Oxidative Stress in Cardiovascular Disease

Transcription

1 Indian Journal of Biochemistry & Biophysics Vol. 46, December 2009, pp Review Oxidative Stress in Cardiovascular Disease S V Vijaya Lakshmi 1, G Padmaja 1, Periannan Kuppusamy 2 and Vijay Kumar Kutala 1 1 Department of Clinical Pharmacology & Therapeutics, Nizam s Institute of Medical Sciences, Hyderabad, India 2 Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio, USA Received 4 August 2009; revised 2 November 2009 Over the last two decades, it has become increasingly clear that reactive oxygen species (ROS), including free radicals are involved in cardiovascular disease. In recent years, there has been a growing interest in the clinical implications of these oxidants. The ROS are common by-products of many oxidative biochemical and physiological processes. They can be released by xanthine oxidase, NAD(P)H oxidase, lipoxygenases, mitochondria, or the uncoupling of nitric oxide synthase in vascular cells. ROS mediate various signaling pathways that underlie vascular inflammation in atherogenesis. Various animal models of oxidative stress support that ROS have causal role in atherosclerosis and other cardiovascular diseases. They are too reactive to be tolerated in living tissue, and aerobic organisms use sophisticated defense system, both enzymatic and non-enzymatic for prevention of overload of free radicals. In a number of pathophysiological conditions, the delicate equilibrium between free-radical production and antioxidant capability can be altered in favor of the former, thus leading to oxidative stress and increased tissue injury. This review focuses on the biochemical evidences concerning involvement of ROS in several cardiovascular diseases, namely atherosclerosis, heart failure, hypertension and ischemia/reperfusion injury. Keywords: Free radicals, Oxidative stress, Cardiovascular diseases, Antioxidants, Reactive oxygen species, Hypertension, Blood pressure Reactive oxygen species (ROS) participate in normal cell signaling as mediators that regulate vascular function 1-5. In the vascular wall, ROS are produced by all layers, including endothelium, smooth muscle, and adventitia 6. ROS include superoxide anion radical (O 2 - ) hydrogen peroxide (H 2 O 2 ), hydroxyl radical (. OH), nitric oxide (NO), and peroxynitrite (ONOO - ) (Figure 1). Under physiological conditions, ROS are produced in low concentrations and act as a signaling molecule that Author for correspondence Tel: ; Fax: kuppusamy.1@osu.edu Abbreviations: ACE, angiotensin converting enzyme; Angio-II, angiotensin-ii; AT1, angiotensin-ii type 1 receptor; ARE, antioxidant response elements; CVD, cardiovascular disease; ECs, endothelial cells; EH, essential hypertension; ED, endothelial dysfunction; GST, glutathione-s-transferase; H 2 O 2, hydrogen peroxide; IL-1β, interleukin-1β; LDL, low-density lipoprotein; LOO., lipid hydroperoxyl radical; MCP1, monocyte chemotactic protein-1; mtdna, mitochondrial DNA; NO, nitric oxide; NOS, nitric oxide synthase; NQ01, NAD(P)H: quinone oxidoreductase; Nrf2, nuclear factor erythroid-2 related factor 2; O -. 2, superoxide; OH, hydroxyl radical; ONOO -, peroxynitrite; OxLDL, oxidized low-density lipoprotein; PDGF, platelet derived growth factor; PKC, protein kinase C; ROCK, Rho-associated kinase; ROS, reactive oxygen species; SOD, superoxide dismutase; TGF-β, transforming growth factor-β; VSMC, vascular smooth muscle cell. regulate vascular smooth muscle cell (VSMC) contraction and relaxation, and participate in VSMC growth 7-9. Under pathophysiological conditions, these free radicals play important roles in various conditions, including atherosclerosis, ischemiareperfusion injury, ischemic heart disease, arrhythmias, cardiomyopathy, congestive heart failure, cancer, and diabetes There is now considerable biochemical, physiological and pharmacological data to support a link between free radicals and cardiovascular tissue injury. Major vascular risk factors, such as hypertension, dyslipidemia, diabetes and smoking are associated with a marked increase in vascular ROS production. There is accumulating evidence, suggesting that disease conditions are directly or indirectly related to oxidative damage and that they share a common mechanism of molecular and cellular damage. As these mechanisms are elucidated, it may be possible to improve the techniques for clinical and pharmacological intervention. The present review focuses on the evidences concerning the involvement of free radicals in cardiovascular diseases and their relationship to specific pathophysiological events.

2 422 INDIAN J. BIOCHEM. BIOPHYS. VOL. 46, DECEMBER 2009 Figure 1 Sources of reactive oxygen species (ROS) generation and their effects on signaling systems in cardiovascular disease [Activated NAD(P)H oxidases, lipoxygeneases and xanthine oxidase generate O 2 -., NOS switches from a coupled state to an uncoupled state and generates O 2 -. with decreased 5,6,7,8-tetrahydrobiopterin (BH 4 ) or L-arginine. Dysfunctional mitochondrial electron-transport chain is another source of O ROS reduce bioavailability of NO, leading to endothelial dysfunction. ROS influence the activity of a variety of cellular signaling pathways ultimately leading the changes in the expression of redox-sensitive genes, which regulate cellular process involved in cellular apoptosis/death that may involved in the pathogenesis of cardiovascular disease] There are several potential sources of ROS production. In cardiovascular disease (CVD); the sources include xanthine oxidase 14, cyclooxygenase 15, lipooxygenase 16, mitochondrial respiration 17,18, cytochrome P450 19, uncoupled nitric oxide synthases 11,20,21 and NAD(P)H oxidase 22. They have been identified as sources of ROS generation in all types of vasculature. These sources may contribute to ROS formation, depending on cell type, cellular activation site and disease context. Numerous studies have shown that various physiological stimuli that contribute to pathogenesis of vascular disease can induce the formation of ROS. For example, a variety of agents, including vasoactive agents such as angiotensin-ii (Ang II), endothelin-1 and thrombin have been shown to activate NAD(P)H oxidase 23. Treatment of VSMCs with Ang II causes an increase in expression of NAD(P)H oxidase as well as ROS production 24,25. Cytokines (IL-1β, TNF-α), growth factors (plateletderived growth factor, PDGF; transforming growth factor-β, TGF-β) and hemodynamic forces (shear stress and cyclic stretch) can regulate the expression and/or activity of the vascular NAD(P)H oxidase Recent studies suggest that intracellular ROS production may also be derived from the mitochondria. The production of mitochondrial superoxide radicals occurs primarily at two discrete points in the electron-transport chain, namely at complex I (NADH dehydrogenase) and at complex III (ubiquinone-cytochrome c reductase) 31. Several intracellular signal events stimulated by ROS have been defined, including the two members of mitogen-activated protein-kinase family (ERK1/2 and big MAP kinase, BMK1), tyrosine kinases (Src and Syk) and different isoenzymes of PKC as redoxsensitive kinases ROS regulation of signal transduction components include the modification in the activity of transcriptional factors such as NF-κB and others that result in changes in gene expression and modifications in cellular responses. The small guanosine triphosphatase (GTPase) Rho works as a switch and plays an important role in various cellular physiologic functions, including actomyosin-based cellular processes such as cell adhesion, migration, motility, cytokinesis and contraction, all of which may be involved in the pathogenesis of atherosclerosis 37.

