Physiol Rev 93: 1659 –1720, 2013
doi:10.1152/physrev.00021.2012
SMALL G PROTEINS IN THE CARDIOVASCULAR
SYSTEM: PHYSIOLOGICAL AND PATHOLOGICAL
ASPECTS
Gervaise Loirand, Vincent Sauzeau, and Pierre Pacaud
INSERM, UMR S1087; University of Nantes; and CHU Nantes, l’Institut du Thorax, Nantes, France
Loirand G, Sauzeau V, Pacaud P. Small G Proteins in the Cardiovascular System:
Physiological and Pathological Aspects. Physiol Rev 93: 1659 –1720, 2013;
doi:10.1152/physrev.00021.2012.—Small G proteins exist in eukaryotes from yeast
to human and constitute the Ras superfamily comprising more than 100 members. This
superfamily is structurally classified into five families: the Ras, Rho, Rab, Arf, and Ran
families that control a wide variety of cell and biological functions through highly coordinated regulation
processes. Increasing evidence has accumulated to identify small G proteins and their regulators as key
players of the cardiovascular physiology that control a large panel of cardiac (heart rhythm, contraction,
hypertrophy) and vascular functions (angiogenesis, vascular permeability, vasoconstriction). Indeed,
basal Ras protein activity is required for homeostatic functions in physiological conditions, but sustained
overactivation of Ras proteins or spatiotemporal dysregulation of Ras signaling pathways has pathological consequences in the cardiovascular system. The primary object of this review is to provide a
comprehensive overview of the current progress in our understanding of the role of small G proteins
and their regulators in cardiovascular physiology and pathologies.
L
I.
II.
III.
IV.
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VI.
VII.
INTRODUCTION
SMALL G PROTEINS OF THE RAS...
RAS PROTEINS IN HEART FUNCTION...
RAS PROTEINS IN VASCULAR...
“RASOPATHIES”-ASSOCIATED...
THE RAS SUPERFAMILY PROTEINS...
CONCLUDING REMARKS
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I. INTRODUCTION
The cardiovascular system is a complicated multiorgan
and multicellular system that constantly and instantaneously integrates, with an extreme accuracy, a considerable amount of input information to adjust blood circulation and therefore nutrient and oxygen delivery to
the whole organism according to its demand. This adjustment involves coordinated control of a great number of
different functions of cardiac and vascular cells and requires an extremely high level of spatial and temporal
regulation of signaling pathways driving these cellular
processes. Over the past two decades, biochemical, functional, and genetic analyses as well as observations from
model organisms and human pathology support the notion that monomeric G proteins of the Ras superfamily
are essential elements of the spatiotemporal fine tuning of
numerous cardiovascular functions and also of cardiovascular development.
Ras proteins act as binary molecular switches able to
regulate a wide range of cellular processes including pro-
liferation, differentiation, metabolism, contraction, motility, survival, and apoptosis. They form complex signaling networks that can be activated by a large array of
external upstream stimuli and can achieve highly specific
cellular effects thanks to a myriad of regulators and effectors. Accordingly, basal Ras protein activity is required for homeostatic functions in physiological conditions, but spatiotemporal dysregulation or sustained
overactivation of Ras protein signaling has pathological
consequences in the cardiovascular system. In this review, we focus on our current understanding of the roles
the different subfamilies of Ras proteins in cardiovascular development, function, and diseases. The studies presented here will help reveal the major challenges in the
field and the duality of Ras proteins, displaying both
beneficial and deleterious effects in the cardiovascular
system.
II. SMALL G PROTEINS OF THE RAS
SUPERFAMILY
Ras proteins (H-, N-, and K-Ras) are the founding members of a large superfamily of monomeric small GTPases
(20 –25 kDa). These proteins are best known for their
ability to serve as molecular switches regulating diverse
cellular processes that include cell cycle progression, cell
survival, actin cytoskeletal organization, cell polarity
and movement, and vesicular and nuclear transport (62,
161, 266). Both unicellular and multicellular organisms
express Ras proteins. The human Ras superfamily is composed of 166 members, which is divided into five major
0031-9333/13 Copyright © 2013 the American Physiological Society
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LOIRAND, SAUZEAU, AND PACAUD
branches: 36 Ras proteins, 20 Rho, 27 Arf/Sar, 1 Ran, 61
Rab, and 11 “unclassified” sequences (TABLE 1). Although the separation of these five protein families was
an evolutionary event that predated the expansion of
eukaryotes, all these proteins present sequence and functional similarities (201, 263, 397).
A. Structures and Domains (Figure 1)
The basic structure of Ras proteins consists of a central
six-stranded mixed ␤-sheet surrounded by five ␣-helices.
The ⬃20 kDa core G domain (corresponding to Ras residues 4 –166) is conserved among all Ras superfamily proteins and is involved in GTP binding and hydrolysis (355).
This domain is comprised of five polypeptides loops designated G1 through G5. The diphosphate-binding loop G1
(also known as P-loop), with the consensus sequence
GXXXXGKS/T, connects the ␤1 strand to the ␣1 helix and
contacts the ␣- and ␤-phosphates of the guanine nucleotide.
The connection between the ␣1 helix and the ␤2 strand
corresponds to G2 and contains a conserved threonine residue (Thr35) involved in Mg2⫹ coordination. The G3 domain, at the NH2 terminus of the ␣2 helix, links the sites for
binding Mg2⫹ and the ␥-phosphate of GTP. The G4 domain
that links the ␤5 strand and the ␣4 helix recognizes the
guanine ring. The G5 loop, located between ␤6 and helix
␣5, reinforces the guanine base recognition site (195, 266,
397). Structurally, the Rho family proteins are distinguished from the Ras family by the presence of an extra
10 –15 amino acids between helix ␣3 and strand ␤5 (47).
The function of this insert region remains to be elucidated.
The transition between the GTP- and the GDP-bound form
of Ras proteins is accompanied by conformational changes
that dramatically affect its affinity for downstream signaling molecules. The crystal and NMR structures, in the active and inactive state, revealed that structural differences
are primarily confined to two highly mobile regions, designated as switch I (corresponding to Ras residues 30 –38)
and switch II (corresponding to Ras residues 59 –76) (195,
266, 397, 523).
Most proteins of the Ras superfamily are localized to membranes by virtue of posttranslational modification with lipid
moieties and posttranslational lipid processing (8). The
only significant differences in the sequence of Ras proteins
resides in COOH-terminal 25 amino acids of the hypervariable domain (HVR) and probably account for diversity of
the biological functions of Ras proteins The HVR contains
the protein sequences that are involved in the association of
Ras proteins with membranes. This domain commonly terminates with a CAAX motif that signals for farnesyl or
geranylgeranyl isoprenoid addition to the cysteine residue.
This process is catalyzed in the cytoplasm by either type I
geranylgeranyltransferase (GGT) or farnesyltransferase (FT).
Farnesyltransferase inhibitors (FTIs), by preventing isopre-
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nylation of normally farnesylated Ras proteins inhibit their
activity. Rab proteins contain a COOH-terminal HVR that
terminates with cysteine-containing motifs that are modified by addition of geranylgeranyl lipids, with some undergoing carboxymethylation (8, 62, 195, 266, 397). Unlike
other members of Ras superfamily, Ran protein is not lipid
modified but contains a COOH-terminal extension that is
essential for its functions (500).
B. The GDP-GTP Cycle and Regulatory
Proteins
Ras proteins are transducers that couple cell surface receptors to intracellular effector pathways. Ras-superfamily
small GTPases act by a conserved mechanism (FIGURE 2)
(47, 266, 283, 284). Ras proteins cycle between “on” and
“off” conformations that are conferred by the binding of
GTP and GDP, respectively. Ras functions require the participation of distinct regulatory proteins to control the
GDP/GTP cycling rate. Indeed, the extent and duration of
Ras activation in cells depends on the interplay between a
variety of negative and positive regulators of the Ras cycle.
Guanine dissociation inhibitors (GDIs) maintain small GTPases in the inactive state. GTP exchange factors (GEFs) promote the active GTP-bound state by facilitating the exchange of GDP for GTP. Ras proteins have only a low
intrinsic GTPase activity that is increased by interaction
with GTPase activating proteins (GAPs).
1. GAPs
The basic function of GTPase-activating proteins is to promote GTP hydrolysis leading to inhibition of the active state
of small G proteins. Based on the consensus sequence of the
typical G domains, the human genome codes for ⬃150
potential GAPs. The sizes of these proteins range from 50 to
250 kDa. GAPs acting on Rho/Rac family of small G proteins are especially abundant. In humans, over 70 potential
GAPs for Rho/Rac proteins have been identified, 38 GAPs
for the Rab family, 15 GAPs for Ras proteins, 31 GAPs for
Arf family, and 1 GAP for the Ran protein. It is interesting
to note that in the Rho family, the number of GAPs is in
almost threefold excess over Rho proteins, while in contrast, in the Ras and Rab families there are approximately
two more G proteins than GAPs (266). In the native state,
several GAPs are in a folded, autoinhibited conformation
that limits the accessibility of the catalytic site. GAPs are
regulated by several different mechanisms including phosphorylation/dephosphorylation, lipid-protein or proteinprotein interactions, and degradation/resynthesis (262,
265, 266).
2. GEFs
GEFs provide a direct link between Ras protein activation and membrane receptors for soluble factors such as
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
Table 1. Ras protein superfamily
Ras (36)
Rho (20)
Family members
H-Ras
N-Ras
K-Ras
E-Ras
M-Ras/R-Ras3
R-Ras
TC21/R-Ras2
Rit1
Rit2/Rin
RalA
RalB
Rap1A
Rap1B
Rap2A
Rap2B
Rap2C
Noey2
Di-Ras1
Di-Ras2
RasD1
RasD2
RRP17
RPR22
Rem1
Rem2
Gem/Kir
Rad
Rerg
Ris
RERGL
RasL11A
RasL11B
Rheb
Rheb2
NKIRas1
NKIRas2
RhoA
RhoB
RhoC
Rnd1
Rnd2
Rnd3
RhoD
Rif
RhoG
RhoH
Rac1
Rac2
Rac3
Cdc42
TC10
TCL
Wrch-1
Wrch-2
RhoBTB1
RhoBTB2
Posttranslational modification
Number of GEFs
Number of GAPs
Number of GDIs
Main downstream effectors
Farnesyl
29
15
1 (GDI-like)
Raf/MAPK
PI3-kinase
Ral-GDS
Farnesyl or geranylgeranyl
⬎80
⬎70
3
Rho kinases
Paks
Rab (64)
Ran (1)
Arf (28)
Other (11)
Rab1A, B
Rab2A, B
Rab3A-D
Rab4A, B
Rab5A-C
Rab6A-C
Rab7A, B
Rab7L1
Rab8A, B
Rab9A, B
Rab10
Rab11 A, B
Rab12
Rab13
Rab14
Rab15
Rab18
Rab17
Rab19
Rab21
Rab22A, B
Rab23
Rab24
Rab25
Rab26
Rab28
Rab27A, B
Rab30
Rab31
Rab32
Rab33A, B
Rab34
Rab35
Rab36
Rab37
Rab38
Rab39A, B
Rab40A-C
Rab41
Rab42
Rab43
RabL4
RasEF
Geranylgeranyl
53
38
3
Ran/TC4
Arf1
Arf3
Arf4
Arf4L
Arf5
Arf6
ArfRP1
ArfRP2
FLJ22595
Arfd1
Arl1
Arl2
Arl2L1
Arl3
Arl4
Arl5
Arl6
Arl7
Arl8
Arl9
Arl10A
Arl10B
Arl10C
Arl11
Sar1b
Sar1a
Arl16
Arl14
RabL2A
RabL2B
RabL5
SRPRB
LOC401884
Arl10C
Arl10B
RabL3
Rab20
Miro1
Miro2
1
1
Myristoyl
18
31
Number of proteins is given in parentheses. See text for definitions.
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A
B
α4
β
6
β6
α4
β5
G4
Rho
Rab
Arf
Ran
β4
α5
G1
β1
G5
α2
β3
Mg2+
α1
G3
(Switch II)
G2
(Switch I)
FIGURE 1. A: typical structure of Ras protein illustrated by the structure of H-Ras in complex with GTP. The
Ras 3-dimensional structure is composed by 6 ␤-sheets and 5 ␣-helices. Conserved sequence motifs (G1–G5)
involved in nucleotide binding are represented. B: similar representation of Rho, Rab, Arf, and Ran protein
structure.
neurotransmitters, cytokines, or growth factors and adhesion molecules (62, 284). The first mammalian Rho
GEF, Dbl, was identified as a transforming gene in an
NIH3T3 focus formation assay using DNA from a cell
line of human diffuse B-cell lymphoma. Since then, over
70 distinct members of this Dbl family have been identified in humans (115). GEFs for the large number of different Ras superfamily proteins vary drastically in structure, but there are significant similarities. GEFs constitute subfamilies specific for the different subclasses of
Ras proteins, characterized by a 20- to 30-kDa catalytic
domain responsible for the GEF activity. Examples of
such GEF domains are CDC25 for the Ras family, Dblhomology (DH) and Pleckstrin-homology (PH) for Rho
family, and Sec7 for the Arf family GEFs.
GEFs are multidomain proteins, and the regulation of their
activity occurs via a variety of strategies. They include protein-protein interactions, lipid binding, interaction with
second messenger molecules [cAMP, diacylglycerol (DAG),
Ca2⫹], and posttranslational modification such as phosphorylation. Basically, when GEF proteins are activated,
they interact with the switch I and switch II domains of
Ras-related proteins, causing a series of side-chain rearrangements. These structural changes induce a 10,000-fold
increase in the rate of GDP release and its replacement by
GTP, the concentration of which in the cytosol is 10 times
higher than that of GDP (400).
3. GDIs
Ras-related proteins are regulated by a third class of
proteins, the guanine nucleotide dissociation inhibitors
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(GDIs) thus named as they were first identified as inhibitors of GDP dissociation (126). GDI proteins have been
identified for geranylgeranylated Rab and Rho GTPases,
but PDE␦ that preferentially solubilizes farnesylated Ras
subfamily proteins, has been qualified as a GDI-like factor (71, 297, 330) (TABLE 1). The most important effects
of GDI are their capacity to solubilize membrane-associated small G proteins in the cytosol (62, 132, 161, 283).
By binding to their prenylated COOH terminus, GDIs are
able to extract GDP-bound small G proteins from the
membrane and to keep them inactive in the cytosol by
protecting the hydrophobic lipid tail from aqueous solvent. Binding of GDIs, GEFs, and GAPs is mutually exclusive, suggesting that cytosolic GDI-bound G proteins
are protected from nucleotide alternation regulated by
GEFs and GAPs (78). The dissociation of GDI from the
small G protein is therefore a prerequisite for GEF- and
GAP-mediated regulation of the activity of small G proteins. Nevetheless, the mechanisms by which GDIs deliver their target G proteins to membranes remain largely
unknown. Current knowledge suggests that a variety of
different mechanisms may be involved, including regulation by candidate GDI-displacement factors (GDFs) for
RabGDI and PDE␦ and phosphorylations for RhoGDIs
(78). GDFs, through allosteric regulation, may squeeze
the lipid out by shrinking the lipid-binding pocket of
GDI, thereby facilitating the release of prenylated G proteins and its translocation to membranes. Contrary to
RabGDI and PDE␦, there is no GDF candidates identified
for RhoGDIs, and accumulating evidence suggests that
the affinity between RhoGDIs and Rho proteins, and thus
their release, is regulated by phosphorylations (132).
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
A
B
Ras
Ras
GTP
GDP
Rho
GTP
GAP
GDP
Effector
Pi
GTP
Rho
Ras
Rho
GDP
GDP
Pi
GDI
GTP
GDP
GDI
C
D
Cargo
GTP
GAP
Effector
GEF
GEF
Rho
GTP
GDI
Importin
Rab
GAP
Ran
GDP
GDF
Nucleus
REP
Importin
Cargo
REP
Rab
Rab
GDP
Ran
GDP
Cytoplasm
Rab
GDP
Rab
GDP
GDP
Ran
GTP
GDP
RCC1
Rab
Rab
GDP
GTP
GAP
GEF
REP
GDP
GTP
Effector
FIGURE 2. Activation cycle of small G proteins and their regulatory proteins. A: Ras GTPase cycle. Inactive
Ras is GDP-bound and localized in the cytosol. Activation of Ras is mediated by a guanine nucleotide exchange
factor (GEF). GTP loading induces the translocation of Ras from the cytosol to the plasmatic membrane and
permits the interaction with effectors. To “turn off” the cycle, a GTPase-activating protein (GAP) accelerates the
intrinsic GTPase activity of Ras, allowing Ras to return to its inactive state in the cytosol. Arf proteins have the
same cycle of activation. B: the GDP-GTP cycle of Rho GTPases is similar to Ras proteins but a new regulator
appears: the guanine nucleotide-dissociation inhibitor (GDI). This protein binds specifically to GDP-bound Rho,
prolonging the inactive state and sequestering the GTPase in the cytosol. C: Ran protein is a regulator of
nuclear import and export. Activation cycle of Ran occurs in the cytoplasm and in the nucleus. Nuclear Ran is
maintained in the GTP-bound state through localization of GAP in the cytosol and the GEF RCC1 in the nucleus.
Ran is the only small GTPase without posttranslational modification. D: Rab proteins include COOH-terminal
prenylation signals. The Rab escort protein (REP) interacts with newly synthesized Rab and allows the Rab
geranylgeranyl transferase (RabGGT) to add two geranylgeranyl lipid groups to the COOH terminus of Rab. Rab
activation follows the prototypical GDP/GTP cycle of Rho proteins. Nevertheless, an additional step is needed
to the localization of Rab. The insertion of Rab in the target membrane is mediated by a GDI dissociation factor
(GDF) that releases the Rab from GDI.
C. Subgroups of Ras Superfamily
1. Ras family
The history of Ras proteins started in the early 1980s by the
discovery that a common set of genes, termed ras (for rat
sarcoma virus), are responsible for the oncogenic properties
of the Harvey and Kirsten sarcoma viruses (97). The Harvey
and Kirsten sarcoma virus-associated oncogenes were thus
named H-ras and K-ras, respectively. Together with the
subsequently discovered neuroblastoma ras oncogene (Nras), products of these genes constitute the founding members of the canonical Ras proteins (149, 478). Hormones,
cytokines, and growth factors through the stimulation of
plasma membrane receptors activate Ras proteins. This activation involves receptors tyrosine kinase and specific
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GEFs (Sos, RasGRP, or RasGRF) (213). The effectors with
which they interact determine the biological effects of Ras
proteins, including gene expression, proliferation, survival,
differentiation, cell cycle entry and cytoskeletal dynamics.
The most important signaling pathways initiated by active
Ras proteins are summarized in other reviews (116, 213,
374, 571).
Dysregulation of these cellular functions is a hallmark of
cancer. In humans, dominant gain-of-function mutations of
these Ras sequences were discovered in many cancers of the
bladder, colon, and lungs (374). The resulting amino acid
replacements were usually found to affect the hot spot residue 12, and also less frequently residues 13 and 61. Oncogenic substitutions in residues G12 and G13 prevent the
formation of van der Waals bonds between Ras and the
GAP proteins, which results in the pronounced attenuation
of GTP hydrolysis. Similarly, oncogenic mutations of Q61
reduce the rate of GTP hydrolysis by affecting the coordination of a water molecule thereby protecting from the
nucleophilic attack on the ␥-phosphate (59, 424).
2. Rho family
The Ras homologous (Rho) family of small GTPases contains 20 members in humans. The most studied members
are RhoA, Rac1, and Cdc42. Rho subfamily proteins act as
molecular switches within extracellular factor-activated intracellular signaling pathways that regulate multiple cellular functions. RhoA, Rac1, and Cdc42 are ubiquitously
expressed, and the major function of these proteins is the
regulation of the cytoskeleton. However, the impact of each
member of Rho family on the cytoskeleton could be different. RhoA activation leads to the formation of actin stress
fibers and focal adhesion, as well as membrane protusion by
activating mDia and driving actin polymerization (162,
391). Stimulation of Rac1 induces actin polymerization at
the cell periphery to generate protrusive actin-rich lamellipodia and membrane ruffling. Cdc42 initiates long parallel
bundles of actin filaments to induce the formation of filopodia. These reorganizations of the actin cytoskeleton induced by Rho proteins have consequences on cell morphology, polarity, motility, and adhesion (47, 62, 161, 283).
In addition to their regulation of the cytoskeleton, Rho
proteins could act as regulators of enzymatic activity. For
example, Rac1 controls the enzyme complex responsible
for superoxide formation (NADPH oxidase) (421),
whereas RhoA regulates the Rho-kinases (Rock1 and
Rock2) to modulate cell contraction (283, 284). Last but
not least, Rho proteins play a major role in additional
process such as gene transcription and cell cycle progression (306, 353, 524).
3. Ran family
The small GTPase Ran has functions in centrosome duplication, microtubule dynamics, chromosome alignment, ki-
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netochore attachment of microtubules, and nuclear-envelope dynamics, in addition to its characterized roles in nucleocytoplasmic transport.
Ran was first identified as an essential cofactor for nuclear
import of nuclear localization signal (NLS)-containing proteins that interacts with transport factors from the importin-␤ superfamily, also known as the karyopherins (240).
The NLS-containing protein-associated importin-␤ binds
directly GTP-bound Ran protein in the nucleus, and this
interaction triggers the release of the NLS-containing protein from the imported complex. The Ran-GTP-importin-␤
complex returns to the cytoplasm, and importin-␤ disassembles from Ran-GTP upon Ran-GAP1-mediated hydrolysis of GTP to GDP. Free Ran-GDP is then imported into
the nucleus by a mechanism that involves the nuclear transport factor-2 (390, 453), where it is loaded with GTP by the
Ran guanine nucleotide-exchange factor RCC1 (46). RCC1
is restricted to the nucleus, while the Ran-GAP1 is concentrated in the cytoplasm. Due to this compartmentalization
of Ran regulators, the concentration of GTP-bound form of
Ran is predominantly localized in the nucleus while the
GDP-bound form is prevalently cytoplasmic and the energy
supplied by GTP hydrolysis in the cytosol drives nuclear
transport (147, 572).
4. Rab and Arf families
Originally described as Ras-like proteins in brain, Rab proteins constitute the largest family of small Ras-like GTPases
with more than 60 members in humans. They are localized
in the membrane of various subcellular organelles (463).
Most intracellular vesicles contain more than one type of
Rab preotein. Analysis of Rab-GTP structures shows the
greatest structural heterogeneity in their switch domains.
These structural differences could explain how different
Rab proteins recruit specific sets of effectors to regulate
precisely their respective pathways. Rab proteins are associated with vesicular trafficking functions, including ensuring transport specificity and demarcating organelle identity
(156, 185, 463).
Rab proteins use the classical GDP-GTP cycle, but use distinct additional regulatory protein. De novo synthesized
Rab is GDP bound and inactive, with a very low affinity
for RabGGT. Newly synthesized Rab protein associates
with Rab escort protein (REP) to interact with RabGGT
and to be prenylated. The insertion of the Rab-GDP into
the target membrane is mediated by a GDF that releases
the Rab from GDI (185).
A large body of evidence indicates that the Arf family is also
implicated in membrane trafficking. The Arf family includes the Arf proteins (ADP-ribosylation factor), the Sar
proteins (secretion and Ras-related protein), and the Arl
(Arf-like) proteins. Arfs are ubiquitously expressed, but
they are mainly concentrated in the Golgi apparatus. In this
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
organelle, Arfs regulate the assembly of coat complexes
around budding vesicles (195, 267).
pressed in the heart cannot compensate for the loss of K-Ras
function in K-Ras-deficient mice.
Recent results suggest a cross talk between Rab and Arf
proteins. For example, it is reported that the Arf6 GTPase
negatively regulates Rab35 activation and hence the fast
endocytic recycling pathway (79).
Nevertheless, the embryonic heart development is normal
in mice in which K-Ras has been replaced by H-Ras (371)
and the introduction of H-Ras transgene in K-Ras-deficient
mice rescued mice from embryonic lethality in association
with correction of thin ventricular walls of the heart of
K-Ras-deficient mice (327). These observations thus suggest that intrinsic activities of H-Ras and K-Ras are similar
and that H-Ras can functionally replace K-Ras in embryonic heart development and cardiomyocyte proliferation
(327). However, during the adult life, K-Ras function is not
entirely rescued by H-Ras as adult mice in which K-Ras has
been replaced by H-Ras develop dilated cardiomyopathy
associated with arterial hypertension, suggesting that KRas has a unique role in cardiovascular homeostasis (371).
III. RAS PROTEINS IN HEART FUNCTION
AND DISEASES
A. Embryonic Heart Development
The heart is the first organ to be formed during embryogenesis in vertebrates. The primordium of this organ is derived
from mesodermal cells that are involved in numerous interactions with cells from several embryonic origins (352). The
constitution of the multichambered heart is a multistep process, starting by linear heart tube formation from paired cardiac fields, then followed by the formation of the cardiac
chambers, heart looping, and septation (352). The epithelialmesenchymal transformation of the endocardial cells and
invasion of the endocardial cells into the cardiac jelly are the
major events involved in the formation of endocardial cushions that play a critical role in valve formation and cardiac
septation, which separates the primitive heart tube into four
chambers (352, 459). After cardiac looping, cardiac myocytes express muscle genes, the specific site of which is characteristic for each chamber, thereby leading to chamberspecific properties. These processes require precise temporal
and region-specific regulation of cell proliferation, migration, death, and differentiation. Among the intracellular
signaling molecules involved in this regulation, small G proteins and their effectors have been recognized as key actors
of cardiac embryogenesis.
1. Ras proteins
A) RAS.
In situ hybridization analysis showed that all three
ras genes (H-ras, K-ras, N-ras) are uniformly expressed in
the embryonic heart (embryonic day E13.5–E18.5) (329).
While H-Ras-deficient mice and N-Ras-deficient mice are
born and grow normally, the K-Ras-deficient embryos die
progressively between E12.5 and term (230). K-Ras-deficient embryos display heart abnormality on E15.5, with
thin ventricular walls that can be reduced to a one-cellcompact layer (230). However, the hearts of the K-Rasdeficient embryos were without septal and trabecular defects, and cardiomyocyte-specific makers were expressed in
ventricular myocytes. It is likely that the thin ventricular
walls in the K-Ras-deficient embryos are not related to abnormal differentiation or cell death but are due to a deficiency in the proliferation of the cardiomyocytes (230). This
indicates that K-Ras is essential for normal heart development and that other Ras members H-Ras and N-Ras ex-
Activation of the Raf/MEK/ERK1/2 signaling pathway in
endothelial cells is a major effector of Ras during cardiac
development. The hearts of endothelial specific ERK1/2
knockout mice are much smaller than that of control mice,
with highly reduced cellularity, and the endocardial layer
was detached from myocardial trabeculae indicating defective heart development and function (458). This phenotype
is associated with a reduced migration and proliferation of
endothelial cells.
The Ras exchange factor of K-Ras and H-Ras Sos1 has been
identified as one potential regulator of Ras activity during
embryonic heart development. Sos1-deficient embryos have
an enlarged heart and distended pericardium (508). The
myocardium of mutant embryos frequently showed reduced trabeculation. The orientation of the heart during
development may also be abnormal in Sos1-deficient mice.
Other aspects of cardiac development in Sos1-deficient mice
appeared relatively normal, including the partitioning of
the heart and the development of endocardial cuffs (508).
This specific phenotype of Sos1-deficient mice has been related to the role of Sos1 downstream of the EGF receptor
(508).
