Endothelial permeability induced by thrombin and histamine is accompanied by actin stress fibre assembly and intercellular gap formation. Here, we investigate the roles of the Ρ family GTPases Rho1, Rac1 and Cdc42 in regulating endothelial barrier function, and correlate this with their effects on F-actin organization and intercellular junctions. RhoA, Rac1 and Cdc42 proteins were expressed efficiently in human umbilical vein endothelial cells by adenovirus-mediated gene transfer. We show that inhibition of Ρ prevents both thrombin- and histamine-induced increases in endothelial permeability and decreases in transendothelial resistance. Dominant-negative RhoA and a Ρ kinase inhibitor, Y-27632, not only inhibit stress fibre assembly and contractility but also prevent thrombin- and histamine-induced disassembly of adherens and tight junctions in endothelial cells, providing an explanation for their effects on permeability. In contrast, dominant-negative Rac1 induces permeability in unstimulated cells and enhances thrombin-induced permeability, yet inhibits stress fibre assembly, indicating that increased stress fibre formation is not essential for endothelial permeability. Dominant-negative Cdc42 reduces thrombin-induced stress fibre formation and contractility but does not affect endothelial cell permeability or responses to histamine. These results demonstrate that Ρ and Rac act in different ways to alter endothelial barrier function, whereas Cdc42 does not affect barrier function.

REFERENCES

Balda
M. S.
,
Matter
K.
(
1998
).
Tight junctions.
J. Cell Sci
111
,
541
547
Balda
M. S.
,
Whitney
J. A.
,
Flores
C.
,
Gonzalez
S.
,
Cereijido
M.
,
Matter
K.
(
1996
).
Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein,.
J. Cell Biol
134
,
1031
1049
Boswell
C.
,
Majno
A. G.
,
Joris
I.
,
Ostrom
K. A.
(
1992
).
Acute endothelial cell contraction in vitro: a comparison with vascular smooth muscle cells and fibroblasts.
Microvasc. Res
43
,
178
191
Braga
V. M.
,
Machesky
L. M.
,
Hall
A.
,
Hotchin
N. A.
(
1997
).
The small 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.
,
Dejana
E.
(
1999
).
Regulation of cadherin function by Rho and Rac: modulation by junction maturation 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.
,
Smith
C. W.
(
2000
).
Analysis of tight junctions during neutrophil transendothelial migration.
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.
,
Smith
C. W.
(
1997
).
Neutrophil transendothelial migration is independent of tight junctions and occurs preferentially at tricellular corners.
J. Immunol
159
,
2893
2903
Carbajal
J. M.
,
Schaeffer
R. C.
(
1999
).
RhoA inactivation enhances endothelial barrier function.
Am. J. Physiol
277
,
955
–.
Draijer
R. D.
,
Atsma
E.
,
van der Laarse
A.
,
van Hinsberg
V. W. M.
(
1995
).
cGMP and nitric oxide modulate thrombin-induced endothelial permeability.
Circ. Res
76
,
199
208
Ehringer
W. D.
,
Edwards
M. J.
,
Miller
F. N.
(
1996
).
Mechanisms of-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.
,
Aepfelbacher
M.
(
1998
).
Thrombin inactivates myosin light chain phosphatase 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.
,
Aepfelbacher
M.
(
1998
).
Pasteurella multocida toxin increases endothelial 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.
,
Siess
W.
(
1999
).
Mildly oxidized low density lipoprotein induces contraction of human endothelial cells through activations of Rho/Rho kinase and inhibition of myosin light chain phosphatase.
J Biol. Chem
274
,
30361
30364
Fallaux
F. J.
,
Kranenburg
O.
,
Cramer
S. J.
,
Houweling
A.
,
van Ormondt
H.
,
Hoeben
R. C.
,
van der Erb
A. J.
(
1996
).
Characterization of 911: a new helper cell line for the titration and propagation 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.
,
Kaibuchi
K.
(
1999
).
Cdc42 and Rac1 regulate the interaction of IQGAP1 with-catenin.
