Molecular pathways of arterial aging

Abstract

The incidence of stroke and myocardial infarction increases in aged patients and it is associated with an adverse outcome. Considering the aging population and the increasing incidence of cardiovascular disease, the prediction for population well-being and health economics is daunting. Accordingly, there is an unmet need to focus on fundamental processes underlying vascular aging. A better understanding of the pathways leading to arterial aging may contribute to design mechanism-based therapeutic approaches to prevent or attenuate features of vascular senescence. In the present review, we discuss advances in the pathophysiology of age-related vascular dysfunction including nitric oxide signalling, dysregulation of oxidant/inflammatory genes, epigenetic modifications and mechanisms of vascular calcification as well as insights into vascular repair. Such an overview highlights attractive molecular targets for the prevention of age-driven vascular disease.

Full text

Clinical Science (2015) 128, 69–79 (Printed in Great Britain) doi: 10.1042/CS20140302
Molecular pathways of arterial aging
Francesco Paneni∗, Sarah Costantino∗and Francesco Cosentino∗
∗Cardiology Unit, Department of Medicine, Karolinska University Hospital, Stockholm, Sweden
Abstract
The incidence of stroke and myocardial infarction increases in aged patients and it is associated with an adverse
outcome. Considering the aging population and the increasing incidence of cardiovascular disease, the prediction
for population well-being and health economics is daunting. Accordingly, there is an unmet need to focus on
fundamental processes underlying vascular aging. A better understanding of the pathways leading to arterial aging
may contribute to design mechanism-based therapeutic approaches to prevent or attenuate features of vascular
senescence. In the present review, we discuss advances in the pathophysiology of age-related vascular dysfunction
including nitric oxide signalling, dysregulation of oxidant/inflammatory genes, epigenetic modifications and
mechanisms of vascular calcification as well as insights into vascular repair. Such an overview highlights attractive
molecular targets for the prevention of age-driven vascular disease.
Key words: arterial aging, inflammation, oxidative stress, pathways, vascular repair
AGING AND CARDIOVASCULAR DISEASE
Aging is the main risk factor and driver of incident cardiovas-
cular disease (CVD) and results in a progressive functional and
structural decline of the vasculature [1]. Arterial aging, through
its impact on physical and mental health, impairs quality of life
and the ability of individuals to carry out the tasks of every-
day life [2]. As the average lifespan of the European population
is increasing, the overall future CVD burden in Europe is pre-
dicted to increase dramatically leading to a pandemic of frailty
syndromes, poor quality of life and high morbidity in the aging
population [3,4]. Since half of all people aged 65 years and older
develop CVD, arterial aging has a profound impact on the health
of populations in Europe [5,6]. Moreover, clustering of chrono-
logical age with other cardiovascular (CV) risk factors clearly
anticipates senescent features of heart and vessels [7]. Intricate
signalling cascades are emerging as determinants of accelerated
arterial aging in the presence of CV risk factors. Accordingly,
there is an unmet need to focus on fundamental processes of
vascular aging enabling early diagnosis and prevention of CVD.
This is important in the context that, although aging is inevitable,
vascular aging is a modifiable risk factor.
Abbreviations: ALDH2, aldehyde dehydrogenase 2; AMPK, AMP-activated protein kinase; AP-1, activated protein 1; ApoE, apolipoprotein E; Arg II, arginase II; BAP, bone alkaline
phosphatase; BH4, tetrahydrobiopterin; CV, cardiovascular; CVD, cardiovascular disease; eNOS, endothelial nitric oxide synthase; EOC, early angiogenic outgrowth cell; EPC,
endothelial progenitor cell; fOXO, Forkhead box O; HIF-1α, hypoxia inducible factor 1α; MCC, myeloid calcifying cell; NF-κB, nuclear factor κB; OC, osteocalcin; PI3K, phosphoinositide
3-kinase; RANKL, receptor activator of NF-κB ligand; ROS, reactive oxygen species; SDF-1, stromal cell-derived factor 1; VSMC, vascular smooth muscle cell; WT, wild-type.
Correspondence: Professor Francesco Cosentino (email francesco.cosentino@ki.se).
