Download Key transcriptional regulators of the vasoprotective effects of shear

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© 2009
Schattauer GmbH
Key transcriptional regulators
of the vasoprotective effects of shear stress
R. A. Boon; A. J. G. Horrevoets
Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam,
The Netherlands
Keywords
Shear stress, transcription factor, KLF2, Nrf2
Summary
Atherosclerotic plaque rupture and subsequent thrombosis
is the main cause of sudden coronary death. Remarkably,
atherosclerosis only develops in certain predisposed areas
of the vasculature. Endothelial cells in these predisposed
areas experience low or oscillatory shear stress, which activates the proinflammatory and procoagulant transcription
factors activator protein 1 (AP-1) and nuclear factor κB
(NFκB), thus inducing a proinflammatory, procoagulant
surface. In contrast, healthy endothelial cells that are exposed to prolonged high laminar shear stress, express antiinflammatory and anticoagulant genes. The key shear
stress-induced transcription factors that govern the expression of these genes are Krüppel-like factor 2 (KLF2) and
nuclear factor erythroid 2-like 2 (Nrf2). Together KLF2 and
Nrf2 govern ~70% of the shear stress-elicited gene sets.
Nrf2 potently induces anti-inflammatory/antioxidant
enzymes, while KLF2 induces anti-inflammatory and anticoagulant proteins, most specifically endothelial Nitric
oxide synthase (eNOS) and thrombomodulin (TM). KLF2
also inhibits proinflammatory and antifibrinolytic genes
through inhibition of the proinflammatory transcription factors AP-1 and NFκB. The widespread beneficial effects of
the key transcription factors KLF2 and Nrf2 on endothelial
phenotype, holds the promise that their targeted modulation might lead to a new class of cardiovascular drugs.
Hämostaseologie 2009; 29: 39–43
Shear stress
sis in 84% of the cases of MI (1). In the remaining cases, either endothelial erosion or
its local, strong procoagulant properties are
thought to be responsible. Despite the systemic nature of the associated risk factors
like smoking, diabetes, hyperlipidemia and
hypertension, it is known that atherosclerosis only develops at predisposed sites
in the arterial tree (2). This is likely due to
the local disturbances in blood flow, as these
sites are always near bends and bifurcations
in the vasculature. Laminar blood flow occurs in straight parts of arteries and near the
outer curvatures of bends and exerts a tangential viscous drag on the vascular endothelium, called shear stress. This laminar
flow that is sensed directly by the endothelium is unidirectional and pulsatile, due to
the cardiac cycle, and reaches shear stress
levels of 15 to 70 dynes/cm2 (3). Near bends
and bifurcations, the flow is still pulsatile,
but highly turbulent and bidirectional, generating oscillatory shear stress of only 0 to
10 dynes/cm2. High shear stress induces an
atheroprotective and anticoagulant endothelial phenotype, while low or oscillatory
shear stress is associated with endothelial
dysfunction and atherosclerosis development. This review focuses on the transcriptional activities of endothelial cells in
response to shear stress.
Effects on endothelial cells
Inverse correlation with
atherosclerosis development
Different transcriptional responses
Atherosclerosis is a prevalent disease in the
Western world and is the underlying cause
for acute myocardial infarction (MI) and
stroke. These acute events are mainly triggered by rupture of the atherosclerotic
plaque and subsequent occlusive thrombo-
In an experimental context, endothelial cells
are mostly cultured in the absence of flow,
whereas healthy endothelial cells in vivo are
exposed to shear stress throughout their entire lifespan. Therefore, it is important to
distinguish between short-term shear stress
exposure (< 24 h) and prolonged shear stress
exposure (> 24 h), as the first endothelial responses to acute shear stress changes will be
less reminiscent of endothelial cell shear
stress exposure in vivo. Nonetheless, shortterm shear stress exposure gives vital clues
about the mechanisms by which endothelial
cells sense shear stress and convey these signals. For example, using shear stress exposure of less than one hour, Tzima and colleagues identified a cell-cell junctional
complex-dependent sensory complex that
detects acute shear stress changes (4). Transcriptional changes specific for an acute
shear response are mainly elicited by the activator protein 1 (AP-1) and nuclear factor
κB (NFκB) transcriptional complexes (5).
These transcription factors are generally
known to induce proinflammatory and procoagulant gene expression and this does not
correlate with the anti-inflammatory and
anti-coagulant effects of shear stress in vivo.
