Circulation Research. 2007;100:460-473
doi: 10.1161/01.RES.0000258450.44413.96
(Circulation Research. 2007;100:460.)
© 2007 American Heart Association, Inc.
Mitochondrial Dysfunction in Atherosclerosis
Nageswara R. Madamanchi,
Marschall S. Runge
From the Carolina Cardiovascular Biology Center, Department of Medicine, University of North Carolina, Chapel Hill.
Correspondence to Marschall S. Runge, MD, PhD, Department of Medicine, 3033 Old Clinic Building, University of North Carolina, Chapel Hill, NC 27599-7005. E-mail mrunge{at}med.unc.edu
This Review is part of a thematic series on the Role of Mitochondria in Cardiovascular Diseases, which includes the following articles:
Mitochondrial Dysfunction in Atherosclerosis
Free Radicals, Mitochondria, and Oxidized Lipids: The Emerging Role in Signal Transduction in Vascular Cells
Defective Mitochondrial Biogenesis: A Hallmark of the High Cardiovascular Risk in Metabolic Syndrome?
Mitochondrial Biology and Vascular Biology
Role of Mitochondria in Insulin Resistance
Marshall S. Runge Guest Editor
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Abstract
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Increased production of reactive oxygen species in mitochondria,
accumulation of mitochondrial DNA damage, and progressive respiratory
chain dysfunction are associated with atherosclerosis or cardiomyopathy
in human investigations and animal models of oxidative stress.
Moreover, major precursors of atherosclerosishypercholesterolemia,
hyperglycemia, hypertriglyceridemia, and even the process of
agingall induce mitochondrial dysfunction. Chronic overproduction
of mitochondrial reactive oxygen species leads to destruction
of pancreatic ß-cells, increased oxidation of low-density
lipoprotein and dysfunction of endothelial cellsfactors
that promote atherosclerosis. An additional mechanism by which
impaired mitochondrial integrity predisposes to clinical manifestations
of vascular diseases relates to vascular cell growth. Mitochondrial
function is required for normal vascular cell growth and function.
Mitochondrial dysfunction can result in apoptosis, favoring
plaque rupture. Subclinical episodes of plaque rupture accelerate
the progression of hemodynamically significant atherosclerotic
lesions. Flow-limiting plaque rupture can result in myocardial
infarction, stroke, and ischemic/reperfusion damage. Much of
what is known on reactive oxygen species generation and modulation
comes from studies in cultured cells and animal models. In this
review, we have focused on linking this large body of literature
to the clinical syndromes that predispose humans to atherosclerosis
and its complications.
Key Words: oxidative stress DNA damage obesity diabetes aging
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Introduction
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Atherosclerotic vascular disease and its clinical sequelae are
the leading causes of morbidity and mortality in the Western
world. An elevated level of low-density lipoprotein (LDL) is
associated with increased risk of coronary artery disease.
1,2 Oxidative modification of LDL, and its transport into the subendothelial
space of the arterial wall at the sites of endothelial damage,
is considered an initiating event for atherosclerosis.
36 Oxidative modification of LDL results from the interaction of
reactive oxygen species (ROS) and reactive nitrogen species,
produced from vascular wall cells and macrophages,
5 with LDL.
The resulting increased oxidative and nitrosooxidative stress
induces endothelial dysfunction by impairing the bioactivity
of endothelial nitric oxide and promotes leukocyte adhesion,
inflammation, thrombosis, smooth muscle cell proliferationall
processes that exacerbate atherosclerosis.
Of the many potential cellular sources of chronic ROS production, mitochondria and nonphagocytic NAD(P)H oxidase are the major sources under physiological conditions.7,8 Increased mitochondrial ROS generation and dysfunction are associated with cardiovascular and many other diseases.911 Aortic samples from atherosclerotic patients had greater mitochondrial DNA (mtDNA) damage than nonatherosclerotic aortic samples from age-matched transplant donors.12 MtDNA damage not only correlates with the extent of atherosclerotic lesions in apolipoprotein E (apoE) knockout mice but precedes atherogenesis in young apoE knockout mice. Mitochondrial dysfunction, resulting from manganese superoxide dismutase (SOD2) deficiency, increased mtDNA damage and accelerated atherosclerosis in apoE knockout mice, consistent with the notion that increased ROS production and DNA damage in mitochondria is an early event in the initiation of atherosclerosis.
Thus, mitochondrial dysfunction may play an important role in the initiation and development of atherosclerosis. In this review, we will discuss (1) mitochondrial oxidative dysfunction, ROS production, regulatory mechanisms and targets of ROS; (2) the mechanisms by which mitochondrial dysfunction and ROS production could lead to vascular dysfunction and atherosclerosis; and (3) the role of mitochondrial dysfunction in various atherosclerotic risk factors.