STRUCTURAL ABNORMALITIES OF ARTERIAL
AGING
Clinical and pre-clinical data have shown that aging is associ-
ated with structural and functional properties of large arteries
[8]. Aging blood vessels become thicker and stiffer, resulting in
a reduced ability to adjust vessel shape and function to chan-
ging tissue demands. In aged healthy humans, these alterations
are represented by luminal dilation, increased arterial stiffness,
endothelial dysfunction and diffuse intimal thickening. A de-
crease in vascular plasticity may be the result of different factors
including enhanced elastin degradation and collagen deposition
in the vascular media [9]. Importantly, remodelling of the vas-
culature is accompanied by key alterations of endothelial ho-
moeostasis [10]. Indeed, seminal studies have clearly shown that
endothelium-dependent vasorelaxation is impaired in aged ves-
sels and this phenomenon is associated with increased vascular
permeability and inflammation, as well as impaired angiogenesis
[1,11]. Moreover, age-related alterations of endothelial cell func-
tionality may, in turn, aggravate media thickness and vascular
fibrosis.
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F. Paneni, S. Costantino and F. Cosentino
NITRIC OXIDE SIGNALLING
Vascular aging, characterized by endothelial dysfunction and in-
creased vascular stiffness, is associated with reduced endothelial
nitric oxide (NO) bioavailability and increased generation of re-
active oxygen species (ROS) [12]. Increased ROS production
in vascular aging derives from enzymatic and non-enzymatic
sources such as mitochondria [13]. Interestingly enough, dysreg-
ulation of the endothelial nitric oxide synthase (eNOS), known as
eNOS uncoupling, results in loss of endothelial NO generation
and increased ROS production [14]. The mechanism of eNOS-
uncoupling seems multiple and includes oxidation of the cofactor
tetrahydrobiopterin (BH4), decreased intracellular availability of
the substrate L-arginine due to either increased arginase activ-
ity or accumulation of endogenous arginine analogues such as
asymmetric dimethyl-L-arginine that competes with L-arginine
for eNOS binding [15].
Tetrahydrobiopterin
The small molecule BH4is a redox cofactor for eNOS and a
pivotal regulator of its catalytic activity [14]. Loss of BH4is
associated with vascular disease states, including aging, and res-
ults in eNOS uncoupling reduced NO release and superoxide
anion (O2−) generation, in turn, leading to production of hydro-
gen peroxide and peroxynitrite (ONOO−) (Figure 1) [12]. The
role of BH4in vascular aging has been demonstrated by sev-
eral studies over the last 10 years. Older animals have reduced
vascular BH4levels due to its oxidation into dihydrobiopterin
(BH2), which is not acting as a cofactor for eNOS. Such a short-
age of cofactor leads to a conformational change in eNOS from
a dimeric to monomeric state resulting in loss of NO produc-
tion and ROS generation [15]. Pharmacological supplementation
of BH4can improve endothelial function in aged humans com-
pared with young subjects [16], suggesting that BH4treatment
might be a rational approach to improve vascular function in
aging [17].
Arginase II
L-Arginine, a major eNOS substrate, is rapidly metabolized by
arginase enzyme to urea and L-ornithine, leading to a decrease
in its availability and, hence, reduced NO synthesis [18]. An
increasing body of evidence suggests that arginase II (Arg II)
is deregulated in aging and participates to early vascular senes-
cence by altering the phenotype of endothelial cells and vascular
smooth muscle cells (VSMCs). A recent study performed in VS-
MCs isolated from human umbilical veins showed that Arg II
overexpression activates p66Shc/p53 signalling, thus triggering
mitochondrial dysfunction and cell apoptosis, key features of
cellular aging. Consistently, gene silencing of Arg II attenuates
senescence of human endothelial cells by maintaining a detri-
mental vicious cycle involving the mammalian target of rapamy-
cin complex 1 (mTORC1)/S6 kinase (S6K1) pathway.
Activation of Arg II may represent an important link between
CV risk factors and the aging process. Indeed, several common
risk factors such as hyperglycaemia, hyperlipidaemia and obesity
have been shown to increase Arg II expression/activity. Genetic
deletion of Arg II reduces vascular inflammation in mice fed on
a high-cholesterol and high-fat diet, prevents atherosclerosis and
ameliorates insulin sensitivity as well as glucose homoeostasis
[19,20]. These findings shed light on the role of Arg II as a
common determinant across the spectrum of metabolic disease,
atherosclerosis and senescence. The clinical impact of this gene is
supported by the notion that arginase inhibition improves brachial
artery vasodilation in subjects with Type 2 diabetes [21]. Phar-
macological suppression of arginase activity may indeed restore
physiological substrate levels, thus suppressing molecular path-
ways involved in endothelial aging and dysfunction (Figure 1).
