rho gtpases in endothelial permeability · 2001-05-03 · rho gtpases in endothelial permeability...

INTRODUCTION

Members of the Rho family of GTPases regulate both actincytoskeletal organization and the integrity of intercellularjunctions (Hall, 1998). Rho itself induces the formation of actinstress fibres in fibroblasts (Ridley and Hall, 1992), epithelialcells (Ridley et al., 1995) and endothelial cells (Wójciak-Stothard et al., 1998), and acts at least in part by increasing thephosphorylation of myosin light chain (MLC), which in non-muscle cells leads to actomyosin contraction (Ridley, 1999).Rho-induced phosphorylation of MLC is mediated by Rhokinases (also known as ROCKs or ROKs), whichphosphorylate and inhibit the activity of MLC phosphatase.Rac and Cdc42 also regulate actin organization, inducing theextension of lamellipodia and filopodia, respectively (Ridley etal., 1992; Nobes and Hall, 1995). In addition, Rho, Rac andCdc42 affect the assembly of E-cadherin-containing adherensjunctions in epithelial cells, and inhibition of either Rho, Racor Cdc42 activity leads to a reduction in E-cadherin-mediatedcell adhesion (Braga et al., 1997; Hordijk et al., 1997; Takaishiet al., 1997; Jou and Nelson, 1998; Fukata et al., 1999;reviewed in Kaibuchi et al., 1999). One mechanism wherebyRac and Cdc42 may enhance adherens junction formation isby repressing the antiadhesive activity of their downstreamtarget IQGAP1 (Fukata et al., 1999). In endothelial cells,however, inhibiting Rho or Rac does not perturb the

localization of endothelial-specific VE-cadherin tointercellular junctions (Braga et al., 1999), suggesting that theregulation of adherens junctions differs according to context orcell type. Expression of either constitutively activated ordominant-negative forms of Rho and Rac also perturbs tightjunction structure and function in epithelial cells (Nusrat et al.,1995; Jou et al., 1998).

There is accumulating evidence that Rho regulatesendothelial permeability, which depends on the integrity ofintercellular junctions and actomyosin contractility (Hordijket al., 1999; Essler et al., 1998a,b; Essler et al., 1999). Thevasoactive mediators thrombin and histamine have long beenknown to increase vascular permeability in vivo (Majno andPalade, 1961; Haraldsson et al., 1986; Wu and Baldwin, 1992)and in vitro (Killackey et al., 1986; Lum et al., 1992; Ehringeret al., 1996). Increased vascular permeability is essential forinflammatory responses, but can also contribute to thedevelopment of pathological conditions such as atherosclerosis(Stemerman et al., 1986; Stender and Hjelms, 1987; Ross,1993; Raines and Ross, 1996) and is accompanied byalterations to cell-cell junctions and the actin cytoskeleton.Thrombin-induced endothelial permeability has been attributedto stress fibre formation and subsequent actomyosin-mediatedcontraction of cells (Lum and Malik, 1996; Van Hinsbergh,1997; Essler et al., 1998a) as well as to changes in thedistribution and phosphorylation of intercellular adhesion

1343

Endothelial permeability induced by thrombin andhistamine is accompanied by actin stress fibre assemblyand intercellular gap formation. Here, we investigate theroles of the Rho family GTPases Rho1, Rac1 and Cdc42 inregulating endothelial barrier function, and correlate thiswith their effects on F-actin organization and intercellularjunctions. RhoA, Rac1 and Cdc42 proteins were expressedefficiently in human umbilical vein endothelial cells byadenovirus-mediated gene transfer. We show thatinhibition of Rho prevents both thrombin- and histamine-induced increases in endothelial permeability anddecreases in transendothelial resistance. Dominant-negative RhoA and a Rho kinase inhibitor, Y-27632, notonly inhibit stress fibre assembly and contractility but alsoprevent thrombin- and histamine-induced disassembly ofadherens and tight junctions in endothelial cells, providing

an explanation for their effects on permeability. Incontrast, dominant-negative Rac1 induces permeabilityin unstimulated cells and enhances thrombin-inducedpermeability, yet inhibits stress fibre assembly, indicatingthat increased stress fibre formation is not essentialfor endothelial permeability. Dominant-negative Cdc42reduces thrombin-induced stress fibre formation andcontractility but does not affect endothelial cellpermeability or responses to histamine. These resultsdemonstrate that Rho and Rac act in different ways to alterendothelial barrier function, whereas Cdc42 does not affectbarrier function.

Key words: Vascular permeability, Rho GTPase, Actin cytoskeleton,Tight junction

SUMMARY

Rho and Rac but not Cdc42 regulate endothelial cellpermeabilityBeata Wójciak-Stothard 1,2, Sandra Potempa 1,3, Thomas Eichholtz 3 and Anne J. Ridley 1,2,*1Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, 91 Riding House Street, London W1W 7BS, UK2Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK 3GlaxoWellcome Research and Development, Gunnels Road, Stevenage, UK*Author for correspondence (e-mail: [email protected])

Accepted 11 January 2001 Journal of Cell Science 114, 1343-1355 © The Company of Biologists Ltd

RESEARCH ARTICLE

1344

proteins such as cadherins and catenins (Rabiet et al., 1996;Drenckhahn and Ness, 1997; Lampugnani and Dejana, 1997).The relative contributions however, of these two responses tothe increase in endothelial permeability are not clear.

In order to investigate the contribution of different signallingpathways to the regulation of endothelial cell permeability, itis essential to be able to inhibit the activity of specificsignalling proteins in the majority of an endothelial cellpopulation. As normal endothelial cells are not transfectedefficiently, this has been achieved primarily by adding cell-permeable pharmacological inhibitors to cells. For example, aninhibitor of Rho kinase can inhibit thrombin-inducedpermeability (Essler et al., 1998a). By using C3 transferase, abacterial exoenzyme that can enter cells and modify Rho, it hasbeen possible to implicate Rho in both thrombin-inducedpermeability and stress fibre formation in endothelial cells(Essler et al., 1998a; Van Nieuw Amerongen et al., 1998;Carbajal and Schaeffer, 1999), but the roles of Cdc42 andRac in regulating permeability have not been described. Toinvestigate the effects of Rho, Rac and Cdc42 on endothelialcell permeability we have developed recombinant adenovirusesas an efficient means to express Rho, Rac and Cdc42 proteinsin endothelial cells. This has allowed us to correlate changesin permeability with alterations in both the actin cytoskeletonand intercellular junctions, using immunofluorescence andtransmission electron microscopy.

We have shown that inhibition of Rho or Rho kinase preventsthrombin- and histamine-induced loss of tight junctions andadherens junctions, as well as inhibiting stress fibre formation,and that Rho is required for both thrombin- and histamine-induced vascular permeability. Inhibiting Rac activity by itselfleads to increased permeability and enhances thrombin-induced permeability, reflecting Rac-induced changes tointercellular junctions and the appearance of intercellular gaps.In contrast, inhibition of Cdc42 has no effect on endothelialpermeability, despite its effects on the actin cytoskeleton.

MATERIALS AND METHODS

ReagentsHuman fibronectin, FITC-phalloidin and histamine were obtainedfrom Sigma-Aldrich (Poole, UK); Y-27632 was from WelfideCorporation (Osaka, Japan); FITC-dextran, tetramethylrhodamineisothiocyanate (TRITC)-dextran, BODIPY-, fluorescein- and TRITC-labelled goat anti-mouse secondary antibodies were from MolecularProbes (Leiden, The Netherlands); mouse monoclonal anti-humancadherin-5 (VE-cadherin) antibody was from BDPharmingen (SanDiego, CA, USA); rabbit anti-occludin antibody was from Zymed(San Francisco, CA, USA); anti-c-myc monoclonal antibody (9E10)was from Santa Cruz Biotechnology (Santa Cruz, CA); Moviol wasobtained from Calbiochem (Nottingham, UK).