3 VIJAYA LAKSHMI et al.: OXIDATIVE STRESS IN CARDIOVASCULAR DISEASE 423 There is growing evidence that Rho-associated kinase (ROCK) (also known as Rho-kinase), the immediate downstream target of the small GTPbinding protein Rho contributes to endothelial dysfunction (ED) and vascular disease The clinical evidence has demonstrated that ROCK is significantly activated in patients with coronary vasospasm 43, hypertension 44, stable-effort angina 45 and in current smoking in healthy subjects 46,47. Evidences indicate that ROCK is significantly associated with the regulation of not only endothelial nitric oxide synthase (enos) expression, but also enos phosphorylation, both of which are important mechanisms for regulating endothelial function and subsequent cardiovascular injury 38-42,48. A significant relationship has been found between ED and increased ROCK activity in smokers 47. The aging and cigarette-smoking are also involved in increase in ROCK activity, which may be partly explained by the significant correlation between ROCK and endothelial function. These observations suggest that activation of ROCK is involved in several aspects of the atherosclerotic process, including ED 44. Nitric oxide (NO) has recently emerged as an important mediator of cellular and molecular events that impact the pathophysiology of myocardial ischemia. NO produced by vascular endothelium is shown to possess potent vasodilatory properties and also an inhibitor of platelet aggregation which may be beneficial to the early stages of focal myocardial ischemia 49. It may also facilitate collateral blood flow to the ischemic territory 44. An increase in intracellular Ca 2+ (resulting from the activation of voltage-gated Ca 2+ channels or ligand-gated Ca 2+ channels or from the mobilization of intracellular Ca 2+ stores) could activate the enzyme NO synthase 44, which catalyzes the synthesis of NO from L-arginine and molecular oxygen. NO may cause cytotoxicity through formation of iron-no complexes with several enzymes including mitochondrial electron-transport chain, oxidation of protein sulfhydryls and DNA nitration 44. It may also mediate cell death through formation of the potent oxidant peroxynitrite (ONOO - ), the reaction product of NO with O Peroxynitrite decomposes to the hydroxyl free radical ( OH) and to radical nitrogen dioxide (NO 2 ), which are potent activator of lipid peroxidation. The Nrf2 (NF-E2-related factor-2)/antioxidant response element (ARE) pathway is a cis-acting sequence that mediates transcriptional activation of genes in cells exposed to oxidative stress The ARE is present in the 5' flanking regions of genes encoding phase II detoxification enzymes and cellular antioxidant proteins including glutathione-stransferase (GST) 54, NAD(P)H: quinone oxidoreductase (NQ01) 55, glucuronosyl transferase 54, heme oxygenase-1 56 and ferritin 57. Nrf2 is the transcription factor that upon activation by oxidative stress binds to the ARE and activates transcription of ARE-regulated genes 58,59. In vascular cells, the ARE pathway is activated by oxldl 60,61 and NO exposure 62. ECs when exposed to prolonged laminar flow show a marked increase in the expression of ARE-mediated genes such as GST, NQO1, HO-1 and ferritin through Nrf2- dependent mechanism 63. Protective pathways in mammalian cells and tissues To prevent overloading of free radicals and peroxides, aerobic organisms use a sophisticated defense system, which operates both in intra- and extracellular aqueous phases and in membranes. Antioxidant defense strategies are committed to counteract the oxidative attack in its early moments, i.e. formation of primary radicals, as well as during the initiation and chain-propagation processes. Antioxidant protection can be viewed as consisting of four sequential levels of defensive activity: preventive, chain-breaking, repairing, and adaptive. The first level of defense, which is largely enzymatic, involving enzymes such as superoxide dismutases (SODs), glutathione peroxidases (GPx) and catalase is concerned with the control of formation and proliferation of primary radical species derived from molecular oxygen. There are three different forms of SODs (manganese, -. copper/zinc, extracellular) that metabolize O 2 to hydrogen peroxide (H 2 O 2 ). Catalase and at least four isoforms of GPx then convert H 2 O 2 into water. Extracellular SOD (ecsod) is produced by VSMCs and not endothelial cells 64,65 and localizes in highest concentrations between the endothelium and VSMCs 66. The second level of defense, which involves vitamins C and E and probably carotenoids is concerned with the prevention of proliferation of secondary radicals in chain reactions, such as lipid peroxidation, initiated and driven by primary radicals. The third level of defense is the enzymatic prevention of formation of secondary radicals from chain-terminated derivatives and enabling the removal of such

4 424 INDIAN J. BIOCHEM. BIOPHYS. VOL. 46, DECEMBER 2009 molecules from an environment in which metalcatalyzed reactions might cause further oxidative damage. Free radicals in cardiovascular diseases Oxidative stress and endothelial dysfunction Endothelium is the bioactive inner layer of the blood vessels, which serves as an important locus on control of vascular and thus other organ functions regulating vascular tone permeability 67. It produces components of extracellular matrix such as collagen and a variety of regulatory mediators, including NO, prostanoids, endothelin-1 (ET-1), angiotensin II (Ang II), tissue-type plasminogen activator (t-pa), von Willebrand factor (vwf), adhesion molecules and cytokines. Endothelial dysfunction (ED) is an early event in atherosclerotic disease, preceding clinical manifestations and complications. Evidences have shown that ED is a strong predictor of future cardiovascular events in patients with cardiovascular risk factors 68. ROS have been implicated as important mechanisms that contribute to ED and may function as intracellular messengers that modulate signaling pathways. Increased ROS production is a major cause of ED in experimental and clinical atherosclerosis 69,70. Among important molecules synthesized by endothelial cells is NO, which is a potent vasodilator 49. The important