The role of Ras in cardiac development has been further
documented through indirect evidence arising from studies
on neurofibrin encoded by the Nf1 gene. Neurofibrin is a
Ras GAP and thus downregulates Ras signaling (294). Loss
of function mutation of Nf1 in human leads to neurofibromatosis type1 (NF1) characterized by the appearance of
benign and malignant tumors and cardiovascular defects
such as pulmonary valvular stenosis (574). Mice in which
the Nf1 gene has been inactivated have been useful to decipher the role of Ras signaling in the embryonic development
of the heart, in particular valve formation. Homozygous
deficient embryos die in mid gestation (E14) and exhibit
signs of cardiac dysfunction with histological abnormalities
such as an overabundance of tissue in endocardial cush-
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LOIRAND, SAUZEAU, AND PACAUD
ions of the outflow tract due to hyperproliferation and
lack of normal apoptosis, and abnormal cardiac morphogenesis including a failure of proper conotruncal rotation
resulting in double outlet right ventricle (247). Endothelial specific inactivation of Nf1 recapitulates the cardiovascular abnormalities involving endothelial cushions
and myocardium (138). In contrast, endothelial specific expression of the Ras GAP-domain of neurofibromin rescued
cardiac development in Nf1⫺/⫺ embryos (190). With the use
of explants of endocardial cushion tissue, it has been also
demonstrated that Nf1⫺/⫺ explants displayed enhanced epithelial-mesenchymal transformation that was corrected by the
dominant negative Ras (Ras-N17) and mimicked by constitutively activated Ras (Ras-L61) in wild-type endocardial
cushion explants (247). This indicates that Ras signaling is
necessary and sufficient for endocardial-mesenchymal
transformation and proliferation.
B) RALA AND RALB.
RalA and RalB are both expressed in the
myocardium and endocardial cushions of developing heart
in mice from E9.5 to E16.5. Higher levels of expression
were seen in the apical edge of endocardial cushions in the
fusion seam of the septa at E12.5 and E13.5, a critical
period for the formation of septa that involved mechanism
of apoptosis in atrioventricular cushions (584).
C) RHEB. Rheb (Ras homolog enriched in brain)-deficient
embryos die around midgestation because an impaired development of the cardiovascular system with thinning of the
ventricular walls (141).
2. Rho proteins
Rho proteins have been found necessary for the development of normal heart. An original experimental approach
consisting of cardiac overexpression of Rho GDI␣ using the
␣-myosin heavy chain promoter, active at embryonic day
E8.0 during morphogenesis on the linear heart tube, has
been used to analyze the role of Rho proteins in cardiac
morphogeneis (520). Cardiac overexpression of RhoGDI␣
led to embryonic lethality. In these embryos, cardiac morphogenesis was disrupted, with incomplete looping, lack of
chamber demarcation, hypocellularity with inhibition of
cell proliferation, and lack of trabeculation. As the activities
of RhoA, Rac1, and Cdc42 were all decreased in this model,
the respective role of these three proteins cannot be determined.
A) RHOA. RhoA transcripts and proteins have been shown to
be markedly upregulated in the heart forming region in
chick embryo (204). SiRNA-mediated RhoA silencing inhibited the fusion of the bilateral heart primordia, suggesting that RhoA is involved in the migration of cardiac precursors to the ventral midline and is necessary for the
proper looping of the heart and the development of normal
heart (204). During development, the initial myocardiumwide expression of RhoA becomes restricted to the right
1666
side of the sinus venosus myocardium including the sinoatrial node, suggesting a role of RhoA in the conduction
system development (501). Rho-kinases appear as key
effectors of RhoA signaling during cardiogenenesis. Rhokinase transcripts are enriched in cardiac mesoderm and
Rho-kinase inhibition induced cardia bifida and precocious expression of early marker genes of cardiomyocytes
differentiation (521). Rock1 and Rock2 genes are expressed in the atrioventricular endocardial cushions at all
phases of cardiac development, and pharmacological
blockade of Rho-kinases inhibits epithelial-mesenchymal
transformation and cell migration, suggesting that Rhokinases play an essential role in endocardial cell differentiation and migration during endocardial cushion development (521, 583). Rho-kinases are also involved in the
formation of initial heart myofibrillogenesis by means of
actin-myosin assembly, and focal adhesion/costamere and
cell-cell adhesion (410). Upstream to RhoA, the Rho GEF
AKAP13 could account, at least in part, for the role of
RhoA activity in the development of the heart (302). Developing hearts of AKAP13-null mice display thin cardiac
walls, with cardiomyocytes showing deficient sarcomere
formation, and mice die at embryonic day 10.5–11.0. This
phenotype is likely to be due to a reduced RhoA-dependent
activation of the transcription factors SRF and MEF2c and
defect in actin assembly (302).
B) RHOB. RhoB is mainly localized on endosomes and regulates cytokine trafficking and cell survival. RhoB levels vary
through the cell cycle, and the rhoB transcript has a half-life
of 30 min, which is substantially shorter than rhoA or rhoC
and indicates that RhoB function requires its expression to
be highly regulated (526). During cardiac embryogenesis,
RhoB is expressed in the developing atrioventricular and
outflow tract endocardial cushions throughout their development (163). Transcripts can first be detected in the endocardial cells overlying the cushions, prior to the delamination of cushion mesenchyme. RhoB increases in expression
as epithelium to mesenchyme transformation begins, and
reaches a peak of expression as the mesenchymal cells migrate into the cardiac jelly. RhoB expression is downregulated at E11.5 as the endocardial cushions begin their differentiation to form the valves and septa of the heart (163).
Expression of RhoB in migrating cells seems likely to be
associated with its role in organization of the actin cytoskeleton.
C) RAC1.
Rac1 is the only Rac gene expressed early in development. In mouse embryo, although the cardiac mesoderm
was specified, the lack of Rac1 is associated with the absence of fusion of the heart at the midline, leading to cardia
bifida (308). This defect has been ascribed to a defect in the
actin remodeling required for the migration of the nascent
mesoderm (308). Targeted deletion of Rac1 in the endothelial cells is sufficient to produce defects in cardiac development. Endothelial Rac1-deficient embryos lack a common
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
ventricular chamber of the heart but seemed to develop the
atrial chamber and the atrioventricular canal (477).
The atypical Rac exchange factor Dock180 was found to
have a major role during cardiovascular development. Deletion of Dock180 in mouse induces major cardiac abnomalities such as submembranous ventricular septal defect and
double outlet right ventricle (413). Mitral valve leaflets
were also thickened and sometimes fused, leading to blood
retention in left atrium. A similar phenotype was obtained
by specific Dock180 deletion in endothelial cells, suggesting
the endothelial origin of the cardiac defect in Dock180deficient mice. This effect has been ascribed to a major role
of Dock180 in the regulation of Rac activation to control
endothelial cell migration during cardiac development, but
not in the epithelial-mesenchymal transformation of cardiac endothelial cells (413).
D) CDC42.
Indirect evidence also suggested that Cdc42 is
required for normal heart formation as the absence of the
Cdc42 effector p21-activated kinase (Pak) 4 led to lethality
by embryonic day 11.5, due to a defect in the fetal heart
associated with a thinning of the myocardium and a dilation
of the atrium and sinus venosus, probably related to the role
of Cdc42/Pak4 signaling in the regulation of cytoskeleton
organization (377).
B. Cardiac Excitation/Contraction Coupling:
Contractility
The transient change in tension and length in a working
cardiac myocyte during the heart beat reflects the intregrated effects of signaling cascades regulating mechanisms controlling the dynamics and intensity of rise in cytoplasmic Ca2⫹ as well as the responsiveness of the sarcomeric proteins to Ca2⫹.
The opening of cardiac voltage-dependent Na⫹ channel
causes initial plasma membrane and t-tubule depolarization
that induces activation of L-type voltage-dependent Ca2⫹
channel (CaV1.2) and Ca2⫹ entry into the cell. The resulting
fast and localized Ca2⫹ rise in the subsarcolemmal space
elicits the opening of sarcoplasmic reticulum Ca2⫹ release
channels (ryanodine receptors, RyRs) leading to an amplified release of Ca2⫹ constituting the Ca2⫹ transient. The
sarcoplasmic Ca2⫹-ATPase SERCA2 is responsible for sarcoplasmic reticulum Ca2⫹ reuptake after systole.
The Ca2⫹-dependent contractile response is regulated by
the binding of cytosolic Ca2⫹ to a single site on cardiac
troponin C (cTnC), which strengthens the affinity of cTnC
for cardiac troponin I (cTnI). Through change in cardiac
troponin T (cTnT), these Ca2⫹-dependent conformational
changes in cTnC-cTnI are transmsitted to tropomyosin, favoring the displacement of the troponin complex/tropomyosin on the thin filament away from the actin-bindin sites for
myosin. Myosin cross-bridges then bind actin, and crossbridge cycling proceeds (229). Phosphorylation of myosin
binding protein C (MyBP-C) and myosin regulatory light
chain (RCL, MLC2) also control the Ca2⫹ sensitivity of the
contraction (455).
Cardiac excitation-contraction coupling is regulated by a
variety of signaling molecules and receptors, one of the
most significant being the ␤-adrenergic stimulation coupled
to cAMP pathway. Emerging evidence indicates that small G
proteins, particularly by controlling intracellular Ca2⫹ signaling and phosphorylation/dephosphorylation processes, contribute to the regulation of cardiac excitation-contraction coupling and contraction.
1. Regulation of Ca2⫹ handling mechanisms
A) RAS PROTEINS. I) Ras. The hypertrophic effect of Ras is
associated with changes in the contractile properties of the
myocardium. The cellular mechanism involved in Ras-mediated cardiac contractile defects has thus been the subject of a
number of studies. Expression of constitutively active Ras (HRas-V12) in cultured neonatal cardiac myocytes reduced the
amplitude and prolonged the decay phase of the contractile
Ca2⫹ transients (177). It also decreased the expression of
SERCA2 and striated myofibrils. This Ras-mediated regulation of intracellular Ca2⫹ in cardiac myocytes was ascribed to
the activation of the Raf-MEK-ERK cascade (177). However,
it was unexpectedly observed that Ca2⫹ loading of the reticulum was increased in H-Ras-V12 cardiomyocytes, suggesting
that H-Ras-V12 affects the excitation-Ca2⫹ release coupling.
In support of this hypothesis, H-Ras-V12 was shown to reduce the amplitude of the L-type calcium channel current in
cardiac myocytes through a Raf-MEK-ERK-dependent decrease in the expression of the pore-forming ␣1C subunit (also
called Cav1.2) of the Ca2⫹ channel (176).
In vivo, direct hemodynamic measurements in mice demonstrated that selective expression of H-Ras-V12 in cardiac
myocytes significantly decreases cardiac contractility as
well as causes diastolic dysfunction associated with interstitial fibrosis and a blunted response to a ␤-adrenergic agonist (585). At the ventricular myocyte level, these changes in
contractility were associated with a reduction and a slowing
of the decay of intracellular Ca2⫹ transient and a decrease
of the sarcoplasmic reticulum Ca2⫹ content, confirming results obtained in cultured myocytes (402, 585). Only a
modest reduction of the expression of SERCA2 was observed, and the expression and activity of the Na⫹/Ca2⫹
exchanger were found to be increased (402, 585). However,
a strong decrease in Ser-16 phosphorylation of the SERCA2
regulator phospholamban (PLB) associated with an increase in the expression of protein phosphatase PP1␣, the
major PLB phosphatase in the heart, without change in
PP2A level was observed in H-Ras-V12 ventricular myocytes (402, 585). These results provide evidence to implicate
Ras signaling in the regulation of cardiac contractility
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LOIRAND, SAUZEAU, AND PACAUD
through modulation of Ca2⫹ handling proteins expression
and activity, including the L-type Ca2⫹ channel and
SERCA2.
B) RAP PROTEINS.
A role of Rap1 protein in cardiac myocyte
excitation-contraction coupling became evident from recent studies. Besides the activation of the canonical PKA
signaling, cAMP activates other signaling molecules, including Rap1 and Rap2 via the recruitment of cAMP-activated exchange factors Epac (52). In cardiac myocytes, activation of Epac increases electrically evoked Ca2⫹ transient
via PLC⑀, the catalytic activity of which is directly regulated
by Rap (350). Furthermore, inhibition of Rap activity by
expressing RapGAP significantly inhibited ␤-adrenoceptordependent stimulation of Ca2⫹ release. Epac-mediated regulation of ␤-adrenergic-dependent stimulation of CICR appears to be mediated by PLC⑀-dependent stimulation of
CAM kinase II and subsequent increase in the phosphorylation of the type 2 RyR (RyR2) and phospholamban (349,
362). Further evidence also suggested that Rap is involved
in the electrical coupling of cardiomyocytes through the
regulation of gap junctions which are essential to coordinate cardiac contractions; Epac-Rap1 signaling redistributes connexin 43, increasing the accumulation at cell-cell
contacts, and leads to the formation of new gap junctions.
This redistribution of connexin 43 is related to Rap-mediated N-cadherin accumulation at the cell-cell contacts, the
major component of adherens junctions that are responsible for mechanical coupling between cardiomyocytes (456).
C) THE RGK SUBFAMILY OF RAS PROTEINS.
A common feature of
the RGK subfamily members (Rad, Rem1, Rem2, Gem/Kir)
is their ability to potently inhibit L-type Ca2⫹ channels in
the heart and to reduce left ventricular systolic function
(324, 509, 552). In addition, it has been recently shown that
Rem prevents the stimulatory action of cAMP/cAMP-activated kinase (PKA) on L-type Ca2⫹ channel, suggesting a
physiological and/or pathophysiological role of RGK proteins in the modulation of ␤-adrenergic regulation of cardiac function (551). This hypothesis is further supported by
the observation that Rad overexpression negated ␤-adrenergic effects on L-type Ca2⫹ current and Ca2⫹ transients
(509). One prevailing explanation of this effect is that RGK
proteins interfere with the trafficking of pore forming subunit ␣1C to the sarcolemma, possibly by buffering endogenous ␤ subunits (␤1, ␤2, and ␤3), thereby reducing the
surface density of the cardiac L-type Ca2⫹ channels (31,
367). However, recent data showing that Rem predominantly inhibits L-type Ca2⫹ channel current by arresting
surface CaV1.2 channels in a low open probability gating
mode emphasize the idea that RGK proteins use diverse
mechanisms and determinants to inhibit L-type Ca2⫹ channel current (551). Similarly, whether or not the RGK proteins should be in the active GTP-bound state and targeted
to the cell membrane to exert its effect on Cav1.2 remains
controversial (89, 551, 561, 562).
1668
D) RHO PROTEINS. I) RhoA. Ventricular myocytes isolated
from transgenic mice carrying a sixfold overexpression of
Rho GDI␣ in the myocardium exhibited a 40% decrease in
the density of the L-type Ca2⫹ channel current (565). As
there was no change in the levels of mRNA and protein of
the L-type Ca2⫹ channel, this inhibition is due to an effect
on the activity of the channel. Expression of a dominant
negative form of RhoA but not a dominant negative Rac1
or Cdc42 mimicked the effect of GDI␣ overexpression, indicating that the L-type Ca2⫹ channel is a downstream target of RhoA in cardiac myocytes (565). The exact molecular
mechanism responsible for the positive action of RhoA on
the L-type Ca2⫹ channel has not been precisely defined;
however, as the density of channels in the plasma membrane
was not modified, it seems to affect the activity of the channel
through an actin-independent signal pathway (565). The progressive atrioventricular conduction defects displayed by
GDI␣ overexpressing mice are in agreement with a loss of the
positive regulatory role of RhoA on L-type Ca2⫹ channel activity (522). However, surprisingly, the ventricular contractile
function was largely preserved in Rho GDI␣ transgenic hearts
(522). In ventricular myocytes, L-type Ca2⫹ current constitute the major pathway for Ca2⫹ entry that initiates excitation-contraction coupling, and pharmacological inhibition
of L-type Ca2⫹ channel expression or activity affects myocyte contractility. This thus suggests that functional compensation exists in GDI␣ overexpressing mice, allowing a
normal Ca2⫹ transient and contractility of ventricular myocytes despite a reduced L-type Ca2⫹ current (522).
RhoA/Rho-kinase signaling also affects intracellular Ca2⫹
store as in contracting neonatal ventricular myocytes, and
pharmacological inhibition of Rho-kinase by Y27632 induces a 150% increase in SERCA2 mRNA expression
(502). This effect has been shown to be due to a repressing
effect of RhoA/Rho-kinase signaling on the SERCA2a promoter activity by transcription factor other than the wellknown RhoA/Rho-kinase-dependent transcription factor
SRF, myocardin, or GATA4 (502).
II) Rac and Cdc42. Although it has been suggested in other
cell types that Rac and CdC42 inhibit L-type calcium current, direct evidence for such an effect in the heart does not
exist (529). Nevertheless, the identification of the Rac/
Cdc42 Pak1 as a major regulator of the catalytic activity of
the protein phosphatase 2A (PP2A) strongly argues for a
role of Rac/Cdc42-Pak1 signaling in the regulation of cardiac contractility. It has been suggested that Pak1 forms a
signaling module with PP2A in cardiomyocytes. Pak1 is
highly expressed in the heart, and, as PP2A, localized to
Z-disc, cell and nuclear membrane and intercalated disc of
ventricular myocytes (220). In the resting state of the Pak1/
PP2A complex, PP2A is inactive and phosphorylated on
Tyr-307 (221, 525). Activation of Rac1/Cdc42 and the subsequent association of the small G protein with the Rho
binding domain of Pak1 promotes its activation and its
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
auto-phosphorylation at Thr-423 inducing a conformational change in PP2 that allows autodephosphorylation of
Tyr-307 and stimulation of its catalytic activity towards its
substrates such as L-type Ca2⫹ channel and Ryr2 (220,
221). Expression of constitutively active Pak1 in single, isolated adult rat ventricular myocytes significantly changes
the decay and peak characteristics of spontaneous [Ca2⫹]i
transients both under resting conditions and after ␤-adrenergic stimulation (441). This occurs without changes in the
Ca2⫹ content of the sarcoplasmic reticulum and in the phosphorylation of phospholomban. Although not demonstrated, it has been suggested that Pak1 activity reduced
Ca2⫹ transient by altering L-type Ca2⫹ channels and/or
RyR2 gating via dephosphorylation by PP2A (220, 441).
In addition, Rac1 controls the electrical coupling of cardiomyocytes. In cardiac myocytes in vitro, activation of Rac1,
downstream of N-cadherin, determined the localization of
connexin 43 at the intercalated disks (300).
E) RAB PROTEINS. Endosomal trafficking represents an important mechanism for maintaining proper plamalemmal expression of ion channels, including L-type Ca2⫹ channel.
Endosomal transport is largely regulated by the Rab family
of small G proteins (463). Among Rab proteins, Rab11b
has been recently identified as a regulator of CaV1.2 L-type
Ca2⫹ channel surface membrane density (37). Rab11b is
expressed in ventricular myocardium and in cardiac myocytes; expression of a dominant-negative form of Rab11b
increases the peak of L-type Ca2⫹ channel current by 98%,
indicating that contrary to that expected, Rab11b limited
rather than promoted the plasma membrane expression of
CaV1.2. It has thus been proposed that, in cardiac myocytes, Rab11b regulates endocytic recycling of CaV1.2 by
targeting the internalized channels toward a degradation
pathway (37).
2. Regulation of contractile proteins
Ras signaling through ERK1/2 to p90RSK
(ribosomal S6 kinase) induces phosphorylation of cTnI on
S23/S24 (194). In protein lysates from cardiomocytes,
phosphorylation of cTnT is inhibited by the Raf inhibitor
GW5074, and in vitro kinase assays indicated that Raf-1
phosphorylated cTnT on T206 that has been linked to myofilament function (363).
A) RAS PROTEINS.
B) RHO PROTEINS. I) RhoA. RhoA through Rho-kinase increases the phosphorylation of the 20-kDa myosin regulatory light chain (RCL), via direct phosphorylation of myosin phosphatase targeting peptide (MYPT2) and consequently, inhibition of the myosin light chain phosphatase
(MLCP) (428). This Rho-kinase-mediated increase in RLC
phosphorylation could participate in the positive inotropic
effects of RhoA activating agent such as ␣-adrenoceptor or
PGE2 receptor agonists (381). More recently, it has been
demonstrated that Rho-kinase also directly phsophorylates
cTnI (on S23, S24, and T144) and cTnT (on S278 and
T287) (493). This effect is associated with a depression of
the maximal tension and a decrease in the Ca2⫹ sensitivity
(493). In addition, RhoA/Rho-kinase signaling has been
proposed to participate in the maturation of myocardial
contractile system through phosphorylation of its molecular targets in Z-discs and intercalated discs (464).
II) Rac1/Cdc42. More recent evidence also suggests that
activation of the Rac1 and Cdc42 effector Pak1 leads to
decreased phosphorylation of TnI and MyBP-C through
interaction with the phosphatase PP2A and stimulation of
PP2A activity (221, 440). In single cardiac myocytes, Pak1
increased the Ca sensitivity of tension, consistent with PP2mediated dephosphorylation Ser-23/24 of cTnI (441). Although through a distinct mechanism, Pak3 has been shown
to also increase the Ca sensitivity of cardiac myofilaments
(61). This effect could be related to direct phosphorylation
of cTnI on a new phosphorylation site identified as Ser-149
in the inhibitory domain, and of cTnC. Taken together,
these studies suggest that Paks form signaling complexes in
the heart that are active in cTnI regulation.
C. Heart Rhythm and Arrhythmia (Table 2)
The normal mechanical functioning of the heart depends on
proper rhythmic electrical activity, originating from specialized “pacemaker” cells in the sinoatrial node (SAN) in
the right atrium. Propagation of this initial excitation wave
through intercellular gap junctions leads to the depolarization of adjacent atrial myocytes, ultimately resulting in excitation of the atria. Next, the excitation wave propagates
via the atrioventricular node (AVN) and the Purkinje fibers
to the ventricles, where ventricular myocytes are depolarized, leading to excitation of the ventricles.
Rhythmic cardiac electrical activity is attributed to the generation of action potentials in individual cardiac cells. In
atrial and ventricular myocytes, the resting potential between each action potential is stable and negative (around
⫺85 mV) due to the high conductance of the IK1 channel
(15). Electrical impulses received from adjacent cells induces opening of Na⫹ channels and the generation of Na⫹
current responsible for the rapid depolarization phase of the
action potential. Transient outward K⫹ current (ITO) is
then responsible for the early repolarization phase. The plateau of the action potential results from a balance between
depolarizing L-type Ca2⫹ channel and outward delayed rectifying K⫹ currents (IKur, IKr, and IKs). The repolarization
phase is due to the closing of L-type Ca2⫹ channels and the
maintenance of outward rectifying K⫹ current and then to
K⫹ efflux through IK1 channels. The P wave and the QRS
complex of the surface electrocardiogram represent atrial
and ventricular excitation, respectively, and the T wave
represents ventricular repolarization.
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LOIRAND, SAUZEAU, AND PACAUD
Table 2. Effect of Ras proteins on cardiac ion channels and heart rhythm
Ras
Protein
Ras
Target
Currents/Channels
IK,Ach
Effect
Inhibition of activation
by muscarinic
receptor stimulation
Effector/Signaling
Involved
Ras-GAP
Mice Models
H-Ras-V12
Increased Gi␣
signaling
Kir2.1
L-type Ca2⫹
channel
Gem
L-type Ca2⫹
channels
Inhibition by decreasing
membrane density of
the channel
Inhibition by decreasing
expression of the ␣1c
subunit of the
channel
Inhibition by decreasing
membrane density of
the channel
Rad
L-type Ca2⫹
channels
Rem
L-type Ca2⫹
channels
Rap1a
IKAch
RhoA
IKAch
Inhibition by decreasing
expression of the ␣1c
subunit of the
channel
Inhibition by
maintaining the
channel in a lox open
probability gating
mode
Antagonize the effect
of Ras on atrial IK,Ach
Constitutive activation
Kv1.2
Inhibition
IKr
IK1
L-type Ca2⫹
channel
Inhibition of hERG
Inhibition of Kir2.1
Stimulation of channel
activity
Rac1
Cdc42
Rab11b
1670
IKr
Stimulation of hERG
Kir2.1
Increased membrane
density of the
channel
Alteration of gating
properties
Alteration of gating
properties
Target the channels
toward degradation
T-type Ca2⫹
channel
T-type Ca2⫹
channel
T-type Ca2⫹
channel
In Vivo Phenotype
Arrhythmia (sinus
arrest,
idioventricular
rhythm, ventricular
tachycardia,
conduction block,
and AF)
Reference
Nos.
402, 566
MAPK
137
Raf-MEK-ERK
176
Adenoviral
delivery of
Gem:
- in the left
ventricle
- in the AV node
Cardiac specific
Rad-DN
overexpression
324
- QT interval
shortening
- PR interval
prolongation
Arrhytmia (sinus node
dysfunction, AV
block)
509, 552
551
Cardiac specific
RhoA
overexpression
Bradychardia with
prolonged PR
intervals, seconddegree AV block
and AF
Rho-kinase
Alteration of electrical
propagation
(decreased
expression of Cx40)
NADPH oxidase
(CTGF, N-cadherin,
Cx43)
Cardiac specific
overexpression
of GDI␣
Bradychardia, AV
block
Cardiac specific
Rac-V12
overexpression
AF
Increased activity of
PP5
65, 407
467
203
522, 565
2, 3
134, 467
54
Pak1/PP2A
220, 441
Pak1/PP2A
223, 441
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
In contrast to atrial and ventricular myocytes, the absence
of IK1 in SAN and AVN myocytes allows a slow depolarization of the resting potential to occur between each action
potential, via, though not exclusively, the activation of the
inward pacemaker currents (If), the T-type Ca2⫹ current,
and Cav1.3-dependent L-type Ca2⫹ current. As a consequence, most Na⫹ channels are inactivated and, in SAN and
AVN myocytes, the depolarization phase of the action potential is mainly achieved by the L-type Ca2⫹ current (290).
Heart rate is physiologically regulated by the autonomic
nervous system that modulates pacemaker currents in SAN
cells. Release of norepinephrine and ␤-adrenergic stimulation following sympathetic stimulation increases the heart
rate, primarily by modulating Ca2⫹ and Na⫹ permeability.
In contrast, parasympathetic fiber stimulation decreases
heart rate through the activation of IK,Ach (generated by
Kir3.1 and Kir3.4) and the decrease of If pacemaker current
and Cav1.3-dependent L-type Ca2⫹ current by acetylcholine-mediated M2 muscarinic receptor activation.
Modifications of the properties, regulation, and/or expression of cardiac ion channels can modify action potential
waveforms, synchronization, and/or propagation, thereby
increasing susceptibility to arrhythmias. Atrial fibrillation
(AF) is the most common cardiac arrhythmia, often associated with many forms of cardiac pathology. However, ventricular arrhythmias are the most common cause of sudden
cardiac death in hypertrophic cardiomyopathy and other
cardiac diseases, including heart failure. Numerous studies
have shown that small G protein regulation of electrical
coupling, ion channel function, location, or expression participates in the generation and maintenance of normal
and/or abnormal heart rhythms (2, 203, 324, 402, 407,
522, 552).
1. Ras proteins
A) RAS.
Ras and Ras-GAP have been found to block IK,Ach
activation by muscarinic receptor (566). This effect occurs
by uncoupling of the receptor from the trimeric G protein
rather than uncoupling of the G protein from the channel. It
has been proposed that binding of GTP-bound Ras to RasGAP causes a conformational change that exposes the SH2SH3 domain of the NH2 terminus of Ras-GAP, allowing
interaction with membrane phosphoproteins and finally
uncoupling of muscarinic receptor from the trimeric G protein (295).