Cell cultureTo purify human umbilical vein endothelial cells (HUVECs),umbilical veins were flushed twice with PBS, then filled with 0.1%collagenase in PBS (Sigma) prewarmed to 37°C, and incubated for 20minutes at 37°C. The collagenase solution was then collected and theveins were washed twice with 20 ml of Medium 199 (LifeTechnologies, Paisley, UK) containing 20% fetal calf serum (FCS) tocollect the remaining cells. The collagenase solution and the washeswere combined together and centrifuged. The cell pellet wasresuspended in 20 ml of Medium 199/20% FCS and cells plated into

a 75 ml culture flask (Nunclon) coated with 10 µg/ml fibronectin.After 3 hours the medium was changed to remove non-adherent cells.Cells were cultured in Medium 199 containing 20% FCS, 100 µg/mlendothelial cell growth supplement (Sigma), 100 µg/ml heparin(Sigma), 1% Nutridoma NS (Boehringer Mannheim Ltd, Lewes, UK)in flasks coated with 10 µg/ml human fibronectin, as previouslydescribed (Wojciak-Stothard et al., 1998). To determine the purity ofthe cell population, the uptake of low-density lipoprotein (LDL) wasmeasured by adding DiI-labelled LDL (Molecular Probes, Leiden,The Netherlands) to cells at 2.5 µg/ml. 98-99% of cells in the thirdpassage labelled positive for diI-LDL. For some experiments,HUVECs kindly provided by Ruggero Pardi (DIBIT, Milan, Italy)were used. For experiments, HUVECs were used between 2 and 5passages. Human thrombin was added to the culture medium at a finalconcentration of 1 U/ml, and histamine at 10 µM.

For confocal microscopy, HUVECs were grown on glass coverslipscoated with 10 µg/ml human fibronectin until confluent. To obtainquiescent HUVECs, the culture medium was replaced by mediumcontaining 10% FCS but no heparin or other growth factors, aspreviously described (Wojciak-Stothard et al., 1998), and the cellswere incubated in this new medium for 16-20 hours beforemicroinjection and/or treatment with different agents.

For permeability studies, HUVECs were grown on fibronectin-coated polyester Transwell-Clear filters (3 µm pore size, 12 mmdiameter; Corning Costar Corporation, Costar Ltd, High Wycombe,UK). The cells were plated at 3×105 cells/well, and after 4 hours non-adherent cells were removed. Filters were used for experiments 4 daysafter plating. The Rho kinase inhibitor Y-27632 (5 µΜ) was added tothe upper chamber 30 minutes before the addition of histamine orthrombin and C3 transferase (15 µg/ml) 1 hour before stimulation.Although the cells grown on filters were visible under the lightmicroscope and were examined for confluency before the experiment,all filters were fixed and then stained with Coomassie Blue after theexperiment to reveal the general appearance of the cell monolayer andto visualize gaps between cells.

Purification and microinjection of recombinant proteinsThe recombinant proteins, V12Rac1, N17Rac1, N17Cdc42 and C3transferase, were expressed in Escherichia colifrom the pGEX-2Tvector as glutathione S-transferase fusion proteins and purified aspreviously described (Ridley et al., 1992). Protein concentrations wereestimated by Bradford assay using a protein assay kit (Bio-Rad).

Recombinant proteins were microinjected into the cytoplasm ofHUVECs together with TRITC-dextran (MW 10,000) (5 mg/ml) toidentify injected cells. C3 transferase was microinjected at aconcentration of 4 µg/ml, V12Rac1 at approximately 0.75 mg/ml,N17Cdc42 at 1 mg/ml and N17Rac1 at 2 mg/ml. Histamine andthrombin were added 15 minutes after microinjection.

Generation of adenoviruses and adenoviral infectionTo generate recombinant adenoviruses, cDNAs encoding amino-terminal myc-tagged N19RhoA, N17Cdc42, N17Rac1 and V12Rac1were subcloned into the admid transfer vectors pCR259 and pCR244.The admid transfer vectors were then transformed into an E. colistraincontaining a vector encoding a transposase and an Ad5-basedadenovirus vector deleted in the E1 and E3 genes (Ad5 ∆E1∆E3).Transposition of the Rho cDNAs from the admid transfer vectors intoAd5 ∆E1∆E3 created the adenoviral vectors Ad-N19RhoA, Ad-N17Rac1, Ad-N17Cdc42 and Ad-V12Rac1, where the transgenes areunder the control of a CMV promotor.

Recombinant adenoviral DNA was purified from E. coli andtransfected into 293 human embryonic kidney cells, which express E1genes, to allow purification of adenoviral particles. Ad-β-Gal,encoding the β-galactosidase protein, was a kind gift from CarolynDent (GlaxoWellcome, Stevenage, UK). The titer of adenovirus stockswas determined by titration on 911 cells (Fallaux et al., 1996).

Confluent HUVECs grown on Transwell filters or glass coverslips

JOURNAL OF CELL SCIENCE 114 (7)

1345Rho GTPases in endothelial permeability

were infected with adenoviruses at a multiplicity of infection (MOI)of 1500 in medium containing 10% FCS. Adenoviruses were addedonly into the top chamber of Transwells. For transmission electronmicroscopy, HUVECs were grown on a flexible transparentsubstratum, melanex (ICI), coated with fibronectin. After incubationfor 4 hours the virus-containing medium was removed and freshmedium containing 10% FCS was added. The cells were thenincubated for a further 18 hours before the addition of thrombin orhistamine.

To detect β-galactosidase activity, Ad-β-Gal-infected cells werefixed with 3.7% formaldehyde for 20 minutes. After washing twicewith PBS, 1 mg/ml of X-gal (5-bromo-4-chloro-3-indoyl galactoside)substrate (Promega, Southhampton, UK) in PBS containing 5 mMpotassium ferricyanide, 5 mM potassium ferrocyanide and 2 mMMgCl2 was added and cells were incubated at 37°C for 1-2 hours.Blue cells were counted to monitor expression of β-galactosidase.

Transendothelial permeability assaysTransendothelial permeability was measured as previously described(Draijer et al., 1995). HUVECs in Transwell chambers were incubatedin Medium 199 with 1% bovine serum albumin (BSA) for 1 hourbefore adding 1 mg/ml FITC-dextran (MW 42,000) in the samemedium together with thrombin and histamine. Samples were takenat 15 minutes, 30 minutes, 1 hour, 2 hours and 3 hours afterstimulation from the lower compartment of the Transwell chambersand an equal volume of Medium 199 with 1% BSA was re-added tothe lower chamber. The amount of FITC-dextran in the upper andlower wells was determined with a fluorometer (RF-5301PC,Shimadzu Corp. Kyoto, Japan), using an excitation wavelength of 492nm, and detecting emission at 520 nm. In experiments withadenovirally infected cells, the flux of FITC-dextran (µg/hour/cm2)was compared with control cells over the first hour after addition of

thrombin or 30 minutes after addition of histamine, as at these timepoints the highest accumulation of FITC-dextran was obtained in thelower wells of the Transwell chambers. The results in Fig. 2Arepresent mean values of fluorescence ± s.d. of three independentexperiments and are presented as percentage of the fluorescence incontrols.

Transendothelial electrical resistance The electrical resistance of HUVEC monolayers cultured onTranswell filters was measured with a Millicell-ERS instrument(Millipore Continental Water Systems, Bedford, MA, USA).Resistance measurements were taken before and 5 minutes afterstimulation with thrombin or histamine. The measured potentialdifference between the upper and lower wells was used to calculatethe electrical resistance in Ω cm2. Transendothelial resistance (TEER)values were then calculated by subtracting the contribution of the filterand the bathing solution. To normalize the results of differentexperiments, the TEER for monolayers of control cells (uninfected)was taken as 100%. Changes in TEER are presented as percentage ofthe control value. Experiments were performed in triplicate and valuesare means ± s.d.

Immunofluorescence and localization of F-actinTo visualise the distributions of VE-cadherin, β-catenin, ZO-1,vinculin and F-actin, HUVECs were fixed with 4% formaldehydedissolved in PBS for 10 minutes at room temperature andpermeabilised for 6 minutes with 0.2% Triton X-100. Cells wereincubated in 0.5% BSA in PBS for 45 minutes to block nonspecificantibody binding and then incubated with 1 µg/ml FITC-phalloidinfor 45 minutes to stain actin filaments or with mouse monoclonalanti-human cadherin-5 (VE-cadherin) antibody (1:400), rabbit anti-ZO-1 antibody (1:200), mouse monoclonal anti-β-catenin antibody

Fig. 1. Changes in the distribution of F-actin, occludin and VE-cadherin inHUVECs treated with thrombin andhistamine. Quiescent HUVECs orHUVECs stimulated with 1 U/mlthrombin or 10 µM histamine for 5minutes were fixed and stained tolocalize F-actin, occludin or VE-cadherin, as indicated. (A,D,G) F-actin(A), occludin (D) and VE-cadherin(G) localization in control, unstimulatedHUVECs. (B,E,H) F-actin (B), occludin(E) and VE-cadherin (H) in thrombin-stimulated HUVECs and (C,F,I) inhistamine-stimulated cells. The arrows inB and C point to gaps in intercellularjunctions. Bar, 20 µm.

