Role of free radical in atherosclerosis, diabetes and dyslipidaemia: larger‐than‐life

Summary

During the past few decades, there have been numerous studies related to free radical chemistry. Free radicals including reactive oxygen species (ROS) and reactive nitrogen species are generated by the human body by various endogenous systems, exposure to different physiochemical conditions, or pathological states, and have been implicated in the pathogenesis of many diseases. These free radicals are also the common by‐products of many oxidative biochemical reactions in cells. When free radicals overwhelm the body's ability to regulate them, a condition known as oxidative stress ensues. They adversely alter lipids, proteins, and DNA, which trigger a number of human diseases. In a number of pathophysiological conditions, the delicate equilibrium between free radical production and antioxidant capability is distorted, leading to oxidative stress and increased tissue injury. ROS which are mainly produced by vascular cells are implicated as possible underlying pathogenic mechanisms in a progression of cardiovascular diseases including ischemic heart disease, atherosclerosis, cardiac arrhythmia, hypertension, and diabetes. This review summarizes the key roles played by free radicals in the pathogenesis of atherosclerosis, diabetes, and dyslipidaemia. Although not comprehensive, this review also provides a brief perspective on some of the current research being conducted in this area for a better understanding of the role free radicals play in the pathogenesis of atherosclerosis, diabetes, and dyslipidaemia. Copyright © 2014 John Wiley & Sons, Ltd.

Introduction

The recent growth in the knowledge of free radicals including reactive oxygen species (ROS) and reactive nitrogen species (RNS) in biology is producing a medical revolution that promises a new age of health and disease management and pathogenesis of various disorders and diseases. A free radical can be defined as any molecular species capable of independent existence that contains an unpaired electron in an atomic orbital. Many radicals are unstable and highly reactive in their present state, which can either accept one electron or donate from other molecules; therefore, they behave as oxidants or reductants, respectively 1. The life span of free radicals ranges from the high reactivity of hydroxyl (*OH), which has a half‐life of 1 ns, to the low reactivity of melanin 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. The formation of free radicals as by‐products of metabolism and cellular respiration is also known to be synthesized by enzyme systems (nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, myeloperoxidases), and by exposure to ionizing radiation, smoking, herbicides, pesticides, fried foods, etc. [3-6]. The most important oxygen‐containing free radicals formed in human body, which are also responsible for different disease states include hydroxyl radical (*OH), superoxide anion radical (O₂*), hydrogen peroxide (H₂O₂), oxygen singlet (O₂*), hypochlorite (ClO*), nitric oxide radical (NO*), and peroxynitrite radical (ONOO*). Collectively, they are called ROS and RNS. ROS also forms different radicals, e.g. R*, ROO*, RO*, RS*, RSO₂*, RSO*, etc. 7. These free radicals are highly reactive species, capable of damaging biologically relevant molecules such as DNA, proteins, carbohydrates, and lipids in the nucleus, and in other cell membranes. They are formed by several known pathways, which may be either a result of homolytic cleavage of covalent bond of molecule or as the result of loss and/or addition of single electron to a stable molecule [8, 9]. These free radicals and the resultant oxidative stress were explored after the discovery of enzyme superoxide dismutase (SOD) 8, which catalyses the dismutation of highly reactive O₂* into H₂O₂ 10 and, thus, acts as an intracellular generator of H₂O₂. As charged O₂* is unable to diffuse through intracellular membranes, cells express distinct SOD isoforms in the cytosol (Cu, Zn‐SOD) and mitochondria (Mn‐SOD), where they locally scavenge O₂* leading to production of H₂O₂. This H₂O₂ is most efficiently scavenged by the enzyme glutathione peroxidase (GPx), which requires glutathione (GSH) as the electron donor. Oxidized glutathione (GSSG or glutathione disulphide) is reduced back to GSH by the enzyme glutathione reductase which uses NADPH as the electron donor in the organism [11, 12]. This oxidized glutathione (GSSG) is accumulated inside the cells, and the ratio of GSH/GSSH is a measure of oxidative stress in humans. Some transition metals (e.g. Fe²⁺, Cu⁺ among others) can break H₂O₂ to the reactive hydroxyl radical (*OH) which can cause alteration of DNA bases, break DNA strands, inactivate proteins, and result in lipid peroxidation. [13-15]. This lipid peroxidation is another pathway 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 lipid peroxyl radical (LOO*). The hydroxyl radical can also remove an electron from polyunsaturated fatty acid and in the presence of molecular oxygen, results in a LOO* 16, and if lipid peroxyl radical is further not reduced by antioxidants, this results in the process of lipid peroxidation 17. LOO* can be reduced within the membrane by the reduced form of vitamin E and can form a lipid hydro‐peroxide and a radical of vitamin E 18. The regeneration of vitamin E occurs with vitamin C (ascorbic acid) where the vitamin E radical is reduced back to vitamin E by ascorbic acid and leaves behind the ascorbyl radical (Asc*) 19. The GSSG and the ascorbyl radical (Asc*) are reduced back to GSH and ascorbate monoanion (AscH*), respectively, cooperating with NADPH and the dihydrolipoic acid (DHLA). This DHLA itself is converted to α‐lipoic acid [20-22] and also regenerates back from α‐lipoic acid using NADPH reaction. Coenzyme Q10 (CoQ10) (ubiquinone), in its reduced form, inhibits lipid peroxidation and can protect mitochondrial inner‐membrane proteins and DNA from oxidative damage. It is the most widely used co‐factor supplement in the treatment of mitochondrial disorders 23.

