Brief Reviews |
From the Gaubius Laboratory TNO-PG (G.P.v.N.A., V.W.M.v.H.), Leiden, and the Department of Physiology, Institute for Cardiovascular Research (G.P.v.N.A., V.W.M.v.H.), Vrije Universiteit, Amsterdam, the Netherlands.
Correspondence to Prof Dr V.W.M. van Hinsbergh, Gaubius Laboratory TNO-PG, PO Box 2215, 2301 CE Leiden, Netherlands. E-mail VWM.VANHINSBERGH{at}PG.TNO.NL
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Key Words: small GTPases vascular disorders
| Introduction |
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| The Rho GTPase Family |
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With the identification of more members and isoforms, a confusing nomenclature has developed. Members of the Rho protein family can be divided into 6 different classes consisting of the following members: Rho (RhoA, RhoB, and RhoC), Rac (Rac1, Rac2, and Rac3, which is also known as Rac1B [RhoG]), Cdc42 (Cdc42Hs, Chp, G25K, and TC10), Rnd (RhoE/Rnd3, Rnd1/Rho6, and Rnd2/Rho7), RhoD, and TTF.23 24 In this list, RhoE is the same as Rnd3, RhoF does not exist, and Cdc42, TC10, and TTF lack the R in their name to identify them as members of the Ras superfamily of proteins.
Rho, Rac, and Cdc42 are the 3 classes for which the most is known. Each has its own specific effects on the actin cytoskeleton, likely resulting from the activation of different protein subsets involved in actin polymerization.20 25 A striking feature of the activation of Rho is the formation of cytoplasmic stress fibers (SFs) in cultured cells that can form SFs and an increase in actomyosin-based contractility in cells that cannot form SFs (such as neuronal cells).2 SFs are long cytoskeletal cables or bundles of actin and myosin II/nonmuscle myosin filaments that can contract and exert tension (see below under SF Formation) and are linked to the plasma membrane at focal adhesions (FAs). Rac and Cdc42 regulate peripheral F-actin assemblies. Rac is involved in the formation of membrane ruffles and lamellipodia,1 whereas Cdc42 induces the formation of radial unipolar bundles termed microspikes or filopodia.26
All 3 protein classes can also regulate the assembly of integrin-containing FA complexes and thus regulate cell-matrix interactions and cell adhesion.17 27 Rho induces the formation of the classical FAs. These integrin-containing complexes are connected to bundles of SFs and are clustered over the basal surface of the cell, maintaining their firm attachment to the underlying substratum. Rac and Cdc42 induce the formation of the smaller focal contact sites at the cell periphery, associated with lamellipodia and filopodia.28
Recently, new members of the Rho family of small GTPases, Rnd1, Rnd2, and Rnd3/RhoE, have been identified.29 Rnd proteins have a close homology to RhoA, but they display a very distinct biochemical behavior. Rnd proteins lack GTPase activity and are constitutively in the active state. Therefore, expression levels of Rnd proteins primarily determine their involvement in signaling. Expression of these proteins in fibroblasts causes cell rounding (Rnd indicates round) and inhibits the formation of SFs, lamellipodia, and FAs and therefore appears to have an antagonistic effect on Rho and Rac.30
| General Outline of Rho-Like Small GTPase Action |
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-adrenergic agonists, sphingolipids, and
purinergic receptor agonists (see
review4 ). The activity of low
molecular weight G proteins is not directly regulated by agonist
binding to GPCRs, as is the case with the
subunit of the
heterotrimeric G proteins, but is indirectly regulated (see below).
Similar to other G proteins, low molecular weight G proteins, with the
exception of the Rnd proteins, are molecular switches, which can bind
either GDP or GTP, which results in a change in conformation. They are
active in their GTP-bound status and inactive in the GDP-bound form. In
the GTP-bound form, Rho GTPases interact with and activate
their target molecules. The capacity to cycle between 2 conformations
enables these molecules to amplify or to temporize upstream signals.
