Review
This Review is in a thematic series on Cardiovascular Aging, which includes the following articles:
The Intersection between Aging and Cardiovascular Disease [Circ Res. 2012;110:1097–1108]
Mitochondria and Cardiovascular Aging [Circ Res. 2012;110:1109 –1124]
Contribution of Impaired Mitochondrial Autophagy to Cardiac Aging: Mechanisms and Therapeutic Opportunities
[Circ Res. 2012;110:1125–1138]
Growth Factors, Nutrient Signaling, and Cardiovascular Aging [Circ Res. 2012;110:1139 –1150]
Telomeres and Mitochondria in the Aging Heart [Circ Res. 2012;110:1226 –1237]
FOXOs and Sirtuins in Vascular Growth, Maintenance, and Aging
Effects of Aging on Angiogenesis
Non-Mammalian Models of Cardiovascular Aging
David Sinclair & Brian North, Guest Editors
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FOXOs and Sirtuins in Vascular Growth, Maintenance,
and Aging
Mark F. Oellerich, Michael Potente
Abstract: Blood vessels form the first organ in the developing embryo and build extensive networks that supply all
cells with nutrients and oxygen throughout life. As blood vessels get older, they often become abnormal in
structure and function, thereby contributing to numerous age-associated diseases including ischemic heart and
brain disease, neurodegeneration, or cancer. First described as regulators of the aging process in invertebrate
model organisms, Forkhead box “O” (FOXO) transcription factors and sirtuin deacetylases are now emerging
as key regulators of mammalian vascular development and disease. The integration of individual FOXO and
sirtuin family members into various aspects of vessel growth, maintenance, and function provides new
perspectives on disease mechanisms of aging, the most important risk factor for medical maladies of the vascular
system. (Circ Res. 2012;110:1238-1251.)
Key Words: Forkhead box O 䡲 sirtuin 䡲 angiogenesis 䡲 metabolism 䡲 aging 䡲 hypoxia signaling 䡲 vascular biology
B
quate tissue perfusion, thereby causing tissue ischemia. Abnormal vessel growth and function are hallmarks of various
disorders, and they contribute to disease progression. For
instance, insufficient vessel growth or enhanced vessel regression cause ischemic heart and brain disease and can also
lead to neurodegeneration or hypertension.3,4 On the other
hand, excessive vascular growth and remodeling promote
cancer, age-related macular degeneration, and inflammatory
diseases.3,4 Many of the above-mentioned vessel-related pa-
lood vessels form large tubular networks that pervade all
tissues in the body. These hierarchically branched networks of arteries, capillaries, and veins are lined by endothelial cells (ECs), which integrate functionally into organs to
support growth, function, and repair.1–3 As all cells in the
body thrive on nutrients and oxygen supplied by these
networks, proper vessel maintenance is essential for tissue
homeostasis throughout life. However, during lifetime, vessels often become dysfunctional and unable to sustain ade-
Original received September 24, 2011; revision received February 14, 2012; accepted February 14, 2012. In February 2012, the average time from
submission to first decision for all original research papers submitted to Circulation Research was 13.77 days.
From the Vascular Epigenetics Group, Institute of Cardiovascular Regeneration, Center for Molecular Medicine, and the Department of Cardiology,
Internal Medicine III, Goethe University, Frankfurt, Germany.
Correspondence to Michael Potente, MD, Vascular Epigenetics Group, Institute of Cardiovascular Regeneration, Center for Molecular Medicine and
Department of Cardiology, Internal Medicine III, Goethe University, Theodor-Stern-Kai 7, D-60590, Frankfurt, Germany. E-mail
[email protected]
© 2012 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.111.246488
1238
Oellerich and Potente
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thologies are common in the elderly and the incidence of
vascular disease correlates with aging, even in the absence of
risk factors. Current concepts suggest that aging individuals
acquire changes in vascular structure and function that
provide the basis for future vascular disease.5– 8
Aging is not solely a passive degenerative process but is in
fact actively regulated by genetic pathways. This realization has
fueled interest in understanding the molecular basis of aging, as
manipulating these pathways could offer therapeutic opportunities to combat age-related disorders.9,10 In the past decade,
research on aging has identified genes and pathways that affect
longevity. Among these are the Forkhead box “O” (FOXO)
family of forkhead transcription factors as well as nicotinamide
adenine dinucleotide (NAD⫹)-dependent protein deacetylases
termed sirtuins that have been described to prolong lifespan in
worms and flies.9,11–13 Whereas the lifespan-extending effects of
sirtuins have been challenged in recent studies,14 it remains
undisputed that FOXOs and sirtuins coordinate a wide range of
cellular responses that are frequently deregulated during aging
including metabolism, stress resistance, cell cycle progression,
and programmed cell death.15–17 Studies in the vascular system
point to the vascular endothelium as a pivotal target tissue for the
physiological functions of FOXO and sirtuin family members,
where they control multiple facets of vessel development and
function. In this review, we summarize recent insights into the
biology of FOXOs and sirtuins in the vessel wall and discuss
their relevance for age-related vascular changes. We will first
provide a general overview of how blood vessels grow, mature,
and age that will serve as a conceptual framework for the second
part, in which we highlight key functions and prototypic principles of FOXO and sirtuin signaling.
Life of a Blood Vessel
Vessel Development and Growth
The first blood vessels in life form by the differentiation of
mesoderm-derived endothelial precursor cells (angioblasts)
that assemble into a primitive vascular plexus (vasculogenesis).1–3 Continuous endothelial proliferation, migration, and
sprouting expand and remodel this primitive plexus into a
highly organized and ramified network (angiogenesis) that
differentiates into arteries and veins (arterio-venous differentiation)1–3 (Figure 1A). Nascent blood vessels recruit mural
cells (pericytes and vascular smooth muscle cells) that enwrap
EC tubules to provide stability and to enable dynamic tissue
perfusion (Figure 1A). Although angiogenesis is the major mode
of vessel growth, the vasculature can also expand its size and
complexity by other mechanisms. The enlargement and remodeling of preexisting collateral vessels between arterial networks
(arteriogenesis) is essential for revascularization of ischemic
tissues.1,3,4 Moreover, the vascular network can be extended
through the splitting of preexisting vessels in a process called
intussuception.1,3,4 Although debated, circulatory and/or vascular
wall resident (endothelial) precursor cells may also contribute to
vessel formation.1,3,4 In the healthy adult, ECs are mostly
quiescent and angiogenesis occurs only in specific organs (eg,
cycling ovary, placenta during pregnancy). However, ECs retain
a remarkable plasticity to switch from a quiescent to a highly
angiogenic phenotype to promote tissue (re)vascularization.
FOXOs and Sirtuins in ECs
1239
Non-standard Abbreviations and Acronyms
CR
EC
ECM
eNOS
FOXO
HIF
MC
miR
NADⴙ
NO
PHD
PTM
ROS
SIR2
SIRT
VEGF
calorie restriction
endothelial cell
extracellular matrix
endothelial nitric oxide synthase
Forkhead box “O” transcription factor
hypoxia-inducible factor
mural cell
microRNA
nicotinamide adenine dinucleotide
nitric oxide
prolyl hydroxylase domain protein
posttranslational modification
reactive oxygen species
silent information regulator 2
sirtuin
vascular endothelial growth factor
Thus, angiogenesis remains an important instigator for tissue
growth and repair throughout life.
Vessel Maturation and Maintenance
Newly formed vessels must become mature and stable to
support tissue homeostasis. Vessel maturation involves a
gradual shift from actively growing vessels toward a quiescent, fully functional tubular network.1,3 Maturation of nascent vessels entails the suppression of endothelial proliferation, the adoption of survival properties, as well as the
establishment of junctional barriers. Vessels also must recruit
mural cell (MCs), generate extracellular matrix (ECM), and
specialize their ECs, MCs, and matrix to tissue-specific
requirements. In mature vessels, long-lived ECs form a
tightly aligned and flattened monolayer with cobblestone-like
appearance that enables efficient tissue perfusion.
Vessel Remodeling and Regression
Vessels do not only sprout, branch, and elongate but also
regress physiologically (pruning) to match perfusion with
tissue metabolic demands3 (Figure 1A). Blood flow plays a
key role in determining whether vessels regress or persist. In
newly formed sprouts, the onset of blood flow stabilizes
vascular connections by activating genes that keep vessels
quiescent, dilated, and antithrombogenic.3 In contrast, disturbed or abrogated flow causes endothelial retraction and
apoptosis. As a consequence, vessels regress, leaving behind
empty basement membrane sleeves. The removal of angiogenic or survival factors also contributes to vascular regression, particularly in immature vessels.3 Abnormally enhanced
regression causes vascular rarefaction, a characteristic feature
of neurodegenerative and hypertensive disorders.3
Vessel Aging
A hallmark of aging in many organs is a gradual decline in
tissue cellularity and function, leading to an impaired capacity to maintain tissue homeostasis, especially under challenging environmental conditions.10 Aging blood vessels, in
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Figure 1. Mechanisms of angiogenic vessel growth. A, Principal steps of angiogenic vessel growth including sprouting and branching, remodeling and regression, stabilization, and maturation as well as arterio-venous differentiation, illustrated on a postnatal day 5
mouse retina stained for an endothelial marker. A indicates artery; V, vein. B, Characteristics of endothelial tip and stalk cells (left) and
model of tip stalk cell selection by DLL4-NOTCH signaling (right). Tip cells extend filopodia toward the source of the angiogenic signal,
whereas stalk cells proliferate and form a lumen of the emerging vessel sprout. Tip and stalk cell specification is controlled by DLL4NOTCH signaling. VEGF promotes the upregulation of DLL4 in tip cells through activation of VEGF receptor 2 (VEGFR2). DLL4 instructs
neighboring ECs to become stalk cells by activating their NOTCH receptors, which leads to the suppression of tip cell behavior.
particular arteries, also experience a well-documented series
of structural and functional alterations that impede their tissue
nourishing functions.5– 8 With advancing age, EC function
declines, resulting in an attenuation of endotheliumdependent dilatation that promotes vascular stiffness. Agerelated changes also limit the plasticity of ECs to switch from
a quiescent to a rapidly proliferating and migrating phenotype
after injury or ischemia. Endothelial barriers become leaky
and vascular smooth muscle cells penetrate into the subendothelial space, where they divide and produce ECM proteins
that cause intimal thickening. As a result, arteries become
stiffer, thicker, and less able to function. The development of
atherosclerotic lesions in such aged vessels further aggravates
vessel narrowing, thereby causing hypertension, vascular
obstruction, and tissue ischemia in certain vascular beds.5– 8
Molecular Pathways in Aging: Insights From
FOXOs and Sirtuins
Aging and the Metabolism Link
The observation that perturbations in specific genetic pathways can either promote or retard the aging process has
spurred research into the molecular details of aging and on
the mechanisms that link aging with age-related illnesses.9 –11 Progress in understanding the molecular basis of
mammalian aging has been hindered by the complexity of
the aging process, the influence of external factors, as well
as the lack of biomarkers to reliably determine the extent
of aging.10 Much our knowledge about the molecular
regulation of aging stems from the analysis of small,
short-lived model organisms such as yeast, worms, and
flies.9,11 Genetic studies in these model organisms unraveled that many lifespan-extending mutations affect metabolic regulators and control circuits. Examples of such
metabolic regulators include the target of rapamycin
(TOR), PPAR␥ coactivator-1␣ (PGC1␣), AMP-dependent
kinase (AMPK), FOXO transcription factors, and sirtuins.9,11,18 The importance of metabolic regulation for
organismal lifespan is best illustrated by the salutary
effects of calorie restriction (CR), the only intervention to
date that has consistently been shown to increase life
expectancy in species ranging from worms to primates.18,19
In this review, we focus on the biology of FOXOs and
Oellerich and Potente
FOXOs and Sirtuins in ECs
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Figure 2. Regulation of FOXO transcription factors by the PI3K-AKT pathway.