functions of NO include anti-platelet and anti-proliferative, permeabilitydecreasing and anti-inflammatory properties 71. NO Inhibits leukocyte adhesion and rolling as well as cytokine-induced expression of VCAM-1 (vascular endothelial cell adhesion molecule) and MCP-1 (monocyte chemotactic protein) 72, effects partly attributable to inhibition of the transcription factor NF-κB 73. ED leads to a rapid decrease in NO production or availability, partly due to inactivation of NO by superoxide. Superoxide reacts rapidly with NO, resulting in the formation of peroxynitrite and loss of NO bioavailability. Decrease in NO bioavailability could also result from reduced expression or activity of enos, increased generation of asymmetric dimethylarginine (ADMA; an endogenous circulating inhibitor of NOS), decreased availability of 6Rtetrahydrobiopterin (BH4; an essential NOS cofactor) or increased inactivation of NO by superoxide 74. Recent studies have shown that ROS, in particular peroxynitrite can oxidize tetrahydrobiopterin 27,75. Polymorphisms have been observed in a variety of genes whose products have been implicated in ED. In NOS3 (enos) gene, more than 15 polymorphisms exist in the promoter region that might influence reduced gene expression. The presence of polymorphisms in other genes include methylene tetrahydrofolate reductase, angiotensin-converting enzyme, p22 phox, glutathione-s-transferase and cytochrome P450, which can cause ED 76. ROS are involved in the endothelial and VSMC pro-inflammatory signaling, particularly in the regulation of VCAM-1 and MCP-1 expression. They also are involved in redox-signaling cascade, leading to vascular pro-inflammatory and prothrombotic gene expression involving the transcription factor NF-κB. Finally, ROS activate matrix metallo-proteinases (MMPs), contributing to plaque instability and rupture 77. Homocysteine (Hcy) and oxidized LDL (oxldl) have been shown to enhance the activity and expression of oxidative stress markers, such as NF-κB and heme oxygenase These results suggest that these proatherogenic stimuli increase oxidative stress in endothelial cells and thus explain the loss of endothelial function associated with the atherogenic process. ED in experimental atherosclerosis could be reversed by administration of superoxide scavengers, suggesting that increased vascular superoxide production represents a major cause of ED 75,79. Tetrahydrobiopterin improves endothelial dysfunction and vascular oxidative stress in microvessels of intrauterine undernourished rats 80. Oxidative stress and atherosclerosis Atherosclerosis originates from ED and inflammation. The importance of oxidative stress in the development of atherosclerosis seems to be widely accepted. The free radicals are involved throughout the atherogenic process, beginning from ED in an otherwise intact vessel wall up to the rupture of a lipid-rich atherosclerotic plaque, leading to acute myocardial infarction or sudden death 81. The development of atherosclerosis is a multi-factorial process in which both elevated plasma cholesterol levels and proliferation of smooth muscle cells play a central role. Atherogenesis is an alteration of the artery wall that includes two major phases: (i) adhesion of monocytes to the endothelium and their migration

5 VIJAYA LAKSHMI et al.: OXIDATIVE STRESS IN CARDIOVASCULAR DISEASE 425 into the sub-endothelial space and differentiation into macrophages. These cells ingest (oxidized) low density lipoproteins (LDL) and through this process they are transformed into "foam cell"; (ii) VSMC migration from the media into the intima and their proliferation with the formation of atherosclerotic plaque. Oxidation of low-density lipoprotein Considerable in vivo evidence from animal and human studies support the important role of oxygen free-radical reactions in atherogenesis and atherosclerotic coronary heart disease While the exact mechanisms for atherogenesis are not completely understood, recent studies suggest that oxidative modification of low-density lipoproteins (LDL) is a critical factor Thus, LDL is the "bad actor" in the free-radical hypothesis of atherosclerosis and may be oxidatively modified by all major cell types of the arterial wall, including endothelial cells, smooth muscle cells and macrophages via their extracellular release of ROS. Hydroxyl radicals may initiate the peroxidation of long-chain polyunsaturated fatty acids within LDL molecule, giving rise to conjugated dienes and lipid hydroperoxy radicals (LOO ). This process is self-propagating, such that LOO can attack adjacent fatty acids until complete fatty acid chain fragmentation occurs. A number of highly reactive products, including malondialdehyde and lysophosphatides then accumulate in the LDL particle. These products interact with the amino side chain of the apoprotein B 100 and modify it to form new epitopes that are not recognized by the LDL receptor. Oxidatively modified LDL (OxLDL) is avidly taken up by sub-endothelial macrophages via the "scavenger" receptor pathway which does not recognize native, unmodified LDL. Through the scavenger receptor, unlimited amounts of modified LDL are ingested by the monocyte/macrophage, which is now a "foam cell" in the arterial intima. Accumulation of LDL-laden foam cells beneath arterial endothelium results in the formation of "fatty streak", the earliest histopathological evidence of the development of atherosclerotic plaque 88,89. Oxidized LDL also stimulates the release of monocyte-derived TNF-α and IL-1β, leading to smooth muscle cell proliferation. Elaboration of collagen and elastin by smooth muscle cells leads to plaque formation and fibrosis 88. Lipid peroxides also inhibit synthesis of prostacyclin, an antiplatelet-aggregation substance, which can result in platelet adherence and aggregation. Platelets release growth factors, subsequently leading to smooth muscle cell proliferation and migration to intima. Besides, this may also lead to thrombosis due to the aggregation of platelets 88. OxLDL has additional atherogenic and many proinflammatory properties. It stimulates the expression of macrophage colony-stimulating factor (M-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF) and monocyte chemotactic protein-1 (MCP-1) by endothelial cells and is also cytotoxic to these cells 90,91. oxldl is chemotactic for monocytes and inhibits the motility of macrophages. It is highly immunogenic, forming immune complexes in the arterial wall that can also be taken by macrophages. Antibodies against oxidized LDL have been detected in rabbit atherosclerotic lesions, and the plasma of rabbits and humans contains autoantibodies that react with several forms of oxidized LDL 92. Atherosclerotic lesions from human aorta contain lipid peroxides, and the peroxide content correlates with the extent of atheroma 93. Detectable levels of oxldl are also found in human plasma, and elevated plasma peroxide levels have been found in diabetics, smokers and patients with coronary disease 92,94,95. Oxidative stress and mitochondrial dysfunction The mitochondria are shown to be sensitive to both ROS-mediated damage and alterations in function Recent studies have shown that intracellular ROS production may also be derived from the mitochondria. The production of mitochondrial ROS occurs primarily at two discrete sites in the electron-transport chain, namely at complex I (NADH dehydrogenase) and at complex III (ubiquinone-cytochrome c reductase). Under pathophysiological conditions, the electron-transport chain may become uncoupled, leading to increase in O 2 -. production 102. Mitochondrial DNA (mtdna) is particularly susceptible to modification by ROS/RNS (reactive nitrogen species) because (i) mtdna is in close proximity to the site of ROS/RNS production, (ii) mtdna lacks histone proteins, which can protect it from oxidative damage 103, and (iii) poor DNA damage-repair activity 104. Damage to mtdna can lead to functional changes in the cell, as it encodes several critical protein components of the mitochondrial respiratory chain. Numerous studies have reported the existence of a correlation between DNA damage and

6 426 INDIAN J. BIOCHEM. BIOPHYS. VOL. 46, DECEMBER 2009 atherosclerosis 105,106. The patients with CVD have shown increased mtdna damage when compared with healthy controls in the both the heart and aorta 18,105,107,108. DNA adduct levels are significantly higher in the thoracic aorta from atherosclerotic patients compared to controls 109. Increased immunoreactivity against 8-oxoG (a product of oxidative DNA damage) in plaques of the human carotid artery compared with the adjacent inner media has been reported 109. Multi-variate analysis reveals that DNA-adduct levels are a significant predictor of stage of atherosclerosis 110. Consistent with these studies is the observation that mtdna damage is increased in vascular tissues in CVD patients 18. Proatherogenic risk factors such as smoking, hypercholesterolemia and obesity are all associated with increased mtdna damage 108. The exogenous ROS-mediated mtdna damage is reported to decrease mtdna-encoded gene transcription in a dose-dependent manner and the extent of atherosclerosis correlates well with mtdna damage in the aorta from humans and mice 18. Atherogenesis is also considered a chronic inflammatory disease of the vessel wall 111. Thus, in this condition, after exposure to endotoxins and certain cytokines (TNF-α, IL-1β, expression of inos in vascular endothelial cells, smooth muscle cells, endocardium and macrophages located within the vessel wall leads to prolonged synthesis of large amounts of NO and also to the endothelial cell damage or dysfunction. The initial hypothesis for deleterious effects of NO has been based on its freeradical nature and its reactivity. NO diffuses out and can reach adjacent cells, where it reacts with the ironsulfur centers of several important enzymes from the mitochondrial electron-transport chain and/or ribonucleotide reductase, the enzyme necessary for DNA synthesis 99,100. Recently, it has been suggested that inos is expressed in aneurysmal atherosclerotic human aorta 112. Besides, studies with NO-donor drugs suggest that overproduction of NO in the human heart might decrease contractility and impair diastolic relaxation. Based on these findings, it may be proposed that the net effect of NO modulation in cardiovascular system probably results from a balance between beneficial hemodynamic effects and cytotoxicity. It remains to be determined, why normal physiological production of NO is protective in cardiovascular system and may prevent atheroma formation, whereas overproduction of NO after inos induction is potentially harmful. Taken together, these studies indicate that ROS are clearly associated with enhanced susceptibility to atherosclerosis. Oxidative stress in hypertension ROS and oxldl may play a critical role in the pathophysiology of hypertension. The studies in experimental hypertension and hypertension in humans have demonstrated increased generation of ROS 13,113. Inactivation of the genes for the enzymes that generate ROS in mice results in lowering of their blood pressure. Antioxidants may reduce blood pressure in animal models of hypertension and prevent target organ damage 12. They also demonstrate some beneficial effects in essential hypertension (EH) in humans The reports suggest that EH is associated with increased superoxide anion and H 2 O 2 production, as well as decreased antioxidant capacity The involvement of reactive oxygen intermediates in EH is also suggested by the increased level of lipid peroxides and decreased concentrations of antioxidant vitamin E in plasma of essential hypertensive patients 117. The underlying mechanism that leads to the oxidative stress in EH remains largely unexplored. Reactive oxygen radicals may play a dual role in EH. On one hand, they may inactivate NO by converting them into peroxynitrite in reaction with superoxide anion, thereby causing arteriolar vasoconstriction and elevation of peripheral hemodynamic resistance 118. On the other hand, enhanced production of free radicals may serve as trigger mechanism for oxidative damage of numerous macromolecules, for example, LDL. The enhanced LDL oxidation has been observed in EH patients 119,120. This conclusion is based on findings obtained in isolated LDL (which appears more prone to oxidation triggered by exogenous stimuli) and on demonstration of autoantibodies directed against epitopes generated during oxidative modification of apoprotein B To understand the mechanism for oxygen free radical formation in hypertension, the cellular source must be identified. The endothelial cell, which is recognized as a source of NO has also been identified as a potential site of ROS production 12,121. Superoxide radicals in and around vascular endothelial cells play a critical role in the pathogenesis of hypertension. Increased superoxide anion and H 2 O 2 production by leukocytes isolated from hypertensive patients

