Review
This Review is in an ongoing thematic series on Mitochondria in Health and Disease, which includes the following articles:
Mitochondrial Control of Cellular Life, Stress, and Death [Circ Res. 2012;111:1198–1207.]
Mitochondria and Mitophagy: The Yin and Yang of Cell Death Control [Circ Res. 2012;111:1208–1221.]
Mitochondria as a Drug Target in Ischemic Heart Disease and Cardiomyopathy [Circ Res. 2012;111:1222–1236.]
Physiological Roles of the Permeability Transition Pore [Circ Res. 2012;111:1237–1247.]
Mitochondria and Endothelial Function
Mitochondria and ROS Generation
Guido Kroemer and Lorenzo Galluzzi, Guest Editors
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Mitochondria and Endothelial Function
Matthew A. Kluge, Jessica L. Fetterman, Joseph A. Vita
Abstract: In contrast to their role in cell types with higher energy demands, mitochondria in endothelial cells
primarily function in signaling cellular responses to environmental cues. This article provides an overview of
key aspects of mitochondrial biology in endothelial cells, including subcellular location, biogenesis, dynamics,
autophagy, reactive oxygen species production and signaling, calcium homeostasis, regulated cell death, and heme
biosynthesis. In each section, we introduce key concepts and then review studies showing the importance of that
mechanism to endothelial control of vasomotor tone, angiogenesis, and/or inflammatory activation. We particularly
highlight the small number of clinical and translational studies that have investigated each mechanism in human
subjects. Finally, we review interventions that target different aspects of mitochondrial function and their effects
on endothelial function. The ultimate goal of such research is the identification of new approaches for therapy. The
reviewed studies make it clear that mitochondria are important in endothelial physiology and pathophysiology. A
great deal of work will be needed, however, before mitochondria-directed therapies are available for the prevention
and treatment of cardiovascular disease. (Circ Res. 2013;112:1171-1188.)
Key Words: apoptosis
■
biogenesis
■
endothelium
A
large body of work has shown that endothelial dysfunction contributes to the pathogenesis of cardiovascular
disease,1 and considerable effort has been made to define
operative mechanisms. Recent work has emphasized the importance of mitochondria for endothelial function. Although
they have long been recognized for their role in bioenergetics, mitochondria participate in a host of other cellular processes. In contrast to cardiac myocytes and other cell types,
energy requirements in the endothelium are relatively low, and
glycolysis is the major source of ATP production.2 It is now
recognized that endothelial mitochondria play a prominent
■
mitochondria
■
mitophagy
role in signaling cellular responses to environmental cues.3 An
important mode of mitochondrial signaling is the regulated
production of reactive oxygen species (ROS).4 Cardiovascular
risk factors are associated with excess mitochondrial ROS
production that promotes inflammation and decreases nitric
oxide (NO) bioavailability.
This article reviews the key aspects of mitochondrial biology
in endothelial cells, including subcellular location, biogenesis,
dynamics, mitophagy, ROS production, calcium homeostasis,
regulated cell death, and heme synthesis. Because many of
these topics are covered in great detail by other articles in
Original received October 31, 2012; revision received February 26, 2013; final version accepted March 1, 2013. In February 2013, the average time from
submission to first decision for all original research papers submitted to Circulation Research was 11.98 days
From the Evans Department of Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA.
Correspondence to Joseph A. Vita, MD, Professor of Medicine, Boston University School of Medicine, 88 East Newton St, C-818, Boston, MA 02118.
E-mail [email protected]
© 2013 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.111.300233
1171
1172 Circulation Research April 12, 2013
Nonstandard Abbreviations and Acronyms
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ABC–me
ALA
AMPK
DRP1
eNOS
ER
FIS1
LC3
MAM
MAO
miR
mitoKATP
MnSOD
MPT
mtDNA
NO
NOX
PGC-1α
ROS
SIRT1/3
TNF-α
UCP
ATP-binding cassette–mitochondrial erythroid
amino levulinic acid
AMP-activated protein kinase
dynamin-related protein-1
endothelial nitric oxide synthase
endoplasmic reticulum
fission 1
microtubule-associated protein 1 light chain 3
mitochondria-associated membrane
monoamine oxidase
microRNA
mitochondrial ATP-sensitive potassium channel
manganese superoxide dismutase
mitochondrial permeability transition
mitochondrial DNA
nitric oxide
NADPH oxidase
peroxisome proliferator–activated receptor-γ coactivator-1α
reactive oxygen species
Sirtuin 1/3
tumor necrosis factor-α
uncoupling protein
this Circulation Research Review Series, we do not provide a
detailed discussion of each area or extensively review findings
in cardiac myocytes or other cell types. Instead, we provide
an introduction to the key concepts and describe experimental
work in endothelial cells. Each section concludes with a review
of the available human studies relating mitochondrial and
endothelial dysfunction in cardiovascular vascular disease.
Our final section describes experimental and clinical studies
of mitochondria-directed interventions and their effects on
endothelial function.
Mitochondrial Content and Subcellular
Localization in Endothelial Cells
Mitochondrial content in endothelial cells is modest compared
with other cell types with higher energy requirements. In the
rat, for example, mitochondria occupy 2% to 6% of cytoplasmic volume in endothelial cells compared with 32% in cardiac
myocytes.5,6 Mitochondrial content varies by vascular bed and
may relate to function. For example, highly active endothelial cells at the blood–brain barrier have a higher mitochondrial content (8%–11%) compared with endothelial cells in
other capillary beds.6 Mitochondrial distribution within the
cell has been predicted to influence mitochondrial signaling
in the endothelium.7 Consistent with this idea, a recent study
demonstrated that perinuclear clustering of mitochondria and
diffusion of mitochondria-derived ROS into the nucleus contribute to the regulation of hypoxia-sensitive genes in rat pulmonary endothelial cells.8
A few translational studies have examined the importance
of subcellular localization for signaling by mitochondria in human subjects. In arterioles isolated from human myocardium,
for example, mitochondria are anchored to the cytoskeleton
and release ROS in response to cell deformation by shear
stress.9 In this setting, ROS release signals NO production and
flow-mediated dilation.
Mitochondrial Biogenesis and PGC-1α in the
Endothelium
Mitochondrial content depends on the balance between mitochondrial biogenesis and mitophagy (Figure 1). The formation
of new mitochondria is a complex and incompletely understood process involving replication of mitochondrial DNA
(mtDNA) and expression of nuclear and mitochondrial genes.
The peroxisome proliferator–activated receptor-γ coactivator1α (PGC-1α) plays a primary role in this process.10,11 PGC-1α
activates nuclear respiratory factor-1 and nuclear respiratory
factor-2 to coordinate expression of nuclear genes required for
biogenesis. PGC-1α also activates transcription factor A mitochondrial (TFAM) and transcription factor B mitochondrial
(TFBM), which regulate the expression of genes encoded by
mtDNA, including genes for essential subunits of the electron
transport chain.
Studies in nonvascular tissues have shown that stimuli for
mitochondrial biogenesis, such as hypoxia, calorie restriction,
exposure to cold, and exercise, act by increasing the expression
and activity of PGC-1α.10,11 Expression of PGC-1α is controlled
by multiple factors, including NO, sympathetic beta receptor
activation, calcineurin, cAMP, AMP-activated protein kinase
(AMPK), p53, and calcium/calmodulin-dependent protein kinase. Posttranslational modifications further regulate PGC-1α
activity. For example, PGC-1α is phosphorylated by AMPK,
p38 mitogen–activated protein kinase, Akt, and glycogen synthase kinase-3. PGC-1α activity also depends on its acetylation
state; activity is increased by sirtuin-mediated deacetylation
and is decreased by the acetyltransferase GCN5.12
In addition to its role in mitochondrial biogenesis, PGC-1α
also coordinates expression of numerous genes related to glucose and lipid metabolism in cardiac myocytes and other cell
types.11,13 In general, it is activated in states of increased energy demand and serves to augment cellular capacity for ATP
production. PGC-1α also regulates expression of vascular endothelial growth factor-1 and stimulates angiogenesis.14 Given
the energy demands in cardiac myocytes, it is perhaps not
surprising that PGC-1α plays an important role in regulating
metabolism, and it is known that genetic deletion causes heart
failure. Furthermore, cardiac PGC-1α expression is altered in
diabetes mellitus and pressure overload and contributes to the
pathogenesis of heart failure in these settings.13,15
Because energy demands are lower, it is interesting to consider that PGC-1α might play a different role in endothelial
cells. It is known that PGC-1α is expressed in endothelial
cells and regulates mitochondrial biogenesis.10 Additionally,
PGC-1α orchestrates cellular defenses against oxidative stress
that include many mitochondrial proteins. In this regard, overexpression of PGC-1α by adenoviral transfection increases
expression of uncoupling protein (UCP)-2 and mitochondrial
antioxidant enzymes, including manganese superoxide dismutase (MnSOD), catalase, and thioredoxin 2, whereas silencing PGC-1α has the opposite effect.16 PGC-1α overexpression
protects against apoptosis, limits inflammatory activation, prevents activation of c-Jun N-terminal kinase, and improves NO
Kluge et al Mitochondria and Endothelial Function 1173
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Figure 1. Conceptual illustration of the mitochondrial life cycle and the contribution of mitochondrial dynamics and mitophagy to
quality control. Biogenesis is regulated by peroxisome proliferator–activated receptor-γ coactivator-1α (PGC-1α), which activates nuclear
respiratory factor (NRF)-1 and NRF-2 and transcription factor A mitochondrial (TFAM) and transcription factor B mitochondrial (TFBM).