For example, it has recently been shown in neuronal cells, by fishing
for GTP-bound RhoA with the Rho-binding domain of Rho kinase, that LPA
induces an increase in GTP-bound Rho in a protein tyrosine
kinasesensitive way via G
12/13, concomitant
with growth cone collapse.35
Activation of Rho is accompanied by an increase of membrane-associated
Rho and decrease of cytosolic
Rho.36 37
The activity of small G proteins is under the direct control
of a large set of other regulatory proteins: specific factors that
activate GTPases, specific factors that turn them off, and
finally, specific factors that keep them in their inactive state (see
Figure 1
). For each of these regulating factors, several
different molecular entities have been identified, and their number
still is growing. Guanine nucleotide exchange factors
(GEFs) enhance or catalyze the exchange of GDP for
GTP.4 The slow intrinsic rate
of GTP hydrolysis of the small GTPases is enhanced by GTPase-activating
proteins, whereas guanine dissociation inhibitors (GDIs)
slow the rate of GDP dissociation from the GTPases, thereby locking the
G proteins into the inactive
state.38 These GDIs bind to
the carboxyl terminus of Rho. In this way, they prevent the
translocation of the GTPases from the cytosol to the plasma membrane
when activated.38
Ezrin, radixin, and moesin (ERM) proteins also have been implicated in
membrane recruitment. In quiescent fibroblasts, Rho, Rac, and ERM
proteins, but not Rho GDIs, are enriched in caveolar membranes compared
with plasma membranes. Stimulation with growth factors results in a
further recruitment of Rho, Rac, and ERM
proteins.39 In
endothelial cells (ECs), RhoA colocalizes with ERM
proteins.40
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Upstream signaling events, which result in the activation of
the small GTPases, are poorly understood. Very recently, 2 possible
mechanisms were identified by which GPCR stimulation results in the
activation of small GTPases. The first was the identification of a
direct link between G
and
p115RhoGEF.3 4 41 42
The second is a ligand-independent activation of the epidermal growth
factor receptor acting upstream from
Rho.43 In fibroblasts, Cdc42,
Rac, and Rho were initially found to act in a hierarchical
cascade,26 which also seems
to be true in some cases in
ECs.44 Later on, it turned
out that this cannot be taken as a general rule, inasmuch as more
recent reports indicate that Rac and Rho also have some mutually
antagonistic
effects.28 45 46
This becomes immediately clear as one compares the cell
extensionpromoting effects of Rac and Cdc42 with the cell
contractionpromoting effects of Rho.
MLC Phosphorylation
Rho GTPases regulate cytoskeletal changes involved in
cell motility, shape, and contraction. The dominant regulatory system
of the nonmuscle and smooth muscle F-actin cytoskeleton involves
activation of myosin by phosphorylation of the myosin
(regulatory) light chains
(MLCs).16 Activated
myosins bundle F-actin, resulting in the formation of F-actin
filaments, of which the stress fibers are the most prominent group.
Evidence is now accumulating that Rho GTPases are important regulators
of MLC phosphorylation and F-actin
organization.
MLC Kinases
The myosin II molecule is composed of 2 heavy and 2
distinct light chains, an essential and a regulatory one.
Phosphorylation of the regulatory MLC increases ATPase
activity of the myosin molecule and regulates myosin motor function of
the actin/myosin system as its primary function. In the
nonphosphorylated folded form, myosin cannot assemble
into filaments. Phosphorylation also promotes myosin
filament assembly by a conformation change in the myosin molecule. MLC
phosphorylation is accomplished by a set of specific
kinases, the classic
Ca2+/calmodulin-dependent MLC
kinases (MLCKs).47 Several
MLCK isoforms, in the range of 130 to 150 kDa and a splice variant of
210 kDa, have been identified. Ser19 is the major site of
phosphorylation induced by agonists that stimulate MLC
phosphorylation. Under conditions of maximal
stimulation, Thr18 also becomes
phosphorylated.
In search of other MLCKs, Gallagher et al48 isolated a developmentally regulated MLCK, which was called embryonic MLCK. Embryonic MLCK seems to be an unique kinase because it is immunologically distinct from the above-mentioned 210-kDa MLCK and is also regulated by Ca2+. ECs significantly express embryonic MLCK. Besides embryonic MLCK, ECs express an endothelium-specific MLCK with a molecular mass of 214 kDa, which seems to have a role in endothelial permeability.49 Recently, evidence was obtained for a Ca2+-independent MLC kinase, distinct from MLCK, involved in Ca2+ sensitization of smooth muscle cell contraction. This kinase phosphorylates MLC on Ser19 or Thr18,50 but its molecular identity remains to be elucidated.