7 VIJAYA LAKSHMI et al.: OXIDATIVE STRESS IN CARDIOVASCULAR DISEASE 427 Figure 2 Rate of superoxide and hydrogen peroxide generation in polymorphonuclear leukocytes (PMNLs) of hypertensive rats [(A) Rate of superoxide generation and (B) rate of hydrogen peroxide generation by PMNLs from Control (n = 18), untreated hypertensive (n= 30), and post-treated hypertensive (n = 18) rats. PMNLs (1 x 10 6 cells/ml) were incubated with and without phorbolmyristate acetate (PMA, 200 ng/ml), nitroblue tetrazolium (NBT, 1%) in PBS (ph 7.4) for 20 min at 37 o C and the blue color formed was read at 560 nm 117. Hydrogen peroxide generated by PMNLs was estimated by horseradish peroxidase method by incubating PMNLs with 1% phenol red containing peroxidase (3.75 U/assay) and the absorbance was read 610 nm 209. All values are expressed as mean ± SD. *p< 0.05 vs control; **p< 0.05 vs untreated hypertension (Adapted from 117 )] (Figure 2) and increased levels of lipid peroxides (Figure 3A) and decreased levels of nitrites in plasma have been observed (Figure 3B) 121,122. Besides, the spontaneously hypertensive rats have shown an elevated number of circulating leukocytes that produce superoxide compared with normotensive control 123. Essential hypertensive patients have been shown to produce excessive amounts of ROS 124,125 and decreased antioxidant capacity 126. Activation of renin-angiotensin system is a major mediator of NAD(P)H oxidase activation and ROS production in human hypertension 127. The molecular basis of hypertension is complex; more than 50 genes have been implicated in the regulation of blood pressure 26. The role of AT1 receptor in regulating hypertension has been investigated in both in vitro and animal models. Ang II modulates hypertension through its effects on the renin-angiotensin system and the stimulation of NAD(P)H oxidase in vascular walls. It also directly regulates NAD(P)H Figure 3 Plasma levels of malondialdehyde (MDA) and nitrite in hypertensive rats [(A) Malondialdehyde (MDA), (B) nitrite (end product of NO) in control (n=18), untreated hypertensive (n = 25), and post-treated hypertensive (n = 18) rats. The amount of lipid peroxidation products (MDA) was determined by thiobarbituric acid (TBA) method and measured at 532 nm 117,210. Plasma nitrite level was measured by using Greiss reagent (1% sulfanilamide in 5% H 3 PO % naphthalene-ethylenediamine dihydrochloride) at 543 nm 211. All values are expressed as mean ± SD. *p< 0.05 vs control; **p< 0.05 vs untreated hypertension (Adapted from 117 )] oxidase activation by enhancing a rapid translocation of small GTPase rac 1 to the cell membrane 29 or by phosphorylating and translocating p47 phox membrane translocation to cell membranes 30. The most important source of ROS in blood vessels appears to be NAD(P)H oxidase 1,12. a multi-subunit enzyme 6,22 having NAD(P)H as electron donor. The best characterized NAD(P)H oxidase is found in phagocytes, neutrophils, monocytes and macrophages 128. Non-phagocytic oxidase is the main source of ROS in blood vessels 6,129, present in the endothelium 130 and VSMCs in the media 129,130. Transgenic mice that express constitutively active rac1 in VSMCs exhibit a hypertensive phenotype; increased ROS production and treatment with antioxidants reverses hypertension 131. The activity of vascular NAD(P)H oxidase is modulated by many different factors that include cytokines, growth factors and vasoactive peptides. Stretch, pulsatile strain and shear stress may activate NAD(P)H oxidase 6,132. Ang II not only stimulates NAD(P)H oxidase, but also enhances the expression of the subunits of NAD(P)H oxidase. It induces ROS generation by endothelial cells, VSMCs and adventitial fibroblasts via stimulation of AT 1 receptors 133. PDGF, TGF-β, TNF-α and thrombin also activate NAD(P)H oxidase in VSMCs

8 428 INDIAN J. BIOCHEM. BIOPHYS. VOL. 46, DECEMBER 2009 Endothelin-1 increases NAD(P)H oxidase activity in human endothelial cells via ET A receptors 119. The statins, antihypertensive drugs, such as β-blockers, calcium channel blockers, angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers decrease the expression of NAD(P)H oxidase subunits and its activity The antihypertensive action of ACE inhibitors and angiotensin receptor blockers might be due to inhibition of NAD(P)H oxidase activity and decreased ROS production 127. The beneficial effects of β-adrenergic blockers (carvedilol) and some calcium channel blockers may be mediated, in part by decreasing vascular oxidative stress 139,144. The genetic models of hypertension, such as SHR (spontaneously hypertensive rat) and stroke-prone SHR exhibit enhanced NAD(P)H oxidase-mediated superoxide generation by increased expression of its subunits (p22 phox and p47 phox ) in conduit (aorta) and resistance arteries (mesenteric) and in the kidney 113,141. Polymorphisms have been identified in the promoter region of the p22 phox gene in SHR, which could contribute to increased NAD(P)H oxidase activity 142. Treatment with antioxidant vitamins, NAD(P)H oxidase inhibitors, SOD blockers, folic acid AT1 receptor blockers decrease vascular superoxide production, lipid-peroxidation products and may decrease development of blood pressure elevation in genetic hypertension 113,116,143. Polymorphisms in p22 phox gene may play a role in altered NAD(P)H oxidase-generation of superoxide in humans, particularly 903(A/G) polymorphism 142. However, in homozygous individuals with the T allele of the C242T CYBA, polymorphism may have reduced vascular oxidative stress 145. Taken together, these observations strongly suggest that oxidative stress is a modulator of hypertension, a potential risk factor for atherosclerosis. Oxidative stress and heart failure Oxidative stress is