Mitochondria undergo cycles of fusion to form elongated mitochondrial networks and fission into smaller individual organelles. Fusion
is mediated by mitofusin (MFN) 1, MFN2, and optic atrophy protein 1 (OPA1). Fission is mediated by dynamin-related protein-1 (DRP1)
and fission 1 (FIS1). During their normal lifespan and in the setting of increased oxidative stress, damage to mitochondrial components
accumulates. Fission provides a mechanism to isolate damaged components for elimination. Mitophagy involves mitochondrial
depolarization, retention phosphatase and tensin homolog–induced putative kinase protein 1 (PINK1) in the mitochondrial membrane, and
recruitment of Parkin, which targets the mitochondria to autophagosome. P62 also plays a role in targeting cargo to the autophagosome
and is subsequently degraded during active autophagy. Assembly of the phagosome involves beclin-1 and conjugation of microtubuleassociated protein 1 light chain 3 (LC3) onto phosphatidylethanolamine to form of LC3-II. (Illustration Credit: Ben Smith).18
bioavailability.16,17 It further has been argued that PGC-1α–
induced mitochondrial biogenesis protects against oxidative
stress by supplying undamaged mitochondria that produce
less ROS.18
Despite the favorable effects on endothelial phenotype, it
is uncertain whether augmenting PGC-1α will prove useful
as an antiatherosclerosis strategy. Unexpectedly, PGC-1α−/−
mice bred onto an apolipoprotein E−/− background did not have
increased lesion formation.19 Interpretation of this finding is
complicated, however, because PGC-1α−/− mice are lean and
hyperactive, which would tend to limit atherogenesis. In
addition, increased expression of PGC-1β may compensate
for the loss of PGC-1α.20 Studies with endothelium-specific
or double knockout animals may be required to sort out
these possibilities.11 A very recent pilot study showed that
endothelial-specific overexpression of PGC-1α protects
against angiotensin II–induced hypertension.21
With regard to clinical studies, it is well-established that
PGC-1α gene expression and mitochondrial mass are lower in
skeletal muscle of patients with diabetes mellitus and are associated with insulin resistance and impaired bioenergetics.22–24
With regard to the endothelial cells, mitochondrial content is
lower in tissue collected from patients with pulmonary hypertension.25 A recent study showed lower mitochondrial mass
in arterioles isolated from patients with diabetes mellitus by
biopsy of subcutaneous fat.26 Epidemiological studies have
shown that PGC-1α polymorphisms are associated with hypertension,27 carotid atherosclerosis,28 and coronary artery
disease,29 suggesting links with vascular disease. However,
such observational studies do not prove decreased mitochondrial mass or function in the endothelium is important, given
that PGC-1α regulates so many other aspects of metabolism.
Overall, however, the available studies suggest that interventions that activate PGC-1α and stimulate mitochondrial biogenesis protect against cardiovascular disease, and efforts are
underway to identify small molecules that may be developed
as drugs.30
Mitochondrial Dynamics in Endothelial Cells
Although traditionally viewed as discrete organelles, it is now
recognized that mitochondria form networks and undergo cycles of fusion and fission in many cell types, including cardiac
myocytes31 and endothelial cells (Figure 1).18 This process of
mitochondrial dynamics has been extensively reviewed.18,32
Briefly, fusion of the outer mitochondrial membrane is mediated by the transmembrane GTPases mitofusin-1 (MFN1)
and mitofusin-2 (MFN2), whereas fusion of the inner membrane is controlled by optic atrophy protein 1 (OPA1). Fission
is mediated by dynamin-related protein-1 (DRP1) and fission-1 (FIS1). FIS1 recruits DRP1 to mitochondria to initiate
fission.33 DRP1 translocation is inhibited by phosphorylation
at serine-656 by AMPK and is activated by the phosphatase
calcineurin.34
Under physiological conditions, fusion and fission are
balanced and mitochondrial networks are present. Fusion
facilitates distribution of metabolites, proteins, and mtDNA
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and helps maintain electrical and biochemical connectivity.
Likewise, fission is important for normal cell function. For
example, fission is required for cell division and movement of
mitochondria within the cell, and is involved in the elimination
of senescent mitochondria.18 Fission is also an adaptive response
to cellular stress that facilitates the isolation and removal of
damaged mitochondrial components by mitophagy.18 Finally,
mitochondrial fission occurs concomitantly with outer
membrane permeabilization and release of cytochrome c during
apoptosis, although it remains controversial whether fission is
a necessary step in mitochondria-dependent apoptosis.32,35
Recent studies suggest that mitochondrial dynamics are relevant to endothelial cell function. For example, silencing mitofusin-1 and inhibiting fusion reduce angiogenic responses
to vascular endothelial growth factor and Akt-dependent activation of endothelial NO synthase (eNOS).36 Networks are
present in endothelial cells under physiological conditions,
but fragmentation is observed after exposure to hydrogen peroxide,37 high glucose,38,39 and ischemia-reperfusion injury.40
Endothelial expression of DRP1 and FIS1 is increased and
expression of optic atrophy protein 1 is decreased in experimental diabetes mellitus.39 Silencing FIS1 or DRP1 under
conditions of high glucose maintains eNOS activity and NO
bioavailability, likely by decreasing mitochondrial ROS.39
There is emerging evidence that mitochondrial dynamics are altered in the endothelium of patients with cardiovascular risk factors. For example, a recent study showed
mitochondrial fragmentation and increased FIS1 in freshly
isolated endothelial cells from patients with diabetes mellitus.39 Polymorphisms in the optic atrophy protein 1 and mitofusin-2 genes are associated with hypertension.41,42 These
studies suggest links between mitochondrial dynamics and
vascular disease, and raise the possibility that interventions
directed toward restoring normal dynamics might have therapeutic benefit.
Autophagy and Mitophagy in Endothelial Cells
Autophagy is a cellular mechanism for controlled degradation
of proteins, macromolecules, and organelles.43–46 Autophagy
provides energy when the cell is deprived of nutrients and eliminates damaged organelles under conditions of cellular stress.
This process involves formation of the double-membrane autophagosome that engulfs and isolates cellular components
targeted for degradation. The autophagosome subsequently
merges with a lysosome, where the contents and inner membrane are degraded by acid hydrolases and recycled for use
by the cell. Mechanisms of autophagy have been ­extensively
reviewed.43–45 In addition, a review by Kubli and Gustafsson46
appears in this Series on Mitochondria in Health and Disease.
Mitophagy refers to the selective autophagy of mitochondria (Figure 1). As mitochondrial damage accumulates, networks undergo rearrangement and fission to yield different
populations of daughter mitochondria. Those with normal
membrane potential and preserved components can be reincorporated into networks, whereas dysfunctional progeny are
targeted for elimination.18,44 It has been suggested that this
process purges the cell of vulnerable mitochondria that otherwise would undergo outer membrane permeabilization and
induce apoptosis.44 It is interesting that stimuli for mitophagy
also activate PGC-1α and biogenesis, thus providing fresh
replacements for eliminated mitochondria.47 Thus, dynamics,
mitophagy, and biogenesis provide a unified mechanism for
mitochondrial quality control.
Membrane depolarization is an important trigger for mitophagy.44 Under physiological conditions, phosphatase and
tensin homolog–induced putative kinase protein-1 (PINK1)
is imported into mitochondria and degraded via a process that
depends on mitochondrial membrane potential. Membrane
depolarization leads to accumulation of PINK1 at the mitochondrial surface and recruitment of the E3 ubiquitin ligase
Parkin, and may derepress beclin-1 to activate mitophagy.
Ubiquitylation of mitochondrial surface proteins leads to
binding and degradation of p62 and targets the mitochondria
for enclosure in an autophagosome and eventual degradation. Autophagosome formation and maturation involves a
number of proteins, including microtubule-associated protein 1 light chain 3 (LC3)-I, which is a ubiquitin-like protein
that conjugates phosphatidylethanolamine to form LC3-II.