Free radicals attack important macromolecules leading to cell damage and homeostatic disruption. There is significant evidence, showing that most of the degenerative diseases that afflict humanity have their origin from deleterious free radical reactions. Cancer, atherosclerosis, inflammatory joint disease, asthma, diabetes, senile dementia, and degenerative eye diseases and ageing are salient ‘free radical’ diseases 24. According to a World Health Organization report, cardiovascular disease (CVD) remains the major cause of mortality throughout the world, typically causing one third of all deaths [25, 26]. The oxidative events may affect CVD; therefore, they have the potential to provide enormous benefits to human well‐being. There are different known causes for cardiovascular disorders including atherosclerosis, diabetes, dyslipidaemia, obesity, and hypertension. The primary cause of CVD is atherosclerosis, which refers to the hardening of arteries 27. Studies on atherosclerosis reveal the probability that the disease may be due to free radical reactions involving diet‐derived lipids in the arterial wall and serum to yield peroxides and other substances. Poly unsaturated fatty acids occur as a major part of low density lipoproteins (LDL) in the blood, and oxidation of these lipid components in LDL plays a vital role in atherosclerosis, which progresses slowly during a person's lifetime and typically begins before adulthood. This slow progression and its complex etiology have challenged attempts to identify the initial atherogenic event so that it remains elusive 28. The other important known cause of CVD is diabetes, which confers a fourfold risk for atherosclerosis, manifested in the vessel wall as intimal thickening, smooth muscle hypertrophy, lipid accumulation, extracellular matrix expansion, and inflammatory cell infiltration 29. There is much known evidence regarding the mechanisms responsible for accelerated atherosclerosis, which occurs in patients with diabetes, and it is believed that proteins in blood containing amadori glucose adducts may play a substantive role and are strongly linked to the pathogenesis of vascular complications of diabetes 30. Hyperlipidaemia or multiple‐type hyper‐lipoproteinemia is the responsible factor for CVD and increases platelet activity. Oxidized LDL is antherogenic and cytotoxic and plays a significant role in the formation of atherosclerosis plaques 31. Atherosclerosis, diabetes, and dyslipidaemia are known as the major causes of CVD, and are linked to have some kind of interconnection with free radicals. Thus, the review emphasizes the relationships between free radicals, atherosclerosis, diabetes, and dyslipidaemia.