Ras is also a negative regulator of the inwardly rectifying
K⫹ channel Kir2.1 via a mechanism involving the MAPK
cascade (137). Active Ras reduces IK1 current by reducing
the amount of available Kir2.1 channel at the cell surface
membrane. This effect is not due to a transcriptional regulation but to an action of the Ras-MAPK pathway on the
trafficking of Kir2.1 molecules.
In 1991, a tight linkage between H-ras gene on chromosome 11p and long QT syndrome in a large Utah family was
reported (222). In agreement with its effect on IK,Ach, H-Ras
is considered as a candidate gene for this autosomal dominant cardiac arrhythmia. However, both sequencing and
more precise definition of the chromosomic region containing the LQT gene has excluded H-ras from the candidate
genes for LQT syndromes (401).
Nevertheless, some studies in transgenic mice model described a role of Ras in heart rhythm regulation and arrhythmia. In addition to cardiac hypertrophy, transgenic
mice with a tamoxifen inducible cardiac specific expression
of H-Ras-V12 also gradually developed severe arrhythymia
(402). Cardiac arrhythmia in H-Ras-V12 transgenic mice
started at around day 7 after tamoxifen induction of the
trangene expression and was more pronounced at day 14.
Electrocardiogram recordings indicated sinus arrest, idioventricular rhythm, ventricular tachycardia, conduction
block, and AF. Severity of arrhythmia in H-Ras-V12 mice
was influenced by gender-specific factors as females demonstrated a more consistent arrhythmia phenotype. The onset
of arrhythmia in H-Ras-V12 mice was temporally correlated with the increase of action potential duration, an altered sarcoplasmic reticulum Ca2⫹ cycling leading to an
increase in Na⫹-Ca2⫹ exchanger current, and a decrease in
outward K⫹ current (402). In addition to these changes in
electrophysiological properties of cardiac cells, H-Ras-V12
expression also led to induction of Gi␣1 expression and
pertussis toxin (PTX) treatment, that blocks Gi␣ activity,
normalized electrophysiological parameters, and reduced
arrhythmias (402). This report thus suggests that contrary
to hypertrophy, the electrophysiological remodeling induced by H-Ras-V12 expression in mouse heart does not
seem to depend on a MAPK-dependent pathway, but
strongly involves upregulation of Gi␣ signaling.
B) RGK FAMILY. The role of RGK family in the regulation of
heart rhythm is directly related to their actions on the L-type
Ca2⫹ channel. In vivo adenovirus-mediated delivery of Gem,
injected in the left ventricular cavity of guinea pig hearts, produces a significant shortening of the electrographic QT interval, due to decreased expression of L-type Ca2⫹ channels and
the resulting abbreviation of action potential duration (324).
Focal delivery of Gem adenovirus to AV node in pig prolongs
the PR interval of the surface electrocardiogram, indicating
slowed conduction in the AV node. Moreover, overexpression
of Gem in the AV node caused a 20% decrease in the ventricular rate during AF (324).
In contrast, cardiac ␣-myosin heavy chain promoter-driven
overexpression of a dominant negative Rad mutant in
mouse produced a prolongation of the QT interval on the
surface electrocardiogram, without changes of others parameters. This was associated with an increased expression
of the ␣1c subunit of the L-type Ca2⫹ channel and a pro-
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longed duration of action potentials recorded from papillary muscle (552). Diverse arrhythmias such as sinus node
dysfunction, atrioventricular block, and ventricular extrasystoles were observed in Rad transgenic mice (552).
C) RAP1A.
A microarray screening performed to search differentially expressed genes in samples of right appendages
from patients with AF or sinusal rhythm has identified
Rap1a as a gene potentially involved in the pathological
process. Rap1a transcript expression is negatively correlated with AF (223). Further supporting the potential effect
of Rap1a in the regulation of heart rhythm is its role as an
antagonist of the inhibitory effect of Ras on atrial muscarinic K⫹ channels (567).
2. Rho proteins
A) RHOA. Development of transgenic mouse models has allowed direct in vivo assessment of the role of RhoA in heart
rhythm. Cardiac specific overexpression of RhoA induces a
marked sinus and AV node dysfunction characterized by
depression of heart rate, wandering pacemaker, prolonged
PR intervals, second-degree AV block, and AF (407). While
a marked positive chronotropic effect was induced by muscarinic receptor blockade by atropine in control mice, this
effect was not observed in RhoA transgenic mice so that the
bradycardia was not reversed by atropine (407). Since muscarinic receptors activate IK,ACh and slow heart rate via
hyperpolarization of pacemaker cells in SA and AV nodes,
an hypothesis to explain this phenotype and the bradycardia observed in RhoA transgenic mice would be that RhoA
overexpression led to constitutive activation of IKACh,
which thus became independent of muscarinic regulation.
Another potential mechanism could involve delayed rectifier potassium channels that participate in the repolarization of the action potential. Supporting this hypothesis, it
has been shown that RhoA physically associates with and
inhibits the delayed rectifier K⫹ channel Kv1.2, originally
cloned from rat heart atrium and found to physically interact with RhoA in mouse atria and ventricles (65, 407).
Overexpression of RhoA would therefore be predicted to
suppress Kv1.2 current leading to prolongation of action
potential thereby decreasing SA nodal rate.
AV conduction abnormalities at a young age, bradycardia,
and severe atrial arrhythmia at old age were also observed
in mice overexpressing the endogenous Rho protein inhibitor GDI␣ specifically in the heart (522). As the overexpression of GDI␣ resulted in the simultaneous reduction of the
activity of RhoA, Rac1, and Cdc42, it is not possible to link
the phenotype observed to a single Rho proteins. Electrocardiography and intracardiac electrophysiological recordings revealed first-degree AV block in GDI␣ mice at 1 wk of
age, which progressed into second-degree AV block at 4 wk
of age (522). These conduction defects were associated with
a dramatic decrease in the expression of connexin 40
(Cx40), a connexin specifically expressed in the atrium and
1672
the conduction system, the ablation of which resulted in AV
and ventricular conduction abnormalities (225). GDI␣
mice displayed normal positive chronotropic response to
␤-adrenergic receptor stimulation, and the AV conduction
defects could be partially rescued by ␤-adrenoceptor stimulation (522). This effect of RhoGDI␣/Rho protein signaling on AV conduction was thus probably due to a primary
effect on the expression and/or activity of cardiac proteins,
including Cx40, involved in the propagation of the electrical activity.
In addition to in vivo studies in transgenic mouse models,
several studies directly analyzed the effects of Rho proteins
on ion channels involved in electrical cardiac activity. An
example of these channels is the human ether-a-go-go related gene K⫹ channels (h-ERG encoded by KCNH2 gene),
responsible for IKr and the reduction of which leads to prolonged cardiac repolarization resulting in the development
of long QT syndrome. The constitutively active RhoA-L63
inhibits h-ERG channels, and it has been suggested that this
effect was due to Rho-kinase either by directly affecting the
channel or indirectly by modulation of protein phosphatase
activity (467).
RhoA is also defined as a negative regulator of the IK1
current. Constitutively active RhoA inhibited Kir2.1, and in
contrast, dominant negative form of RhoA prevented muscarinic receptor-induced inhibition of Kir2.1 (203). This
suggests that RhoA is involved in the physiological regulation of Kir2.1 by activation of muscarinic receptor and that
abnormal overactivation could potentially result in ventricular arrhythmia. This inhibitory effect of RhoA on Kir2.1
could also participate in the loss of the positive chonotopic
effect of muscarinic receptor blockade observed in mice
overexpressing RhoA in the heart (407).
B) RAC.
Evidence for a role of Rac1 in the regulation of heart
rhythm comes from both in vivo and in vitro studies. One of
the strategies used in mice was to overexpress a constitutively active Rac1 (Rac1-V12) under the control of the
␣-MHC promoter (2). Cardiac Rac1-V12 mice, showing a
30-fold increase in cardiac Rac1 activity, spontaneously
developed AF with age. Electrocardiogram indicated that at
the age of 10 and 16 mo, 44 and 75% of Rac1-V12 mice
displayed AF, respectively. Aging of Rac1-V12 mice was
associated with increased size of the atrium, but not the
ventricles. A fourfold increase in atrial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and
a significant enhancement of atrial collagen content was
observed in Rac1-V12 mice. Treatment with statin strongly
reduced the cardiac Rac1 and NADPH activities of Rac1V12 mice, without affecting the atrial dilation and interstitial fibrosis. Interestingly, a fourfold upregulation of Rac1
and NADPH oxidase activities was observed in samples of
the left atrial appendage of patients with AF (2). A more
recent study has suggested that the role of Rac1/NADPH
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
signaling in AF involves upregulation of connective tissue
growth factor (CTGF) and the resulting modulation of Ncadherin and Cx43 that were all upregulated in Rac1-V12
mice and in left atrial myocardium from patients with AF
(3). This study thus suggests that Rac1 and NADPH oxidase contributes to signal transduction during AF. It can be
speculated that statin-induced inhibition of Rac1 could
contribute to the prevention of postoperative AF obtained
by statin treatment (515).
Results obtained in guinea pig SAN pacemaker cells expressing constitutively active Pak1 indirectly suggest a role
of Rac1 and/or Cdc42 in Ca2⫹ and K⫹ channels. Endogenous Pak1 protein is abundant in SAN, atrial, and ventricular tissue (219). Expression of active Pak1 does not modify
the amplitude of the L-type Ca2⫹ current in SAN cells but
strongly reduces the stimulatory effect of ␤-adrenergic stimulation (219). Similarly, in the resting condition, the outward K⫹ current was not changed in SAN cells expressing
active Pak1, but the ␤-adrenergic stimulation-induced increase in K⫹ current amplitude and acceleration of the K⫹
current decay was almost abolished. In whole heart preparations, overexpression of active Pak1 attenuated the positive chronotropic action of ␤-adrenergic stimulation (219).
As the PP2A inhibitor okadaic acid partially reversed the
suppressing effect of active Pak1 on the response to ␤-adrenergic stimulation on Ca2⫹ and K⫹ currents, the effect of
Pak1 is likely to be due to PP2A (219). These observations
thus identify Pak1 as a potential target for therapeutic strategies against tachycardia triggered by cathecholamines in
different pathological conditions.
Several studies performed in non-cardiac cells can help to
define the molecular identity of channels that underlie the
effect of Rac1 on heart rhythm. For example, active Rac1
mutant stimulates ERG channel activity, via a stimulatory
action on the serine-threonine phosphatase PP5 (134, 467).
Dominant negative Rac1 has been shown to strongly increase Kir2.1 current, while the constitutively active Rac1
had no significant effect (54). This effect resulted from
Rac1-mediated reduction of basal internalization of the
channels, leading to an increase in the number of Kir2.1
channels at the plasma membrane, without change of its
global expression level (54). These data thus suggest that
Rac1-mediated regulation of ERG or Kir2.1 channels can
participate in the arrhythmogenic effect of Rac1 activation.
D. Cardiac Hypertrophy/Remodeling and
Heart Failure (Table 3)
The primary response of the heart to increased work load is
cardiomyocyte growth in an attempt to enhance contractility and preserve cardiac function (170). Cardiac hypertrophy is a physiological adaptative response to normal postnatal maturation or physical exercice that is characterized
by the maintenance of a normal myocardial structure and
function. However, cardiac hypertrophy is also a common
response to pathological stressors such as hypertension,
myocardial infarction-induced defects in the efficiency of
the contraction, neurohormonal stimulation, or oxidative
stress. Although it is an initial compensatory response to
maintain cardiac output, it evolves into a maladaptive process and a decompensated state with profound changes in
cardiac structure and function, predisposing to arrythmia
and sudden death, and leading to the development of heart
failure (121, 175). Pathological hypertrophy corresponds
to an abnormal enlargement of the heart muscle originating
from an increase in cell size of myocytes, interstitial fibrosis,
and proliferation of nonmuscle cells. Cardiomyocyte hypertrophy is associated with the enhancement of myofibril organization and change in gene expression including increased expression of immediate early genes (c-jun, c-fos,
egrI) and reexpression of fetal genes such as atrial natriuretic factor (ANF) and B-type natriuretic peptide (BNP) as
well as genes involved in cardiac contractility [␤-myosin
heavy chain (␤-MHC), ␣-skeletal actin].
Since heart failure is the leading cause of hospitalization and
mortality, the signaling mechanisms underlying cardiac hypertrophy and, even more importantly, the processes responsible for the transition between compensated hypertrophy to decompensated heart failure remain the subject of
extensive research. The following section focuses on the
role of small G protein in cardiac hypertrophy and remodeling, in particular Ras and Rho subfamilies, shown to play
a key role in the hypertrophic signaling pathways initiated
by the activation of G protein-coupled receptor.
1. Ras proteins
A) RAS.
Transient upregulation of H-Ras has been observed
in association with reexpression of c-myc in a variety of
experimental models of cardiac hypertrophy in adult rats,
suggesting that it may participate in hypertrophy of heart
(198, 232). In agreement with this observation in animal
models, H-Ras is also upregulated in patients with hypertrophic cardiomyopathy and cardiomyocyte size correlated
with the expression level of H-Ras (205). Moreover, patients suffering from “the RAS/MAPK syndromes” caused
by mutations of molecules in the RAS/MAPK cascade
(Noonan, LEOPARD, Costello, and CFC syndromes and
neurofibromatosis type I) exhibit hypertrophic cardiomyopathy (18).
Both in vitro and animal studies support this role of H-Ras
in cardiac hypertrophy (TABLE 3). In primary neonatal rat
ventricular myocytes, microinjection of active H-Ras-V12
stimulates hypertrophic response, attested by an increase in
cell surface and the induction of the expression of c-fos and
ANF (483). Expression of a H-Ras-V12 mutant that selectively activates Raf or a constitutively active Raf mutant
induces the same phenotype as H-Ras-V12, suggesting that
Raf is the downstream effector involved in the hypertrophic
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Table 3. Genetically modified mice and cardiac hypertrophy
Target
Type of Modification
Ras
Cardiac specific overexpression
(␣-MHC promoter)
Nf1
Myocardial specific deletion
RASSF1A
Deficiency (KO)
Raf-1
Cardiac specific overexpression
(␣-MHC promoter)
Transgene/Mouse
Active H-Ras mutant
(H-Ras-V12)
Dominant-negative Raf1
mutant (DN-Raf1)
Cardiac specific deletion
Rad
Deficiency (KO)
Rab1
Cardiac specific overexpression
(␣-MHC promoter)
Cardiac specific overexpression
(␣-MHC promoter)
RhoA
Rock1
Haploinsuficiency (Rock1⫹/⫺)
Deficiency (Rock1⫺/⫺)
RhoA
Active RhoA mutant
(RhoA-L63)
Phenotype
Reference Nos.
Cardiac muscle hypertrophy, with
increased ventricular mass and
myocardial cell size
Myofibrillar disarray
Fetal cardiac gene expression
Abnormal left ventricular diastolic
function
Normal systolic functions
remained normal
Progressive cardiac hypertrophy,
fibrosis, and cardiac myocytes
enlargement
Hyperactivation of Ras/Erk
signaling in the heart
Massive cardiac hypertrophy after
transverse aortic constriction
Cardiac fibrosis
Left ventricular dilatation
Fetal cardiac gene expression
2 of pressure overlaod-induced
cardiac hypertrophy
2 of pressure overlaod-induced
fetal cardiac gene expression
Increased cardiomyocyte
apoptosis
Decreased ERK1/2 activity and
cell growth
Left ventricular dysfunction and
dilatation
Increased number of apoptotic
cardiac cells
Increased susceptibility to cardiac
hypertrophy
Increased CaM-kinase II activity
Enhanced cardiac fibrosis and
increased CTGF expression
142, 184, 402
Cardiac hypertrophy
542
Premature death
No increase in ventricular mass
407
No increase in cardiomyocyte size
Increased expression of ANF and
␤-MHC
Cardiac conduction defects
No change in heart structure and
function
No change in ANG II- or L-NAMEinduced cardiac hypertrophy
2of ANG II-, L-NAME- or pressure
overload-induced cardiac
fibrosis
2of pressure-overload-induced
cardiac fibrosis
2of pressure-overload-induced
cardiomyocyte apoptosis
548
346
186
554
73
393
563, 582
Continued
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
Table 3.—Continued
Target
Type of Modification
Deficiency (Rock1⫺/⫺)
Cardiac specific overexpression
(␣-MHC promoter)
Rac1
Cardiac-specific overexpression
(␣-MHC promoter)
Cardiac-specific overexpression
(␣-MHC promoter)
Transgene/Mouse
In mice overexpressing
G␣q in
cardiomyocytes
Rock1: in mice
overexpressing G␣q in
cardiomyocytes
Active Cter-truncated
Rock1
Active Rac1 mutant
(Rac1-V12)
Cardiomyocyte-specific Rac1
deficiency (c-Rac1⫺/⫺)
Tamoxifen-inducible Cremediated deletion of
floxed Rac1 gene in
cardiomyocytes
Pak1
Cardiomyocyte-specific Pak1
deficiency (c-Pak1⫺/⫺)
Cre-mediated deletion
of floxed Pak1 gene in
cardiomyocytes
Cdc42
Cardiomyocyte-specific Cdc42
deficiency (c-Cdc42⫺/⫺)
Cre-mediated deletion
of floxed Cdc42 gene
in cardiomyocytes
effect of Ras (177). However, other evidence indicates that
activation of Raf/MEK1/2/ERK1/2 alone is insufficient to
initiate a complete hypertrophic response and that other
Ras effectors are required. Indeed, it has been shown that in
addition to Raf, the Ras effectors RalGDS, an exchange
factor for RalA and RalB, and phosphatidylinositol (PI)
3-kinase participate in Ras-mediated hypertrophic response
(128). RalGDS and Raf are mediators of specific hypertrophic transcriptional responses, whereas PI 3-kinase elicits a
global effect on gene expression in cardiac myocytes (128).
Other signaling molecules have been proposed to participate in Ras-mediated hypertrophy including MEKK/JNK
pathway (383) and Ras-GAP (1).
Ras has emerged as a central signaling protein required for
cardiac hypertrophy induced by different external stimuli
such as mechanical overload or neurohumoral factors in-
Phenotype
No effect on cardiac hypertrophy
Improved survival
Preserved left ventricular
structure and contractile
function
Decreased cardiomyocyte
apoptosis
Accelerate hypertrophic
decompensation
Reference Nos.
443
442
Extensive cardiac fibrosis
563
Neonatal lethal dilated
cardiomyopathy
Transient hypertrophy in young
mice (hypercontractile heart)
2of ANG II-induced cardiac
hypertrophy
473
of pressure-overload- or ANG IIinduced cardiac hypertrophy
of pressure-overload- or ANG IIinduced cardiac fibrosis
of pressure-overload-induced
cardiomyocyte apoptosis
of pressure-overload-induced
cardiac fetal gene expression
vulnerability to transition to heart
failure
of pressure-overload- or ANG II,
PhE-induced cardiac
hypertrophy
of exercice-induced cardiac
hypertrophy
vulnerability to transition to heart
failure
279
415
282
cluding phenylephrine (PhE) or angiotensin II (ANG II) (86,
404, 483, 558). G␣q downstream of ␣1-adrenergic or type 1
ANG II (AT1) receptors is required for Ras activation associated with cardiomyocyte hypertrophic response to PhE
and ANG II, in a protein kinase C-dependent or -independent manner (86, 171, 404). Furthermore, ␤␥-subunits of
Gi protein-dependent activation of Ras has also been shown
to be critical for ANG II-dependent cardiac fibroblast proliferation that also participates in cardiac hypertrophy in
vivo (557). While Ras GEFs linking trimeric G protein to
Ras activation following hypertrophic agonist stimulation
of cardiomyocytes are unknown, ␣1-adrenergic receptormediated cardiac hypertrophy has been ascribed to reactive
oxygen species-dependent Ras activation (512, 545). Stimulation of ␣1-adrenergic receptor causes the oxidative posttranslational modification of Ras leading to the decrease in
free Ras thiols and, thereby, Ras activation (243).
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In mice, cardiac specific expression of H-Ras-V12 leads to
cardiac muscle hypertrophy, with increased ventricular
mass and myocardial cell size, myofibrillar disarray, and
reactivation of ANF gene expression (142, 184) (TABLE 3).
Functionally, mice expressing cardiac H-Ras-V12 display
abnormal left ventricular diastolic function, but basal and
␤-adrenergic stimulated systolic functions remained normal
(184). This hypertrophic action of H-Ras-V12 does not
seem to secondarily result from the overactivation of Ras
signaling during embryonic development because induction
of H-Ras-V12 transgene expression in adult mice heart also
leads to significant myocyte hypertrophy already observed
1 wk after induction as posterior wall thickness of the left
ventricle, increased heart weight and single-cell capacitance, and fetal cardiac gene expression (402). Cardiac hypertrophy thus appears as an immediate downstream effect
of Ras activation in adult hearts. This was confirmed by a
recent study showing the reversibility of Ras-induced cardiac hypertrophy in a mouse model of temporary expression of H-Ras-V12 in myocardium (517).
Myocardial specific deletion of the Ras GAP Nf1 in mice
promotes progressive cardiac hypertrophy, fibrosis, and
cardiac myocytes enlargement associated with an hyperactivation of Ras/Erk signaling in the heart, suggesting a role
of Nf1 to limit Ras activity in cardiomyocytes of the adult
heart (548) (TABLE 3). These findings are in agreement with
observations made in Ras-association domain family protein isoform 1A (RASSF1A) knockout mice, another mice
model with defective Ras signaling (346). RASSF1A is a
potent tumor suppressor ubiquitously expressed that inhibits the Ras-Raf1-ERK1/2 pathway in cardiomyocytes and
prevents PhE-induced hypertrophy (346). RASSF1A knockout mice, after transverse aortic constriction, exhibited
massive cardiac hypertrophy that was almost double that
observed in control mice, associated with cardiac fibrosis
and increased expression of fetal genes and left ventricular
dilatation (346). A role of Ras signaling in cardiac hypertrophy is further supported by the discovery that Ras GAP
and the Ras protein Rheb would be potential targets of
miR1, a miRNA involved in cardiac hypertrophy (423).
Taken together, these data underlie the importance of endogenous molecular systems that maintain a low level of
Ras activity under physiological conditions.
Transgenic mice with cardiac specific expression of a dominant negative form of Raf (DN-Raf-1) has been generated
to define the requirement of Raf-1, downstream to Ras in
cardiac hypertrophy in vivo (TABLE 3). Under basal conditions, DN-Raf-1 mice have normal cardiac structure and
function (155). However, cardiac hypertrophy and hypertrophic gene induction in response to transverse aortic constriction-induced pressure overload are reduced in DN-Raf
mice, associated with an increased cardiomyocyte apoptosis and a decreased ERK1/2 activity and cell growth (155).
A more pronounced phenotype was obtained by cardiac
1676
specific deletion of Raf-1 in mice that, in absence of external
stress, leads to left ventricular dysfunction and dilatation,
with an increased number of apoptotic cells (554). These
data are thus consistent with an antiapoptotic effect of Ras/
Raf signaling, independent of ERK1/2 but related to the
antagonistic action of Raf against the propapoptotic kinase
ASK1 (554). These findings also indicate that the role of
Ras/Raf signaling in cardiac hypertrophy is mainly due to
its effect on cardiomyocyte hypertrophy and apoptosis resistance.
Very recently, a proteomic analysis of heart samples from
the transgenic mouse model of cardiac specific expression of
H-Ras-V12 has suggested that activation of the Wnt/␤catenin canonical pathway occurs secondary to Ras activation in the course of pathogenic myocardial hypertrophic
transformation and may also participate in the hypertrophic effect of Ras activation (518).
addition to its inhibitory role on Ca2⫹ channels,
Rad has been shown to play a protecting role in cardiac
hypertrophy. Rad is abundantly expressed in human
heart, and its expression is strongly decreased in left ventricular myocardium samples from patients with heart
failure that displayed severe hypertrophic morphology
(73). Similarly, Rad has been shown to be decreased in
the model of PhE-induced cardiomyocyte hypertrophy
(73). Silencing of Rad expression by siRNA in rat neonatal cardiomyocytes strongly increased protein synthesis
and ANF expression, whereas overexpression of Rad significantly reduced PhE-induced protein synthesis and
ANF expression, and decreased CaM kinase II activity
(73). In vivo, Rad-deficient mice were more susceptible
than normal mice to cardiac hypertrophy, with increased
CaM kinase II activity (73) and more severe cardiac fibrosis related to an increase in CTGF expression (579).
Rad is thus identified as an endogenous cardioprotective
mediator that prevents cardiac hypertrophy through the
inhibition of CaM kinase II activity and cardiac fibrosis
through the inhibition of CTGF.
B) RAD. In
C) RAP.
In addition to their role in the regulation of excitation-contraction coupling, Rap proteins could also participate in signaling cascades involved in cardiac hypertrophy.
Rap1 has been shown to mediate the activation of B-Raf/
ERK1/2 induced by M1 muscarinic receptor activation, via
the activation of the Rap1 GEF Ca2⫹ and diacylglycerolregulated GEF I (CalDAG-GEFI) (146). In neonatal ventricular myocytes, cAMP/PKA-dependent Rap1 activation is
responsible for ERK1/2 activation and cell growth downstream of the PGE2 receptor EP4 (160). In the same cellular
model, the role of Rap1 in hypertrophy has been ascribed to
cAMP-mediated activation of the Rap1 GEF Epac1, leading
to Rap1-dependent inhibition of ERK5 (106). Nevertheless,
surprisingly, more recent in vivo and in vitro studies suggested that Epac1 hypertrophic effects, including those in-
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
duced by ␤-adrenergic receptor stimulation, are independent of Rap1 but rather involve Ras and Rac, calcineurin,
and CaM kinase II (305, 319). Both Rap1 and Epac1
knockout mice will be useful to decipher the role of Rap1
signaling in cardiac hypertrophy.
D) RAB. Rab1 regulates protein transport, specifically from
the endoplasmic reticulum to the Golgi apparatus. Overexpression of Rab1 induced hypertrophy of neonatal cardiomyocytes in culture (117). Transgenic expression of Rab1
in the myocardium promotes cardiac hypertrophy in mouse
that has been ascribed to an abnormal expression and subcellular localization of proteins, such as PKC␣ (542).
E) RAL.
RalA and RalB are among the closest relatives of
Ras, and Ral proteins have been shown to be recruited by
Ras. Activated Ras binds to the Ras-binding domain of Ral
GEFs such as Ral-GDS, Ral guanine nucleotide exchange
factor-like factor (Rlf), RGL1, and RGL2, leading to its
translocation to the membrane where it produces GDP/
GTP exchange on Ral (299). Since the identification of RalGDS as an effector of Ras involved in Ras-mediated ventricular myocytes hypertrophy (128), further studies supported the role of Ral signaling in cardiac hypertrophy.
Overexpression of Ral-GDS, constitutively active Ral mutant, or Rlf stimulates promoter activity of early and embryonic genes in rat neonatal myocytes, synergically with
activated Ras (218, 370). Dominant-negative Ral partially
inhibited active Ras-induced promoter activation (218).
Stimulation of ␣1-adrenergic receptor induced activation of
Ral in neonatal ventricular myocyte (370), and Ral activity
is increased in hypertrophied heart (218).
2. Rho proteins
A) RHOA. RhoA has been implicated in the morphological
changes and induction of gene expression associated with
hypertrophic response in cardiac myocytes. In vitro, stimulation of cultured neonatal ventricular myocytes with hypertrophic factors such as PhE, endothelin-1, or ANG II
induced activation of RhoA (16, 84). Transfection or infection of neonatal ventricular myocytes with active RhoA
mutant (RhoA-V14) stimulates ANF expression and myofibrillogenesis (16, 181, 406, 484). Conversely, expression
of the dominant negative RhoA-N19, Rho-GDI, or the selective RhoA inhibitor Clostridium botulitum C3 exoenzyme inhibits hypertrophic responses induced by soluble
stimuli such as PhE, ANG II, or lysophosphatidic acid (10,
16, 168, 406) or by mechanical stress (12).