Endothelial nitric oxide synthase
Alteration of eNOS transcription may be involved in age-related
endothelial dysfunction but its biological significance remains
unclear. Some studies found increased eNOS expression in aging
and such observations have been interpreted as a compensatory
but futile mechanism to counter-regulate the loss of NO [2,22].
In contrast, other work in humans and rodents did not show
significant changes in eNOS expression but rather an increase
or decrease in its activating Ser1177 phosphorylation [15,23,24].
Age-dependent impairment of NO release also plays an import-
ant role in the coronary circulation [25]. Indeed, eNOS deregu-
lation is critically involved in microvascular dysfunction and im-
paired ventricular contractility in aged patients [26]. Moreover,
advanced age is associated with less collaterals to the infarct-
related artery in patients with acute myocardial infarction [27].
This abnormality may contribute to the poor prognosis of older
patients with acute coronary syndromes. Collectively, these evid-
ence strongly suggest that modulation of eNOS functionality is a
promising target for prevention of age-related vascular disease.
OXIDATIVE STRESS
A major feature of CV aging is the imbalance between NO
bioavailability and accumulation of ROS, leading to endothelial
dysfunction [28]. Indeed, age-dependent generation of O2−in-
activates NO to form ONOO−, a powerful oxidant that easily
penetrates across phospholipid membranes leading to substrate
nitration [22,29]. Protein nitrosylation blunts activity of antiox-
idant enzymes and eNOS [30]. Generation of ROS into the vessel
wall has been postulated as a major pathophysiological step fa-
vouring arterial aging [1,11]. Among the different aging theories,
the mitochondrial free radical theory has been in the spotlight for
several decades [31]. Accordingly, ROS are considered to be
by-products of aerobic metabolism that induce oxidative cellular
damage [32]. ROS, small highly reactive molecules, are gener-
ated within the cell by means of several metabolic and enzymatic
pathways. The majority of ROS are produced within mitochon-
drial oxidative phosphorylation. During this process, electrons
are extracted from NADH and FADH and transferred to molecu-
lar oxygen through a chain of four enzymatic complexes ensuring
phosphorylation of ADP into ATP and final reduction of molecu-
lar oxygen to water. However, electrons derived from NADH or
FADH can directly react with oxygen or other electron accept-
ors within the mitochondrial electron transport chain upstream
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Molecular pathways of arterial aging
Figure 1 Schematic representation of molecular pathways involved in arterial aging
VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; MCP-1, monocyte chemoattractant
protein-1; MnSOD, manganese superoxide dismutase; ecSOD, extracellular superoxide dismutase.
the last enzymatic complex (complex IV, which is responsible
for the reduction of molecular oxygen to water) and generate
free radicals [33]. Accumulation of ROS causes mitochondrial
disruption leading to cytochrome crelease and subsequent activ-
ation of caspase 3 [1,33]. CV risk factors such as hyperglycaemia
induce a ROS-dependent alteration of the mitochondrial net-
work resembling early signs of vascular aging [28]. Indeed, en-
dothelial cells isolated from middle-age diabetic subjects show
a premature derangement of organelle structure, which correl-
ates with impaired flow-mediated vasodilation of the brachial
artery [34].
NAPDH oxidase
NADPH oxidases of the Nox family are differentially expressed
in the CV system and may critically contribute to oxidative bur-
den and vascular disease [35]. The NOX family consists of seven
members: the classic NOXs, NOX1–NOX5, and the dual oxi-
dases Duox1 and Duox2 [36,37]. A significant expression in the
CV system has been reported for Nox1, Nox2, Nox4 and Nox5
[37]. As a consequence of the interaction between the differ-
ent ROS-generating systems such as mitochondria and eNOS,
NADPH oxidases have been demonstrated to contribute to ROS
formation for almost all risk factor conditions. However, the con-
tribution of NADPH oxidase in aging remains incompletely un-
derstood. This is likely to be due to the fact that the role of
NADPH in cellular senescence has been overlooked, since the
overproduction of ROS associated with aging has mainly been
attributed to leakiness of the mitochondrial respiratory chain.