1346

(1:200) or mouse monoclonal anti-vinculin antibody VIN-11-5(1:200). For occludin staining, cells were pre-extracted, fixed in 95%ethanol for 30 minutes, then rehydrated and incubated with rabbitanti-occludin antibody (1:20) as described (Balda et al., 1996).Primary antibodies were visualised by incubation with BODIPY-,fluorescein- or TRITC-labelled goat anti-mouse or goat anti-rabbitsecondary antibodies for 1 hour at room temperature and thespecimens mounted in Moviol.

Confocal laser scanning fluorescence microscopy andelectron microscopyConfocal laser scanning microscopy was carried out with an LSM 510(Zeiss, Welwyn Garden City, UK), using a ×10 eyepiece, and eithera ×40 NA 1.3 or a ×63 NA 1.4 oil immersion objective (Zeiss). FITCand TRITC were excited at 488 nm and 543 nm and visualised witha 540±25 and a 608±32 nm bandpass filter, respectively, where thelevels of interchannel cross-talk were insignificant. Image files werecollected as a matrix of 1024×1024 pixels describing the average of8 frames scanned at 0.062 Hz.

For transmission electron microscopy (TEM), HUVECs werefixed with 2% paraformaldehyde/2% glutaraldehyde in PBS for30 minutes. Cells were prepared for analysis by TEM by MarkTumaine (Department of Anatomy and Developmental Biology,University College London, UK). After fixation, the cells werewashed three times in 0.1 M sodium cacodylate, then incubated for10 minutes in 0.1 M sodium cacodylate and 1% osmium tetroxide,then washed again in 0.1 M sodium cacodylate and distilled water.The samples were dehydrated by subsequent washes in 25%, 50%and 100% ethanol, then embedded in epoxy resin and viewed inTEM Jeol 1010. In order to identify tight junctions and adherensjunctions, the stage goniometer was used to tilt the specimen andview cross sections of cell membranes at different angles. Thequantitative analysis of the number of tight junctions and adherensjunctions on lateral membranes by TEM was based on themethodology of Burns et al. (Burns et al., 2000). Results arepresented as means ± s.d. from 2-3 separate experiments where 20-25 lateral membranes/monolayer were analysed in threemonolayers/treatment group.


0

1 0 0

2 0 0

3 0 0

4 0 0

5 0 0

6 0 0a b4 0 0

30 t ime (h) 0 3

4 0 0

t i m e ( h )

**

**

*

*

*

**

*

6 0

7 0

8 0

9 0

1 0 0

1 1 0

A

B 1 2 0

*

*

**

* *

Fig. 2.Effects of Rho, Rac and Cdc42 onendothelial barrier function. (A) Roles of Rho, Rhokinase, Rac and Cdc42 in regulating basal andthrombin- and histamine-induced endothelialpermeability. Confluent HUVECs grown on filtersin Transwells were infected with Ad-β-gal, Ad-N19RhoA, Ad–N17Rac1, Ad-V12Rac1 or Ad-N17Cdc42 as indicated. 18 hours later, cells wereeither not stimulated (cross-hatched bars), orstimulated with 1 U/ml thrombin (thr) for 1 hour(black bars) or with 10 µM histamine (his) for 30minutes (grey bars). Alternatively, HUVECs wereincubated for 1 hour with 15 µg/ml C3 transferase,or 5 µM Y-27632 for 30 minutes and thenstimulated with thrombin and histamine.Permeability was measured by determining flux ofFITC-dextran from the upper to lower chamber ofTranswells. Inserts indicate the time course of(a) thrombin- and (b) histamine-induced changes inpermeability in control cells. (B) Effects of Rho andRho kinase inhibitors on changes in TEER inducedby thrombin and histamine. Confluent HUVECs onfilters were treated as described in A. TEER incontrol (unstimulated) cells pretreated with Y-27632, C3-transferase or infected with Ad-N19RhoA was similar to controls (data not shown).The average TEER of unstimulated HUVECs variedbetween 13-18 Ωcm2 (100%). Results are presentedas % of control values and are means ± s.d. *P<0.05** P<0.01.


Statistical analysisData are presented as means ± s.d. Comparisons between>2 groups were made using a one-way ANOVA testfollowed by Tukey post-test for multiple comparisons(TEER and TEM data) or Kruskal-Wallis test followed byDunn’s post-test (endothelial permeability data). Thechoice between parametric and non-parametric tests wasbased on the Bartlett’s test for homogeneity of variances.Statistical significance was accepted for P<0.05 and alltests were performed with GraphPad Instat version 2.01.

RESULTS

Thrombin- and histamine-inducedreorganization of the actin cytoskeleton,tight junctions and adherens junctionscorrelates with changes in endothelialbarrier functionTo investigate the contributions of Rho, Rac andCdc42 to thrombin- and histamine-inducedendothelial permeability, and correlate this withchanges in the actin cytoskeleton and cell-celljunctions, we first characterized the effects ofthrombin and histamine under precisely controlledconditions of HUVEC culture. 4 days after reachingconfluence, HUVECs were maintained in 10% FCSwithout growth supplements for 24 hours prior tostimulation. Under these conditions, HUVECs werepolygonal in shape and had few stress fibrestraversing the body of cells, although there weresome stress fibres localised at the cell periphery(Fig. 1A). VE-cadherin, the major transmembranecomponent of endothelial adherens junctions(Lampugnani and Dejana, 1997), as well asoccludin, a transmembrane component of tightjunctions (Balda and Matter, 1998), were localizedalong the cell margins as previously reported(Lampugnani et al., 1995; Burns et al., 2000) (Fig.1D,G).

Thrombin induced rapid assembly of stress fibres(Fig. 1B) and vinculin-containing focal contacts (data notshown) in quiescent HUVECs. The peak in stress fibreformation was observed within 3-5 minutes of thrombinaddition, and the earliest changes in F-actin distribution wereobserved after 1-2 minutes, consistent with previousobservations on F-actin (Goeckeler and Wysolmerski, 1995)and on the time course of myosin light chain phosphorylation(Van Nieuw Amerongen et al., 1998). Thrombin also induced

disappearance of occludin from cell margins (Fig. 1E) anddisruption of the linear VE-cadherin staining at intercellularjunctions (Fig. 1H). Although VE-cadherin was still localizedto areas of cell-cell contact following thrombin stimulation, thejunctions became segmented and discontinuous. This in partreflected the appearance of gaps between cells (e.g. arrows inFig. 1B), and some cells clearly retracted and became rounded.Thrombin induced similar changes in the actin cytoskeleton,

Fig. 3. Rho and Rho kinase are required for thrombin-and histamine-induced changes to the actin cytoskeletonand intercellular junctions. Quiescent HUVECs wereuntreated (A,B), microinjected with C3 transferase(C,D), infected with Ad-N19RhoA (E-H), or pre-treatedwith 5 µM Y-27632 (J). Cells were subsequentlystimulated as indicated with thrombin (1 U/ml)(A,C,F,I,J) or histamine (10 µM) (B,G,H) for 5 minutes.Cells were fixed and stained to show the distribution ofF-actin (A-C,E,G), VE-cadherin (D,F,H) and occludin(I,J). Injected cells are indicated by arrowheads. Bar,20µm.

1348

cell retraction and intercellular gapformation when endothelial cells weregrown on fibronectin-coated Transwellfilters (data not shown), demonstrating thatthe cells behave similarly on glass and onfilters.

Like thrombin, histamine promoted theformation of stress fibres within 5 minutes ofaddition (Fig. 1C). The earliest changes inthe distribution of actin filaments wereobserved after 1 minute of histaminetreatment, consistent with reports by others(Niimi et al., 1992). In contrast to thrombin,histamine-treated cells did not retract andalthough gaps were observed betweensome cells (Fig. 1C, arrow), they wereconsiderably less abundant and generallymuch smaller than those observed withthrombin (Fig. 1B). Histamine caused fragmentation ofoccludin and VE-cadherin staining (Fig. 1F,I). In contrast tothe response to TNF-α (Wójciak-Stothard et al., 1998), nomembrane ruffling or formation of lamellipodia or filopodiawas detected in either thrombin- or histamine-treated cells.