Rho GTPases and MLC
Phosphorylation
Several mechanisms by which the small G proteins
regulate MLC phosphorylation have been elucidated (see
Figure 2
). The first Rho GTPase target shown to be involved
in MLC phosphorylation was Rho kinase. Rho kinase acts
itself as an MLCK,51
at least in vitro. Although the apparent
Km value
(0.91 µmol/L) of Rho kinase for the MLC is lower than that (52
µmol/L) for MLCK, the molecular activity of Rho kinase for MLC is
3 times lower than that of MLCK. This may be the result of the lower
amount of Rho kinase present in cells compared with the amount of
MLCK, as in blood
platelets,52 or this may
be the result of a different localization within the cell. Therefore,
the Ca2+/calmodulin-dependent
MLCK is thought to be the primary regulator of MLC
phosphorylation at
Ser19.53 54
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Rac and Cdc42 activate another family of kinases
(the p21-activated kinases [Pak]) that is involved in MLC
phosphorylation. Three isoforms of Pak have been
identified: Pak1 (
Pak), Pak2 (
Pak), and Pak3 (ßPak). In HeLa
cells, overexpression of Pak reduces MLCK activity and MLC
phosphorylation,55
and cell spreading was inhibited. In vitro, it has been shown that Pak1
phosphorylates MLCK and inhibits the MLCK activity. This
might in part explain the antagonistic effects of Rac and
Rho. In addition to inhibiting MLCK activity, active Pak2 itself was
shown to increase MLC phosphorylation in
ECs.56 57 Pak2
phosphorylates the MLC on Ser19, in contrast to MLCK and
Rho kinase, which phosphorylate MLC on Ser19 and Thr18. ECs
contract on exposure to Cdc42 or activated Pak2. Pak is
required to initiate
Ca2+/calmodulin-independent cell
retraction. These apparently contradictory findings probably do not
reflect Pak isoform differences, inasmuch as other investigators have
demonstrated that Pak1 increases MLC phosphorylation in
ECs by use of microinjection of active
Pak1.58 A
physiological role of Pak remains to be elucidated,
but Pak seems to be involved in cell
migration.58
Besides phosphorylating MLC directly, Rho GTPasedependent kinases regulate MLC phosphate levels by inhibition of the MLC dephosphorylation. This reaction is as important as the phosphorylation reaction is for controlling the extent of MLC phosphorylation. MLC dephosphorylation is accomplished by a specific myosin phosphatase, myosin phosphatase type 1 (PP1M).59 Initially, it was assumed that the phosphatase activity was a steady one, but now it is known that phosphatase activity is regulated by other factors, including small GTPases. Activity of PP1M is inhibited by phosphorylation of the myosin binding subunit of PP1M by Rho kinase.60 61 62 In many cases, inhibition of PP1M accounts for the major contribution of Rho kinase to the elevation of MLC phosphorylation.52 54 63
SF Formation
One of the most striking actin structures in the
vasculature, which have attracted attention for a long time, are SFs.
In ECs in vivo, SFs occur mainly in large
arteries64 65 and
to a lesser extent in the entire
microcirculation,66 but they
are largely absent in the venous
system.65 67 Many
studies have shown that SFs develop during EC adaptation to unfavorable
or pathological situations, including wound healing,
atherosclerosis, and
hypertension.64 67 68 69 70 71
Remarkably, for each of these conditions, evidence has been obtained
for the involvement of Rho-like small GTPases (see below in Functional
Studies).
As pointed out above, Rho plays an eminent role in the formation of SFs, but how does Rho induce SFs? Many targets of Rho have been identified, including rhoteckin, rhophilin, PRK2, and citron.5 The target that has received major attention, however, is Rho kinase. Activation of Rho kinase results in an increased MLC phosphorylation. This MLC phosphorylation precedes the appearance of SFs and seems to be an early event in SF formation.16 MLC phosphorylation promotes myosin filament assembly and actin-activated myosin ATPase activity, resulting in the bundling of actin filaments and the generation of tension.17
However, other targets of Rho kinase are also involved in
the regulation of SF formation (see
Figure 3
).72 Rho
kinase phosphorylates proteins of the ERM family, promoting
their interaction with actin and transmembrane receptors.