increased in heart failure and may contribute to many of the structural and functional changes that characterize disease progression. There are both indirect and direct evidences of increased oxidative stress in humans with heart failure. In patients with heart failure, the level of the lipid peroxidation product malondialdehyde (MDA) is increased in plasma. Direct evidence of increased myocardial oxidative stress is also obtained from the fact that the level of 8- iso-prostaglandin F 2α (8-isoprostane) is increased in the pericardial fluid of the patients with heart failure. The involvement of oxidative stress in heart failure is further supported by the prevention of the progression of several pathological processes such as cardiac hypertrophy, cardiac myocyte apoptosis, ischemiareperfusion and myocardial stunning, which can lead heart failure in animals models and TNF-α and Ang II-induced hypertrophy in cardiac myocytes is prevented by vitamin E, hydroxyanisole and catalase 146. Overexpression of catalase significantly reduces Ang II-induced hypertrophy and transfection of antisense p22 phox inhibits Ang II-induced H 2 O 2 production. This suggests that NAD(P)H oxidaseinduced oxidative stress leads to the hypertrophy 9. There are a number of potential sources of ROS in the myocardium. Several enzyme systems generate superoxide and among these, the mitochondria appear to be an important source of myocardial ROS in the failing heart. A small fraction of the electrons that pass through the mitochondrial electron transport chain may 'leak', thereby reacting with molecular oxygen to form superoxide. Electron paramagnetic resonance (EPR) spectroscopy with a superoxide spin-trap shows 2.8-fold increase in ROS in mitochondria from failing hearts, together with a decrease in the activity of electron transport complex I, suggesting that in heart failure, a functional uncoupling of the mitochondria contributes to increased ROS formation. Increased production of ROS may decrease NO bioavailability and impair diastolic function 147. In addition, increased peroxynitrite may cause cytokine-induced myocardial contractile failure by inactivating sarcoplasmic Ca 2+ -ATPase and dysregulating Ca 2+ homeostasis 148,149. Evidence also suggests that oxidases may contribute to ROS generation in the myocardium. Xanthine oxidase activity is increased in the failing heart and xanthine oxidase inhibitors improve myocardial energetics in a dog model of heart failure and in humans with heart failure 150. NADPH oxidase, a plasmalemmal enzyme that generates superoxide in the cytosol is another oxidase implicated in myocardial failure. In the failing myocardium of patients with ischemic or dilated cardiomyopathy, NAD(P)H oxidase-derived ROS are upregulated. In patients with heart failure, plasma TNF-α and plateletderived NAD(P)H oxidase activity is also elevated 151. In the failing myocardium, the translocation of regulatory p47 phox from the cytosol to the sarcolemmal

9 VIJAYA LAKSHMI et al.: OXIDATIVE STRESS IN CARDIOVASCULAR DISEASE 429 membrane is recently demonstrated 152. Together, these results suggest that oxidative stress has a role in the pathophysiological cardiac dysfunction in heart failure. Oxidative stress and ischemia-reperfusion myocardial injury Exposure of myocardial tissue to a brief transient ischemia, followed by reperfusion has attracted remarkable attention in recent years. Myocardial ischemia occurs when myocardial oxygen demand exceeds oxygen supply. Unless reversed, this situation results in cell injury, leading to clinical myocardial infarction. Reperfusion of ischemic myocardium is recognized as potentially beneficial, because mortality is directly proportional to infarct size, and the severity and duration of ischemia. Reperfusion of ischemic myocardium can restore oxygen and substrates to the ischemic myocardial cells, but this process may create another form of myocardial damage termed "reperfusion injury" 153,154. Thus, restoration of a normal blood flow in the heart by methods, such as angioplasty, thrombolysis cardiopulmonary bypass can lead to specific lesions (arrhythmias, deficit in contractility, necrosis), the importance of which also depends on the duration of ischemia. The damage to the myocardial cell induced by cycles of ischemia and reperfusion may be due, in part to the generation of toxic ROS such as superoxide radical, H 2 O 2 and hydroxyl radicals The active involvement of free radicals in the ischemia-reperfusion damage is demonstrated by direct and indirect experimental evidences. Direct evidence arises from the possibility of measuring radicals in myocardial tissue by EPR spin-trapping 21 ; indirect evidence by the measurement of the products of free radical attack on biological substrates (usually MDA as a measure of lipid peroxidation) and intracellular and extracellular antioxidant capacity 23. EPR spectroscopy has shown an increased freeradical production in blood after reperfusion of infarct tissue 21. EPR signals are also recorded in blood samples taken from coronary sinus of patients undergoing percutaneous transluminal coronary angioplasty (PTCA), an ideal model of myocardial ischemia-reperfusion 21. In patients undergoing cardiopulmonary bypass, an increased free-radical generation and reduction of blood-antioxidant capacity in plasma have been reported, following aortic declamping 23. Furthermore, the experimental findings suggest an impairment of antioxidant mechanisms in the ischemic tissue 23. Evidence to support this is also obtained from the cardioprotective effect of agents capable of inducing antioxidant enzymes in the heart and from the beneficial effects of several enzymatic free-radical scavengers, antioxidants and iron chelators in reperfused myocardium 158. Reperfusion of the isolated rat heart with oxygenated buffer has been shown to generate free radicals, as detected by EPR spectroscopy 159,160. A burst of free-radical generation, such as of. OH., R. and RO. adducts of 5,5,-dimethyl-1-pyrroline-N-oxide (DMPO) is observed during early period of reperfusion, with peaking occurring within 30 sec of reperfusion (Figure 4). The reperfusion-induced ROS generation is markedly decreased by SOD, suggesting that adduct formed is from ROS. In an another study, pretreatment of heart with the plant-based antioxidants Spirulina and C-phycocyanin significantly attenuates the I/R-induced ROS generation (Figure 5) 161. Figure 4 EPR spectra of effluent of heart perfusate [(A) preischemia, (B) effluent collected 2 min after reperfusion with DMPO (40 mm), showing hydroxyl adduct and alkyl adduct, (C) simulation of DMPO-OH adduct, (D) simulation of DMPO-alkyl adduct, and (E) C + D simulation of both DMPO-OH adduct + stimulation of DMPO-alkyl adduct (compare with 4B)]