Relationship between free radicals and atherosclerosis

In the past, the role of free radicals in the development of atherosclerosis is widely understood 32. The development of atherosclerosis is a multifactorial process in which both elevated plasma cholesterol levels and proliferation of smooth muscle cells (SMC) play central roles 33. Considerable in vivo studies support the role of free radical reactions in atherogenesis and atherosclerotic‐related coronary heart disease. Free radicals are involved throughout the atherogenic process beginning from endothelial dysfunction up to the rupture of a lipid‐rich atherosclerotic plaque, which further leads to acute myocardial infarction or sudden death 34. An increased concentration of plasma LDL cholesterol plays a major role in the development of atherosclerosis. Epidemiological, clinical, and genetic studies convincingly demonstrate that LDL promotes atherosclerosis. However, the precise mechanism(s) by which LDL promotes the development of the early fatty‐streak lesion still remain to be elucidated 35. LDL is composed of a hydrophilic surface layer of phospholipid, free cholesterol, and hepatically derived apoB100 to package the particle and to add stability. The core of the particle includes esterified cholesterol and triglyceride together with the fatty acid tails of the phospholipid. LDL may be oxidative modified by all major cell types of the arterial wall including endothelial cells, SMC, and macrophages via their extracellular release of ROS 35. Additionally, the initiation of peroxidation catalysed by a hydroxyl radical within the LDL molecule gives rise to conjugated dienes, and lipid hydroperoxy radicals (LOO*) 36. Hydrogen peroxide also has been reported to increase vascular permeability, prostacyclin release, and translocation of P‐selectin to the endothelial cell surface [37, 38]. The free radical chain reaction is self‐propagating, so that LOO* can attack adjacent fatty acids until complete fatty acid chain fragmentation occurs 34. During free peroxidation, reactive products then accumulate in the LDL particle, including malondialdehyde (MDA) and lysophosphatides (Figure 1) 4. These products interact with the amino side chain of the apoB100 and modify it to form new epitopes which are not recognized by the LDL receptor 34. Antioxidants like B‐carotene or vitamin E play a vital role in the prevention of various CVD. Whereas apoprotein E (apoE) is reported to impede sub‐endothelial LDL retention, it is an early proatherogenic event and protects against oxidative stress and apoptosis. ApoE has an anti‐oxidant activity and inhibits LDL oxidation; it also prevents LDL retention in the artery wall and delivers lipid antigens to CD1 in dendritic cells for presentation to T‐cells. This also includes apoE‐mediated inhibition of platelet aggregation and smooth‐muscle‐cell migration and proliferation 39. Oxidized and glycated LDL are taken up by the macrophage scavenger receptors such as SRB1, CD36, and TR4 40. Oxidized LDL may also form oxidized LDL–antibody complexes which can be taken up by the macrophage foam cell receptor. Scavenger receptor uptake is not regulated and leads to macrophage cholesterol accumulation and foam cell formation in the arterial intima 35. The foam cell recruits more monocyte/macrophages to convert to foam cells. Accumulating LDL‐laden foam cells beneath arterial endothelium lay the foundation for the fatty streak, the earliest histopathological evidence of the development of atherosclerotic plaque 41. Endothelial dysfunction is a known biomarker of cardiovascular risk factors, and it precedes the development of atherosclerosis. Endothelial dysfunction is also involved in lesion formation by the promotion of both the early and late mechanisms of atherosclerosis including up‐regulation of adhesion molecules, increased chemokine secretion, leukocyte adherence, increased cell permeability, enhanced low‐density lipoprotein oxidation, platelet activation, cytokine elaboration, and vascular SMC proliferation and migration. A dysfunctional endothelium promotes many processes involved in atherogenesis. These include decreased bioavailability of nitric oxide (NO*), migration of SMC, and/or an increase in endothelium‐derived contracting factors and proliferation which contribute to the neointimal formation between the 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, leading to SMC proliferation 44. SMC elaborate collagen and elastin and provide the foundation for plaque formation and fibrosis 45. Oxidized lipids, derived from oxidative modified LDLs, which accumulate in the intima, strongly modulate inflammation‐related gene expression, through involvement of various signalling pathways 46. The oxidative stress factors and oxidative modification of biomolecules are also involved in a number of other known physiological and pathophysiological processes such as aging, inflammation, carcinogenesis, and drug toxicity. Lipid peroxidation is another process which involves a source of secondary free radical; thus, it acts as a second messenger or can directly react with other biomolecules, further enhancing the biochemical lesions. This process mainly occurs in polysaturated fatty acid located on the cell membranes, which further leads to radical chain reaction. Hydroxyl radical is thought to initiate ROS, by removing hydrogen atoms, thus producing lipid radicals, which further converted into conjugate dienes. After the addition of oxygen, this conjugate diene forms a peroxyl radical, which is a highly reactive radical. It then attacks another fatty acid resulting in the formation of lipid hydro‐peroxide (LOOH*) and additional free radical. Lipid peroxides also inhibit the synthesis of prostacyclin, which is used to inhibit platelet aggregation resulting in platelet adherence 47. The aggregation of platelets further lays the foundation for formation of thrombus 42. Platelet release growth factor subsequently leads to SMC proliferation and migration to intima. Measurement of this intima‐media thickness using high resolution B‐mode ultrasonography has emerged as one of the methods of choice for determining the anatomic extent of preclinical atherosclerosis and for assessing cardiovascular risk related to it 48. Increased number of ROS formation in mitochondria results in mitochondrial DNA damage, and progressive respiratory chain dysfunction, which are associated with atherosclerosis or cardiomyopathy in human and animal studies of oxidative stress 49. It has also been reported that, especially in individuals with aging and various types of cancer, DNA is considered to be a major target 50. Free radical‐induced alterations in the endothelial cells of arterial the wall have been implicated in the pathogenesis and progression of atherosclerosis. Endothelial cell injury is measured by decrease count in protein, glyceride, and phospholipid synthesis. The concurrent increases in lipid peroxidation and cholesterol synthesis may explain the relationship between free radical injury and the pathogenesis of atherosclerosis 51. HDL is inherently capable of exerting anti‐atherogenic effects by metabolizing and transporting lipid oxidation products as well as cholesterol from the cells to the liver by virtue of paraoxonase‐1 (PON‐1), lecithin‐cholesterol acyltransferase, phospholipase A‐2, and platelet‐activating factor acetyl hydrolase 52. The oxidative modification of HDL impairs the ‘reverse cholesterol transport’ ability of apoA‐I and also the anti‐inflammatory function of HDL 53. It is assumed that HDL is oxidized essentially by the same oxidants, and also by the same mechanisms as LDL. It has been found that HDL is a major carrier of lipid hydro‐peroxides.

Figure 1

Pathways of ROS formation and lipid peroxidation process O₂•⁻ is dismutated by SOD into H₂O₂ and further, H₂O₂ efficiently scavenged by GPx. Transition metal (Fe²⁺) can breakdown H₂O₂ into •OH which removes an electron from polyunsaturated fatty acid to give rise to a lipid radical (L•) and finally leads to the formation of lipid peroxyl radical (LOO•). Abbreviations: GSH—glutathione, GSSG—oxidized glutathione, H₂O₂—hydrogen peroxide, L•—lipid radical, LO•—lipid alkoxyl radical, LOO•—lipid peroxyl radical, MA—deoxyadenosine adduct, MC—deoxycytidine adduct, MG—deoxyguanosine adduct, MDA—malondialdehyde, NADPH—nicotinamide adenine dinucleotide phosphate, O₂•⁻—superoxide radical, •OH—hydroxyl radical