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Oxidative Phosphorylation
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ROS are produced by oxidative phosphorylation (OXPHOS) pathway
involved in energy production in mitochondria. OXPHOS is composed
of 5 multiple subunit complexes embedded in the inner mitochondrial
membrane. Electrons are transferred from NADH to molecular oxygen
through an electron transport chain (ETC) consisting of complexes
I (NADH dehydrogenase), II (succinate·ubiquinone oxidoreductase),
III (ubiquinol·cytochrome oxidoreductase), and IV (cytochrome
c oxidase) (
Figure 1). Electrons are donated to complex I from
NADH or to complex II via succinate and passed on to ubiquinol
via coenzyme Q and then ubisemiquinone. Ubiquinol donates electrons
to complex III, which, in turn, transfers electrons to cytochrome
c. From cytochrome
c, electrons move to complex IV and in this
process molecular oxygen is reduced to H
2O. The transfer of
electrons through the ETC leads to pumping of protons across
the mitochondrial inner membrane at complexes I, III, and IV,
creating transmembrane electrochemical gradient,


. The proton-motive
force, which drives the reentry of protons into the matrix,
is used by complex V (ATP synthase) to condense ADP and inorganic
phosphate to synthesize ATP. Matrix ATP is then exchanged for
cytosolic ADP by the adenine nucleotide translocase (ANT).

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Figure 1. Oxidative phosphorylation, superoxide production, and scavenging pathways in mitochondria. Electrons (e) from NADH and FADH2 pass through complex I and complex II, respectively, and then to complex III via ubiquinol. Cytochrome c transfers electrons from complex III to complex IV, which reduces O2 to form H2O. Flow of electrons is accompanied by proton (H+) transfer across the inner mitochondrial membrane at complexes I, III, and IV, creating an electrochemical gradient,  . Protons reenter the mitochondrial matrix through complex V, which uses the proton-motive force to generate ATP. The proton-motive force also drives ATPADP exchange by ANT. UCPs allow protons to return to the matrix, reducing ROS formation. Complex I leaks electrons to generate O2 toward the matrix, whereas complex III generates O2 toward both matrix and intermembrane space. P66Shc in the intermembrane space subtracts electrons from cytochrome c to produce O2 . Superoxide is dismutated to H2O2 by Cu, ZnSOD in intermembrane space and by SOD2 in the matrix. H2O2 is reduced to H2O by glutathione peroxidase (GPX) using GSH, and the resultant oxidized glutathione (GSSG) is reduced back to GSH by glutathione reductase. Pi stands for inorganic phosphate.
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It has been estimated that 0.2 to 2.0% of the molecular oxygen consumed by mitochondria is reduced by a single electron transfer from the ETC to form superoxide anion (O2
).13,14 All the subunits of complex II are encoded by nuclear genes, whereas the subunits of the other 4 complexes are encoded by both nuclear and mtDNA. The human mtDNA is a 16 569-bp circular, double-stranded molecule attached to the mitochondrial inner membrane. Most cells contain hundreds of mitochondria, and each mitochondrion contains 5 to 10 copies of mtDNA.11 The mtDNA contains 13 genes coding for polypeptides essential for OXPHOS, 12S and 16S ribosomal RNA (rRNA) genes and 22 transfer RNA (tRNA) genes required for protein synthesis in mitochondria (Figure 2).

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Figure 2. Human mitochondrial genome encodes 37 genes (16 569 bp; 13 polypeptides, 22 tRNAs, and 2 rRNAs). Each mitochondrion contains several copies of double-stranded, circular DNA bound to the inner membrane on the matrix side (D-loop region). Human mtDNA codes for 7 of the 43 subunits of complex I (NADH dehydrogenase), 1 of the 11 subunits of complex III (ubiquinol: cytochrome c oxidoreductase), 3 of the 13 subunits of complex IV (cytochrome c oxidase), and 2 of 16 subunits of complex V (ATP synthase). The tRNA genes are labeled by letters indicating the cognate amino acids. Mutations in complex I lead to higher ROS production. The common 5-kb deletion is increased several hundred fold in hearts from patients with coronary artery disease. A mutation substituting cytidine for uridine immediately 5' to the mitochondrial tRNAIle anticodon causes hypertension and hypercholesterolemia. Cyt b indicates cytochrome b.
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Because human mtDNA lacks protective histones and many of the repair mechanisms of the nuclear genome,1517 and is located proximal to ROS generation (mitochondrial inner membrane), it is vulnerable to damage by ROS. MtDNA mutations and/or mitochondrial dysfunction are associated with cardiovascular diseases. Atherosclerotic occlusion of coronary arteries and subsequent reperfusion is associated with significant increase in mtDNA damage and concomitant compensatory increase in the expression of OXPHOS genes.18,19 In fact, hearts from patients with coronary artery disease had 8 to 2000 times more mtDNA deletions than their age-matched controls.19 MtDNA damage, in turn, leads to increased ROS production and atherogenesis.20 For example, mutations in complex I genes lead to increased mitochondrial ROS production.21,22
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ROS Production in Mitochondria
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In the mitochondrial electron transport chain, complex IV retains
all partially reduced intermediates until full reduction of
oxygen is achieved.
23 Other complexes may leak electrons to
oxygen, partially reducing this molecule to O
2
. Complex I and III are the primary source of
O
2
production in mitochondria
2325 (
Figure 1). Superoxide is released into the matrix from complex
I, whereas it is released into both the matrix and intermembranous
space by complex III.