Phosphorylation of
adducin73 and of the Na-H
exchanger74 by Rho kinase is
also involved in SF formation in a unknown way.
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Rho kinase also phosphorylates LIM kinase.75 76 LIM kinase is a cofilin-inactivating kinase.77 Cofilin exhibits actin-depolymerizing activity. Thus, activation of LIM kinase inhibits depolymerization of F-actin and in this way can promote the formation of SFs. Recently, the picture became even more complicated, inasmuch as LIM kinase was found to be activated also by Pak kinases.78 This means that LIM kinase could also be involved in the formation of actin structures induced by Rac and Cdc42.
Furthermore, Rho kinase activity alone is not sufficient for proper SF formation. Expression of a dominant active mutant of Rho kinase induces the formation of stellate SFs, which differ from the parallel SFs found in normal cells or induced by the activation of Rho.79 Recently, it has been shown that an appropriate balance of Rho kinase activity and Dia (another target of Rho) activity can induce SF formation, which is indistinguishable from Rho-induced SF formation. The expression of a dominant active mutant of Dia alone results in weak formation of parallel SFs.72 79 80 Dia is a profilin-binding protein and probably contributes to SF formation by localizing profilin-bound actin to sites where Rho is active. Phosphatidylinositol 4,5-biphosphate (PIP2) is also likely to be involved in SF formation, inasmuch as intracellular levels of PIP2 can be increased by phosphatidylinositol 4-phosphate 5-kinase after the activation of Rho81 , and PIP2 is known to promote actin polymerization.82
| Functional Studies |
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The dominant regulatory mechanism of nonmuscle and smooth
muscle contraction is a
Ca2+/calmodulin-dependent MLC
phosphorylation.86
However, the
[Ca2+]i is not
always paralleled by the MLC phosphorylation level.
So, other mechanisms in addition to a
Ca2+-dependent MLC
phosphorylation must exist. It is firmly established
now that Rho plays an important role in Ca2+
sensitization. A decade ago, the importance of G proteins in
Ca2+ sensitization was suggested from
experiments with permeabilized blood vessels. At a
constant [Ca2+]i,
nonhydrolyzable GTP analogues or GTP plus
-adrenergic agonists
induced a
contraction.87 88
The involvement of Rho in Ca2+ sensitization
was demonstrated by use of the specific Rho inhibitor
C3-transferase.89 The
Ca2+ sensitization is accompanied by a
translocation of Rho from the cytoplasm to the cell
membrane.90
The first evidence that Rho kinase mediates the effects of Rho in VSMC contraction came from a study of Uehata et al,84 who developed a specific inhibitor of Rho kinase, Y-27632, and showed that Rho kinase is involved in Ca2+ sensitization induced by a variety of agonists, including phenylephrine, thrombin, serotonin, endothelin-1, and the thromboxane agonist U-46619. This finding was confirmed by other studies.91 92 RhoA and Rho kinase were shown to be present in a variety of VSMCs.93 An interesting observation was that MLCK activity is not necessarily required for Rho kinaseinduced contraction.94 Inhibition of Rho kinase did not affect the basal blood pressure, vessel tone, or heart rate.84,85 In vitro studies indicated that Rho kinase can increase MLC phosphorylation by inhibition of PP1M and by direct MLC phosphorylation.51 60 In a swine model of coronary artery spasm, it has recently been demonstrated in vivo with the use of hydroxyfasudil (another new inhibitor of Rho kinase) that Rho kinase was involved in agonist-induced hyperphosphorylation of MLC at Ser19 and Thr18.85
Rho kinase is also involved in VSMC migration and proliferation. These processes are essential for the remodeling of the vessel wall, which also contributes to the development of hypertension, (re)stenosis, and atherosclerosis (see below).95 96 Interestingly, the Rho-related protein Rnd1 inhibits Ca2+ sensitization of rat smooth muscle and acts as a natural antagonist of Rho in Ca2+ sensitization.30 The expression of Rnd1 in VSMCs was increased by the sex hormones estradiol and progesterone. These hormones are known to reduce vascular contractility.97
Endothelial
Permeability
The endothelium is the main barrier
that regulates the extravasation of blood constituents to the