10 430 INDIAN J. BIOCHEM. BIOPHYS. VOL. 46, DECEMBER 2009 Figure 5 Myocardial oxygen free radical formation at reperfusion with antioxidants, Spirulina (SP) and C-phycocyanin (PC) [(A) Time course and (B) Quantitation of 5,5,-dimethyl-1- pyrroline-n-oxide (DMPO) adducts of radicals at 1 min of reperfusion. Free radical generation was measured using spin-trap (DMPO) with and without SP and PC in hearts subjected to 30 min ischemia and 45 min of reperfusion. DMPO (40 mm final concentration) was infused through sidearm during reperfusion, coronary effluent was collected at min, and DMPO adduct formation was measured by electron spin resonance spectroscopy. Values are mean ± SD from 3 independent experiments. *p < 0.05 vs control (Adapted from 161 ) The oxygen tension during the initial reperfusion of ischemic myocardium may modulate early ROS generation 162. In hearts reperfused with 2% O 2 for the first 5 min, followed by 95% O 2, ROS generation peaks in the first 2 min of reperfusion (Figure 6) Furthermore, the magnitude of ROS generation is significantly higher in the 2% O 2 group, when compared to the 95% O 2 or 21% O 2 (Figure 6). The failure of all energy-dependent mechanisms leads to deterioration of membrane ion gradients, opening of selective and unselective ion channels and equilibration of most intracellular and extracellular ions. As a consequence of this "anoxic depolarization", K + ions leave the cell, while Na + and Ca 2+ ions enter. Cellular accumulation of ions causes formation of cytotoxic edema. Intracellular Ca 2+ Figure 6 Myocardial oxygen free radical formation at reperfusion with different O 2 concentrations [The free radicals were measured as DMPO adducts in the effluent using spintrapping EPR spectroscopy. The DMPO adduct peaked in the first 2 min in all groups and was significantly elevated in the most hypoxic reperfusion group (2% O 2 ) compared with the 20% and 95% O 2 reperfusion groups (*p < versus 95% O 2 and 20% O 2, n=4/group). (Adapted from 162 )] overload can also set off a cascade of events which may lead to the formation of ROS. The elevated Ca 2+ concentration activates proteases that can convert xanthine dehydrogenase to xanthine oxidase. During reoxygenation, xanthine oxidase can use O 2 as an electron acceptor, leading to formation of superoxide anion and H 2 O 2, which can react to produce OH radicals. These reactive species are responsible for the tissue damage. ROS produced from xanthine oxidase play a major role in causing tissue damage in ischemia-reperfusion injury, as evidenced by the ability of inhibitors of xanthine oxidase in protecting against such damage in experimental models of myocardial infarction 163. Another source of ROS generation is the intramitochondrial electron-transport chain. Free radicals produced in mitochondria may also cause point mutations, DNA cross-link and DNA strand breaks in mitochondrial genes. The damage to mitochondrial genome results in impaired respiration, increasing further the possibility of oxygen-radical production. Impaired mitochondrial function and increased production of superoxide are very common reperfusion-associated events. The activities of components of mitochondrial respiratory chain are markedly reduced during post-ischemic reperfusion or post-hypoxic reoxygenation 164. Experimental studies suggest that mitochondrial dysfunction results in increased production of superoxide by this organelle after exposure of cardiac muscle to ischemiareperfusion 165.

11 VIJAYA LAKSHMI et al.: OXIDATIVE STRESS IN CARDIOVASCULAR DISEASE 431 Reperfusion after an ischemia period is associated with impaired bioavailability of NO, most likely due to enhanced inactivation of NO by superoxide and reduced production of NO. Several studies on NO and NOS inhibition in models of I/R have yielded mixed results. Studies on isolated rat heart have shown that L-arginine and several NO donors can attenuate postischemic reperfusion damage 166,167,168. On the other hand, NO and its reaction products have also been demonstrated to cause detrimental effects on the reperfused heart 169. The treatment of heart with NO donors, such as S-nitroso-N-acetylpenicillamine (SNAP) or 3-morpholinosydnonimine hydrochloride (SIN-1) increases the formation of ONOO and exacerbates the myocardial oxidative damage after I/R 170. Interaction between NO and superoxide during the early phase of reperfusion has been demonstrated to form peroxynitrite, an important determinant of post-ischemic myocardial function 171. Peroxynitrite causes cellular damage by lipid peroxidation, oxidation of sulfhydryl groups and inhibition of signaling pathways by nitration of tyrosine residues and DNA strand breaks Increased levels of nitrotyrosine (an indicator of peroxynitrite production) have also been reported in control heart subjected to I/R. Heart pretreated with tempol combined with NO donor NCX-4016 significantly attenuates the peroxynitrite production (Figure 7) 175. Increased formation of ROS, following hypoxiareoxygenation is associated with low antioxidant capacity of myocardial tissue. The catalase activity was reported to be low in myocytes and endothelial cells and most of it is compartmentalized within peroxisomes. This subcellular localization prevents catalase from being an efficient scavenger of H 2 O 2, resulting from SOD activity in the cytosol 176. Insufficient antioxidant capacity of tissue to scavenge the increased content of ROS, following hypoxia/reoxygenation appears to be an important contributing factor to tissue dysfunction, restenosis of bypass grafts and post-balloon angioplasty. These complications may worsen the efficiency of interventions used in the treatment of coronary artery disease 177. The