26,27 ROS production depends on the metabolic
state of mitochondria. For example, O
2
production is greater during state IV (low electron flow
and ATP synthesis, low ADP levels, high NADH/NAD
+ ratio, and
low oxygen consumption) respiration than in state III respiration
(high electron flow, fast ATP synthesis, partial depolarization,
and a decreased NADH/NAD
+ ratio).
28 In the absence of ADP, electrons
derived from succinate (FADH
2-linked complex II substrate) can
reversely flow to complex I generating increased O
2
production, and, for this reason, complex I
is considered the major physiologically and pathologically relevant
ROS-generating site in mitochondria.
26,29
Superoxide anions in the mitochondrial matrix are quickly dismutated to hydrogen peroxide (H2O2) by SOD2 (MnSOD), whereas those in the intermembranous space are converted by SOD1 (Cu, ZnSOD)30 (Figure 1). Hydrogen peroxide can be reduced to highly reactive hydroxyl radical in the presence of reduced transition metals.31 In mitochondria, H2O2 is reduced to water by the enzymes glutathione peroxidase or catalase.13 Glutathione peroxidase catalyzes the 2-electron reduction of H2O2 using reduced glutathione (GSH) as the hydrogen donor. GSH, a tripeptide consisting of glutamate, cysteine, and glycine, is synthesized in cytosol and transported into mitochondria. The process of reducing H2O2, to produce oxidized glutathione, results in oxidation of GSH. Oxidized glutathione is reduced to yield GSH by the enzyme glutathione reductase using NADPH as the substrate. However, with the exception of mitochondria from the heart, catalase is not present in mitochondria from other tissues.32
Nitric oxide (NO) is produced in mitochondria33,34 and is an important modulator of O2
production, as the ETC contains several NO
reactive-redox metal centers.35 At physiological concentrations, NO
modulates mitochondrial oxygen consumption by inhibiting cytochrome c oxidase in a reversible process.36,37 Also, NO
undergoes radicalradical reaction with O2
at near diffusion-limited rates forming peroxynitrite (ONOO2
), an oxidant capable of irreversible nitration of proteins, inactivation of enzymes, DNA damage, and disruption of mitochondrial integrity.3842
A recent report suggested the involvement of p66Shc in mitochondrial ROS production.43 This protein forms a molecular complex with cytochrome c, subtracting electrons to catalyze the partial reduction of oxygen to form O2
(Figure 1). P66Shc, partially localized in the intermitochondrial membrane space, is a down stream target of p53 and is indispensable for increase in ROS production, cytochrome c release, dissipation of mitochondrial transmembrane potential and apoptosis.44,45 However, p66Shc does not affect mitochondrial transmembrane potential under steady-state conditions, indicating the existence of 2 distinct functional states: an inactive basal state and an active proapoptotic state. In support of this hypothesis, it has been demonstrated that mitochondrial p66Shc exists as a high-molecular-weight complex that includes mitochondrial heat shock protein (mtHSP) 70, and, following proapoptotic signals, p66ShcmtHSP70 complex is destabilized, releasing monomeric p66Shc to interact with cytochrome c.44 The importance of p66Shc in regulating oxidative stress burden is underlined by the observation that p66Shc/ mice have increased resistance to paraquat and a 30% increase in life span.46 Furthermore, p66Shc/ cells have decreased basal and stress-induced ROS levels,45,47 and this was attributed to reduced mitochondrial oxidative phosphorylation.48 That the expression of p66Shc might be relevant in cardiovascular function is evident from the observations that p66Shc/ mice are protected against ROS-dependent, age-related endothelial dysfunction,49 high-fatinduced atherosclerosis,50 as well as susceptibility to hindlimb ischemia.51
ROS produced initially in mitochondria or by enzyme sources such as NAD(P)H oxidase in the cell act in a positive feedback, leading to more ROS production from mitochondria in a process termed ROS-induced ROS release.52,53 In cardiac myocytes, photodynamically triggered ROS production in mitochondria led to subsequent increased mitochondrial ROS generation through induction of mitochondrial permeability transition.52 Support for ROS-induced ROS release was provided by the observation that angiotensin IIinduced cardioprotection against ischemic/reperfusion injury in the rat myocardium is mediated by increased mitochondrial ROS production.54 The protective effect of angiotensin II was eliminated by pretreatment with 5-hydroxydecanoate (an inhibitor of mitochondrial ATP-sensitive potassium channels) and apocynin (an NAD[P]H oxidase inhibitor), and, also, 5-hydroxydecanoate inhibited angiotensin IIinduced ROS formation.
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Mitochondria Are Targets of ROS
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In addition to being a major site of ROS production, mitochondria
are compromised by severe and/or prolonged oxidative stress.
55,56 Oxidative modifications of mitochondrial proteins, lipids, and
mtDNA result in loss of function. Mitochondrial enzymes or enzyme
complexes that are sensitive to inhibition by ROS include aconitase,

-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and complexes
I, II, and III.
57 Oxidative inactivation of mitochondrial DNA
polymerase

could slow mtDNA replication and eventually lead
to inhibition of oxidative phosphorylation.
58 Oxidative inactivation
of ANT would diminish oxidative phosphorylation and the supply
of ATP to the detriment of all energy-dependent processes in
the cell.