Mitochondria also can be targeted for autophagy through the
action of NIX, which associates with the mitochondrial membrane and interacts with LC3.44
A growing body of work suggests that impaired autophagy
and mitophagy contribute to the pathogenesis of disease.45
PINK1 and Parkin-mediated mitophagy have received considerable attention because impairment of this mechanism
in dopaminergic neurons plays a role in hereditary forms of
Parkinson’s disease.43 Dysregulation of autophagy in a variety
of tissues also has been linked to diabetes mellitus, atherosclerosis, and reduced lifespan.18,48 Impaired autophagy in cardiac myocytes contributes to the pathogenesis of hypertensive
heart disease.49 Depending on the specific setting and degree
of impairment, loss of autophagy may be protective or have
pathological effects with regard to atherosclerotic cardiovascular disease.50,51 Overall, however, autophagy seems to be an
adaptive response in the endothelium, and interventions that
promote autophagy tend to improve vascular function.48
Several recent studies have specifically examined mitophagy in endothelial cells under conditions of oxidative stress and
energy deprivation. Exposure of cultured endothelial cells to
hydrogen peroxide or the glycolysis blocker 2-deoxyglucose
activates AMPK and stimulates autophagosome formation
as reflected by conversion of LC3-I to LC3-II.52 Oxidative
damage induced by mitochondria-targeted irradiation of endothelial cells promotes Parkin translocation to depolarized
mitochondria and increases LC3-II formation.47 Exposure of
endothelial cells to hemin promotes lipid peroxidation, leads
to mitochondrial depolarization, and stimulates mitophagy.53
Interestingly, beclin-1/LC3-II–mediated autophagy is also a
mechanism for clearance of oxidized low-density lipoprotein
in endothelial cells.54 Thus, autophagy and mitophagy seem to
be important responses to oxidative stress in the endothelium.
Altered mitochondrial quality control has been related to
endothelial dysfunction in aging. For example, disordered
mitochondrial dynamics and loss of membrane potential
are observed in cell culture models of senescence.55
Overexpression of the autophagy proteins autophagy-related
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protein-5 (ATG5), autophagy-related protein-12 (ATG12),
and LC3B extends the lifespan of cultured endothelial cells,
and these effects are associated with improved mitochondrial
fitness, as evidenced by higher membrane potential, increased
ATP production, and decreased damage to mtDNA.47 A recent
study demonstrated higher p62 (suggesting an impairment of
p62 degradation that normally occurs during autophagosome
turnover), lower beclin-1 expression, and impaired autophagic
flux, as well as higher superoxide production and blunted
endothelium-dependent dilation in aortic tissue of aged
mice. These abnormalities were reversed by treatment of the
animals with trehalose, a dietary supplement that stimulates
autophagy.56
Relatively few human studies have examined autophagy in
relation to cardiovascular disease. Expression of autophagy
markers, including beclin-1, ATG5, and LC3-II is altered in
myocardial tissue from patients with dilated cardiomyopathy,
and these markers are restored by mechanical unloading.57
Supporting previous work in mice,58 a recent study showed
that exercise activates autophagy in skeletal muscle obtained
by biopsy from older overweight women.59 Although it is
well-known that exercise improves endothelial function,1 it
remains uncertain whether enhanced autophagy contributes to
this effect. A recent translational study showed that p62 protein expression was higher and beclin-1 expression was lower
in endothelial cells taken by biopsy from older healthy human
subjects, consistent with impaired autophagy.56 Furthermore,
lower beclin-1 expression correlated with impaired endothelium-dependent dilation in the forearm. Overall, the available
experimental and human data suggest that enhancing autophagy in the endothelium would be protective against vascular
disease.
Mitochondrial ROS in Endothelial Cells
Sources of Mitochondrial ROS
Given their importance for cell signaling and vascular disease,
there is considerable interest in the sources and regulation of
mitochondrial ROS in endothelial cells (Figure 2). Although
the primary site of oxygen consumption in mitochondria is
cytochrome c oxidase (complex IV), it has been estimated that
superoxide anion formation at complexes I and III accounts
for 0.1% to 2% of the total.4,60,61 These estimates are based on
studies of isolated mitochondria, however, and the percentage attributable to ROS production in endothelial cells in vivo
remains uncertain.61
In addition to complexes I and III, several other sources of
mitochondrial ROS have been identified. For example, nicotinamide adenine dinucleotide phosphate oxidase (NOX) 4 is
highly expressed in endothelial cells and has been localized to
mitochondria in other tissues, although mitochondrial localization in endothelial cells remains uncertain.62 As has been
recently reviewed, NOX4 serves as an important convergence
point for ROS signaling and contributes to endothelial cell senescence, migration, angiogenesis, and adaptive responses to
hypoxia, inflammation, and oxidative stress.63
The monoamine oxidase (MAO) family of enzymes is localized to the outer mitochondrial membrane and generates
ROS during catabolism of catecholamines. ROS produced by
MAO-A has been implicated in maladaptive hypertrophy and
apoptosis of cardiac myocytes exposed to norepinephrine.64
MAO-derived ROS are also associated with adverse cardiac
remodeling and heart failure in response to pressure overload
in mice.64 Hydrogen peroxide produced by MAO-A in vascular smooth muscle cells contributes to serotonin-induced
vasoconstriction.65 Although endothelial cells are known to
express MAO,66 its importance for endothelial function is
poorly understood.
The growth factor adaptor protein p66Shc is another source
of ROS that functions in mitochondrial signaling. p66Shc
generates hydrogen peroxide by oxidizing cytochrome c.67–70
Under physiological conditions, p66Shc may be contained
within a high-molecular-weight inhibitory protein complex
within mitochondria or the cytoplasm. Specific proapoptotic
signals induce release and migration of p66Shc into the mitochondrial intermembrane space. p66Shc is also activated
when the concentration of reduced cytochrome c is relatively
high, such as when complex IV activity is reduced by hypoxia
or because of physiological inhibition by NO, as discussed
below. Furthermore, p66Shc is activated via phosphorylation
by protein kinase CβII in the setting of high glucose and contributes to the development of diabetic endothelial dysfunction.68,69 Thus, p66Shc transduces environmental signals into
ROS that signal apoptosis and other cellular responses.
Another regulator of mitochondrial ROS is the mitochondrial ATP-sensitive potassium channel (mitoKATP). A consensus structure for this inner membrane channel is lacking but
remains the focus of intense study.71 Studies in cardiac myocytes have shown that mitoKATP is activated by superoxide
and hydrogen peroxide and is regulated in a redox-dependent
manner by reactive nitrogen species.72 This channel is perhaps best known for its role in facilitating ROS-mediated
ischemic preconditioning in cardiac myocytes, possibly by
acting as an uncoupling agent that decreases membrane potential and mitochondrial calcium overload, although the
precise mechanism remains unclear.73 In endothelial cells,
mitoKATP is less well-studied, but pharmacological mitoKATP
activation protects against ischemic cell death in cultured
endothelial cells and preserves endothelial vasodilator function in Langendorff-perfused guinea pig hearts subjected to
ischemia-reperfusion.74,75 Inhibition of mitoKATP channels also
blunts high-glucose–induced endothelial cell apoptosis.76
In healthy human volunteers and patients with atherosclerosis, 20 minutes of ischemia followed by reperfusion produces
a marked impairment in endothelium-dependent dilation in
the forearm. This impairment can be prevented by short cycles of cuff occlusion and release before the ischemic insult.
The benefit of such ischemic preconditioning is blocked by
the nonselective KATP channel blocker glibenclamide and is
mimicked by the mitoKATP opener diazoxide.77,78 These human
studies are limited by the nonselective and off-target effects of
these pharmacological interventions but strongly suggest that
mitoKATP activation may contribute to ischemic preconditioning in the human endothelium.
It is interesting that mitochondrial ROS production
can be triggered by ROS from other enzymatic sites by a
process termed ROS-induced ROS release.4,79 For example,
ROS produced by NOX can stimulate mitochondrial ROS
1176 Circulation Research April 12, 2013
Cytosol
H2 O2
catecholamines
Endothelial Cell
OMM
MAO
AO
H2 O2
High glucose
IM Space
O2.-
H+
H+
H+
p66Shc
H+
NO
(Low [O2]) H+
ALA
K+
CytC
CytC
U
UCP2
CP2
Co Q
CoQ
II
IIII
II
IV
IV
V
Cx43
Cx43
ABCme
mitoKATP
mitoKATP
IMM
II
II
H+
NADH
H+
High glucose,
PGC-1α, ↓ROS
AMPK,
Adiponecn
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Matrix
NAD+
H+
FADH2 FAD
O2-.
NO
O2-.
O2
Signaling,
Damage,
Detoxificaon
MnSOD
ONOO-.