J. Biol. Chem
274
,
26044
26050
Goeckeler
Z. M.
,
Wysolmerski
R. B.
(
1995
).
Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation.
J. Cell Biol
130
,
613
627
Hall
A.
(
1998
).
Rho GTPases and the actin cytoskeleton.
Science
279
,
509
514
Haraldsson
B.
,
Zackrisson
U.
,
Rippe
B.
(
1986
).
Calcium dependence of histamine-induced increases in capillary permeability in isolated perfused rat hindquarters.
Acta Physiol. Scand
128
,
245
258
Hordijk
P. L.
,
van Klooster
J. P.
,
van der Kammen
R. A.
,
Michiels
F.
,
Oomen
L. C.
,
Collard
J. G.
(
1997
).
Inhibition of invasion of epithelial cells by Tiam1-Rac signaling.
Science
278
,
1464
1466
Jou
T.-S.
,
Nelson
W. J.
(
1998
).
Effects of regulated expression of mutant RhoA and Rac1 small GTPases on the development of epithelial (MDCK) cell polarity.
J. Cell Biol
142
,
85
100
Jou
T.-S.
,
Schneeberger
E. E.
,
Nelson
W. J.
(
1998
).
Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases.
J. Cell Biol
142
,
101
115
Kaibuchi
K.
,
Kuroda
S.
,
Fukata
M.
,
Nakagawa
M.
(
1999
).
Regulation of cadherin-mediated cell-cell adhesion by the Rho family GTPases.
Curr. Opin. Cell Biol
11
,
591
596
Killackey
J. J. F.
,
Johnston
M. G.
,
Movat
H. Z.
(
1986
).
Increased permeability of microcarrier-cultured endothelial monolayers in response to histamine 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.
,
Schwartz
M. A.
(
1999
).
A Role for p21-Activated Kinase in Endothelial Cell Migration.
J. Cell Biol
147
,
831
844
Lampugnani
M. G.
,
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.
,
Dejana
E.
(
1995
).
The molecular organization of endothelial cell to cell junctions: differential association of plakoglobin,-catenin, and -catenin with vascular endothelial cadherin (VE-cadherin).
J. Cell Biol
129
,
203
217
Langeler
E. G.
,
van Hinsbergh
V. W. M.
(
1991
).
Norepinephrine and iloprost improve barrier function of human endothelial cell monolayers: role of cAMP.
Am. J. Physiol
260
,
1052
–.
Leung
T.
,
Chen
X. Q.
,
Tan
I.
,
Manser
E.
,
Lim
L.
(
1998
).
Myotonic dystrophy kinase-related Cdc42-binding kinase acts as a Cdc42 effector in promoting cytoskeletal reorganization.
Mol. Cell. Biol
18
,
130
40
Lum
H.
,
Ashner
J. L.
,
Phillips
P. G.
,
Fletcher
P. W.
,
Malik
A. B.
(
1992
).
Time course of thrombin-induced increase in endothelial permeability: relationship to Ca2+and inositol polyphosphates.
Am. J. Physiol
263
,
219
–.
Lum
H.
,
Malik
A. B.
(
1996
).
Mechanism of increased endothelial permeability.
Can. J. Physiol. Pharmacol
74
,
787
800
Madara
J. L.
(
1998
).
Regulation of the movement of solutes across tight junctions.
Annu. Rev. Physiol
60
,
143
159
Moy
A. B.
,
Van Engelenhoven
J.
,
Bodmer
J.
,
Kamath
J.
,
Keese
C.
,
Glaeve
I.
,
Shasby
S.
(
1996
).
Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces.
J. Clin. Invest
97
,
1020
1027
Moy
A. B.
,
Shasny
S. S.
,
Scott
B. D.
,
Shasby
D. M.
(
1993
).
The effect of histamine and cyclic adenosine monophosphate on myosin light chain phosphorylation in human umbilical vein endothelial cells.
J. Clin. Invest
92
,
1198
1206
Niimi
N.
,
Noso
N.
,
Yamamoto
S.