59 Similarly, nitration of active-site Tyr34 in SOD2
results in the inactivation of SOD2,
60 and, consistent with
this, increase in 3-nitrotyrosine adduct levels of this enzyme
were correlated with decreased activity in vivo.
61
Cardiolipin, a phospholipid present almost exclusively within the mitochondrial inner membrane, is an early target of ROS either because of its high content of unsaturated fatty acids or proximity to ETC.62 This phospholipid plays an important role in mitochondrial bioenergetics, as it optimizes the activity of some ETC complexes and ANT by binding to cytochrome c.63,64 ROS-induced cardiolipin oxidation impairs complex I activity62 and induces cytochrome c release.65 Thus, oxidative modification of proteins and lipids along with ROS-induced mtDNA damage can alter the cellular energetics impacting the pathophysiology.
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Regulation of ROS Production in Mitochondria
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ROS generation in mitochondria is regulated by a number of factors,
such as oxygen concentration, efficiency of ETC, availability
of electron donors including NADH and FADH
2, and activity of
uncoupling proteins (UCPs) and cytokines.
23,56,66 Mitochondrial
O
2
production increases linearly
with increase in oxygen concentration.
67 By this equation, formation
of ROS should decrease with hypoxia. Yet, a paradoxical increase
in mitochondrial ROS generation was reported under moderately
hypoxic conditions.
68,69 Under hypoxic conditions, NO

produced
at low concentration may bind and inhibit cytochrome
c oxidase,
which results in the reduction of upstream electron transport
complexes and formation of O
2
at low oxygen concentrations.
23,70,71 Peroxynitrite can induce
O
2
formation by inhibiting
respiratory complexes I and II, enhancing the build-up of semiubiquinone
and culminating in apoptosis via the activation of mitochondrial
permeability transition.
72 Ramachandran et al demonstrated that
chronic exposure to high levels of NO

decreases activity and
protein levels of respiratory complexes I, II, and IV, all of
which were accompanied by an increase in cellular
S-nitrosothiol
levels, modification of cysteine residues, and an increase in
the labile iron pool.
73 Taken together, the data that oxygen
and NO

are important regulators of mitochondrial respiration
and ROS formation are compelling.
UCPs are another set of important physiological regulators of mitochondrial ROS production.74,75 Activation of these inner mitochondrial membrane anion transporters allows protons to leak back into the mitochondrial matrix, decreasing the mitochondrial membrane potential and ROS generation.76,77 The regulatory role of UCPs in atherogenesis is inferred from the observation that transplantation of bone marrow from UCP-2deficient mice to LDL receptordeficient mice markedly increased atherosclerotic lesion size and increased nitrotyrosine staining in plaques.78 Consistent with this observation, UCP-2 overexpression inhibited ROS production and apoptosis induced by linoleic acid and lysophosphatidylcholine.79 Conversely, increased O2
production, hypertension, and dietary arthrosclerosis were reported with inducible expression of UCP-1 in aortic smooth muscle cells,80 indicating tissue-specific function of different UCPs. Superoxide, in turn, activates UCPs, lowering proton motive force and attenuating O2
production in a feedback regulation.74,81
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Mitochondrial Dysfunction and Pathophysiological Mechanisms of Atherosclerosis
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Low aerobic capacity is a strong predictor of mortality in subjects
with or without cardiovascular diseases.
8284 On a practical
basis, this can be manifested as poor exercise tolerance and/or
performance with exercise testing. Regardless of the presence
or absence of known cardiac or pulmonary dysfunction, or other
comorbidities, this simple finding correlates significantly
with increased risk of death within 1 year. Expression of OXPHOS
genes is coordinately decreased and is associated with low aerobic
capacity (
Figure 3).
85

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Figure 3. Atherogenic mechanisms of mitochondrial dysfunction. Various agents associated with vascular pathophysiology induce mitochondrial dysfunction and increase ROS production. Mitochondrial dysfunction leads to decreased aerobic capacity, a strong predictor of mortality. Increased mitochondrial ROS production causes endothelial dysfunction/apoptosis and VSMC proliferation/apoptosis, leading to the development of atherosclerosis. Apoptosis of VSMCs and macrophages caused by enhanced ROS production affects atherosclerotic lesion progression and may cause plaque rupture. The figure also depicts the interplay between mitochondrial dysfunction and ROS production. CVD indicates cardiovascular disease.