Indeed, most of the available studies suggest that arterial aging
is rather the result of impaired antioxidant enzyme expression,
eNOS uncoupling, or increased ROS production by mitochon-
dria [1]. However, few studies support a putative involvement of
NOXs in arterial aging. A study has demonstrated that silencing
of NOX4 in human endothelial cells leads to delayed replicat-
ive senescence and preserves cell functionality [38]. In line with
these findings, we have recently shown that Nox2 and Nox4 are
significantly up-regulated in the vasculature of aged mice, as well
as only Nox2 increases in peripheral blood monocytes isolated
from older individuals [39]. Furthermore, NADPH oxidase is a
key mediator of endothelin-1-induced vasoconstriction, as well as
cerebrovascular damage in aged rodents [40,41]. In contrast, a
previous study showed that activation of NADPHsubunits p47phox
and p67phox remain unchanged in aged endothelial cells, suggest-
ing that age-related oxidative stress mechanistically differs from
endothelial dysfunction seen in the context of other CV risk
factors [42]. Taken together, these results suggest that further re-
search is needed to better understand the role of NADPH oxidase
in arterial aging.
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Activated protein-1 transcription factor JunD
Recently, the activated protein-1 (AP-1) transcription factor JunD
has emerging as an important modulator of age-driven mitochon-
drial oxidative stress [43]. AP-1 is a collection of dimeric com-
plexes made by different members of three families of DNA-
binding proteins: Jun, Fos and activating transcription factor
(ATF)/cAMP-response-element-binding protein-binding protein
(CREB) [44]. These members assemble to form AP-1 transcrip-
tion factors with activities that are strongly influenced by their
specific components and their cellular environment. JunD, the
most recent gene of the Jun family, regulates cell growth and sur-
vival and protects against oxidative stress by modulating genes
involved in antioxidant defence and ROS production [45]. Our
recent work has shown that JunD is a longevity gene implicated
in the preservation of vascular homoeostasis during life [39] (Fig-
ure 1). We found that JunD is down-regulated by aging both in
mouse aorta and peripheral blood monocytes of old as compared
with young healthy individuals. Interestingly, age-dependent re-
duction in JunD expression was explained by a specificepigenetic
signature, namely reduced methylation of CpG dinucleotides on
the JunD promoter [39]. This latter finding strengths the no-
tion that epigenetic signatures may critically participate in early
phenotypes of vascular disease during the lifetime [46,47]. In our
study, young JunD−/−mice showed early endothelial dysfunction
and vascular senescence, which were comparable with the one
observed in aged wild-type (WT) mice. JunD deletion was in-
deed associated with up-regulation of the aging markers p53 and
p16INK4a, reduced telomerase activity and mitochondrial DNA
damage [39]. Interestingly enough, transient overexpression of
JunD was able to rescue endothelial dysfunction in aged mice.
Mechanistically, we found that JunD is required for the expres-
sion of mitochondrial antioxidant enzymes such as manganese
superoxide dismutase (MnSOD) and aldehyde dehydrogenase 2
(ALDH-2). This latter enzyme was almost abolished in young
JunD−/−mice compared with age-matched littermates. The rel-
evance of this finding is supported by the notion that ALDH-2
protects against ischaemia/reperfusion injury [48] and cardiac
arrhythmias [49]. Moreover, JunD participates to eNOS tran-
scription, thus contributing to preserve NO availability during
aging [50]. The transcription factor also modulates the expression
and activity of NADPH oxidase in the vasculature [39]. Indeed,
JunD−/−mice display a premature up-regulation of the NADPH
isoforms p47phox, Nox2 and Nox4 leading to increased vascu-
lar oxidative stress already in early stages of life. Moreover, in
our study JunD expression negatively correlated with NADPH
subunits in aged individuals. In line with our findings, lack
of JunD promotes pressure-overload-induced apoptosis, hyper-
trophic growth and angiogenesis in the heart [51]. Collectively,
these findings indicate that JunD down-regulation may represent
a common molecular event linking premature senescence with
CVD development.