The rapid time course of stress fibre formation and celljunction changes induced by histamine and thrombincorrelated with a decrease in endothelial barrier function,measured either by transendothelial electrical resistance(TEER) or passage of FITC-dextran through HUVECmonolayers grown on Transwell filters. Thrombin andhistamine induced a decrease in TEER within the first 5minutes of stimulation (Fig. 2B). These small but consistentlyobserved changes in TEER were in the range reported by others(Langeler and van Hinsbergh, 1991; Westendorp et al., 1994).Loss of endothelial barrier function was also assayed bymeasuring endothelial permeability to FITC-dextran. FITC-dextran was added to the upper chamber of Transwells, and theamount of FITC-dextran that had accumulated in the lowerchamber was determined at different time points afterstimulation. The passage rate of FITC-dextran through control,unstimulated HUVECs was 20-25 µg/hour/cm2 (measuredover the first hour after addition of FITC-dextran), and was 10-

to 15-fold lower than through filters without cells. Thrombininduced an increase in FITC-dextran flux, and this was highest(60-70 µg/hour/cm2) when measured over the first hour afterstimulation (Fig. 2A, insert a), reflecting the fact that thrombininduces a rapid decrease in endothelial barrier function, whichthen gradually returns to basal levels (Rabiet et al., 1996; VanNieuw Amerongen et al., 1998; Burns et al., 2000). Withhistamine, the maximal rate of FITC-dextran flux (30-40µg/hour/cm2) was observed during the first 30 minutes afterstimulation (Fig. 2A, insert b), consistent with the moretransient increase in permeability reported previously forhistamine compared to thrombin (Rabiet et al., 1996; VanHinsbergh et al., 1997; Andriopoulou et al., 1999). As changesin FITC-dextran flux were larger and more easily measurablethan the relatively small changes in TEER, this was used as ameasure of endothelial permeability in the majority ofsubsequent experiments.

Effects of Rho, Rac and Cdc42 on thrombin- andhistamine-induced endothelial permeability In order to assess the roles of Rho, Rac1 and Cdc42 inregulating thrombin- and histamine-induced changes inendothelial permeability, confluent HUVECs on Transwell


Fig. 4. Inhibition of Rho or Rho kinase preventsdisassembly of intercellular junctions inHUVECs in thrombin-stimulated HUVECs.Representative TEM micrographs are shown ofintercellular junctions in quiescent, unstimulatedHUVECs expressing Ad-β-gal (A); cells pre-treated with 5 µM Y-27632 for 30 minutes(B); cells stimulated with thrombin (C); cellspretreated with Y-27632 and stimulated withthrombin (D); cells expressing Ad-N19RhoA(E). The arrowheads point to electron-densematerial within the junctions, indicative ofintercellular junctions (B,D,E). The junctionsshown in A are representative of both Ad-β-gal-expressing and untreated, control cells.(F) Higher magnification examples of tightjunctions (‘kissing points’) in Ad-N19RhoA-expressing cells (top) and thrombin-stimulatedcells pretreated with Y-27632 (bottom). Bar, 500nm (except for C, 1 µm; F, 30 nm).


filters were infected with the recombinantadenoviruses to express the dominant-negativemutants N19RhoA, N17Rac1 and N17Cdc42.In addition, we constructed an adenoviruscontaining constitutively active V12Rac1, asexpression of V12Rac1 has been reported toenhance adherens junction assembly and/orstability in epithelial cells (Hordijk et al., 1997;Takaishi et al., 1997; Sander et al., 1998;Stöffler et al., 1998), but to perturb epithelialtight junctions (Jou et al., 1998). All proteinswere tagged at the amino terminus with a mycepitope to facilitate analysis of their expressionand localization. The infection efficiency ofHUVECs was initially evaluated using Ad-β-gal, encoding the reporter protein β-galactosidase. By 18 hours after adenoviralinfection 80±10% of cells expressed detectablelevels of β-galactosidase (data not shown).Expression of the Rho GTPases wassubsequently monitored by western blotting andimmunofluorescence, and was detectable at 6hours after infection, although it increasedseveral-fold by 18 hours (data not shown).

Infection with Ad-β-gal had no effect onendothelial permeability in either unstimulatedor stimulated cells (Fig. 2). Both the thrombin-and histamine-induced increase in permeabilitywas significantly reduced in cells infected withAd-N19RhoA (P<0.05) (Fig. 2A). Incubation ofcells with C3 transferase, an exoenzyme fromClostridium botulinumthat specifically inhibitsRho by ADP-ribosylating it (Machesky andHall, 1996), also inhibited histamine- andthrombin-induced permeability (P<0.05) (Fig.2A). Similarly, Y-27632, an inhibitor of Rhokinase acting downstream of Rho, significantlyinhibited thrombin- and histamine-inducedpermeability (P<0.05).

Ad-V12Rac1 and Ad-N17Rac1 bothsignificantly increased endothelial permeabilityin unstimulated cells at 18 hours after infection(P<0.05), and Ad-N17Rac1 enhanced theresponse to thrombin (P<0.05) (Fig. 2A). Incontrast, expression of N17Cdc42 had nosignificant effect on control or thrombin/histamine-induced permeability. Taken together,these results indicate that Rho and Rho kinaseactivity is required for thrombin- and histamine-induced increased permeability whereasperturbation of Rac activity in itself inducesincreased permeability.

In addition to inhibiting thrombin- andhistamine-induced endothelial permeability asmeasured by FITC-dextran flux, expression of N19RhoA orpretreatment of cells with C3 transferase or Y-27632 preventedthe transient drop in TEER induced by thrombin and histamine(Fig. 2B). N17Rac1 and N17Cdc42 did not have a significanteffect on TEER (data not shown). These data are consistentwith a role for Rho in mediating thrombin- and histamine-induced decreases in endothelial barrier function.

Rho and Rho kinase are required for both thrombin-and histamine-induced formation of stress fibresand changes in intercellular adhesionsAs inhibition of Rho function using either N19RhoA, C3transferase or the Rho kinase inhibitor Y-27632 reducedthrombin- and histamine-induced permeability, we investigatedthe effects of these Rho inhibitors on F-actin organization and

Fig. 5.Effects of Rac1 mutants on intercellular junctions in HUVECs and thelocalization of β-catenin and ZO-1 in quiescent HUVECs. Confluent HUVECs wereinfected with Ad-β-gal (A,B), Ad-N17Rac1 (D-F) or Ad-V12Rac1 (C,G,H). Cellswere fixed 18 hours after infection and stained to show localization of β-catenin(A,E,G) or ZO-1 (B,F,H). Arrows in E and G point to intercellular gaps. Thearrowhead in E points to one of the areas where the membranes of two neighbouringcells expressing N17Rac1 are overlapping. TEM images of intercellular junctions incells expressing Ad-V12Rac1 are shown in C and Ad-N17Rac1 in D. Bar, 20 µm(450 nm in C and D).

1350

intercellular junctions in thrombin- and histamine-stimulatedcells. As expected, microinjection of C3 transferase intoHUVECs completely prevented thrombin- and histamine-

induced formation of stress fibres (Fig. 3A-C and data notshown). The microinjected cells remained well spread andwere almost completely devoid of stress fibres or actin

bundles, although F-actin was still associated withintercellular junctions. Similarly, infection ofcells with Ad-N19RhoA inhibited thrombin- andhistamine-induced stress fibre formation (Fig.3E,G), and prevented thrombin-inducedcontractility so that intercellular gaps were notobserved (Fig. 3E, compare with Fig. 3A). Inaddition, C3 transferase and N19RhoA inhibited thefragmentation of VE-cadherin staining induced bythrombin and histamine (Fig. 3D,F, compare withFig. 1H,I). Control Ad-β-gal had no effect onthrombin and histamine responses. Y-27632 actedsimilarly to Ad-N19RhoA and C3 transferase ininhibiting thrombin- and histamine-induced changesto the actin cytoskeleton and adherens junctions(data not shown). Y-27632 also prevented loss ofoccludin from intercellular borders induced bythrombin (Fig. 3I,J). Ad-β-gal and Ad-N19RhoAdid not affect the distribution of F-actin or junctionalproteins in unstimulated cells (Fig. 5A,B and datanot shown).