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Increased production of ROS in mitochondria damages lipids, proteins, and mtDNA. Of these, it is likely that mtDNA is the most sensitive to physiologically relevant ROS-mediated damage. Preferential increase in mtDNA damage (compared with transcriptionally inactive nuclear ß-globin gene), decrease in steady-state levels of mtDNA-encoded mRNA transcripts, mitochondrial protein synthesis, membrane potential, and total cellular ATP pools were observed in vascular smooth muscle cells (VSMCs) and endothelial cells exposed to ROS in cell cultures.39 4-Hydroxynonenal, an end product of membrane lipid peroxidation implicated in the pathogenesis of atherosclerosis, induces VSMC apoptosis through mitochondrial dysfunction and increased production of ROS.86,87 In contrast, increased ROS production attributable to haploinsufficiency of SOD2 isoform reduced aconitase activity in both basal and agonist-stimulated conditions and increased VSMC proliferation.88
Mitochondrial dysfunction is also involved in the increased susceptibility to ischemic injury, certainly, in part, because of opening of the permeability transition pore (PTP)8991 (Figure 4). The molecular components of PTP include ANT in the inner mitochondrial membrane, voltage-dependent anion channel in the outer mitochondrial membrane, cyclophilin D in the matrix, and regulatory molecules such as benzodiazepine receptor, hexokinase, and creatine kinase.92 Transient PTP opening causes depolarization of mitochondrial membrane potential, whereas longer opening leads to matrix swelling and outer mitochondrial membrane rupture. The latter causes the release of proapoptotic molecules within the intermembrane space, leading to cell death via caspase-dependent and caspase-independent mechanisms.93 Consistent with this, mitochondrial depolarization has been implicated in hyperglycemia-induced apoptosis of human aortic endothelial cells (Figure 3).94 Decrease in ANT activity associated with ischemia and inhibition of both ANT activity and oxidative phosphorylation evident during reperfusion may contribute to cardiac failure.95 Chen et al reported that overexpression of SOD2 offered protection against ischemia/reperfusion injury,96 whereas heterozygous deficiency of this enzyme impaired postischemic recovery of the myocardium97 in mice. Together these data support the role of mitochondrial function in protection against ischemia/reperfusion injury.
Various pharmacological inhibitors of mitochondrial energy metabolism significantly increase mitochondrial ROS production and impair endothelium-dependent vascular relaxation.98100 Rotenone (which inhibits electron transport at flavin mononucleotide) abolished acetylcholine-induced, endothelium-dependent relaxation of rat and mouse carotid arteries100 and rat and rabbit aortas.101103 Similarly, antimycin A (which inhibits electron transport at cytochrome bc1) and oligomycin (which inhibits mitochondrial F1ATPase) inhibit the production of endothelial NO in rabbit aorta.103 However, rotenone did not affect vascular relaxation induced by NO donors,102 which suggests that intact mitochondrial function plays an important role in the production of NO in endothelial cells.
Together, these data illustrate that mitochondrial dysfunction impairs aerobic capacity and endothelial function/viability and induces VSMC proliferation or apoptosis, leading to the development of atherosclerosis (Figure 3).
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Dyslipidemia
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Apoptotic endothelial cells, VSMCs, T lymphocytes, and macrophages
are present in atherosclerotic lesions,
104,105 and their number
increases significantly with lesion progression, suggesting
that apoptosis plays a critical role in plaque erosion and rupture.
106,107 Oxidized LDL (oxLDL) induces apoptosis of all cells involved
in atherogenesis
108110 (
Figure 3), and mitochondrial-dependent
pathways play a critical role in this process. OxLDL-induced
apoptosis of human umbilical vein endothelial cells (HUVECs)
is mediated by dysfunction of mitochondrial membrane potential
and the release of cytochrome
c into cytosol and suppression
of apoptosis by cyclosporin A, an antiatherogenic agent, correlated
with the prevention of mitochondrial dysfunction.
111 Recently,
it was shown that oxLDL-induced vascular cell apoptosis involves
2 distinct calcium-dependent mitochondrial pathways.
112 The
first is mediated by activation of the cysteine protease calpain;
release of tBid, a truncated form of the proapoptotic Bcl-2
family member Bid; opening of the mitochondrial PTP; release
of cytochrome
c; and subsequent activation of caspase-3. The
second pathway is mediated by the release of apoptosis-inducing
factor, which is cyclosporine insensitive and caspase independent
(
Figure 4). Interestingly, it was also shown that mitochondrial-derived
O
2
is essential for oxidation
of LDL in vitro.
113
Macrophages in advanced atherosclerotic lesions accumulate excess free cholesterol, which is a potent inducer of their death.114,115 Free-cholesterol loading of mouse peritoneal macrophages induced apoptosis by decreasing mitochondrial membrane potential, inducing cytochrome c release, activating caspase-9, and increasing the levels of the proapoptotic protein Bax.116 OxLDL also induced lysis of human macrophages by promoting mitochondrial dysfunction and scavengers of peroxide radicals that restored mitochondrial membrane potential and prevented macrophage lysis.117 Furthermore, increase in oxidative stress in mitochondria is evident from induction of transcription and expression of SOD2 in human macrophages incubated with oxLDL.118 Consistent with this in vitro observation, SOD2 activity and GSH concentration were higher in atherosclerotic intima compared with the media of the aorta of heritable hyperlipidemic rabbits, but a significant inverse correlation of these 2 with lesion size was also observed. TUNEL-positive nuclei were present in the macrophages of these atherosclerotic aorta and exposure to oxLDL induced increased apoptosis in human macrophages. Hypercholesterolemia significantly increased mtDNA damage and protein nitration of heart homogenates, indicating that atherosclerotic risk factors induce mitochondrial damage and dysfunction.61 MtDNA copy number in leukocytes is redox sensitive119 and low in hyperlipidemic patients.120 Together, these data suggest that dyslipidemia-induced mitochondrial damage and dysfunction not only induce atherosclerotic lesion formation but also affect lesion composition/progression.
