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FOCUS SEMINAR: OXIDATIVE STRESS AND CARDIOVASCULAR DISEASE

STATE-OF-THE-ART REVIEW

Oxidative Stress and Cardiovascular Risk:Obesity, Diabetes, Smoking, and PollutionPart 3 of a 3-Part Series

Bernd Niemann, MD,a Susanne Rohrbach, MD,b Mark R. Miller, PHD,c David E. Newby, MD,c

Valentin Fuster, MD, PHD,d,e,f Jason C. Kovacic, MD, PHDd

ABSTRACT

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Oxidative stress occurs whenever the release of reactive oxygen species (ROS) exceeds endogenous antioxidant capacity.

In this paper, we review the specific role of several cardiovascular risk factors in promoting oxidative stress: diabetes,

obesity, smoking, and excessive pollution. Specifically, the risk of developing heart failure is higher in patients with

diabetes or obesity, even with optimal medical treatment, and the increased release of ROS from cardiac mitochondria

and other sources likely contributes to the development of cardiac dysfunction in this setting. Here, we explore the role

of different ROS sources arising in obesity and diabetes, and the effect of excessive ROS production on the development

of cardiac lipotoxicity. In parallel, contaminants in the air that we breathe pose a significant threat to human health. This

paper provides an overview of cigarette smoke and urban air pollution, considering how their composition and biological

effects have detrimental effects on cardiovascular health. (J Am Coll Cardiol 2017;70:230–51)

© 2017 by the American College of Cardiology Foundation.

O xidative stress is a pervasive aspect of car-diovascular disease (CVD) and occurswhenever the release of reactive oxygen

species (ROS) exceeds endogenous antioxidant capac-ity. Although physiological levels of ROS are impor-tant signaling molecules, prolonged exposure orinappropriate subcellular localization of ROS canhave detrimental effects. Previously in this review se-ries, we covered the basic biology of oxidative stress,telomeres, and telomere dysfunction in Part 1 (1), andthe role of oxidative stress in both heart failure and

m the aDepartment of Adult and Pediatric Cardiovascular Surgery, Univers

ysiology, Justus-Liebig University, Giessen, Germany; cBHF/University o

een’s Medical Research Institute, University of Edinburgh, Edinburgh, U

rdiovascular Institute, Icahn School of Medicine at Mount Sinai, New Yor

vascular Health Center, Icahn School of Medicine at Mount Sinai, New Y

aciones Cardiovasculares, Madrid, Spain. Dr. Rohrbach has received resea

TG1566, SFB1213). Drs. Miller and Newby have received funding from g

266, SP/15/8/31575, and FS/16/14/32023) and chair (CH/09/002) awards f

eived research support from the National Institutes of Health

SFRN20490315 and 14SFRN20840000), and The Leducq Foundation (Tra

thors have reported that they have no relationships relevant to the conten

ller, and Newby contributed equally to this work. Kathy Griendling, PhD,

nuscript received January 16, 2017; revised manuscript received April 25,

vascular disease in Part 2 (2). Here in Part 3, weaddress the role of several specific and importantrisk factors, namely, diabetes, obesity, smoking, andexcessive pollution, and how they increase cardiovas-cular risk via increased oxidative stress.

OBESITY, DIABETES, AND OXIDATIVE STRESS

METABOLISM OF THE OBESE AND THE DIABETIC

HEART. The metabolic phenotypes of the diabeticand the obese heart have many similarities: fatty acid

ity Hospital Giessen, Giessen, Germany; bInstitute of

f Edinburgh Centre for Cardiovascular Science, The

nited Kingdom; dThe Zena and Michael A. Wiener

k, New York; eMarie-Josée and Henry R. Kravis Car-

ork, New York; and the fCentro Nacional de Inves-

rch support from the German Research Foundation

rants (PG/10/042/28388, RG/10/9/28286, FS/10/024/

rom the British Heart Foundation. Dr. Kovacic has

(R01HL130423), the American Heart Association

nsatlantic Network of Excellence Award). All other

ts of this paper to disclose. Drs. Niemann, Rohrbach,

served as Guest Editor for this paper.

2017, accepted May 10, 2017.

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http://dx.doi.org/10.1016/j.jacc.2017.05.043

AB BR E V I A T I O N S

AND ACRONYM S

AGE = advanced glycation end-

product

C-DEAP = combustion-derived

environmental air pollution

CVD = cardiovascular disease

DE = diesel exhaust

DM = diabetes mellitus

ETC = electron transfer chain

IRS = insulin receptor

substrate

NOX = nicotinamide adenine

dinucleotide phosphate

oxidase

ROS = reactive oxygen species

J A C C V O L . 7 0 , N O . 2 , 2 0 1 7 Niemann et al.J U L Y 1 1 , 2 0 1 7 : 2 3 0 – 5 1 Role of Oxidative Stress and CV Risk Factors

231

uptake and fatty acid oxidation (FAO) are increased,as are levels of intramyocardial and circulating tri-acylglycerol and circulating free fatty acids (FFAs),and glucose uptake and glucose oxidation arereduced (3). Despite reduced glucose uptake, there isincreased flux through accessory pathways of glucosemetabolism, such as the polyol pathway or the hex-osamine biosynthetic pathway (4). In the healthyheart, utilization of FFAs and glucose is well-balanced, and enables the heart to switch betweenenergy sources according to their availability and inresponse to environmental stimuli. This provides theheart with a high degree of flexibility in substrateutilization. The inability of the obese or diabetic heartto appropriately use glucose results in a reliance onFAO and reduced metabolic flexibility. The concur-rent cardiac inefficiency is related to increased mito-chondrial uncoupling induced by fatty acids, as wellas the low oxygen (O2) utilization efficiency of FAO(5), resulting in decreased adenosine triphosphate(ATP) production despite fuel oxidation. Metabolicalterations observed in the obese or diabetic heart,such as increased FAO, mitochondrial dysfunction,glucose autoxidation, impaired polyol metabolism,or increased hexosamine metabolism, can causeincreased ROS release. Accordingly, metabolite-generated ROS play a major role in the developmentof various diabetes-related cardiovascular complica-tions. The major metabolic changes in the obese anddiabetic heart are summarized in Figure 1.

ROLE OF MITOCHONDRIAL ROS IN OBESITY AND

DIABETES. Across various organs, including theheart (reviewed in Szendroedi et al. [6]), impairedmitochondrial respiration and changes in mitochon-drial morphology have been consistently observed ininsulin resistance and type 2 diabetes mellitus (DM).Patients with type 2 diabetes have significantly lowercardiac phosphocreatine/ATP ratios (7,8), decreasedcardiac oxidative capacity, and increased mitochon-drial ROS emission (9). Obesity results in disturbedmitochondrial biogenesis and function (respiratorychain complex I), which occurs prematurely inyounger patients with obesity (10). Preclinical studiessuggest early ROS up-regulation during diabetes-induced cardiac remodeling, but analogous prospec-tive studies in patients with diabetes are lacking.Mitochondrial dysfunction, increased mitochondrialROS release, and mitochondria-dependent cell deathhave been reported in the diabetic human heart(9,11–14). Mitochondrial dysfunction appears to bepresent mainly in cardiac subsarcolemmal but not ininterfibrillar mitochondria of patients with type 2diabetes (13). Interestingly, impaired mitochondrial

function and contractile dysfunction wereobserved in patients with diabetes, but not inpatients with obesity (14). Anderson et al. (9)also demonstrated increased levels of4-hydroxynonenal– and 3-nitrotyrosine–modified proteins, together with a reductionin the ratio of reduced to oxidized gluta-thione in diabetic human hearts, indicatingpersistent oxidative stress in these samples.

Under physiological conditions, electrontransport to O2 is tightly coupled to ATPsynthesis. ROS generation by mitochondriaoccurs through electron leakage from theelectron transport chain (ETC) due to adecreased rate of mitochondrial phosphory-lation. Normally, <1% of total oxygen con-sumption leaks from the ETC to generate
ROS. Nevertheless, mitochondria are the primarysource of cellular ROS production in cardiomyocytes,and increased mitochondrial ROS are a major causeof oxidative stress associated with DM. Thehyperglycemia-induced overproduction of superox-ide (O2
�) by the ETC is recognized as a major cause ofthe clinical complications associated with diabetesand obesity (15). Indeed, attenuation of mitochon-drial ROS release results in completely preservedinsulin sensitivity despite a high-fat diet (16,17).Accordingly, mice with overexpression of amitochondrially-targeted catalase show reduced ROSrelease and do not develop insulin resistance despitea high-fat diet (16). Recently, it was also shown that amitochondria-targeted antioxidant prevents insulinresistance and diastolic dysfunction, suggesting thatmitochondrial oxidative stress may be involved inboth conditions (18).