H+
H2O
ADP
+ Pi
H+ ATP
K+
↓ I/R Injury
↓ apoptosis
120-160
mV
Diazoxide
MtDNA damage,
Risk Factors:
H2 O2
Vascular
mtDNA
Protein
modificaons
High ALA
glucose
Age,
Smoke exposure,
Hypercholesterolemia,
Hyperglycemia
Figure 2. Illustration of mitochondrial membrane components in endothelial cells related to reactive oxygen species (ROS)
generation. I, II, III, IV, and V indicate complexes I–V of the electron transport chain; ABC–me, ATP-binding cassette–mitochondrial
erythroid; ALA, amino levulinic acid; AMPK, AMP-activated protein kinase; CoQ, coenzyme Q10/ubiquinone; Cx43, connexin 43; CytC,
cytochrome C; OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane; IM space, intermembrane space; MAO,
monamine oxidase; mV, millivolts; mitoKATP, mitochondrial ATP-sensitive potassium channel; MnSOD, manganese superoxide dismutase;
mtDNA, mitochondrial DNA; NO, nitric oxide; PGC-1α, peroxisome proliferator–activated receptor-γ coactivator-1α; UCP2, uncoupling
protein-2; and VEGF, vascular endothelial growth factor.
production by a mechanism that involves opening of the
mitoKATP channel, membrane depolarization, and the
mitochondrial permeability transition (MPT) pore.80,81 In this
situation, inhibiting mitoKATP may be protective and preserves
NO production in endothelial cells exposed to angiotensin
II.81 Thus, ROS produced by NOX, uncoupled NO synthase,
neighboring mitochondria, and other sources can stimulate
mitochondrial ROS production in a multidirectional fashion.
ROS-induced ROS release may amplify and target ROS signals
under physiological conditions but, when excessive, may
contribute to the pathogenesis of endothelial dysfunction.81,82
Mitochondrial ROS Signaling
Mitochondria-derived hydrogen peroxide, which is generated
via dismutation of superoxide by MnSOD or produced directly by NOX4 or p66Shc, can diffuse across membranes and
function in physiological signaling, as has been extensively
reviewed.3,4,8,52 For example, experimental studies have shown
that shear stress–induced vasodilation, hypoxia signaling, autophagy, and proinflammatory activation depend on mitochondrial ROS. In humans, however, there are very limited data
about mitochondrial ROS in physiological cell signaling in the
vasculature. The lack of benefit and, at times, harmful effects of
high-dose nonselective antioxidants as treatment for cardiovascular disease in clinical trials might be interpreted as indirect
evidence that ROS play a physiological role in the vasculature.83
A few clinical studies have more directly implicated ROS
signaling in endothelial function. Flow-mediated dilation in
arterioles isolated from human myocardium depends on the
production of ROS by the electron transport chain.9 In healthy
human volunteers, flow-mediated dilation of the brachial artery is blunted by oral antioxidant treatment, which might
reflect scavenging of mitochondrial ROS.84 A study of arterioles isolated from adipose tissue of patients with coronary
artery disease demonstrated that mitochondrial ROS compensate for the loss of NO and promote flow-mediated dilation.85
Interpretation of those results is complicated, however, because
hydrogen peroxide scavengers, such as N-acetylcysteine, improve endothelium-dependent dilation in coronary arteries of
patients with atherosclerosis.86 The relative importance of mitochondrial ROS for endothelial cell signaling is likely to vary
according to vascular bed and to depend on risk factor burden.
Excess Mitochondrial ROS Production
Altered mitochondrial membrane potential is an important
factor that drives excess mitochondrial ROS production in the
setting of risk factors. Membrane depolarization may activate
ROS production by increasing the activity of complexes I
and III. Membrane hyperpolarization also leads to higher
ROS production and may be particularly relevant to metabolic
disease states in which there is nutrient excess and low demand
for ATP. These conditions are associated with an increase in the
NADH/NAD+ ratio and a decrease in the rate of electron flow
that prolongs the lifespan of reactive intermediates at complexes
I and III. These intermediates promote the reduction of oxygen
to superoxide anion.4,61 It has been suggested that high glucose
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and fatty acid levels in obesity and diabetes mellitus drive
mitochondrial ROS production by this mechanism.87
It is known that nutrient excess alters mitochondrial metabolism in cardiac myocytes and contributes to heart failure.88,89 In
endothelial cells, where energy requirements are lower, excess
substrate stimulates mitochondrial ROS production to signal
a change in endothelial phenotype. For example, exposure of
endothelial cells to high glucose or fatty acid concentrations
decreases NO bioavailability and activates nuclear factorκB and protein kinase C.87,90 These responses are prevented
by mitochondrial membrane depolarization, inhibitors of the
electron transport chain, and overexpression of UCP1, which
may lower membrane potential.90 Interestingly, p66Shc-derived ROS have been implicated in glucose-induced endothelial dysfunction in endothelial cells. p66Shc-null mice made
diabetic by streptozotocin injection are protected against the
development of endothelial dysfunction and display increased
expression of eNOS and heme oxygenase-1.69 Overexpression
of p66Shc in diabetes mellitus is epigenetically regulated by
promoter hypomethylation and histone acetylation, leading to
endothelial dysfunction that may persist despite restoration of
normal glucose levels.68
ROS-induced modifications to mitochondrial components,
including mtDNA, proteins, and lipids, may further exacerbate
ROS production and set-up a vicious cycle that contributes to
vascular disease.88,91 mtDNA damage may lead to decreased
expression of electron transport chain components or expression of defective components that produce more ROS.92 Risk
factors, including older age, cigarette smoking, hypercholesterolemia, and hyperglycemia, are associated with increased
mtDNA damage in cardiovascular tissues.93 mtDNA damage
correlates with the extent of atherosclerosis in mouse models
and human tissues.94 It has recently been proposed that glycation of mitochondrial proteins might also alter their function
and increase ROS in diabetes mellitus.95
Modified mitochondrial proteins accumulate in disease
states. In the early stages of disease, the combination of dynamics, mitophagy, and biogenesis may replace damaged
mitochondrial components and maintain normal mitochondrial function. However, impairment of these quality-control
mechanisms leads to the retention of dysfunctional mitochondria that produce excess ROS and promote vascular disease.
It is interesting to note that methods are available to measure
mtDNA damage in human tissues.91 Oxidative damage to
mtDNA in circulating leukocytes has considerable potential
as a reliable marker of mitochondrial dysfunction that can be
made in readily available blood cells. However, large-scale
application of this approach has not been reported.
Only a few human studies have specifically implicated mitochondria as the source of excess ROS in patients. In patients
with diabetes mellitus, circulating leukocytes and arterioles
isolated from subcutaneous fat display higher mitochondrial ROS production and membrane hyperpolarization.26,96
Furthermore, impaired endothelium-dependent vasodilation
in freshly isolated arterioles from diabetic individuals is reversed by mild membrane depolarization or mitochondria-targeted antioxidants.26 Interestingly, a recent study showed that
low glucose concentrations also stimulate mitochondrial ROS
production, membrane hyperpolarization, and endothelial
dysfunction in cultured cells and isolated human arterioles.97
These findings might explain clinical trials suggesting that
tight glucose control actually might be harmful in patients
with diabetes mellitus and suggest that mitochondria-derived
ROS from any cause can induce endothelial dysfunction.97
Genetic studies suggest a link between mitochondrial proteins and hypertension. Polymorphisms in mitochondrial
genes, including a subunit of complex IV, correlate with blood
pressure in the Framingham Heart Study.98 In addition, there
is a maternal link in the heritability of blood pressure that may
implicate the mitochondrial genome.99 However, the consequences of these polymorphisms for ROS production and their
specific importance for endothelial function remain unknown.