Relationship between free radicals and diabetes

Diabetes is a group of chronic diseases characterized by hyperglycaemia. Indeed, coronary heart disease and peripheral vascular disease are the leading causes of morbidity and mortality due to diabetes mellitus 54. Diabetes is associated with the formation of the ROS by different pathogenic pathways 55. Free radical activity has been previously implicated in the development of diabetic vascular complications in type‐ I diabetes mellitus. It plays an important role in both micro‐vascular and macro‐vascular complications during diabetes mellitus, which is a known metabolic disorder, characterized by hyperglycaemia resulting from a deficiency in insulin action or secretion and associated with several vascular complications. Long‐term complications of diabetes mellitus are associated with various oxidative reactions, increased free radical generation, and subsequent increase in oxidative stress 56, particularly those which are catalysed by de‐compartmentalized transition metals playing a significant role 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 cellular dysfunction which may become irreversible over time by a process called glucose toxicity 61. Multiple biochemical pathways and mechanisms of action for glucose toxicity include glucose autoxidation, protein kinase C activation, methyl‐glyoxal formation and glycation, hexosamine metabolism, sorbitol formation, and oxidative phosphorylation. There are many potential mechanisms whereby excess glucose metabolites travelling along these pathways might cause beta cell damage. However, all of these pathways have in common the formation of ROS that, in excess and over time, cause chronic oxidative stress, which in turn can result in defective insulin gene expression and insulin secretion as well as increased apoptosis 62. The destruction of β cells by autoimmune cells occurs not only in type‐I diabetes, but also in type‐II diabetes. Insulin resistance in type‐II diabetes mellitus may be the consequence of abnormal production of anti‐insulin receptor antibodies, but is generally due to post‐receptor defects [63-65].

Also, a recent study has shown that thyroid ROS are elevated in experimental diabetic rats, which is a consequence of low‐serum thyroid‐stimulating hormone and insulin, but is also related to hyperglycaemia 66. Toxic amounts of reactive oxygen intermediates are released by endothelial cells and infiltrating macrophages during islet inflammation 63. It is known that islet cells have insufficient defence systems against free radical attacks, which makes them susceptible to reactive oxygen intermediates, leading to the destruction of these cells 8. Macrophages are innate immune cells derived from monocytes, which have many important roles in the innate and adaptive immune response, as well as in tissue homeostasis. These macrophages are known to be central players in diabetes, which act in two different ways. The first one triggers inflammatory responses which initiate insulitis and pancreatic β cell death during type‐I diabetes, whereas the other regulates type‐I diabetes negatively, by decreasing the glucose level hyperglycaemia, insulitis, and inflammation in the pancreas, thereby negatively regulating type‐I diabetes 67. These oxygen radicals not only play a significant role in the pathogenesis of diabetes, but are also involved in some of the complications seen in long‐term diabetes 56, 68. In autoimmune diabetes, the interactions between CD4‐T cells and macrophages are critical to the development and progression of inflammatory events in the pancreatic islet lesion. Cytokines and chemokine produced by auto reactive CD4‐T cells lead to recruitment and activation of macrophages, which, upon activation, produce additional inflammatory mediators and become effector cells during pathogenesis 69. In patients with diabetes mellitus, LDL oxidation by macrophages is also increased due to the activation of several pro‐oxidant systems, as well as due to the depletion of antioxidants such as the paraoxonases (PONs), which protect against atherogenesis by exerting a protective role against diabetes mellitus development by stimulating insulin secretion from β cells, and also by their unique antioxidant properties 70. ROS also regulates macrophage scavenger receptor in type‐I diabetes, but not in type‐II, in the human monocyte cell line. Oxidative stress also contributes to increased activity in macrophage scavenger receptor (MSR) by stabilizing MSR‐I mRNA 71. Further, hyperglycaemia also increases the expression of growth factors and cytokines such as transforming growth factor‐β, vascular endothelial growth factor, platelet‐derived growth factor, insulin‐like growth factor, and TNF‐α. ROS are found to be important arbitrator factors involved in all these events, and they activate intracellular signal transduction and transcription cascades in which mitogen activated protein kinases (MAPKs) and nuclear factor kappa B play the most significant roles [17, 72-74]. Proteins, lipids, and nucleic acids are damaged by oxidation (Figure 2). Osmotic stress from sorbitol accumulation has also been postulated as an underlying mechanism in the development of diabetic microvascular complications, including diabetic retinopathy and nephropathy 75. During hyperglycaemia, the polyol pathway has increased activity, and these pathways are responsible for the reduction of glucose to sorbitol via the aldose reductase enzyme, utilizing the cofactor NADPH 72. This reduction reaction leads to increased sorbitol accumulation in the cells and results in cellular and organ damage 76. Additionally, excessive accumulations of sorbitol result in the decrease of myo‐inositol in the peripheral nerves. When myo‐inositol is decreased, there is a resulting decrease in Na+, or K+‐ATPase activity, which is essential for nerve conduction 77. It is known that increase in the production of ROS leads to more oxidative stress, which further simulates the dysfunction and destruction of β cell, and leads to the impairment of insulin function 78. Auto‐oxidative glycosylation is important in explaining free radical formation and protein damage in diabetes 79. High glucose concentration facilitates the eicosanoid pathway and, consequently, production of the superoxide. In the endothelial cells, NO is synthesized from arginine by NO synthase and is quenched by superoxide, resulting in peroxynitrite anion formation. Inhibition of the nitric oxide action reduces vasodilatation and enhances endothelial dysfunction in diabetes 80. Hyperglycaemia also elicits oxidative stress by directly impairing the cellular mechanisms, which, in turn, elicits endothelial dysfunction 81. Moreover, hyperglycaemia is also responsible for the generation of NO* and O* in excess through activation of nitric oxide synthases (NOSs) and NADPH oxidase, respectively. O₂* reacts with NO* to produce OONO*, another oxidant which increases oxidative stress and elicits endothelial dysfunction by promoting tissue injury 82. O₂* is converted to H₂O₂ by SODs, which not only increases oxidative stress, but also generates endothelial dysfunction by modulating intracellular signalling and transcription factors [82, 83]. In addition to O₂*, hyperglycaemia also stimulates the synthesis of NO via increased enzymatic activity of endothelial and inducible isoforms of NOS [81-84]. According to Desco et al. (2002), ketone, especially acetoacetate, is involved in free radical formation in type‐I diabetes 79. The production of ketone bodies is especially important in brain metabolism, which, under normal circumstances, depends mainly on glucose [85, 86]. Glucose is not toxic at physiological conditions; however, at higher concentrations, it undergoes auto‐oxidation, and noncyclic proteins are non‐enzymatically glycated by high levels of noncyclic glucose. Glucose is also able to modify proteins by the attachment of its oxidation derived aldehydes, thus leading to the development of novel protein fluorophores and fragment protein via free radical mechanisms 67. However, the metal chelators and free radical scavengers can inhibit the fragmentation of these proteins 87. Transition elements (for example iron) are involved in free radical production (Fe²⁺« Fe³⁺), and iron (II) ions are also known to induce oxidation of ascorbic acid and glucose 88. The simultaneous episodes of glycation and oxidation further support the hypothesis that tissue damage is associated with diabetes [87, 89, 90]. Xanthine oxidase, a superoxide‐generating enzyme, is also involved in free radical production in patients with type‐I diabetes, which can be protected by allopurinol 77. Generation of high levels of ONOO* ions initiates the onset of different processes like mitochondrial dysfunction, which is a part of premature aging of the endothelium 91. Hyperglycaemia‐induced overproduction of superoxide by the mitochondrial electron transport chain activates the four major pathways of hyperglycaemic damage by inhibiting glyceraldehyde phosphate dehydrogenase (GAPDH) activity 72, which includes (1) hexosamine biosynthetic pathway 92, (2) sorbitol‐aldose reductase pathway 93, (3) MAPK 94, and (4) protein kinase C 95. GADPH is a classic glycolytic enzyme that also mediates cell death by its nuclear translocation under oxidative stress 96. Hyperglycaemia‐induced mitochondrial superoxide production further inhibits GAPDH by activating PARP 72. Abnormally high levels of free radicals and the simultaneous decline of antioxidant defence mechanisms can lead to damage of cellular development of insulin resistance. The consequences of this oxidative stress also promote the development of diabetes mellitus and its complications 97.