FOXOs control various cellular responses,
ranging from apoptosis, DNA repair, and
metabolism to ROS detoxification and cell
proliferation, by driving the expression of
FOXO target genes. FOXOs are key
downstream effectors of the PI3K-AKT
pathway that blocks FOXO target gene
expression. On stimulation of PI3K AKT
signaling by growth factors, AKT phosphorylates FOXOs on 3 conserved residues, which leads to their cytoplasmic
sequestration and inactivation. Asterisk
denotes the bidirectional regulation of cell
death and DNA repair by FOXOs: depending on the stress signal and posttranslational modification, FOXOs can shift their
transcriptional responses away from apoptosis and toward DNA repair.
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sirtuins and their role in ECs during vascular development,
maintenance, and aging.
FOXOs Control Lifespan in Model Organisms
The first pathway shown to affect aging in model organisms
was the insulin/insulin-like growth factor 1 (IGF1) pathway.9,18 In the nematode Caenorhabditis elegans, mutations
that reduce signaling by the insulin IGF1 receptor (DAF2) or
its downstream effectors phosphatidylinositol-3-kinase
(PI3K) and AKT increase worm lifespan.9,18 Activation of
PI3K-AKT signaling induces AKT-dependent phosphorylation of the transcription factor DAF16, the C. elegans
homolog of FOXO (Figure 2). After phosphorylation by
AKT, DAF16/FOXO becomes transcriptionally inactive and
translocates to the cytoplasm (Figure 2). The increased
lifespan of DAF2 mutants depends, at least in part, on this
transcription factor as deletion of DAF16 abrogates the
longevity phenotype in DAF2 mutant worms.9,18 Activation
of stress-responsive pathways also leads to a DAF16dependent lifespan extension, which requires the interaction
of DAF16 with the sirtuin deacetylase SIR-2.1 and the
adaptor protein 14 to 3–3.20 The lifespan-extending function
of DAF16 FOXO is conserved in more complex organisms
such as the fruit fly Drosophila melanogaster.9,18 Understanding the role of FOXOs in mammalian aging is complicated by the existence of several FOXO isoforms as well as
the severe developmental defects in FOXO mutants mice,
which preclude the analysis of aging phenotypes.9,18
Functions of Mammalian FOXOs
FOXOs belong to the family of forkhead proteins and are
characterized by a highly conserved DNA binding domain
(forkhead box).15,21 By binding to promoters that contain the
FOXO consensus motif TTGTTTAC, FOXOs can act as
transcriptional activators and repressors.15,21 Four FOXO
isoforms have been identified: FOXO1, FOXO3, FOXO4,
and FOXO6. FOXO1, FOXO3, and FOXO4 are ubiquitously
expressed; however, their expression levels are dynamic and
vary between tissues and developmental stages. On the
contrary, expression of FOXO6 is confined to the adult brain.
Studies in FOXO loss-of-function mutant mice revealed
decisive regulatory roles of FOXOs in a great variety of
developmental and physiological programs (Table 1). Global
genetic inactivation of FOXO1 in mice causes embryonic
lethality, as emerging blood vessels are unable to grow
properly.22,23 FOXO3 knockout mice are viable but display
age-dependent infertility due to ovarian follicle activation.22,24 In contrast, FOXO4 knockout mice exhibit no
apparent phenotype under normal conditions.22 FOXOs govern these diverse functions by transcriptionally integrating
signals from key developmental pathways that control tissue
differentiation, growth, and maintenance15,21 Among the prototypical FOXO targets are genes that have pivotal roles in
Table 1.
Gene
FOXO1
Phenotypes of FOXO Mutant Mice
Knockout Phenotype
Reference
Lethal at E10.5 due to deregulated vascular
development: malformed aorta and irregular
intersomitic vessels, lack of properly formed
branches of the internal carotid artery and
vasculature in the yolk sac
Hosaka et al,22
2004; Furuyama
et al,23 2004
Conditional inactivation (Mx-Cre) in adult
mice: liver hemangiomas and angiosarcomas
Paik et al,48
2007
Viable, age-dependent infertility due to global
ovarian follicle activation and early oocyte
depletion
Hosaka et al,22
2004; Castrillon
et al,24 2003
Enhancement of postnatal vessel formation in
models of ischemia-induced vessel growth
Potente et al,44
2005
FOXO4
Viable, indistinguishable from wild-type
littermates
Hosaka et al,22
2004
FOXO1,
FOXO3,
FOXO4
Conditional inactivation (Mx-Cre) in adult
mice
Paik et al,48
2007
FOXO3
Premature mortality due to large
hemangiomas in the uterus, liver, skeletal
muscle
Lymphoblastic thymic lymphomas
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Figure 3. The Sirtuin family of protein deactylases. A, Sirtuins are NAD⫹-dependent deacetylases that target histone and nonhistone proteins
(left upper box) to regulate a wide range of cellular functions such as cellular senescence, survival,
DNA repair, metabolism, and cell cycle progression. The deacetylase reaction catalyzed by sirtuins depends on the cofactor NAD⫹ and generates a deacetylated protein, NAM and 2⬘-O-acetylADP ribose. NAM can be recycled to NAD⫹ by a
salvage pathway that requires the enzyme NAMPT.
B, Sirtuin activity is coupled to metabolism via
NAD⫹ levels, which rise in conditions of nutrient
shortage. Because sirtuins require NAD⫹ for their
catalytic activity, their enzymatic activity is higher
in situations of energy distress.
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cell cycle progression (p21, p27), reactive oxygen species
(ROS) detoxification (MnSOD), programmed cell death
(FAS, BIM), and glucose metabolism (G6PC)15,21 (Figure 2).
The transcriptional output of FOXOs is controlled by a range
of posttranslational modifications (PTMs) that alter transcriptional activity, DNA binding, subcellular localization, and
protein stability. As discussed above, FOXOs are crucial
effectors of the PI3K-AKT signaling branch and are inactivated on AKT-mediated phosphorylation15,21,25 (Figure 2).
Besides AKT, other kinases can phosphorylate and inactivate
FOXOs, including SGK, CK1, DYRK1a, CDK2, ERK, and
IKK.15,21,25 In contrast, phosphorylation of FOXOs by JNK
and the energy-sensor kinase AMPK facilitates FOXO activation.15,21,25 FOXOs are also regulated by reversible acetylation. Depending on the FOXO target gene promoter, acetylation can either promote or diminish FOXO transcriptional
activity.15,21,25,26 Acetylation can also increase FOXO protein
stability by protecting FOXOs from poly-ubiquitination and
degradation. Accordingly, deacetylation of FOXOs by SIRT1
or class II histone deacetylases affects FOXO target gene
expression, subcellular localization, and protein stability.15,21,25–28 Besides SIRT1, other sirtuins similarly regulate
FOXO activity, including SIRT2 and SIRT3.29 –31 FOXO
factors also become methylated and monoubiquitinated/
polyubiquitinated, which leads to their signal-dependent activation or inhibition.21 In addition to PTMs, protein-protein
interactions of FOXOs with SMADs and ␤-catenin play
important roles in specifying FOXO transcriptional
responses.15,21,25
Sirtuins: Sustainers of Tissue Homeostasis
Sirtuins are NAD⫹-dependent deacetylases, which modulate
a wide range of biological processes, spanning from DNA
repair and oxidative stress responses to energy metabolism.16,17,32 A connection between sirtuins and aging was
described more than a decade ago on the basis of studies in
model organisms. Overexpression of the sirtuin member
Silent Information Regulator 2 (SIR2) was reported to extend
lifespan in yeast.32 Subsequent studies in C. elegans and in D.
melanogaster revealed that overexpression of SIR2 orthologues can also promote longevity in these species.32 How-
ever, recent conflicting results about the influence of SIR2 on
lifespan extension in C. elegans and Drosophilia have challenged the view of SIR2 as a lifespan determinant and
kick-started a debate on aspects of sirtuin biology associated
with organismal aging.14,33–35 Beyond controversy, sirtuins
play critical roles in mounting adaptive responses to diverse
forms of cellular stress that fuel tissue degeneration and
age-associated diseases.16,17,32,35 Along these lines, mice
overexpressing SIRT1, the closest SIR2 orthologue, are
protected against age-associated diseases (cancer, diabetes)
and reveal an improved health span but do not live longer.36
These findings suggest that mammalian sirtuins have key
homeostatic functions that delay age-related tissue degeneration but may not control the aging process per se.
In mammals, 7 sirtuins exist (SIRT1–SIRT7).16,17,32 Despite sharing a conserved catalytic domain, sirtuins have
different biological functions and subcellular localizations.
SIRT1, SIRT6, and SIRT7 are nuclear proteins, whereas
SIRT2 is found primarily in the cytosol.16 SIRT3, SIRT4,
and SIRT5 are mitochondrial sirtuins.16 Of note, sirtuins can
shuttle between different subcellular compartments. SIRT1,
for example, is localized in the cytoplasm of certain tissues37
but has also been detected in mitochondria.38 Most sirtuins
exert their biological function through the deacetylation of
acetylated proteins, which comprise histone and nonhistone
proteins16,17,32 (Figure 3A). The deacetylation reaction requires the cofactor NAD⫹ and involves the transfer of an
acetyl group from an acetylated protein substrate to the
ADP-ribose moiety of NAD⫹. This reaction leads to a
deacetylated protein, 2⬘-acetyl-ADP ribose and nicotinamide
(NAM), a noncompetitive inhibitor of sirtuin activity39 (Figure 3A). Many of the physiological functions of sirtuins result
from the adaptation of cell signaling and gene expression to
the energetic state of the cell, which is sensed through NAD⫹
levels.16,17,32 NAD⫹ serves as an electron carrier in cell
metabolism (glycolysis, mitochondrial oxidative phosphorylation), and the levels of NAD⫹ are elevated in situations of
energy distress (Figure 3B). Because sirtuins require NAD⫹
for their catalytic activity, their activity is higher in situations
of nutrient scarcity such as CR.16,17,32
Oellerich and Potente
By removing acetyl groups, sirtuins can either promote or
inhibit the activity of their protein targets.16,17,32 For example,
SIRT1 and SIRT6 can deacetylate specific lysines on histone
tails to promote transcriptional silencing. SIRT1 also deacetylates many nonhistone proteins such as p53, FOXOs,
SMRT/NCOR, and PGC1␣.16,17,32 SIRT3 targets mitochondrial enzymes involved in metabolism, ROS detoxification,
and mitochondrial function including LCAD, IDH2, SOD2,
and cyclophilin D.40,41 Other enzymatic reactions catalyzed
by selected sirtuins are the transfer of an ADP-ribosyl group
from NAD⫹ to an acceptor protein (SIRT1, SIRT4, and
SIRT6)16,17,32 or the demalonylation and desuccinylation of
modified proteins (SIRT5).42 However, the biological relevance of these reactions is only beginning to be unveiled.
