Role of free radical in atherosclerosis, diabetes and dyslipidemia: larger-than-life

Role of free radical in atherosclerosis, diabetes anddyslipidaemia: larger-than-life

Randhir Singh1*Sushma Devi1

Rakesh Gollen2

1MM College of Pharmacy, MaharishiMarkandeshwar University, Mullana,Ambala, India2Department of PharmaceuticalSciences, Long Island University,Brooklyn, New York, NY 11201, USA

*Correspondence to: Randhir Singh,Associate Professor, MaharishiMarkandeshwar College ofPharmacy, MaharishiMarkandeshwar University,Mullana, Ambala, India.E-mail: [email protected]

Abstract

During the past few decades, there have been numerous studies related to the Q1

field of free radical chemistry. Free radicals including reactive oxygen species(ROS) and reactive nitrogen species are generated by the human body by variousendogenous systems, exposure to different physiochemical conditions, orpathological states, and thus, they participate in the pathogenesis of increasingnumbers of diseases. These free radicals are also the common by-products ofmany oxidative biochemical reactions in cells. In the past few years, there havebeen more attention and focus towards the field of free radical chemistry. If freeradicals overwhelm the body’s ability to regulate them, a condition known asoxidative stress ensues. Thus, they adversely alter lipids, proteins, and DNA,which triggers a number of human diseases. In a number of pathophysiologicalconditions, the delicate equilibrium between free radical production andantioxidant capability is distorted leading to oxidative stress and increased tissueinjury. ROS which are mainly produced by vascular cells are implicated aspossible underlying pathogenic mechanisms in a progression of cardiovasculardiseases including ischemic heart disease, atherosclerosis, cardiac arrhythmia,hypertension, and diabetes. This review summarizes the key roles played by freeradicals in the pathogenesis of atherosclerosis, diabetes, and dyslipidaemia.Although not comprehensive, this review will also provide a brief perspectiveon some of the current research being conducted in this area for a better under-standing of the role free radicals play in the pathogenesis of atherosclerosis,diabetes, and dyslipidaemia. Copyright © 2014 John Wiley & Sons, Ltd.

Keywords free radicals; ROS; atherosclerosis; diabetes; dyslipidaemia

Introduction

The recent growth in the knowledge of free radicals including reactive oxygenspecies (ROS) and reactive nitrogen species (RNS) in biology is producing amedical revolution that promises a new age of health and disease managementand pathogenesis of various disorders and diseases. A free radical can be de-fined as any molecular species capable of independent existence that containsan unpaired electron in an atomic orbital. Many radicals are unstable andhighly reactive in their present state, which can either accept one electron ordonate from other molecules; therefore, they behave as oxidants or reductants,respectively [1]. The life span of free radicals ranges from the high reactivity ofhydroxyl (*OH), which has a half-life of 1 ns, to the low reactivity of melanin

REVIEW ARTICLE

Received: 25 September 2013Revised: 17 April 2014Accepted: 29 April 2014

Copyright © 2014 John Wiley & Sons, Ltd.

DIABETES/METABOLISM RESEARCH AND REVIEWSDiabetes Metab Res Rev 2014Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/dmrr.2558

Journal Code Article ID Dispatch: 21.05.14 CE: Mary Charlane PayuanD M R R 2 5 5 8 No. of Pages: 14 ME:

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with the half-life of a few day, whereas nitric oxide (NO*)and ubisemiquinone have half-life of ~10 s and 10 ms,respectively, and show intermediate reactivity [2]. Theformation of free radicals as by-products of metabolismand cellular respiration is also known to be synthesizedby enzyme systems (nicotinamide adenine dinucleotidephosphate (NADPH) oxidase, myeloperoxidases), andby exposure to ionizing radiation, smoking, herbicides,pesticides, fried foods, etc. [3–6]. The most importantoxygen-containing free radicals formed in human body,which are also responsible for different disease statesinclude hydroxyl radical (*OH), superoxide anion radical(O2*), hydrogen peroxide (H2O2), oxygen singlet (O2*),hypochlorite (ClO*), nitric oxide radical (NO*), andperoxynitrite radical (ONOO*). Collectively, they arecalled ROS and RNS. ROS also forms different radicals,e.g. R*, ROO*, RO*, RS*, RSO2*, RSO*, etc. [7]. Thesefree radicals are highly reactive species, capable ofdamaging biologically relevant molecules such as DNA,proteins, carbohydrates, and lipids in the nucleus, and inother cell membranes. They are formed by several knownpathways, which may be either a result of homolyticcleavage of covalent bond of molecule or as the result ofloss and/or addition of single electron to a stable mole-cule [8,9]. These free radicals and the resultant oxidativestress were explored after the discovery of enzyme super-oxide dismutase (SOD) [8], which catalyses thedismutation of highly reactive O2* into H2O2 [10] and,thus, acts as an intracellular generator of H2O2. Ascharged O2* is unable to diffuse through intracellularmembranes, cells express distinct SOD isoforms in thecytosol (Cu, Zn-SOD) and mitochondria (Mn-SOD),where they locally scavenge O2* leading to productionof H2O2. This H2O2 is most efficiently scavenged by theenzyme glutathione peroxidase (GPx), which requiresglutathione (GSH) as the electron donor. Oxidized gluta-thione (GSSG or glutathione disulphide) is reduced backto GSH by the enzyme glutathione reductase which usesNADPH as the electron donor in the organism [11,12].This oxidized glutathione (GSSG) is accumulated insidethe cells, and the ratio of GSH/GSSH is a measure of oxida-tive stress in humans. Some transition metals (e.g. Fe2+,Cu+ among others) can break H2O2 to the reactive hydroxylradical (*OH) which can cause alteration of DNA bases,break DNA strands, inactivate proteins, and result in lipidperoxidation. [13–15]. This lipid peroxidation is anotherpathway for the formation of ROS, where, lipid hydro-peroxide reacts quickly with Fe2+ to form a lipid oxyl radical(LO*) or much more slowly with Fe3+ to form a lipidperoxyl radical (LOO*). The hydroxyl radical can alsoremove an electron from polyunsaturated fatty acid and inthe presence of molecular oxygen, results in a LOO* [16],and if lipid peroxyl radical is further not reduced by antiox-idants, this results in the process of lipid peroxidation [17].