Mitochondrial Antioxidant Enzymes
The first line of defense against mitochondrial superoxide is
MnSOD. MnSOD is located in the mitochondrial matrix and
catalyzes the conversion of superoxide anion to hydrogen peroxide. Consistent with a protective role in the endothelium,
MnSOD+/− mice exhibit impaired endothelium-dependent vasodilation.100 In addition, apolipoprotein E−/− MnSOD+/− mice
display earlier mtDNA damage and accelerated atherosclerosis compared with apolipoprotein E−/− MnSOD+/+ mice.94
Levels of hydrogen peroxide are regulated by antioxidant
enzymes, including catalase and several mitochondrial and
cytosolic peroxidases. Catalase is located in cytosolic peroxisomes. Important mitochondrial peroxidases include thioredoxin-2, peroxiridoxin-3, and glutaredoxin-2. Glutathione
peroxidase-1 is located both in mitochondria and in the cytosol in endothelial cells.4,101 As noted, increased expression of
these enzymes is signaled by AMPK and PGC-1α in response
to hydrogen peroxide and other forms of oxidative stress in
endothelial cells.16,17 Studies in experimental models have
shown that reduced expression of mitochondrial antioxidant
enzymes can induce mitochondrial damage, endothelial dysfunction, and atherogenesis.94,100,102 Conversely, overexpression of these proteins is protective against the development of
vascular disease.103
Uncoupling Proteins
There is considerable interest in mitochondrial UCPs and
their role in the regulation of ROS production.104,105 UCPs
are localized to the inner mitochondrial membrane and
facilitate movement of protons down their concentration
gradient into the matrix, thus making membrane potential
less negative and uncoupling electron transport chain activity
from ATP production. UCP1 is expressed in brown fat and
plays a role in adaptive thermogenesis. UCP2, which is the
primary isoform in endothelial cells, and UCP3 have high
sequence homology with UCP1 but are expressed at lower
levels. UCP2 and UCP3 contribute to the regulation of
mitochondrial membrane potential when they are activated by
superoxide and act in a compensatory fashion to decrease ROS
production (Figure 2). There is controversy about how ROS
dynamically activate UCP2 and UCP3. In this regard, studies
have implicated protein modification by 4-hydroxynonenal
and reversible glutathionylation.105–107 Although UCPs may
function in an adaptive manner in most circumstances, it also
has been suggested that activation of UCPs in myocytes under
conditions of nutrient excess may increase ROS production in
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the electron transport chain. This effect may lead to increased
myocardial oxygen consumption and reduced cardiac effi­
ciency in type 2 diabetes mellitus.108
Experimental studies confirm the importance of UCP2 in
endothelial cells. Decreasing expression with antisense oligonucleotides increases mitochondrial membrane potential and
ROS production.104 Conversely, UCP2 overexpression reduces
ROS production and inflammatory activation in cultured cells
and improves endothelium-dependent dilation in isolated rat
aorta.109 Expression of UCP2 is dynamically regulated, and
exposure to ROS, nutrient excess, and other forms of cellular
stress stimulate UCP2 expression as part of a stress response
coordinated by AMPK and PGC-1α.16,17 In vivo studies have
shown increased atherosclerosis, ischemic stroke, and impaired
endothelium-dependent vasodilation in UCP2-null mice.110–112
There is epidemiological evidence that UCP2 may relate
to human cardiovascular disease. For example, the 866 G>A
variant is associated with increased UCP2 expression and a
decrease in the incidence of cardiovascular disease events.113
Polymorphisms at this locus also may be associated with carotid artery atherosclerosis.114 Interpretation of these findings
is complicated by the observation that UCP2 and UCP3 gene
variants are also linked to incident type 2 diabetes mellitus.115
The direct importance of UCP for the regulation of endothelium-dependent vasodilation or other aspects of endothelial
function has not been directly examined in humans.
NO
NO is another important regulator of mitochondrial bioenergetics and oxidant production. The effects of NO are complex and
depend on the relative availability of oxygen and NO, as well as
the cellular redox state.116 NO inhibits complex IV (cytochrome
c oxidase) by competing with oxygen binding at the binuclear
heme a3/CuB center. In endothelial cells with physiological
levels of bioactive NO, this interaction inhibits mitochondrial
respiration and facilitates ROS signaling.4,116 When cellular levels of reduced glutathione are relatively low in states of oxidative stress, NO has a chronic inhibitory effect on complex I
via S-nitrosylation in a manner that blunts mitochondrial ROS
production. High levels of NO, which may occur in acute inflammatory states, reduce mitochondrial reserve capacity in endothelial cells, which may reflect a decreased ability to respond
to cellular stress.117 Conditions that reduce endothelial NO
bioavailability may promote excess mitochondrial ROS production by withdrawing the inhibitory effects of NO.4,97 Under
such conditions, peroxynitrite formed by the reaction of NO
and superoxide may modify complexes I and III in a manner
that further increases ROS production.81 Antioxidant enzymes,
including MnSOD, are also inactivated by peroxynitrite, further
exacerbating oxidative stress within mitochondria.118
Mitochondria and Calcium Homeostasis in the
Endothelium
Cytosolic calcium levels regulate many aspects of endothelial function. For example, receptor-dependent agonists,
such as acetylcholine and serotonin, activate eNOS by increasing cytosolic calcium and stimulating the binding of
calcium/calmodulin.119 Calcium activates calcium/calmodulin-dependent protein kinase II, which plays a role in eNOS
gene expression and phosphorylation state, and regulates actin
cytoskeletal elements that influence endothelial cell shape,
motility, and barrier function.120 Calcium is also involved in
vascular endothelial growth factor signaling and angiogenic
functions of the endothelium.121
As has been extensively reviewed,122–125 mitochondria and
the endoplasmic reticulum (ER) cooperate in the regulation of
calcium trafficking and thereby control key aspects of endothelial function. In brief, mitochondria serve as calcium buffer
sites and regulate calcium uptake and release by the ER.123,124
Although the ER is the major storage site, ≈25% of cellular
calcium is localized to mitochondria. During cell stimulation,
continuous flux of calcium through mitochondria is needed
for store-operated entry and ER calcium store refilling.126
Localized calcium uptake as well as the position of mitochondria in relation to the plasma membrane produce calcium microdomains that are important in these processes.124,125,127
Calcium movement in and out of mitochondria is highly
regulated.124,125 Generally speaking, the availability of multiple
uptake and extrusion channels serves to buffer mitochondrial
calcium levels over the physiological range of cytosolic calcium. When calcium load exceeds the buffering threshold,
mitochondrial calcium increases rapidly and may trigger
mitochondrial-dependent apoptosis. In endothelial cells, hydrogen peroxide–induced increases in mitochondrial calcium
may depend, in part, on decreased calcium extrusion via inhibition of the sodium/calcium exchanger.128 Because calcium
flux through mitochondria is important for ER calcium stores,
inhibition of calcium extrusion by hydrogen peroxide may
deplete ER calcium stores and thereby trigger the unfolded
protein response and apoptosis.125,126
The ER maintains specific domains for direct interaction
with mitochondria known as the mitochondria-associated
membrane (MAM). As has been recently reviewed, the MAM
tethers the ER to mitochondria and is enriched with proteins
relevant to calcium and lipid metabolism.129 MAMs facilitate
calcium exchange that is important for ER regulation of mitochondrial energetics and apoptosis.129
A recent study suggests that MAMs are relevant to pulmonary hypertension. In pulmonary arterial vascular smooth
muscle cells, disruption of mitochondria–ER interactions by
the reticulon protein Nogo-B impairs hypoxia-induced apoptosis, leading to the typical proliferative lesions seen in pulmonary hypertension.130 Interestingly, Nogo-B expression is
also increased in endothelial cells in pulmonary hypertension,
and it has been suggested that endothelial cells also may assume a proliferative apoptosis-resistant phenotype in this condition. However, the specific importance of Nogo-B and other
aspects of MAM biology have not been extensively studied in
endothelial cells.
Physiological changes in mitochondrial and cytosolic
calcium concentrations have important regulatory effects
on many aspects of mitochondrial function, including ROS
production, energetics, motility, dynamics, and biogenesis.4,123
Calcium activates Krebs cycle enzymes and oxidative
phosphorylation, thus increasing ATP production.124,129
Cytosolic calcium regulates mitochondrial motility through
the Miro-Milton protein complex, which interacts with the
outer mitochondrial membrane and couples mitochondria
to microtubules.125,131 Miro acts as a calcium sensor and is
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responsible for the retention of mitochondria in regions of highcalcium levels. Cytosolic calcium plays a role in mitochondrial
dynamics through calcineurin, which activates DRP1 and
stimulates mitochondrial fission.18,124,131 Finally, mitochondrial
calcium is an important regulator of mitochondrial biogenesis
and increases expression of PGC-1α.124
There is evidence that altered mitochondrial calcium contributes to endothelial responses in pathological states. For
example, high glucose increases cytosolic and mitochondrial
calcium and alters histamine signaling in cultured human
endothelial cells.38 Mitochondrial calcium uptake and ROS
production are required for the shedding of cell membrane
receptors for tumor necrosis factor-α (TNF-α) in response to
TNF-α stimulation. This compensatory effect blunts the extent
of endothelial activation under proinflammatory conditions.132
A recent human study examined the transient receptor potential vanilloid type 4 (TRPV4) channel, a cell membrane
mechanosensitive calcium channel, in myocardial arterioles
isolated from patients with coronary artery disease.133 In that
study, activation of transient receptor potential vanilloid type 4
channels stimulated calcium entry, mitochondrial ROS release,
and endothelium-dependent vasodilation.133 It has been suggested that transient receptor potential vanilloid type 4 channels are localized in close proximity to mitochondria associated
with cytoskeletal elements9,133 and may provide an example of
how calcium microdomains contribute to mitochondrial ROS
signaling in humans. Overall, there is a paucity of translational
studies of mitochondrial calcium flux in the endothelium.
Mitochondrial Regulation of Endothelial Cell Death
Mitochondria are implicated in cell death pathways, including apoptosis and programmed necrosis (necroptosis).46,134
Necroptosis is initiated by death receptors, such as TNF receptor 1, and involves the activities of receptor-interacting
proteins 1 and 3.135,136 Studies suggest that mitochondria
contribute to necroptosis execution. For example, TNFα–induced necrosis requires the formation of protein complexes that include receptor-interacting protein 1 (RIP1) and
receptor-interacting protein 3 (RIP3), and the mitochondrial
phosphoglycerate mutase-5/protein phosphatase (PGAM5)
at the mitochondrial outer membrane.137 Interestingly, phosphoglycerate mutase-5 activates DRP1 by dephosphorylation,
thus facilitating mitochondrial fragmentation.137 Nitration of
mitochondrial complex I and membrane depolarization also
contribute to necroptosis in endothelial cells.138 Although
myocardial cell necroptosis contributes to ischemic injury and
heart failure,135 relatively little is known about the importance
of this process in the endothelium.