Hyperglycemia increases ETC flux, resulting inmitochondrial hyperpolarization and O2

� generation(19). Hyperglycemia-induced mitochondrial over-production of ROS activates 4 major pathwaysinvolved in the pathogenesis of cardiovascular com-plications, including the increased production ofadvanced glycation end-products (AGEs), the polyolpathway, the hexosamine pathway, and proteinkinase C–dependent signal transduction (15). Mito-chondrial ROS production can be blocked by certainETC inhibitors or uncoupling agents (19). Uncouplingproteins (UCPs) such as UCP2 and UCP3, the majorhuman cardiac UCP isoforms (20), can dissipatethe electrochemical gradient and thus attenuate ROSproduction (21) at the expense of decreased cardiacefficiency. Up-regulation of cardiac UCP2 and/orUCP3 has been reported in some, but not inall studies using preclinical models of obesity andDM (22).

FIGURE 1 Metabolic Changes in the Obese and Diabetic Heart

Overnutrition results in increased levels of circulating fatty acids and glucose. Fatty acids induce an activation of cardiac peroxisome

proliferator-activated receptor alpha (PPARa), which enhances the expression of genes that control fatty acid uptake and oxidation as well as

glucose oxidation (pyruvate dehydrogenase kinase 4 [PDK4]). The consequent switch from glucose to free fatty acid oxidation results in

metabolic inflexibility and a decrease in cardiac efficiency. The accompanying mitochondrial dysfunction contributes to increased reactive

oxygen species (ROS) production, uncoupling of the electron transport chain (ETC), and reduced adenosine triphosphate (ATP) production.

Accumulation of toxic lipids and lipid derivatives such as ceramides can directly alter cellular structures and induce cardiomyocyte

dysfunction and cell death, collectively called lipotoxicity. Fatty acids also contribute to the activation of serine/threonine kinases, which

inhibit insulin signaling via insulin receptor substrate (IRS), finally resulting in reduced glucose uptake. Toxic effects of glucose (gluco-

toxicity) include formation of advanced glycation end-products (AGEs), a group of modified proteins and/or lipids with damaging potential,

as well as increased activity of the hexosamine and polyol pathways.

Niemann et al. J A C C V O L . 7 0 , N O . 2 , 2 0 1 7

Role of Oxidative Stress and CV Risk Factors J U L Y 1 1 , 2 0 1 7 : 2 3 0 – 5 1

232

FFAs may increase ROS generation in mitochondriafunctioning in the forward electron transport modeby slowing down the rate of electron flow throughcomplexes I and III of the ETC due to interactionswithin the complex subunit structure (23). Further-more, their effect on the electron flow betweencomplexes III and IV can increase ROS generation dueto the release of cytochrome c from the inner mito-chondrial membrane (23,24). However, a minordepolarization of the inner mitochondrial membranewas suggested to abolish mitochondrial ROS genera-tion, called the mild uncoupling concept (25). FFAscan also decrease ROS generation due to theiruncoupling action, albeit only under conditions ofreverse electron transport (succinate as substrate)(23). Initially, enhanced fatty acid–induced mito-chondrial uncoupling of the obese and diabetic heartmay therefore represent an adaptation to increasedfatty acid–induced ROS production. However, thisdoes not completely compensate for the increasedROS production, as evidenced by the increasedROS-related damage seen in obesity or DM (22). Over

the long term, uncoupling significantly contributesto the energetic deficit, with a decreased cardiacphosphocreatine/ATP ratio in diabetic hearts (8).

Mitochondria are not only a source, but also atarget of ROS. In particular, mitochondrial deoxy-ribonucleic acid (DNA) appears to have increasedsusceptibility to oxidative damage, which may beattributable to low mitochondrial DNA repair capacityand lack of histones, the close proximity to the ETC,or oxidative damage to mitochondrial proteins andlipids. This increased susceptibility has beenobserved in patients or animals with diabetes orobesity and in vitro models (10,22,26,27). Mitochon-drial ROS also impair mitochondrial respirationvia oxidative post-translational modifications ofcomplexes I and II of the ETC, whereas scavenging ofmitochondrial ROS inhibited cardiac hypertrophy andimproved diastolic function in a preclinical model ofobesity (28). The molecular mechanisms linking ROSwith cardiac hypertrophy and diastolic dysfunctioninclude oxidative modification and inhibition ofsarcoplasmic reticulum calcium ATPase and

FIGURE 2 Major ROS Sources in Cardiomyocytes

Most ROS in cardiomyocytes are produced at the mitochondrial ETC and by nicotinamide adenine dinucleotide phosphate oxidase (NOX) 2. In

addition, other cytosolic sources, such as cyclooxygenase (COX) or xanthine oxidase (XO), contribute to ROS production in cardiomyocytes

from patients with obesity or diabetes. Other mitochondrial proteins, such as p66shc, monoamine oxidases A (MAO A), and NOX4, are also

emerging as major ROS producers. Nitric oxide synthase (NOS) can become a powerful ROS generator, when uncoupled. BH4 ¼ tetrahy-

drobiopterin; e� ¼ electron; Hþ ¼ proton; H2O2 ¼ hydrogen peroxide; O2� ¼ superoxide; ONOO� ¼ peroxynitrite; other abbreviations as

in Figure 1.


233

impairment of the function of complex II throughoxidative post-translational modification (28–30).

In addition to allosteric interactions that modulateenzyme activities, metabolic intermediates, such asacetyl coenzyme A or ROS, are involved in controllingthe balance between glucose and fatty acid oxidation.In vitro acetylation of cardiac mitochondria increasesROS production and inhibits pyruvate oxidation (31),suggesting that acetylation of mitochondrial proteinsis also involved in the modulation of metabolicflexibility.

Finally, the p66Shc protein, a redox enzyme thatuses reducing equivalents of the ETC to generatemitochondrial ROS (hydrogen peroxide [H2O2]) (32), isinvolved in obesity and DM. It is activated by hyper-glycemia (33), and H2O2 generated by p66Shc wasshown to be involved in DM-related complications invarious organs (34). In addition, p66Shc is involved inthe regulation of glucose transport into cells (35) andis critical in maintaining insulin-dependent signaling(36). The phosphorylation of p66Shc on Ser36, which isinduced via mitochondrial ROS and results in afurther increase in ROS production, is known to beactivated in various pathologies associated withoxidative stress, including DM and obesity (34). Thisp66Shc-mediated ROS production can cause the

oxidation of specific phosphatases involved in insulinsignal transduction, such as phosphatase and tensinhomolog or protein tyrosine phosphatase 1B, result-ing in their inactivation (37,38). Deletion of p66Shc

prevents cardiac stem cell aging and development ofheart failure in diabetic animals (39), suggesting thattargeting p66Shc may lead to beneficial therapiesfor diabetic cardiomyopathy and other ROS-relatedcardiac pathologies. The major sources of ROSgeneration in cardiomyocytes are summarizedin Figure 2.

ROLE OF MITOCHONDRIAL ANTIOXIDANT DEFENSE

IN OBESITY AND DIABETES. Due to the susceptibilityof mitochondria to oxidative damage, ROS detoxi-fying systems including manganese-dependentsuperoxide dismutase, glutathione peroxidases(GPX1 and GPX4), thioredoxin reductases (TrxR2),thioredoxin 2, glutaredoxin (Grx2), and peroxiredox-ins (Prdx3 and Prdx5) are located directly within themitochondria. In addition to increased ROS produc-tion by mitochondria or other sources, cardiacexpression or activity of many antioxidant enzymesis reduced in obese or diabetic hearts (40), alongwith a concomitant reduction in circulatingantioxidant enzymes (reviewed in Savini et al. [41]).