surrounding tissues. The most prevalent type of dysfunction of this
barrier, which can result in vascular leakage, involves actin-myosin
interaction at the margins of ECs via a
Ca2+/calmodulin-dependent
activation of the MLCK (see
reviews98 99 ). This
process has many characteristics in common with smooth muscle cell
contraction. Rho proteins have been implicated in increased
endothelial permeability. Comparable to the involvement
of Rho in the tonic component of VSMC contraction, Rho is involved in
increased endothelial
permeability.100 101
Initial evidence of the involvement of Rho proteins in cell barrier (dys)function came from studies involving epithelial cell monolayers.102 The first experiments in ECs using the nonselective Clostridium difficile toxin B, which inhibits Rho, Rac, and Cdc42, showed that Rho proteins are essential for a proper endothelial barrier function.103 Additional experiments showed that the specific Rho inhibitor C3 exoenzyme inhibited thrombin- but not histamine-induced endothelial hyperpermeability and MLC phosphorylation in human umbilical vein ECs.100 101 Furthermore, it has been demonstrated that thrombin directly activates RhoA, but not Rac1, in ECs.104 Other Rho-like small G proteins may be involved.105 However, it is less likely that small GTPases such as Rac and Cdc42 are involved in endothelial barrier hyperpermeability, inasmuch as they stimulate cell extension instead of cell contraction. A model was hypothesized in which the transient Ca2+-dependent increase in endothelial permeability can be prolonged or sensitized by activation of RhoA and Rho kinase, similar to Ca2+ sensitization in VSMCs. Earlier studies have indicated the importance of inactivation of myosin phosphatase by thrombin.106 107 Essler et al100 have shown that transient inhibition of PP1M by thrombin is Rho dependent. Rho-mediated endothelial retraction is not restricted to thrombin-induced endothelial permeability but seems to be involved in many more cases of increased endothelial permeability. Pasteurella multocida toxin, endotoxin, and minimally oxidized LDL also induce an endothelial barrier dysfunction via Rho/Rho kinase, even without an increase in [Ca2+]i.108 109 Leukocytes probably use the same mechanism to transmigrate through endothelial monolayers (see Transmigration of Circulating Cells). Interestingly, Siess et al110 have shown that LPA, a well-known Rho activator, is probably a major active component of oxidized LDL with respect to endothelial activation and accumulates in atherosclerotic plaques, and LPA has been shown to increase endothelial permeability.111 Endothelial barrier dysfunction is a hallmark of the early atherosclerotic lesion development. This suggests an important contribution of the Rho-mediated endothelial permeability increase to the development of atherosclerosis.
In addition to the inhibition of the myosin phosphatase by Rho, other mechanisms of Rho action may be involved in endothelial barrier dysfunction. In the case of peroxyvanadate-induced endothelial hyperpermeability, a Rho-mediated activation of the (endothelial) MLCK by tyrosine phosphorylation of the MLCK has been reported.112 It remains to be investigated whether vasoactive compounds, such as thrombin and LPA, induce such an activation of MLCK by tyrosine phosphorylation and whether Rho kinase is involved in MLCK activation.
Another function of Rho proteins, which may be
involved in the regulation of endothelial barrier
function, is the regulation of cell-cell interactions. Such a mechanism
has been demonstrated in epithelial
cells.113 114
However, Braga et al115 have
shown that ECs are exceptional in this sense. They have demonstrated
that in contrast to activity in other cell types, Rho activity is not
necessary for cadherin-based endothelial cell-cell
interaction and that vascular endothelial cadherin
localization was insensitive to the inhibition of either Rho or Rac.
Furthermore, Essler et al100
have shown that inhibition of Rho by C3-transferase does not prevent
the thrombin-induced dissociation of catenins from the cytoskeleton.
Wojciak-Stothard et al44 have
shown that the Cdc42-, Rac-, and Rho-dependent tumor necrosis
factor-
induced stress fiber formation is also accompanied by an at
least partly Cdc42-, Rac-, and Rho-independent dispersion of vascular
endothelial cadherin from intercellular junctions.