(
1992
).
The effect of histamine on cultured endothelial cells. A study of the mechanism of increased vascular permeability.
Eur. J. Pharmacol
221
,
325
331
Nobes
C. D.
,
Hall
A.
(
1995
).
Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibres, 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.
,
Madara
J. L.
(
1995
).
Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia.
Proc. Natl. Acad. Sci. USA
92
,
10629
10633
Rabiet
M. J.
,
Plantier
J. L.
,
Rival
Y.
,
Genoux
Y.
,
Lampugnani
M. G.
,
Dejana
E.
(
1996
).
Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization.
Arterioscler. Thromb. Vasc. Biol
16
,
488
496
Raines
E. W.
,
Ross
R.
(
1996
).
Multiple growth factors are associated with lesions of atherosclerosis: specificity or redundancy?.
BioEssays
18
,
271
282
Ridley
A. J.
,
Hall
A.
(
1992
).
The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibres in response to growth factors.
Cell
70
,
389
399
Ridley
A. J.
,
Paterson
H. F.
,
Johnston
C. L.
,
Diekmann
D.
,
Hall
A.
(
1992
).
The small GTP-binding protein Rac regulates growth factor-induced membrane ruffling.
Cell
70
,
401
410
Ridley
A. J.
,
Comoglio
P. M.
,
Hall
A.
(
1995
).
Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac and Rho proteins in MDCK cells.
Mol. Cell. Biol
15
,
1110
1122
Ross
R.
(
1993
).
Atherosclerosis: current understanding of mechanisms and future 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.
,
Collard
J. G.
(
1998
).
Matrix-dependent Tiam1/Rac signalling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase.
J. Cell Biol
143
,
1385
1398
Sells
M. A.
,
Boyd
J. T.
,
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.
,
Lees
R. S.
(
1986
).
Local variation in arterial wall permeability to low-density lipoprotein in normal rabbit aorta.
Arteriosclerosis
6
,
64
69
Stöffler
H.-E.
,
Honnert
U.
,
Bauer
C. A.
,
Höfer
D.
,
Schwarz
H.
,
Muller
R. T.
,
Dreckhahn
D.
,
Bähler
M.
(
1998
).
Targeting of the myosin-I myr 3 to intercellular adherens type junctions induced by dominant active Cdc42 in HeLa cells.
J. Cell Sci
111
,
2779
2788
Takaishi
K.
,
Sasaki
T.
,
Kotani
H.
,
Nishioka
H.
,
Takai
Y.
(
1997
).
Regulation of cell-cell adhesion by Rac and Rho small G proteins in MDCK cells.
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 Nieuw Amerongen
G. P.
,
Draijer
R.
,
Vermeer
M. A.
,
Van Hinsbergh
V. W. M.
(
1998
).
Transient and prolonged increase in endothelial permeability induced by histamine and thrombin. Role of protein kinases, calcium and RhoA.
Circ. Res
83
,
1115
1123
Vouret-Craviari
V.
,
Boquet
P.
,
Pouyssegur
J.
,
Obberghen-Schilling
E.
(
1998
).
Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function.
Mol. Biol. Cell
9
,
2639
2653
Westendorp
R. G.
,
Draijer
R.
,
Meinders
A. E.
,
van Hinsberg
V. W.
(
1994
).
Cyclic-GMP-mediated decrease in permeability of human umbilical and pulmonary artery endothelial cell monolayers.
J. Vasc. Res
31
,
42
51
Wojciak-Stothard
B.
,
Entwistle
A.
,
Garg
R.
,
Ridley
A. J.
(
1998
).
Regulation of TNF--induced reorganization of the actin cytoskeleton and cell-cell junctions by Rho, Rac, and Cdc42 in human endothelial cells.
J. Cell. Physiol
176
,
150
165
Wu
N. Z.
,
Baldwin
A. L.
(
1992
).
Transient venular permeability increase and endothelial gap formation induced by histamine.
Am. J. Physiol
262
,
1238
–.
This content is only available via PDF.