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Epidemiological studies reported low plasma vitaminE concentrations to be associated with an increasedrisk of developing type 2 diabetes (42). The tran-scription factor Nrf2, which regulates the expressionof key antioxidant enzymes, including glutathioneperoxidase, superoxide dismutase, peroxiredoxin,thioredoxin, or thioredoxin reductase (43), wasreported to be induced by lipid peroxidation prod-ucts. This suggests that ROS products (lipid peroxide)contribute to the induction of antioxidant systems viaNrf2 in cardiomyocytes by a lipid peroxide–induced“pre-conditioning” cardioprotection, but with amitochondrial hormetic response (44). Accordingly,some studies reported increased antioxidant defensesin obese or diabetic hearts (45).

Although numerous studies have documentedsigns of increased oxidative stress or an alteredantioxidant defense in obese or diabetic hearts, therelationship between the degree of obesity andantioxidant defenses or systemic oxidative stress inhumans is still an open question. No correlation at all,a positive correlation, or a link with obesity-relateddiseases have been described (46–48). Furthermore,the causality of the relationship between mitochon-drial function and insulin sensitivity has beenchallenged (49). If oxidative stress is a majorcontributor to the progression of diabetes, strategiesto reduce ROS, such as antioxidant supplementation,should be protective against diabetes-induced cardiacremodeling. Although the use of antioxidants in ani-mal models of diabetes provided promising results,the clinical translation of this approach has not beenstraightforward, with randomized controlled trials ofvitamin E or C supplementation failing to demon-strate a clinically significant benefit (50–52) (see alsoPart 2 of this review series [2]).

ROLE OF OTHER ROS SOURCES. Nonmitochondrialsources of ROS include: nicotinamide adenine dinu-cleotide phosphate oxidase (NOX), xanthine oxidase(XO), uncoupled endothelial nitric oxide synthase(eNOS), mitochondrial monoamine oxidase-A(MAO-A), lipoxygenase, cyclooxygenase, and otherhemoproteins (53). Among these, the NOX family,which comprises 7 family members with distinctcatalytic subunits that generate ROS through electrontransfer from nicotinamide adenine dinucleotidephosphate to molecular oxygen (54), plays a pivotalrole in ROS production in the diabetic and obese heart.Induction of NOX subunits and ROS production havebeen reported in patients with metabolic syndrome,and hyperinsulinemia was suggested to contribute tooxidative stress in these patients through activation ofNOX (55). The activity of NOX is enhanced in the

hearts of obese animals (40). NOX inhibition abolishescardiac O2

�production in obese animals, andmay evenimprove left ventricle function (40,56). High glucoseexposure activates Rac1GTP and induces p47phox

translocation to the plasma membrane, resulting inNOX2 activation and increased ROS production incardiomyocytes, which can be prevented by activationof adenosinemonophosphate (AMP)-activated proteinkinase (57,58). Chronic hyperglycemia increases AGEformation (59). Accordingly, high levels of AGEs havebeen found in the tissues of patients with diabetes(60), and the AGE signaling pathway may act as acommon upstream stimulus for ROS generation.Furthermore, NOX can function cooperatively withother ROS-producing pathways, thereby promotingmitochondrial “ROS-induced ROS release” and exac-erbating overall oxidative stress in cardiomyocytes(61). Indeed, AGE binding to the receptor for advancedglycation end-products (RAGE) leads to activation ofNOX and increased cytosolic ROS, which facilitatesmitochondrial O2

� production in hyperglycemic envi-ronments (62,63). The AGE–RAGE interaction alsoactivates nuclear factor kappa-light-chain-enhancer ofactivated B cells, leading to up-regulation of RAGEitself and further ROS generation (64,65). In line withthe diverse interactions between NOX and mitochon-dria, the mitochondria-targeted antioxidant agentmito-TEMPO attenuated myocardial dysfunction indiabetic mice and reduced messenger ribonucleic acidexpression of components of NOX (66), whereas thesuperoxide dismutasemimetic agent tempol induced asignificant reduction in cardiac fibrosis and ROSproduction while increasing antioxidant enzymecapacity in diabetic rats (67). Sustained activation ofnicotinamide adenine dinucleotide oxidase indiabetes may also diminish intracellular nicotinamideadenine dinucleotide phosphate, an essential cofactorfor eNOS and several antioxidant systems (68). How-ever, ROS formation by high glucose-stimulated NOXmay up-regulate antioxidant enzymes (68).

The saturated fatty acid palmitate, which circulatesin higher concentrations in the blood of patients withdiabetes or obesity, induces mitochondrial ROS,which is amplified by NOX2, causing greater mito-chondrial ROS generation and mitochondrialdysfunction (69). Deficiency of the fatty acid trans-porter CD36 prevents cardiac steatosis, and increasesinsulin sensitivity and glucose utilization, butreduces fatty acid uptake and oxidation, NOX activ-ity, and palmitate-induced ROS production in agenetic mouse model of obesity (ob/ob mouse) (70).

Other important sources of cardiac ROS include XOand MAO-A. XO catalyzes the oxidation of its sub-strates, hypoxanthine and xanthine, to uric acid using


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oxygen as an electron receptor, and produces O2� and

H2O2. DM is characterized by increased myocardialand serum XO activity, which can be attenuated withthe XO inhibitor allopurinol, resulting in reducedcardiac fibrosis and improved systolic and diastoliccardiac performance of treated diabetic animals (71).Accordingly, the Western diet, with its excess fat andfructose, increases uric acid production and promotescardiomyocyte hypertrophy, oxidative stress,myocardial fibrosis, and impaired diastolic relaxation,which can all be improved with allopurinol-inducedreduction in cardiac XO and serum uric acid levels(72). The outer mitochondrial membrane serotonin-degrading enzyme MAO-A is another importantsource of H2O2 in the heart. MAO-A shows greateractivity in diabetic cardiomyocytes, and MAO-Ainhibition results in improved contractile functionin preclinical DM models, despite persistent hyper-glycemia and hyperlipidemia (73). However, nodifference in MAO expression or MAO-related oxida-tive stress was recently observed in right atrialappendages from cardiac surgery patients with orwithout DM (74).

EXCESSIVE ROS PRODUCTION AND CARDIAC

LIPOTOXICITY. In the obese or diabetic heart, thesupply of substrates exceeds the need for ATPsynthesis. However, the ability of cardiomyocytes torespond to an increased fatty acid load is significantlyreduced with age and obesity, resulting in accumu-lation of lipids and ceramide, mitochondrialdysfunction, increased ROS production, and possiblyreduced cell viability (75–77). Specifically, underthese conditions, excess lipids are shunted into non-oxidative pathways, resulting in the generation oftoxic lipid intermediates, such as ceramides (75,78).These toxic lipid intermediates promote mitochon-drial dysfunction, induce changes in signal trans-duction, and increase apoptosis; this phenomenon iscalled lipotoxicity (78). Markers of oxidative stress,such as protein carbonyl content and 8-hydroxy-2ʹ-deoxyguanosine, are significantly elevated incardiomyocytes isolated from young patientswith obesity (10). In addition, telomere length, asensitive indicator of cumulative oxidative stress inpost-mitotic cells such as cardiomyocytes (see Part 1of this review series [1]), is significantly reduced incardiomyocytes from young subjects with obesity(10). Ectopic lipid accumulation in the heart isassociated with cardiac hypertrophy, cardiacdysfunction, and apoptosis (77), and a strong associ-ation between increased cardiac fatty acid uptake andcardiomyopathy has been demonstrated in variouspreclinical models (79,80). Interestingly, cardiac lipid

accumulation appears to be reversible in humans,with mechanical unloading by ventricular assistdevice implantation being shown to correct metabolicderangements and myocardial lipotoxicity inadvanced heart failure (81).

ROS-INDUCED CHANGES IN INSULIN SIGNALING . Insulin-stimulated glucose uptake is impaired in obese andinsulin-resistant animals and humans. Furthermore,the metabolic syndrome and insulin resistance areassociated with abnormal left ventricular diastolicfunction and structure, which occurs independentlyof age, sex, blood pressure, and fasting plasmaglucose, and is mainly associated with the state ofinsulin resistance (82,83).