On the contrary, activation of NFκB and
AP-1 (consisting of a dimer of ATF2 and
c-Jun) by Jun NH2-terminal kinase (JNK) is
known to occur specifically in endothelial
cells exposed to oscillatory shear stress
(6–8). Furthermore, the downstream target
genes of these transcription factors, like
MCP-1, E-Selectin, ICAM-1 and tissue factor (TF) are known to be expressed by inflamed endothelial cells (9). This phenomenon has also lead to the historical misinterpretation of the so-called shear stress responsive element (SSRE) in the promoters of
these genes, which turned out to be the NFκB
binding site (9). With hindsight, this is not
surprising since these experiments were all
done with short shear stress stimulation.
Long-term shear stress exposure
(> 24 h), on the other hand, does confer antiinflammatory and anticoagulant properties
to cultured endothelial cells (10). Wellknown examples of genes that are induced
by long-term shear stress are endothelial
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40
Boon, Horrevoets
nitric oxide synthase (eNOS) and thrombomodulin (TM) (11, 12). These proteins are
potent anti-inflammatory and anticoagulant
molecules and are thought to account for a
large part for the anti-inflammatory and
anticoagulant properties of healthy endothelial cells.
In the past several studies were performed to identify the differences on the transcriptomic level of endothelial cells exposed to high shear stress compared to control conditions. One of the first studies on
this subject showed that in pig aortas, endothelial cells in (atheroprotected) regions of
high shear stress have lower expressions of
both pro- and anti-inflammatory genes (13).
Using a similar full-genome approach,
Dekker et al. (14) identified the transcrip-
A)
B)
tion factor Krüppel-like factor 2 (KLF2) to
be specifically induced by laminar shear
stress in endothelial cells and, importantly,
KLF2 was found not to be induced by other
stimuli that govern the endothelial transcriptome. Another study identified nuclear
factor erythroid 2-like 2 (Nrf2) as the shearinduced transcription responsible for antioxidant gene expression (15). Both of these
transcription factors have been shown to be
induced in protected regions of the vasculature in vivo as well (16, 17).
More recently it was shown that KLF2
improves the nuclear localization of Nrf2
and the combined actions of these two factors constitute about 70% of the shear
stress-induced endothelial gene expression
(Fig. 1a) (18).
Fig. 1
70% of the gene sets that
are regulated by shear
stress can be contributed to
the coordinated activation
of Nrf2 and KLF2, which
tips the endothelial balance
to an anticoagulation and
anti-inflammatory state.
A) Shear stress activates the
MEK5/ERK5 MAPK cascade,
leading to an activation of
MEF2 at the promoter of
KLF2. This increases KLF2
mRNA and protein levels
and transcriptional activity.
Shear stress also releases
Nrf2 from Keap1, through
generation of ROS, after
which Nrf2 translocates to
the nucleus, which in turn is
aided by KLF2. Together
these transcription factors
regulate the gene expression of the majority of
shear
stress-regulated
vasoprotective genes.
B) Prolonged
laminar
shear stress induces anticoagulant and antiinflammatory gene expression through the activation of the transcription
factors KLF2 and Nrf2.
Oscillatory shear stress or
short-term shear stress
stimulation induces expression of pro-inflammatory and procoagulant
genes.
Upstream regulation of Nrf2 and KLF2
Nrf2 is activated by shear stress through two
independent mechanisms. Phosphoinositide-3-kinase (PI3K) and Akt signaling improve the nuclear localization of Nrf2,
thereby augmenting its transcriptional activity (16, 19). In the absence of activating
stimuli, Nrf2 is sequestered in the cytoplasm by Keap1 (Kelch-like ECH-associated protein 1), which promotes the degradation of Nrf2 by the proteasome.
Modification by shear stress-induced
electrophiles of cysteine residues in Keap1,
disrupts its ability to target Nrf2 to the proteasome, thereby allowing nuclear accumulation of Nrf2 and antioxidant responsive
(ARE)-dependent gene expression (20). A
pharmacological inducer of Nrf2 activity,
tert-butyl hydroquinone (tBHQ), stabilizes
Nrf2 in a similar manner (21).
Shear stress induces KLF2 by raising
protein levels through transcriptional activation at the KLF2 promoter, as well as
through KLF2 mRNA stabilization (14, 22).
Implicated in the transcriptional activation
at the KLF2 promoter is myocyte enhancer
binding factor 2 (MEF2), which binds to an
evolutionary conserved MEF2-binding site
(23, 24). MEF2 is activated by the upstream
mitogen activated protein kinase (MAPK)
signaling cascade consisting of MAPK kinase 5 (MEK5) and extracellular-signalregulated kinase 5 (ERK5, BMK1) (18).
Cofactors that have been implicated in the
regulation of MEF2 transcriptional activity
at the KLF2 promoter are histone methyl
transferases (HMTs) and histone de-acetylases (HDACs), as well as nucleolin (25).