cascade of events associated with I/R injury, besides free-radical generation includes release of cytokines and growth factors, leukocyte adhesion, platelet aggregation, smooth muscle proliferation, and mechanical injury 178. Studies have suggested that reperfusion of the ischemic myocardium results in cardiomyocyte apoptosis and necrosis in human 179,180 and in animal models of I/R injury 177. Although necrosis represents the classic manifestation of hypoxia-induced cell damage, myocyte apoptosis appears to be an early event in cardiac I/R injury 181. The I/R-induced apoptosis is mediated by different apoptotic signaling cascades that are mediated by free radicals and oxidative stress 182. The activation of mitochondria-initiated pathway plays an important role in the apoptosis in hearts subjected to I/R 180. Ischemia-reperfusion results in the release of cytochrome c from the mitochondria and the activation of caspase-9 in isolated perfused hearts 183. Several caspase inhibitors have been demonstrated to attenuate apoptosis in myocardial I/R injury Prolonged reperfusion after ischemia leads to down-regulation of the antiapoptotic protein Bcl Myocytes lacking the proapoptotic Bax gene reduces I/R injury through the blocking of necrotic and apoptotic pathways 187. Recently, involvement of mitogen activated protein kinases (MAPKs) has been demonstrated in ischemic injury 188,189. The stressinduced p38 MAP kinase pathway is activated in Figure 7 Nitrotyrosine in coronary effluents from isolated perfused hearts subjected to IR [Nitrotyrosine (an indicator of peroxynitrite production) was measured using a microplate fluorimeter with excitation/emission filters 320/410 nm. Upper: time course of nitrotyrosine formation in coronary effluent from hearts subjected to IR. Lower: Nitrotyrosine formation at 1 min of reperfusion. Data are expressed as percentage of pre-ischemic baseline represented as mean ± SD (n = 3). *p<0.001 vs preischemia; **p<0.01 vs control (IR) (Adapted from 175 )]

12 432 INDIAN J. BIOCHEM. BIOPHYS. VOL. 46, DECEMBER 2009 cardiac myocytes exposed to I/R and plays a role in the induction of apoptosis Inhibition of p38 MAPK decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion 185,193. Previous studies have demonstrated that activation of ERK1/2 after reperfusion is cardioprotective The PI3K-Akt signaling is an important mediator of cell survival and promotes the survival of cardiomyocytes in vitro and in vivo. In addition, it protects against acute I/R-injury in the mouse heart 197,198. The cytoprotective effects of sodium orthovanadate, adrenomedullin, vasodilatory peptide, mexiletine derivative (H-2693) etc. on I/R injury are reported to be mediated by the activation of Akt 199,200. C-phycocyanin, a plant-based antioxidant attenuates I/R-induced injury through antioxidant and antiapoptotic actions and modulation of p38 MAPK and ERK1/ An anti-ischemic drug trimetazidine (TMZ) derivatized with a pyrroline group (TMZ-Ǿ- NH) significantly protects heart against I/R-mediated injury by enhancing the pro-survival Akt activity, without significant effect of p38 MAPK and ERK1/2 (Figure 8) 201. The protective effect of TMZ derivatives could be due to the combined effects of anti-ischemic protection by trimetazidine and ROS scavenging during I/R. ROS have been reported to induce transcription factor NF-κB 202. Inhibition of NF-κB by antioxidants further supports a role of ROS in the activation of NF-κB 203. Figure 8 Akt, ERK1/2 and p38 MAPK phosphorylation in heart subjected to IR [The phosphorylation of Akt, ERK1/2 and p38 MAPK was measured in hearts subjected to ischemia (30 min) and after 10 min reperfusion, without and with trimetazidine (TMZ) and trimetazidine derivatives TMZ-NH and TMZ-φ-NH (50 µm). Phosphorylated Akt, ERK1/2 and p38 MAPK was detected by Western blot analysis (Adapted from 175 ) Strategies for inhibition of oxidative stress in CVD Despite the evidence for association of increased oxidative stress with various vascular diseases, using antioxidant therapy to prevent cardiovascular diseases has produced mixed results 204,205. Natural antioxidant α-tocopherol at a dose of either 400 or 800 IU/day causes a significant reduction in the combined primary end point of cardiovascular death and non-fatal myocardial infarction 206. In the antioxidant supplementation in atherosclerosis prevention (ASAP) study, a combination α- tocopherol (272 IU/day) and slow-release aspirin has been shown to significantly decrease carotid intimamedia thickness in hypercholesterolemic males 207. The vitamin C (359 mg/day) supplementation is also associated with a significant reduction in non-fatal and fatal myocardial infarction 208. In contrast, several antioxidant supplementation studies have not shown any effect on primary end points of cardiovascular events 204. The inability of some of the antioxidants to prevent CVD may be attributable to several reasons: (i) ineffectiveness could relate to optimum dose and type of antioxidants, (ii) complexity of redox reactions in vivo, (iii) inability to target specific redox pathways, although some existing drugs exert some of their effects through redox-depending signaling pathways, for example, statins, angiotensinconverting enzyme inhibitors and protein kinase C inhibitors. Future treatments may need to target redox pathways in cell, tissue and pathway-specific manner. Perhaps, the most exciting prospect is the development of specific targeting strategies to deliver redox-active molecules to the mitochondrion, development of specific ROCK inhibitor or agents which can upregulate Nrf2. Conclusion With drastic changes in the life style pattern, increasing number of subjects is at risk of vascular disease and there is preponderance of evidence for the association of increased oxidative stress with various vascular diseases. These cause premature death from angina, heart attack, stroke, peripheral artery disease, hypertension, ischemia and thrombosis. The loss of control of free-radical formation from the mitochondrion can contribute to the pathology of CVD through a number of mechanisms including damage to mtdna, enzyme degradation, and apoptosis and thus contribute to human disease. However, a better understanding of the ROS-