LOO* can be reduced within the membrane by the reducedform of vitamin E and can form a lipid hydro-peroxide and aradical of vitamin E [18]. The regeneration of vitamin Eoccurs with vitamin C (ascorbic acid) where the vitamin Eradical is reduced back to vitamin E by ascorbic acid andleaves behind the ascorbyl radical (Asc*) [19]. The GSSGand the ascorbyl radical (Asc*) are reduced back to GSHand ascorbatemonoanion (AscH*), respectively, cooperatingwith NADPH and the dihydrolipoic acid (DHLA). ThisDHLA itself is converted to α-lipoic acid [20–22] and alsoregenerates back from α-lipoic acid using NADPH reaction.Coenzyme Q10 (CoQ10) (ubiquinone), in its reduced form,inhibits lipid peroxidation and can protect mitochondrialinner-membrane proteins and DNA from oxidative damage.It is the most widely used co-factor supplement in the treat-ment of mitochondrial disorders [23].

Free radicals attack important macromolecules leadingto cell damage and homeostatic disruption. There issignificant evidence, showing that most of the degenerativediseases that afflict humanity have their origin from delete-rious free radical reactions. Cancer, atherosclerosis, inflam-matory joint disease, asthma, diabetes, senile dementia,and degenerative eye diseases and ageing are salient ‘freeradical’ diseases [24]. According to a World Health Organi-zation report, cardiovascular disease (CVD) remains themajor cause of mortality throughout the world, typicallycausing one third of all deaths [25,26]. The oxidativeevents may affect CVD; therefore, they have the potentialto provide enormous benefits to human well-being. Thereare different known causes for cardiovascular disordersincluding atherosclerosis, diabetes, dyslipidaemia, obesity,and hypertension. The primary cause of CVD is atheroscle-rosis, which refers to the hardening of arteries [27]. Studieson atherosclerosis reveal the probability that the diseasemay be due to free radical reactions involving diet-derivedlipids in the arterial wall and serum to yield peroxides andother substances. Poly unsaturated fatty acids occur as amajor part of low density lipoproteins (LDL) in the blood,and oxidation of these lipid components in LDL plays a vitalrole in atherosclerosis, which progresses slowly during aperson’s lifetime and typically begins before adulthood.This slow progression and its complex etiology havechallenged attempts to identify the initial atherogenic eventso that it remains elusive [28]. The other important knowncause of CVD is diabetes, which confers a fourfold risk foratherosclerosis, manifested in the vessel wall as intimalthickening, smooth muscle hypertrophy, lipid accumula-tion, extracellular matrix expansion, and inflammatory cellinfiltration [29]. There is much known evidence regardingthemechanisms responsible for accelerated atherosclerosis,which occurs in patients with diabetes, and it is believedthat proteins in blood containing amadori glucose adductsmay play a substantive role and are strongly linked to thepathogenesis of vascular complications of diabetes [30].

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Copyright © 2014 John Wiley & Sons, Ltd. Diabetes Metab Res Rev 2014DOI: 10.1002/dmrr

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Hyperlipidaemia or multiple-type hyper-lipoproteinemia isthe responsible factor for CVD and increases platelet activity.Oxidized LDL is antherogenic and cytotoxic and plays asignificant role in the formation of atherosclerosis plaques[31]. Atherosclerosis, diabetes, and dyslipidaemia areknown as the major causes of CVD, and are linked to havesome kind of interconnection with free radicals. Thus, thereview emphasizes the relationships between free radicals,atherosclerosis, diabetes, and dyslipidaemia.

Relationship between free radicals andatherosclerosis

In the past, the role of free radicals in the development ofatherosclerosis is widely understood [32]. The develop-ment of atherosclerosis is a multifactorial process in whichboth elevated plasma cholesterol levels and proliferationof smooth muscle cells (SMC) play central roles [33].Considerable in vivo studies support the role of free radi-cal reactions in atherogenesis and atherosclerotic-relatedcoronary heart disease. Free radicals are involvedthroughout the atherogenic process beginning from endo-thelial dysfunction up to the rupture of a lipid-rich athero-sclerotic plaque, which further leads to acute myocardialinfarction or sudden death [34]. An increased concentra-tion of plasma LDL cholesterol plays a major role in thedevelopment of atherosclerosis. Epidemiological, clinical,and genetic studies convincingly demonstrate that LDLpromotes atherosclerosis. However, the precise mecha-nism(s) by which LDL promotes the development of theearly fatty-streak lesion still remain to be elucidated[35]. LDL is composed of a hydrophilic surface layer ofphospholipid, free cholesterol, and hepatically derivedapoB100 to package the particle and to add stability.The core of the particle includes esterified cholesteroland triglyceride together with the fatty acid tails of thephospholipid. LDL may be oxidative modified by all majorcell types of the arterial wall including endothelial cells,SMC, and macrophages via their extracellular release ofROS [35]. Additionally, the initiation of peroxidationcatalysed by a hydroxyl radical within the LDL moleculegives rise to conjugated dienes, and lipid hydroperoxyradicals (LOO*) [36]. Hydrogen peroxide also has beenreported to increase vascular permeability, prostacyclinrelease, and translocation of P-selectin to the endothelialcell surface [37,38]. The free radical chain reaction isself-propagating, so that LOO* can attack adjacent fattyacids until complete fatty acid chain fragmentation occurs[34]. During free peroxidation, reactive products then ac-cumulate in the LDL particle, including malondialdehyde(MDA) and lysophosphatides (FigureF1 1) [4]. These prod-ucts interact with the amino side chain of the apoB100and modify it to form new epitopes which are not