Mitochondrial adaptor p66Shc
The identification of molecular pathways modulating the en-
dothelial cell redox state is crucial in understanding the mech-
anisms linking vascular aging to endothelial dysfunction and
atherosclerosis. In view of its role on the cellular redox state, mi-
tochondrial p66Shc adaptor protein has been regarded as part of
a putative transduction pathway relevant to endothelial integrity
[11,52]. Intracellular free radicals are reduced in cells lacking the
p66Shc gene (p66Shc−/−cells), and both systemic as well as intra-
cellular free radicals are diminished in a p66Shc−/−mouse model
exposed to high oxidative stress [53,54]. The p66Shc adaptor pro-
tein functions as a redox enzyme implicated in mitochondrial
ROS generation and translation of oxidative signals into apop-
tosis (Figure 1) [53,55–57]. Several chronic stimuli activate the
protein kinase C βII (PKCβII) isoform to induce Ser36 phos-
phorylation of p66Shc, allowing transfer of the protein from the
cytosol to the mitochondrion where it catalyses ROS produc-
tion via cytochrome coxidation [53]. This latter event leads to
mitochondrial disruption and cell death. Indeed, increased ROS
generation alters mitochondrial permeability facilitating the re-
lease of proapoptotic proteins such as cytochrome c[53]. Once
into the cytosol, cytochrome cis responsible for activation of
the apoptosis execution enzyme caspase-3. Accordingly, mice
lacking the p66Shc gene (p66Shc−/−) display prolonged lifespan,
increased resistance to oxidative stress and apoptosis. We have
previously reported that endothelium-dependent relaxations in
response to acetylcholine were age-dependently impaired in WT
mice but not in p66Shc−/−mice [55]. Accordingly, p66Shc−/−mice
were protected against the age-related decline in NO release.
This study implies that p66Shc signalling is required to induce
a ROS-driven vascular senescent phenotype. Importantly, p66Shc
activation is thought to be upstream of the NADPH and mam-
malian target of rapamycin pathway, two important determinants
of vascular damage [58]. Activation of p66Shc also promotes the
heart senescent phenotype and development of heart failure in
diabetic mice [59]. Accordingly, diabetic p66Shc−/−mice are pro-
tected against myocardial oxidative stress, apoptosis and telomere
shortening. Despite these studies provided interesting insights
into the role of p66Shc in CV aging, it remains unclear whether
protein inhibition may reverse vascular disease phenotype. In this
regard, we have demonstrated for the first time that in vivo RNA
interference blunts vascular p66Shc expression, ROS generation
and endothelial dysfunction in diabetic mice [60]. Moreover, ex-
perimental studies have recently shown that genetic deletion of
p66Shc prevents age-related CV disease such as myocardial in-
farction and stroke [56,61]. Of interest, p66Shc expression is also
affected by CV risk factors such as oxidized low-density lipopro-
tein and blood pressure [62,63]. These latter findings imply that
the adaptor p66Shc may represent a critical molecular intermediate
between CV risk factors and premature aging. The clinical relev-
ance of p66Shc is supported by the notion that p66Shc gene expres-
sion is increased in mononuclear cells obtained from patients with
Type 2 diabetes and coronary artery disease [64,65]. A study has
shown that p66Shc expression is higher in fibroblasts isolated from
centenarians [66]. This finding probably indicates that p66Shc
expression may increase in a time-dependent manner. In con-
trast, early gene up-regulation due to risk factors may anticip-
ate features of CV aging in middle-age individuals [64,67]. On
the whole, the evidence reported so far supports the concept
that modulation of p66Shc expression and activity may be a
novel and effective approach for the treatment of age-related
CVD.
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INFLAMMATION
Inflammatory processes are known to contribute to age-related
vascular disease, myocardial infarction, stroke and heart failure
[32]. The molecular events underpinning age-dependent inflam-
mation are not completely understood. Inflammatory alterations
in aging include induction of cellular adhesion molecules, an in-
crease in endothelial–leucocyte interactions and alterations in the
secretion of autocrine/paracrine mediators, which are pivotal to
inflammatory responses.