FOXOs and Sirtuins in Blood Vessel Growth
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FOXOs Are Essential Regulators of Vascular Growth
Vascular networks arose in evolution as organismal size and
metabolic activity increased in species and outgrew the
capacity to supply all cells with oxygen and nutrients by
diffusion. It is therefore not surprising that the vasculature
constitutes the first organ in mammalian development, whose
adequate function is essential for growth and morphogenesis.
FOXO1 plays an essential role for vascular development.22,23
FOXO1 knockout mice show no apparent defects until
embryonic day E9.0, and endothelial differentiation from
angioblasts and the formation of an initial vascular labyrinth
(vasculogenesis) appear normal.22,23 However, subsequent
angiogenic remodeling into a hierarchically organized network is severely deregulated in FOXO1 mutants, leaving
behind a primitive, dysfunctional vascular plexus that causes
embryonic lethality at E10.5.22,23 At that stage, FOXO1 is
predominantly expressed in the endothelial lining suggesting
an essential cell-autonomous function of FOXO1 in
ECs.22,23,43 Consistent with such a model, FOXO1 overexpression in surrogate assays of angiogenesis inhibits endothelial migration and sprouting, whereas FOXO1 knockdown
increases angiogenic behavior of ECs.44 Similarly, ECs derived from FOXO1-deficient embryonic stem cells display
aberrant morphological responses after vascular endothelial
growth factor (VEGF) stimulation.45 Collectively, these studies suggest that FOXO1 acts as a critical negative regulator of
endothelial angiogenic behavior, whose activity is required to
coordinate vessel morphogenesis.
The observation that FOXO1 restricts endothelial angiogenic behavior might, at first sight, appear paradoxical, as
genetic inactivation of FOXO1 does not enhance vessel
formation but instead impairs vascular development. However, it is crucial to point out that the growth of a functional
vessel network requires the coordination of proangiogenic
and antiangiogenic activities of ECs.2,3,46,47 The current understanding of the sprouting process rests on the realization
that the populations of ECs that compose sprouting vessels
are heterogeneous in their angiogenic behavior and signaling:
When stimulated by proangiogenic signals (eg, VEGF) only a
subset of ECs acquires an explorative phenotype. These so
called “tip cells” are located at the leading end of the vessel
sprout and extend numerous filopodia toward the source of
the angiogenic signal46,47 (Figure 1B). The cells that follow
FOXOs and Sirtuins in ECs
1243
tip cells, termed “stalk cells,” extend less filopodia, and are
less motile but proliferate and define the base of the sprout46
(Figure 1B). Uncoordinated tip and stalk cell behavior causes
malformed and dysfunctional blood vessels that often result
in embryonic lethality.46 Although the precise mechanisms
through which FOXO1 regulates endothelial angiogenic behavior remain unresolved, it is tempting to speculate that
FOXO1 codetermines tip stalk cell specification to enable a
balanced vascular response to proangiogenic signals.
Cooperative Regulation of Vessel Growth by FOXOs
Although FOXO3 and FOXO4 are dispensable for embryonic
vascular development, both of them enhance the growthsuppressive activity of FOXO1 in adult mice.22,24,48 Successive somatic inactivation of FOXO3 and FOXO4, in addition
to FOXO1, results in more pervasive endothelial overproliferation and hemangioma formation in adult mice when
compared with the deletion of FOXO1 alone.48 Further
support for a critical role of FOXOs in vascular growth stems
from the observation that FOXOs are upregulated in ECs of
ischemic muscles and that FOXO3 deficiency enhances
vessel density after ischemic stress in mice.44,49 The cell
responses and target genes controlled by different FOXOs in
ECs are overlapping but not entirely redundant.44,48,50 –52 For
example, overexpression of a constitutively active FOXO1 or
FOXO3 mutant inhibits endothelial sprouting and migration,
whereas forced expression of FOXO4 has no discernible
effect.44 Likewise, whereas FOXO1 and FOXO3 cooperate in
controlling EC responses, overexpression of FOXO3 cannot
restore the abnormal function of FOXO1-deficient ECs.45
Together, these studies suggest that FOXOs function as key
negative regulators of endothelial angiogenic behavior,
whose activity is required for vessel development. Within this
family, FOXO1 is the most potent regulator of vascular
growth, with lesser but physiologically significant contributions from the other FOXO members.
SIRT1 Regulates Revascularization of Ischemic Tissues
Targeted mutations of sirtuins in mice have shown that few
developmental processes are absolutely dependent on sirtuins16,17 (Table 2). A cardinal function of sirtuins is the
adaptation of cellular responses to environmental signals to
maintain tissue homeostasis. Consequently, their functions
become eminent under conditions of stress.16,17 One such
example is the role of SIRT1 in vascular growth. Genetic
inactivation of SIRT1 in ECs of mice is compatible with life
and exhibits no severe vascular defects during embryonic
development. However, SIRT1 deficiency impairs tissue
revascularization in response to ischemia.53 SIRT1 deficiency
causes impaired EC branching and proliferation, leading to
reduced blood vessel density. In zebrafish, knockdown of
SIRT1 compromises intersomitic vessel formation, resulting
in a sparse and misguided vessel network.53 SIRT1 inactivation in embryonic stem cells does not affect endothelial
differentiation but abrogates the ability to form vascular-like
networks.54,55 The abnormal angiogenic behavior of SIRT1deficient ECs is in part caused by an unrestrained activity of
FOXO1. SIRT1 deacetylates endothelial FOXO1, thereby
limiting its antiangiogenic activity.53 Recent studies demonstrate that the Notch pathway is a pivotal target of SIRT1 in
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Table 2.
Phenotypes of Sirtuin Mutant Mice
Gene
Knockout Phenotype
Reference
SIRT1
Most mice die perinatally, smaller in
size, eye abnormalities, bone,
cardiac, and pancreatic defects,
infertility
McBurney et al,134
2003
Impairment of postnatal retinal
angiogenesis and ischemia-induced
neovascularization in endothelialrestricted SIRT1 knockout
mice (Tie2cre; SIRT1flox/⫺)
Cheng et al,135 2003
Potente et al,53 2007
Guarani et al,55
2011
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SIRT2
Developmentally normal, cancer
prone
Kim et al,136 2010
SIRT3
Developmentally normal
Lombard et al,137
2007
SIRT4
Developmentally normal
Haigis et al,16 2010
SIRT5
Developmentally normal
Nakagawa et al,138
2009
SIRT6
Death at 4 wk, premature aging
phenotype (kyphosis, osteopenia,
loss of subcutaneous fat,
lymphopenia), hypoglycemia
SIRT7
Reduced lifespan, premature aging
phenotype (kyphosis, loss of
subcutaneous fat), degenerative
cardiac hypertrophy, and
inflammatory cardiomyopathy
Mostoslavsky
et al,139 2006
Vakhrusheva et al,140
2008
ECs.55 In the vascular system, Notch signaling coordinates
critical steps of vessel morphogenesis such as the specification of tip and stalk cells46,47 (Figure 1B). Tip cells express
higher levels of the activating Notch ligand Delta-like 4
(DLL4), which limits the explorative tip cell behavior in
adjacent stalk cells through binding to the NOTCH1 receptor,
thereby allowing coordinated vessel sprouting and branching46,47 (Figure 1B). In stalk cells, excessive Notch activity is
associated with compromised sprouting, whereas weak Notch
activity promotes vessel sprouting by (re)inducing tip cell
behavior.46,47 By limiting endothelial Notch signaling, SIRT1
modulates tip and stalk behavior.55 The NOTCH1 intracellular domain (NICD), released on NOTCH1 receptor activation, is deacetylated by SIRT1, which causes its destabilization and proteasomal degradation. SIRT1-deficient ECs are
thus sensitized to Notch signaling and preferentially adopt
stalk cell phenotypes. Notably, SIRT1 can inhibit Notch
responses also by epigenetic mechanisms.56 SIRT1 associates
with the demethylase LSD1 in chromatin-associated complexes that repress Notch target gene expression through
histone demethylation and deacetylation. The consequences
of SIRT1 activation on Notch signaling may, however,
depend on the cellular and environmental context. Studies in
aging neuronal cells revealed that SIRT1 can increase Notch
activity indirectly through RXR-dependent regulation of
ADAM10.57
FOXOs and Sirtuins at the Vascular-Metabolic Interface
The ability of blood vessels to adjust nutrient and oxygen
delivery to changing metabolic demands of tissues is vital for
development and homeostasis. It is therefore not surprising
that metabolism and blood vessels reciprocally affect each
others’ function.58 How this vascular-metabolic cross-talk
occurs and how their functions are coordinated is not well
understood. Emerging evidence indicates that key regulators
of cellular bioenergetics play major roles in vessel formation,
particularly in situations of ischemia. For example, the
metabolic master regulator PGC1␣ not only boosts oxidative
metabolism but also augments ischemia-induced blood vessel
growth through an HIF-independent induction of VEGF.59
Other examples of angiogenesis-boosting regulators of metabolism are AMPK, LKB1, and SIRT1.58 AMPK and its
upstream activating kinase LKB1 similarly promote ischemia-induced vessel formation by controlling VEGF levels,
whereas SIRT1 restrains the antiangiogenic activity of
FOXO1 through deacetylation.53,60 – 62 FOXOs themselves
appear to coregulate metabolism and angiogenesis toward
stress tolerance and cell protection.15 FOXOs are key effectors of insulin action in metabolic processes, including
hepatic glucose production.15 In the liver, FOXO1 promotes
transcription of glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase (PCK1), the rate-limiting
enzymes in hepatic glycolysis and gluconeogenesis, respectively.15 In situations of energy distress, FOXO1 is upregulated and more active, thereby contributing to the maintenance of blood glucose levels.15 Under such conditions, the
restriction of angiogenesis by FOXOs might help to redirect
glucose and nutrient supply to tissues that are indispensable
for life.
Intriguingly, many of the above-mentioned “vascular-metabolic regulators” are controlled by acetylation. Indeed,
PGC1␣, LKB1, and FOXOs are acetylated proteins that are
deacetylated by SIRT1 in many tissues including ECs.
Metabolism itself codetermines protein acetylation as the
metabolic flux through glycolytic pathways and the tricarboxylic acid cycle supplies the acetyl-groups (acetyl-CoA)
required for acetylation reactions.63 These observations raise
the possibility that changes in lysine acetylation provide an
important regulatory mechanism in the cross-talk of angiogenesis and metabolism signaling. By deacetylating nodal regulators of angiogenesis and metabolism, SIRT1 may act as an
integrator of vascular-metabolic signaling to support tissue
revascularization in situations of nutrient and oxygen scarcity.