Thus, at the moment, there is no firm support for the role of Rho
proteins in the direct regulation of adherens junction organization in
ECs.
Future studies will be necessary to verify whether a similar Rho-induced Ca2+-sensitization mechanism underlies the increased permeability that is enhanced by leukocytes and humoral factors circulating in patients with prolonged edema as well as in the increased permeability in arterial segments after stent implantation.116
Platelet Activation
Similar to VSMC contraction, Rho/Rho kinase signaling
has been implicated in MLC phosphorylation in
activated blood
platelets.52 79 117 118
In fact, Rho kinase was first isolated from
platelets.119
Phosphorylation at Ser19 of platelet MLC increases
an actomyosin contractile response that is involved in platelet
shape change and secretion. Inhibition of Rho kinase prevented ATP
secretion.52 Several reports
indicated that the agonist-induced Ca2+ rise
is not required for platelet shape
change.120 121
Rho signaling is also involved in platelet adhesion to
fibrinogen.122 Activation of
Rho kinase is accompanied by a translocation of Rho kinase to the actin
cytoskeleton.123 There is
evidence that Rho kinase contributes to a great extent to the
platelet secretion induced by agonists and at low concentrations of
thrombin (a strong agonist) but not at high concentrations of
thrombin.117 120
The shape changes induced by weak agonists are fully prevented by the
inhibition of Rho kinase.
Cardiac Myocyte Hypertrophy
Several
reports124 125 126
have indicated that the Rho/Rho kinase pathway is important for
hypertrophic signaling in cultured cardiac myocytes induced by
adrenergic agonists, angiotensin II, and endothelin-1 (see
review127 ). A role for the
Rho GTPases in myocyte hypertrophy is supported by several
recent studies demonstrating the effects of active and inactive forms
of RhoA on hypertrophic target gene
expression.124 126 128 129 130
Similarly, expression of active Rac1 stimulated the hypertrophic
program, whereas expression of inactive Rac was
inhibitory.125 129
The effects of RhoA on the morphological and cytoskeletal aspects of
the hypertrophy were less clear, with recent reports giving
conflicting
results.126 128 131
Cardiac-specific overexpression of RhoA in mice could not unequivocally
confirm a major role of RhoA in cardiac
hypertrophy.132
Overexpression of RhoA resulted in atrial, but not
ventricular, enlargement and was accompanied by contractile
failure. Interestingly, it has been suggested that RhoA regulates
cardiac sinus and atrioventricular nodal function.
Cardiac-specific overexpression of active Rac1 resulted in 2 different
phenotypes: a (lethal) dilated
cardiomyopathy and a resolving transient cardiac
hypertrophy in juvenile
mice.133 134
Involvement of Rho GTPases in Cell
Motility
Vascular Smooth Muscle Migration
Many vascular remodeling processes depend on the
motility of vascular cells, which requires a coordinated rearrangement
of the actin cytoskeleton and cell-matrix interactions. In
cardiovascular diseases, such as hypertension,
atherosclerosis, and restenosis after
angioplasty, vascular remodeling requires changes in the VSMC
cytoskeleton.
The migratory response can be induced by signals from chemoattractants and growth factors as well as by mechanical wounding.24 In a large variety of cell types, an essential role of Rac in cell migration has been established.24 135 On the one hand, Rac is important for the formation of protrusion of lamellipodia at the leading edge of the cells and forward movement. On the other hand, Rac seems to be involved in cell retraction at the trailing edge via Pak.58 136 Cdc42 has been shown to be involved in chemoattractant gradient sensing; ie, Cdc42 regulates cell polarity and the direction of migration.137 138 Data concerning the role of Rho in cell migration are still conflicting and may depend on the extent of Rho activation. A low degree of Rho activity is necessary for the generation of adhesive forces and probably for cell retraction.135 139 A high degree of Rho activity seems to inhibit migration in some cases through the formation of strong FAs,24 135 but these studies were performed in nonvascular cells. In VSMCs, Rho/Rho kinase signaling is involved in cell migration in wound healing assays.73 95 Recent preliminary data indicate that the inhibition of Rho kinase reduces neointimal formation in several animal models and underscore the importance of RhoA/Rho kinase signaling in VSMC migration.140 141 How the responses of the different GTPases are coordinated remains an interesting area for future research.