Cellular insulin signaling occurs through 2 keypathways: the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) and mitogen-associated proteinkinase (MAPK) pathways (84). It also includes tyro-sine phosphorylation of insulin receptor substrate(IRS) proteins (84). The metabolic responses,including glucose uptake by translocation of glucosetransporter type 4 to the cell membrane ofcardiomyocytes, are mainly elicited via PI3K-mediated Akt activation. Among the defects thathave been suggested as underlying mechanisms forinsulin resistance are increased serine phosphoryla-tion of IRS proteins by kinases such as inhibitor ofnuclear factor kappa-B kinase subunit beta, c-JunN-terminal kinase, p38MAPK, extracellular signal-related kinase, mechanistic target of rapamycin, andS6K, resulting in an attenuation of engagement of IRSand PI3K (Figure 3) (85,86). ROS play a major role inaltered activation of many of the aforementionedkinases, and have therefore been proposed as animportant link between impaired mitochondrialfunction and insulin resistance (87,88). Indeed, ROSare known activators of stress-activated proteins p38MAPK and c-Jun N-terminal kinase, which inhibitinsulin signal transduction by phosphorylating IRSproteins (89). In addition, mitochondrial ROS stimu-late proinflammatory signaling by activation ofinhibitor of nuclear factor kappa-B kinase subunitbeta and other kinases that phosphorylate IRS-1 atserine residues (90). Fatty acids (lipid infusion) canalso lead to the accumulation of diacylglycerol andother lipid derivatives that activate protein kinase C,which, in turn, increases serine phosphorylation ofIRS proteins and leads to inhibition of insulinsignaling (91).

Excessive ROS release also impairs protein foldingand post-translational modifications that occur in theendoplasmic reticulum (ER). Compromised ER func-tion and ER stress contribute to altered insulin

FIGURE 3 ROS-Induced Changes in Insulin Signaling

Insulin signaling pathways are initiated by insulin binding to the extracellular alpha subunit of the insulin receptor (IR), followed by a

conformational change in the beta subunit of the IR, which has intrinsic tyrosine kinase activity. Receptor activation results in tyrosine

phosphorylation of the IRS and subsequent activation of phosphatidylinositol 3-kinase (PI3K). Activation of PI3K leads to stimulation of various

downstream serine kinases, including Akt, which is involved in the translocation of the major glucose transporter glucose transporter type 4.

Among the mechanisms underlying insulin resistance in cardiomyocytes from patients with obesity or diabetes is increased serine phos-

phorylation of IRS proteins. Phosphorylation of IRS proteins at particular serine residues inhibits the interaction of IRS proteins with the

insulin receptors, resulting in a reduction in activity of the abovementioned signaling pathway and impaired cardiomyocyte glucose uptake.

Lipid derivatives stimulate c-Jun N-terminal kinase (JNK), protein kinase C (PKC), and IkB kinase (IKK)-mediated phosphorylation of IRS-1 at

serine residues. In addition, mitochondrial dysfunction increases ROS production, which causes activation of serine/threonine kinases,

including p38MAPK, JNK, IKKb, and extracellular signal–regulated kinase (ERK), which increase serine phosphorylation of IRS proteins.

Overnutrition, resulting in increased free fatty acids, also contributes to impaired insulin signaling through mechanistic target of rapamycin

(mTOR)–S6 kinase 1 (S6K1)-mediated serine phosphorylation of IRS. MAPK ¼ mitogen-activated protein kinase; other abbreviations as in

Figure 1.



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signaling by activation of serine/threonine kinases(92). Insulin signaling is also involved in the regula-tion of myocardial autophagy (93). Hyperinsulinemiawas shown to suppress myocardial autophagy viaAkt/mechanistic target of rapamycin signaling,whereas fasting-induced low insulin levels induceautophagy (93). Recently, it was shown that NOX2-derived O2

�, but not mitochondrial O2� production,

induces impaired autophagic flux in response topalmitate in cardiomyocytes (94). This maycontribute to impaired cardiac autophagy in responseto lipid overload in insulin resistant states (94).

INVOLVEMENT OF ROS IN CARDIAC METABOLIC

MEMORY. Prolonged hyperglycemia induces meta-bolic changes that alter tissue homeostasis, even afterglucose normalization, a phenomenon called meta-bolic memory (95). Epigenetic mechanisms contributeto the development and maintenance of cardiacmetabolic memory. Specific enzymes modify discreteresidues on histone tails (writers), other enzymes canremove these marks (erasers), and still others recog-nize these histone marks (readers), enabling gene

expression to proceed (96). Chromatin marks consistof epigenetic post-replicative methylation of DNA atcytosine residues and various post-translationalmodifications mainly at arginine and lysine resi-dues. Long-term epigenetic effects, such as histoneand DNA methylations, are relatively stable and canbe transferred as memory to offspring cells. Further-more, maternal diet and in utero environment caninduce epigenetic changes and can be inherited byfuture generations, triggering diseases such asobesity or DM (97). This chromatin remodeling en-ables cardiomyocytes to respond to different stimuliby controlling DNA accessibility and thus geneexpression.

Hyperglycemia induces long-lasting activation ofinflammatory and oxidative stress pathways, resultingin long-lasting or even irreversible epigenetic changesthat contribute to the abnormal character of bloodcells, endothelial cells, vascular smooth muscle cells,or cardiomyocytes in patients and animals withobesity or diabetes (98). Experimental evidence alsosuggests hyperglycemia-mediated ROS is a majordriver of glycemic memory in endothelial cells (99).

CENTRAL ILLUSTRATION Oxidative Stress and Cardiovascular Risk Factors

Damaging consequences for cardiovascular health

Increased ROS production

Endothelial dysfunction

Activation of autonomic nervous system

Exacerbation of thrombosis

Increased susceptibility of the myocardiumto ischemia-reperfusion damage

Passage of particles into circulation

Air pollution and/or smoking

Increased reactive oxygen species (ROS) production

‘Metabolic memory’ in cardiomyocytes

Accumulation of toxic lipids leads to lipotoxicity

Increased ceramide contentleads to cardiomyocyte death

Increased fatty acid utilization

Mitochondrial dysfunction

Decreased glucose utilization leads to glucotoxicity

Altered insulin signaling

Diabetes and/or obesity

Niemann, B. et al. J Am Coll Cardiol. 2017;70(2):230–51.

Obesity, diabetes, smoking, and pollution are prominent causes of oxidative stress in the cardiovascular system; these mechanisms are increasingly appreciated as

playing a major role in disease pathogenesis.


237

So far, only sparse data on potential molecular mech-anisms for a metabolic memory in cardiomyocyteshave been reported. Recently, it was shown that highglucose levels induce increased levels of the inflam-matory cytokine interleukin (IL)-6 and decreasehistone-3 methylation at the IL-6 promoter incardiomyocytes, which was irreversible after removalof high glucose (100). Thus, the high glucose-inducedincreased inflammatory gene expression incardiomyocytes was due to a loss of repressive epige-netic histone modifications (100). High glucose-induced mitochondrial dysfunction and apoptosisdid not appear to be responsible for the metabolicmemory in cardiomyocytes (100). Furthermore, highglucose induces epigenetic regulation of the insulin-like growth factor-1 receptor in cardiomyocytes (101).

In addition to in vitro analyses, epigenome-wideassociation studies have investigated the effect ofobesity or DM on epigenetic changes in patients. In arecent large study, increased obesity in adults wasassociated with increased methylation at the hypoxiainducible factor 3A (HIF3A) locus in blood cells and

adipose tissue, but not in skin (102). Apparently,epigenetic markers show strong tissue and cell-typespecificity (98,103,104). Therefore, the transferabilityof epigenetic signatures from easily obtainable bloodcells of patients with diabetes or obesity to car-diomyocytes needs to be investigated. Finally, meta-bolic intermediates, such as those of the tricarboxylicacid cycle, glycolysis, FAO, or the hexosaminebiosynthetic pathway, are cofactors for chromatin-modifying enzymes (104). Appreciating how alter-ations in metabolism and nutrition modify epigeneticgene regulation of eukaryotic cells through metabolicintermediates might significantly contribute to thera-peutic innovation in obesity and DM.