FOXOs and Sirtuins in Vascular Maintenance
and Homeostasis
In healthy adults, most vessels are quiescent, forming stable
conduits with a streamlined inner surface.3,4 Genetic studies
in adult mice revealed that FOXOs are essential regulators of
vascular quiescence48 (Figure 4). Conditional mutant mice
that globally lack FOXO1, FOXO3, and FOXO4 spontaneously develop massive EC overproliferation that leads to
widespread hemangiomas, hemorrhage, and premature death
of the animals.48 These findings suggest that ECs are exquisitely sensitive to alterations in FOXO signaling and that
adequate FOXO activity is required to keep endothelial
quiescence. FOXOs control endothelial quiescence by governing the expression of genes with key roles in endothelial
growth and morphogenesis including the general tyrosine
kinase inhibitor SPROUTY2.48
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Figure 4. Cellular functions controlled by FOXO1 in ECs. The
scheme shows the cellular functions controlled by FOXO1 in
ECs including the regulation of vessel growth, maintenance (survival, quiescence, barrier function), and remodeling. FOXO1
activity is negatively regulated by VEGF, ANG1, and shear
stress through activation of PI3K-AKT signaling. SIRT1 can also
limit the antiangiogenic effects of FOXO1 in ECs through FOXO1
deacetylation. Depending on the target gene promoter, SIRT1
can promote or repress FOXO-dependent gene expression.
In mature vessels, ECs have to form barriers between the
blood and the surrounding tissue to control the exchange of
fluids and to regulate the entry of immune cells. Essential for
this barrier function is the ability of ECs to regulate cell-cell
adhesions between themselves and neighboring cells.64 Several transmembrane adhesive proteins have been identified
including vascular endothelial (VE) cadherin at adherens
junctions (AJs), and claudin-5 at tight junctions (TJs).64 The
formation, maintenance and remodeling of these intercellular
contacts requires a functional interaction between AJs and
TJs. Interestingly, this interaction involves FOXO1 which has
been shown to repress claudin-5 expression in a ␤-catenin–
dependent manner65 (Figure 4). This repressive effect is
antagonized by VE-cadherin–mediated EC adhesion, which
promotes nuclear exclusion of FOXO1 via activation of the
PI3K pathway.
ECs must adopt survival properties in quiescent vessels to
maintain the integrity of the inner vessel lining. In addition to
hemodynamic forces, autocrine and paracrine survival signals
from ECs and MCs protect the vessel from environmental
stress signals. The PI3K-AKT pathway is a pivotal endothelial survival pathway that inhibits apoptosis in part by
blocking the expression of proapoptotic FOXO target genes
(Figure 4). Endothelial survival cues such as angiopoietin-1
(ANG1), VEGF, or shear stress inactivate FOXOs through
activation of the PI3K-AKT pathway, thereby blocking the
expression of FOXO target genes involved in apoptosis
(TRAIL, SURVIVIN) and vessel destabilization (MMP7,
ANG2).44,50,51,66 –70
FOXOs and Sirtuins Are Controlled by Blood Flow
Blood vessels are constantly subjected to hemodynamic
forces induced by pulsatile blood pressure and flow. ECs are
in direct contact with the flowing blood and bear most of the
hemodynamic forces, which in turn also modulate their
functions.71 For instance, laminar flow upregulates genes that
protect vessels from atherosclerosis and keeps them quies-
FOXOs and Sirtuins in ECs
1245
cent, dilated and free of clots.71 One of the key transcription
factors that govern the expression of these flow-responsive
genes is Krüppel-like factor 2 (KLF2), which induces vasodilatory, anti-inflammatory, and anticoagulant proteins such
as endothelial nitric oxide synthase (eNOS) and thrombomodulin.72 Recent studies suggest that KLF2 is regulated by
SIRT1.73 Treatment of ECs with resveratrol, a polyphenol
compound that (indirectly) activates SIRT1, enhances the
expression of KLF2 and its target genes by promoting
MEK5/MEF2 signaling. Remarkably, SIRT1 expression itself is enhanced by flow resulting in increased SIRT1mediated deacetylation and activation of eNOS.74 By synergistically modulating KLF2 and its effector eNOS, SIRT1 is
able to potently protect vessels from inflammatory and
procoagulant stress signals (Figure 5).
Emerging evidence indicates that FOXOs are an integral
component of the signaling network that transduces the
endothelial response to flow68 –70 (Figure 4). Studies in
cultured ECs revealed that laminar flow prompts PI3K-AKTdependent inactivation of FOXO1.68,69,70 AMPK has also
been implicated in flow-mediated inhibition of FOXO1.68
Whether this inhibitory effect requires the direct phosphorylation of FOXO1 by AMPK must be determined.
Sirtuins and the Response to Hypoxia
In resting vessels, ECs acquire barrier functions and rarely
migrate or proliferate to enable efficient tissue perfusion. This
phenotype with cobblestone-like appearance has been termed
“phalanx” phenotype because of its resemblance to the
ancient Greek military formation.75 The phalanx phenotype is
regulated by prolyl hydroxylase domain protein 2 (PHD2), an
oxygen sensor that controls signaling by hypoxia-inducible
factors (HIFs).75 Three HIF isoforms exist (HIF-1␣, HIF-2␣,
FIH), which orchestrate adaptive responses to changes in
oxygen tension by regulating the expression of genes involved in cell survival, metabolism, and angiogenesis.76 In
normoxia, HIFs are hydroxylated by PHDs in an oxygendependent manner, which primes HIFs for proteasomal degradation. Under hypoxic conditions PHDs become progressively inactive allowing HIFs to escape degradation.76 Partial
inactivation of PHD2 streamlines the abnormal endothelium
in tumor vessels that otherwise impairs perfusion.75 This
endothelial normalization toward the phalanx phenotype depends, in part, on the HIF-2␣–mediated induction of the
VEGF decoy receptor VEGFR1 (inhibiting EC sprouting)
and the junctional protein VE-cadherin (stabilizing vessels).
Intriguingly, PHD2 and HIF-2␣ are regulated by SIRT1.77,78
SIRT1 activates HIF-2␣ through deacetylation, while promoting PHD2 degradation, together resulting in enhanced
HIF-2␣ signaling (Figure 6). Since SIRT1 expression and
activity increase during hypoxia in an HIF-dependent manner,79 SIRT1 activation might favor an endothelial phenotypic shift toward a phalanx-like phenotype in hypoxic
conditions. Whether such a cooperative regulation of HIF
signaling is operational in ECs remains to be elucidated.
However, given the well-established function of SIRT1 as a
sensor of cellular metabolism,63,80,81 it is tempting to speculate that SIRT1 integrates metabolic and hypoxia signaling to
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April 27, 2012
Figure 5. Roles and targets of SIRT1 in
blood vessels. Schematic illustration of
the regulatory circuits and downstream
effector pathways of SIRT1 in ECs. In the
nucleus, SIRT1 binds and deacetylates
several transcriptional (co)factors, including p53, FOXO, NF␬B, and NICD to control their transcriptional activity and protein stability. SIRT1 also targets histone
proteins for deacetylation (histone H3 on
the p66SHC promoter) to control gene
expression epigenetically. In the cytoplasm, SIRT1 deacetylates enzymes such
as LKB1 and eNOS. Expression of SIRT1
is enhanced by eNOS-derived NO and
downmodulated by the microRNAs
miR-34 and miR-217. TF indicates transcription factor.
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coordinate the vascular response to oxygen- and
nutrient-deprivation.
Several other sirtuins have been shown to interfere with
HIF signaling76 (Figure 6). For example, the chromatinassociated deacetylase SIRT6 acts as a corepressor of HIF-1␣
by deacetylating histones at HIF-1␣–responsive promoters.82
The mitochondrial-localized sirtuin, SIRT3, attenuates
HIF-1␣ activity indirectly by controlling intracellular ROS
Figure 6. Regulation of HIF signaling by sirtuins. In normoxia,
PHDs hydroxylate and inactivate HIFs. Hypoxia inhibits PHD
activity, leading to HIF-1␣ and HIF-2␣ stabilization. HIF-1␣ and
HIF-2␣ form heterodimers with HIF-1ß to control a hypoxiaregulated transcriptional program that affects cell survival,
angiogenesis, and metabolism. Sirtuins control HIF signaling at
multiple levels: SIRT1 inhibits HIF-1␣ while activating HIF-2␣
through deacetylation. By promoting PHD2 degradation, SIRT1
facilitates signaling through the HIF-2␣ pathway. SIRT6 associates with HIF-1␣ to act as a transcriptional corepressor of
HIF-1␣ via H3K9 deacetylation. SIRT3 lowers HIF-1␣ signaling
indirectly by promoting HIF-1␣ degradation in a ROS-dependent
manner.
levels.83 Unlike HIF-2␣, HIF-1␣ activity is inhibited by
SIRT1 in a deacetylation-dependent manner.84 Together,
these results suggest a central regulatory function of sirtuins
in the cellular response to hypoxia.
FOXOs and Sirtuins in the Aging Vasculature
Aging blood vessels become thicker, stiffer, and less plastic,
resulting in a reduced ability to adjust vessel shape and
function to changing tissue demands. Hallmarks of vascular
aging are endothelial senescence and inflammation that compromise vasomotor function as well as vessel growth and
repair. Underlying these hallmarks are increasing oxidative
stress, telomere shortening, DNA damage, and mitochondrial
dysfunction, which set in motion a detrimental cycle of
endothelial damage. Although it is not clear whether FOXOs
and sirtuins control vascular aging directly, it is an intriguing
possibility that alterations in FOXO and sirtuin signaling
pathways contribute to the functional decline of aged blood
vessels.
Endothelial Aging Phenotypes Are Regulated by Sirtuins
and FOXOs
Senescence, particularly in the endothelial lining, is intimately linked to many of the age-associated phenotypes in
the vascular wall. Senescence is a stress response characterized by an inhibition of cell proliferation, which often
becomes irreversible and independent of the initiating stress
signal.85 Senescence occurs in most cell types after prolonged
propagation in culture and is a consequence of telomere
attrition after repeated cell divisions.85 In addition to telomere
shortening, stressors such as genomic instability, DNA damage, and oxidative stress cause a similar cell cycle arrest and
trigger senescence.85 Senescent cells exhibit alterations in
morphology and gene expression that extinguish essential
cellular functions. In ECs, these alterations evoke a phenotype that boosts inflammation, thrombosis, and atheroscle-
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rotic lesion formation while weakening the vasodilatory,
angiogenic, and regenerative properties of ECs.5,6
Several studies have demonstrated that SIRT1 prevents the
onset of senescence in multiple cell types by interfering with
senescence-promoting stress signaling. For instance, SIRT1
inhibits telomere erosion (by interacting with telomere repeats),86 reduces the production of ROS (by regulating
MnSOD through FOXO3), and promotes DNA repair (by
activating NBS1 and WRN helicase).86 – 88 In ECs, forced
expression of SIRT1 prevents oxidative stress–induced premature senescence, whereas SIRT1 inhibition provokes
senescence-like phenotypes.89 These effects of SIRT1 in ECs
depend on its ability to inhibit p53, a major cellular stress
sensor that induces growth arrest and senescence in response
to telomere dysfunction, DNA damage, and oxidative stress89
(Figure 5). Moreover, SIRT1 safeguards ECs from senescence by preventing prolonged LKB1 AMPK signaling
through LKB1 deacetylation and degradation.90 Interestingly,
AMPK enhances SIRT1 activity by elevating NAD⫹ levels,91,92 suggesting that LKB1 AMPK and SIRT1 engage in a
negative feedback loop that prevents sustained LKB1 AMPK
activation (Figure 5). Recent studies also illustrated that
SIRT1 suppresses the expression of p66SHC, an adaptor
protein whose inactivation protects mice from agingassociated vascular disease.93 SIRT1 associates with the
p66SHC promoter to reduce histone H3 acetylation and to
block p66SHC transcription (Figure 5).