Figure 2

ROS‐induced DNA damage activates PARP and modifies GAPDH and mechanism of hyperglycaemia induced cellular damage. In case of diabetes and hyperlipidaemia, the level of glucose increases which further increases the level of free radicals. The increased level of ROS is responsible for activation of PARP which is involved in modification of GAPDH. Modified GAPDH is involved in different pathological mechanisms like activation of protein kinase C and increased production of advanced glycation end‐products and hexamine pathways, etc.; these pathways along with free radicals play a significant role in the pathogenesis of diabetic complications. Abbreviations: AGEs—advanced glycation end products, GAPDH—glyceraldehyde phosphate dehydrogenase, NAD+‐nicotinamide adenine dinucleotide, PARP—poly (ADP‐ribose) polymerase, PKC—protein kinase C, ROS—reactive oxygen species, ADPR—ADP ribose, NA—nicotinamide, PW—pathway

Relationship between free radicals and dyslipidaemia

It is well known that free radicals play a crucial role in the pathogenesis of fatty liver disease and increase metabolic alterations 98. As discussed earlier, high lipid or cholesterol (LDL) levels are important factors responsible for various cardiac diseases since they lead to development of hyperlipidaemia, atherosclerosis, and ischemic heart disease. ROS are known to be associated with the formation of oxidized LDL that, in turn, stimulates the release of ROS and enhanced adhesion molecule expression as well as platelet activity with pro‐atherogenic effects in the vasculature 99. Hyperlipidaemia is characterized by increased LDL and triglyceride concentrations, which is often accompanied by decreased HDL 100. Type‐II hyper‐cholesterolemia causes changes in the structure and fluidity of erythrocyte plasma membranes since the excess cholesterol affects the normal rheology of blood through its interaction with erythrocytes. Hyper‐cholesterolemia may also decrease the deformability of red blood cells, which impairs their haemorheological behaviour and thus promotes atherosclerosis. It also impairs the function and structure of plasma membrane proteins 101. Evolution of hyper‐cholesterolemia is also associated with endothelial cell dysfunction, a near complete abrogation in vascular NO bioavailability, elevated oxidative stress, and creation of pro‐inflammatory condition; these are symptoms which can culminate in profound impairments/alterations to vascular reactivity 9. Dyslipidaemia, with or without atherosclerosis, is one of the major metabolic/CVD states in which the enhanced formation of ROS is of pivotal pathogenic importance 102. Hyperlipidaemia with elevated levels of triglycerides, chylomicron remnants, and free fatty acids results in oxidative stress and inflammation and may independently potentiate the adverse effects of hyperglycaemia 103. It is well known that NO rapidly reacts with O₂* to form the cytotoxic species (ONOO*), and hyperlipidaemia has been shown to increase production of ROS including ONOO* in the vasculature, which results in a deterioration of cardiac function. Hyperlipidaemia also increases the plasma nitro‐tyrosine level, which is a marker for systemic ONOO* generation 104. Some known cytotoxic effects of ONOO* include lipid peroxidation, nitration of tyrosine residues, oxidation of sulfhydryl groups, tissue injury, and DNA‐strand breakage 105. The detrimental effects of increased oxidative stress result in dysfunction of endogenous NO*, production and inactivation of NO* by oxygen‐derived radicals (eliciting the formation of the cytotoxic and genotoxic ONOO*), or altered function of NOS 106. Excessive formation of ONOO* is associated with a significant decrease in cellular glutathione content and, subsequently, of NOS as well as of soluble guanylate cyclase activity (e.g. through oxidation of thiol groups {−SH} 107. Enhanced lipid peroxidation and decreased antioxidant enzyme activity also represent early events in the development of 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 antioxidant enzymes that may lead to an imbalance between antioxidant and oxidant systems 109. This oxidant–antioxidant status has a major impact, not only on the rate of LDL oxidation, but also on the development of atherosclerosis 110. It is possible that a potential risk of atherosclerosis in the hyperlipidaemia population is associated with LDL oxidation and decreased antioxidant enzyme activity. Further hypertriglyceridemia and hypercholesterolemia are associated with LDL peroxidation, protein glycation, glucose auto‐oxidation, and thus lead to excess production of free radicals, which may further elevate the oxidative stress in hyperlipidaemia population 111.