Mitochondria are also central mediators of apoptosis.32,139,140
Briefly, intrinsic apoptosis is initiated by cellular stressors, including hypoxia, ROS, oxidized low-density lipoprotein, and
DNA damage. Such stimuli activate BH3-only proteins, which
inhibit antiapoptotic factors, including B-cell lymphoma 2
(BCL-2), and allow activation of BCL2-associated X protein
(BAX) and BAK. It is generally held true that these proapoptotic factors induce mitochondrial outer membrane permeabilization and depolarization, opening of the MPT pore and
the release of mitochondrial proteins into the cytosol.32,123,139
However, recent studies have questioned the role of MPT
pore opening in intrinsic apoptosis.141 The intrinsic apoptosis
pathway also involves mitochondrial fission, because DRP1
and BAX/BAK cooperate in the induction of outer membrane
permeabilization and fission.32,140,142 Release of cytochrome c,
second mitochondria-derived activator of caspases/DIABLO,
HtrA2/Omi, and other mitochondrial proteins activate caspases and apoptosis.139
The MPT pore has received considerable attention because
of its possible role in cell death pathways as well as its role in
ischemia-reperfusion injury and ischemic preconditioning. As
recently reviewed, the MPT pore is believed to be a multicomponent and relatively large nonselective channel.143 Although
the essential and nonessential components of the MPT pore
remain highly controversial, both adenine nucleotide translocase and cyclophilin D serve regulatory functions, and the
roles of the voltage-dependent anion channel and BAX have
been established as nonessential.143–145 Under physiological
conditions, the pore is functionally closed. In vitro experiments with endothelial cells suggest that short MPT openings may serve a protective function, dissipating membrane
potential in response to oxidative stress or calcium overload,
whereas lengthy MPT openings may trigger apoptosis.80,123,141
However, it remains uncertain whether short or prolonged
MPT openings contribute to physiological or pathological
processes in vivo.
In the endothelium, cardiovascular risk factors can trigger
apoptosis by multiple mechanisms. Apoptosis is observed in
cells exposed to high glucose and in animal models of diabetes mellitus, and under these conditions, apoptosis is inhibited
by interventions that decrease ROS or inhibit MPT pore opening.146,147 Hyperglycemia-induced apoptosis has been shown to
involve activation of nuclear factor-κB and protein kinase C,
which, as discussed, can activate p66Shc and trigger the MPT
pore.67,68 Downregulation of connexin 43 in the inner mitochondrial membrane in retinal endothelial cells also contributes to apoptosis under high glucose conditions.148 In cardiac
myocytes, connexin 43 is known to play a role in ischemic preconditioning,149 possibly by functioning as an uncoupling protein or potassium channel to decrease mitochondrial ROS.150
Another reported stimulus for apoptosis in endothelial cells
and renal podocytes is phosphorylation of DRP1 at serine-600
by rho-associated coiled-coil containing protein kinase 1
(ROCK1).34,151 This mechanism may have clinical relevance
because rho-associated coiled coil-containing protein kinase 1
inhibitors are in development as drugs.151 As discussed, DRP1
is also activated by dephosphorylation at serine-656 by calcineurin in response to increased cytosolic calcium, suggesting
multiple regulatory mechanisms that link mitochondrial dynamics and calcium signaling with apoptosis.
In addition to diabetes mellitus, other risk factors may
provoke apoptosis in endothelial cells. Senescent endothelial
cells are more sensitive to proapoptotic stimuli,152 which may
be attributable in part to decreased expression of BCL-2.153
Relevant to hypertension, angiotensin II induces apoptosis
in cultured endothelial cells.154 As discussed, angiotensin II–
stimulated apoptosis represents an example of ROS–induced
ROS signaling and is mediated by NOX, protein kinase
C, mitochondrial ATP-sensitive potassium channels, and
peroxynitrite formation.81 The effects of angiotensin II are
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blunted by overexpression of the mitochondrial peroxidase
thioredoxin 2, further suggesting that mitochondrial ROS
play a role in this response.103 Relevant to dyslipidemia and
atherosclerosis, oxidized low-density lipoprotein has been
shown to induce apoptosis in endothelial cells by increasing
mitochondrial ROS and activating p53, which leads to BAX
translocation to the mitochondrial membrane and release of
cytochrome c.155,156
Several human studies suggest that endothelial apoptosis
may be important in vascular disease. For example, serum
from patients with heart failure induced apoptosis in cultured
endothelial cells, as reflected by DNA fragmentation and mitochondrial cytochrome c release.157 In addition, circulating endothelial microparticles from patients with heart failure display
markers of apoptosis. Similarly, serum from patients with acute
coronary syndrome induces endothelial cell apoptosis, and the
effect was blunted by treatment with an angiotensin-converting enzyme inhibitor.158 Although the circulating factors that
induced endothelial cell apoptosis were not identified in those
studies, similar changes were induced in isolated endothelial
cells by TNF-α, which is known to be increased in patients
with heart failure and coronary artery disease.157 Finally, study
of surgical specimens from patients undergoing carotid endarterectomy demonstrated endothelial apoptosis in regions of
low shear stress, which are known to be prone to atherosclerosis.159 Overall, these limited human studies show associations
between mitochondria-regulated cell death pathways and cardiovascular disease, and may suggest targets for therapy.
Heme Synthesis and Metabolism in the
Endothelium
The mitochondrion is the site of heme synthesis.160 Heme is essential for the function of electron transport chain complexes
and a variety of other proteins, including eNOS. Heme synthesis begins with the formation of amino levulinic acid (ALA)
by ALA synthase-1. ALA is then transported from the mitochondrion to the cytoplasm, where it is converted to coproporphyrinogen III. This intermediate is transported back into the
mitochondrion and converted to protoporphyrin IX. Ferrous
iron (Fe2+) is incorporated into protoporphyrin IX to form heme.
In addition to providing enzyme prosthetic groups, heme
synthesis may function to reduce oxidative stress by sequestering redox active iron that would otherwise promote ROS
formation. Inhibition of heme synthesis in rats is associated
with decreased NO production and impaired endothelium-dependent vasodilation.161 Interestingly, expression of ALA synthase-1 is regulated by PGC-1α,162 possibly coordinating heme
synthesis with other cytoprotective responses, such as mitochondrial biogenesis and expression of antioxidant enzymes.
The mitochondrial inner membrane transport protein ATPbinding cassette–mitochondrial erythroid (ABC–me) plays a
role in heme synthesis and reduction of oxidative stress. ABC–
me expression is regulated by heme and was initially recognized because of its role in erythropoiesis.163,164 ABC–me is
believed to function as an ALA transporter and to stabilize mitoferrin 1, a mitochondrial iron importer. Interestingly, cardiac
ischemia-reperfusion injury in mice heterozygous for ABC–
me (ABC–me+/−) is associated with increased ROS production,
oxidative damage to mitochondria and the sarcoplasmic/ER
calcium/ATPase pump, impaired mitochondrial respiration,
and more severe cardiac dysfunction.164 These effects may be
attributable to altered heme synthesis and mitochondrial iron
homeostasis. A preliminary study demonstrated that ABC–me
is expressed in endothelial cells and that high glucose lowers
ABC–me expression, which might inhibit heme synthesis and
thereby contribute to mitochondrial ROS production and vascular dysfunction.165
Heme is metabolized by heme oxygenase-1, which forms
biliverdin and carbon monoxide, and releases iron. Biliverdin
is subsequently converted to bilirubin. It has been argued
that heme oxygenase may have both pro-oxidant and antioxidant effects in the cell.160,166 Release of redox active iron may
increase the formation of ROS, and carbon monoxide inhibits complex IV in a manner that increases ROS.167 On the
contrary, bilirubin is an antioxidant and carbon dioxide has
vasodilator properties that mimic the generally favorable effects of NO via activation of guanylyl cyclase.168 Heme oxygenase expression is induced by AMPK via the transcription
factor nuclear respiratory factor-2.169 A recent study showed
that endothelial-selective constitutive activation of AMPK
prevented the development of endothelial dysfunction in a
heme oxygenase–dependent manner in a mouse model of
diabetes mellitus.169 Heme oxygenase also has been shown
to have antiapoptotic effects in endothelial cells.170
Several clinical studies have examined the links between
heme metabolism and human cardiovascular disease. For example, patients with diabetes mellitus have reduced red blood
cell ALA synthase activity.171 A large number of observational
studies have shown correlations between heme oxygenase-1
expression in circulating blood cells and coronary artery disease and diabetes mellitus.166 Many of these studies show
higher levels of heme oxygenase-1 in patients with risk factors or vascular disease. These findings might implicate heme
oxygenase in disease pathogenesis or could represent a compensatory response. It is impossible to distinguish these possibilities or to draw conclusions about the utility of targeting
heme oxygenase for therapy, given the observational design of
these studies. The specific importance of heme synthesis and
heme oxygenase activity for endothelial function has not been
directly examined in humans.