Other SIRT1 targets known to affect ROS production,
DNA repair, and stress resistance might similarly contribute
to the protective effects of SIRT1 in ECs. In fibroblasts,
SIRT1 binds to FOXO3 in oxidative stress conditions to shift
its transcriptional responses away from apoptosis and toward
stress resistance.94 FOXO3 controls the expression of genes
involved in antioxidant defense (MnSOD, catalase, sestrins,
and selenoprotein P) and DNA repair (GADD45a), thereby
opposing the age-promoting effects of ROS.94 –96 In ECs,
FOXO3 also transactivates ROS-scavenging enzymes such as
MnSOD through interacting with PGC1␣.97 Remarkably,
FOXO3 itself appears to be a determinant of organismal
aging, as single-nucleotide polymorphisms in the FOXO3
gene have been associated with longevity in humans.98 –101
These single-nucleotide polymorphisms lie within in an
intronic site of the FOXO3 gene that is coregulated by p53.102
FOXO3 and p53 intersect at several levels. FOXO3 can bind
to p53 and promote p53 stabilization and activation.103,104,105
p53, in turn, can inhibit FOXO3 transcriptional activity in
conditions of oxidative stress and prompt FOXO3 degradation.106,107 Together, these considerations suggest that
FOXO3 is part of a transcriptional network that has pivotal
roles during aging.
Nitric Oxide Signaling in Aging Vessels Is Regulated
by SIRT1
When ECs age, levels of endothelial-derived nitric oxide
(NO) diminish. This decline in NO availability is a consequence of an impaired eNOS activity as well as of a
progressive NO inactivation through increasing levels of
superoxide anions.108,109 As a result, aged vessels exhibit
compromised vasodilatory properties that cause increased
FOXOs and Sirtuins in ECs
1247
vascular resistance and impaired perfusion. Moreover, reduced NO bioavailability prompts vascular inflammation and
atherogenesis and impedes endothelial repair.7 ECs therefore
must be equipped with a molecular machinery to maintain
proper eNOS function. Not surprisingly, given the important
antisenescent effects of SIRT1 in ECs, SIRT1 is part of this
machinery (Figure 5). Indeed, SIRT1 has been shown to
deacetylate eNOS, thereby augmenting NO production.110
Notably, the reverse regulation is also true with NO modulating SIRT1. eNOS-derived NO increases on CR and augments SIRT1 expression indicating that SIRT1 and eNOS
form a signaling circuit to preserve EC function.111
Regulation of FOXOs and Sirtuins in Aging
Blood Vessels
The vital functions of (selected) FOXO and sirtuin members
in the vascular wall imply that deregulation of their expression and/or activity could accentuate or even trigger aging
phenotypes. An example is the modulation of SIRT1 expression by microRNAs (miRs). miR-217 expression is progressively enhanced in aging ECs, in which it promotes senescence and impairs angiogenesis signaling.112 These effects
are mediated by a miR-217– dependent downregulation of
SIRT1 (Figure 5). Another miR that targets SIRT1 is miR-34,
which induces apoptosis and endothelial senescence in a
SIRT1-dependent manner113,114 (Figure 5). SIRT1 expression
is also extensively regulated by several transcriptional complexes (HIC1, CtBP, p53, E2F), and its activity is modulated
by posttranslational modifications (phosphorylation, sumoylation) and through protein-protein interactions (DBC1,
AROS).16,17 Whether these mechanisms modulate SIRT1
during aging remains to be elucidated.
Changes in FOXO activity might similarly contribute to
endothelial aging. In healthy vessels, FOXO activity must be
balanced and tightly controlled. Indeed, ECs must maintain a
certain level of FOXO signaling to keep quiescence, while at
the same time prevent exceeding FOXO activation that
causes apoptosis. Stress-induced changes in the extent and
dynamics of FOXO signaling might thus shift EC responses
toward apoptosis and vessel dysfunction. In this context, it is
interesting to note that FOXOs are strongly regulated by
oxidative stress, which is considered to be a main driver of
the aging process. Oxidative stress can alter FOXO activity
through a multitude of PTMs including phosphorylation (by
JNK, ERK), acetylation (p300, CBP, SIRT1), and ubiquitylation (MDM2, SKP2).115 Furthermore, FOXOs can be directly oxidized on cysteine residues under oxidative stress
conditions, thereby altering their acetylation status and transcriptional activity.116
SIRT1 in Atherosclerotic Disease
Atherosclerosis is a chronic, age-associated disease of the
arterial wall that involves complex cell-cell interactions and
alterations in cholesterol homeostasis that leads to vessel
narrowing and ischemia.117 Studies in mouse models of
atherosclerosis have suggested that SIRT1 acts as a potent
antiatherogenic factor. Transgenic overexpression in ECs or
pharmacological activation by resveratrol significantly reduced atherosclerotic plaque formation.118,119 SIRT1 counteracts atherogenesis by cell-autonomous and nonautonomous
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effects. In ECs, SIRT1-mediated activation of eNOS110 and
inhibition of proinflammatory NF␬B signaling120 reduce the
susceptibility for atherosclerotic lesion formation110,120 (Figure 5). Cell-nonautonomous effects of SIRT1 on atherogenesis include effects on glucose and lipid metabolism. SIRT1
improves glucose metabolism and insulin resistance in type 2
diabetes, another pivotal risk factor for atherosclerosis.
SIRT1 increases insulin sensitivity through repression of the
tyrosine phosphatase PTP1B121–125 and promotes insulin secretion in pancreatic ␤ cells by suppression of UCP2.126
Moreover, SIRT1 facilitates cholesterol clearance from the
vessel wall by deacetylating the nuclear receptors LXR127 and
FXR,128 2 key regulators of reverse cholesterol transport.
Contrary to these findings, a recent study reported a
proatherogenic lipid profile in mice that ubiquitously overexpress SIRT1, which fueled atherosclerotic lesion formation.129 Thus, SIRT1 has pleiotropic effects on atherosclerosis
and may affect atherosclerotic plaque formation in a tissueand context-dependent manner. The potential impact of
FOXOs on atherosclerosis remains enigmatic, as their role in
this process has not been specifically investigated.
Dietary Restriction, Vascular Health, and the Connection
to Sirtuins and FOXOs
In addition to genetics, environmental factors play major
roles in determining whether vessels remain healthy and
plastic or become diseased and age prematurely. A prime
example for the environmental influence on vascular health is
nutrition. Although overeating calories is an eminent risk
factor for almost all vascular diseases, restricting calorie
intake is beneficial and prevents many age-related diseases
including hypertension, atherosclerosis and ischemic heart
disease. CR, a dietary regime of reduced food intake without
malnutrition, leads to an increase in life and health span in a
broad range of species, including yeast, flies, worms, fish,
and rodents.18,130 Many of these favorable effects also apply
to nonhuman primates and humans. A study in rhesus
monkeys revealed that long-term CR delays mortality and the
onset of diabetes, cardiovascular disease, cancer, and neurodegeneration.131 How CR extends lifespan is still not well
understood, but several studies point to the importance of
nutrient-sensing proteins pathways including AMPK, mTOR,
and sirtuins as well as the insulin pathway.16,18,130 Studies in
various organisms including mice have shown that CRinduced lifespan extension and health improvement depend in
part on the presence of sirtuins. SIRT1 is upregulated in
response to CR in both mice and humans, and enforced
SIRT1 expression mimics beneficial effects of CR.16,18,132
Besides sirtuins, FOXOs mediate cellular responses to CR.
By acting as downstream effectors of the insulin and AMPK
pathway, FOXOs drive the expression of stress resistance
genes and adapt metabolism to nutrient scarcity.18,130
made it challenging to generate a unifying model of how
FOXOs operate. Existing evidence suggests that ECs reiteratively utilize FOXO signaling at various stages of development and even in the adult, to control endothelial growth,
morphogenesis, and survival. How these diverse functions are
coordinated and how specificity is achieved cannot be easily
reconciled at this point. However, an intriguing possibility is
that combinations of PTMs specify FOXO responses by
determining selective cofactor recruitment and target gene
selection.25 Defining such a “molecular code” of FOXO
regulation will provide insights into how FOXOs respond to
a combination of environmental signals they encounter and
will aid the understanding of signal integration during vessel
growth and maintenance.
The numerous proteins deacetylated by SIRT1 also pose
hurdles to the identification of those targets that are relevant
for SIRT1’s effects on blood vessels. The ever-growing list of
SIRT1-deacetylated proteins suggests that SIRT1 does not
exert its function through profound regulation of a single
target but rather through modulation of multiple targets that
sometimes act at different levels of a pathway. In this sense,
SIRT1 appears to act as a “fine tuner” that adjusts the activity
of pathways that are inappropriate for a particular environmental setting. Surprisingly, the role of other sirtuins in
vascular physiology has not been investigated thus far. As
highlighted above, a number of sirtuins have emerged as key
regulators of metabolism and stress resistance and are likely
to have shared functions in the vasculature as well. Examples
include SIRT3, a mitochondrial sirtuin associated with the
development of metabolic syndrome133 as well as SIRT6, a
chromatin-associated sirtuin, which transmits its signals
through histone deacetylation.82 Identifying their roles in
normal and diseased blood vessels will provide new perspectives on the emerging cross-talk between vessels and metabolism, and we expect rapid progress in this field of research
in the coming years.
Based on the above outlined findings, the implications for
numerous angiogenesis- and vascular-related pathologies,
including ischemic heart and brain disorders, blinding eye
disease, and cancer, are apparent, and future studies on the
roles of FOXOs and sirtuins in these disease settings are
warranted. These studies also must address whether abnormal
FOXO and/or sirtuin signaling is a cause or consequence of
aging and how such dysregulation is linked to the onset and
progression of the above-mentioned age-related maladies.
Finally, a better understanding of FOXO and sirtuin biology
will undoubtedly aid the development of strategies to target
FOXOs and sirtuins therapeutically in the context of proangiogenic and antiangiogenic medicine.
Acknowledgments
Conclusions and Perspectives
We apologize to those authors whose work could not be referenced
because of space limitations.
Despite advances in understanding the physiological roles of
FOXOs and sirtuins in vascular development and disease, our
knowledge of how individual FOXOs and sirtuins function is
far from complete, and numerous questions remain. For
instance, the many demonstrated roles of FOXOs in ECs have
The research of M.P. is supported by the Deutsche Forschungsgemeinschaft (SFB 834), the Excellence Cluster Cardiopulmonary
System (EXC 147/1) and the Cluster of Excellence Macromolecular
Complexes (EXC115).
Sources of Funding
Oellerich and Potente
Disclosures
None.
28.
References
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
1. Adams RH, Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol. 2007;8:464 – 478.
2. Herbert SP, Stainier DY. Molecular control of endothelial cell behaviour
during blood vessel morphogenesis. Nat Rev Mol Cell Biol. 2011;12:
551–564.
3. Potente M, Gerhardt H, Carmeliet P. Basic and therapeutic aspects of
angiogenesis. Cell. 2011;146:873– 887.
