER was paralleled by intercellular gap formation, inactivation of Rac 1 and activation of Rho A at 5 and 15 min whereas baseline conditions where re-established following 60 min. Both, F/R and O-Me-cAMP completely blocked the thrombin-induced barrier breakdown. F/R completely abolished thrombin-induced Rac 1 inactivation and Rho A activation whereas O-Me-cAMP only partially blocked Rac 1 inactivation. Taken together, these results indicate that Rac 1 activation likely contributes to the barrier-stabilizing effects of cAMP in microvascular endothelium and that these effects may in part be mediated by Epac/Rap 1.

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Acknowledgments

We are grateful to Nadja Niedermeier and Lisa Bergauer for skilful technical assistance and to Wolfgang-Moritz Heupel and Nicolas Schlegel for helpful discussion and technical advice. We thank Moritz Bünemann and Ruth Werthmann (both Institute for Pharmacology and Toxicology, University of Würzburg) for using amaxa nucleofector technology. Cytotoxic necrotizing factor (CNF-1) was supplied by Gundula Schmidt (Department of Pharmacology and Toxicology, University of Freiburg). The phosho-S157-VASP antibody was a kind gift from Ulrich Walter (University of Wuerzburg; Institute for Clinical biochemistry and Pathobiochemistry). The EFGP-Rac 1 plasmids were a kind gift of Kozo Kaibuchi (University of Nagoya, Japan). These studies were supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 688, TP A4).

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  1. Institute of Anatomy and Cell biology, University of Wuerzburg, Koellikerstraße 6, 97070, Würzburg, Germany

    Y. Baumer, D. Drenckhahn & J. Waschke

  2. Institute of Anatomy and Cell Biology, Julius-Maximilians-University, Koellikerstr. 6, 97070, Würzburg, Germany

    J. Waschke

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  1. Y. Baumer
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Correspondence to J. Waschke.

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Baumer, Y., Drenckhahn, D. & Waschke, J. cAMP induced Rac 1-mediated cytoskeletal reorganization in microvascular endothelium. Histochem Cell Biol 129, 765–778 (2008). https://doi.org/10.1007/s00418-008-0422-y

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