Transmission electron microscopy (TEM)indicated that the cells infected with Ad-β-gal aswell as uninfected cells had intercellular junctionsbut there were few tight junctions (Fig. 4A), aspreviously reported in HUVECs in the absence ofgrowth supplements (Burns et al., 1997). Tightjunctions were identified as points where theouter leaflets of lateral membranes betweenadjacent endothelial cells appeared to fuse (Fig.4F; Burns et al., 1997). In control cells, tightjunctions were detected in 20±2% of lateralmembranes between adjacent cells, and this levelwas not significantly different from that in cellstreated with Y-27632 or in cells expressingN19RhoA (Table 1). Adherens junctions lackedmembrane fusion and were identified as areas ofclose apposition of cell membranes associatedwith electron-dense material. The percentage oflateral membranes where adherens junctionswere detected was also similar in control,N19RhoA-expressing and Y-27632-pretreatedcells (Table 1).

TEM analysis of regions of intercellular contactbetween thrombin-stimulated endothelial cellsshowed that there were often wide gaps betweencells, and intercellular junctions were rarelyobserved (Fig. 4C, Table 1). Pretreatment of cellswith Y-27632 prevented the loss of tight junctionsand adherens junctions induced by thrombin(Table 1, Fig. 4D,F). Similarly, Y-27632 preventedhistamine-induced loss of tight junctions andadherens junctions (Table 1).

Rho and its downstream target Rho kinase aretherefore required both for changes in the actincytoskeleton and to intercellular junctions inducedby thrombin and histamine. In particular, the abilityof N19RhoA and Y-27632 to prevent loss of tight


Fig. 6.Rac1 is required for the reorganization of actin and intercellular junctionsinduced by thrombin and histamine. HUVECs were microinjected withN17Rac1 protein (A,B) or infected with Ad-N17Rac1 (C-F) or Ad-V12Rac1(G,H). They were subsequently stimulated as indicated with thrombin(A,C,D,G,H) or histamine (B,E,F) for 5 minutes. Cells were then fixed andstained with FITC-phalloidin to show F-actin (A-C,E,G), or with anti-VE-cadherin antibodies to show adherens junctions (D,F,H). Microinjected cells areindicated by arrowheads. The microinjected cells in A and B are lacking stressfibres. The arrow in C points to an intercellular junction. Bar, 20 µm.


junctions in thrombin- and histamine-stimulated cells providesan explanation for their inhibitory effect on increasedendothelial permeability.

N17Rac1 and V12Rac1 alter intercellular junctionsand induce intercellular gap formation in quiescentHUVECs

As expression of both N17Rac1 andV12Rac1 increased endothelial permeabilityeven in unstimulated HUVECs, we analysedtheir effects on intercellular junctions. VE-cadherin and β-catenin in cells infected withAd-N17Rac1 localized to intercellularborders, but in many places formed ameshwork-like pattern (Fig. 5E,F). Thoughalso occasionally observed in control cells(e.g. Figs 1G, 5A), this pattern of stainingwas much more widespread in N17Rac1-expressing cells. Levels of ZO-1 atintercellular junctions appeared reduced inmost N17Rac1-expressing cells, and ZO-1was absent or had a fragmented localizationalong some intercellular borders (Fig. 5F,compare to Fig. 5B), suggesting loss of tightjunctions. Intercellular gaps were observedbetween some cells (e.g. arrow in Fig. 5E).

TEM analysis of HUVECs expressingN17Rac1 revealed that lateral membranesfrom adjacent cells often came into closeapposition in only a few places and betweenthese there were large intercellular gaps (Fig.5D), which were not observed in control cells(Fig. 4A). N17Rac1-expressing cells showeda significant decrease in the percentage ofendothelial cell borders with tight junctionsand adherens junctions (Table 1). Takentogether, these results indicate that adherensjunctions and tight junctions were weakenedin cells expressing N17Rac1. This is likely tobe responsible for the increased permeabilityobserved in these cells.

We have previously observed thatmicroinjection of V12Rac1 protein rapidlyinduces lamellipodium extension inHUVECs (Wójciak-Stothard et al., 1998). Incontrast, AdV12Rac1-infected HUVECs didnot show lamellipodia at 18 hours afterinfection, probably because lamellipodiumextension is a transient response to Rac inconfluent quiescent cells. However, someV12Rac1-expressing HUVECs hadincreased levels of stress fibres (data notshown), as also observed at later time pointsafter V12Rac1 protein injection (Wójciak-Stothard et al., 1998). Some cells expressinghigh levels of V12Rac1 retracted to leaveintercellular gaps, and in these places VE-cadherin and β-catenin were not detected atthe plasma membrane (Fig. 5G, arrow). ZO-1 staining at intercellular junctions wasconsiderably more fragmented than incontrol cells (Fig. 5H), indicating disruptionof tight junctions. Consistent with this,analysis of TEM micrographs revealed thatcells expressing V12Rac1 had significantly

Fig. 7.Cdc42 is required for thrombin- but not histamine-induced changes to the actincytoskeleton and intercellular junctions. Quiescent HUVECs were untreated (A,B),microinjected with N17Cdc42 protein (C,D; microinjected cells are marked witharrowheads), or infected with Ad-N17Cdc42 (E-H). They were subsequently stimulatedas indicated with thrombin or histamine for 5 minutes, then fixed and stained to show F-actin (A-E,G) or VE-cadherin localization (F,H). Bar, 15 µm.

1352

fewer tight junctions and adherens junctions than control cells(Table 1, Fig. 5C). In some cases, lateral membranes ofadjacent cells had only occasional sites of close contact (datanot shown) as in N17Rac1-expressing cells (Fig. 5D). Overall,these results suggest that the increase in endothelialpermeability observed in V12Rac1-expressing cells is aconsequence of loss of tight junctions and intercellular gapformation.

Rac1 is required for thrombin- and histamine-induced formation of stress fibres and changes inintercellular adhesionsExpression of N17Rac1 not only induced permeability ofquiescent HUVECs, but also enhanced the permeability ofthrombin-stimulated HUVECs (Fig. 2A). To determinewhether this reflects changes in the actin cytoskeleton and/orto intercellular junctions, cells were infected with Ad-N17Rac1or microinjected with N17Rac1 protein before stimulation withthrombin or histamine. Interestingly, thrombin- and histamine-induced stress fibre formation was inhibited both in cellsinjected with N17Rac1 protein (Fig. 6A,B) and in Ad-N17Rac1-infected cells (Fig. 6C,E). Rac1 is best-characterizedfor its role in regulating membrane ruffling and lamellipodiumformation, but in some situations can act upstream of Rho tomediate stress fibre formation, for example in PDGF-stimulated fibroblasts (Ridley et al., 1992) and TNF-α-stimulated endothelial cells (Wójciak-Stothard et al., 1998). Inthrombin-stimulated cells expressing N17Rac1, gaps betweencells were still occasionally detected (Fig. 6C, arrow) althoughthey were less pronounced than in thrombin-stimulated controlcells. VE-cadherin staining in N17Rac1-expressing cells wasless fragmented than in uninfected thrombin- or histamine-treated cells but showed a meshwork-like pattern similar to thatin unstimulated N17Rac1-expressing cells (Fig. 6D,F; comparewith Fig. 5E). These results suggest that Rac1 acts upstream

or in parallel with Rho to mediate thrombin- and histamine-induced stress fibre formation. However, in contrast to theeffects of inhibiting Rho, inhibition of stress fibre formation byN17Rac1 does not correlate with decreased endothelialpermeability (Fig. 2). Instead, the increased permeabilityinduced by N17Rac1 correlates with loss of intercellularjunctions.

Although expression of V12Rac1 increased the permeabilityof quiescent HUVECs, it did not alter histamine- or thrombin-induced permeability (Fig. 2). Consistent with this, V12Rac1did not prevent thrombin- or histamine-induced fragmentationof adherens junctions or stress fibre formation, and gaps werestill observed between cells (Fig. 6G,H; and data not shown).