4. Carmeliet P, Jain RK. Molecular mechanisms and clinical applications
of angiogenesis. Nature. 2011;473:298 –307.
5. Brandes RP, Fleming I, Busse R. Endothelial aging. Cardiovasc Res.
2005;66:286 –294.
6. Minamino T, Komuro I. Vascular aging: insights from studies on
cellular senescence, stem cell aging, and progeroid syndromes. Nat Clin
Pract Cardiovasc Med. 2008;5:637– 648.
7. Ungvari Z, Kaley G, de Cabo R, Sonntag WE, Csiszar A. Mechanisms
of vascular aging: new perspectives. J Gerontol A Biol Sci Med Sci.
2010;65:1028 –1041.
8. Wang M, Monticone RE, Lakatta EG. Arterial aging: a journey into
subclinical arterial disease. Curr Opin Nephrol Hypertens. 2010;19:
201–207.
9. Kenyon CJ. The genetics of ageing. Nature. 2010;464:504 –512.
10. Sahin E, Depinho RA. Linking functional decline of telomeres, mitochondria and stem cells during ageing. Nature. 2010;464:520 –528.
11. Greer EL, Brunet A. Signaling networks in aging. J Cell Sci. 2008;121:
407– 412.
12. Bauer JH, Morris SN, Chang C, Flatt T, Wood JG, Helfand SL. dSir2
and Dmp53 interact to mediate aspects of CR-dependent lifespan
extension in D. melanogaster. Aging (Albany NY). 2009;1:38 – 48.
13. Rizki G, Iwata TN, Li J, Riedel CG, Picard CL, Jan M, Murphy CT, Lee
SS. The evolutionarily conserved longevity determinants HCF-1 and
SIR-2.1/SIRT1 collaborate to regulate DAF-16/FOXO. PLoS Genet.
2011;7:e1002235.
14. Burnett C, Valentini S, Cabreiro F, et al. Absence of effects of Sir2
overexpression on lifespan in C. elegans and drosophila. Nature. 2011;
477:482– 485.
15. Salih DA, Brunet A. FOXO transcription factors in the maintenance of
cellular homeostasis during aging. Curr Opin Cell Biol. 2008;20:
126 –136.
16. Haigis MC, Sinclair DA. Mammalian sirtuins: biological insights and
disease relevance. Annu Rev Pathol. 2010;5:253–295.
17. Finkel T, Deng CX, Mostoslavsky R. Recent progress in the biology and
physiology of sirtuins. Nature. 2009;460:587–591.
18. Houtkooper RH, Williams RW, Auwerx J. Metabolic networks of longevity. Cell. 2010;142:9 –14.
19. Bishop NA, Guarente L. Genetic links between diet and lifespan: shared
mechanisms from yeast to humans. Nat Rev Genet. 2007;8:835– 844.
20. Berdichevsky A, Viswanathan M, Horvitz HR, Guarente L. C. elegans
Sir-2.1 interacts with 14 –3-3 proteins to activate Daf-16 and extend life
span. Cell. 2006;125:1165–1177.
21. van den Berg MC, Burgering BM. Integrating opposing signals toward
forkhead box O. Antioxid Redox Signal. 2011;14:607– 621.
22. Hosaka T, Biggs WH III, Tieu D, Boyer AD, Varki NM, Cavenee WK,
Arden KC. Disruption of forkhead transcription factor (FOXO) family
members in mice reveals their functional diversification. Proc Natl Acad
Sci U S A. 2004;101:2975–2980.
23. Furuyama T, Kitayama K, Shimoda Y, Ogawa M, Sone K, Yoshida-Araki
K, Hisatsune H, Nishikawa S, Nakayama K, Ikeda K, Motoyama N, Mori
N. Abnormal angiogenesis in FOXO1 (Fkhr)-deficient mice. J Biol Chem.
2004;279:34741–34749.
24. Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA. Suppression of ovarian follicle activation in mice by the transcription factor
FOXO3a. Science. 2003;301:215–218.
25. Calnan DR, Brunet A. The FOXO code. Oncogene. 2008;27:2276–2288.
26. Banks AS, Kim-Muller JY, Mastracci TL, Kofler NM, Qiang L,
Haeusler RA, Jurczak MJ, Laznik D, Heinrich G, Samuel VT, Shulman
GI, Papaioannou VE, Accili D. Dissociation of the glucose and lipid
regulatory functions of FOXO1 by targeted knockin of acetylationdefective alleles in mice. Cell Metab. 2011;14:587–597.
27. Mihaylova MM, Vasquez DS, Ravnskjaer K, Denechaud PD, Yu RT,
Alvarez JG, Downes M, Evans RM, Montminy M, Shaw RJ. Class IIa
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
FOXOs and Sirtuins in ECs
1249
histone deacetylases are hormone-activated regulators of FOXO and
mammalian glucose homeostasis. Cell. 2011;145:607– 621.
Wang B, Moya N, Niessen S, Hoover H, Mihaylova MM, Shaw RJ,
Yates JR III, Fischer WH, Thomas JB, Montminy M. A hormonedependent module regulating energy balance. Cell. 2011;145:596 – 606.
Zhao Y, Yang J, Liao W, Liu X, Zhang H, Wang S, Wang D, Feng J, Yu
L, Zhu WG. Cytosolic FOXO1 is essential for the induction of autophagy
and tumour suppressor activity. Nat Cell Biol. 2010;12:665–675.
Wang F, Nguyen M, Qin FX, Tong Q. SIRT2 deacetylates FOXO3a in
response to oxidative stress and caloric restriction. Aging Cell. 2007;6:
505–514.
Sundaresan NR, Gupta M, Kim G, Rajamohan SB, Isbatan A, Gupta
MP. SIRT3 blocks the cardiac hypertrophic response by augmenting
FOXO3a-dependent antioxidant defense mechanisms in mice. J Clin
Invest. 2009;119:2758 –2771.
Guarente L. Franklin h. Epstein lecture: sirtuins, aging, and medicine.
N Engl J Med. 2011;364:2235–2244.
Viswanathan M, Guarente L. Regulation of Caenorhabditis elegans
lifespan by SIR-2.1 transgenes. Nature. 2011;477:E1–E2.
Kaeberlein M, Powers RW III. Sir2 and calorie restriction in yeast: a
skeptical perspective. Ageing Res Rev. 2007;6:128 –140.
Lombard DB, Pletcher SD, Canto C, Auwerx J. Ageing: Longevity hits
a roadblock. Nature. 2011;477:410 – 411.
Herranz D, Munoz-Martin M, Canamero M, Mulero F, Martinez-Pastor B,
Fernandez-Capetillo O, Serrano M. SIRT1 improves healthy ageing and
protects from metabolic syndrome-associated cancer. Nat Commun.
2010;1:3.
Tanno M, Sakamoto J, Miura T, Shimamoto K, Horio Y. Nucleocytoplasmic shuttling of the NAD⫹-dependent histone deacetylase SIRT1.
J Biol Chem. 2007;282:6823– 6832.
Aquilano K, Vigilanza P, Baldelli S, Pagliei B, Rotilio G, Ciriolo MR.
Peroxisome proliferator-activated receptor gamma co-activator 1alpha
(PGC-1alpha) and sirtuin 1 (SIRT1) reside in mitochondria: possible
direct function in mitochondrial biogenesis. J Biol Chem. 2010;285:
21590 –21599.
Bitterman KJ, Anderson RM, Cohen HY, Latorre-Esteves M, Sinclair
DA. Inhibition of silencing and accelerated aging by nicotinamide, a
putative negative regulator of yeast Sir2 and human SIRT1. J Biol
Chem. 2002;277:45099 – 45107.
Zhong L, Mostoslavsky R. Fine tuning our cellular factories: sirtuins in
mitochondrial biology. Cell Metab. 2011;13:621– 626.
Hafner AV, Dai J, Gomes AP, Xiao CY, Palmeira CM, Rosenzweig A,
Sinclair DA. Regulation of the mPTP by SIRT3-mediated deacetylation
of CypD at lysine 166 suppresses age-related cardiac hypertrophy. Aging
(Albany NY). 2010;2:914 –923.
Du J, Zhou Y, Su X, Yu JJ, Khan S, Jiang H, Kim J, Woo J, Kim JH,
Choi BH, He B, Chen W, Zhang S, Cerione RA, Auwerx J, Hao Q, Lin
H. SIRT5 is a NAD-dependent protein lysine demalonylase and desuccinylase. Science. 2011;334:806 – 809.
Villarejo-Balcells B, Guichard S, Rigby PW, Carvajal JJ. Expression
pattern of the FOXO1 gene during mouse embryonic development. Gene
Expr Patterns. 2011;11:299 –308.
Potente M, Urbich C, Sasaki K, Hofmann WK, Heeschen C, Aicher A,
Kollipara R, DePinho RA, Zeiher AM, Dimmeler S. Involvement of
FOXO transcription factors in angiogenesis and postnatal neovascularization. J Clin Invest. 2005;115:2382–2392.
Matsukawa M, Sakamoto H, Kawasuji M, Furuyama T, Ogawa M.
Different roles of FOXO1 and FOXO3 in the control of endothelial cell
morphology. Genes Cells. 2009;14:1167–1181.
Phng LK, Gerhardt H. Angiogenesis: a team effort coordinated by
Notch. Dev Cell. 2009;16:196 –208.
Adams RH, Eichmann A. Axon guidance molecules in vascular patterning. Cold Spring Harb Perspect Biol. 2010;2:a001875.
Paik JH, Kollipara R, Chu G, Ji H, Xiao Y, Ding Z, Miao L, Tothova Z,
Horner JW, Carrasco DR, Jiang S, Gilliland DG, Chin L, Wong WH,
Castrillon DH, DePinho RA. FOXOs are lineage-restricted redundant
tumor suppressors and regulate endothelial cell homeostasis. Cell. 2007;
128:309 –323.
Milkiewicz M, Roudier E, Doyle JL, Trifonova A, Birot O, Haas TL.
Identification of a mechanism underlying regulation of the antiangiogenic forkhead transcription factor FOXO1 in cultured endothelial
cells and ischemic muscle. Am J Pathol. 2011;178:935–944.
Daly C, Wong V, Burova E, Wei Y, Zabski S, Griffiths J, Lai KM, Lin
HC, Ioffe E, Yancopoulos GD, Rudge JS. Angiopoietin-1 modulates
1250
51.
52.
53.
54.
55.
56.
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
Circulation Research
April 27, 2012
endothelial cell function and gene expression via the transcription factor
Fkhr (FOXO1). Genes Dev. 2004;18:1060 –1071.
Abid MR, Shih SC, Otu HH, Spokes KC, Okada Y, Curiel DT, Minami
T, Aird WC. A novel class of vascular endothelial growth factorresponsive genes that require forkhead activity for expression. J Biol
Chem. 2006;281:35544 –35553.
Czymai T, Viemann D, Sticht C, Molema G, Goebeler M, Schmidt M.
FOXO3 modulates endothelial gene expression and function by classical
and alternative mechanisms. J Biol Chem. 2010;285:10163–10178.
Potente M, Ghaeni L, Baldessari D, Mostoslavsky R, Rossig L, Dequiedt
F, Haendeler J, Mione M, Dejana E, Alt FW, Zeiher AM, Dimmeler S.