Mechanisms linking extracellular stimuli to RhoA activation in neonatal myocytes are still not firmly established.
Trimeric G proteins play a key role in the activation of
RhoA by agonists of G protein-coupled receptor, probably
through activation of Rho GEFs that remain to be identified. G␣q was initially found to mediate RhoA activation
following PhE-induced ␣1-adrenergic receptor stimulation
(171, 406), but G␣12/13 was also shown to be responsible
for this activation (296). ANG II-induced RhoA activation
depends on G␣12/13 signaling (337), whereas activation of
RhoA by lysophosphatidic acid involves G␣i (168). In response to mechanical stretch, activation of RhoA depends
on PKC␣ (356).
Rho-kinases have been identified as the RhoA effector mediating RhoA-dependent hypertrophic responses both in
vitro and in vivo. In neonatal ventricular myocytes, pharmacological inhibition of Rho-kinase (fasudil or Y27632)
or transfection of dominant negative forms of Rho-kinase
prevents morphological and cytoskeleton changes, protein
synthesis, and ANF expression associated with response
induced by hypertrophic factors such as PhE, endothelin-1,
or leptin (181, 245, 576). In neonatal ventricular myocytes,
Ras activation by hypertrophic factors is also essential to
their hypertrophic effects (see above), suggesting that Ras/
MAPK and RhoA induce a hypertrophic response by separate, complementary, and synergistic pathways (58, 292).
In fact, RhoA/Rho-kinase-mediated actin cytoskeleton remodeling has been shown to be necessary for the translocation of ERK to the nucleus, providing a mechanism for the
synergy between RhoA and Ras signaling pathways regulating gene transcription and inducing hypertrophy. The precise
downstream mechanisms by which RhoA/Rho-kinase activate
the hypertrophic gene program remain obscure, however. Activation of the cardiac transcription factor GATA-4 is identified as downstream nuclear mediators of Rho-kinase during
myocardial cell hypertrophy (560). Recently, myocardin-related transcription factor A (MRTF-A), and the subsequent
transcriptional activation of serum response factor (SRF)-dependent fetal cardiac genes, have been defined as critical
downstream mediators of RhoA/Rho-kinase-dependent hypertrophic response in cardiac myocytes (244). Another RhoA
effector, protein kinase N (PKN), has also been involved in the
SRF-dependent transcriptional responses associated with cardiomyocytes hypertrophy (321).
In vivo, pressure overload induces a rapid activation of
Rho-kinases in the adult rat myocardium, suggesting that it
could participate in mechanisms of initial as well as chronic
adaptative responses of cardiac myocytes to mechanical
stress (488). While both Rock1 and Rock2 are expressed in
the myocardium, the increased Rho-kinase activity induced
by pressure overload has been ascribed to Rock1 (582). In
Dahl salt-sensitive hypertensive rats, the ventricular hypertrophy and function are significantly ameliorated by Rhokinase inhibition possibly through an upregulation of the
downregulated endothelial nitric oxide synthase (eNOS)
and the reduction of oxidative stress through the inhibition
of NAD(P)H oxidase and LOX-1 expression (228, 314).
Chronic Rho-kinase inhibition also markedly limits pathological cardiac hypertrophy induced by pressure overload in
rats and improves diastolic function in rats (23, 364), but
has no effect on chronic swimming-induced physiological
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LOIRAND, SAUZEAU, AND PACAUD
cardiac hypertrophy (23). Several neurohormonal factors,
such as ANG II, are suspected to play a role in ventricular
hypertrophy and the transition to heart failure. Long-term
treatment with the Rho-kinase inhibitor fasudil reduces the
ANG II-induced cardiomyocyte hypertrophy in mice (166,
514). Rho-kinase activity is also involved in the pathogenesis of left ventricular remodeling after myocardial infarction (158). All these findings support the involvement of
RhoA/Rho-kinase signaling in the development of cardiac
hypertrophy in vivo.
Genetically modified mouse models have then been useful
to further refine the role of RhoA and Rho-kinases in cardiac remodeling and hypertrophy (TABLE 3). Surprisingly,
although ventricular expression of some hypertrophic
marker genes such as ANF and ␤-MHC are increased in
mice that overexpressed RhoA in a cardiac specific manner,
they did not develop an obvious cardiac hypertrophy (407).
In contrast, they display progressive left ventricular chamber dilation and decreased contractility. The role of RhoA
during cardiac development and the potential of compensatory mechanisms could explain why in vivo overexpression of RhoA did not mimic its in vitro hypertrophic effect.
Haploinsufficiency of Rock1 in mice does not alter cardiac
structure and function under basal conditions (393). All
indexes of cardiac hypertrophy, i.e., wall thickness, left ventricular mass, cardiomyocyte size, or expression of ANF,
induced by ANG II or L-NAME were similar in control and
Rock1⫹/⫺ mice (393). However, a marked reduction of
cardiac fibrosis is observed in Rock1⫹/⫺ mice chronically
treated with ANG II or L-NAME, or after transaortic constriction, compared with control mice (393). This decreased
fibrosis is associated with a reduced expression of markers
of fibrosis including transforming growth factor-␤ (TGF␤), CTGF, and type III collagen. Similar reduction of fibrosis has been observed in Rock1 knockout mice (582), together with a decreased cardiomyocyte apoptosis following
pressure overload by aortic banding (563). These results
thus indicate that in vivo, Rock1 contributes to the development of cardiac fibrosis but not hypertrophy in response
to various pathological conditions. This was recently confirmed in a transgenic mice expressing a truncated activate
form of Rock1 in cardiomyocytes (563) These mice displayed extensive cardiac fibrosis due to upregulation of
TGF-␤ and cytokine release in cardiomyocytes that promoted myofibroblast differentiation (563).
In a mouse model with cardiac specific overexpression of
G␣q, that develops compensated cardiac hypertrophy at
young ages and progressing to heart failure, transgenic
overexpression of Rock1 increases cardiomyocyte apopotosis and accelerates hypertrophic decompensation (443).
In contrast, although it does not prevent the development of
cardiac hypertrophy, deletion of Rock1 improves survival,
inhibits cardiomyocytes apoptosis, and preserves left ventricular structure and function in old G␣q mice (442, 443).
1678
These data thus support the absence of a role of Rho-kinase
in pathological hypertrophy but provide evidence for a crucial role of Rock1 in the transition from cardiac hypertrophy to heart failure.
The difference in the cardiac phenotype resulting from genetic modifications of RhoA or Rock1 in mice could be
related to the relative independence of Rock1 activation
from RhoA activity. Rock1 can be constitutively activated
by caspase-3-mediated proteolytic cleavage of its inhibitory
COOH terminus (432). Accumulation of this COOH terminus-truncated form of Rock1 has been described in failing human heart and genetic murine models of heart failure
(72). This active truncated Rock1 stimulates caspase-3 and
phosphatase and tensin homolog deleted on chromosome
ten (PTEN) activities. The resulting repression of the PI
3-kinase/Akt cell survival pathway thus sets on a positive
feed-forward regulatory loop that favors cardiomyocyte
apoptosis (72). Because cardiomyocyte apoptosis is a major
cellular event involved in the transition from compensated
hypertrophy to dilated cardiomyopathy and failure, this
mechanism can support the observed important role of
Rock1 in hypertrophic decompensation (443).
Finally, the inhibitory effect of pharmacological inhibition
of Rho-kinases on cardiac hypertrophy is not mimicked by
Rock1 deletion. That might suggest that Rock2 could be
involved in cardiac hypertrophy or the antihypertrophic
effect of Rho-kinase inhibitors did not result from Rhokinase inhibition. Cardiac specific deletion of Rock2 in mice
would be useful to address this question.
B) RAC1. As for other small G proteins, first evidence for a
role of Rac1 in cardiac hypertrophy was provided by in
vitro analysis in neonatal ventricle myocytes. Expression of
active V12-Rac1 results in sarcomeric reorganization, increase in cell size, and atrial natriuretic peptide (ANP) secretion (372). In contrast, the dominant negative N17-Rac1
expression inhibits the morphological changes and the increased protein synthesis induced by PhE (372). In agreement with this observation, hypertrophic factors including
PhE, ANG II, and endothelin-1 have been shown to induce
rapid Rac1 activation in cardiac myocytes, and N17-Rac1
inhibits the generation of reactive oxygen species (ROS) and
the stimulation of ERK cascade, but not the activation of
p38MAPK induced by these stimuli (84, 93, 172). Mechanical stretch also induces Rac1 activation in cardiomyocytes
via a signaling involving PKC␦ (84, 356). In contrast to that
observed after stimulation with soluble hypertrophic factors, expression of dominant negative form of Rac1 prevents stretch-induced p38MAPK activation, increased protein synthesis, and intracellular ROS generation (13).
Stretch-induced p38MAPK kinase activation is also inhibited by treatment with the antioxidant N-acetyl-L-cysteine
(NAC), indicating that stretch-induced Rac1-dependent activation of p38MAPK was mediated by ROS production
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
(13). Indeed, the stimulation of the superoxide generating
NADPH oxidase by Rac1 and the subsequent generation
ROS generation are important mediators of the cellular
responses downstream of Rac1. The use of Rac1-V12 and
Rac1-N17 mutant expression in cardiomyocytes has demonstrated that, in addition to p38MAPK, Rac1-mediated
ROS is responsible for the activation of the transcription
factor NF␬B. It was further shown that this activation involved ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1) and that this pathway is essential for
Rac1-induced hypertrophic effects (167). This Rac1-ROS
signaling pathway has been also shown to be upstream to
JNK activation and cardiomyocyte death, in particular in
the context of ␤-adrenoceptor-stimulated apoptosis (192).
In vivo, transaortic constriction-induced cardiac hypertrophy in mice is associated with activation of Rac1 NADPH
oxidase in left ventricular myocardium (93). To further address the role of Rac1 activation in the heart, transgenic
mice that express constitutively activated Rac1 specifically
in the myocardium have been developed (473). These mice
display two different cardiac phenotypes: a neonatal lethal
dilated cardiomyopathy and a transient hypertrophy in
young mice that resolved with age. Hypertrophic hearts do
not show signs of myofibril disarray and are hypercontractile in working heart analyses (473). These results thus unexpectedly suggest that both phenotypes result from deregulation of signaling by Rac1 and Pak, followed by alteration of cellular adhesion. The use of mice harboring
cardiomyocyte-specific, tamoxifen-inducible Cre recombinase circumvented the problem of the embryonic lethality
of Rac1 deletion to generate cardiomyocyte-specific Rac1
knockout (c-Rac1⫺/⫺) mice (415). Cardiac specific deletion of Rac1 prevents ANG II infusion-induced myocardial
oxidative stress, NADPH activation, and cardiac hypertrophy. In agreement with in vitro studies, this mouse model
supports the notion that Rac1-mediated NADPH oxidase
activity and ROS production, but not some other effects of
Rac1, are the primary regulators of cardiac hypertrophy in
response to ANG II (415). This idea has been recently supported by data obtained in cardiomyocyte-specific Pak1
knockout mice (c-Pak1⫺/⫺) (279). These mice develop enhanced cardiac hypertrophy and fibrosis in response to
pressure overload or ANG II infusion, compared with control, and they are more vulnerable to the transition to heart
failure. This phenotype is associated with an increase in
cardiomyocyte apoptosis and fetal gene expression. In
c-Pak1⫺/⫺ mice myocardium, pressure overload does not
induce the activation of MKK4/MKK7-JNK pathway,
whereas activation of p38MAPK and ERK1/2 are normal.
In ANG II-treated mice, ROS production is comparable in
control and c-Pak1⫺/⫺ mice (279). These mice thus allow
to uncover the cadioprotective role of Pak1/JNK pathway
in attenuating cardiac hypertrophy and halting the transition to heart failure. In addition, the opposed phenotype of
c-Rac1⫺/⫺ and c-Pak1⫺/⫺ mice provides further evidence
that Pak1 is not a major effector of Rac1 in cardiomyocytes
and supports an important role of ROS in the hypertrophic
effect of Rac1. This also suggests that upstream activators
of Pak1 other that Rac1 regulate Pak1 activity in cardiomyocytes, with Cdc42 as a good candidate.
All these in vivo data, together with the threefold increase of
Rac1 activity and the upregulation of NADPH oxidasemediated ROS release observed in myocardium of patients
with ischemic or dilated cardiomyopathy, suggest that
Rac1/ROS signaling contributes to the pathophysiology
(286).
C) CDC42. Very little is known about the function of Cdc42 in
cardiomyocytes. In fact, in vitro, Cdc42 was found to have
hypertrophic effects in neonatal cardiomyocytes that appear morphologically distinct from those exerted by Rac1
or RhoA (328). Expression of dominant active mutant of
Cdc42 promoted myofibril assembly in series and increased
the length/width ratio of cardiomyocytes while RhoA and
Rac1 induced parallel assembly of sarcomeric units and did
not affect the length/width ratio (328). Dominant active
mutant of Cdc42 expression also increased p38MAPK activity in neonatal cardiomyocytes (13). An increase in
Cdc42 activity is rapidly observed (10 min) in cardiomyocytes stimulated by hypertrophic stimuli such as leukocyte
inhibitory factor (LIF), ANG II, PhE, or isoproterenol
(288), and expression of a dominant negative form of
Cdc42 inhibits LIF-induced sarcomere organization in series and length-to-width ratio increase (328). Furthermore,
silencing of Cdc42 by shRNA also reduces MEKK1/
MKK4/7 and JNK activation induced by LIF or isoproterenol in neonatal cardiomyocytes (288). The observation
that dominant negative Cdc42 suppressed stretch-induced
p38MAPK activation indirectly suggests that mechanical
stretch also activates Cdc42 (13). These data thus support
pro-hypertrophic effect of Cdc42 that may involve
MEKK1/MKK4/7/JNK and p38MAPK pathways. However, this pro-hypertrophic effect of Cdc42 observed in
vitro has not been confirmed by in vivo studies. In fact, mice
with cardiac-specific deletion of Cdc42 developed exacerbated pressure overload hypertrophy, greater cardiac hypertrophy in response to neuroendocrine agonist infusion
(ANG II and PhE), and enhanced exercise-induced hypertrophy and sudden death (288). They transitioned more
quickly from hypertrophy to heart failure than control
mice. This phenotype is associated with the suppression of
MEKK1/MKK4/7/JNK pathway activity, resulting in the
upregulation of calcineurin-nuclear factor of activated T
cells (NFAT) signaling (288). The causal role of the suppression of the JNK pathway in the cardiac phenotype of
c-Cdc42⫺/⫺ mice has been demonstrated by the reversion
of the enhanced cardiac growth when they were crossed
with MKK7 transgenic mice (288). These results thus identify Cdc42 as a antihypertrophic and protective mediator
during stress stimulation. Interestingly, the phenotype of
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LOIRAND, SAUZEAU, AND PACAUD
c-Cdc42⫺/⫺ mice is very similar to that of c-Pak1⫺/⫺
mice, thereby supporting the idea that Cdc42 is the endogenous activator of Pak1 and that the Cdc42/Pak1/JNK signaling pathway is cardioprotective, in limiting hypertrophy
and transition to heart failure.
Other clues speaking in favor of a role of Cdc42 in cardiac
hypertrophy were recently supplied by studies of microRNAs.
Cdc42 was identified as a target of, and negatively regulated
by miR-1 and miR-133 (that also targets RhoA), and a decreased expression of both miR-133 and miR-1 was observed
in mouse and human models of cardiac hypertrophy (66, 376).
E. Ischemia/Reperfusion Damage,
Preconditioning
Myocardial ischemia, due to a reduced blood supply, induces severe tissue injury in the heart, and large effort has
been made to understand the mechanisms causing cellular
damage during ischemia. It is now recognized that upon
reperfusion, exposure of ischemic myocardium to molecular oxygen initiates a cascade of events that paradoxically
augments myocardial cell dysfunction and death, with ROS
as essential mediators of ischemia/reperfusion injury. Nevertheless, the heart and cardiac myocytes have developed
powerful endogenous mechanisms to protect themselves
against ischemia/reperfusion injury which can be activated
by brief period of ischemia and reperfusion preceding a
more severe one through a phenomenon known as ischemic
preconditioning. Signaling mechanisms mediating both
ischemia/reperfusion injury and cardioprotection are the
subject of intense investigation as they may represent molecular targets that could be used to mimic ischemic preconditionning by pharmacological interventions. Members of
the Ras superfamilly of small G proteins represent potential
good candidates for such an objective. However, despite
intensive research in this field, data are still inconsistent.
Furthermore, studies are essentially performed in rat or
mice models of cardiac ischemia/reperfusion while the temporal and spatial development of myocardial ischemia/reperfusion injury in these animal species is different from that
seen in humans (165). Therefore, although small rodent
heart models are useful to decipher molecular mechanisms
and identify potentially interesting targets, the confirmation
of such novel mechanisms in larger animal (such as pig or
dog) models of myocardial ischemia/reperfusion that more
closely resembles the human situation (124) appears mandatory before translation to the clinic.
1. Ras proteins (Figure 3)
A) RAS. Oxidative stress activates Ras in the cardiomyocytes
and in the heart (85, 469). Numerous in vitro and in vivo
studies clearly identified the Ras/Raf/MEK1/2/ERK1/2 pathway as a prosurvival pathway that protects cardiomyocytes from apoptotic death following oxidative stress,
1680
IR/ROS
Ras
↑ Raf-1
Rad
↑ Rassf1A
↑ MEK
↓ ASK1, Mst2
↑ Mst1
↑ ERK1/2
↓ JNK/p38
↑ JNK/p38
↓ apoptosis
cardioprotection
↑apoptosis
FIGURE 3. Involvement of Ras proteins in ischemia/reperfusioninduced myocardium injury. Ras and Rab signaling activated by ishemia/reperfusion (IR) or reactive oxygen species (ROS) leading to
decrease myocardial cell survival and increase apoptosis pathways.
thus identifying ERK1/2 as major components of the reperfusion injury salvage kinase (RISK) pathway (11, 325,
399, 573). Results from pharmacological studies confirmed these data by showing that activation of ERK1/2
signaling significantly contributes to the cardioprotective
effects of ␤-adrenergic receptor antagonists that are
largely used in the clinical management of human ischemic heart diseases (237).
Genetic manipulations of MEK1/2, ERK1/2, and Raf-1 in
mice nicely demonstrated the anti-apoptotic action of the
Raf/MEK1/2/ERK1/2 signaling leading to cardioprotection
during reperfusion (155, 269). However, cardiac specific
disruption of the c-raf-1 gene indicated that the beneficial
effect of Raf-1 on cardiomyocytes survival is not mediated
by the MEK1/2-ERK1/2 pathway but involves repression of
ASK1, Mst2, and JNK-p38MAPK activity (76, 325, 554).
More recent data also suggest that Ras, through the activation of its other downstream effector Rassf1A, has a proapoptotic effect through the kinase Mst1 already known to
be activated by ischemia/reoxygenation in the heart (347,
556). Rassf1A has been identified as an endogenous activator of Mst1 in the heart, and the Rassf1A/Mst1 pathway
promoted apoptosis in cardiomyocytes (98).
It therefore appears that although Ras signaling exerts potent cardioprotective effects through Raf-dependent and
-independent mechanisms, stimulation of Raf-independent
apototic signaling also occurred downstream of Ras. Accordingly, the dynamic balance of this dual signaling would
be critical in determining cardiomyocyte fate subsequent to
reperfusion injury. The observation that inhibition of Ras
proteins by FTIs limits ischemia/reperfusion-induced injury
even suggests that the Raf-independent pro-apoptotic Ras
effects, probably through Mst1 and JNK, predominates
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
over its Raf-ERK1/2-mediated cardioprotective action (35,
358).
B) RAD.
Although its physiological or pathophysiological
role has not been yet assessed, it has been recently shown in
vitro in neonatal rat ventricular myocytes that Rad induces
cardiomyocyte apoptosis through p38MAPK activation
(472).
2. Rho proteins (Figure 4)
A) RHOA. RhoA has been initially described as both a mediator of survival and a trigger of cell death. High levels of
expression of activated RhoA in cardiomyocytes initially
elicit a hypertrophic response, but sustained expression
leads to the development of apoptosis, via a mechanism
involving Rho-kinase, p53-dependent upregulation of the
pro-apoptotic protein Bax and the consequent induction of
the mitochondrial death pathway (100). A special role has
been ascribed to Rock1 as this isoform, cleaved by caspase
3, becomes irreversibly active and leads to a positive feedback on caspase 3 activity, activation of PTEN, and inhibition of Akt in cardiomyocytes (72). This truncated form is
absent in normal heart but was found in human failing
heart (72). In contrast to this pro-apoptotic effect of RhoA/
Rho-kinase signaling, moderate expression of activated
RhoA, at levels close to physiological levels of RhoA activation, protects cardiomyocytes from ROS-induced apo-
IR
Rac-1
ROS
RhoA
↑ Rock1
↑ FAK
↑ PDK
↑ PKN
↑ Caspase 3
↑ PTEN
↓ Akt
↑ apoptosis
↓ survival
↑ PI3-K
↑ Akt
↓ apoptosis
↑ survival
FIGURE 4. Involvement of Rho proteins in ischemia/reperfusioninduced myocardium injury. RhoA and Rac signaling activated by
ishemia/reperfusion (IR) or reactive oxygen species (ROS) leading to
decrease myocardial cell survival and increase apoptosis pathways.
ptosis through FAK phosphorylation, association of FAK
with PI 3-kinase, and finally, Akt activation (99). Confirming these findings, inhibition of endogenous RhoA by C3
exoenzyme has been shown to decrease Akt activity and
stimulate cardiomyocyte apoptosis (238). Thereafter, several studies have been designed to investigate in vivo
whether RhoA signaling is beneficial or detrimental to cardiomyocyte survival during ischemia/reperfusion.
The first in vivo data reported were based on the effects of
the pharmacological inhibition of the RhoA effector Rhokinase on heart damages caused by ischemia/reperfusion in
rats or mice. RhoA/Rho-kinase activity is not modified by
ischemia, but an early and sustained activation is observed
in the ischemic myocardium during reperfusion (25, 150).
Rho-kinase inhibition with Y27632 or fasudil reduces ischemia/reperfusion-induced cardiomyocytes apoptosis and
improves postischemic cardiac function through the activation of PI 3-kinase/Akt/NO signaling (25, 150, 538) and the
inhibition of JNK (580). All these pharmacological data
thus support a deleterious effect of Rho-kinase activity in
myocardial ischemia/reperfusion injury. However, recent
results obtained in mice with cardiomyocyte-specific conditional expression of low levels of activated RhoA (RhoAL63) unexpectedly reveal that RhoA protects the heart
against ischemia/reperfusion injury (544). Both in perfused
heart experiments and in vivo, expression of active RhoA
strongly reduced ischemia/reperfusion-induced myocardial
cell damage, evidenced by a reduction of the infarct size and
lactate dehydrogenase and cytochrome c release, thus
showing that active RhoA in cardiomyocytes is protective
against ischemia/reperfusion injury (544). Conversely, in
mice with cardiomyocyte specific deletion of RhoA, infarct
size and lactate dehydrogenase release following ischemia/
reperfusion are significantly increased compared with control mice, thus confirming the beneficial effect of RhoA in
cardiomyocytes (544). Downstream of RhoA, all the potential effectors involved such as Akt, FAK, ERK1/2, or PKN
are not modified in hearts expressing active RhoA compared with control hearts, while PKD activity is markedly
increased (544). A causal role of the PKD activation in the
cardioprotective effect of RhoA is supported in vitro by the
similar potentiation of H2O2-induced cardiomyocyte death
by RhoA and PKD inhibition, and in vivo by the complete
reversion of the cardioprotection conferred by active RhoA
expression by PKD blockade (544). It is of interest to mention recent data regarding the other RhoA effector PKN.
Hypotonic swelling of cardiomyocytes, a condition found
in pathological contexts such as ischemia-reperfusion, leads
to RhoA and PKN activation that mediates cardiac myocyte
survival (206). In vivo, cardiomyocyte specific expression
and deletion of the RhoA effector PKN in mice demonstrate
that activation of PKN plays a cell-protective role in cardiac
myocytes during ischemia/reperfusion (475). Although a
direct link with RhoA was not made, this study is however
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in agreement with a cardioprotective role of RhoA against
ischemia/reperfusion injury in vivo.
This discrepancy between pharmacological studies and genetic manipulation of RhoA could result from multiple reasons, including 1) the targeting of Rho-kinase in pharmacological studies and RhoA in mice models, 2) the specific
manipulation of RhoA signaling in cardiomyocytes in genetically modified mice while pharmacological inhibition
affects every cell types with potential effects on neurohumoral feedback loops, and 3) the possible lack of selectivity
of the pharmacological inhibitors.
Similarly, inconsistent results have been reported regarding
the role of RhoA/Rho-kinase in preconditioning. Ischemic
preconditioning reduces the activity of Rho-kinase in the
myocardium, while, in contrast, the ERK1/2 signaling is
increased (578). This downregulation of the RhoA/Rhokinase signaling as well as the decrease in cardiomyocyte
apoptosis induced by ischemic preconditioning is lost after
ERK1/2 inhibition (578). These data suggest a relationship
between ERK1/2 and RhoA/Rho-kinase, such that ERK1/2
activity is required in ischemic preconditioning to oppose
the effect of RhoA/Rho-kinase signaling on apoptosis. This
is in agreement with the observation that inhibition of Rhokinase by Y27632 or high dose of fasudil mimicks the effect
of ischemic preconditioning in rats (101, 102). However, in
opposition to these results, it has also been suggested that
RhoA/Rho-kinase activity participates in the cardioprotective effect of ischemic preconditioning since inhibition of
Rho-kinase by Y27632 in perfused hearts or by low doses
of fasudil in vivo in rats reduces the protective effects of
ischemic preconditioning (101, 409). This cardioprotective
role of RhoA is also supported by its involvement in the
preconditioning effect of adenosine, coupling A3 adenosine
receptor to phospholipase D activation (259, 322).
In short, these data highlight the difficulties to definitively
define whether RhoA signaling is deleterious or protective
during ischemia/reperfusion and preconditioning, and
much remains to be done before considering RhoA/Rhokinase inhibition as a potential therapy of ischemia/reperfusion injury.
B) RAC. As a component of the NADPH complex, Rac1 has
been suggested to play a dominant role in ROS generation
after ischemia/reperfusion. Nevertheless, a limited number
of studies have addressed the role of Rac in cardiac ischemia/reperfusion injury, and there are no data on cardiac
preconditioning. Expression of the dominant negative
Rac1-N17 mutant results in protection of cardiomyocytes
from hypoxia/reoxygenation-induced cell death (224). In
vivo, ischemia/reperfusion increases Rac1 in the heart and
ROS production, and ischemia/reperfusion-induced ROS
production and apoptosis are strongly decreased in hearts
from mice with cardiac specific deletion of Rac1, compared
1682
with controls, resulting in a limitation of the infarct size and
preservation of myocardial function (439). Pharmacological inhibition of Rac1 by NSC23766 gives the same results
(439), thus providing evidence that Rac1 is involved in
myocardial damage produced by ischemia/reperfusion and
that strategy aiming at decreasing Rac1 activity in these
circumstances could represent new potential therapy.