Cdc42 is required for responses to thrombin but nothistamineExpression of N17Cdc42 did not affect endothelialpermeability (Fig. 2A), but we have previously shown thatmicroinjection of N17Cdc42 protein prevents TNF-α-inducedstress fibre formation and the appearance of intercellulargaps in HUVECs (Wójciak-Stothard et al., 1998). Similarly,microinjection of HUVECs with N17Cdc42 protein orinfection with Ad-N17Cdc42 inhibited thrombin-inducedcontractility and reduced stress fibre formation (Fig. 7A,C,E).Ad-N17Cdc42 did not alter the distribution of F-actin orjunctional proteins in quiescent, unstimulated HUVECs (datanot shown), consistent with the lack of effect of N17Cdc42 onendothelial barrier function (Fig. 2A). In contrast, N17Cdc42did not appear to affect histamine-induced stress fibreformation or alterations to adherens junctions (Fig. 7B,D,G,H).These results are consistent with a model where Cdc42 actsto enhance contractility and intercellular gap formation inresponse to thrombin. As the response of HUVECs tohistamine differs from that to thrombin in that histamine doesnot induce cell contraction or the appearance of largeintercellular gaps (Fig. 1B,C), this may explain why Cdc42 isnot involved in histamine-induced changes to F-actin andadherens junctions.

DISCUSSION

Vasoactive agents such as thrombin and histamine rapidlyinduce vascular permeability, and this has been attributed toincreased actomyosin contractility leading to the formation ofintercellular gaps and/or to modification of intercellularjunctions (van Hinsbergh, 1997; Lampugnani and Dejana,1997). Rho, Rac and Cdc42 are prime candidates forintracellular signalling molecules regulating endothelialpermeability, as they influence both actin cytoskeletalorganization and the integrity of intercellular junctions. Wehave found that inhibition of Rho and Rac but not Cdc42significantly affects thrombin- and histamine-inducedendothelial permeability, and that this correlates with effectson intercellular junctions.

We have observed that inhibition of Rho using either C3transferase or dominant-negative RhoA expression, or ofRho kinase using Y-27632, prevented thrombin-inducedpermeability, consistent with previous observations (Essler etal., 1998; Carbajal and Schaeffer, 1999). That Rho and Rhokinase are also required for increased permeability induced by


Table 1. The Rho kinase inhibitor Y-27632 prevents loss oftight junctions and adherens junctions in thrombin- and

histamine-treated HUVECs, whereas N17Rac1 andV12Rac1 induce disassembly of intercellular junctions

Treatment Adherens junctions (%) Tight junctions (%)

None (control) 89±18 20±2Thrombin 22±8 2.8±1.5Y-27632+thrombin 85±15* 16±2*Histamine 60±9 4±3Y-27632+histamine 88±17* 19±8*Y-27632 96±16 22±5N19RhoA 101±12 18±3V12Rac1 35±11* 6±3*N17Rac1 24±1.8* 2.8±1.6*

HUVECs infected with Ad-N19RhoA, Ad-N17Rac1 or Ad-V12Rac1, ortreated with Y-27632, were stimulated as indicated with thrombin orhistamine for 5 minutes. Cells were then fixed and processed for TEManalysis. The percentage of cell-cell borders with adherens junctions and tightjunctions was determined using TEM.

Values are means ± s.d. from 2-3 separate experiments, where 20-25 lateralmembranes/monolayer from each of 3 monolayers/treatment group wereanalysed.

Comparisons between more than two groups were made using one-wayANOVA test followed by Tukey post-test for multiple comparisons. *P<0.05,based on comparisons between thrombin\histamine-stimulated cells and Y-27632-pretreated thrombin/histamine-stimulated cells or between controlcells and cells expressing Ad-V12Rac1\Ad-N17Rac1 as appropriate.


histamine (our observations), Pasteurellatoxin (Essler et al.,1998b), and oxidized low-density lipoprotein (Essler et al.,1999), demonstrates that Rho and Rho kinase are importantgenerally for regulating endothelial permeability. Tightjunctions play a central role in regulating permeability ofepithelial and endothelial cell monolayers, and we demonstratefor the first time that inhibition of RhoA and Rho kinaseprevents the loss of tight junctions induced by thrombin andhistamine. The requirement for Rho and Rho kinase inmediating increased endothelial permeability also correlateswith their involvement in stress fibre formation (Essler et al.,1998; Vouret-Craviari et al., 1998; Wójciak-Stothard et al.,1998; Ridley, 1999), and Rho may therefore play a dual rolein endothelial cells, concomitantly regulating stress fibreformation and destabilizing intercellular junctions.

How Rho affects intercellular junctions is not known, but itis interesting that in epithelial cells inhibition of Rho reducestight junction function (Nusrat et al., 1995; Jou et al., 1998)whereas it preserves tight junction function in endothelialcells. This indicates that the regulation of tight junctionintegrity differs between epithelial cells and endothelial cells,similar to what has been observed with adherens junctions,where inhibition of Rho or Rac leads to loss of cadherins fromjunctions in epithelial cells but not in endothelial cells(Braga et al., 1999). Rho might directly affect tight andadherens junction assembly/disassembly by regulating thephosphorylation status of junctional proteins via Rho kinase.Rho could also affect intercellular junctions indirectly throughits effects on actomyosin contractility. It has been suggestedthat Rho might affect tight junction structure and function inepithelial cells by altering the contractility of the cortical actinbelt (Nusrat et al., 1995; Jou et al., 1998; Madara, 1998). Inendothelial cells, Rho-mediated stress fibre assembly inducedby thrombin and histamine would lead to increased tensionexerted on junctional regions, and this could contribute tojunction disassembly.

Both thrombin and histamine rapidly induce the assembly ofstress fibres and focal contacts in HUVECs. Thrombin,however, induces the appearance of consistently largerintercellular gaps compared to histamine, and the staining ofVE-cadherin is fragmented or even lost from intercellularjunctions in thrombin-stimulated cells, whereas in histamine-stimulated cells interjunctional staining of VE-cadherin is lessdisrupted and cells do not retract. This is consistent withdifferences in endothelial cell responses to histamine andthrombin reported previously. For example, thrombin but nothistamine was shown to increase isometric tension andcontractility, and thrombin induces higher levels of myosinlight chain (MLC) phosphorylation and causes a moreprolonged increase in endothelial permeability than histamine(Boswell et al., 1992; Moy et al., 1993; Moy et al., 1996; vanHinsbergh, 1997). The molecular basis for these differencesbetween thrombin and histamine responses is not known, butit is interesting in this respect that dominant-negative Cdc42has no effect on histamine responses but reduces thrombin-induced stress fibre formation and prevents the appearance ofintercellular gaps. This suggests that Cdc42 might contributeto the increased cell contractility observed in thrombin-compared to histamine-treated cells (Moy et al., 1996). Onepossibility is that Cdc42 enhances and/or prolongs theactivation of Rho and thereby MLC phosphorylation. Indeed,

introduction of constitutively activated Cdc42 induces stressfibre formation and gap formation in HUVECs, indicating thatCdc42 can induce Rho activation (Wójciak-Stothard et al.,1998). Cdc42 may also activate actomyosin-based contractilityindependently of Rho, as PAKs and myotonic-dystrophyrelated kinase MRCKα, which are downstream targets of Racand Cdc42, can induce increased MLC phosphorylation(Leung et al., 1998; Kiosses et al., 1999; Sells et al., 1999).However, the fact that dominant-negative Cdc42 prevents gapformation between cells treated with thrombin yet has no effecton permeability supports a model where permeability canincrease even when there are no microscopically detectablegaps between cells (Andriopoulou et al., 1999).

Rac1 has previously been shown to regulate the integrity ofadherens and tight junctions in epithelial cells (reviewed inKaibuchi et al., 1999), and it is interesting that in endothelialcells either inhibition or activation of Rac1 increasespermeability. This is likely to be a consequence of changes tointercellular junctions rather than to stress fibres, as dominant-negative Rac1 and constitutively active Rac1 have opposingeffects on stress fibres. With dominant-negative Rac1, althoughVE-cadherin is still localized to intercellular borders, aspreviously reported (Braga et al., 1999), TEM analysis revealsthat junctional areas are often reorganized so that small areasof close contact are interspersed by large intercellular gaps, andfar fewer tight junctions are present. This suggests that bothadherens junctions and tight junctions in endothelial cells areweakened by expression of dominant-negative Rac1, asobserved in epithelial cells (Takaishi et al., 1997; Braga et al.,1997; Jou et al., 1998). A reduction in tight junctions andadherens junctions is also observed with constitutively activeRac1, suggesting that Rac activity needs to be preciselycontrolled to maintain the integrity of endothelial celljunctions.