CONCLUSIONS REGARDING OBESITY, DIABETES,

AND OXIDATIVE STRESS. Increased oxidative stressin the heart and cardiomyocytes arises via multiplemechanisms, including mitochondrial dysfunctionand uncoupling, increased FAO, enhancedNOX activity, and reduced antioxidant capacity(Central Illustration). Recent data suggest that



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metabolic memory is an important contributor toCVD, as cells remember exposure to hyperglycemiaand oxidative stress. Pharmacological ROS scav-enging has been used to improve myocardial energymetabolism and insulin responsiveness and to reducecardiac dysfunction in preclinical models of obesityor DM, as well as in some patient studies (105).However, despite overwhelming evidence of thedamaging consequences of oxidative stress in obesityand DM, large-scale clinical trials using antioxidanttherapies for the treatment of CVD have failed todemonstrate benefit. Vitamin E treatment, forexample, had no apparent effect on cardiovascularoutcomes in the HOPE (Heart Outcomes PreventionEvaluation) study, a randomized, placebo-controlled,double-blind clinical trial involving more than 8,000patients with CVD or DM (50). ROS differ in terms oftheir reaction kinetics, diffusion parameters, site ofproduction, and degradation kinetics. Rather thanmerely scavenging reactive radicals, a more compre-hensive approach may be required that prevents ROSgeneration while also promoting ROS scavenging inparticular cellular compartments. Importantly, thecomplex interactions among the various ROS sourceswithin the cell and mitochondria, and the mecha-nisms responsible for the increase in ROS formationin patients with diabetes or obesity, remain to beelucidated. Future work will also need to addresswhether the epigenetic signatures associated withobesity and DM can be reverted by drugs that canmodify the epigenome.

ROLE OF OXIDATIVE STRESS IN THE

CARDIOVASCULAR EFFECTS OF

AIR POLLUTION AND SMOKING

INTRODUCTION. Few would argue that our entitle-ment to breathe clean air is a basic and essentialhuman right. Yet, the air we breathe is far from clean.Even in the most isolated environments, theatmosphere is a diverse array of substances fromaerosolization of the ground, dusts carried in thewind, natural gases, biological material such aspollens and spores, as well as viruses and bacteria.We have evolved complex defense mechanisms toprotect our bodies against natural pollutants, but thesame cannot be assumed for man-made sources of airpollution, such as those from industry, households,and traffic. In most modern societies, these pollutantsare ubiquitous and, for many, exposure is unavoid-able. The dramatic mortality that accompaniedepisodes of high air pollution, such as that ofthe Meuse Valley fog (Belgium, 1930; 60 deaths fromthe persistence of industrial emissions over 3 days),

the Donora air inversion (United States, 1948; 3 daysof high air pollution led to ill-health in over one-thirdof the town’s population) and the 1952 London smog(United Kingdom, 1952; 4- to 5-day period believed tocause between 4,000 and 10,000 deaths), has cemen-ted the need for regulatory control. The introductionof legislation (e.g., the 1956 Clean Air Act or nationalregulations such as the 1977 and 1990 U.S. Air PollutionControl Act, 2008 European Union directives) haseffectively reduced levels of many pollutants. Yet,present-day air pollution continues to be a seriouspublic health issue, with increasing industrializationand the rapid expansion of urban environments.Recent estimates suggest air pollution is responsiblefor between 3 and 7million deaths worldwide per year,accompanied by staggering levels of morbidity (3.1%of global disability-adjusted life-years) and associatedeconomic risks ($1 to U.S. $3 trillion U.S. dollars/yearworldwide) (106–108). Indeed, a recent report placedboth indoor and outdoor air pollution within the top10 risk factors for all-cause disease, greater than thatcaused by risk factors such as sedentary lifestyle orhigh cholesterol (108).

Given these disturbing figures, it is perhapssurprising that we continue to expose ourselves to airpollutants for pleasure, by the smoking of cigarettesor other tobacco products. Smoking is believed to kill6 million people each year worldwide, with 480,000and 96,000 deaths each year in the United States andUnited Kingdom, respectively (109–111). More than 16million people in the United States are believed to beliving with a disease caused by smoking, with esti-mated annual costs in the region of $300 billion in theUnited States and V380 billion in Europe (110,112).Although the ban of smoking in public places hasbeen one of the major successful health interventionsof recent years (reduced rates of myocardial infarc-tion by 17%) (113), smoking remains prevalent(currently estimated to be over 1 billion personsworldwide). Thus, although current trends suggestthat the prevalence of smoking is declining overall,the decline is slow, use is increasing in many low- andmiddle-income countries, and the global health costsare expected to rise over the next decade (114,115).

There are clear differences in the chemicalcomposition of environmental pollution and smok-ing. However, the counter is also true: both derivefrom the combustion of complex carbon-rich mate-rials, with notable similarities in the compositions ofthese fumes (Figure 4). More importantly, the conse-quences to health are also comparable, and areparticularly well-exemplified in relation to CVD.Notably, in terms of mortality, the cardiovascular ef-fects of both smoking and air pollution outweigh that

FIGURE 4 Similarities Between the Chemical Composition of Tobacco Smoke and Traffic-Derived Air Pollution

As shown, there are many similarities between the chemical composition of tobacco smoke and traffic-derived air pollution. Although certain

differences exist, the consequences of these pollutants on cardiovascular health are broadly similar. CO ¼ carbon monoxide; CO2 ¼ carbon

dioxide; HCN ¼ hydrogen cyanide; NO2 ¼ nitrogen dioxide; O3 ¼ ozone; PAH ¼ polyaromatic hydrocarbon; SO2 ¼ sulfur dioxide;

SVOCs ¼ semi-volatile organic compounds.


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of death from pulmonary conditions (108). The bio-logical pathways that link pulmonary exposure totheir cardiovascular actions remain the subject ofongoing research, although oxidative stress is are-emerging mechanism, and one that undoubtedlycontributes to the progression of disease.

We provide an overview of cigarette-related prod-ucts (C-RPs) and combustion-derived environmentalair pollution (C-DEAP) (principally urban air pollu-tion), to highlight where the similarities existbetween their physicochemical properties and themeans through which they impair cardiovascularhealth. We describe the potential biological pathwaysunderlying these actions, with a focus on the role ofoxidative stress and how this cellular event couldhave implications for interventional strategies(Central Illustration).

SOURCES, COMPOSITION, PARTICLES, AND EXPOSURE.

Smoking . Cigarettes are the most commonly usedtobacco products, and despite falling use, cigarettemanufacture is substantial (6.35 trillion cigarettes

smoked worldwide in 2012) (115). Other tobaccoproducts, such as cigars and tobacco pipes, follow asimilar trend, albeit with markedly lower numbers.Water pipes (hookah, shisha) remain a popular meansto inhale tobacco smoke in Middle Eastern countries.There is overwhelming evidence that all these formsof smoking have major detrimental health effects(116,117). Electronic (e)-cigarettes have surged inpopularity recently, largely attributed to the uncon-firmed assumption that they have lesser adversehealth effects, a discussion of which can be foundelsewhere (e.g., Benowitz et al. [118], Kaisar et al.[119], and Morris et al. [120]).

Tobacco smoke comprises a mixture of gases,aerosolized liquids, and small particles. The compo-sition is highly complex, with more than 4,000chemicals in C-RP smoke, of which 250 are knownto be harmful and 50 more are known to cause cancer(109). The gas/vapor phase of C-RP smoke containsnitrogen, carbon dioxide (CO2), and carbon monoxide(CO), with smaller but significant amounts ofhydrogen cyanide, nitric acid, methane, benzene,



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acrolein, acetone, hydrogen sulfide, and a wideselection of hydrocarbons, aldehydes, carbonyls, andnitrosamines (121). The particulates within C-RPfumes are predominantly carbon-based, composed ofa mixture of semicombusted material that containstar and nicotine. Elemental carbon within the sootparticle provides a scaffold for adsorbed organic car-bon species and liquids, including phenols, carboxylicacid, paraffin waxes, and nitrosamines among manyothers. The polyaromatic hydrocarbons (PAHs),quinone species, and transition metals within C-RPparticulates have a high capacity to instigate redoxreactions in cells.Ai r pol lut ion . Environmental air pollution derivesfrom a large number of both man-made (e.g.,industry, power plants, traffic, household combus-tion and cooking, construction, mechanical wear, andagriculture) and environmental sources (e.g., forestfires, volcanic eruptions, aerosolization of soil anddusts, pollen, and molds). Indoor air pollution(e.g., from cooking, heating, dust, candles, andaccumulation of outdoor pollutants) is a pressingconcern, but one that has received less attention thanits outdoor counterpart. The majority of attention hasfallen on outdoor urban air pollution due to the highincidence of exposure in dense urban populations.Pollution of urban air will depend on the extent of thesources listed previously, as well as geographical andmeteorological conditions. Traffic-derived emissions(e.g., diesel exhaust [DE]) are a prominent source ofurban pollution, and are under particular scrutinydue to their ubiquity and increasing prevalenceworldwide.