SIRT1 controls endothelial angiogenic functions during vascular
growth. Genes Dev. 2007;21:2644 –2658.
Ou X, Chae HD, Wang RH, Shelley WC, Cooper S, Taylor T, Kim YJ,
Deng CX, Yoder MC, Broxmeyer HE. SIRT1 deficiency compromises
mouse embryonic stem cell hematopoietic differentiation, and embryonic
and adult hematopoiesis in the mouse. Blood. 2011;117:440–450.
Guarani V, Deflorian G, Franco CA, et al. Acetylation-dependent regulation of endothelial Notch signalling by the SIRT1 deacetylase.
Nature. 2011;473:234 –238.
Mulligan P, Yang F, Di Stefano L, Ji JY, Ouyang J, Nishikawa JL, Toiber D,
Kulkarni M, Wang Q, Najafi-Shoushtari SH, Mostoslavsky R, Gygi SP, Gill G,
Dyson NJ, Naar AM. A SIRT1-LSD1 corepressor complex regulates Notch
target gene expression and development. Mol Cell. 2011;42:689–699.
Donmez G, Wang D, Cohen DE, Guarente L. SIRT1 suppresses betaamyloid production by activating the alpha-secretase gene Adam10.
Cell. 142:320 –332.
Fraisl P, Mazzone M, Schmidt T, Carmeliet P. Regulation of angiogenesis by oxygen and metabolism. Dev Cell. 2009;16:167–179.
Arany Z, Foo SY, Ma Y, Ruas JL, Bommi-Reddy A, Girnun G, Cooper M,
Laznik D, Chinsomboon J, Rangwala SM, Baek KH, Rosenzweig A,
Spiegelman BM. HIF-independent regulation of VEGF and angiogenesis by
the transcriptional coactivator PGC-1 alpha. Nature. 2008;451:1008–1012.
Ouchi N, Shibata R, Walsh K. AMP-activated protein kinase signaling
stimulates VEGF expression and angiogenesis in skeletal muscle. Circ
Res. 2005;96:838 – 846.
Ylikorkala A, Rossi DJ, Korsisaari N, Luukko K, Alitalo K, Henkemeyer M, Makela TP. Vascular abnormalities and deregulation of
VEGF in LKB1-deficient mice. Science. 2001;293:1323–1326.
Ohashi K, Ouchi N, Higuchi A, Shaw RJ, Walsh K. LKB1 deficiency in
Tie2-Cre-expressing cells impairs ischemia-induced angiogenesis. J Biol
Chem. 2010;285:22291–22298.
Guarente L. The logic linking protein acetylation and metabolism. Cell
Metab. 2011;14:151–153.
Cavallaro U, Dejana E. Adhesion molecule signalling: not always a
sticky business. Nat Rev Mol Cell Biol. 2011;12:189 –197.
Taddei A, Giampietro C, Conti A, Orsenigo F, Breviario F, Pirazzoli V,
Potente M, Daly C, Dimmeler S, Dejana E. Endothelial adherens
junctions control tight junctions by VE-cadherin-mediated upregulation
of Claudin-5. Nat Cell Biol. 2008;10:923–934.
Abid MR, Guo S, Minami T, Spokes KC, Ueki K, Skurk C, Walsh K,
Aird WC. Vascular endothelial growth factor activates PI3K/AKT/
forkhead signaling in endothelial cells. Arterioscler Thromb Vasc Biol.
2004;24:294 –300.
Potente M, Fisslthaler B, Busse R, Fleming I. 11,12-Epoxyeicosatrienoic
acid-induced inhibition of FOXO factors promotes endothelial proliferation
by down-regulating p27Kip1. J Biol Chem. 2003;278:29619–29625.
Dixit M, Bess E, Fisslthaler B, Hartel FV, Noll T, Busse R, Fleming I.
Shear stress-induced activation of the AMP-activated protein kinase
regulates FOXO1a and angiopoietin-2 in endothelial cells. Cardiovasc
Res. 2008;77:160 –168.
Goettsch W, Gryczka C, Korff T, Ernst E, Goettsch C, Seebach J,
Schnittler HJ, Augustin HG, Morawietz H. Flow-dependent regulation
of angiopoietin-2. J Cell Physiol. 2008;214:491–503.
Chlench S, Mecha Disassa N, Hohberg M, Hoffmann C, Pohlkamp T,
Beyer G, Bongrazio M, Da Silva-Azevedo L, Baum O, Pries AR,
Zakrzewicz A. Regulation of FOXO-1 and the angiopoietin-2/Tie2
system by shear stress. FEBS Lett. 2007;581:673– 680.
Gimbrone MA Jr, Topper JN, Nagel T, Anderson KR, Garcia-Cardena
G. Endothelial dysfunction, hemodynamic forces, and atherogenesis.
Ann N Y Acad Sci. 2000;902:230 –239.
Boon RA, Horrevoets AJ. Key transcriptional regulators of the vasoprotective effects of shear stress. Hamostaseologie. 2009;29:39 – 40.
Gracia-Sancho J, Villarreal G Jr, Zhang Y, Garcia-Cardena G. Activation of SIRT1 by resveratrol induces Klf2 expression conferring an
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
endothelial vasoprotective phenotype. Cardiovasc Res. 2010;85:
514 –519.
Chen Z, Peng IC, Cui X, Li YS, Chien S, Shyy JY. Shear stress, SIRT1, and
vascular homeostasis. Proc Natl Acad Sci U S A. 2010;107:10268–10273.
Mazzone M, Dettori D, Leite de Oliveira R, et al. Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via
endothelial normalization. Cell. 2009;136:839 – 851.
Majmundar AJ, Wong WJ, Simon MC. Hypoxia-inducible factors and
the response to hypoxic stress. Mol Cell. 2010;40:294 –309.
Dioum EM, Chen R, Alexander MS, Zhang Q, Hogg RT, Gerard RD,
Garcia JA. Regulation of hypoxia-inducible factor 2alpha signaling by the
stress-responsive deacetylase sirtuin 1. Science. 2009;324:1289–1293.
Rane S, He M, Sayed D, Vashistha H, Malhotra A, Sadoshima J, Vatner
DE, Vatner SF, Abdellatif M. Downregulation of miR-199a derepresses
hypoxia-inducible factor-1 alpha and sirtuin 1 and recapitulates hypoxia
preconditioning in cardiac myocytes. Circ Res. 2009;104:879 – 886.
Chen R, Dioum EM, Hogg RT, Gerard RD, Garcia JA. Hypoxia
increases sirtuin 1 expression in a hypoxia-inducible factor-dependent
manner. J Biol Chem. 2011;286:13869 –13878.
Yu J, Auwerx J. The role of sirtuins in the control of metabolic homeostasis. Ann N Y Acad Sci. 2009;1173(Suppl 1):E10 –E19.
Guarani V, Potente M. SIRT1-a metabolic sensor that controls blood
vessel growth. Curr Opin Pharmacol. 2010;10:139 –145.
Zhong L, D’Urso A, Toiber D, et al. The histone deacetylase SIRT6
regulates glucose homeostasis via HIF1alpha. Cell. 2010;140:280 –293.
Finley LW, Carracedo A, Lee J, Souza A, Egia A, Zhang J, TeruyaFeldstein J, Moreira PI, Cardoso SM, Clish CB, Pandolfi PP, Haigis
MC. SIRT3 opposes reprogramming of cancer cell metabolism through
HIF1alpha destabilization. Cancer Cell. 2011;19:416 – 428.
Lim JH, Lee YM, Chun YS, Chen J, Kim JE, Park JW. SIRT1 modulates
cellular responses to hypoxia by deacetylating hypoxia-inducible factor
1 alpha. Mol Cell. 2010;38:864 – 878.
Collado M, Blasco MA, Serrano M. Cellular senescence in cancer and
aging. Cell. 2007;130:223–233.
Palacios JA, Herranz D, De Bonis ML, Velasco S, Serrano M, Blasco
MA. SIRT1 contributes to telomere maintenance and augments global
homologous recombination. J Cell Biol. 2010;191:1299 –1313.
Yuan Z, Zhang X, Sengupta N, Lane WS, Seto E. SIRT1 regulates the
function of the Nijmegen breakage syndrome protein. Mol Cell. 2007;
27:149 –162.
Li K, Casta A, Wang R, Lozada E, Fan W, Kane S, Ge Q, Gu W, Orren
D, Luo J. Regulation of WRN protein cellular localization and
enzymatic activities by SIRT1-mediated deacetylation. J Biol Chem.
2008;283:7590 –7598.
Ota H, Akishita M, Eto M, Iijima K, Kaneki M, Ouchi Y. SIRT1
modulates premature senescence-like phenotype in human endothelial
cells. J Mol Cell Cardiol. 2007;43:571–579.
Zu Y, Liu L, Lee MY, Xu C, Liang Y, Man RY, Vanhoutte PM, Wang
Y. SIRT1 promotes proliferation and prevents senescence through targeting LKB1 in primary porcine aortic endothelial cells. Circ Res.
2010;106:1384 –1393.
Fulco M, Cen Y, Zhao P, Hoffman EP, McBurney MW, Sauve AA,
Sartorelli V. Glucose restriction inhibits skeletal myoblast differentiation by activating SIRT1 through AMPK-mediated regulation of
NAMPT. Dev Cell. 2008;14:661– 673.
Canto C, Gerhart-Hines Z, Feige JN, Lagouge M, Noriega L, Milne JC,
Elliott PJ, Puigserver P, Auwerx J. AMPK regulates energy expenditure
by modulating NAD⫹ metabolism and SIRT1 activity. Nature. 2009;
458:1056 –1060.
Zhou S, Chen HZ, Wan YZ, Zhang QJ, Wei YS, Huang S, Liu JJ, Lu YB,
Zhang ZQ, Yang RF, Zhang R, Cai H, Liu DP, Liang CC. Repression of
p66Shc expression by SIRT1 contributes to the prevention of hyperglycemiainduced endothelial dysfunction. Circ Res. 2011;109:639–648.
Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, Tran H,
Ross SE, Mostoslavsky R, Cohen HY, Hu LS, Cheng HL, Jedrychowski
MP, Gygi SP, Sinclair DA, Alt FW, Greenberg ME. Stress-dependent
regulation of FOXO transcription factors by the SIRT1 deacetylase.
Science. 2004;303:2011–2015.
Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer PJ,
Huang TT, Bos JL, Medema RH, Burgering BM. Forkhead transcription
factor FOXO3a protects quiescent cells from oxidative stress. Nature.
2002;419:316 –321.
Tran H, Brunet A, Grenier JM, Datta SR, Fornace AJ Jr, DiStefano PS,
Chiang LW, Greenberg ME. DNA repair pathway stimulated by the
Oellerich and Potente
97.
98.
99.
100.
101.
102.
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
115.
116.
117.
118.
forkhead transcription factor FOXO3a through the Gadd45 protein.
Science. 2002;296:530 –534.
Olmos Y, Valle I, Borniquel S, Tierrez A, Soria E, Lamas S, Monsalve
M. Mutual dependence of FOXO3a and PGC-1 alpha in the induction of
oxidative stress genes. J Biol Chem. 2009;284:14476 –14484.
