Elsevier

Vascular Pharmacology

Volume 39, Issues 4–5, November 2002, Pages 187-199
Vascular Pharmacology

Review
Rho GTPases and the regulation of endothelial permeability

https://doi.org/10.1016/S1537-1891(03)00008-9Get rights and content

Abstract

Endothelial permeability depends on the integrity of intercellular junctions as well as actomyosin-based cell contractility. Rho GTPases have been implicated in signalling by many vasoactive substances including thrombin, tumour necrosis factor α (TNF-α), bradykinin, histamine, lysophosphatidic acid (LPA), vascular endothelial growth factor (VEGF), and hepatocyte growth factor (HGF). Two Rho family GTPases, Rho and Rac, have emerged as key regulators acting antagonistically to regulate endothelial barrier function: Rho increases actomyosin contractility, which facilitates breakdown of intercellular junctions, whereas Rac stabilizes endothelial junctions and counteracts the effects of Rho. In this review, we present evidence for the opposing effects of these two regulatory proteins and discuss links between them and other key signalling molecules such as cyclic AMP (cAMP), cyclic GMP (cGMP), phosphatidylinositide 3-kinases (PI3Ks), mitogen-activated protein kinases (MAPKs), and protein kinases C (PKCs). We also discuss strategies for targeting Rho GTPase signalling in therapies for diseases involving altered endothelial permeability.

Introduction

The endothelium is the inner lining of blood vessels and constitutes a barrier between blood and interstitium regulating extravasation of fluids, plasma proteins, and leukocytes. Endothelial permeability is controlled by a variety of chemical stimuli derived from blood or surrounding tissues as well as by mechanical stress exerted by pulsatile blood flow. Changes in the structural integrity of the endothelial monolayer are essential for vascular repair or inflammatory responses, but, if induced inappropriately, contribute to pathological conditions such as vascular leakage, septic shock, edema, atherosclerosis, hypertension, or cancer.

Breakdown of endothelial integrity is a consequence of an increased centripetal tension created by actomyosin contractility and decreased intercellular adhesion (tethering forces) Drenckhahn and Ness, 1997, Michel and Curry, 1999. It has been well documented that endothelial barrier function is controlled by the stability of intercellular junctions as well as tensile force within the monolayer maintained by the actomyosin cytoskeleton. Rho family GTPases regulate both actin cytoskeletal organization and the integrity of intercellular junctions Ridley, 2001, Braga, 2002 and have been implicated in intracellular signalling induced by several vasoactive substances (Table 1).

Section snippets

Rho GTPases—general overview

Rho GTPases belong to the Ras superfamily of proteins and are approximately 25% identical to Ras. Since Rho was first identified in 1985, a number of other related GTPases have been characterized, and so far, 21 members have been identified in mammals, Rho (A, B, C), Rac (1, 2, 3), Cdc42 (Cdc42Hs/G25K, TC10, Tcl), RhoD, RhoG, Chp (1,2), Rnd (RhoE/Rnd3, Rnd1/Rho6, Rnd2/Rho7), RhoH/TTF, Rif, Wrch1, and RhoBTB (1,2) Ridley, 2001, van Nieuw Amerongen and van Hinsbergh, 2001. Some of these members

Tools used in studies of Rho protein function

To study the roles of Rho proteins in cellular processes, several approaches have been used. The bacterial toxins described above that selectively inactivate particular GTPases have provided much useful information of Rho proteins, and the most commonly used are C3 transferase (Aktories and Hall, 1989) and toxin B (Hippenstiel et al., 1997). One of the most extensively used inhibitors of Rho signalling is the pyridine derivative Y-27632, which acts as an inhibitor of Rho kinase Uehata et al.,

Measurement of Rho protein activity

The levels of activated GTP-bound Rho, Rac, and Cdc42 can be measured in “pulldown assays.” In these assays, cell lysates are incubated with Sepharose beads with derivatized recombinant domains of target proteins of Rho, Rac, and Cdc42 (p21-activated kinase [PAK] CRIB for Rac/Cdc42, WASp-CRIB for Cdc42, rhoteckin Rho-binding domain for Rho) Sander et al., 1999, Ren and Schwartz, 2000. As these domains only bind GTP-loaded Rho proteins, the active proteins are being literally “pulled down” by

Rho proteins and actomyosin contractility

Increased actomyosin contractility is manifested by the formation of stress fibres, bundles of actin filaments associated with nonmuscle myosin II. Stress fibres in endothelial cells are formed in vivo and in vitro in response to many vasoactive agents (see Table 1 and Fig. 1). The best-characterized signal triggering formation of stress fibres is phosphorylation of the regulatory part of the myosin molecule, myosin light chain (MLC), which enables the myosin molecule to change conformation,

Rac and enhancement of endothelial barrier function

Although actomyosin contractility induced by RhoA is usually required for increased endothelial permeability, as described above, stress fibre formation does not always correlate with increased permeability. Both toxin B, an inhibitor of Rho, Rac, and Cdc42, and dominant negative Rac1 decrease stress fibres yet impair endothelial barrier function Hippenstiel et al., 1997, Wojciak-Stothard et al., 2001. In addition, Rho activation with bacterial toxins is not sufficient to increase endothelial

Signalling pathways upstream of Rho proteins

Enzymes regulating lipid metabolism have been implicated in the responses of several vasoactive substances including TNF-α, thrombin, histamine, bradykinin, HGF as well as shear stress and oxygen radicals Lum and Malik, 1994, Liu et al., 2002. Of particular relevance to Rho family proteins are the PI3Ks. PI3Ks become activated by GPCRs or tyrosine kinase receptors (Katso et al., 2001) and produce 3′ phosphoinositide lipids. The lipid products of PI3Ks, PI(3,4,5)P3, and PI(3,4)P2 act as second

Pharmacological intervention in vivo and future directions

Most research on Rho and Rac involvement in endothelial permeability has been carried out in vitro on cultured endothelial cells. A major challenge for the future will be to translate these findings into animal models for diseases involving impaired endothelial barrier function. So far, a few studies indicate that Rho signalling is a potential target for therapeutical intervention. The Rho kinase inhibitor, Y-27632, has been used in several animal models mainly because it is a small

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