In summary, our observations suggest that in endothelialcells Rho activation is important for thrombin- and histamine-induced endothelial cell permeability and that this correlateswith the ability of Rho to mediate disassembly of adherens andtight junctions. Although increased permeability generallycorrelates with increased stress fibre formation (van Hinsbergh,1997), the fact that dominant-negative Rac1 enhancesendothelial permeability yet blocks stress fibre formationindicates that the two responses are separable. Through itseffects on endothelial permeability, the Rho signalling pathwayrepresents a potential target for the treatment of pathologicalconditions involving the impairment of endothelial barrierfunction.

This work was supported by a European Community ConcertedAction grant no. QLG1-1999-01036 and the British Heart Foundation.We are particularly grateful to Ritu Garg for purification ofrecombinant proteins, to Mark Turmaine (University College London,UK) for processing HUVECs for electron microscopy, to LaurencePearl (Institute of Cancer Research, London, UK) for use of hisfluorimeter, to Caroline Dent (GlaxoWellcome Research Centre,Stevenage, UK) for providing the β-gal adenovirus and for experttuition in working with adenoviruses and to Welfide Corporation forproviding the Rho kinase inhibitor, Y-27632. We thank Ruggero Pardi(DIBIT-Scientific Institute San Rafaele, Milan, Italy) for providingHUVECs for some experiments, and Peter Clarke, J. Anthony Firth(Imperial College, London, UK) and Paul Martin (University College,London, UK) for advice on electron micrographs.

1354

REFERENCES

Andriopoulou, P., Navarro, P., Zanetti, A., Lampugnani, M. G. andDejana, E. (1999). Histamine induces tyrosine phosphorylation ofendothelial cell-to-cell adherens junctions. Atheroscler. Thromb. Vasc. Biol.19, 2286-2297.

Balda, M. S. and Matter, K. (1998). Tight junctions. J. Cell Sci.111, 541-547.

Balda, M. S., Whitney, J. A., Flores, C., Gonzalez, S., Cereijido, M. andMatter, K . (1996). Functional dissociation of paracellular permeability andtransepithelial electrical resistance and disruption of the apical-basolateralintramembrane diffusion barrier by expression of a mutant tight junctionmembrane protein, J. Cell Biol.134, 1031-1049.

Boswell, C., Majno, A. G., Joris, I. and Ostrom, K. A. (1992). Acuteendothelial cell contraction in vitro: a comparison with vascular smoothmuscle cells and fibroblasts. Microvasc. Res.43, 178-191.

Braga, V. M., Machesky, L. M., Hall, A. and Hotchin, N. A. (1997). Thesmall GTPases Rho and Rac are required for the establishment of cadherin-dependent cell-cell contacts. J. Cell Biol.137, 1421-1431.

Braga, V. M. M., Del Mashio, A., Machesky, L. and Dejana, E. (1999).Regulation of cadherin function by Rho and Rac: modulation by junctionmaturation and cellular context. Mol. Biol. Cell 10, 9-22

Burns, A. R., Bowden, R. A., MacDonell, S. D., Walker, D. C., Odebunmi,T. O., Donnachie, E. M., Simon, S. I., Entman, M. L. and Smith, C. W.(2000). Analysis of tight junctions during neutrophil transendothelialmigration. J. Cell Sci. 113, 45-57.

Burns, A. R., Walker, D. C., Brown, E. S., Thurmon, L. T., Bowden, R. A.,Keese, C. R., Simon, S. I., Entman, M. L. and Smith, C. W. (1997).Neutrophil transendothelial migration is independent of tight junctions andoccurs preferentially at tricellular corners. J. Immunol.159, 2893-2903.

Carbajal, J. M. and Schaeffer, R. C. (1999). RhoA inactivation enhancesendothelial barrier function. Am. J. Physiol.277, C955-C964.

Draijer, R. D., Atsma, E., van der Laarse A. and van Hinsberg, V. W. M.(1995). cGMP and nitric oxide modulate thrombin-induced endothelialpermeability. Circ. Res.76,199-208.

Drenckhahn, D. and Ness, W. (1997). The endothelial contractile system. In:Vascular Endothelium: Physiology, Pathology and TerapeuticOpportunities, New Horizon Series 3, (ed. G. V. R. Born and C. J. Schwartz),pp. 1-25. Stuttgart: Schattauer.

Ehringer, W. D., Edwards, M. J. and Miller, F. N. (1996). Mechanisms ofα-thrombin, histamine, and bradykinin induced endothelial permeability. J.Cell Physiol.167, 562-569.

Essler, M., Amano, M., Kruse, H. J., Kaibuchi, K., Weber, P. C. andAepfelbacher, M. (1998a). Thrombin inactivates myosin light chainphosphatase via Rho and its target Rho kinase in human endothelial cells.J. Biol. Chem.273, 21867-21874.

Essler, M., Hermann, K., Amano, M., Kaibuchi, K., Heesemann, J., Weber,P. C. and Aepfelbacher, M.(1998b). Pasteurella multocida toxin increasesendothelial permeability via Rho kinase and myosin light chain phosphatase.J. Immunol.161, 5640-5646.

Essler, M, Retzer, M., Bauer, M., Heemskerk, J. W., Aepfelbacher, M. andSiess, W. (1999). Mildly oxidized low density lipoprotein inducescontraction of human endothelial cells through activations of Rho/Rhokinase and inhibition of myosin light chain phosphatase. J Biol. Chem.274,30361-30364.

Fallaux, F. J., Kranenburg, O., Cramer, S. J., Houweling, A., vanOrmondt, H., Hoeben, R. C. and van der Erb, A. J. (1996).Characterization of 911: a new helper cell line for the titration andpropagation of early region 1-deleted adenoviral vectors. Hum. Gene Ther.7, 215-222.

Fukata, M., Kuroda, S., Nakagawa, M., Kawjiri, A., Itoh, N., Shoji, I.,Matsuura, Y., Yonehara, S., Fujisawa, H., Kikuchi, A. and Kaibuchi, K.(1999). Cdc42 and Rac1 regulate the interaction of IQGAP1 with β-catenin.J. Biol. Chem.274, 26044-26050.

Goeckeler, Z. M. and Wysolmerski, R. B. (1995). Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometrictension, actin polymerization, and myosin phosphorylation. J. Cell Biol.130, 613-627.

Hall, A. (1998). Rho GTPases and the actin cytoskeleton. Science279, 509-514.

Haraldsson, B., Zackrisson, U. and Rippe, B. (1986). Calcium dependenceof histamine-induced increases in capillary permeability in isolated perfusedrat hindquarters. Acta Physiol. Scand.128, 245-258.

Hordijk, P. L., van Klooster, J. P., van der Kammen, R. A., Michiels, F.,

Oomen, L. C. and Collard, J. G. (1997). Inhibition of invasion of epithelialcells by Tiam1-Rac signaling. Science 278, 1464-1466.

Jou, T.-S. and Nelson, W. J. (1998). Effects of regulated expression of mutantRhoA and Rac1 small GTPases on the development of epithelial (MDCK)cell polarity. J. Cell Biol. 142, 85-100.

Jou, T.-S., Schneeberger, E. E. and Nelson, W. J. (1998). Structural andfunctional regulation of tight junctions by RhoA and Rac1 small GTPases.J. Cell Biol.142, 101-115.

Kaibuchi, K., Kuroda, S., Fukata, M. and Nakagawa, M. (1999).Regulation of cadherin-mediated cell-cell adhesion by the Rho familyGTPases.Curr. Opin. Cell Biol. 11, 591-596.

Killackey, J. J. F., Johnston, M. G. and Movat, H. Z. (1986). Increasedpermeability of microcarrier-cultured endothelial monolayers in response tohistamine and thrombin. A model for the in vitro study of permeability. Am.J. Pathol. 122, 50-61.