ROLE AND MOLECULAR TARGETS OF
SIRTUINS IN THE AGED VASCULATURE
The family of nicotinamide adenine dinucleotide-dependent
proteins termed sirtuins has recently emerged as an import-
ant regulator of arterial aging. SIRT1 is considered a ma-
jor gatekeeper against oxidative stress and tissue inflam-
mation [68]. Increased SIRT1 activity confers resistance to
many of the CV sequelae associated with aging [69]. Ac-
tivation of SIRT1 in endothelial tissues may be of bene-
fit in preserving endothelial cell function during aging. Mice
with endothelial specific SIRT1 overexpression on an apoli-
poprotein E (ApoE)−/−genetic background exhibit attenuated
development of atherosclerotic lesions [70]. In contrast, SIRT1
insufficiency results in greater foam cell formation and ather-
osclerosis [71]. In the human endothelium, overexpression of
SIRT1 prevents oxidative stress-induced senescence, whereas its
inhibition leads to a premature senescence-like phenotype. In-
terestingly, immunosuppressant drugs, like sirolimus and ever-
olimus, induce endothelial cellular senescence via SIRT1 down-
regulation [72]. SIRT1 inhibition also impairs eNOS functional-
ity, whereas its activation improves endothelial NO availability
[73]. Hence, the SIRT1/NO axis may represent a relevant tar-
get against vascular senescence [74,75]. Interestingly enough,
hyperglycaemia, obesity and hypertension are able to reproduce
age-related effects on SIRT1 expression, thus strengthening a
common role of SIRT1 in CVD and early aging.
SIRT1/p53/p66Shc axis
SIRT1-mediated deacetylation modulates the function of pro-
teins via transcriptional and post-translational changes. A previ-
ous study reported that vascular p66Shc gene transcription may
be the result of decreased promoter deacetylation due to down-
regulation of SIRT1 [76]. Expression of p66Shc gene transcript
and protein was significantly increased by different kinds of class
III histone deacetylase inhibitors in human endothelial cells. Con-
sistently, SIRT1 overexpression inhibited high glucose-induced
p66Shc up-regulation, whereas SIRT1 knockdown exerted op-
posite effects. Moreover, endothelium-specific SIRT1 transgenic
mice had blunted p66Shc gene and protein expression and im-
proved endothelial function, as well as reduced accumulation of
oxidative stress markers, compared with WT littermates [76].
The study by Zhou et al. [76] demonstrated that SIRT1 binds to
the p66Shc promoter (−508 to −250 bp) where it deacetylates
histone 3, thus suppressing gene transcription of the mitochon-
drial adaptor. Decreased SIRT1-dependent deacetylation is the
main epigenetic mark responsible for p66Shc overexpression in
the vasculature (Figure 1). Therefore, one can certainly pos-
tulate that SIRT1 and p66Shc stand along the same molecular
pathway involved in the modulation of vascular and myocardial
integrity during aging (Figure 1). Moreover, SIRT1 and p66Shc
have molecular circuits with the tumour suppressor p53, crit-
ically involved in age-dependent mitochondrial disruption and
apoptosis (Figure 1) [74]. Indeed, SIRT1 inhibition increases p53
acetylation and transcriptional activity [77]. In this regard, p53
is a master regulator of p66Shc transcription [78]. Accordingly,
down-regulation of p66Shc expression as well as inhibition of p53
function in mice restored impairment of acetylcholine-induced
vascular relaxations and increased NO bioavailability [78]. Taken
together, these observations strongly suggest that p53 is a crit-
ical intermediate between the upstream regulator SIRT1 and its
downstream target p66Shc (Figure 1).
SIRT-1 dependent regulation of energy balance
Age-dependent down-regulation of SIRT1 also favours acetyla-
tion of nuclear factor κB(NF-κB) p65, leading to its nuclear
translocation and transcription of inflammatory genes [71]. The
maintenance of sirtuin homoeostasis (SIRT1 and 3) is funda-
mental to repress detrimental pathways of arterial aging such
as forkhead box O (FOXO) [75]. Inactivation of sirtuins during
aging favours FOXO acetylation and subsequent transcription of
FOXO-dependent genes favouring cellular apoptosis, cell cycle
arrest and accumulation of ROS as well as metabolic derange-
ments [79]. FOXOs are key downstream effectors of the phos-
phoinositide 3-kinase (PI3K)/Akt pathway that blocks FOXO
target gene expression. Following stimulation of PI3K/Akt sig-
nalling by growth factors, Akt phosphorylates FOXOs on three
conserved residues, which leads to their cytoplasmic sequestra-
tion and inactivation [75]. By contrast, Akt is inactive in the
aged vasculature thus promoting FOXO-dependent transcrip-
tional programmes, culminating with endothelial senescent phen-
otypes (Figure 1). Genetic studies in experimental animal mod-
els have clearly shown that many lifespan-extending mutations
affect metabolic regulators and control circuits [80,81]. In this
regard, particular attention deserves the regulation of LKB/AMP-
activated protein kinase (AMPK) pathway by SIRT1 [82]. In the
cytoplasm, SIRT1 deacetylates LKB1 leading to activation of the
final effector enzyme AMPK, a central energy regulator involved
in glucose homoeostasis, maintenance of cellular ATP levels and
endothelial integrity via regulation of eNOS activity [74,75].