Relationship of nitric oxide with atherosclerosis, diabetes, and dyslipidaemia

NO is an endogenous anti‐atherogenic molecule. The dual role of NO as a protective and toxic molecule depends on several factors. Primarily, it depends on the isoform of NOS involved, and there are three main isoforms of the enzyme: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). They differ in their dependence on Ca²⁺, as well as in their expression and activities 112. The important factor includes eNOS, which are the only NOS isoform expressed in normal coronary arteries, and it offers protection against atherosclerosis 113. iNOS is proatherogenic, so it decreases atherosclerotic plaque areas 114. Additionally, nNOS might modulate atherosclerosis by directing the flow of NO* toward particular targets 115. The important isoform in the regulation of insulin resistance is iNOS. Understanding the molecular mechanisms regulating the iNOS pathway in normal and hyperglycaemic conditions would help to explain some of the vascular abnormalities observed in type‐II diabetes mellitus 116. The other factors include, but are not limited to, the concentration of NO* type cells in which NO* is synthesized, the availability of the substrate L‐arginine, generation of guanosine 3,5′‐cyclic monophosphate (cGMP) from guanosine triphosphate catalyse by soluble guanylate cyclise (sGC), and the overall extra and intracellular environment in which NO* is produced. NOS activation as a result of trauma (calcium influx) or infection leads to NO* production, which activates its downstream receptor sGC to synthesise cGMP and/or leads to protein nitrosylation. This may lead to one or more systemic effects including altered neurotransmission, which can be protective or toxic vaso/broncho dilatation in the cardiovascular and respiratory systems, and enhanced immune activity against invading pathogens 117. NO* also regulates critical lipid membranes and lipoprotein oxidation events either by contributing to the formation of more potent free radicals from superoxide (i.e. ONOO*) or by its antioxidant properties through termination reactions with lipid radicals to potentially less reactive secondary nitrogen‐containing products [118, 119]. In addition to being a potent vasodilator, it may inhibit a number of key processes in atherogenesis, including vascular SMC proliferation 120, platelet adherence or aggregation 121, generation of oxygen‐derived free radicals 122, and monocyte adherence and infiltration (possibly by inhibiting the expression or activity of endothelial adhesion molecules or chemotactic proteins) [123-126]. Protein‐bound nitrotyrosine (NO₂Tyrosine), a post‐translational modification specific for protein oxidation by NO‐derived oxidants, is markedly enriched within human atheroma. Further, recent clinical studies demonstrate that systemic levels of protein‐bound NO₂Tyrosine serve as an independent predictor of atherosclerotic risk 127.

The impairment of NO* activity by hypercholesterolemia, diabetes mellitus, or hypertension plays a significant role in the initiation of atherosclerosis 124. For example, in a traumatic arterial injury, the loss of endothelial influence facilitates platelet plug formation and vasoconstriction, thereby promoting homeostasis 126. This becomes a pathophysiologic mechanism when the endothelium is dysfunctional due to a systemic disorder (hyperlipidaemia). Further, platelet adherence to the dysfunctional endothelium promotes thrombosis and the growth of vascular lesions 127. Deficiency of nitric oxide is responsible for reduced nerve blood flow and response in the development of endoneurial ischemia in diabetic rats 128. There is an increased production of NO* observed immediately after the onset of diabetes. NO* may be responsible for the glomerular hyper filtration observed in diabetic kidneys by inducing vasodilation of the afferent arteriole [129, 130]. In addition, hypercholesterolemia is associated with the loss of nitric oxide‐induced vasodilation and the subsequent increase in blood pressure 131.