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Hypertension
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Hypertension is next in importance to dyslipidemia as a risk
factor for atherosclerosis, and mitochondrial dysfunction has
been implicated in increased arterial blood pressure. Mitochondrial
energy deficiency and calcium overload play a role in the pathogenesis
of arterial hypertension.
121 Similarly, decrease in mitochondrial
energy metabolism and abnormality of calcium metabolism was
reported in hypertrophied myocardium of spontaneously hypertensive
rats.
122,123 Mitochondrial antioxidant system is also important
in protection against hypertension because arterial blood pressure
increased with aging or high-salt diet in SOD2-deficient mice.
124 Mild respiratory uncoupling in arterial smooth muscle cells
increased oxidative stress, hypertension, and dietary atherosclerosis
in mice.
80 Recently, it was shown that a mutation in mitochondrial
tRNA results in hypertension, hypercholesterolemia, and hypomagnesemia.
125 Because cholesterol concentration and blood pressure increase
at approximately 30 years of age in these patients, it is hypothesized
that this gene mutation interacts with environmental or age-related
decline in mitochondrial function in the development of hypertension
and hypercholesterolemia.
126 Together, these findings indicate
a potential role of mitochondrial dysfunction in hypertension.
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Diabetes
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Diabetes mellitus is a major risk factor for coronary artery
disease morbidity and mortality,
127,128 and people with type
2 diabetes experience higher rates of ischemic events and death
after a first myocardial infarction.
129,130 Worldwide, the incidence
of diabetes mellitus continues to increase at rapid rates because
of lifestyle and dietary changes. Understanding mechanisms by
which diabetes mellitus contributes to atherosclerotic risk
is, thus, of great importance.
Insulin resistance is a common occurrence in patients with type 2 diabetes and in subjects with impaired glucose tolerance.131 Insulin resistance causes hyperglycemia as lack of insulin signaling decreases transport of glucose into muscle and fat, while increasing glucose production by the liver.132,133 Hyperglycemia-induced increases in production of O2
by the mitochondrial ETC in endothelial cells has been implicated in glucose-mediated vascular damage.134136 Normalizing mitochondrial ROS levels by an inhibitor of electron transport complex II, by an uncoupler of oxidative phosphorylation, by overexpression of UCP-1 or SOD2 each prevented glucose-induced activation of protein kinase C (PKC), formation of advanced glycation end products (AGE), and activation of the polyol pathway, which results in sorbitol accumulation and nuclear factor
B activationall of which have been implicated in hyperglycemia-induced vascular dysfunction, including atherosclerosis134 (Figure 5). Activation of nuclear factor
B induces expression of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 in aortic endothelial cells stimulating atherogenesis.137

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Figure 5. Insulin resistance and hyperglycemia-induced increased mitochondrial superoxide production activates atherogenic signaling pathways. Overproduction of mitochondrial O2 , caused by high glucose flux through endothelial cells, results in DNA strand breaks and activation of poly(ADP ribose) polymerase (PARP). Poly(ADP ribose) polymerase ribosylates and inactivates glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The upstream metabolites of disrupted glycolytic pathway are processed through polyol pathway, hexosamine pathway, PKC pathway, or AGE pathway. Glycosylation of transcription factor Sp1, by the addition of N-acetylglucosamine (GlcNAc), causes transactivation of proatherogenic genes such as plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor ß1 (TGF-ß1). Activation of transcription factor nuclear factor B (NF- B), downstream of AGE pathway, induces transactivation of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1). Increased flux of FFAs, released from insulin-resistant adipocytes through arterial endothelial cells, also activates hexosamine, PKC, and AGE pathways. In addition, FFA-induced excess mitochondrial O2 production inactivates antiatherogenic enzymes such as prostacyclin (PGI2) synthase and eNOS.