Endothelial Migration and
Angiogenesis
Comparable to VSMC migration, RhoA/Rho kinase signaling
has been implicated in endothelial
migration.142 143 144 145
Angiogenesis, the formation of new blood vessels from existing ones, is
a process that depends not only on proliferation but also on the
migration and invasion of ECs. Lee et
al146 recently identified
sphingosine-1-phosphate as a new angiogenic factor and showed that
sphingosine-1-phosphateinduced angiogenesis was completely blocked by
the inhibition of Rho with C3-transferase. Similarly, the inhibition of
RhoA/Rho kinase signaling reduced tube formation in the Matrigel
assay147 and angiogenesis in
the chick embryo.147
Interestingly, Rho is involved in activation of the vascular
endothelial growth factor receptor
VEGFR2.148 The involvement
of similar signal transduction cascades seems appropriate for the
mutual interaction between angiogenesis and endothelial
permeability. Vascular leakage can be an early manifestation of
angiogenesis and results in the extravasation of a fibrinous exudate,
providing a provisional matrix for the ingrowth of ECs. It now seems
that Rho activation is an ongoing process in angiogenesis and that
initial alterations in actinnonmuscle myosin interaction prepare the
ECs for migration and ingrowth. When the importance of Rac/Pak
signaling in EC migration is
considered,58 it is likely
that Rac activation is also involved in
angiogenesis.
Transmigration of Circulating Cells
For lymphocyte transmigration, a multistep model of
lymphocyte-EC recognition and recruitment of lymphocytes from the blood
has been proposed in which the activation of Rho GTPases plays a
central role.149 This model
involves (1) contact through microvillous receptors and rolling of
lymphocytes and (2) activation of lymphocytes through G proteinlinked
receptors, which trigger (3) integrin adhesion to vascular ligands in
seconds through an intracellular pathway involving the small
GTP-binding protein Rho, followed by (4) diapedesis. This general model
implicates changes in Rho GTPase activity in the migrating and in the
barrier-forming (endothelial) cell. This model also
seems to be applicable to leukocyte transmigration and tumor cell
invasion.150 151 152
Rho, Rac, and Cdc42 regulate the actin cytoskeleton dynamics necessary for chemotaxis of circulating cells.137 153 Stimulation of leukocytes with fMLP or interleukin-8 induces a rapid activation of RhoA. In human neutrophils (and eosinophils), fMLP also induces a very rapid and transient activation of Rac.154 Inhibition of Rho by C3 exoenzyme blocks the adhesion of neutrophils to fibrinogen,155 and inhibition of Rho kinase completely inhibits chemotactic peptideinduced MLC phosphorylation and neutrophil migration.156 Whereas activation of Rho GTPases is clearly critical for the adhesion and migration of circulating cells, the regulation of their activity and the relative individual contribution of each of the distinct GTPases are far from being resolved. Comparable to transmigration of leukocytes and lymphocytes, tumor cell invasion involves RhoA/Rho kinase signaling.150 157 158
Evidence that adhesion of circulatory cells to the endothelium directly activates Rho signaling in the endothelium, without the involvement of an intermediate inflammatory mediator, is now accumulating. In the multistep model outlined above, lymphocyte integrin clustering and adhesion to the counterreceptors on the endothelium takes a central place. Integrin-mediated adhesion can also activate Rho signaling in the EC and thus cause local endothelial retraction. Activation of Rho in ECs in this way might facilitate the transmigration of these cells across the endothelium,159 160 by creating small pores in the endothelial barrier comparable to those involved in the passage of macromolecules,161 and it is accompanied by an increased MLC phosphorylation162 163 164 165 and the formation of SFs159 161 163 166 (see Endothelial Permeability). In contrast to the activation of circulatory cells by chemoattractants, in which Rho, Rac, and Cdc42 are involved, activation of the endothelium by circulatory cells probably involves the activation of Rho but not of Rac and Cdc42.166
| Pharmacological Modulation of Rho-Like Small GTPase Signaling |
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Rho proteins are targets for covalent modification by toxins of many pathogenic bacteria (see reviews10 172 173 ). This suggests an important role for Rho in vivo. Among the bacterial toxins, several specific activators and inhibitors of Rho function are currently known. Toxin B from Clostridium difficile is a general inhibitor of Rho, Rac, and Cdc42. C3-transferase from Clostridium botulinum has a high specificity toward Rho.172 173 A new development is the in vivo application of C3 transferase in mice via an osmotic minipump.174 Cytotoxic necrotizing factor-1 from Escherichia coli and Pasteurella multocida toxin are specific activators of Rho.108 175