Relationship of free radicals with atherosclerosis, diabetes, and dyslipidaemia

The relationship between oxidant generation, oxidative stress, oxidative damage, and various disorders like atherosclerosis, diabetes, dyslipidaemia, ischemic pain, etc. is now well known (Figure 3). In many scientific publications, oxidative stress is solely used to describe impaired ROS or RNS systems which may develop secondary hypertension, atherosclerosis, or hyperglycaemia 132. There is greater risk of developing atherosclerosis and its complications: stroke, myocardial infarction, and peripheral vascular disease in diabetic populations as compared to non‐diabetic ones 68, 133. Artherogenesis has been associated with lipid disorders characterized by hypertriglyceridemia, and increased levels of VLDL and LDL without diabetes. However, diabetic populations with relatively normal plasma lipid and lipoprotein concentrations still have an increased level of atherosclerosis 134. Therefore, increased incidence of vascular diseases in diabetic patients may involve dyslipidaemia or other factors unique to diabetes. Several risk factors have been proposed to explain the increased risk of CVD with diabetes, e.g. hyperglycaemia, dyslipidaemia 132, accelerated formation of advanced glycation end (AGEs) products, increased oxidative stress, and other genetic factors 135. Advanced oxidative stress is generated by hyperglycaemia and, in many diabetic patients, also by dyslipidaemia which accelerates endothelial dysfunction as the first step in the development of angiopathy 80. In the diabetic state, the accelerated formation of amadori‐modified glycated serum proteins and lipoproteins fosters the pathogenesis of atherosclerosis 15. MDA formation, which is a marker of oxidative stress, is altered in different stages of development of hyperlipidaemia and is also an important risk factor of atherosclerosis [136, 137]. According to Renard et al., (2004) diabetes increases lesion initiation, and diabetes‐induced dyslipidaemia leads to a progression of atherosclerotic lesions, and their transformation into more severe condition of lesions which are prone to rupture and thrombosis 138. Many epidemiological studies have also established that risk factors for diabetes and atherosclerosis coexist 139. Further, endothelial dysfunction supports atherosclerosis 34. Glucose and free fatty acids overwhelm the Krebs cycle, stimulate excess production of NAD+ which outpaces the capacity for oxidative phosphorylation and creates free radicals 140. Postprandial in diabetes is complex and involves a variety of factors including hyperinsulinemia, insulin resistance, hyperglycaemia, and disturbed fatty acid metabolism. Postprandial oxidative stress triggers a number of atherogenic changes including increases in inflammation, sympathetic tone, vasoconstriction, thrombogenicity, and oxidation of LDL in diabetic and non‐diabetic populations 141. Numerous clinical studies have shown that postprandial dyslipidaemia is associated with endothelial dysfunction in type‐II diabetes and with alterations in other surrogate markers in the cascade of atherosclerosis 142.

Figure 3

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

Type‐I diabetes is associated with early impairment of common carotid artery structure and function, and this diabetic state may be the main risk factor for common carotid artery wall stiffening and thickening, which are of considerable concern as possible early events in the genesis of atheroma 143. Substantial clinical and experimental evidence suggest that both diabetes and insulin resistance cause a combination of endothelial dysfunctions, which may diminish the anti‐atherogenic role of the vascular endothelium. Oxidative stress due to high glucose can also lead to a number of proatherogenic events 56. The elucidation of the mechanisms of oxidative stress in diabetes and their relationship with atherosclerosis could potentially identify molecular targets of therapy for this condition and its cardiovascular consequences 135. Blood flow restriction is achieved by vasoconstriction despite increased production of NO*, and the vasodilation effects of which are overridden by catecholamine (and also by angiotensin‐II and endothelia). Decreased blood flow reduces the availability of oxygen, provoking massive glycolysis (hyperglycaemic conditions), which results in the production of lactate which is exported to the liver for further processing 144. However, this produces local acidosis, which elicits the rapid dissociation of oxyhaemoglobin, freeing bursts of oxygen in localized zones of the tissue. The excess oxygen (and nitric oxide) induces the production of ROS, which deeply affects the endothelial, blood, and adipose cells, inducing oxidative and nitrosative damage, and eliciting an increased immune response, which translates into inflammation, and then leads to atherosclerosis 145. Cholesterol‐enriched diet‐induced hyperlipidaemia results in an increase in cardiac ONOO* formation and decrease in the bioavailability of NO*, both of which contribute to the deterioration of cardiac performance and may lead to further cardiac pathologies 104. Control of hyperglycaemia thus remains the best way to improve endothelial function and to prevent atherosclerosis and other cardiovascular complications of diabetes 81. Dehydro‐ascorbic acid (oxidized form of vitamin C) is transported into mammalian cells via facilitative glucose transporters. Hyperglycaemia inhibits this process by competitive inhibition. This inhibited transport may promote oxidative stress and contribute to the increase in atherosclerotic CVD observed in patients with diabetes mellitus 146.

Micro‐albuminuria is now considered to be an atherosclerotic risk factor and predicts future CVD risk in diabetic patients 144. A wide range of micro‐albuminuria cut‐off values are currently used for diagnosing the early stages of nephropathy in patients with type‐II diabetes. However, in the same early stage of diabetic nephropathy, there is a significant correlation between different levels of micro‐albuminuria and markers of oxidative stress. Serum conjugated dienes have emerged as the main marker for evaluating 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 may be mediated by increased glycosaminoglycan deposition and monocyte recruitment 139. Maternal hypercholesterolemia during pregnancy is known to be responsible for cascade of pathogenic events. It is also associated with greatly increased fatty streak formation in human foetal arteries and accelerated progression of atherosclerosis during the early stages of life 148. Impaired lipid and glucose metabolism is a typical feature in diabetes. Diabetes is associated with formation of ROS by different pathogenic pathways. Advanced generation of ROS may further accelerate glycation, sorbitol, protein kinase C, or hexosamine pathways which are activated by high glucose and lipids. These pathogenic mechanisms are closely linked to oxidative stress, which has been recognized as the main cause for development of the endothelial dysfunction 149. Further endothelial dysfunction increases permeability of endothelial cell monolayers, pro‐coagulant activity, expression of adhesion molecules and intracellular oxidative stress 132, 150. Additionally, they serve as the nidus for atherosclerotic lesion formation. Increased levels of oxidants also function as a link between diabetes, dyslipidaemia, and free radicals, which respond to atherosclerosis 151. A major mechanism appears to be the hyperglycaemia/hyperlipidaemia induced intracellular ROS, produced by the proton electromechanical gradient generated by the mitochondrial electron transport chain and resulting in increased production of superoxide 152. Two other mechanisms have been proposed to elucidate the role of hyperglycaemia in ROS formation. One mechanism involves the transition metal‐catalysed autoxidation of free glucose; glucose itself initiates an auto‐oxidative reaction and free radical production, yielding superoxide anion and hydrogen peroxide 153. The other mechanism involves the transitional metal‐catalysed autoxidation of protein‐bound amadori products, which yield superoxide and hydroxyl radicals and highly reactive di‐carbonyl compounds 132, 154.