Both activation of the HMT P300/cAMPresponse element-binding protein-binding
protein-associated factor (PCAF) and nucleolin requires PI3K signaling to enhance
MEF2 transcriptional activity on the KLF2
promoter (26). Recruitment of HDACs 4
and 5, however, have been implicated in the
inhibition of MEF2-dependent KLF2 transcription by NFκB after stimulation with the
inflammatory cytokines IL1 and TNFα
(27).
Pharmacological inducers of KLF2 are
the well-known 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, also
known as statins (22, 28, 29). The mech-
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41
Key transcriptional regulators
anism behind this induction is that statins
inhibit the addition of a crucial geranyl
geranyl pyrophosphate moiety to Rho,
thereby relieving the inhibitory effect of
Rho on the MEK5/ERK5/MEF2 pathway
(29). On the other hand, statins do not stabilize KLF2 mRNA levels like shear stress
stimulation does and therefore the statinmediated induction of KLF2 is not as potent
as the shear stress-mediated induction of
KLF2 in evoking stable eNOS and TM expression, especially in the presence of
TNFα (22).
Knock-out models
Nrf2–/– mice are seemingly normal, but
show a reduced capacity to cope with oxidative insults (30), while Keap1–/– mice die
postnatally due to constitutively active Nrf2
(31). Studies concerning endothelial-specific Nrf2–/– in the context of atherosclerosis
are still lacking. KLF2–/– mice have been
generated more than a decade ago, but these
mice die around embryonic day E13.5 due
to blood vessel malformation (32). More recent results show that lack of endothelial
KLF2 results in improper recruitment of
smooth muscle cells to form a stable blood
vessel, giving rise to a lack of vessel tone
and ultimately leading to cardiac overload
and embryonic lethality (33, 34). InterestTab. 1 Proteins implicated in coagulation and fibrinolysis and their regulation by KLF2: Many of the upregulated
target genes of the shear stress-induced transcription factor
KLF2 are implicated in anticoagulation, while the downregulated genes are implicated in procoagulation and antifibrinolysis.
protein
upregulated
downregulated
references
eNOS
24, 40–42
TM
24, 40, 42
VWF
42
PAI1
24, 40–42
TFPI2
42
IL8
24, 42
TF
24, 40
PAR-1
24, 40
VWF
40
ingly, the upstream MAPKs MEK5 and
ERK5 that induce KLF2 expression have
also been found to be essential for proper
endothelial function (35, 36).
In addition, MEF2C is a critical mediator
in vascular development and MEF2A loss of
function mutations have been found to be associated with cardiovascular disease (37, 38).
The viable hemizygous KLF2 knockout
mouse (KLF2+/–) does not have an apparent
endothelial phenotype, which is probably due
to compensatory upregulation of KLF4 (39).
Still, when crossed to the apolipoprotein
E-deficient background, KLF2+/– do have aggravated atherosclerosis development, possibly through increased foam-cell formation.
Target gene expression and
function
Transcription factors regulate gene expression by directly binding to promoters of
target genes. Nrf2 binds to the antioxidant
response element (ARE) present in the promoters of many antioxidant enzymes like
heme oxygenase 1 (HO1) and NAD(P)H
dehydrogenase quinone 1 (NQO1). These
direct target genes can also affect the expression of other genes, which therefore
constitute indirect targets. In the case of
Nrf2 activation, the direct targets enhance
the antioxidant capacity of the cell, resulting
in less oxidative stress and subsequently in
less inflammation.
Direct targets for KLF2 have also been
identified, even though a consensus KLF2
binding sequence in promoters of target
genes has not yet been identified. The general GC rich KLF binding site 5’-CACC-3’
seems not very specific and can in principle
be bound by all 20-odd KLF family
members, as well as by other transcription
factors such as SP-1. Such GC rich KLF
binding sites are present in most of the
identified direct targets of KLF2, like eNOS
and TM, which are known to be involved in
endothelial homeostasis (40, 41). These two
proteins both have essential functions in
keeping the endothelium anticoagulant and
the shear stress-mediated and also the statin-mediated induction of these proteins was
shown to be dependent on KLF2 expression
(17, 29, 40).
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Boon, Horrevoets
The KLF2-regulated transcriptome
probably contains a large amount of indirect
targets as well, because KLF2 was reported
to regulate the expression of over a thousand
genes (24, 42). Most of the anti-inflammatory effects of KLF2 (apart from direct
eNOS induction) are probably indirect. For
instance, KLF2 was shown to recruit the
essential cofactor cyclic AMP response element-binding protein (CBP/p300) away
from NFκB, thereby inhibiting the transcriptional activity of NFκB, leading to attenuation of inflammatory gene expression
(41).