Perturbation of SIRT1/LKB/AMPK pathway leads to energy im-
balance, cellular stress and activation of the apoptotic machinery
thus contributing to arterial aging (Figure 1) [75,83].
Nuclear factor κB
Increasing evidence suggests that activationof the redox-sensitive
transcription factor NF-κB plays a key role in endothelial activ-
ation and vascular inflammatory changes in aging [31]. NF-κB
is an important transcription factor expressed in all mammalian
cell types [84]. It regulates expression of genes controlling cell
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F. Paneni, S. Costantino and F. Cosentino
Figure 2 Mechanisms underlying age-dependent impairment of vascular healing
spred1, Sprouty-related, EVH1 domain-containing protein 1.
adhesion, proliferation, inflammation and redox state. Activa-
tion of NF-κB mediates vascular and myocardial inflammation
in metabolic and age-related diseases [84]. A previous study
has clearly demonstrated that endothelial suppression of NF-κB
prolongs lifespan in mice and ameliorates obesity-induced en-
dothelial insulin resistance [85]. Impaired insulin signalling is
indeed an important hallmark linking metabolic disease with
premature CV aging [86]. The relevance of these findings is
supported by the notion that NF-κB protein is up-regulated in
vascular endothelial cells isolated from obese and aged adults as
compared with normoweight and young controls [87]. Moreover,
age-dependent NF-κB activation is associated with systemic in-
flammation and impaired endothelial function [88]. Taken to-
gether, these data validate SIRT1 as a key orchestrator of arterial
aging via modulation of oxidative stress, energy balance and vas-
cular inflammation (Figure 1).
ECTOPIC VASCULAR CALCIFICATION
Vascular calcification represents an inevitable hallmark feature
of vascular aging which favours atherothrombosis [89]. Global
measures of coronary artery calcification were shown to inde-
pendently predict both CV events and mortality [90,91]. Current
investigations are now focusing on the possibility that ectopic cal-
cification is mediated by cellular elements acquiring osteogenic
phenotypes. Previous work clearly demonstrated that circulating
osteoblastic cells isolated from human peripheral blood are able
to calcify in vitro and in vivo [92]. These cells, which express
the bone protein osteocalcin (OC) and bone alkaline phosphatase
(BAP), have been considered circulating osteoprogenitor cells
and might participate to vascular calcification and atherosclero-
sis. Indeed, preliminary clinical studies found that coronary ath-
erosclerosis and arterial stiffening are associated with activation
of an osteogenic programme in bone-marrow-derived cells [93].
A study has demonstrated that OC+/BAP+cells originate from
the myeloid lineage and retain monocyte/machrophage markers
[94]. These cells, described as ‘myeloid calcifying cells’ (MCCs),
can be differentiated from pheripheral blood mononuclear cells
and ectopic calcifications in vivo. Interestingly, MCCs are sig-
nificantly increased in patients with Type 2 diabetes and athero-
sclerotic lesions [94], thus providing an important link between
CV risk factors and accelerated arterial aging. Among putative
pathways involved, activation of the transcription factor Runx2
seems to play a major role in osteogenic differentiation (Figure 1).
In conditions of oxidative stress, Akt activates Runx2 which in
turn binds to the promoter of receptor activator of NF-κB ligand
(RANKL), thus favouring changes of VSMC phenotype as well
as differentiation of machrophages into osteoclast-like cells [95].
The Runx2/RANKL pathway is particularly active in senescent
VSMCs, suggesting the importance of this signalling in age-
related calcification and stiffening [96]. In summary, circulating
(MCCs) and resident (VSMCs and machropahges) cells may sig-
nificantly contribute to accelerate ectopic vascular calcification
in aging (Figure 1).
AGE-DEPENDENT DECLINE OF VASCULAR
HEALING PROCESS: CELLULAR AND
MOLECULAR TARGETS
Recovery after ischaemia or infarction in any organrequires blood
vessel growth [97]. The incidence of stroke, claudication and
myocardial infarction all increase in older patients, and they have
worse outcomes when ischaemia and infarction occurs [6]. Un-
derstanding the mechanisms involved in vascular repair is an
important challenge to reduce CVD in aged individuals.