Urban pollution is a complex cocktail of chemicalsthat can also be broadly characterized into gases,semivolatile liquids, and particles (122). Numerousgases are found within C-DEAP, such as sulfur diox-ide, CO2, and CO, with recent attention focused onozone (O3) and nitrogen dioxide (NO2) in particular.O3 readily oxidizes other air pollutants and biological/cellular material, both directly and through freeradical generation. NO2 also acts as an oxidizingagent, as well as modulating cell function via nitricacid formation, nitrosative reactions, and free radicalreactions (123). The effects of O3 and NO2 are likely tobe additive; however, shared sources and the dy-namic relationship with other pollutants makes dis-entangling their actions challenging.

A plethora of semivolatile organic compounds formthe “liquid” phase of air pollution. These includemethane, benzene, naphthalene, formaldehyde, andalkanes, as well as a range of PAHs, polychlorinatedbiphenyls, and polybrominated diphenyl ethers.The close interaction of semivolatile chemicals with

gaseous (e.g., interplay between methane and ozone)and particulate (absorption to the carbonaceoussurface) components often results in the “liquid”phase of C-DEAP being grouped within the gases or,more usually, as “particulate matter” (PM) for toxi-cological purposes.

The PM in air pollution is a mix or organic andinorganic particulates. Environmental PM is catego-rized into 3 groups according to sampling conventions:coarse particles (particles with a diameter #10 mm[PM10]), fine particles (diameter #2.5 mm PM2.5), orultrafine particles (diameter <100 nm [PM0.1], alsoreferred to as nanoparticles). Airborne PM is regulatedon the basis of PM10 and PM2.5; at present, PM0.1

cannot be measured through existing environmentalmonitoring networks. Different sources of PM havevarious size ranges, compositions, and reactivities.C-DEAP particulate is rich in carbon, with the mixtureof elemental and organic carbon depending on the fuelsource and efficiency of combustion. They carry acocktail of harmful chemical species on their surfacesthat include unburned hydrocarbons, carcinogenicorganic carbon species (PAHs, alkanes, quinones), andredox-active transition metals. The biological toxicityof PM is greatly dependent on the composition ofthe particulate; however, in general, the small sizefractions exert greater effects due to their largereactive surface area for a given mass and their abilityto penetrate deep into the alveoli of the lungs, andpotentially into the bloodstream.

MECHANISMS OF INDUCTION OF CVD. Epidemio-logical studies have shown clear associations betweenvarious CVDs and both smoking and air pollution,including for coronary artery disease (124–127),peripheral arterial disease (128,129), heart failure(130,131), cerebrovascular disease (126,132–134),cardiac arrhythmia/arrest (135,136), and venousthromboembolism (137) (for overviews, see Morriset al. [120] for C-RP and Miller et al. [138] and Newbyet al. [139] for C-DEAP). There is now a substantialbody of experimental work demonstrating that thesepollutants impair cardiovascular function (140,141).In particular, controlled exposure studies in humansand animals have demonstrated that pollutants havethe capacity to impinge on almost all aspects ofcardiovascular function (Figure 5A).

Vascular d i sease and pol lut ion . Air pollution isassociated with elevated blood pressure and,accordingly, many sources of C-DEAP increase bloodpressure in humans and animals (142,143). Elevatedblood pressure is primarily accounted for by alteredvascular function, with C-DEAP exposure generallypromoting vasoconstriction and decreasing

FIGURE 5 Biological Pathways Through Which Inhaled Pollutants Could Induce Cardiovascular Morbidity and Mortality

(A) Overview of the 3 main hypotheses by which inhaled pollutants could cause cardiovascular effects and their multiple detrimental actions

on the blood, vasculature, and heart that lead to cardiovascular morbidity and mortality. Oxidative stress plays a role in exacerbating

numerous aspects of each pathway. (B) Section of mouse lung following instillation of ultrafine particles. Note the presence of macrophages

densely-loaded with particles (arrows), and the thin cellular barrier between the alveolar space and the pulmonary arterioles. (C) The 3 main

hypotheses for how inhaled pollutants cause cardiovascular effects overlaid on a transmission electron micrograph of a rat lung (micrograph

reprinted with permission from Lehnert [259]): 1) pollutants induce an inflammatory response in the lungs, leading to release of cytokines

and other mediators that spill over into the systemic circulation; 2) constituents within pollutants may translocate across the alveolar wall and

directly interact with the cardiovascular system; and 3) pollutants may activate the autonomic nervous system through sensory receptors on

the alveolar surface. Note the thin barrier of alveoli with pulmonary capillaries (shaded in red) and the presence of an alveolar macrophage

(green outline) on the pulmonary surface overlying a capillary. Adapted with permission from Miller et al. (207). aas ¼ alveolar air space;

pc ¼ pulmonary capillary.


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vasodilator responses. Controlled inhalation of diluteDE in humans (at levels representative of busy road-side exposure) impairs the skin microvasculatureresponse (144) and produces a marked attenuation ofvasodilation of forearm blood flow (145). Interest-ingly, the magnitude of the attenuation of forearmvasodilation is similar to that in a lifelong smokercompared with a nonsmoker (146,147). Vascular im-pairments are apparent at 2 h after exposure and arestill evident 24 h later (148). Increases in arterialstiffness are also found after acute exposure toC-DEAP (149,150). Altered baroreceptor sensitivitymay also contribute to the vascular impairmentfollowing C-DEAP exposure (151,152). Epidemiological

studies have clearly demonstrated that exposure tourban air pollution is associated with atherosclerosis(139,153,154), and data from animal studies supportsthat C-DEAP can increase the size and, potentially,the vulnerability of plaques (155–157). Finally, thereare indications that DE has the capacity to alterischemia-induced angiogenesis (158).Blood, c i r culat ing factors , and pol lut ion . Expo-sure to air pollution is frequently, although incon-sistently, accompanied by increases in circulatinginflammatory markers (159–161). Animal models haveshown that inhalation of C-DEAP promotes theadherence of leukocytes to the vascular wall, an earlyevent in atherogenesis (162). There have been mixed



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findings regarding whether C-DEAP increases coagu-lation pathways (163–167). However, inhalation of DEin healthy volunteers potentiated the formation ofthrombus in an ex vivo model of arterial injury,predominantly through platelet hyper-reactivity(168). Similar platelet activating effects are seen incigarette smokers (169). Fibrinolytic pathways arealso affected through impaired release of tissueplasminogen activator from endothelial cells(145–147,170). The particulate components of DEalone have the capacity to exacerbate thrombosis andinhibit fibrinolysis (171). C-DEAP also induces alter-ations in circulating stem cell populations (172,173),potentially disrupting vascular repair (174).Card iac d i sease and pol lut ion . In addition to itseffects on the pulmonary and peripheral vasculature,C-DEAP has also been shown to have detrimentaleffects on the coronary circulation in animal models(175–178). Pulmonary exposure to DE particlesincreases the susceptibility of the myocardium toischemia-reperfusion damage in rats (179), and inha-lation of dilute DE is associated with greatermyocardial ischemia (ST-segment depression duringexercise) in patients with ischemic heart disease(170). The PM of C-DEAP has been shown to increasethe incidence and duration of arrhythmia (179–181),and there are extensive (but somewhat inconsistent)published reports showing a trend between airpollution exposure and measures of heart rate vari-ability (182,183). Finally, epidemiological studieshave found consistent associations between airpollution and heart failure (131), with animal modelsdemonstrating that long-term exposure to PM pro-motes myocardial hypertrophy and loss of cardiacfunction (184).Pol lut ion and other systems . Briefly, it is worthhighlighting that C-DEAP has recently been shown tohave other systemic effects that adversely affect thecardiovascular system. These include impaired renalblood flow and an increased incidence of renal disease(185,186), exacerbation of metabolic syndrome/diabetes (187–190), changes to the placental circulation(191–193), and epigenetic alterations that may affectthe cardiorespiratory health of offspring (194,195).