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Hyperglycemia-induced increase in mitochondrial O2
production also decreases glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity and increases hexosamine pathway activity in aortic endothelial cells.138 Activation of the hexosamine pathway causes increased glycosylation and subsequent transactivation of transcription factor Sp1, resulting in increased expression of Sp1-dependent genes such as transforming growth factor-ß1 and plasminogen activator ihibitor-1 (Figure 5). Elevated plasma levels of plasminogen activator ihibitor-1 are strongly associated with increased risk of ischemic heart disease,139,140 whereas transforming growth factor-ß1 plays a key role in early atherosclerosis and restenosis.141 Recently it was shown that the activation of the major pathways of hyperglycemic damage in endothelial cells induced by enhanced mitochondrial O2
production is mediated via inhibition of glycolytic enzyme, GAPDH.136 The GAPDH inhibition is caused by poly(ADP-ribosyl)ation by poly(ADP ribose) polymerase, which is activated by nuclear DNA strand breaks produced by mitochondrial O2
overproduction. Inhibition of GAPDH increases the entry of upstream glycolytic metabolites into pathways of glucose overuse, including increased flux through AGE and PKC glucotoxic pathways.135,142
Central obesity is associated with insulin resistance and is a risk factor for type 2 diabetes and atherosclerosis.143 Central obesity is characterized by increased cytosolic triglyceride levels in adipose and nonadipose tissues.144,145 In tissues, triglycerides are the source of long-chain acylcoenzyme A esters (LCACs), the metabolically active form of fatty acids. In central obesity, LCAC levels are increased because of a steady-state equilibrium with triglycerides.146 Impairment of glucose utilization by LCACs induces insulin resistance in skeletal muscles.144 High concentrations of LCACs decrease intramitochondrial ADP concentration, perhaps by inhibiting ANT,147 leading to increased ROS production.148,149 Incidentally, long-chain fatty acids not only induce hyperplasia in normal islet ß cells but also lower the threshold for glucose-induced insulin secretion.146,150 Thus, insulin resistance may first lead to hyperinsulinemia.148 However, increased levels of free fatty acids (FFAs) will cause progressive deterioration of ß-cell function/apoptosis, resulting in insulin deficiency. This progression from initial hyperinsulinemia to insulin deficiency is characteristic of the development of type 2 diabetes.150152
Incubation of endothelial cells with high concentrations of FFAs, similar to those found in insulin-resistant subjects, has been shown to increase O2
production several fold.153 The increase in ROS production was inhibited by overexpression of either UCP-1 or SOD2, which indicates that mitochondrial ETC is the source of FFA-induced ROS production. Mitochondrial function is also necessary for the progressive oxidation of LDL in endothelial cultures.113 In addition, small dense LDL particles associated with central obesity are more prone to oxidation.143,155 Consistent with this, central obesity and enhanced FFA levels are strongly correlated with clinical, as well as subclinical, coronary heart disease and atherosclerosis in renal transplant recipients.156,157 Together, these data indicate that prolonged enhanced mitochondrial ROS production leads to destruction of ß-cells, dysfunction of endothelial cells, and increased subendothelial LDL oxidationall factors that promote atherosclerosis. Atherosclerotic lesions in brain microvessels from Alzheimers patients and a mouse Alzheimers model have increased mtDNA deletions and mitochondrial abnormalities, demonstrating that mitochondria in the vascular wall are central targets of oxidative stressinduced damage.158
Insulin resistance increases plasma FFA levels because of enhanced lipolytic activity of adipocytes.159 Increased oxidation of FFAs by aortic endothelial cells would lead to accelerated production of O2
by ETC, leading to activation of hexosamine, PKC, and AGE pathways, resulting in the activation of proatherogenic inflammatory pathways135,153,160 and inactivation of the antiatherogenic enzymes prostacyclin synthase153,161,162 and endothelial NOS (eNOS)153,163 (Figure 5). The relevance of these 2 enzymes in atherogenesis is evident from gene knockout models: both apoE//prostacyclin receptor/164 and apoE//eNOS/163 mice showed accelerated atherosclerosis compared with apoE/ mice. Inactivation of prostacyclin synthase and eNOS was prevented by either blocking FFA release from adipocyte tissue or inhibition of mitochondrial fatty acid oxidation and by reduction of O2
levels, which supports the role of enhanced mitochondrial metabolism in accelerated atherosclerosis in people with insulin resistance.153
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Aging
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The incidence and prevalence of coronary heart disease and other
vascular diseases increases with age, and adults >65 years
of age are 4 times more likely to experience coronary heart
disease than are those between 40 to 49 years of age.
165 A large
body of evidence supports the hypothesis that impaired mitochondrial
oxidation is a major contributor to aging.
55,166168 Several
human and animal studies have demonstrated an age-related decline
of mitochondrial respiratory function and ATP synthase activity.
169172 The mitochondrial theory of aging
173 postulates that ROS production,
mtDNA damage, and respiratory chain dysfunction are linked to
each other to create a "vicious cycle" that leads to progressive
decline in mitochondrial function and impairment of cell viability.
It is also true that oxidative mtDNA mutations accumulate with
age in human and animal organs
167,174 and that oxidative mtDNA
damage is inversely correlated to maximal life span.
175
Further support for mitochondrial dysfunction in aging is obtained using genetically engineered mice containing a point mutation in mtDNA polymerase-
(POLG).176,177 The mutant POLG has normal DNA polymerase activity but lacks 3'
5' exonucleolytic proofreading activity, and homozygous POLG mutant mice show an increased load (3- to 8-fold) of somatic mtDNA point mutations that accumulate with increasing age. Consistent with this increase in mtDNA mutations, the mutant mice showed decline in respiratory chain enzyme activities and the production of mitochondrial ATP.176 The mutant mice were found to have reduced life spans and to show symptoms of accelerated aging such as weight loss, hair loss, curvature of the spine, and heart enlargement. However, no increase in ROS levels was observed in these mice, arguing against a direct role of mitochondrial oxidative stress in aging. Instead, it has been suggested that respiratory chain dysfunction per se178 or increased apoptosis177 caused by accumulation of mtDNA mutations may play a central role in aging.