Endothelial progenitor and angiogenic outgrowth
cells
Endothelial progenitor cells (EPCs) and early angiogenic out-
growth cells (EOCs) significantly contribute to endothelial re-
pair, a phenomenon which is less efficient in aging [98]. EPCs
are thought to directly mediate endothelial regeneration, whereas
EOCs represent a heterogeneous pool of cell precursors, mostly
of myeloid origin, which favour endothelial healing via a parac-
rine mechanism [99]. Although we are still far from a clear
understanding of these processes, available knowledge support
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Figure 3 Unmet scientific needs and future applications
BM, bone marrow; MRI, magnetic resonance imaging.
the notion that aging impairs the function of ex vivo-expanded
EPCs [100]. Age-related EPC dysfunction is mediated by the im-
balance between factors promoting growth, migration/survival
and those enhancing oxidative stress/senescence. Hypoxia indu-
cible factor 1α(HIF-1α) induces the expression of stromal cell-
derived factor 1 (SDF-1) that enhances the recruitment of EPCs
in injured or ischaemic tissues in mice. HIF-1α/SDF-1 signalling
is impaired in aging and contributes to altered vascular repair
(Figure 2) [101]. Interestingly, CV risk factors mirror the aging
process by impairing EPCs functionality and, hence, vascular re-
pair [102,103]. Indeed, the aging gene p66Shc is up-regulated in
EPCs isolated from diabetic subjects and contributes to impaired
migration and tube formation [104]. By contrast, vascular repair
capacities are preserved in EPCs isolated from p66Shc−/−diabetic
mice [104]. Hence, targeting p66Shc may contribute to rejuvenate
EPCs thus improving cell functionality and angiogenic properties
(Figure 2).
microRNAs
Recent work suggests that microRNAs (miRs) may be involved
in the pathogenesis of age-related EPCs dysfunction (Figure 2)
[105]. These small non-coding RNAs orchestrate EPCs func-
tionality by regulating gene expression at the post-transcriptional
level. In order to map the microRNA/gene expression signa-
tures of EPCs senescence, Zhu et al. [106] performed a mi-
croRNA profiling and microarray analysis in lineage-negative
bone marrow cells from young and aged as well as ApoE−/−
mice. The analysis found miR-10A∗and miR-21, and their com-
mon target gene high-mobility group AT-hook 2 (Hmga2)as
critical regulators of EPCs senescence. Indeed, overexpression of
miR-10A∗and miR-21 in young EPCs suppressed Hmga2, leading
to increased p16INK4a/p19ARF expression and impaired EPCs
angiogenesis in vitro and in vivo. In contrast, suppression of
miR-10A∗and miR-21 in aged EPCs increased Hmga2 expres-
sion and rejuvenated EPCs, thus improving angiogenesis [106].
Furthermore, miR-34a was found to inhibit EPC-mediated an-
giogenesis by suppressing SIRT1 [107]. Recent studies showed
that reprogramming angiomiR-126 in EOCs and circulating EPCs
restores endothelial homeostasis and favours vascular healing
[99,108]. These novel data suggest the possibility that in vitro
reprogramming of human EPCs may improve vascular repair.
CONCLUSIONS
In the present paper, we have addressed key molecular mech-
anisms implicated in arterial aging and CVD. Premature ac-
tivation of aging genes such as p66Shc and NF-κBaswellas
down-regulation of lifespan determinants JunD and SIRT1 may
trigger senescence features leading to early CVD. Dysregula-
tion of protective and detrimental genes caused by aging and
other CV risk factors suggests the possibility to reprogramme
such modifications in circulating and resident vascular wall cells.
In this perspective, ex vivo reprogramming of autologous EPCs
may rescue age-related cell dysfunction and restore angiogenic
and reparative capacities after myocardial infarction or stroke.
Future experimental and clinical research should address unmet
scientific needs and provide insights for their clinical application
in the context of CV aging (Figure 3).
FUNDING
Our own work was supported by the Swiss Heart Foundation and
the Italian Ministry of Education, University and Research [grant
number PRIN 2010–2011 (to F.C.).] F.P is the recipient of a PhD
programme fellowship in Experimental Medicine at the University
of Rome “Sapienza”.
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Received 13 May 2014/27 June 2014; accepted 22 July 2014
Published on the Internet 5 September 2014, doi: 10.1042/CS20140302
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