The strategies indicated above are based on the inhibition of all of the functions of either one or more Rho-like small GTPases. Specificity might be achieved by interfering with the function of specific downstream effector or upstream regulator molecules. The latter principle has been applied in the development of peptides based on Rac GTPase-activating protein molecules, which are effective in the inhibition of Rac-mediated oxidant production.167 176 The former principle was used in the development of several inhibitors with high specificity for Rho kinase compared with MLCK and protein kinase C: Y-27632 and related compounds84 177 and fasudil and its hydroxyl derivative.85 Both compounds can be used in vivo without major effects on basal heart rate and blood pressure, and no changes in blood and urine chemistry have been reported sofar.84 85 151 Y-27632 reduced elevated blood pressure in several animal models of hypertension.84 Hydroxyfasudil reduced coronary artery vasospasm in a swine model.85 SCH51344 has been shown to inhibit certain downstream activities of Rac, including regulation of the cytoskeleton and transformation, but it also interferes with Ras signaling.178 179
Posttranslational lipid modifications are important for interactions with GEFs and for downstream functions of Rho and are subject to regulation.13 An exciting new development with therapeutic consequences is the use of statins as inhibitors of Rho function. Statins are inhibitors of the enzyme ß-hydroxy-ß-methylglutaryl coenzyme A reductase and are used in lipid-lowering therapy. Statins prevent isoprenylation of Rho proteins as a side effect of inhibition of the ß-hydroxy-ß-methylglutaryl coenzyme A reductase. Isoprenylation is necessary for targeting of RhoA to the plasma membrane.37 180 181 This may contribute to the nonlipid-related beneficial effects of statins, including reduced smooth muscle cell proliferation,182 reduced inflammation and endothelial permeability,183 184 stroke protection,185 and increased fibrinolytic activity.186 However, relatively high concentrations of statins in the micromolar range are necessary to prevent isoprenylation of Rho in vitro.37 180 187 188 The reduction of stroke size by statins in an experimental mouse model via this mechanism underscores the importance of this recognition.174 185 Specificity could be obtained by the use of inhibitors of protein isoprenylation, ie, inhibitors of farnesyltransferase and geranylgeranyltransferases, as was successful in blocking the oncogenic properties of Ras.189 Whether this alternative really will be an advantage in the case of inhibition of Rho-like small GTPases remains to be seen, because the statin drugs themselves are generally regarded as safe.
| Perspective |
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However, our knowledge regarding the precise role of Rho GTPases in these disorders remains fragmentary, and we are in an early stage of learning how these processes are integrated and what the initial triggers are. Furthermore, one has to be aware that similar processes can be regulated differently in distinct cell types. This is demonstrated by the following examples: thrombin-induced Ca2+ mobilization is blocked by the inhibition of Rho in fibroblasts but not in ECs100 190 ; Rho is involved in cadherin function in epithelial cells but not in ECs115 191 ; and phosphorylation of MLC is induced by Pak in ECs, whereas MLC kinase activity is reduced by Pak in HeLa cells.55 56 58
Studies involving the function of Rho-like GTPases have resulted in the identification of new targets for pharmacological intervention. The detailed present knowledge of the structure of these proteins will facilitate the development of additional drugs with higher specificity. The discovery that statin drugs inhibit Rho function will obtain clinical application. It is a future challenge to apply the present knowledge of Rho-like GTPase function in the specific treatment(s) of vascular disorders.
| Acknowledgments |
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Received April 25, 2000; accepted August 22, 2000.
| References |
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12/13 subunits in neuronal cells:
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