IV. RAS PROTEINS IN VASCULAR
FUNCTION AND DISEASES
A. Embryonic Vascular Development
(Vasculogenesis and Angiogenesis)
Vascular development in vertebrates is a complex phenomenon, initiated in the embryo at different places and times,
and underlaid by two processes, vasculogenesis and angiogenesis. The first blood vessels of the mouse begin to form in
the yolk sac at E6 – 6.5. Later in development, vasculogenesis is initiated with the embryo proper, with blood vessels
appearing in the following order: endocardium, primary
vascular network lateral to the midline, paired dorsal aortae, head, and cardinal vessels. Vasculogenesis corresponds
to de novo formation of vessels from endothelial cell precursors or angioblasts that differentiate into endothelial
cells and form lumenized tubes organized in primitive continuous vascular network (after E8.5 in mouse embryo).
Angiogenesis is then responsible for the formation of secondary vessels by endothelial cells sprouting from the primary vessels. Endothelial cell proliferation, migration, and
differentiation, essential to form mature vasculature are
guided by extracellular cues that include soluble growth
factors, extracellular matrix, and cell-cell interactions (249,
425). When the endothelial cells are assembled into vascular tubes, they become surrounded by mural cells of the
smooth muscle cell lineage, referred to as pericytes (in capillaries, small venules, and immature blood vessels) and vascular smooth muscle cells (in mature and large blood vessels). Pericytes and smooth muscle cells differentiate from
the mesenchyme and migrate around the growing blood
vessels. Signaling molecules such as small G proteins that
regulate differentiation, proliferation, and migration have
been shown to play important role in vasculogenesis and
angiogenesis.
1. Ras proteins
A) RAS. Mouse embryos lacking p120-Ras GAP exhibited
major defects in their circulatory system organization. The
yolk sac of p120-RasGAP deficient embryos was abnormal,
with delayed organization of endothelial cells that aggregated into a honeycombed pattern but subsequently failed
to organize into a vascular network (164). Within p120RasGAP deficient embryos, the dorsal aorta was thinned
with aberrant ventral branches that resemble intersegmen-
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
tal arteries. In late stage embryo (E9.5), the embryonic vascular exhibited local ruptures causing leakage of blood into
the body cavity. This phenotype is likely to be due to abnormal endothelial cell migration and organization, related
to inappropriate regulation of Ras activity by upstream signaling that controlled Ras activity through p120-RasGAP.
The important role of p120-RasGAP in the regulation of
Ras activity during vasculogenesis/angiogenesis has been
further supported by the discovery of mutations in RASA1,
the gene encoding p120-RasGAP in human (108). These
mutations, leading to the loss of the GAP activity towards
Ras proteins, cause capillary and arteriovenous malformations (CVM-AVM). This indicates that p120-RasGAP
plays specific roles in the regulation of Ras signaling during
vessel development that cannot be compensated for by
other RasGAPs such as RAS2, RASAL, and NF1.
R-Ras GAP (also called GAP1IP4BP), which is membrane
associated and displays a stronger GAP activity against RRas than H-Ras, has also been described as an important
regulator of embryonic vascular development, in particular
in blood barrier establishment (197). Homozygous mice
expressing a R-Ras GAP mutant lacking its catalytic activity
die at E12.5–13.5 of massive subcutaneous and intraparencymal bleeding, probably due to underdeveloped adherens
junctions between capillary endothelial cells, suggesting
that Ras proteins are involved in the implementation of
cell-cell junctions during vascular development.
Mechanisms and/or exchange factors upstream to Ras that
ensure the control of Ras activity during the vascular development have been little addressed. Nevertheless, deletion of
the H-Ras and K-Ras exchange factor Sos1 induces embryo
death at mid-gestation with cardiovascular defects (508). In
addition to an enlarged heart, the major vessels of Sos1deficient embryos are distended, with areas of extensive
hemorrhage. However, whether these changes are a secondary consequence of an earlier developmental failure or a
direct result of loss of Sos1 protein has not been determined.
Effect of Ras during vascular development may involve at
least in part its effector Braf identified as a critical signaling
factor in the formation of the vascular system (537). Brafdeficient embryos die of vascular defects during mid-gestation. They had enlarged and irregularly shaped large blood
vessels that were incompletely lined with endothelial cells.
Apoptotic cells were particularly abundant in the endothelium of Braf-deficient embryos. Braf thus plays a critical and
indispensable role downstream of Ras in the integration of
mechanisms that regulate the differentiation of angioblasts,
development and integrity of large vessels, and the survival
of cells of the endothelial lineage (537).
In fact, inactivation of Erk1 and Erk2 in endothelial cells
during embryonic development results in embryonic lethality due to strongly altered angiogenesis with a reduced vas-
cular complexity of the vascular network, with only large
unbranched vessels (458). This phenotype has been ascribed to a redundant role of ERK1 and ERK2 in the regulation of endothelial cell proliferation and migration, two
processes that are critical for angiogenesis. The transcription factors Ets1 and Ets2, defined as targets of ERK signaling could mediate, at least in part, the effect of Ras/Raf/
ERK1/2 signaling during angiogenesis (519). However, the
rescue of the abnormal blood vessel organization in K-ras
knock-down by Akt provided evidence that PI 3-kinase/Akt
also plays an important role in mediating Ras signaling
during embryonic vascular development (275).
Downstream to Ras, the Ras interacting protein 1 (Rasip1/
Rain), identified as an endomembrane Ras effector, has
been shown to be exclusively expressed in the endothelium
of the developing vasculature in both frog and mice and
involved in vasculogenesis (550). Although angioblasts
emerged relatively normally, blood vessel development was
severely inhibited in the Rasip1 knock-down frog embryo,
due to a defect in branching. This suggests a role of Ras/
Rasip1 signaling in angioblasts and endothelial cells after
their initial angioblast specification, possibly during their
migration, cord formation, or tubulogenesis. However, as
rescue experiments demonstrate that failure of tubulogenesis in the absence Rasip1, can be fully restored by dominant-negative RhoA or siRhoA treatment, it appears that
Rasip1 suppresses RhoA (known to inhibit lumen formation, see below) and promotes activation of Cdc42 and
Rac1 (both known to positively mediate lumen formation,
see below) probably secondary to the downregulation of
RhoA activity (550).
B) RAP1. Total deletion of either Rap1a or Rap1b in mice
leads to an inhibition of angiogenesis in vivo that has been
ascribed to an endothelial cell defect (80, 81, 559). This
defective angiogenesis in Rap1b-knockout mice resulted
from an altered VEGF response as Rap1 has been shown to
be required for VEGF-mediated VEGFR2 activation, in
part via an integrin ␣v␤3-dependent mechanism (248).
A role in the stabilization of endothelial cell-cell junctions is
also ascribed to Rap1 and its specific effector KRIT1 (274).
Mouse homozygous null mutations of the Krit1 gene are
embryonic lethal due to vascular defects including dilation
of precursor vessels in the brain, enlargement and increased
endothelial proliferation of the caudal dorsal aorta, as well
as variable narrowing of the branchial arch arteries and
proximal dorsal aorta (528). In humans, loss-of-function
mutations of this gene causes autosomal dominant familial
cerebral cavernous malformations (246). Mechanistically,
it has been demonstrated that Rap1/KRIT1 interaction
leads to the translocation of microtubule-sequestrated
KRIT1 to cell-cell junctions, thereby supporting junctional
integrity of endothelial cells and vascular development
(139, 274). Interestingly, a recent study points to a role of
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LOIRAND, SAUZEAU, AND PACAUD
the Rap-activated GAP ARAP3 in developmental angiogenesis (131). ARAP3 is a GAP for both Arf6 and RhoA, and its
deletion in mice resulted in a major defect in developmental
sprouting angiogenesis. ARAP3 could thus represent the
molecular link between Rap1/KRIT1 and RhoA/Rho-kinase signaling, involving the repression of RhoA/Rho-kinase activity normally exerted by KRIT1 (see below).
Upstream to Rap1 protein, the guanine exchange factor
C3G has been identified as a key regulator of vascular
development. C3G stimulates guanine nucleotide exchange predominantly on Rap1 and, to a lesser extent, on
R-Ras (187). C3G is a molecular link between Rap1 and
cell-cell junction molecules such as cadherins, and induces activation of Rap1 upon dissociation of junctions
(24). C3G mutant embryos died of vascular defect
around E11.5 due to hemorrhage and loss of vascular
integrity (503). Vascular endothelial cells appeared to be
functioning normally, and the defects were related to
abnormal blood vessel maturation with aberrant recruitment/migration, adhesion, and differentiation of mural
cells. These data thus suggest that the role of Ras protein
during vascular development is not restricted to the regulation of endothelial cell functions.
2. Rho proteins
A) RHOA. Part of the evidence for a role of RhoA in murine
embryonic vascular development indirectly arises from the
analysis of experimental models of cerebral cavernous malformation (CCM). CCM disease results from autosomal
dominant loss of function mutations in the genes KRIT1
(CCM1), CCM2 (malcavernin, MGC4607), or PDCD10
(CCM3). Homozygous null mutations in KRIT1 or CCM2
results in lethal vascular and cardiac phenotype in mice and
zebrafish, ascribed to essential dysfunction of endothelium
causing embryonic lethality at midgestation cells (270, 408,
527). While initial vascular patterning is normal, targeted
deletion of CCM2 in endothelial cells severely affects angiogenesis in mice, leading to morphogenic defects in the
major arterial and venous blood vessels (53). KRIT1 and
CCM2 (and also CCM3) physically interact, suggesting
that they converge on the same downstream signaling/effector and that the mechanisms altered by the mutations in
these three genes causing the pathological phenotype are the
same (504, 575). Indeed, KRIT1, CCM2, or CCM3 mutations induce activation of RhoA/Rho-kinase signaling, responsible for reorganization of endothelial cell actin cytoskeleton, loss of endothelial cell junctions, and increased
vascular permeability (51, 466). The phenotype of endothelial cells from CCM2 mutant mice was rescued by pharmacological inhibition of Rho protein by simvastatin (466,
527). RhoA and Rho-kinase activity in endothelial cells
thus appears to have a leading role during vascular morphogenesis and that a tight spatial and temporal inhibition of
this pathway in endothelial cells is essential for normal cell
migration, lumen formation, and normal angiogenesis. This
1684
concept is in agreement with in vitro studies that provide
evidence that RhoA and Rho-kinase need to be actively
inhibited during endothelial tube lumen formation and tube
maintenance events (96). The role of RhoA/Rho-kinase axis
in the developmental angiogenesis was further supported
recently by the observation that homozygous mice deficient
in both isoforms of Rho-kinase (Rock1⫺/⫺;Rock2⫺/⫺) display defective vascular remodeling in the yolk sac and die in
utero before E9.5 (208). These results also suggest a redundant role of Rho-kinase isoform functions in vascular development during embryogenesis.
Loss-of-function studies in zebrafish and mouse point to a
specific role of the RhoA GEF Syx (also called Tech,
GEF720, PLEKHG5) in angiogenic sprouting in the developing vascular bed. Syx is not involved in vasculogenesis and
angioblast differentiation, but in vascular sprouting. In zebrafish, Syx depletion induced the truncation and even the
absence of intersomitic vessels (133). In syx knockout mice,
arterial networks are sparse, with deficiency in small-diameter
vessels and capillaries, whereas major arteries are intact (133).
These phenotypes suggest evolutionay conservation of Syx
function. At the molecular level, Syx has been identified as a
regulator of the directional endothelial cell migration required
for angiogenic sprouting through its interaction with angiomotin (Amot) to precisely localize RhoA activity to the leading front of migrating cells (110, 133).
Among RhoA activity regulators, a recent study pointed out
a role of the RhoA GAP ARAP3, activated by PI 3-kinase
and Rap. Deleting Arap3 in the mouse causes embryonic
death in mid-gestation due to an endothelial defect in
sprouting angiogenesis (131). However, as ARAP3 is also a
GAP for Arf6, in addition to altered RhoA activity, change
in the activity of Arf6 can also participate in the effect of
Arap3 deletion on angiogenesis.
In addition to its role in endothelial cells, RhoA/Rho-kinase
signaling in vascular smooth muscle cells is important for
their recruitment to nascent vessels and vessel stabilization.
Adapted regulation of RhoA and Rho-kinase activity is required for normal coverage of endothelial tubes with vascular smooth muscle, through the control of vascular
smooth muscle cell contraction, spreading, polarity, and
direct migration (317).
B) RHOB.
RhoB plays a critical role in vascular development.
Retinas of newborn RhoB-deficient mice display signs of
retardation in the outgrowth of the primary plexus (6). The
mechanism responsible for this effect is related to RhoBmediated regulation of Akt stability and trafficking into the
nucleus of endothelial cells. This regulation of Akt confers
on RhoB a stage-specific role in the survival of sprouting
endothelial cells that contributes to blood vessel assembly
during development (6).
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
C) RAC1. The role of Rac1 in vascular development has
been addressed thanks to the generation of mice with
endothelial-specific deletion of Rac1. Endothelial-specific Rac1-deficient mouse embryos die at midgestation
(around E9.5). They show an alteration of the development of major vessels, and small branched vessels were
completely absent both in the embryos and their yolk sacs
(477). In vitro functional studies of Rac1-deficient endothelial cells functions demonstrated the essential role of
Rac1 in the transduction of the effects of vascular endothelial growth factor (VEGF) such as migration, tube
formation, adhesion, and permeability. Rac1-deficient
endothelial cells are not able to form lamellipodia structures and focal adhesions, or to reorganize their cell-cell
contacts (477). Rac1 is not strictly required for the initial
differentiation or recruitment of angioblasts from the
mesoderm and blood islands but essential for subsequent
migration of angioblasts to promote the establishment of
capillary-like networks (477). These data support an important role of Rac1 in developmental angiogenesis
rather than in vasculogenesis. Furthermore, Rac1 is necessary for the transition from blood endothelial cells to
lymphatic endothelial cells required for endothelial cells
to separate and form lymphatic vasculature and is therefore an essential part of lymphangiogenesis (94).
D) CDC42.
Ablation of Cdc42 in mouse embryonic stem cells
does not affect endothelial lineage differentiation but completely blocks vascular network assembly because of an
impaired directional migration (375). PKC␫ and GSK-3␤
have been identified as downstream effectors of Cdc42 during vascular morphogenesis (375). In zebrafish Cdc42 is
involved in intracellular vacuole formation, the fusion of
which plays a fundamental role in endothelial cell lumen
formation of intersegmental vessels (207).
These data are in agreement with results obtained in mice
lacking the specific Cdc42 effector Pak4 that showed abnormalities in vasculature throughout the extraembryonic
tissue and the epiblast (486). In the knockout embryos,
large vessels could still be detected, but smaller branching
vessels were completely absent. The Pak4 knockout yolk
sac did stain positively for endothelial cells, showing the
presence of the vascular plexus, but did not have organized
vessels. These data suggest that Cdc42/Pak4 signaling affects angiogenesis and branching, rather than the initial
formation of vessels (486).
3. Other Rho signaling molecules
Pak2a, another member of the Pak family that can be activated by Cdc42 or Rac1, also participates in embryonic
vessel formation. In zebrafish, Pak2a was identified as a
downstream effector of the Rac1 and Cdc42 exchange factor ␤Pix (273). Loss of ␤Pix or Pak2 leads to loss of vascular
integrity and hemorrhage in the head but not in other vascular bed. The ␤Pix/Pak2a axis is thus involved in cerebro-
vascular stabilization, an effect that is likely to be due to a
role in support cells (vascular smooth muscle cells and pericytes) surrounding the cranial vasculature (273).
B. Blood Pressure Regulation and
Hypertension
In general, three major mechanisms contribute to long-term
blood pressure control: the contraction of small arteries, the
control of blood volume in which the kidney is a key player,
and the regulation of the cardiac output. The first part of
this section focuses on the involvement of Ras proteins and
their regulators in the control of vascular tone, with a focus
on their role in arterial smooth muscle cell contractility,
either directly or indirectly through modulation of endothelial cell functions. The following sections will address the
role of Ras proteins in the central control of blood pressure,
and in the kidney-mediated regulation of water-electrolytes
balance.
1. Role of Ras protein superfamily members in
arterial smooth muscle cell contractility and vascular
tone
The regulation of arterial contractility is essential for blood
pressure control and represents a typical multicellular function regulated by Rho signaling pathways. Phosphorylation
of 20-kDa myosin light chain (MLC) is the key event of the
regulation of vascular smooth muscle contraction (373).
MLC is phosphorylated by the Ca2⫹/calmodulin-activated
MLC kinase (MLCK) and dephosphorylated by the Ca2⫹independent MLC phosphatase (MLCP). Agonists (ANG
II, norepinephrine, endothelin-1, thromboxane A2 . . .) that
bind to G protein-coupled receptors produce contraction by
increasing both 1) the cytosolic Ca2⫹ concentration and 2)
the Ca2⫹ sensitivity of the contractile apparatus. The rise in
cytosolic Ca2⫹ concentration results from the stimulation
of Ca2⫹ entry through L-type voltage-dependent Ca2⫹
channels and Ca2⫹ release from the sarcoplasmic reticulum
through IP3-gated Ca2⫹ channels activated by PLC-generated inositol 1,4,5-trisphosphate (IP3). The increased sensitivity of vascular smooth muscle toward Ca2⫹ results from
inhibition of MLCP activity leading to increased MLC
phosphorylation and tension at a constant Ca2⫹ concentration. In contrast, vasorelaxing agents decrease both the cytosolic Ca2⫹ concentration and the Ca2⫹ sensitivity of the
contractile apparatus. Surprisingly, while Rho family proteins emerged as key regulators of smooth muscle cell contraction, the role of the other subfamilies of Ras proteins in
the regulation of vascular smooth muscle contraction has
not been extensively addressed.
2. Ras protein-mediated regulation of smooth muscle
cell contraction, vascular tone and blood pressure
(Figure 4)
A) RAS.
The first demonstration of a role of Ras in the regulation of smooth muscle contraction was provided by the
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observation that active H-Ras induces tension of permeabilized arteries by increasing the Ca2⫹ responsiveness in a
concentration-dependent manner (416). Downstream of
Ras, ERK1/2 activation triggers MLCK phosphorylation,
leading to an increase in its sensitivity to calmodulin and,
consequently, its ability to phosphorylate MLC (227). In
addition to this stimulation of MLCK activity, the RasERK1/2 signaling was also shown to positively regulate the
expression of MLCK in arterial smooth muscle by stimulating MLCK promoter (151, 152). This signaling pathway is
activated by ANG II, both in vitro and in vivo in rats (152),
and is also involved in the upregulation of MLCK in the
arteries of spontaneously hypertensive rats (SHR) (151).
The attenuation of high blood pressure produced by Ras
FTIs in SHR or in ANG II-induced hypertension in rats
suggested that activation of Ras signaling can contribute to
the development of hypertension (34, 326).
All these data, suggesting a vasoconstricting and hypertensive action of Ras-ERK1/2 pathway, have been confirmed in
vivo in genetically modified mice. Knock-in mice in which
K-Ras has been replaced by H-Ras develop arterial hypertension (371). More recently, high blood pressure was also
observed in a strain of genetically modified mice carrying
the activating G12V mutation within their endogenous HRas locus (H-Ras-V12) (427). In contrast, H-Ras⫺/⫺ mice
show lower blood pressure than control animals, probably
related to an upregulation of the NO-cGMP pathway (70).
B) RAL.
Although a direct role of Ral proteins has not been
provided, recent evidence suggests that RalA and RalGDS
Rap1
but not RalB is involved in the coupling of AT1 receptor to
the activation of IP3 signal transduction (140).
C) RAP. A recent study reported the existence of a cross-talk
between Rap1 and RhoA signaling pathways in vascular
smooth muscle. Activation of Rap1 causes relaxation by
decreasing the Ca2⫹ sensitization of force in smooth muscle
through downregulation of RhoA activity (588). Rap1 activation is triggered by the exchange factor Epac, activated
by the rise in cAMP induced by the stimulation of vasorelaxing receptors such as ␤-adrenergic or prostglandine I2
receptors. The mechanism by which Rap1 inactivates RhoA
has not been identified but, by analogy to that found in
endothelial cells and in neurons, it was suggested that a
Rap1-activated RhoGAP such as RA-RhoGAP or ARAP3,
shown to be directly stimulated by Rap1 (239, 553), may
downregulate RhoA activity in smooth muscle cells (588).
3. Rho protein-mediated regulation of smooth
muscle cell contraction, vascular tone and blood
pressure (Figure 5)
A) RHOA. The major role of RhoA in smooth muscle contrac-
tion has been unequivocally demonstrated over the past 15
years. The Ca2⫹-sensitizing effect of vasoconstrictors is ascribed to RhoA activation and the subsequent stimulation
of its target Rho-kinase that phosphorylates MYPT-1, the
regulatory subunit of MLCP, and inhibits its activity (490).
While Rock1 and Rock2 are both expressed in vascular
smooth muscle cells, Rock2 plays a prominent role in the
regulation of vascular smooth muscle contractility (513).
This mechanism of Rho-kinase-mediated Ca2⫹ sensitiza-
RhoA
(Rock2)
H-Ras
(ERK1/2)
RalA
Vasodilation
MLC
MLCP
MLCK
↑ Cai
IP3
P-MLC
Vasoconstriction
Rac1
(Pak1)
Caldesmon
Rac1
(Pak3)
FIGURE 5. Role of Ras proteins in the regulation of vascular smooth muscle cell contraction. Contraction is
determined by the level of myosin light chain (MLC) phosphorylation (P-MLC), controlled by the activity of the
MLC kinase (MLCK) and the MLC phosphatase (MLCP).
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
tion is thus responsible for the tonic component of vascular
smooth muscle cell contraction. For in-depth coverage of
RhoA/Rho-kinase signaling-mediated Ca2⫹ sensitization
and regulation of smooth muscle contraction, the reader is
referred to more comprehensive reviews (282, 373, 457).
Following the discovery of the key role of RhoA/Rho-kinase
signaling in vascular smooth muscle cell contraction, numerous studies have addressed the role of this pathway in vivo in
the control of blood pressure and hypertension (283). In SHR
or in rats and mice treated with ANG II, L-NAME, or DOCAsalt, the development of hypertension is associated with an
increase in RhoA and Rho-kinase activity in arteries (145,
216, 431, 435), thus showing that RhoA/Rho-kinase signaling
activation is a common component for the pathogenesis of
hypertension (282, 587). The causal relationship between the
activation of RhoA and Rho-kinase and the increase in blood
pressure has been indirectly addressed by the use of pharmacological Rho-kinase inhibitors. Rho-kinase inhibitors such as
Y27632, fasudil, or SAR407899 reduce blood pressure in animal models of hypertension [ANG II (281); L-NAME (216,
281, 435); DOCA-salt (281, 490); SHR (241, 323)]. In addition to their blood pressure-lowering effect, Rho-kinase inhibitors
also prevent the hypertension-associated cardiovascular remodeling, fibrosis, and inflammation (166, 189, 210, 216, 323).
All vasoconstrictors acting through G protein-coupled receptors induce RhoA activation, and different types of hypertension are all associated with an increased arterial
RhoA activity. It is therefore likely that different Rho GEFs
couple vasoconstrictor receptors to RhoA activation and
are differently involved in the overactivation of Rho during
hypertension. Nevertheless, only a few studies have addressed Rho GEFs expression, regulation, and activation in
vascular smooth muscle cells. They mainly focused on the
RGS-containing Rho GEF subfamily of Rho GEFs grouping
Arhgef1 (p115-RhoGEF), Arhgef11 (PDZ-RhoGEF), and
Arhgef12 (Larg) that are supposed to link G protein-coupled receptors to RhoA activation. All three RGS-containing Rho GEFs are expressed in arteries (169, 202, 569).
Arhgef11 was described as the most expressed RGS-RhoGEF in rat aorta and mesenteric artery, while in mice aorta,
the more abundant RGS-Rho GEF was Arhgef12 (531).
Arhgef12 transcript and protein are expressed in vascular
smooth muscle of murine embryos from E14 to later stages
of development and in adulthood (30). The specific RhoA
GEF p63Rho GEF is the most expressed Rho GEF in arterial smooth muscle cells (67, 316).
It has been recently demonstrated both in vivo and in vitro
that Arhgef1 is involved in ANG II-induced RhoA activation and contraction in vascular smooth muscle cells and is
activated in arteries from ANG II-infused rats or mice
(145). ANG II-induced Arhgef1 activation depends on AT1
receptor stimulation and subsequent JAK2-mediated Arhgef1 phosphorylation (145). Two other RhoGEFs,
p63RhoGEF and Arhgef11, have also been shown to participate in the early RhoA activation induced by ANG II
stimulation of vascular smooth muscle cells (543, 568). The
RhoGEF p63RhoGEF is also involved in RhoA activation
and contraction induced by PhE and endothelin-1, by selectively coupling G␣q/11 protein to RhoA activation (316).
The RhoA exchange factors Lbc and Arhgef12 have been
identified as mediator of RhoA activation in vascular
smooth muscle cells stimulated by 5-HT and sphingosine
1-phosphate, respectively (28, 303) (FIGURE 6).
Genetic targeting of Rho protein signaling in mice shed light
on the role of Rho GEFs and RhoA/Rho-kinase signaling in
the regulation of blood pressure and hypertension. Rock1
haploinsufficient and deficient mice have normal basal
blood pressure, and the rise in blood pressure induced by
ANG II or L-NAME treatment is similar to that observed in
control mice but vascular fibrosis is reduced (393, 582).
Given the effect of Rho-kinase inhibitors in ANG II- and
L-NAME-treated animals, this observation might suggest
that Rock2 is the main isoform of Rho-kinase mediating the
vasoconstricting effect of RhoA signaling in vascular
smooth muscle cells. Unfortunately, since Rock2 deficiency
is embryonically lethal, the cardiovascular phenotype of
Rock2-deficient mice has not been assessed. Upstream to
RhoA and Rho-kinase, genetic ablation of Arhgef1 and Arhgef12 has confirmed in vivo the role of these Rho GEFs in
the regulation of vascular tone and blood pressure and
highlighted their specific involvement in different pathways. While specific Arhgef1 deficiency in smooth muscle
renders the mice resistant to ANG II- and L-NAME-induced
hypertension (145), deletion of Arhgef12 induces insensitivity to salt-dependent hypertension (531).
B) RAC1.
Rac1 is rapidly activated in vascular smooth
smooth muscle cells in response to stimulation with various
vasoconstrictors such as ANG II, endothelin-1, and thromboxane A2. Nevertheless, in contrast to RhoA, its role in the
regulation of smooth muscle contraction and the control of
vascular tone remains unclear (452). The Rac effector Pak1
has been shown to phosphorylate and inhibit MLCK,
thereby decreasing MLC phosphorylation and smooth
muscle cell contraction (412, 532). This suggests that Rac
action antagonizes the effect of RhoA on MLC phsophorylation, and negatively regulates vascular smooth muscle
tone. However, opposite effects have also been reported as
the other Rac effector Pak3 has been described to enhance
smooth muscle cell Ca2⫹ sensitivity and contraction
through phosphorylation of the thin filament regulatory
protein caldesmon (119).
In vivo, chronic ANG II infusion induces Rac1 activation,
essentially described in cardiac tissue (385, 415) but probably also in arteries (516). By triggering the activation of
NADPH oxidase and redox signaling, Rac1 is a major mediator of the cardiac and vascular hypertrophic effect of
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LOIRAND, SAUZEAU, AND PACAUD
PhE
Ang II
α1R
AT1R
Arhgef1
Arhgef11
ET1
5HT
S1P
Lbc
Arhgef12
ETAR
p63RhoGEF
RhoA
Rock2
MLCP
MLC
P-MLC
Vasoconstriction
FIGURE 6. RhoA GEFs involved in the activation of RhoA and Rock2 by the stimulation of G protein-coupled
receptor by vasoconstrictors: ANG II and type 1 ANG II receptor (AT1R), phenylephrine (PhE) and ␣1-adrenoceptor
(␣1R), endothelin-1 (ET1) and endothelin receptor A (ETAR), serotonin (5HT), and sphingosine 1-phosphate.