recognized by the LDL receptor [34]. Antioxidants likeB-carotene or vitamin E play a vital role in the preventionof various CVD. Whereas apoprotein E (apoE) is reportedto impede sub-endothelial LDL retention, it is an earlyproatherogenic event and protects against oxidative stressand apoptosis. ApoE has an anti-oxidant activity andinhibits LDL oxidation; it also prevents LDL retention inthe artery wall and delivers lipid antigens to CD1 indendritic cells for presentation to T-cells. This alsoincludes apoE-mediated inhibition of platelet aggregationand smooth-muscle-cell migration and proliferation [39].Oxidized and glycated LDL are taken up by the macro-phage scavenger receptors such as SRB1, CD36, and TR4[40]. Oxidized LDL may also form oxidized LDL–antibodycomplexes which can be taken up by the macrophagefoam cell receptor. Scavenger receptor uptake is not regu-lated and leads to macrophage cholesterol accumulationand foam cell formation in the arterial intima [35]. Thefoam cell recruits more monocyte/macrophages to convertto foam cells. Accumulating LDL-laden foam cells beneatharterial endothelium lay the foundation for the fattystreak, the earliest histopathological evidence of thedevelopment of atherosclerotic plaque [41]. Endothelial

Figure 1. Pathways of ROS formation and lipid peroxidationprocess O2•

� is dismutated by SOD into H2O2 and further, H2O2 effi-ciently scavenged by GPx. Transition metal (Fe2+) can breakdownH2O2 into •OH which removes an electron from polyunsaturatedfatty acid to give rise to a lipid radical (L•) and finally leads to theformation of lipid peroxyl radical (LOO•). Abbreviations: GSH—glutathione, GSSG—oxidized glutathione, H2O2—hydrogenperoxide, L•—lipid radical, LO•—lipid alkoxyl radical, LOO•—lipidperoxyl radical, MA—deoxyadenosine adduct, MC—deoxycytidineadduct, MG—deoxyguanosine adduct, MDA—malondialdehyde,NADPH—nicotinamide adenine dinucleotide phosphate,O2•

�—superoxide radical, •OH—hydroxyl radical

Role of Free Radicals in Atherosclerosis, Diabetes and Dyslipidaemia 3


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dysfunction is a known biomarker of cardiovascular riskfactors, and it precedes the development of atherosclerosis.Endothelial dysfunction is also involved in lesion formationby the promotion of both the early and late mechanisms ofatherosclerosis including up-regulation of adhesion mole-cules, increased chemokine secretion, leukocyte adherence,increased cell permeability, enhanced low-density lipopro-tein oxidation, platelet activation, cytokine elaboration,and vascular SMC proliferation and migration. A dysfunc-tional endothelium promotes many processes involved inatherogenesis. These include decreased bioavailability ofnitric oxide (NO*), migration of SMC, and/or an increasein endothelium-derived contracting factors and prolifera-tion which contribute to the neointimal formation betweenthe endothelium and the inner elastic lamina [42,43].Oxidized LDL also stimulates the release of monocyte-derived tumour necrosis factor-α (TNF-α) and IL-1β, leadingto SMC proliferation [44]. SMC elaborate collagen andelastin and provide the foundation for plaque formationand fibrosis [45]. Oxidized lipids, derived from oxidativemodified LDLs, which accumulate in the intima, stronglymodulate inflammation-related gene expression, throughinvolvement of various signalling pathways [46]. Theoxidative stress factors and oxidative modification ofbiomolecules are also involved in a number of other knownphysiological and pathophysiological processes such asaging, inflammation, carcinogenesis, and drug toxicity.Lipid peroxidation is another process which involves asource of secondary free radical; thus, it acts as a secondmessenger or can directly react with other biomolecules,further enhancing the biochemical lesions. This processmainly occurs in polysaturated fatty acid located on the cellmembranes, which further leads to radical chain reaction.Hydroxyl radical is thought to initiate ROS, by removinghydrogen atoms, thus producing lipid radicals, whichfurther converted into conjugate dienes. After the additionof oxygen, this conjugate diene forms a peroxyl radical,which is a highly reactive radical. It then attacks anotherfatty acid resulting in the formation of lipid hydro-peroxide(LOOH*) and additional free radical. Lipid peroxides alsoinhibit the synthesis of prostacyclin, which is used to inhibitplatelet aggregation resulting in platelet adherence [47].The aggregation of platelets further lays the foundationfor formation of thrombus [42]. Platelet release growthfactor subsequently leads to SMC proliferation andmigration to intima. Measurement of this intima-mediathickness using high resolution B-mode ultrasonographyhas emerged as one of the methods of choice for determin-ing the anatomic extent of preclinical atherosclerosis andfor assessing cardiovascular risk related to it [48]. Increasednumber of ROS formation in mitochondria results in mito-chondrial DNA damage, and progressive respiratory chaindysfunction, which are associated with atherosclerosis orcardiomyopathy in human and animal studies of oxidative

stress [49]. It has also been reported that, especially inindividuals with aging and various types of cancer, DNA isconsidered to be a major target [50]. Free radical-inducedalterations in the endothelial cells of arterial the wall havebeen implicated in the pathogenesis and progression ofatherosclerosis. Endothelial cell injury is measured bydecrease count in protein, glyceride, and phospholipidsynthesis. The concurrent increases in lipid peroxidationand cholesterol synthesis may explain the relationshipbetween free radical injury and the pathogenesis of athero-sclerosis [51]. HDL is inherently capable of exerting anti-atherogenic effects by metabolizing and transporting lipidoxidation products as well as cholesterol from the cells tothe liver by virtue of paraoxonase-1 (PON-1), lecithin-cholesterol acyltransferase, phospholipase A-2, and platelet-activating factor acetyl hydrolase [52]. The oxidativemodification of HDL impairs the ‘reverse cholesterol trans-port’ ability of apoA-I and also the anti-inflammatoryfunction of HDL [53]. It is assumed that HDL is oxidizedessentially by the same oxidants, and also by the samemechanisms as LDL. It has been found that HDL is a majorcarrier of lipid hydro-peroxides.