Another mechanism by which KLF2
confers its anti-inflammatory actions is
through inhibition of nuclear localization of
phosphorylated ATF2, which is essential for
inflammatory gene expression in endothelial cells (6). ATF2 can form a transcriptional complex together with c-Jun, called
AP-1 and c-Jun phosphorylation is also inhibited by KLF2 (43). AP-1 is well-known
to induce pro-inflammatory and procoagulant gene expression, and can be activated
by p38 and JNK MAPK signalling, suggesting that KLF2 interferes with these proinflammatory MAPK pathways. Furthermore, KLF2 inhibits pro-inflammatory
signalling through the thrombin receptor,
and by inhibiting TGF-β signalling, thus potently preventing the expression of antifibrinolytic PAI-1. The latter occurs through a
simultaneous induction of the inhibitory
Smad7 and inhibition of the above-mentioned AP-1, which is an essential co-factor
for TGF-β signalling, leading to a decrease
of Smad4 transcriptional activity and downstream gene expression (43). Other indirect
anti-inflammatory effects of KLF2 are elicited through Nrf2 and its antioxidant
enzyme target genes, as KLF2 improves the
nuclear localization and transcriptional activity of Nrf2 (18).
The changes in expression of many of the
KLF2 modulated genes can be classified as
anti-coagulant and pro-fibrinolytic (Tab. 1).
Especially, the coordinated upregulation of
TM and downregulation of the thrombin receptor (F2R, PAR-1) results in a KLF2-mediated hundred-fold lower affinity for
thrombin (42, 44). As a consequence, von
Willebrand factor (VWF) release from
Weibel-Palade bodies is also markedly re-
duced by KLF2, even though a slight controversy still exists whether KLF2 induces
or represses vWF expression on the mRNA
level (Tab. 1) (40, 42). Other procoagulant
proteins like tissue factor (TF), TF pathway
inhibitor 2 (TFPI2) and IL8 are also
markedly down-regulated by KLF2, as well
as antifibrinolytic proteins like plasminogen activator inhibitor 1 (PAI1). Many
of these anticoagulant genes also contain
AP-1 sites but not an archetypal KLF-site
(CACC), likely indicating indirect modulation by KLF2 (6), while the direct KLF2
targets TM and eNOS do have GC-rich KLF
consensus binding sites (40, 41).
Future directions, conclusion
Endothelium is in a constant balance between a pro- or anti-inflammatory and a proor anticoagulant state. Prolonged laminar
shear stress tips the balance to the antiinflammatory and anticoagulant side
through activation of KLF2 and Nrf2, while
oscillatory shear stress and short shear
stress exposure tips the balance to the proinflammatory and procoagulant state via
NFκB and AP-1 (Fig. 1b). Several key issues are still unresolved.
● It remains to be established whether
ectopic expression or activation of the
antithrombotic and vasoprotective transcription factors KLF2 and Nrf2 in endothelial cells exposed to atheroprone
shear stress would indeed tip the balance
towards an anti-inflammatory state. As
such, this might protect against atherosclerosis development or even arterial
thrombosis.
● Even the reverse experiment, determining if experimental loss of endothelial
Nrf2 and KLF2 leads to aggravation of
atherosclerosis in vivo turned out quite a
challenge. Inducible endothelial-specific
Nrf2–/– or KLF2–/– mice still need to be
developed and crossed into atherosclerosis model mice. Moreover, the non
inducible endothelial-specific KLF2–/–
mouse is embryonic lethal and the inducible specific ERK5–/– mouse dies shortly
after gene ablation, perhaps through loss
of KLF2 expression (34, 45).
●
Since Nrf2 and KLF2 govern approximately 70% of the gene sets that are
regulated by shear stress, which other
factors regulate the expression of the residual 30% of gene sets, and do these affect KLF2 and Nrf2 function? This question is probably not easy to answer and it
is likely not a single factor that is responsible for this remaining part.
In conclusion, for KLF2 and Nrf2 to be used
in anti-thrombosis or anti-atherosclerosis
therapies, specific inducers need to be developed. In principle, for KLF2 these can be
molecules similar to statins and for Nrf2
compounds analogous to tBHQ. Furthermore, activators of the upstream signaling cascades for these transcription factors
could also be used. In the case of KLF2, activators of the MEK5/ERK5/MEF2 signaling cascade might augment KLF2 and inhibit atherogenesis. Still, given the widespread beneficial effects of these key transcription factors on the endothelial phenotype, their targeted modulation might lead
to a new class of cardiovascular drugs.
Conflict of interest
The authors declare that they have no conflicts of
interest.
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Correspondence to:
Anton J. G. Horrevoets
Department of Molecular Cell Biology and Immunology
VU University Medical Center , van der Boechorststraat 7,
1081 BT Amsterdam, The Netherlands
Tel. +31/(0)20/444 81 61/80 80;
Fax +31/(0)20/444 80 81
E-mail: [email protected]
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