BIOLOGICAL MECHANISMS. Lung to card iovascu larsystem. The exact mechanisms by which inhaledpollutants lead to effects on the cardiovascularsystem remain to be determined. However, 3 theoriescurrently predominate (Figure 5) (see also Miller[196]). The classical hypothesis is that inhaledpollutants activate inflammatory cells in the lung,leading to the release of inflammatory mediators thatpass into the circulation to influence cardiovascular

function (197). A number of constituents of airpollution and tobacco smoke have the capacity toinduce pulmonary inflammation and oxidative stress(see later discussion), and parallel pathways can beup-regulated in the systemic circulation and organsystems. At present though, inconsistencies in sys-temic biomarkers of inflammation between studiesand dissociation across the time course of responsesuggest that this pathway alone cannot fully explainthe multiple cardiovascular effects of either expo-sure. The second theory is that inhaled pollutantsactivate alveolar receptors, stimulating sensoryafferents that alter cardiovascular function viachanges in autonomic balance or neuroendocrineregulation (127,198). This pathway is very plausible interms of the effects of C-DEAP on cardiac electro-physiology, and potentially for some of the rapid(<2 h) cardiovascular effects after exposure. Finally,pollutants themselves (or the constituents releasedfrom pollutants) may cross the alveolar wall into thepulmonary circulation, and directly interact with thecardiovascular system (199). Ultrafine particles and/ortheir constituents have been shown to translocatefrom the lung to the blood in animal models;however, the fate of these constituents and themechanisms by which very low levels of translocatedparticles/constituents can produce widespreadcardiovascular impairment requires further research.Overall, evidence exists for all 3 theories, and it islikely that all 3 act in concert to produce the wide-spread cardiovascular effects of inhaled pollutants(196). The cellular signaling at each stage is subject toongoing research; however, oxidative stress is arecurring observation and is likely a driver of all 3pathways.

INDUCTION OF OXIDATIVE STRESS. Cohort studieshave found that exposure to high levels of air pollu-tion is associated with a number of biomarkers ofoxidative stress, including oxidation of plasma pro-teins and lipids (e.g., malonaldehyde and protein2-aminoadipic semialdehyde) (200,201), urinaryisoprostanes (202), and oxidative DNA adducts(8-hydroxy-2ʹ-deoxyguanosine) (203–206). In manycases, indications of oxidative stress coincide withthat of markers of inflammation, although there isconsiderable variability in both (207).

Controlled exposure studies in humans have sug-gested a role for oxidative stress after acute exposureto C-DEAP. DE inhalation attenuates vasodilation inresponse to both endothelium-dependent and nitricoxide donor drugs, but not in response to vasodilatorsacting via smooth muscle cell receptors; a profile thatis suggestive of NO scavenging by O2

� free radicals


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(145,208). Exposure to C-DEAP is known to cause acompensatory increase and to eventually depleteantioxidant concentrations in respiratory tract liningfluids and pulmonary cells (209). Likewise, inhalationof DE alters the expression of several antioxidantpathways in peripheral blood monocytes (210).Furthermore, DE particles induce a greater inflam-matory response in individuals with geneticdeficiencies in various antioxidant systems (211–213).

There are extensive published data from cellularand animal models showing that different types ofC-DEAP induce oxidative stress (seeMiller et al. [207]).Animal models are especially useful in assessing themechanisms of action of air pollution in chronic dis-ease processes such as atherosclerosis. Mouse modelshave shown that the proatherosclerotic effects ofC-DEAP are closely associated with oxidation ofcirculating lipids and arachidonic acid metabolites(214–216), increased urinary isoprostanes (217), nitro-tyrosine staining (an indirect marker of oxidativestress) (218), compensations in antioxidant expression(156,218–220), and markers of oxidative stress inplaques/vascular wall (221,222). Furthermore, adiverse array of oxidative pathways are emerging toaccount for these observations, including an inabilityof high-density lipoprotein (HDL) cholesterol toprotect against low-density lipoprotein (LDL)cholesterol oxidation (219), up-regulation of NOXexpression/activity (223,224), eNOS uncoupling (225),and signaling through lectin-like oxidized LDLreceptors (214).

Both gaseous and particulate pollutants have thecapacity to initiate oxidative reactions in the body.O3 and NO2 are both oxidizing agents, and areknown to induce oxidative stress in pulmonary cells(123). The lung lining fluids surrounding pulmonaryepithelial cells in vivo provide an early defenseagainst acute exposure to inhaled pollutants. How-ever, prolonged or repeated exposure leads todepletion of antioxidants in cell surfactants andchanges in pulmonary cell function (209). The gaseswithin C-DEAP can modify blood constituents in thepulmonary circulation, although the precise mecha-nism by which the oxidative “signal” is carried ortransferred to peripheral organs requires furtherinvestigation (161). Particulates in C-DEAP can alsodiminish pulmonary defenses and induce oxidativestress in most cell types of the lung (226). Particlesthemselves generate free radicals in the absence ofbiological tissue (196,227) (Figure 6), and directlyoxidize isolated DNA, lipids, and enzymes in vitro(see Miller et al. [207]). There is some debate as towhether the in vitro oxidative potential of particu-lates alone is a useful predictor of toxicity in vivo

(209), given that it will underestimate the contribu-tion of oxygen free radicals generated from particle-induced activation of cellular enzymes. However,should particles translocate into the circulation,their oxidative capacity is likely to be an importantdeterminant of their systemic effects. Levels ofmetals and organic carbon in C-DEAP play a crucialrole in the oxidizing and free radical–generating ca-pacity (122,212). Finally, both gaseous and particu-late pollutants can induce inflammatory responses inthe lungs and blood, which provide a means ofamplification of the oxidative stress induced by C-DEAP. It is notable that, almost without exception,all of the mechanisms described earlier for C-DEAPhave been also proposed as potential mediators forthe detrimental effects of smoking on the cardio-vascular system, especially for oxidative stresspathways (140).

INTERVENTIONS TO REDUCE EXPOSURE, OXIDATIVE

STRESS, AND CVD. Reducing sources of air pollutionwill undoubtedly remain the principal means ofpreventing its adverse health effects (113,138). How-ever, although the levels of many air pollutants havefallen dramatically in many countries over the lastfew decades, there are concerns as to whether we areregulating the correct pollutants. For example PM10

and PM2.5 are currently used for measuring particu-late air pollution in the environment, but thesemeasures are greatly skewed by larger particles andare not reliable indicators of ultrafine particles(e.g., those from vehicle exhaust), which have a muchhigher potential to affect health (228). Furthermore,current epidemiological evidence suggests that evenair pollution at levels below those recommended bycurrent regulatory guidelines is associated withdetrimental health effects (222,229).

Awareness of the adverse health effects of airpollution is growing around the world, and there arean increasing number of avenues to reduce it(see Miller et al. [138] and Laumbach et al. [230]).Industrial emissions are tightly regulated, and relo-cation to nonresidential areas and stringent healthand safety measures for workers have reduced humanexposure. Modern vehicle engines and fuels in gen-eral produce lower emissions than their predecessors,and there is a slow but persistent movement towardalternative energy fuels, such as biodiesel, hydrogencells, and electric vehicles. Emerging scientific evi-dence has shown that alternative fuels (231), fueladditives to improve the efficiency of combustion(232), and exhaust filters to remove particles fromemissions (233) have the potential to greatly reducethe cardiovascular actions of vehicle exhaust.