Contraction of vascular smooth muscle cells is determined by the level of myosin light chain (MLC) phosphorylation
(P-MLC), controlled by the activity of the MLC kinase (MLCK) and the MLC phosphatase (MLCP).
ANG II (385, 421). Furthermore, it has been recently demonstrated that Rac1 is responsible for the oxidative stress
and vascular dysfunction induced by mechanical stress of
the arterial wall caused by high intraluminal pressure levels
(499). Mechanical stress activates via integrin-linked kinase
1 and the Rac1 GEF betaPIX. Adenoviral transfection of a
dominant-negative Rac1 decreases high blood pressure-induced NADPH oxidase activity and oxidative stress in carotid arteries, and normalizes vascular function. These data
thus support a role of Rac1 in oxidative stress and its consequent vascular damages associated with hypertension
(499). This role of Rac1 has been confirmed in mice with
specific overexpression of the constitutively active mutant
of Rac1 (Rac1-V12) in smooth muscle cells. These mice
develop moderate hypertension compared with wild-type
littermates, caused by enhanced superoxide production
(157). Conversely, mice with heterozygous inactivation of
Rac1 in endothelial cells (EC-Rac1⫹/⫺) develop moderate
hypertension and display decreased eNOS expression and
1688
activity as well as impaired endothelium-dependent vasorelaxation (422). These observations suggest that endothelial
Rac1 indirectly participates in the regulation of vascular
tone and blood pressure through its effects on eNOS. Pharmacological strategies aiming to enhance endothelial Rac1
function might be of therapeutic interest for preserving endothelial function and normal blood pressure.
4. Rho protein-dependent endothelial-smooth muscle
cells crosstalk
The maintenance of vascular homeostasis is largely dependent on the functional crosstalk existing between endothelial and smooth muscle cells within the arterial wall. The
endothelium is a key determinant of vascular tone and resistance that acts by releasing vasoactive factors among
which endothelium-derived nitric oxide (NO) is the most
important. Both endothelial NO production and NO effects
in vascular smooth muscle cells are regulated by RhoA and
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
Rac1 signaling (283). RhoA, through Rho-kinase activation, downregulates eNOS expression by destabilizing
eNOS mRNA (256, 587) and decreases eNOS catalytic activity by inhibiting and stimulating PI 3-kinase/Akt pathway (538) and arginase activity (313), respectively. Rac1
antagonizes RhoA/Rho-kinase effects on eNOS. Rac1 upregulates eNOs expression by increasing eNOS mRNA stability and Pak1-mediated stimulation of transcription, and
stimulates eNOS activity (422, 436).
In vascular smooth muscle cells, it has been shown that
cGMP-dependent protein kinase (PKG) inhibits RhoA/
Rho-kinase signaling by phosphorylating Ser188 of RhoA
(417). This inhibitory phosphorylation, by decreasing the
Ca2⫹ sensitivity of contractile proteins, plays a major role in
the vasodilator effect of NO (417). However, recent findings indicate that Rac1 participates in the control of cGMP
level in vascular smooth muscle cells. Activation of Rac1
increases intracellular cGMP concentration via Pak1-mediated inhibition of the activity of type 5 phosphodiesterase
(PDE5) (418). The reduced cGMP and vasodilatory response to NO donors observed in arteries from Vav2-deficient mice supports a role of the Rho GEF Vav2 and the
tyrosine kinase Src as the upstream regulators of Rac1 in
vascular smooth muscle cells (418).
5. Role of Rho protein superfamily members in
central blood pressure regulation
The baroreceptor reflex is one of the main physiological
mechanism that contributes to the stability of blood pressure (365). The specific areas involved in this nervous regulation of blood pressure are located in the brain stem
(365). Afferent fibers from arterial baroreceptors project in
the nucleus tractus solitarii (NTS). Signals are transmitted
from the NTS to the rostral ventrolateral medulla (RVLM)
from which sympathetic nerves conduct them to heart and
vessels (365).
adenovirus vector encoding a dominant-negative Rac1
reduces the increase in blood pressure induced by ANG II
(589). Similarly, the intracisternal infusion of the Rhokinase inhibitor Y27632 reduces the central pressure action of ANG II (405). These observations thus provide
evidence that ANG II-mediated Rac1 and RhoA/Rhokinase activation in the brain stem contributes to hypertension induced by ANG II infusion.
6. Role of Ras protein superfamily members on
kidney-mediated blood pressure regulation
The kidney has a central role in the control of blood pressure by regulating the maintenance of water-electrolyte balance, mainly through the reabsorption of filtered Na⫹. Na⫹
flux through epithelial Na⫹ channels (ENaCs) expressed in
the apical plasma membrane of the renal collecting duct is
the final renal adjustment step for Na⫹ balance. ENaC thus
plays a critical role in modulating the homeostasis of Na⫹
and blood pressure. This channel is an end-effector in the
renin-angiotensin-aldosterone system, and several regulatory pathways, some of them involving Ras proteins, modulate its activity and its expression (FIGURE 7).
7. Role of Ras proteins in the regulation of ENaC
A) RAS.
In renal epithelia, aldosterone induces expression
and activation of K-Ras (465). Moreover, K-Ras is both
necessary and sufficient for activation of ENaC (465). KRas increases the activity of channels already present in the
High-salt
Aldosterone
↑ MR
↑ SGK1
↓ AS160
In vivo studies in the rat revealed that RhoA/Rho-kinase
signaling in the NTS is involved in the sympathetic nervous
system-dependent regulation of basal blood pressure. Inhibition of Rho-kinase in the NTS by adenoviral transfection
of dominant-negative Rho-kinase or local infusion of the
Rho-kinase inhibitor Y27632 reduces mean blood pressure
and urinary excretion of norepinephrine (191). Blockade of
Rac1 signaling in the NTS does not affect mean blood pressure or urinary norepinephrine excretion, although it decreases NADPH oxidase activity (344). These data suggest
that RhoA- but not Rac1-dependent pathways in the
NTS participate in the physiological regulation of blood
pressure via the central nervous system. In contrast, both
proteins play a role in ANG II-dependent hypertension.
ANG II infusion increases NADPH oxidase activity and
superoxide production and activates RhoA/Rho-kinase
in the brain stem through an AT1 receptor-dependent
mechanism (405, 590). Transfection into the brain of
K-Ras
Rac1
Rab
RhoA
↑ Rock
↑ PI3-kinase
↑ WAVE1/2
↑ PI(4)P5-kinase
↑ PI(4,5)P2
↑ ENAc activity
↑ ENAc expresssion
↑ Na+ reabsorption
↑ Blood pressure
FIGURE 7. Ras protein-mediated regulation of blood pressure
through modification of activity and/or expression of the renal epithelial Na⫹ channels (ENaC).
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LOIRAND, SAUZEAU, AND PACAUD
membrane by increasing channel open probability (461,
462, 465). Inhibition of PI 3-kinase by wortmannin blocks,
while in contrast overexpression of active PI 3-kinase mimics the stimulation of ENaC induced by K-Ras (461, 462).
This wortmannin-sensitive stimulation of ENaC is also observed in cells expressing an effector-specific mutant of Ras
capable of only activating PI 3-kinase (462). These data
indicate ENaC is activated by K-Ras in a PI 3-kinase signaling-dependent manner and that this K-Ras-dependent pathway may participate in the control of systemic Na⫹ balance
and blood pressure.
B) RAB. Rab proteins have been recently identified as a component of aldosterone-dependent ENaC trafficking, thus
regulating the ENaC density the apical membrane of epithelial cell of the collecting duct and Na⫹ absorption (63,
264). Under basal conditions, the nonphosphorylated RabGAP AS160, by maintaining Rap proteins in an inactive
GDP-bound state, stabilizes ENaC within an intracellular
compartment (264). Aldosterone stimulation of Na⫹ transport, associated with increased apical ENaC density, involves the transcriptional induction of serum- and glucocorticoid-induced kinase (SGK1), which phosphorylates
AS160 and inhibits its GAP activity leading to Rab activation. This relieves the AS160-mediated intracellular retention of ENaC and permits forward trafficking of the channel to the apical surface to augment Na⫹ absorption (264).
As ENaC recycles in polarized kidney epithelial cells, it is
likely that the channel traverses a number of Rab-dependent compartments en route to the apical membrane, involving different Rab proteins. In fact, Rab 4, 11, and 27
have been shown to participate in ENaC regulation, and
recent data indicate the absolute requirement for Rab11b to
deliver ENaC to the apical membrane (63, 64).
8. Role of Rho proteins in the regulation of ENaC
A) RHOA.
As for Ras proteins, evidence for a role of RhoA in
the regulation of ENaC has been obtained in cell sytems in
vitro. Expression of wild-type RhoA and constitutively active V14-RhoA markedly increases ENaC activity, while in
contrast the dominant negative RhoA-N19 decreases it
(460). The effects of RhoA are primarily to increase the
density of ENaC on the plasma membrane. Activation of
ENaC in response to RhoA is sensitive to inhibition of Rhokinase and disruption of PI(4,5)P2 synthesis and is mimicked by overexpressing phosphatidylinositol-4-phosphate
5-kinase [PI(4)P5-kinase], indicating that activation of
Rho-kinase and its known effector PI(4)P5-kinase, and the
consequent increase in PI(4,5)P2 levels mediate the increase
in membrane levels of ENaC (460). It was further demonstrated that activation of RhoA rapidly increases the membrane levels of ENac by promoting translocation and insertion of the channel. In addition to PI(4,5)P2, RhoA-induced
targeting of vesicles containing ENaC to the plasma membrane also involves cytoskeletal actin and tubulin filaments
(214, 366).
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B) RAC1.
Expression of wild-type Rac1 or constitutively active Rac1 significantly increases ENaC activity, while
CdC42 has no effect (360, 460). In contrast, the Rac inhibitor NSC23766 markedly decreases ENaC activity in cortical collecting duct (215). Knock-down of Rac1 also decreases ENaC-mediated Na⫹ reabsorption by decreasing
the number of channels at the apical membrane (215). The
effect of Rac1 on ENaC depends on WAVE1 and WAVE2,
two members of the Wiskott-Aldrich syndrome proteins
known to stimulate Arp2/3-mediated actin polymerization
(215). The Rac1/WAVE signaling complex might thus play
a central role in blood pressure control in the kidney, although the exact mechanisms involved are not defined yet.
In support of such a role of Rac1, however, it has been
recently suggested that Rac1 in kidneys is essential for saltsensitive hypertension, via a mineralocorticoid receptor-dependent pathway (123, 446). High-salt loading triggers
Rac1 activation in the kidneys of the Dahl rat model of
salt-sensitive hypertension, and NSC23766 treatment significantly reduces the blood pressure of the rats, indicating
a causal role of Rac1 activity in the salt-dependent elevation
of blood pressure (446). Along with the repression of Rac1
activity, the treatment of Dahl rats with NSC23766 also
significantly reduces salt loading-induced increase in mineralocorticoid receptor expression as well as SGK1 upregulation, indicating that the effect of Rac1 activation on blood
pressure involved the activation mineralocorticoid receptor
signaling in the kidney (446, 447).
9. Role of Rho proteins on sodium-hydrogen
exchanger
In addition to the role of ENaC in the renal collecting duct,
the sodium-hydrogen exchanger isoform 3 (NHE3) expressed in the apical membrane of the proximal convoluted
tubules is one of the major transporters involved in the renal
reabsorption of sodium under physiological conditions.
Inhibition of Rho proteins by toxin B from Clostridium
difficile or Rho-kinase by Y27632 results in reduced NHE3
activity, due to a marked decrease in the surface expression
of NHE3 and relocation of the exchanger to an intracellular
compartment (159). In fact, RhoA and Rho-kinase, by controlling the phosphorylation of MLC and, ultimately, the
organization of the actin cytoskeleton, are important for
NHE3 association with ezrin and actin, and this interaction
is required for maintenance of the exchanger at the apical
surface (159, 474). In SHR, hypertension is associated with
a marked increase in NHE3 activity and a decreased Na⫹
excretion (338). Inhibition of Rho-kinase by Y27632 reduces apical NHE3 activity and enhances Na⫹ excretion
and total urine volume (338). Because, in general, a natriuresis in patients with hypertension reduces blood pressure,
it has been suggested that the natriuresis induced by
Y27632 may contribute to its blood pressure lowering effect (474).
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
10. Evidence for a role of Rho protein signaling in
high blood pressure in humans
prevent or limit vascular remodeling associated with cardiovascular diseases.
Both pharmacological data and genetic studies indirectly
support a potential role of Rho proteins in the generation of
high blood pressure in humans. The first data suggesting a
role of RhoA/Rho-kinase in the pathogenesis of human hypertension were obtained by showing that the increase in
forearm blood flow and the decrease in forearm vascular
resistance produced by the Rho-kinase inhibitor fasudil are
significantly stronger in patients with essential hypertension
than in the normotensive group of patients, suggesting that
hypertension-associated vascular dysfunction depended, at
least in part, on Rho-kinase activation (298). The altered
vasodilation and increased resistance of the forearm observed in patients with heart failure has been also ascribed
to Rho-kinase activation (226). More recently, data obtained in healthy male subjects indicated that the Rho-kinase activity correlates with oxidative stress and aortic stiffness (340).
1. Ras proteins
In addition to these functional evidences, genetic analyses
have shown that Rho-kinase polymorphism influences
blood pressure. This has been demonstrated for the nonsynonymous Thr431Asn Rock2 variant (430). A high basal
blood pressure and increased systemic vascular resistance is
associated with the Asn/Asn genotype (compared with Asn/
Thr or Thr/Thr). More recently, a lower risk of high blood
pressure is associated with a major haplotype block at the
Rock2 locus in a recessive manner (384). In addition, recent
data from genome-wide association studies have identified
the association between diastolic blood pressure and common variants in eight loci, with one of them containing two
genes related to Rho protein signaling: 1) RTKN2 encoding
the RhoA effector rhotekin-2 and 2) RHOBTB1 encoding
RhoBTB (333). Nevertheless, the potential role of these two
gene products in the regulation of blood pressure is completely unknown.
C. Vascular Remodeling
Vascular remodeling occurs during normal development
and participates in various physiological processes. However, vascular smooth muscle cell hypertrophy, proliferation, and migration also occur in hypertension, atherosclerosis, and restenosis after angioplasty or stent implantation,
leading to pathophysiological remodeling of the arterial
wall. These processes are possible because, in contrast to the
majority of differentiated cells, smooth muscle cells retain
the capacity to modulate their phenotype, to lose their contractile properties, to proliferate, and to migrate in response
to a variety of pathological stimuli. By making analogies
between vascular proliferative disorders and cancer, studies
of Ras proteins have led to the discovery of their roles in cell
processes underlying pathological arterial wall remodeling,
and suggested that targeting these small G proteins may
A) RAS.
A role for Ras protein in the regulation of vascular
smooth muscle cell proliferation has been proposed more
than 20 years ago. H-Ras is expressed in smooth muscle
cells, with a peak expression in proliferating cells in mid to
late G1 phase (403). Growth factors such as PDGF activate
the Ras-MAPK pathway in vascular smooth muscle cells,
and Ras inhibition by FTI or expression of dominant negative H-Ras mutant reduces proliferation (235, 491). In contrast, introduction of the dominant active H-Ras-V12 in
vascular smooth muscle cells induces a growth arrest with
phenotypic characteristics of cellular senescence, suggesting
that H-Ras signaling contributes to age-related vascular disorders (312). The other member of the Ras family,
KiRas2A, is identified as a mediator of ANG II-induced
vascular smooth muscle cell proliferation and senescence
(310, 311). Accordingly, it has been proposed that different
levels of Ras expression and activity may be one of the
mechanisms that regulate the switch between growth/proliferation and senescence signaling in vascular cells (309).
Evidence for a role of Ras signaling in the regulation of
vascular smooth muscle cell in vivo was then provided by
studies showing that plasmid- or virus-mediated local expression of H-Ras dominant negative mutant significantly
reduced neointimal formation in balloon-injured rat carotid arteries (188, 491) or pig coronary artery (541). In the
same in vivo models, prevention of posttranslational modifications and thereby, inhibition of Ras activity by FTIs or
S-adenosylhomocysteine hydrolase inhibitors (433, 540),
or S-trans,trans-farnesylthiosalicylic acid (136) has the
same inhibitory action on balloon injury-induced neointimal proliferation.
Genetically modified mice models have further confirmed
the in vivo role of Ras signaling in vascular remodeling and
vascular cell proliferation, in particular models of haploinsufficiency or tissue specific deletion. Haploinsufficient nf1
mice (nf1⫹/⫺) have increased neointima formation, vascular
inflammation, excessive vessel wall cell proliferation, and
ERK activation after vascular injury (252, 253). A similar
phenotype is observed in mice with smooth muscle cell specific deletion of nf1 but is rescued by smooth muscle cell
expression of the GAP domain of NF1, indicating the causative role of excessive Ras activity in the increased vascular
smooth muscle cell proliferation produced by nf1 deletion
(549).
Upstream to Ras, the Grb2 adaptator, linking growth factor
receptors to the Ras exchange factor Sos, has been shown to
be expressed in vascular smooth muscle cells and recruited
to the membrane by stimulation with PDGF, ANG II, or
mechanical stretch, suggesting that Grb2/Sos could be re-
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LOIRAND, SAUZEAU, AND PACAUD
sponsible for the activation of Ras (33, 182, 268). This
hypothesis is supported in vivo by the resistance to the
development of neointima thickening in injured carotid of
heterozygous Grb2⫹/⫺ mice (581).
The Ras/MAPK pathway is also the target of physiological
processes that inhibit vascular smooth muscle cell proliferation and maintain vascular wall homeostasis. Vascular
smooth muscle cells expressed endogenous negative regulator of Ras that, under normal conditions, maintain a low
activity of the Ras-Raf-MAPK signaling, thus preserving
differentiation and quiescence. Such a regulation is responsible for the antiproliferative effect of the contractile phenotype marker SM22␣. SM22␣ binds Ras, resulting in
downregulation of Ras-Raf-MAPK signaling and blockade
of G1-S phase cycle progression in vascular smooth muscle
cells (107). In vivo, this mechanism is responsible for
SM22␣ overexpression-induced inhibition of neointimal
hyperplasia induced by balloon injury (107). In the same
way, R-Ras is a member of Ras protein family that antagonizes H-Ras signaling (88, 438). R-Ras is abundantly expressed in normal mature blood vessels but significantly
downregulated in hyperplastic neointimal smooth muscle
cells (231). R-Ras knockout mice develop exaggerated neointimal thickening in injured femoral artery due to sustained smooth muscle cell proliferation (231). These results
thus indicate the in vivo role of R-ras as a negative regulator
of vascular smooth muscle cell hyperplasia, probably
through inhibition of H-Ras signaling. A similar role has
been ascribed to mitofusin-2, initially called hyperplasia
suppressor gene (HSG). Mitofusin expression is decreased
in hyperplastic smooth muscle cells from SHR arteries and
from balloon-injured rat carotids compared with controls
(77). It is also reduced in vitro upon stimulation of vascular
smooth muscle cells by mitogenic factors such as PDGF,
FGF, or ET-1 (77). Overexpression of mitofusin-2 by adenoviral infection almost totally prevents balloon injuryinduced neointima formation and restenosis in rat carotid
arteries (77). Mechanistically, mitofusin-2 binds Ras leading to inhibition of Raf-MAPK signaling and G1/G0 cell
cycle arrest (77).
B) RAD. Rad expression has been shown to be increased
during vascular lesion formation after balloon injury (122).
Adenovirus-mediated in vivo Rad gene delivery in carotid
artery induces a marked reduction in neointimal formation
after balloon injury, while in contrast, expression of a dominant negative form of Rad increases neointimal thickening.
Rad was thus identified as an endogenous inhibitor of vascular lesion formation, acting by reducing migration of vascular smooth muscle cells, through at least in part, inhibition of Rho-kinase signaling (122).
C) RAP.
Rap1a and Rap1b proteins have been shown to be
upregulated by PDGF in vascular smooth muscle cells, and
this upregulation during the smooth muscle cell cycle is
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directly linked to cell proliferation (378). PDGF and FGF-2
also induce an increase in Rap2 during late S phase and
G2/M phase of the smooth muscle cell cycle (368). Silencing
of Rap2 indicated that Rap2 is required for migration but
not for vascular smooth muscle cell proliferation. Despite
these data, the role of Rap proteins in vascular smooth
muscle cells and vascular remodeling is not known, and in
vivo studies are now necessary to address this question.
2. Rho proteins
A) RHOA.
Numerous studies provide evidence for a direct
link between RhoA/Rho-kinase signaling and pathological
vascular remodeling, by interfering with several processes
such as vascular smooth muscle cell differentiation, migration, and proliferation. RhoA/Rho-kinase signaling has
been demonstrated to be a critical mechanism for controlling smooth muscle differentiation through the regulation
of the actin cytoskeleton that selectively controls differentiation marker gene expression by modulating SRF-dependent transcription (272, 287).
In vitro, most mitogen factors for vascular smooth muscle
cells activate RhoA and Rho-kinase, and inhibition of Rhokinase reduces vascular smooth muscle cell proliferation
induced by mechanical stretch or by several growth factors
and vasocontrictors including PDGF, thrombin, ANG II,
and 5-HT (209, 210, 280, 345, 420, 429, 555, 577). RhoA/
Rho-kinase activation accelerates cell cycle progression
through downregulation of p27Kip1 expression (257, 420).
Conversely, the upregulation of p27Kip1 expression induced
by Rho-kinase inhibitors accounts for their antiproliferative effects (210, 420). In addition to this effect on p27Kip1,
the necessary role of Rho-kinase for the nuclear translocation of ERK1/2 may also participate in Rho-kinase-mediated regulation of smooth muscle cell proliferation (280,
345, 577). As RhoA and Rho-kinase positively regulate two
incompatible processes, differentiation and proliferation, it
is possible that functions of RhoA/Rho-kinase signaling depend on the differentiation status of smooth muscle cells.
This hypothesis is supported by the differential effect of
Rho-kinase inhibition on the migration of proliferative and
differentiated arterial smooth muscle cells. Inhibition of the
RhoA/Rho-kinase signaling by pharmacological agents or
expression of dominant negative limits vascular smooth
muscle cell migration induced by several factors such as
PDGF, thrombin, or lysophosphatidic acid (9, 420, 431),
while in contrast, it increases cell motility of differentiated
aortic smooth muscle cells (74).
In vivo studies have confirmed the role of RhoA/Rho-kinase
in vascular remodeling. Rho-kinase inhibitors reduce neointimal formation induced by balloon injury in rat (420,
444) and pig (114, 332) or by stent implantation in pig
(301). In these models, both an antiproliferative action
through downregulation of the cyclin-dependent kinase inhibitor p27Kip1 (420) and stimulation of apoptosis (444,
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
445) have been involved in the beneficial effect of Rhokinase inhibitors. Hypertension-associated medial hypertrophy and perivascular fibrosis of small coronary artery of
SHRs are also suppressed by long-term treatment with Rhokinase inhibitors (323). Heterozygous deletion of Rock1
and Rock2 alleles further demonstrated the major role of
Rock2 in arterial remodeling. Neointima formation induced by carotid ligation was markedly reduced in
Rock1⫹/⫺ mice compared with wild-type or Rock2⫹/⫺ mice
(341). This correlates with decreased vascular smooth muscle cell migration, proliferation, and survival and reduced
leukocyte infiltration (341).
The RhoA/Rho-kinase signaling also participates in ageinduced arterial wall remodeling and stiffening developing
with ageing. A threefold increase in RhoA expression and
activity has been observed in artery of old (19 mo) compared with young (2 mo) rats (307). Indirect evidence suggested that this upregulation also exists in human artery as
it has been observed that RhoA/Rho-kinase-mediated coldinduced cutaneous vascoconstriction is augmented with age
(482). Increased RhoA and Rho-kinase activity could thus
be part of the mechanisms underlying the hypersensitive
response of the vessel wall to injury and its increased susceptibility to develop hypertension and advanced atherosclerotic lesions with age. Indeed, it has been recently demonstrated that age-dependent increase in extracellular matrix stiffness enhances vascular cell contractility through
RhoA/Rho-kinase activation (186). In endothelial cells, this
process leads to mechanical destabilization of cell-cell junctions, increasing endothelial permeability and thus susceptibility to atherosclerosis.
B) RHOB.
While RhoB is very similar to RhoA and shares its
downstream effectors, in particular Rho-kinase, the role of
RhoB in the vascular wall is, so far, almost not investigated.
However, recently a role of RhoB in vascular smooth muscle cell migration induced by PDGF has been reported.
Stimulation of the PDGF receptor-␤ on primary vascular
smooth muscle cells results in RhoB-dependent trafficking
of endosome-bound Cdc42 from the perinuclear region to
the cell periphery, where the Rho GEF Vav2 and Rac are
also recruited to drive formation of circular dorsal and peripheral ruffles necessary for cell migration (183). Analysis
of vascular remodeling in RhoB-deficent mice would be
certainly of great interest.
C) RAC1.
Compared with RhoA, the function of Rac1 in
vascular remodeling is less well understood. However,
through its role in NADPH oxidase-dependent ROS generation, Rac1 plays an important role in the development of
vascular smooth muscle hypertrophy and migration (180,
285). This role of Rac1 is involved, in particular, in vascular
remodeling associated with increased oxidative stress as
observed in high blood pressure (499). Moreover, Pak1 also
appears as a downstream effector of Rac1 in vascular
smooth muscle as expression of a dominant negative Rac1
suppresses ANG II-induced Pak1 activation, hypertrophy
(539), and migration (173). Activation of the Rac1 and the
subsequent engagement of Pak1 and JNK also participate in
the EGF-induced vascular smooth muscle cell proliferation
(32). Only a few studies have addressed the role of Rac1 in
vascular remodeling in vivo. Transgenic overexpression of
dominant active Rac1-V12 confirmed the role of Rac1 as a
regulator of the redox state of blood vessel (157). In the
balloon injury model of vascular remodeling, Pak1 is found
to be activated in neointima of injured rat carotid artery,
and a marked reduction of the neointimal thickening is
observed in dominant-negative Pak1 or Rac1 adenovirustreated artery (49, 173). In diabetes-induced vascular injury
linked to an increased oxidative stress and Rac1/Pak1 activity, dominant-negative Rac1 gene transfer has also been
shown to be protective (498). In short, all these data suggest
that Rac1/Pak1 activation participates in pathophysiological vascular remodeling.
While Rac2 was initially found to have a restricted expression in hematopoietic cells, Rac2 expression and activity are
induced upon stimulation of vascular smooth muscle cell
with inflammatory cytokines (485). Retroviral overexpression of Rac2 in vascular smooth muscle cells leads to increased migration, activation of the NADPH oxidation cascade, and increased activation of Pak1 and its proximal
effectors, ERK1/2, and p38MAPK. Rac2 was thus identified as a potential mediator of inflammation-driven vascular smooth muscle response to injury (485). Further studies,
in particular in vivo analyses, are now required to clarify the
role of Rac isoforms in arterial remodeling.
D) CDC42.
Almost nothing is known regarding the role of
Cdc42 in the arterial wall. A very recent work reports that
the regulation of collagen I secretion by PKC␦ in arterial
smooth muscle cells is dependent on Cdc42 (261). Collagen
type I is the most abundant component of extracellular
matrix in the arterial wall, and a tight regulation of collagen
is critical to homeostasis of the arterial wall. These data
thus suggest that Cdc42, in addition to RhoA and Rac proteins, could also participate in the control of arterial wall
structure.