Relationship between free radicals anddiabetes

Diabetes is a group of chronic diseases characterized byhyperglycaemia. Indeed, coronary heart disease andperipheral vascular disease are the leading causes ofmorbidity and mortality due to diabetes mellitus [54].Diabetes is associated with the formation of the ROS bydifferent pathogenic pathways [55]. Free radical activityhas been previously implicated in the development ofdiabetic vascular complications in type- I diabetes mellitus.It plays an important role in both micro-vascular andmacro-vascular complications during diabetes mellitus,which is a known metabolic disorder, characterized byhyperglycaemia resulting from a deficiency in insulin actionor secretion and associated with several vascular complica-tions. Long-term complications of diabetes mellitus areassociated with various oxidative reactions, increased freeradical generation, and subsequent increase in oxidativestress [56], particularly those which are catalysed by de-compartmentalized transition metals playing a significantrole in diabetic tissue damage [57,58]. In hyperglycaemia,ROS is produced by various processes: glucose oxidation,glucose toxicity [59], and oxidative phosphorylation [60].Chronic exposure to hyperglycaemia can lead to cellulardysfunction which may become irreversible over time by aprocess called glucose toxicity [61]. Multiple biochemicalpathways and mechanisms of action for glucose toxicityinclude glucose autoxidation, protein kinase C activation,methyl-glyoxal formation and glycation, hexosamine

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metabolism, sorbitol formation, and oxidative phosphor-ylation. There are many potential mechanisms wherebyexcess glucose metabolites travelling along thesepathways might cause beta cell damage. However, allof these pathways have in common the formation ofROS that, in excess and over time, cause chronic oxida-tive stress, which in turn can result in defective insulingene expression and insulin secretion as well asincreased apoptosis [62]. The destruction of β cells byautoimmune cells occurs not only in type-I diabetes,but also in type-II diabetes. Insulin resistance in type-IIdiabetes mellitus may be the consequence of abnormalproduction of anti-insulin receptor antibodies, but isgenerally due to post-receptor defects [63–65].

Also, a recent study has shown that thyroid ROS areelevated in experimental diabetic rats, which is a conse-quence of low-serum thyroid-stimulating hormone andinsulin, but is also related to hyperglycaemia [66]. Toxicamounts of reactive oxygen intermediates are releasedby endothelial cells and infiltrating macrophages duringislet inflammation [63]. It is known that islet cells haveinsufficient defence systems against free radical attacks,which makes them susceptible to reactive oxygen inter-mediates, leading to the destruction of these cells [8].Macrophages are innate immune cells derived frommonocytes, which have many important roles in theinnate and adaptive immune response, as well as in tissuehomeostasis. These macrophages are known to be centralplayers in diabetes, which act in two different ways. Thefirst one triggers inflammatory responses which initiateinsulitis and pancreatic β cell death during type-I diabe-tes, whereas the other regulates type-I diabetes nega-tively, by decreasing the glucose level hyperglycaemia,insulitis, and inflammation in the pancreas, therebynegatively regulating type-I diabetes [67]. These oxygenradicals not only play a significant role in the pathogenesisof diabetes, but are also involved in some of the complica-tions seen in long-term diabetes [56,68]. In autoimmunediabetes, the interactions between CD4-T cells and macro-phages are critical to the development and progression ofinflammatory events in the pancreatic islet lesion. Cyto-kines and chemokine produced by auto reactive CD4-Tcells lead to recruitment and activation of macrophages,which, upon activation, produce additional inflammatorymediators and become effector cells during pathogenesis[69]. In patients with diabetes mellitus, LDL oxidationby macrophages is also increased due to the activation ofseveral pro-oxidant systems, as well as due to the deple-tion of antioxidants such as the paraoxonases (PONs),which protect against atherogenesis by exerting a protec-tive role against diabetes mellitus development by stimu-lating insulin secretion from β cells, and also by theirunique antioxidant properties [70]. ROS also regulatesmacrophage scavenger receptor in type-I diabetes, but

not in type-II, in the human monocyte cell line. Oxidativestress also contributes to increased activity in macrophagescavenger receptor (MSR) by stabilizing MSR-I mRNA[71]. Further, hyperglycaemia also increases the expres-sion of growth factors and cytokines such as transforminggrowth factor-β, vascular endothelial growth factor, plate-let-derived growth factor, insulin-like growth factor, andTNF-α. ROS are found to be important arbitrator factorsinvolved in all these events, and they activate intracellularsignal transduction and transcription cascades in whichmitogen activated protein kinases (MAPKs) and nuclearfactor kappa B play the most significant roles [17,72–74].Proteins, lipids, and nucleic acids are damaged by oxida-tion (Figure F22). Osmotic stress from sorbitol accumulationhas also been postulated as an underlying mechanism inthe development of diabetic microvascular complications,including diabetic retinopathy and nephropathy [75].During hyperglycaemia, the polyol pathway has increasedactivity, and these pathways are responsible for the reduc-tion of glucose to sorbitol via the aldose reductaseenzyme, utilizing the cofactor NADPH [72]. This reduc-tion reaction leads to increased sorbitol accumulation inthe cells and results in cellular and organ damage [76].Additionally, excessive accumulations of sorbitol result inthe decrease of myo-inositol in the peripheral nerves.