One of the several explanations that could account for the lack of increased ROS production in POLG mutant mice is that mutations in POLG may be downstream from mechanisms that generate ROS and the damage to POLG that renders the enzyme error prone might be the result of protein damage by ROS.179 Consistent with this hypothesis, it was shown that exogenous addition of ROS significantly inhibits the activity of POLG.58 It was also not clear what effect the respiratory chain dysfunction in these mice had on the vascular aging because pharmacological inhibition of mitochondrial energy metabolism could contribute to endothelial dysfunction and increase susceptibility to atherosclerosis.100 In contrast to POLG mutant mice, overexpression of human catalase in mitochondria of mice delayed cardiac pathology, reduced oxidative damage and mtDNA deletions and increased median and maximal life span by
20%.181 At present, increases in the incidence of atherosclerosis and mitochondrial damage and dysfunction with increasing age are merely correlative. Detailed cardiovascular phenotyping of the above mice and other accelerated aging mutants to be developed in the future will help ascertain the causal relationship between mitochondrial dysfunction and aging-associated atherosclerosis.
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Cigarette Smoking
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Cigarette smoking and secondhand smoke significantly increase
the risk of early atherosclerosis, the risk burden being cumulative
and exceeding the active smoking period.
182,183 Atherogenic
effects of cigarette smoking include endothelial injury, platelet
activation, oxidation of LDL, and oxidative DNA damage.
183,184 Consistent with the cumulative and residual risk of cigarette
smoking on atherosclerosis, a significant increase in mtDNA
content was reported in the saliva of smokers and former smokers
compared with never smokers
185 and increase in mtDNA content
is accompanied by mtDNA deletions, evidence of oxidative damage,
decreased transcription of mitochondrial-specific proteins,
and apoptosis.
186188 Significant decrease in complex
IV activity of mitochondrial respiratory chain and increase
in lipid peroxidation of lymphocyte membranes were observed
in circulating lymphocytes from smokers compared with nonsmokers.
189
Further evidence that cigarette smokeinduced mitochondrial dysfunction is important in the initiation and progression of atherosclerotic lesions was obtained from a number of cell culture and animal studies. Exposure to tobacco smoke filtrate caused loss of mitochondrial membrane potential, apoptosis, or necrosis in human monocytes and endothelial cells.190 VSMCs isolated from rats treated with benzo(a)pyrene, a prooxidant in the cigarette smoke, exhibit proliferative phenotype associated with experimentally induced atherogenesis and upregulation of mtDNA transcripts.191 Rats exposed to low concentrations of passive smoke exhibited impaired mitochondrial oxidative function and increased sensitivity of hearts to ischemia/reperfusion injury.192 Similarly, exposure to passive cigarette smoke impaired oxidative phosphorylation, diminished cytochrome oxidase activity, increased mitochondrial F1ATPase protein levels, and decreased coenzyme Q levels in rabbit cardiomyocytes.193 Acute tobacco smoke exposure, as might occur in social settings, increases the susceptibility of rat cardiac mitochondria to calcium and promotes mitochondrial permeability transition.194 Second-hand smoke significantly increased aortic mtDNA damage, decreased ANT activity, and increased nitration and inactivity of SOD2 in mice.61 Exposure to second-hand smoke in the background of hypercholesterolemia increased atherogenesis and synergistically enhanced mitochondrial damage. Furthermore, prenatal exposure to environmental tobacco smoke significantly enhanced mtDNA damage and atherosclerotic lesion development in adult male apoE/ mice.195 Together, these data indicate that cigarette smoking enhances atherogenesis by affecting mitochondrial function, and this effect could be synergistic in the backdrop of other atherosclerotic risk factors.
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HIV and Atherosclerosis
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Highly active antiretroviral therapy (HAART), which involves
a combination of antiretroviral drugs (ie, protease inhibitors
[PIs], nucleoside reverse transcriptase inhibitors [NRTIs],
nonnucleoside reverse transcriptase inhibitors [NNRTIs], nucleotide
reverse transcriptase inhibitors [NtRTIs]) has substantially
reduced morbidity and mortality in patients with human immunodeficiency
virus type-1 (HIV-1) infection. With the increase in the survival
of HIV patients receiving these medications, it is now clear
that there is a relationship between either HAART or HIV-1,
or both, and accelerated atherosclerotic cardiovascular disease.
196200 Because mitochondrial toxicity is a common adverse effect of
HAART, a commonly accepted theory is that the accelerated atherosclerosis
in HAART-treated HIV-1infected patients is attributable
to mitochondrial dysfunction. Phosphorylated NRTIs compete with
endogenous deoxyribonucleotides for incorporation into nascent
mtDNA chains and ultimately inhibit mtDNA POLG, resulting in
mtDNA depletion, altered OXPHOS enzyme activities, and mitochondrial
ultrastructural changes.
201203 Exposure to NRTIs impaired
endothelium-dependent vasorelaxation and increased endothelial
O
2
production in mice.
204 PI therapy can alter lipid metabolism in HIV patients, by either
increasing lipid release or inhibiting chylomicron uptake, and
the resulting hyperlipidemia can, in turn, affect mitochondrial
function and accelerate atherosclerosis.
205 In fact, changes
in atherogenic lipid proteins and endothelial dysfunction have
been associated with PI therapy in HIV patients.
206 In addition,
near-clinical plasma levels of the PI ritonavir caused endothelial
mtDNA damage and cell death.
207 The above data indicate that
mitochondri