Willcox BJ, Donlon TA, He Q, Chen R, Grove JS, Yano K, Masaki KH,
Willcox DC, Rodriguez B, Curb JD. FOXO3a genotype is strongly associated
with human longevity. Proc Natl Acad Sci U S A. 2008;105:13987–13992.
Anselmi CV, Malovini A, Roncarati R, Novelli V, Villa F, Condorelli G,
Bellazzi R, Puca AA. Association of the FOXO3a locus with extreme
longevity in a southern Italian centenarian study. Rejuvenation Res.
2009;12:95–104.
Flachsbart F, Caliebe A, Kleindorp R, Blanche H, von Eller-Eberstein H,
Nikolaus S, Schreiber S, Nebel A. Association of FOXO3a variation
with human longevity confirmed in German centenarians. Proc Natl
Acad Sci U S A. 2009;106:2700 –2705.
Pawlikowska L, Hu D, Huntsman S, et al. Association of common
genetic variation in the Insulin/IGF1 signaling pathway with human
longevity. Aging Cell. 2009;8:460 – 472.
Renault VM, Thekkat PU, Hoang KL, White JL, Brady CA, Kenzelmann Broz D, Venturelli OS, Johnson TM, Oskoui PR, Xuan Z,
Santo EE, Zhang MQ, Vogel H, Attardi LD, Brunet A. The prolongevity gene FOXO3 is a direct target of the p53 tumor suppressor.
Oncogene. 2011;30:3207–3221.
Nemoto S, Fergusson MM, Finkel T. Nutrient availability regulates SIRT1
through a forkhead-dependent pathway. Science. 2004;306:2105–2108.
Wang F, Marshall CB, Yamamoto K, Li GY, Plevin MJ, You H, Mak
TW, Ikura M. Biochemical and structural characterization of an intramolecular interaction in FOXO3a and its binding with p53. J Mol Biol.
2008;384:590 – 603.
You H, Yamamoto K, Mak TW. Regulation of transactivationindependent proapoptotic activity of p53 by FOXO3a. Proc Natl Acad
Sci U S A. 2006;103:9051–9056.
Miyaguchi Y, Tsuchiya K, Sakamoto K. p53 negatively regulates the
transcriptional activity of FOXO3a under oxidative stress. Cell Biol Int.
2009;33:853– 860.
Fu W, Ma Q, Chen L, Li P, Zhang M, Ramamoorthy S, Nawaz Z,
Shimojima T, Wang H, Yang Y, Shen Z, Zhang Y, Zhang X, Nicosia
SV, Zhang Y, Pledger JW, Chen J, Bai W. MDM2 acts downstream of
p53 as an E3 ligase to promote FOXO ubiquitination and degradation.
J Biol Chem. 2009;284:13987–14000.
Matsushita H, Chang E, Glassford AJ, Cooke JP, Chiu CP, Tsao PS.
eNOS activity is reduced in senescent human endothelial cells: preservation by hTERT immortalization. Circ Res. 2001;89:793–798.
van der Loo B, Labugger R, Skepper JN, Bachschmid M, Kilo J, Powell
JM, Palacios-Callender M, Erusalimsky JD, Quaschning T, Malinski T,
Gygi D, Ullrich V, Luscher TF. Enhanced peroxynitrite formation is
associated with vascular aging. J Exp Med. 2000;192:1731–1744.
Mattagajasingh I, Kim CS, Naqvi A, Yamamori T, Hoffman TA, Jung
SB, DeRicco J, Kasuno K, Irani K. SIRT1 promotes endotheliumdependent vascular relaxation by activating endothelial nitric oxide
synthase. Proc Natl Acad Sci U S A. 2007;104:14855–14860.
Nisoli E, Tonello C, Cardile A, Cozzi V, Bracale R, Tedesco L, Falcone
S, Valerio A, Cantoni O, Clementi E, Moncada S, Carruba MO. Calorie
restriction promotes mitochondrial biogenesis by inducing the
expression of eNOS. Science. 2005;310:314 –317.
Menghini R, Casagrande V, Cardellini M, Martelli E, Terrinoni A,
Amati F, Vasa-Nicotera M, Ippoliti A, Novelli G, Melino G, Lauro R,
Federici M. MicroRNA 217 modulates endothelial cell senescence via
silent information regulator 1. Circulation. 2009;120:1524 –1532.
Yamakuchi M, Ferlito M, Lowenstein CJ. MIR-34a repression of SIRT1
regulates apoptosis. Proc Natl Acad Sci U S A. 2008;105:13421–13426.
Ito T, Yagi S, Yamakuchi M. MicroRNA-34a regulation of endothelial
senescence. Biochem Biophys Res Commun. 2010;398:735–740.
de Keizer PL, Burgering BM, Dansen TB. Forkhead box O as a sensor,
mediator, and regulator of redox signaling. Antioxid Redox Signal.
2011;14:1093–1106.
Dansen TB, Smits LM, van Triest MH, de Keizer PL, van Leenen D,
Koerkamp MG, Szypowska A, Meppelink A, Brenkman AB, Yodoi J,
Holstege FC, Burgering BM. Redox-sensitive cysteines bridge p300/Cbpmediated acetylation and FOXO4 activity. Nat Chem Biol. 2009;5:664–672.
Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature. 2011;473:317–325.
Zhang QJ, Wang Z, Chen HZ, Zhou S, Zheng W, Liu G, Wei YS, Cai
H, Liu DP, Liang CC. Endothelium-specific overexpression of class III
119.
120.
121.
122.
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
FOXOs and Sirtuins in ECs
1251
deacetylase SIRT1 decreases atherosclerosis in apolipoprotein
E-deficient mice. Cardiovasc Res. 2008;80:191–199.
Do GM, Kwon EY, Kim HJ, Jeon SM, Ha TY, Park T, Choi MS.
Long-term effects of resveratrol supplementation on suppression of
atherogenic lesion formation and cholesterol synthesis in apo E-deficient
mice. Biochem Biophys Res Commun. 2008;374:55–59.
Yeung F, Hoberg JE, Ramsey CS, Keller MD, Jones DR, Frye RA,
Mayo MW. Modulation of NF-kappa B-dependent transcription and cell
survival by the SIRT1 deacetylase. EMBO J. 2004;23:2369 –2380.
Milne JC, Lambert PD, Schenk S, et al. Small molecule activators of
SIRT1 as therapeutics for the treatment of type 2 diabetes. Nature.
2007;450:712–716.
Baur JA, Pearson KJ, Price NL, et al. Resveratrol improves health and
survival of mice on a high-calorie diet. Nature. 2006;444:337–342.
Lagouge M, Argmann C, Gerhart-Hines Z, Meziane H, Lerin C, Daussin
F, Messadeq N, Milne J, Lambert P, Elliott P, Geny B, Laakso M,
Puigserver P, Auwerx J. Resveratrol improves mitochondrial function
and protects against metabolic disease by activating SIRT1 and Pgc-1
alpha. Cell. 2006;127:1109 –1122.
Banks AS, Kon N, Knight C, Matsumoto M, Gutierrez-Juarez R,
Rossetti L, Gu W, Accili D. SIRT1 gain of function increases energy
efficiency and prevents diabetes in mice. Cell Metab. 2008;8:333–341.
Sun C, Zhang F, Ge X, Yan T, Chen X, Shi X, Zhai Q. SIRT1 improves
insulin sensitivity under insulin-resistant conditions by repressing
PTP1b. Cell Metab. 2007;6:307–319.
Moynihan KA, Grimm AA, Plueger MM, Bernal-Mizrachi E, Ford E,
Cras-Meneur C, Permutt MA, Imai S. Increased dosage of mammalian
Sir2 in pancreatic beta cells enhances glucose-stimulated insulin
secretion in mice. Cell Metab. 2005;2:105–117.
Li X, Zhang S, Blander G, Tse JG, Krieger M, Guarente L. SIRT1
deacetylates and positively regulates the nuclear receptor LXR. Mol
Cell. 2007;28:91–106.
Kemper JK, Xiao Z, Ponugoti B, Miao J, Fang S, Kanamaluru D, Tsang
S, Wu SY, Chiang CM, Veenstra TD. FXR acetylation is normally
dynamically regulated by p300 and SIRT1 but constitutively elevated in
metabolic disease states. Cell Metab. 2009;10:392– 404.
Qiang L, Lin HV, Kim-Muller JY, Welch CL, Gu W, Accili D.
Proatherogenic abnormalities of lipid metabolism in SIRT1 transgenic mice
are mediated through CREB deacetylation. Cell Metab. 2011;14:758–767.
Fontana L, Partridge L, Longo VD. Extending healthy life span: from
yeast to humans. Science. 2010;328:321–326.
Colman RJ, Anderson RM, Johnson SC, Kastman EK, Kosmatka KJ,
Beasley TM, Allison DB, Cruzen C, Simmons HA, Kemnitz JW,
Weindruch R. Caloric restriction delays disease onset and mortality in
rhesus monkeys. Science. 2009;325:201–204.
Civitarese AE, Carling S, Heilbronn LK, Hulver MH, Ukropcova B,
Deutsch WA, Smith SR, Ravussin E. Calorie restriction increases muscle
mitochondrial biogenesis in healthy humans. PLoS Med. 2007;4:e76.
Hirschey MD, Shimazu T, Jing E, et al. SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the
metabolic syndrome. Mol Cell. 2011.
McBurney MW, Yang X, Jardine K, Hixon M, Boekelheide K, Webb JR,
Lansdorp PM, Lemieux M. The mammalian SIR2alpha protein has a role in
embryogenesis and gametogenesis. Mol Cell Biol. 2003;23:38–54.
Cheng HL, Mostoslavsky R, Saito S, Manis JP, Gu Y, Patel P, Bronson
R, Appella E, Alt FW, Chua KF. Developmental defects and p53
hyperacetylation in SIR2 homolog (SIRT1)-deficient mice. Proc Natl
Acad Sci U S A. 2003;100:10794 –10799.
Kim HS, Vassilopoulos A, Wang RH, et al. SIRT2 maintains genome
integrity and suppresses tumorigenesis through regulating APC/C
activity. Cancer Cell. 2011;20:487– 499.
Lombard DB, Alt FW, Cheng HL, et al. Mammalian SIR2 homolog
SIRT3 regulates global mitochondrial lysine acetylation. Mol Cell Biol.
2007;27:8807– 8814.
Nakagawa T, Lomb DJ, Haigis MC, Guarente L. SIRT5 deacetylates
carbamoyl phosphate synthetase 1 and regulates the urea cycle. Cell.
2009;137:560 –570.
Mostoslavsky R, Chua KF, Lombard DB, et al. Genomic instability and
aging-like phenotype in the absence of mammalian SIRT6. Cell. 2006;
124:315–329.
Vakhrusheva O, Smolka C, Gajawada P, Kostin S, Boettger T, Kubin T,
Braun T, Bober E. SIRT7 increases stress resistance of cardiomyocytes
and prevents apoptosis and inflammatory cardiomyopathy in mice. Circ
Res. 2008;102:703–710.
FOXOs and Sirtuins in Vascular Growth, Maintenance, and Aging
Mark F. Oellerich and Michael Potente
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Circ Res. 2012;110:1238-1251
doi: 10.1161/CIRCRESAHA.111.246488
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