Figure 2. ROS-induced DNA damage activates PARP and modifiesGAPDH and mechanism of hyperglycaemia induced cellulardamage. In case of diabetes and hyperlipidaemia, the level ofglucose increases which further increases the level of free radicals.The increased level of ROS is responsible for activation of PARPwhich is involved in modification of GAPDH. Modified GAPDH isinvolved in different pathological mechanisms like activation ofprotein kinase C and increased production of advanced glycationend-products and hexamine pathways, etc.; these pathways alongwith free radicals play a significant role in the pathogenesis of dia-betic complications. Abbreviations: AGEs—advanced glycation endproducts, GAPDH—glyceraldehyde phosphate dehydrogenase,NAD+-nicotinamide adenine dinucleotide, PARP—poly (ADP-ribose) polymerase, PKC—protein kinase C, ROS—reactive oxygenspecies, ADPR—ADP ribose, NA—nicotinamide, PW—pathway



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When myo-inositol is decreased, there is a resultingdecrease in Na+, or K+-ATPase activity, which is essentialfor nerve conduction [77]. It is known that increase in theproduction of ROS leads to more oxidative stress, whichfurther simulates the dysfunction and destruction of βcell, and leads to the impairment of insulin function[78]. Auto-oxidative glycosylation is important inexplaining free radical formation and protein damage indiabetes [79]. High glucose concentration facilitates theeicosanoid pathway and, consequently, production of thesuperoxide. In the endothelial cells, NO is synthesizedfrom arginine by NO synthase and is quenched by super-oxide, resulting in peroxynitrite anion formation. Inhibi-tion of the nitric oxide action reduces vasodilatation andenhances endothelial dysfunction in diabetes [80].Hyperglycaemia also elicits oxidative stress by directlyimpairing the cellular mechanisms, which, in turn, elicitsendothelial dysfunction [81]. Moreover, hyperglycaemiais also responsible for the generation of NO* and O* inexcess through activation of nitric oxide synthases (NOSs)and NADPH oxidase, respectively. O2* reacts with NO* toproduce OONO*, another oxidant which increases oxida-tive stress and elicits endothelial dysfunction by promot-ing tissue injury [82]. O2* is converted to H2O2 bySODs, which not only increases oxidative stress, but alsogenerates endothelial dysfunction by modulating intracel-lular signalling and transcription factors [82,83]. In addi-tion to O2*, hyperglycaemia also stimulates the synthesisof NO via increased enzymatic activity of endothelialand inducible isoforms of NOS [81–84]. According toDesco et al. (2002), ketone, especially acetoacetate, isinvolved in free radical formation in type-I diabetes [79].The production of ketone bodies is especially important inbrain metabolism, which, under normal circumstances,depends mainly on glucose [85,86]. Glucose is not toxic atphysiological conditions; however, at higher concentra-tions, it undergoes auto-oxidation, and noncyclic proteinsare non-enzymatically glycated by high levels of noncyclicglucose. Glucose is also able to modify proteins by theattachment of its oxidation derived aldehydes, thus leadingto the development of novel protein fluorophores andfragment protein via free radical mechanisms [67]. How-ever, the metal chelators and free radical scavengers caninhibit the fragmentation of these proteins [87]. Transitionelements (for example iron) are involved in free radicalproduction (Fe2+« Fe3+), and iron (II) ions are also knownto induce oxidation of ascorbic acid and glucose [88]. Thesimultaneous episodes of glycation and oxidation furthersupport the hypothesis that tissue damage is associatedwith diabetes [87,89,90]. Xanthine oxidase, a superoxide-generating enzyme, is also involved in free radical produc-tion in patients with type-I diabetes, which can be protectedby allopurinol [77]. Generation of high levels of ONOO* ionsinitiates the onset of different processes like mitochondrial

dysfunction, which is a part of premature aging of the endo-thelium [91]. Hyperglycaemia-induced overproduction ofsuperoxide by the mitochondrial electron transport chainactivates the four major pathways of hyperglycaemicdamage by inhibiting glyceraldehyde phosphate dehydroge-nase (GAPDH) activity [72], which includes (1) hexosaminebiosynthetic pathway [92], (2) sorbitol-aldose reductasepathway [93], (3) MAPK [94], and (4) protein kinase C[95]. GADPH is a classic glycolytic enzyme that alsomediatescell death by its nuclear translocation under oxidative stress[96]. Hyperglycaemia-induced mitochondrial superoxideproduction further inhibits GAPDH by activating PARP[72]. Abnormally high levels of free radicals and the simulta-neous decline of antioxidant defencemechanisms can lead todamage of cellular development of insulin resistance. Theconsequences of this oxidative stress also promote the devel-opment of diabetes mellitus and its complications [97].

Relationship between free radicals anddyslipidaemia

It is well known that free radicals play a crucial role in thepathogenesis of fatty liver disease and increase metabolicalterations [98]. As discussed earlier, high lipid or choles-terol (LDL) levels are important factors responsible forvarious cardiac diseases since they lead to developmentof hyperlipidaemia, atherosclerosis, and ischemic heartdisease. ROS are known to be associated with the forma-tion of oxidized LDL that, in turn, stimulates the releaseof ROS and enhanced adhesion molecule expression aswell as platelet activity with pro-atherogenic effects inthe vasculature [99]. Hyperlipidaemia is characterizedby increased LDL and triglyceride concentrations, whichis often accompanied by decreased HDL [100]. Type-IIhyper-cholesterolemia causes changes in the structure andfluidity of erythrocyte plasma membranes since the excesscholesterol affects the normal rheology of blood throughits interaction with erythrocytes. Hyper-cholesterolemiamay also decrease the deformability of red blood cells,which impairs their haemorheological behaviour and thuspromotes atherosclerosis. It also impairs the function andstructure of plasma membrane proteins [101]. Evolutionof hyper-cholesterolemia is also associated with endothelialcell dysfunction, a near complete abrogation in vascular NObioavailability, elevated oxidative stress, and creation ofpro-inflammatory condition; these are symptoms whichcan culminate in profound impairments/alterations tovascular reactivity [9]. Dyslipidaemia, with or withoutatherosclerosis, is one of the major metabolic/CVD statesin which the enhanced formation of ROS is of pivotalpathogenic importance [102]. Hyperlipidaemia withelevated levels of triglycerides, chylomicron remnants, andfree fatty acids results in oxidative stress and inflammation