D. Vascular Permeability
In addition to its role in the regulation of vascular tone and
blood flow, a major function of the vascular endothelium is
to constitute a semi-permable barrier that controls passage
of macromolecules and fluids between circulating blood
and the body tissues. Endothelial barrier integrity is governed by the junctions between adjacent endothelial cells
that line blood vessels. Both tight junctions that are particularly developed in the blood-brain barrier and adherens
junctions are responsible for endothelial cell-cell adhesion.
Most of the factors affecting vascular permeability modify
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the endothelial barrier function through the regulation of
endothelial cell junctions. This regulation is particularly
important during physiological processes that require transient breakdown of the endothelial barrier followed by resealing such as acute inflammation or angiogenesis (see sect.
IVA). In contrast, dysregulated endothelial barrier function
characterizes a pathological situation such as chronic inflammatory diseases and atherosclerosis (see sect. IVE).
Vascular endothelial (VE)-cadherin plays a key role in the
regulation of adherens junctions. VE-cadherin is a transmembrane protein that forms homotypic interactions between neighboring endothelial cells. Its intracellular part
interacts with the actin cytoskeleton through both ␣- and
␤-catenin and p120catenin (29). Because Rho proteins are
potent regulators of the actin cytoskeleton organization,
numerous studies have addressed the role of RhoA, Rac1,
and Cdc42 in the control of endothelial permeability (369,
536). Rap1 has also been recognized as a regulator of the
endothelial barrier, and more recently, a role of other small
G proteins such as Ras has also been described (359, 369).
1. Ras proteins
A) RAS. In endothelial cells, endogenous R-Ras physically
interacts with filamin A, a member of the nonmuscle actin
binding protein family (143). Knockdown of either R-Ras
or filamin A promotes increased vascular permeability and
disorganization of VE-cadherin at adherens junctions,
through an increase in Src-dependent phosphorylation of
VE-cadherin at Tyr731 (143). R-Ras, through its interaction with filamin A, is therefore required for maintaining
endothelial barrier function. An analysis of tumor blood
vessel formation in R-Ras knockout mice has confirmed its
critical role in vessel integrity and function and particularly
in the regulation of vascular permeability (419). Plasma
leakage is increased, endothelial cell-cell junctions are disrupted, and VE-cadherin immunostaining is decreased in
tumor vessels in R-Ras knockout mice. It was further demonstrated that expression of the constitutively active R-RasV38 in confluent endothelial cell cultures induced accumulation of VE-cadherin and ␤-catenin at the cell-to-cell interface, thus improving endothelial barrier function (419).
This effect has been ascribed to the suppression of VEcadherin internalization through inhibition of its phosphorylation on Ser665. Expression of the dominant negative
R-Ras-N43 or knockdown of endogenous R-Ras has opposite effects (419).
In contrast, in vitro in human umbilical endothelial cells,
H-Ras signaling has been shown to disrupt endothelial
barrier integrity (148, 437). H-Ras/Raf1-dependent activation of MEK/ERK signaling results in increased phosphorylation of MLC, which leads to the recruitment of
Src to the VE-caherin, Src-mediated VE-cadherin phosphorylation, and dissociation of adherens junctions
(148). Nevertheless, activation of PI 3-kinase/Akt path-
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way downstream of H-Ras is also described to be sufficient for H-Ras-induced vascular permeability (437).
B) RAP1.
In vitro activation of Epac1/Rap1 in endothelial
monolayer decreases basal and thrombin-induced permeability by potentiating VE-cadherin-mediated endothelial
cell-cell contacts (92, 125, 234). This Epac1/Rap1 signaling
pathway participates in the cAMP-dependent increase in
endothelial cell-cell contacts induced by physiological factors such as prostaglandins or ANP (44, 45). In vivo, activation of the Epac/Rap1 signaling strongly reduces the
baseline and platelet-activating factor-induced acute increase in endothelial permeability in intact microvessels and
stabilizes the endothelial-endothelial junction complex (5).
Through this regulation of the endothelial barrier function,
the Epac1/Rap1 signaling pathway was thus identified as an
essential mediator of the anti-inflammatory effects of cAMP
and cAMP-elevating agents.
In addition to the AMPc/Epac1 activating system, Rap1 is
also activated by homophilic VE-cadherin interaction upon
cell-cell contact formation by the Rap1 GEF PDZ-GEF1 via
the protein adaptator MAGI (411). This result thus reveals
a reciprocal interaction between Rap1 and VE-cadherin,
with Rap1 stabilizating VE-cadherin-dependent adhesion
and cell-cell contact inducing Rap1 activation. Recently, it
has been described that Rap1 isoforms differentially regulated endothelial cell junctions. Although Rap1B accounts
for more than 90% of the total Rap1 expressed in endothelial cells, Rap1A is the predominant isoform involved in the
formation and maintenance of endothelial cell barrier properties, and Rap1B is not able to substitute to Rap1A for
these functions (534).
Downstream of Rap1, several different effectors are involved in the effects on endothelial barrier function. Among
them is Rac1 as activation of Epac1/Rap1 in endothelial
cells results in the activation of the Rac1 GEFs Tiam1 and
Vav2 (44, 45). The resulting activation of Rac1 leads to
enhancement of peripheral actin cytoskeleton and adherens
junctions. CCM1 was also identified as a Rap1 effector for
endothelial cell-cell junction regulation (139). Activated
Rap1 interacts with CCM1, which targets CCM1 to cellcell junctions through physical interaction with junction
proteins (␤-catenin and the junctional scaffold protein
AF-6) and finally leads to stabilization of endothelial junctions (139). Interestingly, AF6 is also considered as a Rap1
effector as it specifically binds Rap1-GTP (468) and preferentially the Rap1A isoforms (534).
Recently, Raf1 has been identified as an essential component of Epac1/Rap1-mediated endothelial cell cohesion.
Upon Rap1 activation, Raf1 is recruited to VE-cadherin
complexes, which is required to bring Rock2 to VE-cadherin containing junctions to provide a relevant control of
actomyosin activity in these structures (530).
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2. Rho proteins
A) RHOA.
RhoA was first described for its role in endothelial
cell contraction and hyperpermeability. Both in vitro and in
vivo data demonstrated that RhoA activation plays an essential role in the barrier-disrupting effect of various agents
such as thrombin, tumor necrosis factor (TNF)-␣, lipopolysaccharirde (LPS), and lysophosphatidic acid (LPA) (83,
111, 112, 495, 496, 535). In addition, RhoA and its target
Rho-kinase also have a negative role in the regulation of
basal endothelial barrier function. Inhibition of Rho-kinase
reduced baseline vascular permeability in vivo and in different cultured endothelial cells in vitro (4). Generation of
actomyosin contractility and the resulting isometric tension
in endothelial cells are the main mechanisms through which
RhoA/Rho-kinase signaling induces junctional breakdown
and induction of permeability. RhoA-stimulated Rho-kinase activity phosphorylates and inhibits MLCP, leading to
an increased phosphorylation of MLC and acto-myosin interactions (111).
In contrast, it was also reported in confluent endothelial cell
cultures that intrinsic basal activity of Rho-kinase is required for barrier maintenance through an action on VEcadherin endosomal recycling (494). Nevertheless, the relevance of these data has not been demonstrated in vivo.
Several mechanisms have been proposed to trigger the activation of RhoA by agents that increase endothelial permeability. It has been first described that thrombin/PAR-1induced RhoA activation resulted from PKC␣-mediated
Rho-GDI phosphorylation and inhibition of its activity,
leading to increased RhoA-GDP/GTP turnover and RhoA
activation (304). Arhgef1 has then been identified as the
RhoA GEF mediating activation of RhoA in response to
thrombin/PAR-1 receptor stimulation in a G␣12/13- and
PKC␣-dependent fashion (40, 179). This first phase of Arhgef1-mediated RhoA activation is followed by a second
phase, dependent on microtubule disassembly. It has been
proposed that, in addition to its role on actomyosin contractility, Rho-kinase phosphorylates the microtubule regulatory protein tau, causing peripheral microtubule disassembly and the subequent release of microtubule-associated
GEFs such as p190-RhoGEF and Arghef2 (GEF-H1) (39,
40). These events induce a more sustained RhoA activation,
cytoskeletal changes, and endothelial barrier dysfunction in
response to thrombin (39, 40). More recently, Arghef1 was
also identified as the RhoA GEF mediated-RhoA activation
and permeability induced by TNF-␣ (361) and LPS (546).
Activation of Arhgef1 by TNF-␣ is mediated by PKC␣
phosphorylation of Arhgef1 (361).
On the other hand, concomitantly to Rac1/Cdc42 activation, downregulation of RhoA activity is essential for both
the maintenance of basal endothelial barrier function and
recovery after disruption. The GAP p190-RhoGAP that inactivates RhoA has been recognized as a key signaling mol-
ecule for this process. The activity of p190-RhoGAP is regulated by Src-mediated tyrosine phosphorylation, and in
the basal condition, the tyrosine phosphatase SHP2 participates in the maintenance of a low RhoA activity by regulating p190-RhoGAP phosphorylation (144). Phosphorylation of p190-RhoGAP on serine/threonine residues also
participates in the regulation of its activity (318), and kinases such as PKC␦ and FAK also positively control p190RhoGAP activity to limit basal RhoA activity and to stop
RhoA activation during thrombin stimulation, respectively
(154, 178). Regulation of p190-RhoGAP expression provides an additional level of regulation of RhoA activity as
the enhancement of endothelial barrier function induced by
cAMP elevating agents involves downregulation of RhoA
activity through stimulation of p190RhoGAP-A expression
at a transcriptional level (75).
B) RAC1 AND CDC42.
Contrary to RhoA, Rac and Cdc42 reinforce endothelial barrier function. Rac1 is required for the
formation and the maturation of endothelial junctions. Rac1
activity is increased during junction assembly, and Rac1-deficient endothelial cells are unable to form cell-cell contacts
(250, 343, 477). Consistent with this role of Rac1 for maintaining cell-cell junctions, expression of dominant negative
Rac1 increases endothelial permeability and disturbs tight and
adherens junctions (27, 535). Inhibition of Rac1, in association with RhoA activation, is involved in the increase in endothelial permeability and loss of cell-cell junctions induced by
thrombin (505, 535). Thrombin-induced Rac1 inhibition results from proteinase-activated receptor 1/G␣i-mediated inactivation of adenylate cyclase, decrease of cAMP level, and
subsequent inhibition of the Epac1/Rap1/Vav2, Tiam/Rac1
cascade (41, 507). On the other hand, factors that increase
endothelial barrier function such as mechanical forces, sphingosine 1-phosphate, ANP, prostacyclin, oxidized phospholipids, or hepatocyte growth factor are thought to act via activation of Rac1 downstream of either Epac1/Rap1 or PI3-kinase
signaling pathways (42, 45, 271, 449, 506). In addition to
these mechanical and soluble factors, “ouside-in” signaling
from VE-cadherin also regulates Rac1 activity by a positive
feedback mechanism that is essential for maximal endothelial
barrier function and recovery of endothelial cell monolayer
integrity (43).
Cdc42 does not participate in the effect of thrombin on
endothelial permeability, but it contributes to the increased
actomyosin contractility subsequent to thrombin stimulation as dominant negative Cdc42 prevents thrombin-induced stress fiber formation (535). Moreover, Cdc42 is activated at sites of junctional complex formation, suggesting
its role in the regulation of cadherin junctions (343). Cdc42
has then been shown to promote association of ␣-catenin
with E-cadherin/␤-catenin complex and its consequent interaction with the actin cytoskeleton (56). In vivo expression of a dominant active Cdc42 prevents RhoA-dependent
increase in endothelial permeability, thus protecting barrier
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function (382). Adherens junctions themselves control
Cdc42 activity (369). Following loss of homotypic interaction, the cytoplasmic domain of VE-cadherin triggers the
activation of Cdc42 (369). The mechanisms responsible for
this VE-cadherin effect are not identified but might involve
either a direct activation via a specific Cdc42 GEF or an
indirect mechanism involving the recruitement of p190RhoGAP by p120-catenin in disassembled junctions, the
inactivation of RhoA followed by the activation of Cdc42
(57, 236). This activation of Cdc42 by junction disassembly
plays an essential role in the restoration of endothelial barrier integrity.
inactivates cofilin, which finally leads to accumulation of
cortical actin (506). Focal adhesion proteins have also been
proposed to participate in Rac1/Cdc42-mediated regulation of endothelial permeability. Rac1/Cdc42 activation allows redistribution of focal adhesion proteins and association with adherens junction protein complexes through
paxillin/␤-catenin interactions (42). Although the molecular mechanism involved is not completely understood, it
might involve Pak-induced phosphorylation of paxillin on
Ser273 (331).
E. Atherosclerosis, Atherogenesis (Figure 8)
The mechanisms by which Rac1 and Cdc42 contribute to
junction assembly are still speculative but probably involve
IQGAP1. Active Rac and Cdc42 bind IQGAP1, thus releasing ␤-catenin from IQGAP and allowing ␤-catenin/VE-cadherin association and stabilization of the junctional protein
complex (242, 369). Another possibility is that Rac1 and
Cdc42 indirectly control adherens junction through local
actin cytoskeleton regulation (271, 369). Active Rac1 recruits Pak and its target Lim kinase that phosphorylates and
Monocyte
LDL
In recent years, great efforts have been devoted to unraveling the basic mechanisms of atherosclerosis, the underlying
pathology of cardiovascular diseases. An imbalanced lipid
metabolism with increased circulating levels of low-density
lipoprotein (LDL) and a maladaptive immune response entailing a chronic inflammation of the arterial wall are clearly
identified as major triggering factors. Atherogenesis typically starts with dysfunction and activation of endothelial
T-cell
Adhesion receptor
expression
Permeability
increase
Ras
RhoA
Rac1
RhoA
Rac1
Cdc42
ROS formation
Rac1
LDL oxidation
Cytokine/
chemokine
release
Endothelium
Chemotaxis,
migration
T cell
proliferation
RhoA
Rac1
Cdc42
RhoA
Cholesterol
efflux
RhoA
Cdc42
oxLDL uptake
RhoA
Contraction
RhoA
Foam cell
formation
Macrophage
retention
RhoA
Rac1
Smooth muscle cells
FIGURE 8. Involvement of Ras/Rho proteins in atherosclerotic lesion formation. Protein names are indicated
under a brief description of the process in which they are involved in a green background for a positive effect
and a red background for a negative action.
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
cells that triggers recruitment of proinflammatory circulating cells. The resulting local inflammation induces leukocyte chemotaxis and adhesion to the damaged endothelium,
leading in turn to an increased vascular permeability for
plasma lipids. Monocytes located in the arterial intima accumulate these lipids and transform into macrophage-derived foam cells, thus constituting early plaques. By secreting cytokines and growth factors that stimulate vascular
cells, these early plaques generate their own growth and
progress into mature plaques. Therefore, in addition to vascular wall cells, circulating immune cells including monocytes, lymphocytes, and platelets contribute to the disease
initiation and progression. By regulating numerous functions in these different cell types, members of Ras protein
superfamily, in particular Rho proteins, are involved in this
complex pathological process.
1. Ras proteins
A) RAS. Cellular senescence and inflammation are classical
features of atherosclerosis. The enhancement of vascular
inflammation and senescence induced in vivo by adenovirus-mediated expression of dominant active H-Ras in rat
arteries has led to the suggestion of a potential role of Ras in
atherogenesis (312). This is in agreement with the attenuation of atherosclerotic plaques in ApoE⫺/⫺ mice treated by
FTI, in association with a reduced Ras activity in arteries
(135, 471). FTI has no effect on plaque T lymphocyte and
macrophage content, but vascular cell adhesion protein-1
(VCAM-1) and NF␬B expression, and oxidative stress are
markedly reduced in treated ApoE⫺/⫺ mice compared with
controls (135, 471). This suggests that Ras activity has a
pro-atherogenic action, via mechanisms that are not elucidated. It has been proposed that the downstream Ras effector early growth response-1 (Egr-1) that regulates transcription of proinflammatory and procoagulant genes could be
involved. Oxidized LDL induces expression of Egr-1 in endothelial and smooth muscle cells and in macrophages at
least in part via the Ras-ERK1/2 pathway (153).
Recently, pieces of evidence supporting a role of Ras proteins in atherosclerosis in human have been provided by
genome-wide association studies (109). They identified a
coronary artery disease locus in M-Ras. The gene encodes
for the M-Ras protein that is widely expressed in all tissues,
with a very high expression in the cardiovascular system.
Although these data do not allow anticipating the role of
M-Ras in atherogenesis, it could be related to its involvement in adhesion signaling (570).
2. Rho proteins
Given the key role of Rho proteins in the regulation of
vascular cell migration and proliferation, the recognition of
their involvement, in particular RhoA and its effector Rhokinase, in atherosclerosis is not surprising. However, recent
data also revealed that Rho proteins contribute to vascular
inflammation, leukocytes infiltration, and lipid uptake/efflux in macrophages.
A) RHOA. RhoA expression and activity increase with cellular
infiltration during the progression of atherosclerotic lesions
(351). Downstream RhoA, Rho-kinases have been also
shown to be upregulated at inflammatory arteriosclerotic
lesions in both a porcine model of coronary artery spasm
(211) and arteriosclerotic human arteries (212). In agreement with this observation, in vitro studies have shown that
oxidized LDL and its compound LPA activate RhoA in
vascular smooth muscle and endothelial cells, suggesting
that hyperlipidemia could contribute to the increased RhoA
activity in the wall of atherosclerotic arteries (130, 470). A
causal role of RhoA/Rho-kinase signaling in the progression of plaques was first provided by pharmacological analyses that showed that in vivo chronic treatment with Rhokinase inhibitors strongly reduced atherosclerotic lesions
and spasms in pig (320) and in ldlr⫺/⫺ mice (289). NF-␬B
activation downstream to RhoA/Rho-kinase and modulation of T-lymphocyte proliferation have been suggested to
contribute to the pro-atherosclerotic effect of RhoA/Rhokinase signaling (289). Together with the indirect evidence
that inhibition of Rho protein isoprenylation mediates, at
least in part, the anti-inflammatory and anti-arteriosclerotic
properties of statins, these observations thus confirm the
pro-inflammatory and pro-arteriosclerotic effects of RhoA/
Rho-kinase signaling (334).
Upregulation of RhoA in atherosclerotic vascular wall
could also account for endothelial cell activation and dysfunction associated with atheroma plaque formation. Activation of RhoA/Rho-kinase signaling decreases expression
and activity of eNOS and, by increasing endothelial cell
contractile forces, enhances permeability, thus allowing
easier passage of molecules, such as LDL, and leukocytes
from the bloodstream into the vessel wall intimal layer
(186, 254, 533). Many different mechanisms are likely to be
involved in the overactivation of RhoA, including the increased oxidative stress and ROS production in atherosclerotic arteries. Effects of ROS on Rho proteins may be cell
specific and depend on the localization and the activity of a
particular Rho protein. Indeed, both ROS-mediated activation and inhibition of RhoA have been described (7, 336).
Under conditions where ROS are generated at increased
pathological levels, ROS can directly activate RhoA by oxidation of cysteine 16 and 20 (7).
Leukocyte adhesion is further favored by RhoA-mediated
direct positive regulation of leukocyte adhesion to the endothelium. RhoA controls both the expression of leukocyte
adhesion receptors such as E-selectin, VCAM-1, and intercellular adhesion molecule-1 (ICAM-1) on endothelial cell
membrane at least in part through NF-␬B signaling, and
downstream signals from these receptors (69). In fact, en-
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LOIRAND, SAUZEAU, AND PACAUD
gagement of ICAM-1 following leukocyte adhesion induces
RhoA activation and recruitment of ERM (ezrin/radixin/
moesin) to sites of ICAM-1 clustering. ERM proteins thus
participate to further increase RhoA activity in endothelial
cells by binding to and recruiting the Rho GEF Dbl, and by
binding to RhoGDI which releases its inhibition of RhoA
(533). Furthermore, RhoA/Rho-kinase activation also favors recruitment of circulating cells into atherosclerotic lesions by inducing release of cytokines and chemokines such
as monocyte chemotactic protein-1 (MCP-1, also known as
chemokine ligand 2, CCL2) and interleukin-6 by vascular
cells (129, 193).
On the other hand, adhesion to the endothelium also induces upregulation of Rock1 in leukocytes (120), and accumulating evidence also suggests that RhoA signaling in infiltrating leukocytes, in particular in monocyte/macrophages, plays a key role in the progression of atheroma
plaques. Indeed, atherosclerosis is decreased in mutant mice
with specific Rock1 deficiency in bone marrow-derived cells
on a LDL receptor deficiency background (510). Lack of
Rock1 in bone marrow-derived cell decreases macrophages, T cells, and lipid accumulation in the atherosclerotic lesions. In vitro, Rock1-deficient macrophages exhibit
impaired chemotaxis and reduced oxidized LDL uptake
and foam cell formation. By promoting macrophages chemotaxis and lipid accumulation, Rock1 in macrophages is
therefore critical to the development of atherosclerosis.
Whether or not Rock2 plays a similar role remains to be
determined. Recently, although the involvement of RhoA
activation has not been addressed, it has been shown that
the Vav family of Rho GEFs regulate CD36-dependent uptake of oxidized LDL in macrophages and foam cell formation (380).
The pro-atherosclerotic effect of RhoA/Rho-kinase signaling and its positive action of foam cell formation could also
be related to its inhibitory effect on macrophage cholesterol
efflux. RhoA, by inhibiting PPAR␥ activity, negatively controls ABCA1 expression and ApoA1-mediated cholesterol
efflux, thus favoring foam cell formation (21, 293, 564).
B) RAC1.
As RhoA, Rac1 expression and activity is increased in arteries from atherogenic animal models (196,
255, 351). Interestingly, Rac1 expression and activity are
reduced by estrogen in vascular smooth muscle and
monocytes, suggesting that this effect could participate in
the atheroprotective action of estrogen (255). The involvement of Rac1 activation in atherogenesis has not
been directly assessed, although a number of results suggest it could play a role. Rac1 is part of the NADPH
oxidases NOX1 and NOX2, which play a major role in
ROS formation and subsequent processes involved in
atheroma plaque formation such as vascular smooth
muscle hypertrophy and migration (180, 285) and VEcadherin-dependent endothelial cell-cell adhesion (29,
1698
497). Rac1 also regulates ICAM-1 and VCAM-1 expression in endothelial cells and could thus participate in the
recruitment of inflammatory cells (69). Moreover,
VCAM-1 activation induces Rac1 signaling in endothelial cells leading to NADPH-derived ROS-dependent loss
of cell-cell junction that facilitates paracellular leukocyte
transendothelial migration (497, 533).
A tight control of temporal and spatial activation of
RhoA and Rac proteins is essential to coordinate leukocyte and macrophage migration. Active Rac at the leading edge of lamellipodia mediates local actin reorganization, producing lamellar extension and forward cell
movement, while active RhoA at the rear produces contraction and detachement of the tail. The balance between the opposing activities of these proteins is therefore a key determinant of cell morphology and movement
that is also regulated by Rac1-mediated ROS generation
(336). Rac1 induces downregulation of RhoA activity
through production of ROS that inhibits the low-molecular-weight protein phosphatase and then increases the
tyrosine phosphorylation and activation of p190RhoGAP which in turn inactivates RhoA (336). This
mechanism is essential for the induction and maintenance of lamellipodia in migrating cells (336).
Macrophage chemotaxis is induced by chemokines and cytokines produced during local inflammation at sites of
plaque formation. Macrophage migration triggered by such
chemotactic factors, including MCP-1 and macrophage colony-stimulating factor (M-CSF), is dependent on Rac1 activation with Pak1 and ERK1/2 as downstream Rac1 effectors (392, 454).
Although local activation of Rac1 in the lamellipodia is
required for cell movement, strong enhancement of Rac
activity and imbalanced Rac1/RhoA activities disrupt cell
migration (14). Such a mechanism has been recently identified as responsible for the inability of macrophage foam
cells to leave the artery, thus causing progression of
atheroslerosic lesions. Cholesterol loading of macrophages
leads to increased plama membrane localization and activation of Rac1, and abrogation of migration toward various chemotactic stimuli (354). Conversely, it has been suggested that the ability of high-density lipoproteins (HDL) to
promote regression of atherosclerosis resulted from the inhibition of this mechanism and consequent emigration of
macrophages from lesions. HDL, in conjunction with the
ATP-binding cassette transporters ABCA1 and ABCG1,
serve to prevent formation of cholesterol-rich domains on
the inner side of the plasma membrane of cholesterolloaded macrophages and thus limit Rac1 activity (354).
This antiatherogenic effect is further reinforced by the inhibitory action of HDL lysosphingolipids and type 3 sphingosine 1-phosphate receptors on Rac1 activity, ROS generation, and MCP-1 production by vascular smooth muscle
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
cells, which limits macrophage entry into atherosclerotic
lesions (487).
A role for Rac signaling in human atherosclerosis has
been recently provided by a genetic association mapping
study showing associations of single nucleotides polymorphisms (SNPs) in the KALRN gene with early onset
of coronary arterial disease (511). Kalirin, the product of
the KALRN gene, is a GEF for Rac (and RhoG). The
strongest association was found for the SNP rs9289231
that resides in the first intron of an alternative transcript
of the KALRN gene. Furthermore, the risk allele of
rs9289231 was significantly correlated with atherosclerosis burden in human aortas (511). Strinkingly, the same
study also identified a SNP in the CDGAP gene encoding
for the Rac/Cdc42 GAP CDGAP that displays evidence of
association with coronary artery disease. These data thus
strongly suggest that dysregulation of Rac signaling contributes to the coronary artery disease susceptibility
linked to KALRN SNP. Functional analyses of the consequence of kalirin mutations are now required to understand its role in the development of atherothrombotic
diseases.
C) CDC42. Again, like for Rac, the role of Cdc42 in atherosclerosis has never been directly assessed, although several observations suggest that it could participate in various processes involved in atherogenesis. First, Cdc42
activity is essential for endothelial barrier repair, adherens junctions stability, and restoration of permeability
(57). Therefore, dysregulated Cdc42 activity might contribute to the failure to restore endothelial barrier function associated with atheroslerosis. The mechanisms involved in this Cdc42 function are not fully understood,
although it probably involves its effect on actin remodeling. Upstream receptors as well as Cdc42 regulators
and effectors engaged during endothelial barrier repair
remain to be identified. Second, although in contrast to
Rac and Rho, Cdc42 is not required for cell locomotion,
it is essential for polarizing in the direction of the chemotactic gradient. Macrophages expressing a dominant
negative Cdc42 mutant are able to migrate but are unable
to polarize in the direction of the gradient of MCSF and
move randomly (14). Cdc42 activity is thus probably
involved in chemottractant-induced monocyte recruitment to sites of vascular inflammation during the formation and the progression of atherosclerosis. Third, Cdc42
is involved in cholesterol movement in macrophages. As
mentioned above, HDL exerts atheroprotection via multiple mechanisms, but one of the most important is cholesterol efflux. Cholesterol efflux from the cells is the
rate-limiting step in regulating the intracellular cholesterol content as well as raft structure in the plasma membrane. By this mechanism, that involved ABCA1 and
ABCG1, excess cellular cholesterol is released from cells
and transferred to HDL particles. This is the first step of
reverse cholesterol transport that carries cholesterol
from peripheral tissues to the liver for eventual elimination. The role of Cdc42 in cholesterol efflux has been
discovered thanks to studies on Tangier disease, a hereditary HDL deficiency caused by Abca1 mutations characterized by the presence of defective cellular cholesterol
efflux and development of premature atherosclerosis
(174). Expression of Cdc42 is markedly decreased in
macrophages, and also in fibroblasts from patients with
Tangier disease. Expression of a dominant negative
Cdc42 mutant reduces while in contrast, expression of an
active Cdc42 mutant increases lipid efflux, suggesting
that defective Cdc42 signaling is enough to alter lipid
efflux (174). It was further demonstrated that binding of
apoA-I, the major HDL apolipoprotein, to the plasma
membrane induced activation of Cdc42 and JNK has
been identified as a downstream Cdc42 effector involved
in apoA-I/HDL-induced cholesterol efflux (174, 339). By
promoting rearrangement of actin cytoskeleton and activation of JNK, Cdc42 activation could allow trafficking
of cholesterol vesicles from the intracellular site to the
plasma membrane where ABCA1 assembles apoA-I and
cholesterol for its efflux from cells. Cdc42 thus plays an
important role in cellular cholesterol efflux, and its dysregulation might lead to the development of atherosclerotic cardiovascular disease
All these data point out the need to combine in vitro and in
vivo approaches using relevant animal models to advance
our understanding of Cdc42 role in the initiation and the
progression of atherosclerosis.