FIGURE 6 Mechanisms Through Which Inhaled Pollutants Can Induce Cellular Oxidative Stress

1. Innate generation of free radicals by particles in the absence of biological tissue. Free radicals may be produced by redox reactions between

different chemicals on the surface of the particulate, as well as interactions with other constituents within air pollution. 2. Release of

cytokines and oxidative mediators from pollutant-induced activation of inflammatory cells. 3. Free radical generation from interaction

between pollutants and cells (e.g., from stimulation of enzymes such as nicotinamide adenine dinucleotide phosphate [NADPH] oxidase,

xanthine oxidase [XO], uncoupling of endothelial nitric oxide synthase [eNOS], induction of inducible nitric oxide synthase [iNOS],

exacerbation of free radicals from mitochondrial inefficiency, or depletion of antioxidant defenses). These pathways may be stimulated by

particles themselves, through release of soluble constituents on the particle surface, or intermediate chemical reactions of different

constituents within biological fluids. Adapted with permission from Miller et al. (207).



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Prevention at the individual level also meritsattention, particularly for those believed to be espe-cially susceptible to air pollution (e.g., the young,elderly, and those with pre-existing cardiopulmonarydisease). The availability of regularly updated airquality data and low-cost personal measuring devicesis providing individuals with the means to avoid pro-longed exposure on high air pollution days (122).Furthermore, face masks may be a simple and cost-effective means to reduce exposure to some pollut-ants that may result in benefits to cardiovascularhealth (234).

One subject of debate in the scientific communityis whether or not pharmacological agents or supple-ments can limit the harmful effects of air pollution(recently reviewed by Tong [235]). Although phar-macological agents are unlikely to ever represent apractical means of primary prevention of the healtheffects of air pollution, their use in scientific

investigations provides insight into the biologicalmechanisms involved. Beta-blockers or antagonists ofalveolar sensory receptors can attenuate the cardiaceffects of C-DEAP in rodent models (179,236). Addi-tionally, endothelin receptor antagonists (177,237),inhibitors of the renin/angiotensin system (238), andstatins (239,240) can reduce the vascular andatherosclerotic effects of air pollutants.

Given the clear involvement of oxidative stress inthe cardiovascular actions of air pollution, it ispossible that antioxidant agents could represent auseful preventative strategy. Cell culture studieshave demonstrated that vitamin C, N-acetylcysteine(NAC), trolox, and inhibitors of iron-mediatedfree radical generation can prevent the actions ofC-DEAP particles in vascular cells (241,242) and mac-rophages (243). Supplementation of the endogenousantioxidant superoxide dismutase attenuates thevascular effects of C-DEAP particles in isolated blood


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vessels (207). Furthermore, emerging data from ani-mal models suggests that several antioxidant (-rich)compounds, including curcumin (244), NAC (245),NAD(P)H oxidase inhibitors (246), vanillic acid (247),emodin (248), tempol (237), and even dark chocolate(249), can protect against some cardiovascular actionsof C-DEAP. Regrettably, these findings have not beenreplicated in human studies, with antioxidant-richfoods or supplements having inconsistent effects onPM2.5-induced heart rate variability (250,251) andvascular dysfunction (252–254). Of concern, anddespite the fact that in chronic smokers vitamin C hasbeen shown to improve endothelium-dependentforearm blood flow (254), in another study, thevasoconstrictor effects of DE were actually greater involunteers receiving vitamin C or NAC (255). Thesefindings mirror those of the largely negative results oflarge-scale antioxidant trials for CVD in general (256),and may reflect the inability of current compounds toreach and reduce oxidative stress at key biologicalareas. Nevertheless, the identification of individualswith polymorphisms of antioxidant genes as beingparticularly susceptible to the effects of air pollution(212) suggests that this avenue of research still de-serves further attention in hypothesis-driven studieswith long-term intervention.

Finally, although targeting human toxins that arepotentially even broader in their range than those insmoking and air pollution, including the full gamut oflifetime environmental exposures, comment isrequired regarding chelation therapy and TACT (Trialto Assess Chelation Therapy) (257). TACT was aNational Institutes of Health–sponsored, double-blind, placebo-controlled, 2 � 2 factorial randomizedtrial enrolling 1,708 patients 50 years of age or olderwho had experienced a myocardial infarction at least6 weeks prior and had serum creatinine levels of2.0 mg/dl or less. Participants were recruited across134 U.S. and Canadian sites. Patients were randomizedto receive 40 infusions of a chelation solution(comprising disodium ethylenediaminetetraaceticacid, ascorbate, B vitamins, electrolytes, procaine, andheparin; n ¼ 839) versus placebo (n ¼ 869), and an oralvitamin-mineral regimen versus an oral placebo. Theputative active chelating agents (the infusions ofvitamins and disodium ethylenediaminetetraaceticacid), are believed to act by binding divalent and sometrivalent cations, including calcium,magnesium, lead,cadmium, zinc, iron, aluminum, and copper, facili-tating their urinary excretion (257). The pre-specifiedprimary endpoint was a composite of total mortality,recurrent myocardial infarction, stroke, coronaryrevascularization, or hospitalization for angina.Importantly, the primary endpoint occurred in

222 (26%) of the chelation group versus 261 (30%) of theplacebo group (hazard ratio: 0.82 [95% confidenceinterval: 0.69 to 0.99]; p ¼ 0.035). This somewhatunexpected result, published in 2013, was met with adegree of skepticism (258), and vigorous debateensued. Regardless of these controversies, the efficacyof chelation therapy should soon be definitivelyshown, as the TACT2 trial (NCT02733185), with asimilarly rigorous design as its predecessor, beganrecruitment in October 2016, with a target enrollmentof 1,200 subjects. The TACT2 trial should prove orrefute the efficacy and clinical role of this approach.CONCLUSIONS REGARDING POLLUTION, SMOKING, AND

OXIDATIVE STRESS. There is now overwhelming evi-dence that air pollution is associated with CVD,with expert opinion suggesting it should be formallyrecognized as a risk factor in the same way that smok-ing of tobacco products has been for many years. Thereare striking similarities between the physicochemicalcomposition of cigarette smoke and combustion-derived air pollution, and both are associated withmultiple detrimental cardiovascular effects.

A number of unresolved issues in the field requireattention. First, although it is assumed that certainindividuals are more susceptible to air pollution thanothers (e.g., children, the elderly, and those with pre-existing cardiorespiratory disease), this has not beendefinitively shown and requires further elaborationso that practical guidance can be provided to those atrisk (e.g., altered activity, dietary supplementation/medication, or interventions such as facemasks or airpurifiers). Additionally, although the particulates andsome gases (e.g., NO2) are highly likely to be detri-mental constituents of urban air pollution, moreprecise identification of the components that drivethe cardiovascular action of air pollution are required(e.g., particle sizes and sources, and the degree ofchemical interaction between gases and particulates).Smoking, although declining in prevalence, is alsonot without its outstanding health issues. The currentpopularity of “smoking alternatives,” such ase-cigarettes, should continue to be a topic of research,especially to establish the constituents of the fumesthat could be associated with health effects.

Although the biological mechanisms underlyingthe cardiovascular actions of smoking and air pollu-tion must still be fully established, there is a clear rolefor oxidative stress as a key mediator that will exac-erbate and potentially instigate the disease process.In particular, the biological pathways that link theinitial lung response to air pollutants to that of thesubsequent cardiovascular actions remain an impor-tant undetermined area for future research. Preciseidentification of the mechanisms at play will be

https://clinicaltrials.gov/ct2/show/NCT02733185



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extremely useful for identifying which constituentsof air pollution are especially harmful and who isparticularly susceptible to their effects. At present,though, reducing the prevalence and exposure toboth these environmental risk factors remain the keymeans to preventing the significant burden that theyinflict on health.

ADDRESS FOR CORRESPONDENCE: Dr. Jason C.Kovacic, Cardiovascular Institute, Mount Sinai Hospital,

One Gustave L. Levy Place, Box 1030, New York, NewYork 10029. E-mail: [email protected]. ORDr. Susanne Rohrbach, Institute of Physiology, Justus-Liebig University Giessen, Aulweg 129, 35392 Giessen,Germany. E-mail: [email protected]. OR Prof. David E. Newby, BHFCentre for Cardiovascular Science, University ofEdinburgh, Chancellor’s Building, 49 Little FranceCrescent, Little France, Edinburgh, United Kingdom,EH16 4SB. E-mail: [email protected].

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KEY WORDS air pollution, atherosclerosis,diabetes, exhaust, inflammation, metabolicstress, obesity, particulate matter, tobacco
























































































































































































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