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and may independently potentiate the adverse effects ofhyperglycaemia [103]. It is well known that NO rapidlyreacts with O2* to form the cytotoxic species (ONOO*),and hyperlipidaemia has been shown to increase productionof ROS including ONOO* in the vasculature, which results ina deterioration of cardiac function. Hyperlipidaemia alsoincreases the plasma nitro-tyrosine level, which is a markerfor systemic ONOO* generation [104]. Some known cyto-toxic effects of ONOO* include lipid peroxidation, nitrationof tyrosine residues, oxidation of sulfhydryl groups, tissueinjury, and DNA-strand breakage [105]. The detrimentaleffects of increased oxidative stress result in dysfunction ofendogenous NO*, production and inactivation of NO* byoxygen-derived radicals (eliciting the formation of thecytotoxic and genotoxic ONOO*), or altered function ofNOS [106]. Excessive formation ofONOO* is associatedwitha significant decrease in cellular glutathione content and,subsequently, of NOS as well as of soluble guanylate cyclaseactivity (e.g. through oxidation of thiol groups {�SH} [107].Enhanced lipid peroxidation and decreased antioxidant en-zyme activity also represent early events in the developmentof hyperlipidaemia in humans [108]. In high lipid conditions,decreased activities of SOD and GPx are observed. Thus,there is insufficient detoxification of these ROS by antioxi-dant enzymes thatmay lead to an imbalance between antiox-idant and oxidant systems [109]. This oxidant–antioxidantstatus has a major impact, not only on the rate of LDL oxida-tion, but also on the development of atherosclerosis [110]. Itis possible that a potential risk of atherosclerosis in the hyper-lipidaemia population is associated with LDL oxidation anddecreased antioxidant enzyme activity. Further hypertriglyc-eridemia and hypercholesterolemia are associated with LDLperoxidation, protein glycation, glucose auto-oxidation, andthus lead to excess production of free radicals, which mayfurther elevate the oxidative stress in hyperlipidaemiapopulation [111].

Relationship of nitric oxide withatherosclerosis, diabetes, anddyslipidaemia

NO is an endogenous anti-atherogenic molecule. The dualrole of NO as a protective and toxic molecule depends onseveral factors. Primarily, it depends on the isoform ofNOS involved, and there are three main isoforms of theenzyme: neuronal NOS (nNOS), inducible NOS (iNOS),and endothelial NOS (eNOS). They differ in their depen-dence on Ca2+, as well as in their expression and activities[112]. The important factor includes eNOS, which are theonly NOS isoform expressed in normal coronary arteries,and it offers protection against atherosclerosis [113].iNOS is proatherogenic, so it decreases atheroscleroticplaque areas [114]. Additionally, nNOS might modulate

atherosclerosis by directing the flow of NO* towardparticular targets [115]. The important isoform in theregulation of insulin resistance is iNOS. Understandingthe molecular mechanisms regulating the iNOS pathwayin normal and hyperglycaemic conditions would help toexplain some of the vascular abnormalities observed intype-II diabetes mellitus [116]. The other factors include,but are not limited to, the concentration of NO* type cellsin which NO* is synthesized, the availability of thesubstrate L-arginine, generation of guanosine 3,5′-cyclicmonophosphate (cGMP) from guanosine triphosphate ca-talyse by soluble guanylate cyclise (sGC), and the overallextra and intracellular environment in which NO* isproduced. NOS activation as a result of trauma (calciuminflux) or infection leads to NO* production, whichactivates its downstream receptor sGC to synthesise cGMPand/or leads to protein nitrosylation. This may lead to oneor more systemic effects including altered neurotransmis-sion, which can be protective or toxic vaso/broncho dila-tation in the cardiovascular and respiratory systems, andenhanced immune activity against invading pathogens[117]. NO* also regulates critical lipid membranes andlipoprotein oxidation events either by contributing to theformation of more potent free radicals from superoxide(i.e. ONOO*) or by its antioxidant properties throughtermination reactions with lipid radicals to potentially lessreactive secondary nitrogen-containing products [118,119].In addition to being a potent vasodilator, it may inhibit anumber of key processes in atherogenesis, including vascu-lar SMC proliferation [120], platelet adherence or aggrega-tion [121], generation of oxygen-derived free radicals [122],and monocyte adherence and infiltration (possibly byinhibiting the expression or activity of endothelial adhesionmolecules or chemotactic proteins) [123–126]. Protein-bound nitrotyrosine (NO2Tyrosine), a post-translationalmodification specific for protein oxidation by NO-derivedoxidants, is markedly enriched within human atheroma.Further, recent clinical studies demonstrate that systemiclevels of protein-bound NO2Tyrosine serve as an indepen-dent predictor of atherosclerotic risk [127].

The impairment of NO* activity by hypercholesterol-emia, diabetes mellitus, or hypertension plays a significantrole in the initiation of atherosclerosis [124]. For example,in a traumatic arterial injury, the loss of endothelialinfluence facilitates platelet plug formation and vasoconstric-tion, thereby promoting homeostasis [126]. This becomes apathophysiologic mechanism when the endothelium isdysfunctional due to a systemic disorder (hyperlipidaemia).Further, platelet adherence to the dysfunctional endothe-lium promotes thrombosis and the growth of vascularlesions [127]. Deficiency of nitric oxide is responsible forreduced nerve blood flow and response in the developmentof endoneurial ischemia in diabetic rats [128]. There is anincreased production of NO* observed immediately after



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the onset of diabetes. NO* may be responsible for theglomerular hyper filtration observed in diabetic kidneys byinducing vasodilation of the afferent arteriole [129,130].In addition, hypercholesterolemia is associated with theloss of nitric oxide-induced vasodilation and the subsequentincrease in blood pressure [131].