Interventions That Improve Mitochondrial
Function in the Endothelium
As discussed, a variety of risk factors induce mitochondrial
dysfunction in the endothelium, which may contribute to the
pathogenesis of atherosclerosis. This observation prompts
speculation that interventions that restore mitochondrial
function might be protective.172 A list of potential interventions
that might affect vascular disease is provided in Table 1. Also
refer to the review by Walters et al73 that focuses on these and
other interventions that affect mitochondrial function in the
myocardium.73
Mitochondria-Directed Antioxidants
There is considerable interest in the therapeutic potential of
mitochondrial-directed antioxidants. Because mitochondrialderived ROS are important for signaling, a strategy that reduces
ROS to physiological levels rather than completely eliminates
Kluge et al Mitochondria and Endothelial Function 1181
Table 1. Potential Interventions to Improve Mitochondrial
Function in the Endothelium
Intervention
Effect
Mitochondrial-targeted
compounds
Scavenge mitochondrial ROS
MitoQ
Mito-α tocopherol
Mito-tempol
S-nitroso-TPP
Mitochondrial-targeted NO donor
Lipoic acid
ROS scavenger, cofactor for Krebs cycle
enzymes
Carnitine/acetyl-l-carnitine
Fatty acid transport into mitochondria
Activators of AMPK/PGC-1α
Activate biogenesis
Calorie restriction
Inhibit apoptosis
Exercise
Decrease ROS
2-Deoxyglucose
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Metformin
Thiazolidinediones
Resveratrol
Sirtuin activators
Activate eNOS
Improve angiogenesis
Promote mitochondrial biogenesis
Reduce mitochondrial ROS
Improve energetics
Mdivi-1
Inhibit DRP1 and mitochondrial fission
FCCP, dinitrophenol
Membrane depolarizers (current agents are
limited by toxicity)
AMPK indicates AMP-activated protein kinase; DPR1, dynamin-related
protein-1; eNOS, endothelial nitric oxide synthase; FCCP, carbonyl cyanide
p-(trifluoromethoxy) phenylhydrazone; Mdivi, mitochondrial division inhibitor-1;
MPT, mitochondrial permeability transition; NO, nitric oxide; PGC-1α, peroxisome
proliferator–activated receptor-γ coactivator-1α; ROS, reactive oxygen species;
TPP, triphenylphosphonium.
ROS, likely will be important.173 One strategy to target mitochondrial ROS is to link antioxidant compounds with a lipophilic cation, such as triphenylphosphonium.172 Mitoquinone
(MitoQ) is a triphenylphosphonium-modified ubiquinone that
is reduced to ubiquinol within the mitochondria matrix. A similar approach has been used to direct α-tocopherol or the SOD
mimetic tempol to mitochondria.
MitoQ had favorable effects in preclinical studies of ischemia-reperfusion injury, sepsis, adriamycin toxicity, and
Parkinson’s disease.172 Regarding endothelial function, MitoQ
was shown to inhibit oxidative damage and apoptosis in cultured endothelial cells,174 blunt amyloid light chain–induced
dysfunction in isolated arterioles,175 and improve endothelial function and lower blood pressure in hypertensive rats.176
Mito-Tempol lowers blood pressure and improves endothelium-dependent vasodilation in mouse models of hypertension.82 Clinical studies have examined MitoQ for the treatment
of Parkinson’s disease177 and chronic hepatitis C178 with mixed
results. At the present time, it remains unknown whether
MitoQ or other triphenylphosphonium-linked compounds will
have favorable effects on endothelial function or cardiovascular disease in humans.
Lipoic Acid and Acetyl-l-Carnitine
Lipoic acid is a mitochondrial antioxidant and a coenzyme required for the activity of the Krebs cycle. l-carnitine plays an
important role in energy production by conjugating fatty acids
for transport into mitochondria. Oral carnitine supplementation in humans is frequently formulated as acetyl-l-carnitine,
which enters mitochondria and improves mitochondrial function to a greater extent than l-carnitine.179 In animal models of
aging, diabetes mellitus, and hypertension, the combination
of lipoic acid and acetyl-l-carnitine reverses abnormalities
of mitochondrial membrane potential, ATP production, and
ROS production in a variety of tissues.180 In cultured endothelial cells, lipoic acid improves NO production, blunts inflammatory activation, and increases the antiapoptotic protein
BCL-2.181–183 Dietary supplementation with lipoic acid or
acetyl-l-carnitine improves NO bioavailability and reduces
atherosclerosis lesion formation in animal models.184,185
Lipoic acid, l-carnitine, and acetyl-l-carnitine are available
over-the-counter as dietary supplements, and their effects on
vascular function have been examined. Lipoic acid improves
endothelial function in patients with metabolic syndrome
or diabetes mellitus, and l-carnitine blunts free fatty acid–
induced endothelial dysfunction.186–188 It remains unclear,
however, whether the beneficial effects of these compounds
on endothelial function can be attributed to an effect on mitochondrial function.
Interventions That Activate AMPK and PGC-1α
Interventions that increase the activity of AMPK and PGC-1α
have been consistently shown to improve endothelial function
in animals. For example, short-term calorie restriction reverses
endothelial dysfunction and oxidative stress in old mice.189
Calorie restriction also promotes mitochondrial biogenesis
and increases expression of MnSOD.189,190 The glycolysis
inhibitor 2-deoxyglucose activates AMPK, induces autophagy,
and protects against cell death in cultured endothelial cells.52
The thiazolidinediones, including pioglitazone, also have
been reported to activate PGC-1α and increase mitochondrial
biogenesis in endothelial cells.191 Metformin, which activates
AMPK, has been shown to inhibit MPT pore opening and
endothelial apoptosis, and to prevent the development of
endothelial dysfunction in experimental models.17,147
With regard to clinical studies, it is well-established that
exercise and weight loss improve endothelial vasodilator
function in patients with obesity, diabetes mellitus, and other
risk factors.1 A weight loss and exercise program increased
expression of PGC-1α in skeletal muscle of elderly women.59
Metformin reverses endothelial dysfunction in patients with
diabetes mellitus,192 although it remains unclear whether
metformin actually activates AMPK in vivo. Given the many
favorable effects of these interventions on risk factors and metabolism, mechanisms beyond improved mitochondrial function may be important.
Sirtuins
The sirtuins play important roles in the regulation of lifespan
and metabolism, with implications for mitochondrial function,
and may be targets for therapy, as was recently reviewed.193,194
Sirtuins regulate gene expression and enzyme activity via their
activity as NAD+-dependent deacetylases. Sirtuin 1 (SIRT1)
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deacetylates and activates PGC-1α, leading to favorable effects on glucose and lipid metabolism and expression of mitochondrial genes.193,195–197 SIRT3 is specifically localized to the
mitochondrial matrix and controls mitochondrial energetics
by deacetylating mitochondrial enzymes.193,198 Interestingly,
SIRT3 also deacetylates and activates MnSOD, and thus may
reduce mitochondrial superoxide levels.199
With regard to the cardiovascular system, there is extensive evidence that SIRT1 has protective effects against ischemic injury and hypertrophy in the heart.193 SIRT1 is known
to deacetylate and activate eNOS in the endothelium and
to regulate angiogenesis.193,200,201 In a study of elderly human subjects, SIRT1 expression was reduced in endothelial
cells isolated from the brachial artery and correlated with
­endothelium-dependent vasodilation.202
There is limited evidence that the mitochondrial SIRT3
may have favorable effects in endothelial cells, although it is
unclear whether such effects are mediated through alterations
in mitochondrial function. For example, endothelial-specific
inhibition of nuclear factor-κB in a mouse model is associated with reduced inflammation, improved insulin sensitivity,
enhanced mitochondrial biogenesis, improved blood flow, and
increased lifespan.203 In vascular tissue from these animals,
there is upregulation of SIRT3.203 In humans, aging is associated with reduced expression of SIRT3 in skeletal muscle of
sedentary, but not endurance-trained, individuals. To date, the
effects of specific SIRT3 activation on endothelial function
have not been examined in experimental models or human
subjects. However, there is currently great interest in the possibility that activators of SIRT1 and SIRT3 will have benefits
against metabolic and cardiovascular disease.194
Resveratrol
Resveratrol (3,4′,5-trihydroxystilbene) is a polyphenolic compound found in a variety of foods, including grapes and red
wine. Initial experimental studies reported that resveratrol activates SIRT1.204 More recent evidence suggests that the beneficial effects of resveratrol also involve activation of AMPK.205
In animal models, resveratrol mimics the beneficial effects of
calorie restriction on insulin sensitivity, mitochondrial biogenesis, and longevity.204
Resveratrol induces mitochondrial biogenesis in endothelial cells by increasing PGC-1α and nuclear respiratory factor-1
in a manner that depends on SIRT1 and eNOS activation, and
has similar effects in the aorta of diabetic (db/db) mice.206 In
endothelial cells, resveratrol has antioxidant effects and has
been shown to increase the expression of MnSOD and to reduce cellular hydrogen peroxide levels.207 Furthermore, resveratrol has cytoprotective properties and prevents induction
of apoptosis in the setting of oxidative stress by upregulation
of BCL-2.208 This constellation of atheroprotective effects has
generated significant interest for therapeutic applications. In
obese humans, oral resveratrol treatment was shown to acutely
improve endothelium-dependent vasodilation.209 A number of
other ongoing clinical trials are examining resveratrol treatment for metabolic and vascular end points but, to date, the
effects of long-term resveratrol treatment on vascular function, mitochondrial function, or cardiovascular disease remain
unknown.