Discussions and future prospective

The development of procedures to ameliorate undesirable ROS production may be one of the central issues in the field of research studying aging and other oxidative stress‐related diseases 18, 155. Now, it has become possible to accurately quantify in vivo and ex vivo ROS concentrations under different pathophysiological and experimental conditions by electron paramagnetic resonance or electron spin resonance spectroscopy 156. There are some encouraging reports regarding the use of SOD/CAT mimetic in certain experimental systems. Dietary antioxidants are widely used to ameliorate excessive oxidative stress, but scientific proof of their efficacy is scarce. There is, nevertheless, a strong possibility that the process of senescence and disease‐related wasting results, at least to some extent, from a progressive shift in biochemical conditions that may not be irreversible in principle 18. Hyperlipidaemia stimulates the generation of ONOO* in the heart, which further leads to myocardial dysfunction. Targeting ONOO* with pharmacological active agents may be an exciting new strategy to protect the heart and the vasculature in the event of hyperlipidaemia 104. Hyperglycaemia induces endothelial cell dysfunction, which further activates multiple pathways including enhanced glycolysis (ROS), the build‐up of glycolytic intermediates (polyol pathway, hexosamine pathway, PKC activation, and AGE formation), and AGE modification of proteins (ROS, PKC). Given the interdependent nature of these pathways, it is not surprising that inhibitors targeting one of them can profoundly affect hyperglycaemia‐induced alterations in endothelial cell function 157. Apo E is a well‐known blood circulating protein with pleiotropic atheroprotective properties that have emerged as a strong candidate for treating hyper‐cholesterolemia and other cardiovascular disorders 40.

Mitochondria play an important role in controlling the bioenergetics status of cells in physiological conditions. Consequently, mitochondrial dysfunction also leads to oxidative stress that subsequently contributes to an increase in the amount of free radicals (Figure 4). In recent developments, mitochondria‐targeted antioxidants (in particular mitochondria‐targeted vitamin E) are under investigation and are in development due to their potential for the treatment of CVD 158. Within the mitochondrial phospholipid bilayer, the fat‐soluble antioxidants vitamin E and coenzyme Q‐10 both prevent lipid peroxidation, while coenzyme Q‐10 also recycles vitamin E and is itself regenerated by the respiratory chain. With the exception of α‐lipoic acid studies in diabetic neuropathy, data from clinical trials are limited for antioxidants. Thus, there is a need to further investigate the pathophysiology of oxidative stress, and to explore the role of antioxidant therapy that will lead to appropriately designed clinical trials showing the promise of antioxidant therapy 159. Besides minerals and vitamins etc., there is also considerable interest in determining the total phenolic content 160, and antioxidant capacities of different vegetables, fruits, spices, medicinal plants, and microalgae [161-167]. Phenolic compounds can reduce high blood glucose levels by restoring hepatic glycogen and increasing insulin and glucokinase levels. Therefore, herbal plants with phenolic compounds have therapeutic implications [168, 169]. Recently, researchers have been interested in, for example, antioxidant enzyme systems such as the heme‐oxygenase/biliverdin reductase system, which are modulated by dietary antioxidant molecules, including polyphenols and β‐carotene and if antioxidants are taken in excess, the risk of originating diseases instead of preventing them is quite high 170. Carnitines, α‐tocopherol, α‐lipoic acid, co‐enzyme Q10, and selenomethionine used as antioxidant supplementation in healthy volunteers, resulting in a significant increase in plasma antioxidant status by causing a decrease in blood peroxide levels and, thus, a reduction in the formation of ROS at the mitochondrial level 171. It is important to note that, as for all drugs, antioxidants may have significant side effects if not used correctly or if used in combination with other drugs. Also, vitamin A, E, and β‐carotene, for instance, have been shown to have pro‐oxidant effects at higher doses or under certain conditions 170. The use of natural antioxidants to augment physiological antioxidant mechanisms is particularly attractive, but potential difficulties exist, for example in situations where most of the diseases where oxidative damage has been demonstrated, free radical generation is not the primary cause of the disease; rather, it plays a role in further complicating/worsening the disease condition. The possibility of antioxidant therapy may also be harmful which can be a potential possibility is for any synthetic agent, but it may also be true for natural antioxidant agents taken in excess which is needed to be investigated further.

Figure 4

Oxidative cellular damage mechanism. Abbreviations: CAT—catalase, GPx—glutathione peroxidise, SOD—superoxide dismutase, GSH—glutathione, GSSG—oxidized glutathione

Conflict of interest

None declared.