Relationship of free radicals withatherosclerosis, diabetes, anddyslipidaemia

The relationship between oxidant generation, oxidativestress, oxidative damage, and various disorders like athero-sclerosis, diabetes, dyslipidaemia, ischemic pain, etc. is nowwell known (FigureF3 3). In many scientific publications,oxidative stress is solely used to describe impaired ROS orRNS systems which may develop secondary hypertension,atherosclerosis, or hyperglycaemia [132]. There is greaterrisk of developing atherosclerosis and its complications:stroke, myocardial infarction, and peripheral vasculardisease in diabetic populations as compared to non-diabeticones [68,133]. Artherogenesis has been associated with

lipid disorders characterized by hypertriglyceridemia, andincreased levels of VLDL and LDL without diabetes.However, diabetic populations with relatively normal plasmalipid and lipoprotein concentrations still have an increasedlevel of atherosclerosis [134]. Therefore, increased incidenceof vascular diseases in diabetic patients may involvedyslipidaemia or other factors unique to diabetes. Severalrisk factors have been proposed to explain the increased riskof CVD with diabetes, e.g. hyperglycaemia, dyslipidaemia[132], accelerated formation of advanced glycation end(AGEs) products, increased oxidative stress, and othergenetic factors [135]. Advanced oxidative stress is generatedby hyperglycaemia and, in many diabetic patients, also bydyslipidaemia which accelerates endothelial dysfunction asthe first step in the development of angiopathy [80]. Inthe diabetic state, the accelerated formation of amadori-modified glycated serum proteins and lipoproteins fostersthe pathogenesis of atherosclerosis [15]. MDA formation,which is a marker of oxidative stress, is altered in differentstages of development of hyperlipidaemia and is also animportant risk factor of atherosclerosis [136,137]. Accordingto Renard et al., (2004) diabetes increases lesion initiation,and diabetes-induced dyslipidaemia leads to a progression

Figure 3. Relationship between free radicals, atherosclerosis, diabetes, and dyslipidaemia. Diabetes and hyperlipidaemia triggerprejudicial pathways like PKC, AGE, and polyol, leading to excessive generation of ROS. As a consequence of redox imbalance, oxidativestress is enhanced leading to increased prevalence of atherosclerosis and other diabetic complications. Abbreviations: FFA—free fattyacid, SMC—smooth muscle cell, PKC—protein kinase C, AGE—advanced glycation end-products, ROS—reactive oxygen species

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of atherosclerotic lesions, and their transformation intomoresevere condition of lesions which are prone to rupture andthrombosis [138]. Many epidemiological studies have alsoestablished that risk factors for diabetes and atherosclerosiscoexist [139]. Further, endothelial dysfunction supportsatherosclerosis [34]. Glucose and free fatty acids overwhelmthe Krebs cycle, stimulate excess production of NAD+whichoutpaces the capacity for oxidative phosphorylation andcreates free radicals [140]. Postprandial in diabetes is complexand involves a variety of factors including hyperinsulinemia,insulin resistance, hyperglycaemia, and disturbed fatty acidmetabolism. Postprandial oxidative stress triggers a numberof atherogenic changes including increases in inflammation,sympathetic tone, vasoconstriction, thrombogenicity, andoxidation of LDL in diabetic and non-diabetic populations[141]. Numerous clinical studies have shown that postpran-dial dyslipidaemia is associated with endothelial dysfunctionin type-II diabetes and with alterations in other surrogatemarkers in the cascade of atherosclerosis [142].

Type-I diabetes is associated with early impairment ofcommon carotid artery structure and function, and thisdiabetic state may be the main risk factor for commoncarotid artery wall stiffening and thickening, which areof considerable concern as possible early events in thegenesis of atheroma [143]. Substantial clinical and exper-imental evidence suggest that both diabetes and insulinresistance cause a combination of endothelial dysfunc-tions, which may diminish the anti-atherogenic role ofthe vascular endothelium. Oxidative stress due to highglucose can also lead to a number of proatherogenicevents [56]. The elucidation of themechanisms of oxidativestress in diabetes and their relationship with atherosclerosiscould potentially identify molecular targets of therapy forthis condition and its cardiovascular consequences [135].Blood flow restriction is achieved by vasoconstrictiondespite increased production of NO*, and the vasodilationeffects of which are overridden by catecholamine (and alsoby angiotensin-II and endothelia). Decreased blood flowreduces the availability of oxygen, provoking massiveglycolysis (hyperglycaemic conditions), which results inthe production of lactate which is exported to the liver forfurther processing [144]. However, this produces localacidosis, which elicits the rapid dissociation ofoxyhaemoglobin, freeing bursts of oxygen in localizedzones of the tissue. The excess oxygen (and nitric oxide)induces the production of ROS, which deeply affects theendothelial, blood, and adipose cells, inducing oxidativeand nitrosative damage, and eliciting an increased immuneresponse, which translates into inflammation, and thenleads to atherosclerosis [145]. Cholesterol-enriched diet-induced hyperlipidaemia results in an increase in cardiacONOO* formation and decrease in the bioavailability ofNO*, both of which contribute to the deterioration ofcardiac performance and may lead to further cardiac

pathologies [104]. Control of hyperglycaemia thus remainsthe best way to improve endothelial function and to preventatherosclerosis and other cardiovascular complications ofdiabetes [81]. Dehydro-ascorbic acid (oxidized form ofvitamin C) is transported into mammalian cells via facilita-tive glucose transporters. Hyperglycaemia inhibits thisprocess by competitive inhibition. This inhibited transportmay promote oxidative stress and contribute to the increasein atherosclerotic CVD observed in patients with diabetesmellitus [146].

Micro-albuminuria is now considered to be an atheroscle-rotic risk factor and predicts future CVD risk in diabeticpatients [144]. A wide range of micro-albuminuria cut-offvalues are currently used for diagnosing the early stagesof nephropathy in patients with type-II diabetes. However,in the same early stage of diabetic nephropathy, there is asignificant correlation between different levels of micro-albuminuria and markers of oxidative stress. Serumconjugated dienes have emerged as the main marker forevaluating kidney damage in diabetic nephropathy [147].Numerous experimental studies suggest that diabetes/hyperglycaemia accelerates atherosclerotic lesion initiation,but not progression of pre-existing lesions. This effect maybe mediated by increased glycosaminoglycan depositionand monocyte recruitment [139]. Maternal hypercholester-olemia during pregnancy is known to be responsible forcascade of pathogenic events. It is also associated withgreatly increased fatty streak formation in human foetalarteries and accelerated progression of atherosclerosisduring the early stages of life [148]. Impaired lipid andglucose metabolism is a typical feature in diabetes. Diabetesis associated with formation of ROS by different pathogenicpathways. Advanced generation of ROS may further accel-erate glycation, sorbitol, protein kinase C, or hexosaminepathways which are activated by high glucose and lipids.These pathogenic mechanisms are closely linked to oxida-tive stress, which has been recognized as the main causefor development of the endothelial dysfunction [149].Further endothelial dysfunction increases permeability ofendothelial cell monolayers, pro-coagulant activity, expres-sion of adhesionmolecules and intracellular oxidative stress[132,150]. Additionally, they serve as the nidus for athero-sclerotic lesion formation. Increased levels of oxidants alsofunction as a link between diabetes, dyslipidaemia, and freeradicals, which respond to atherosclerosis [151]. A majormechanism appears to be the hyperglycaemia/hyperlipid-aemia induced intracellular ROS, produced by the protonelectromechanical gradient generated by the mitochondrialelectron transport chain and resulting in increased produc-tion of superoxide [152]. Two other mechanisms have beenproposed to elucidate the role of hyperglycaemia in ROSformation. One mechanism involves the transition metal-catalysed autoxidation of free glucose; glucose itself initiatesan auto-oxidative reaction and free radical production,