Inhibition of Mitochondrial Fission
Clinical studies suggest that diabetes mellitus and other
pathological states are associated with increased mitochondrial fission and impaired autophagy in the endothelium.39,56
Mitochondrial division inhibitor-1 (Mdivi-1) is an inhibitor of
the fission protein DRP1 and has been shown to decrease mitochondrial outer membrane permeabilization, cytochrome c release, and apoptotic cell death.210 Mdivi-1 also has been shown
to limit ischemia reperfusion injury in isolated myocytes and
to reduce infarct size in a mouse model.211 The effects of this
compound on endothelial function are unknown.
Mitochondrial Membrane Depolarizing Agents
UCPs, including UCP2, act to induce mild membrane depolarization and reduce mitochondrial ROS production.104
Overexpression of UCP2 improves endothelial function in a
rat model,109 suggesting that an uncoupling agent might have
therapeutic potential. In arterioles isolated from patients with
diabetes mellitus, the uncoupling agent carbonyl cyanide p(trifluoromethoxy) phenylhydrazone lowers membrane potential and improves endothelial function.26 2,4-dinitrophenol is
another uncoupling agent that has been used in humans for
the treatment of obesity.172 Clinical trials with this compound
were stopped because of high toxicity, but it remains possible
that less potent, self-limiting uncoupling agents might prove
useful mitochondria-directed therapeutics.172
MicroRNAs
There is recent appreciation of the importance of microRNAs
(miRs) in the coordination of many biological processes.212 In
pulmonary endothelial cells, miR-210 has been identified as a
repressor of iron–sulfur cluster assembly proteins 1/2, which
are important for the expression of components of the electron
transport chain and Krebs cycle.213 Under conditions of
hypoxia and activation of hypoxia-inducible factor-1α, miR210 is upregulated, thereby repressing mitochondrial iron–
sulfur enzymes and mitochondrial respiration in endothelial
cells. This response may be important for the Pasteur effect
and the mammalian adaptation to hypoxia.213 Given the recent
interest in miRs as therapeutic targets,212 these results suggest
that targeting miR-210 might be an approach to broadly
regulate mitochondrial respiration and endothelial function.
Conclusions
This article reviews the key aspects of mitochondrial function
in the endothelium. Mitochondrial content in endothelial cells
is relatively modest, and subcellular localization relative to the
nucleus, cytoskeletal elements, calcium channels, and the ER
is important for ROS and calcium signaling. In addition to regulating biogenesis, PGC-1α orchestrates a cellular response
to oxidative stress by increasing the expression of mitochondrial antioxidant enzymes and UCPs. Mitochondrial qualitycontrol mechanisms, including mitochondrial dynamics and
autophagy/mitophagy, are needed for normal angiogenic
and vasodilator function of endothelial cells. Mitochondrial
ROS are important for signaling physiological responses to
nutrient status, hypoxia, and shear stress. An array of antioxidant enzymes, UCPs, and other mechanisms in mitochondria
combine to modulate and direct ROS signals. Mitochondria
Kluge et al Mitochondria and Endothelial Function 1183
Table 2. Translational Methods for Study of Mitochondria and Endothelial Function in Humans
Method/Approach
Examples
Lead Author
Isolated arterioles
Mitochondria anchored to cytoskeleton are involved in shear stress–mediated NO
release
Liu9
ROS mediates flow-induced vasodilation in adipose arterioles in patients with
coronary artery disease
Phillips85
Increased mitochondrial ROS and membrane potential in diabetes mellitus
Endothelial dysfunction reversed by FCCP and mito-tempol in adipose arterioles
Kizhakekuttu26
Calcium microdomains important for flow-mediated dilation of coronary arterioles
Bubolz133
Decreased mitochondrial mass in pulmonary hypertension
Xu25
Increased FIS1 and mitochondrial fragmentation in diabetes mellitus
Shenouda39
Increased p62, lower beclin-1 in elderly subjects
Larocca56
Decreased SIRT1 in elderly subjects
Donato202
Increased mtDNA damage
Ballinger94
Apoptosis in endothelial cells from low-shear stress regions adjacent to carotid
plaques
Tricot159
PGC-1α associated with hypertension, carotid, and coronary atherosclerosis
Oberkofler,27 Iglseder,28 Zhang,29
OPA1, MFN2 associated with hypertension
Wang,41 Jin42
Polymorphisms in mitochondria-encoded genes associated with blood pressure and
blood glucose
Liu98
Polymorphisms in UCP2 related to coronary disease events
Cheurfa113
Endothelial cells obtained by biopsy
Excised atherosclerotic lesions
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Genetic polymorphisms
Endothelium-dependent vasodilation Beclin-1 correlated with impaired endotheliumdependent vasodilation
Intervention studies
Larocca56
Ischemic preconditioning and diazoxide prevent ischemia-reperfusion injury in the
forearm, implicating mitoKATP channels
Broadhead,77 Loukogeorgakis78
Flow-mediated dilation inhibited by ROS scavengers in healthy volunteers, possibly
implicating ROS in signaling
Richardson84
Lipoic acid, l-carnitine, metformin, and resveratrol treatment improve endothelial
function
Heinisch,186 Sola,187 Shankar,188 Mather,192
Wong209
FCCP indicates carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone; FIS1, fission 1; MFN2, mitofusin-2; mtDNA, mitochondrial DNA; mitoKATP, mitochondrial
ATP-sensitive potassium channel; NO, nitric oxide; OPA1, optic atrophy protein 1; PGC-1α, peroxisome proliferator–activated receptor-γ coactivator-1α; ROS, reactive
oxygen species; UCP2, uncoupling protein-2.
interact with the ER to regulate calcium signaling, which is
essential for the key aspects of endothelial function, including eNOS activation, motility, barrier function, and angiogenesis. In general, physiological ROS signaling and calcium
signaling activate short-term or long-term functional changes
in endothelial cells to maintain blood flow to tissues through
control of vascular tone and angiogenesis. Physiological ROS
signaling that promotes inflammation is important for normal
immune function. Such signals are also involved in regulated
cell death pathways, which eliminate senescent and irreversibly damaged cells. Excessive ROS and disordered calcium
signaling, however, contribute to the pathogenesis of vascular
disease. Finally, we reviewed the importance of mitochondria
in heme synthesis and metabolism, which, in addition to generating heme for use as an enzymatic cofactor, may be emerging as another aspect of mitochondrial function that regulates
cellular redox state in the endothelium.
Although a large body of experimental work has shown that
mitochondria are important for endothelial function, only a
relatively small number of translational studies have shown the
clinical relevance of these mechanisms in humans (Table 2). As
described, studies of isolated vascular tissue and endothelial
cells from patients with diabetes mellitus and elderly humans
indicate the importance of subcellular location, biogenesis,
dynamics, autophagy, and pathological ROS levels. Only a few
studies have investigated the signaling aspects of ROS and calcium, and little is known about the specific importance of endothelial PGC-1α, regulated cell death, and heme metabolism
in humans. Epidemiological studies have shown correlations
between human disease and polymorphisms of genes related
to mitochondrial function. Blood markers of mitochondrial
dysfunction, such as mtDNA damage and mitochondrial oxygen consumption in circulating leukocytes or platelets, may
facilitate studies of systemic abnormalities of mitochondrial
function and their relation to cardiovascular disease. The presented studies suggest that mitochondria-directed therapies
have potential for the prevention and management of cardiovascular vascular disease, in part by improving mitochondrial
function in the endothelium.‍
Sources of Funding
J.L. Fetterman receives support from T32 HL007224. J.A. Vita is supported by National Institutes of Health grants HL081587, HL083801,
HL102299, and HL083781 from the National Heart, Lung, and Blood
Institute.
Disclosures
None.
1184 Circulation Research April 12, 2013
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Mitochondria and Endothelial Function
Matthew A. Kluge, Jessica L. Fetterman and Joseph A. Vita
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Circ Res. 2013;112:1171-1188
doi: 10.1161/CIRCRESAHA.111.300233
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