Kiosses, W. B., Daniels, R. H., Otey, C., Bokoch, G. M. and Schwartz, M.A. (1999). A Role for p21-Activated Kinase in Endothelial Cell MigrationJ. Cell Biol. 147, 831-844

Lampugnani, M. G. and Dejana, E. (1997). Interendothelial junctions:structure, signalling and functional roles. Curr. Opin. Cell Biol. 9, 674-682.

Lampugnani, M. G., Corada, M., Caveda, L., Breviario, F., Ayalon, O.,Geiger, B. and Dejana, E. (1995). The molecular organization ofendothelial cell to cell junctions: differential association of plakoglobin, β-catenin, and α-catenin with vascular endothelial cadherin (VE-cadherin). J.Cell Biol. 129, 203-217.

Langeler, E. G. and van Hinsbergh, V. W. M. (1991). Norepinephrine andiloprost improve barrier function of human endothelial cell monolayers: roleof cAMP. Am. J. Physiol. 260, C1052-C1059.

Leung, T., Chen, X. Q., Tan, I., Manser E. and Lim, L. (1998). Myotonicdystrophy kinase-related Cdc42-binding kinase acts as a Cdc42 effector inpromoting cytoskeletal reorganization. Mol. Cell. Biol.18, 130-40.

Lum, H., Ashner, J. L., Phillips, P. G., Fletcher, P. W. and Malik, A. B.(1992). Time course of thrombin-induced increase in endothelialpermeability: relationship to Ca2+ and inositol polyphosphates. Am. J.Physiol.263, L219-L225.

Lum, H. and Malik, A. B. (1996). Mechanism of increased endothelialpermeability. Can. J. Physiol. Pharmacol.74, 787-800.

Machesky, L. M. and Hall, A. (1996). Rho: a connection between membranereceptor signalling and the cytoskeleton. Trends Cell Biol. 6, 304-310.

Madara, J. L. (1998). Regulation of the movement of solutes across tightjunctions. Annu. Rev. Physiol.60, 143-159.

Majno, G. and Palade, G. E. (1961). Studies of inflammation. I. Effect ofhistamine and serotonin on vascular permeability: an electron microscopicstudy. J. Biophys. Biochem. Cytol.11, 571-605.

Moy, A. B., Van Engelenhoven, J., Bodmer, J., Kamath, J., Keese, C.,Glaeve, I. and Shasby, S. (1996). Histamine and thrombin modulateendothelial focal adhesion through centripetal and centrifugal forces. J. Clin.Invest.97, 1020-1027.

Moy, A. B., Shasny, S. S., Scott, B. D. and Shasby, D. M. (1993). The effectof histamine and cyclic adenosine monophosphate on myosin light chainphosphorylation in human umbilical vein endothelial cells. J. Clin. Invest.92, 1198-1206.

Niimi, N., Noso, N. and Yamamoto, S. (1992). The effect of histamine oncultured endothelial cells. A study of the mechanism of increased vascularpermeability. Eur. J. Pharmacol.221, 325-331.

Nobes, C. D. and Hall, A. (1995). Rho, Rac, and Cdc42 GTPases regulate theassembly of multimolecular focal complexes associated with actin stressfibres, lamellipodia, and filopodia. Cell 81, 53-62.

Nusrat, A., Giry, M., Turner, J. R., Colgan, S. P., Parkos, C. A. Carnes,D., Lemichez, E., Boquet, P. and Madara, J. L. (1995). Rho proteinregulates tight junctions and perijunctional actin organization in polarizedepithelia. Proc. Natl. Acad. Sci. USA92, 10629-10633.

Rabiet, M. J., Plantier, J. L., Rival, Y., Genoux, Y., Lampugnani, M. G.and Dejana, E. (1996). Thrombin-induced increase in endothelialpermeability is associated with changes in cell-to-cell junction organization.Arterioscler. Thromb. Vasc. Biol.16, 488-496.

Raines, E. W. and Ross, R. (1996). Multiple growth factors are associatedwith lesions of atherosclerosis: specificity or redundancy? BioEssays18,271-282

Ridley, A. J. (1999). Stress fibres take shape. Nature Cell Biol. 1, E64-E66.Ridley, A. J. and Hall, A. (1992). The small GTP-binding protein rho

regulates the assembly of focal adhesions and actin stress fibres in responseto growth factors. Cell 70, 389-399.

Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D. and Hall, A.



(1992). The small GTP-binding protein Rac regulates growth factor-inducedmembrane ruffling. Cell 70, 401-410.

Ridley, A. J., Comoglio, P. M. and Hall, A. (1995). Regulation of scatterfactor/hepatocyte growth factor responses by Ras, Rac and Rho proteins inMDCK cells. Mol. Cell. Biol. 15, 1110-1122.

Ross, R. (1993). Atherosclerosis: current understanding of mechanisms andfuture strategies in therapy. Transplant Proc. 25, 2041-2043.

Sander, E. E., van Delft, S., ten Klooster, J. P., Reid, T., van der Kammen,R. A., Michiels, F. and Collard, J. G. (1998). Matrix-dependent Tiam1/Racsignalling in epithelial cells promotes either cell-cell adhesion or cellmigration and is regulated by phosphatidylinositol 3-kinase. J. Cell Biol.143, 1385-1398.

Sells, M. A., Boyd, J. T. and Chernoff, J. (1999). p21-activated kinase 1(Pak1) regulates cell motility in mammalian fibroblasts. J. Cell Biol.145:837-49.

Stemerman, M. B., Morrel, E. M., Burke, K. R., Colton, C. K., Smith, K.A. and Lees, R. S. (1986). Local variation in arterial wall permeability tolow-density lipoprotein in normal rabbit aorta. Arteriosclerosis6, 64-69.

Stender, S. and Hjelms, E. (1987). In vivo transfer of cholesterol from plasmainto human aortic tissue. Scand. J. Clin. Lab. Invest. Suppl. 186, 21-29.

Stöffler, H.-E., Honnert, U., Bauer, C. A., Höfer, D., Schwarz, H., Müller,R. T., Dreckhahn, D. and Bähler, M. (1998). Targeting of the myosin-Imyr 3 to intercellular adherens type junctions induced by dominant activeCdc42 in HeLa cells. J. Cell Sci. 111, 2779-2788.

Takaishi, K., Sasaki, T., Kotani, H., Nishioka, H. and Takai, Y. (1997).Regulation of cell-cell adhesion by Rac and Rho small G proteins in MDCKcells. J. Cell Biol.139, 1047-1059.

Van Hinsbergh, V. (1997). Endothelial permeability for macromolecules.Mechanistic aspects of pathophysiological modulation. Arterioscler.Thromb. Vasc. Biol. 17, 1018-1023.

Van Hinsbergh V. W. M., van Nieuw Amerongen G. P. and Draijer R.(1997). Regulation of the permeability of human endothelial monolayers. InVascular Endothelium. Physiology, Pathology and TherapeuticOpportunities(ed. ?. Born, ?. Schwartz and ?. Schattauer), pp. 61-77. NewYork: Stuttgart.

Van Nieuw Amerongen, G. P., Draijer, R., Vermeer, M. A. and VanHinsbergh, V. W. M. (1998). Transient and prolonged increase inendothelial permeability induced by histamine and thrombin. Role of proteinkinases, calcium and RhoA. Circ. Res.83, 1115-1123.

Vouret-Craviari, V., Boquet, P., Pouysségur, J. and Obberghen-Schilling,E. (1998). Regulation of the actin cytoskeleton by thrombin in humanendothelial cells: role of Rho proteins in endothelial barrier function. Mol.Biol. Cell 9, 2639-2653.

Westendorp, R. G., Draijer, R., Meinders, A. E. and van Hinsberg, V. W.(1994). Cyclic-GMP-mediated decrease in permeability of humanumbilical and pulmonary artery endothelial cell monolayers. J. Vasc. Res.31, 42-51.

Wójciak-Stothard, B., Entwistle, A., Garg, R. and Ridley, A. J. (1998).Regulation of TNF-α-induced reorganization of the actin cytoskeleton andcell-cell junctions by Rho, Rac, and Cdc42 in human endothelial cells. J.Cell. Physiol.176, 150-165.

Wu, N. Z. and Baldwin, A. L. (1992).Transient venular permeability increaseand endothelial gap formation induced by histamine. Am. J. Physiol. 262,H1238-H1247.

rho gtpases in endothelial permeability · 2001-05-03 · rho gtpases in endothelial permeability...

Documents