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yielding superoxide anion and hydrogen peroxide [153]. Theother mechanism involves the transitional metal-catalysedautoxidation of protein-bound amadori products, whichyield superoxide and hydroxyl radicals and highly reactivedi-carbonyl compounds [132,154].

Discussions and future prospective

The development of procedures to ameliorate undesirableROS production may be one of the central issues in thefield of research studying aging and other oxidativestress-related diseases [18,155]. Now, it has becomepossible to accurately quantify in vivo and ex vivo ROSconcentrations under different pathophysiological andexperimental conditions by electron paramagnetic reso-nance or electron spin resonance spectroscopy [156].There are some encouraging reports regarding the use ofSOD/CAT mimetic in certain experimental systems.Dietary antioxidants are widely used to ameliorate exces-sive oxidative stress, but scientific proof of their efficacy isscarce. There is, nevertheless, a strong possibility that theprocess of senescence and disease-related wasting results,at least to some extent, from a progressive shift inbiochemical conditions that may not be irreversible inprinciple [18]. Hyperlipidaemia stimulates the generationof ONOO* in the heart, which further leads to myocardialdysfunction. Targeting ONOO* with pharmacologicalactive agents may be an exciting new strategy to protectthe heart and the vasculature in the event of hyperlipi-daemia [104]. Hyperglycaemia induces endothelial celldysfunction, which further activates multiple pathwaysincluding enhanced glycolysis (ROS), the build-up ofglycolytic intermediates (polyol pathway, hexosaminepathway, PKC activation, and AGE formation), and AGEmodification of proteins (ROS, PKC). Given the inter-dependent nature of these pathways, it is not surprisingthat inhibitors targeting one of them can profoundly affecthyperglycaemia-induced alterations in endothelial cellfunction [157]. Apo E is a well-known blood circulatingprotein with pleiotropic atheroprotective properties thathave emerged as a strong candidate for treating hyper-cholesterolemia and other cardiovascular disorders [40].

Mitochondria play an important role in controlling thebioenergetics status of cells in physiological conditions.Consequently, mitochondrial dysfunction also leads to ox-idative stress that subsequently contributes to an increase inthe amount of free radicals (Figure F44). In recent develop-ments, mitochondria-targeted antioxidants (in particularmitochondria-targeted vitamin E) are under investigationand are in development due to their potential for the treat-ment of CVD [158]. Within the mitochondrial phospholipidbilayer, the fat-soluble antioxidants vitamin E and coen-zymeQ-10 both prevent lipid peroxidation, while coenzymeQ-10 also recycles vitamin E and is itself regenerated by therespiratory chain.With the exception of α-lipoic acid studiesin diabetic neuropathy, data from clinical trials are limitedfor antioxidants. Thus, there is a need to further investigatethe pathophysiology of oxidative stress, and to explore therole of antioxidant therapy that will lead to appropriatelydesigned clinical trials showing the promise of antioxidanttherapy [159]. Besides minerals and vitamins etc., there isalso considerable interest in determining the total phenoliccontent [160], and antioxidant capacities of differentvegetables, fruits, spices, medicinal plants, and microalgae[161–167]. Phenolic compounds can reduce high bloodglucose levels by restoring hepatic glycogen and increasinginsulin and glucokinase levels. Therefore, herbal plantswith phenolic compounds have therapeutic implications[168,169]. Recently, researchers have been interested in,for example, antioxidant enzyme systems such as theheme-oxygenase/biliverdin reductase system, which aremodulated by dietary antioxidant molecules, includingpolyphenols and β-carotene and if antioxidants are takenin excess, the risk of originating diseases instead ofpreventing them is quite high [170]. Carnitines, α-tocoph-erol, α-lipoic acid, co-enzyme Q10, and selenomethionineused as antioxidant supplementation in healthy volunteers,resulting in a significant increase in plasma antioxidantstatus by causing a decrease in blood peroxide levels and,thus, a reduction in the formation of ROS at the mitochon-drial level [171]. It is important to note that, as for all drugs,antioxidants may have significant side effects if not usedcorrectly or if used in combination with other drugs. Also,vitamin A, E, and β-carotene, for instance, have been shownto have pro-oxidant effects at higher doses or under certain

Figure 4. Oxidative cellular damage mechanism. Abbreviations: CAT—catalase, GPx—glutathione peroxidise, SOD—superoxidedismutase, GSH—glutathione, GSSG—oxidized glutathione

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conditions [170]. The use of natural antioxidants toaugment physiological antioxidant mechanisms is particu-larly attractive, but potential difficulties exist, for examplein situations where most of the diseases where oxidativedamage has been demonstrated, free radical generation isnot the primary cause of the disease; rather, it plays a rolein further complicating/worsening the disease condition.The possibility of antioxidant therapy may also be harmful

which can be a potential possibility is for any syntheticagent, but it may also be true for natural antioxidant agentstaken in excess which is needed to be investigated further.

Conflict of interest

None declared.

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Role of free radical in atherosclerosis, diabetes and dyslipidemia: larger-than-life

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