V. “RASOPATHIES”-ASSOCIATED
CARDIOVASCULAR DYSFUNCTIONS
The Rasopathies form a group of nine developmental syndromes caused by germline mutations in genes encoding
Ras proteins as well as Ras protein regulators or effectors,
leading to Ras/MAPK pathway dysregulation. (Table 4 is
available online as supplementary data due to its size.)
Therefore, although each syndrome displays unique phenotypic features, they share a common pathophysiology and
many clinical signs including characteristic facial features,
heart defects, skin anomalies, mental retardation or learning difficulties, growth deficit, and an increased risk of developing tumors. This part thus deals with the cardiovascular features of the major rasopathies.
A. Noonan Syndrome
1. Clinical features
Noonan syndrome (NS; OMIM 163950) is a relatively
common autosomal dominant inherited disorder with an
estimated prevalence of 1 in 1,000 to 1 in 2,500 live births
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(342). The NS phenotype, which becomes less pronounced
with age, includes a typical dysmorphic appearance, developmental delay and learning problems, visual abnormalities, auditory problems, orthopedic defects, and short stature (103, 398, 481). Congenital heart defects are found in
50 – 80% of patients with NS (60, 291, 398). Newborns
and children suspected of developing NS need a careful
cardiac evaluation. Pulmonary valve stenosis and hypertrophic cardiomyopathy are the most common forms of cardiac disease. Atrial septals and partial atrioventricular canal
defects are also associated with the NS. Fifty percent of
patients with NS have an unusual electrocardiographic pattern characterized by left-axis deviation, an abnormal R/S
ratio over the left precordial precordium, and an abdnormal
Q wave (379).
2. Molecular pathogenesis
Tartaglia et al. (479) identified missense mutations in the
Ptpn11 gene in ⬃50% of NS patients (479). It is more
prevalent among NS patients with pulmonary valve stenosis
and short stature, and less common in subjects with hypertrophic cardiomyopathies and/or severe cognitive deficits. The
Ptpn11 gene encodes Src homology 2 containing protein tyrosine phosphatase 2 (SHP2), a nonreceptor protein tyrosine phosphatase. This ubiquitous protein is involved in
the effects of numerous physiological mediators such as
growth factors, hormones, or cytokines. In particular, by
modulating phosphorylation of proteins within multiple
signaling pathways, including the Ras/MAPK cascade,
SHP2 controls intracellular transduction mechanisms that
regulate cell cycle progression, growth, and migration (38,
95). Most of the mutations of Ptpn11 in NS patients disrupt
an autoinhibitory interaction between the NH2-terminal
domain and the catalytic phosphatase domain. These
amino acid substitutions promote SHP2 gain-of-function
leading to increased binding affinity for signaling partners
of Ras/MAPK pathway. Various strains of mice have been
generated to understand the role of SHP2 in cardiac development and physiology. Mice expressing a constitutively
active form of SHP2 (Ptpn11 D61G allele) exhibit defects
similar to those seen in NS patients including short stature,
facial dysmorphia, valvular hyperplasia, and septal defects
(20). By studying an inducible Ptpn11 allele, they demonstrated that the expression of mutant SHP2 only in endocardium is sufficient to induce all cardiac defects in NS (19).
The cardiac-specific deletion of Ptpn11 rapidly induces a
compensated dilated cardiomyopathy in mice without an
intervening hypertrophic phase (233). In this study, the authors demonstrated in vitro and in vivo an abnormal ERK/
MAPK and RhoA/Rho-kinase activation in SHP2-deleted
cardiomyocytes. Ex vivo, inhibition of these signaling pathways rescues the dilated phenotype, suggesting a critical
role of RhoA and ERK/MAPK in the dilated cardiomyopathy of NS patients. Recently, Langdon et al. (251) have
confirmed this hypothesis by introduction of Noonan
SHP-2 mutation (SHP-2N308D) into Xenopus embryos. Em-
1700
bryos expressing this mutation are characterized by an over
activity of Rho-kinase leading to abnormal arrangement
and polarity of cardiac actin fibers, and consequently cardiac development defects.
The second most frequent gene mutated identified in NS
patient (20%) is Sos1 (258, 395, 480). The Sos1 gene encodes the Ras and Rac GEF Son of Sevenless 1 protein.
Mutations in Sos1 affect principally two domains, the
Pleckstrin homology (PH) and the Ras exchanger motif
(REM) domains, which are not implicated in the exchange
activity but rather in the intrinsic autoinhibitory function of
Sos1. These mutations lead to the maintenance of Sos1 in a
noninhibited form, resulting in a permanent activation of
the Rac and Ras/ERK pathways. Although some GEFs have
been found to be mutated or aberrantly expressed in human
cancer, NS-associated germline mutations of Sos1 were the
first described example of activating GEF mutations linked
with a human genetic disease (395, 480). The extent to
which Sos1 gain-of-function mutations affect different Rasdependent or Rac-dependent signals remains to be elucidated.
In 2006, a germline K-Ras mutation was found in a NS
patient who presented a severe clinical phenotype. Subsequently, K-Ras mutations were identified in ⬍2% of NS
patients but generally associated with more significant
learning issues and developmental delays (68, 426). Cardiac
involvement was present in up to 85% of affected individuals, including pulmonary stenosis, hypertrophic cardiomyopathy, atrial septal defect, patent ductus arteriosus, and
ventricle septal defect. Most mutations of K-Ras cluster in
the first and second coding exons, which are shared by the
two K-Ras isoforms K-RasA and K-RasB. Mutations affecting exon 6 encoding residues at the COOH terminus of
K-RasB but not K-RasA have also been reported. NS-associated K-Ras mutations affect highly conserved amino acid
residues and are assumed to confer mild gain of function.
In 2007, heterozygous missense mutations in Raf1 were
reported in 20% of NS patients (357). Whereas hypertrophic cardiopathies are rare (18%) in NS caused by Ptpn11
or Sos1 mutations, they are frequently observed (76%) in
association with Raf1 mutations. These observations highlight differences in genotype/phenotype correlations, in particular for hypertrophic cardiopathies, and suggest an important role of Raf1 in pathophysiological modulation of
cardiac hypertrophy. However, the role of Raf1 in cardiac
hypertrophy obtained with animal experimental models remains controversial. In mice, cardiac expression of dominant-negative Raf1 was shown to prevent ERK activation
and to strongly reduce pressure overload-induced cardiac
hypertrophy (155). In contrast, cardiac-specific deletion of
Raf1 had no effect on ERK expression/activation or heart
growth (554).
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
B. LEOPARD Syndrome
2. Molecular pathogenesis
1. Clinical features
Aoki et al. (17) identified heterozygous de novo missense
mutations of H-Ras gene in CS patients. The patient missense mutations result in amino acid substitutions of a glycine residue in position 12 or 13 of the protein product.
These particular amino acids are located at the GTP binding
site, and mutations at these sites have previously been
shown to cause constitutive activation of H-Ras, in turn
causing increased activation of downstream effectors in
MAPK signaling pathways controlling cell proliferation
and differentiation. Interestingly, these H-Ras germline mutations affect the same amino acid residues that are mutated
in cancer.
LEOPARD syndrome (LS; OMIM 151100) is an acronym
for multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, growth retardation, and sensorineural
deafness. LS was first reported by Zeisler and Becker in
1936 (577). Diagnostic clues to LS include multiple lentigines, café-au-lait macules, deafness, and hypertrophic cardiomyopathy. LS patients show retardation of growth and a
delayed puberty. Electrocardiographic anomalies and progressive conduction anomalies are the most common heart
defects, including left or biventricular hypertrophy, often in
association with Q waves, prolonged QTc, and repolarization abnormalities. Hypertrophic cardiomyopathy is the
most frequent anomaly (80%), which in general is asymmetric. Less frequent heart defects are pulmonary stenosis
25% and aortic/mitral valve abnormalities in some LS patients (414).
2. Molecular pathogenesis
It has recently been reported that LS is related to a missense
mutation in the Ptpn11 gene (105). As described in the
previous paragraph, germinal mutations in the Ptpn11 gene
are also responsible for ⬃40 –50% of NS. But biochemical
in vitro studies point to a decrease of SHP-2 catalytic activity for LEOPARD-associated mutations, in contrast to the
enhanced SHP-2 activity found for NS. Curiously, LS and
NS are associated with the same gene and lead to similar
clinical phenotype. To date, no explanation is provided to
understand how the gain of function or the loss of function
of SHP-2 could induce the same phenotype. In addition,
Raf1 gene mutations were identified in LS patients (357,
426), indicating that LEAOPARD syndrome cannot simply
be understood by invoking reduced Ras/MAPK signaling.
A strain of mice carrying a germline G12V mutation within
their endogenous H-Ras locus has been generated to shed
light on the role of this gain of function of H-Ras in the
developmental and physiological defects associated with CS
(427). H-RasG12V mice are viable and closely phenocopy
some of the defects observed in CS patients, including a
number of cardiovascular dysfunctions. Four-month-old
mutant mice develop cardiomyopathy characterized by
general enlargement of all heart chambers, concentric left
ventricle hypertrophy, and fibrosis. The authors have observed consistent thickening of the aortic valve without any
alterations in the pulmonary or atrioventricular valves. In
addition, H-RasG12V mice exhibit systemic arterial hypertension resulting from activation of the ANG II pathway.
High blood pressure is not common in CS patients, but it
has been described in 10% of CS patients with the mutation
34G (113). H-RasG12V mice thus appear as a good animal
model to understand the etiology of cardiovascular CS
symptoms and to evaluate potential therapeutic strategies.
D. Cardio-Facio-Cutaneous Syndrome
1. Clinical features
C. Costello Syndrome
1. Clinical features
Costello syndrome (CS; OMIM 218040) was first described
in 1971 by Costello. The clinical manifestations included
postnatal growth retardation, distinct facial appearance
with large head, curly hair, nasal papillomata, hyperextensible fingers with loose integuments of the hands and feet,
and hyperpigmented skin (22, 90, 91). This rare congenital
disorder affects multiple organ systems encompassing severe failure to thrive, cardiac anomalies, tumor predisposition, and cognitive impairment. More than 60% of patients
develop cardiac defects including hypertrophic cardiomyopathies, congenital malformations, usually pulmonary
valve stenosis, and arrhythmia, especially atrial tachycardia
(103).
The cardio-facio-cutaneous syndrome (CFCS; OMIM
115150) is a rare sporadic occurring disorder (389). Short
stature, mental retardation, global developmental defects,
and delayed language acquisition are found in most patients
(⬎75%) (103, 217, 394). Children with CFC have typical
facial characteristics similar to those noted in NS: high forehead with bitemporal constriction, palpebral ptosis, and a
short nose with relatively broad nasal base. Cutaneous and
adnexal abnormalities detected in CFCS are similar to those
reported in the NS and CS. Cardiac abnormalities are detected in 75% of patients. Affected individuals often present
with pulmonary stenosis (45%), hypertrophic cardiomyopathy (40%), and atrial septal defect (22%). Life expectancy
of CFC patients is probably shortened on average, due to
the severe cardiovascular defects.
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2. Molecular pathogenesis
CFCS has been first considered as a severe form of NS and
CS because these three syndromes share several manifestations, and “borderline” cases do exist, usually in infants.
However, Ptpn11 and H-Ras mutations were not found in
CFC patients, indicating a specific etiology and genetic entity of this syndrome. The final evidence came in 2006, with
the discovery among 23 patients with the CFC, of 11 different mutations of the B-Raf gene in 18 of them, mutations
of MEK1 in 2 and of MEK2 in 1 patient (396). These
mutations have then been rapidly confirmed by other authors (335). Two clusters of mutations were identified in the
B-Raf gene: in the cysteine-rich domain in the exon 6 and in
the protein kinase domain. All of them are missense mutations, suggesting a gain-of-function effect and a consequent
over-activation of the B-Raf/MEK1/2 signaling.
A mouse strain that expresses a constitutive active form of
B-Raf (B-RafV600E) has been recently generated (492). This
animal model mimics some characteristic defects found in
CFC patients, including reduced life span, small size, facial
dysmorphism, and epileptic seizures. In addition, B-RafV600E
mice develop cataracts and upregulation of some sympathetic functions. With regard to the cardiovascular system,
no gross alterations in the histological cardiac structure
(auricules, ventricles, aortic and pulmonary valves) and sign
of tissue fibrosis are observed in mutant mice. However, a
cardiomegaly is detected, due to an increase in the total
number of cardiomyocytes. Heart function from
B-RafV600E mice is altered, including lower end-systolic and
end-diastolic volumes. Systolic arterial pressure is not modified in the mutant animals, despite a significant increase in
the heart ejection fraction. The other cardiovascular alterations typical of CFC patients, such as pulmonary valve
stenosis as well as septal and aortic defects are not observed
in the mutant mice. B-RafV600E mice are a suitable animal
model of the RASopathy CTC and may serve as a tool to
evaluate the potential therapeutic efficacy of B-Raf inhibitor, such as PLX4032 described recently as a potent inhibitor of B-Raf kinase activity (48, 118).
E. Neurofibromatosis 1
1. Clinical features
Neurofibromin is encoded by a large gene spanning over
350 kb in the chromosomal region 17q11.2 (26, 434). The
protein product of this gene functions as a Ras GTPaseactivating protein (GAP) to negatively regulate Ras activity
(294, 547). More than 240 different mutations have been
described within the Nf1 gene (386). The majority of mutations lead to a truncated or no protein product. Reduction
or complete loss of neurofibromin induces activation of
Ras, which in turn activates mitogenic signaling leading to
increased cellular proliferation or differentiation. For this
1702
reason, neurofibromin is considered as a tumor suppressor
(260). Neurofibromatosis type 1 (NF1), also known as von
Recklinghausen disease (OMIM 611431), is the condition
most commonly associated with mutations in the Nf1 gene.
It is a dominant disorder affecting 1 in 3,000 individuals.
Most adults with NF1 develop café-au-lait macules, neurofibromas, optic glioma, osseous lesions, Lisch nodules, and
axillary or inguinal freckling. In addition, NF1 patients
present prominent cognitive impairments (attention deficit,
hyperactivity disorder, spatial deficit, and language delay)
and musculoskeletal abnormalities (scoliosis, long bone
dysplasia, and osteopenia). NF1 is also remarkable for its
association with occlusive (stenoses) or aneurysmal arterial
disease affecting predominantly the aorta and renal, mesenteric and carotid-vertebral arteries. One of the more common vascular lesions in NF1 patients is renal artery stenosis
with consequent hypertension (200, 253, 348). The frequency of vasculopathy among NF1 patients in general has
not been determined because most of the time NF1-associated vascular lesions are asymptomatic.
2. Molecular pathogenesis
NF1 vasculopathy lesions are characterized by an accumulation of vascular smooth muscle cells within the intima
layer of the blood vessel resulting in neointima formation.
The pathogenesis of this vascular lesion formation remains
to be undertsood, whereas they contribute to excess morbidity and mortality of young NF1 patients (387).
To shed light on the role of neurofibromin in vascular lesions, murine models of NF1 vasculopathy have been generated. Homozygous deficient embyros die in mid gestation
(approximately E13.5). Mutant embryos display hyperplasia of neural crest-derived sympathetic ganglia and exhibit
signs of cardiac dysfunction including over abundant tissue
that accumulates and obstructs blood flow in the hearts due
to hyperproliferation and lack of normal apoptosis of endocardial cushions (55, 199, 247). Endothelial-specific inactivation of Nf1 recapitulates the complete null cardiovascular phenotype providing evidence that neurofibromin is
essential for endocardial cushion formation by modulating
Ras activity in endothelial cells (138). In contrast, no apparent defects were observed in mice with specific Nf1 deletion in smooth muscle, and no structural abnormalities
were detected in the vascular system (549). Nevertheless,
the loss of Nf1 in vascular smooth muscle leads to an exaggerated vascular remodeling in response to vascular injury
(carotid artery ligation) ascribed to an over-activation of
Ras and its downstream effectors, ERK, S6K, and mTOR
(549). Surprisingly, the same results were obtained in
Nf1⫹/⫺ mice that also display vascular inflammation (252)
but not in heterozygous inactivation of Nf1 in vascular
smooth muscle cells or endothelial cells alone (253). These
results suggest the role of another cell lineage in NF1 vascular diseases. This hypothesis has been confirmed by showing that specific heterozygous inactivation of Nf1 in bone
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
marrow-derived cells was sufficient for increased neointima
formation in response to vascular injury (253). In vitro
studies demonstrate that direct contact of Nf1⫹/⫺ macrophages with vascular smooth muscle cells stimulates vascular smooth muscle cell proliferation through a Ras/Erkdependent pathway, and this effect is further increased with
Nf1⫹/⫺ vascular smooth muscle cells (253). This observation underlies the potential role of inflammatory cells in the
pathogenesis of NF1 vasculopathy. This hypothesis is reinforced by the fact that NF1 patients have increased levels of
circulating monocytes and proinflammatory cytokines
known as biomarkers linked to human vasoocclusive disease (IL-1␤ and IL-6) compared with the healthy population (253). In short, these recent data provide evidence for
an important role of vascular inflammation in NF1 patients
and provide a framework for understanding the pathogenesis of NF1 vasculopathy and developing potential therapeutic and/or diagnostic strategies.
VI. THE RAS SUPERFAMILY PROTEINS
AND EFFECTORS AS
PHARMACOLOGICAL TARGETS
As frequently mentioned throughout this review, pharmacological compounds that with a more or less good speci-
ficity targeting Ras proteins or their regulators have been
extensively used in vitro and in animal models and have
proven efficient to demonstrate the involvement of Ras protein signaling in numerous studies. Among these compounds, some of them are already used against cardiovascular diseases in humans, and development of new inhibitors targeting these signaling pathways for a potential
therapeutical application is the subject of intensive research.
A. Targeting Ras Protein Lipidation
Ras protein activity absolutely requires their anchoring to cellular membranes via a lipophyllic prenyl group covalently
bound to the COOH terminus of these proteins (FIGURE 9).
For instance, enzymatic attachment of farnesyl isoprenoid
by the action of FT is required for Ras to bind membranes,
transform cells, and induce tumorigenesis. Over the past
decade, intensive efforts have been devoted to developing
FTIs as anticancer agents (36, 489). To date, four types of
FTIs have been developed: peptidomimetics, farnesylpyrophosphate analogs, bisubstrate analogs that combine the
two former features, and small-molecule inhibitors. In cells,
FTIs inhibit H-Ras farnesylation and membrane association, block the activation of mitogen-activated protein ki-
Acetyl CoA
HMG CoA
HMG CoA
reductase
statins
mevalonate
Geranyl pyrophosphate
Farnesyl
pyrophosphate
synthase
FTI
farnesyl pyrophosphate
Farnesyl
transferase
Geranylgeranyl
pyrophosphate
synthase
Ras proteins
RhoB
Rho kinase
inhibitors
GGTI
geranylgeranyl pyrophosphate
Rho proteins
Rab proteins
Effectors
Geranylgeranyl
transferase
FIGURE 9. Biosynthesis pathway of isoprenoids leading to prenylation of Ras proteins. Statins are specific
inhibitors of HMG-CoA reductase (the rate-limiting enzyme of cholesterol biosynthesis). After prenylation, the
proteins can move to the cellular membranes where they exert their signaling function. Inhibition of their activity
could thus be obtained by farnesyl transferase inhibitors (FTI) or geranylgeranyl transferase inhibors (GGTI).
Alternatively, Ras protein signaling pathways can be interrupted by specific inhibition of their target proteins,
as shown for Rho-kinase inhibitors.
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LOIRAND, SAUZEAU, AND PACAUD
nase and PI 3-kinase/Akt pathways, and reduce the growth
of a wide variety of human cancer cell lines (388). However,
results obtained from nearly 75 clinical trials of 4 different
FTIs (lonafarnib, tipifarnib, BMS-214662, and L-778123)
reveal that these agents have no or minimal efficacy in the
treatment of hematopoietic cancers or solid tumors (36).
With regard to the cardiovascular system, unwanted cell
growth is involved in the pathogenesis of cardiovascular
diseases. Proliferation of vascular smooth muscle cells participates in the development of atherosclerotic lesions and is
the main mechanism leading to restenosis of the coronary
artery after balloon angioplasty. Thus inhibition of vascular
smooth muscle cell proliferation would logically have beneficial effects in these pathological contexts. Although Ras
proteins are important, geranylgeranylated proteins such as
Rho proteins may also be required for the proliferation of
these cells (188). Accordingly, GGT has also been defined as
a relevant target for the development of inhibitors of
smooth muscle cell proliferation (FIGURE 9). While a great
number of preclinical studies showed that FTI and GGTI
inhibit vascular smooth muscle growth, and despite the possibility of local delivery with drug releasing stents in the case
of prevention of restenosis (540), they did not lead to clinical trials (87). These compounds thus appear to be far from
reaching clinical application for cardiovascular diseases.
Nevertheless, the therapeutic interest of inhibition of Ras
protein lipidation for cardiovascular diseases is strongly
supported by the effect of statins (FIGURE 9). Statins are
considered as the gold standard therapy for lowering serum
LDL-cholesterol and improving cardiovascular morbidity/
mortality. Interestingly, results from recent experimental
and clinical studies suggest that their cholesterol-lowering
effects could not account for all their beneficial effects on
the cardiovascular system (50). Actually, inhibition of the
isoprenoid synthesis and the resulting downregulation of
Ras protein signaling pathways are responsible for most of
the cholesterol-independent pleiotropic effects of statins
(586). Although the involvement of inhibition of the Ras
subfamily in the beneficial effect of statins is not documented, numerous studies point out the important role of
Rho protein inhibition. Indeed, statins, at clinically relevant
concentrations that are used to reduce LDL-cholesterol,
have been shown to inhibit RhoA isoprenylation (82) and
Rho-kinase activity in humans (476). Furthermore, a recent
clinical study demonstrates that therapeutic doses of statins
mainly exhibit a pleiotropic effect through the inhibition of
Rac1 (385). The analogies between the effects of RhoA/Rhokinase and Rac1 inhibition observed in preclinical/basic studies and the effects of statins in humans, including increased
expression of atheroprotective genes, inhibition of pro-inflammatory mediators, endothelial protective effects, enhanced stability of atherosclerotic plaques, and inhibition
of vascular smooth muscle proliferation, strongly argue of
an important role of statin-mediated inhibition of Rho proteins in the benefits of statin treatments. In fact, measure-
1704
ment of leukocyte Rho-kinase activity is currently being
utilized to identify patients at cardiovascular risk and to
gauge statin efficacy on vascular function that is independent of cholesterol lowering effects (276 –278).
On the basis of these observations, Rho protein inhibitors
are thus considered as promising treatment for cardiovascular diseases, and the current search for small-molecule
inhibitors of RhoA could be highly valuable for developing
clinically useful agents (104).
B. Rho-Kinase Inhibitors
As the major downstream RhoA effector and a key regulator of vascular smooth muscle cell contraction but also cell
migration, proliferation, differentiation, apoptosis and survival, and gene transcription, Rho-kinase is identified as an
interesting target for cardiovascular pharmacology, and
strong efforts are made for the development of Rho-kinase
inhibitors.
Numerous studies have demonstrated the potential beneficial effects of Rho-kinase inhibitors in animals and humans
(282, 451). They have been shown to be effective for the
treatment of vascular smooth muscle hyperconstriction-induced disorders such as coronary and cerebral vasospasm
as well as systemic and pulmonary hypertension. Some data
suggested that in addition, Rho-kinase inhibitors might be
useful for treating arteriosclerotic diseases, including arteriosclerosis, restenosis, cardiac allograft vasculopathy and
vein graft disease ischemia-reperfusion injury, CCM, myocardial hypertrophy, and heart failure.
The Rho-kinase inhibitor fasudil was initially developed as
an intracellular calcium antagonist. Fasudil has been approved in Japan since 1995 for the treatment of cerebral
vasospasm after aneurysmal subarachnoid hemorrhage and
to improve blood flow after acute ischemic stroke (448).
More recently, clinical trials have been performed to assess
the effectiveness of fasudil in other cardiovascular diseases
such as stable effort angina pectoris where it was found to
reduce myocardial ischemia (315, 450). Intra-arterial administration of fasudil was also shown to decrease the forearm vascular resistance in patients with heart failure (226).
Moreover, intravenous fasudil treatment significantly reduces pulmonary vascular resistance in patients with severe
pulmonary hypertension (127).
Clinical trials with Rho-kinase inhibitors are in progress in
the United States, Europe, and Japan, in various indications, including angina, pulmonary hypertension, atherosclerosis, and reperfusion injury (451).
VII. CONCLUDING REMARKS
Recent findings have considerably increased our understanding of the role of Ras proteins in cardiovascular phys-
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SMALL G PROTEINS IN THE CARDIOVASCULAR SYSTEM
iology and physiopathology. These proteins are not only
oncogenes but, in physiological situation, are major regulators of cardiovascular cell functions, so long as activation of
Ras proteins occurs at the right time and at the right place.
Sustained or dysregulated spatiotemporal activation of Ras
proteins is most of the time deleterious, leading to pathological situations. Thus, while these proteins are identified
as potential targets for the development of new therapeutic
strategies in cardiovascular medicine, efforts should be
made to understand the mechanisms and identify the molecular partners that regulate their activity. They are likely
to be different according to the cell types, upstream activating factors, and even not the same in diverse subcellular
compartments. Much remains to be done to understand the
role of the numerous Ras proteins expressed in the cardiovascular system. However, thanks to the rapidly increasing
availability of mouse models of deletion or expression of
active mutants of Ras proteins and regulators with inducible and tissue-selective targeting, our knowledge on the
role of Ras protein signaling in cardiovascular functions
and diseases should continuously improve, with the potential discovery of targets for the development of new therapeutic strategies.
ACKNOWLEDGMENTS
We thank Dr. Flavien Charpentier (UMR_S1087, Nantes)
for critical reading of the manuscript and Dr. Agnès
Quemener (UMR_S892, Nantes) for the Ras superfamily
proteins 3D structure.
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Address for reprint requests and other correspondence: G. Loirand, Inserm UMR_S1087, IRS-UN, 8 quai Moncousu, 44007,
Nantes, France (e-mail: gervaise.loirand@univ-nantes.fr).
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GRANTS
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This work was supported by French Agence Nationale de la
Recherche Grants ANR-08-GENO-040-01, ANR-09-JCJC0115-01, and ANR-11-BSV1-013-01 and by Fondation pour
la Recherche Médicale Grant DEQ20090515416.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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