Modulation of oxidative stress-induced changes in hypertension and atherosclerosis by antioxidants

Abstract

An imbalance between reactive oxygen species and antioxidant reserve, referred to as oxidative stress, results in the altered structure and function of proteins, lipids and DNA. Oxidative stress is associated with hypertension and atherosclerosis, but it is unknown whether it is a causative or resultant factor. The authors suggest that insulin resistance is the key element in the pathogenesis of these diseases, and leads to abnormal glucose and lipid metabolism with an increase in reactive aldehydes. These aldehydes react with the sulfhydryl and amino groups of proteins to form advanced glycation end products, adversely affecting body proteins, including antioxidant enzymes. This leads to oxidative stress. Advanced glycation end products and reactive oxygen species perpetuate a pro-oxidant state, producing the changes that are characteristic of hypertension and atherosclerosis. Antioxidants have been shown to modulate these changes. An ideal therapy for these diseases includes antioxidants, which attenuate insulin resistance, the source of oxidative stress.

Reactive oxygen species (ROS) are normally found in biological systems, and their levels are controlled by various enzymes and antioxidants. When there is a decreased antioxidant capacity or an increased generation of ROS, the result is an imbalance referred to as oxidative stress. There is substantial evidence that oxidative stress contributes to the progression of hypertension and atherosclerosis (1–4). ROS-induced modification of proteins and lipids has the capacity to produce changes that are characteristic of these two vascular disorders. In the present review, we discuss the possible mechanisms of these oxidative stress-induced changes and their modulation by antioxidants.

ROS AND OXIDATIVE STRESS

For the purpose of the present article, we will use the term ‘ROS’ to encompass several oxygen- and nitrogen-derived radicals, including superoxide radical (O₂⁻), H₂O₂, hydroxyl radical (·OH), peroxynitrite (ONOO⁻) and peroxynitrous acid (ONOOH). Under normal circumstances, ROS are generated from various sources. In mitochondrial respiration, leakage of electrons can occur from the electron transport chain, reducing molecular oxygen to form O₂⁻. The enzyme NADPH oxidase is associated with membranes of cells, including endothelial and vascular smooth muscle cells (VSMCs), and is another major contributor of ROS, catalyzing the production of O₂⁻ using NADPH. When produced at this site, ROS may regulate vascular function by modulating cell growth, migration, inflammation and extracellular matrix protein production (4–6). Leukocyte NADPH oxidase also generates ROS as part of the immune response. Prostaglandin synthesis and purine metabolism account for minor production of radicals. The enzyme nitric oxide synthase (NOS), which acts on the substrate arginine to produce the vasodilator nitric oxide (NO), can become uncoupled, resulting in the production of O₂⁻ (7,8). NO is itself considered a radical, and its reaction with O₂⁻ forms more reactive radicals, namely, ONOO⁻ and ONOOH (3).

ROS are controlled, in part, by enzymatic decomposition (9). Superoxide dismutase converts O₂⁻ to H₂O₂, which is further converted to nonradical products via the enzyme catalase. H₂O₂ can also undergo reduction to ·OH in the presence of metal-containing molecules. Glutathione peroxidase (GPx) catalyzes the reduction of H₂O₂ and lipid radicals using reduced glutathione (GSH). Oxidized glutathione is then enzymatically regenerated into its reduced form by glutathione reductase (GRed). Alternatively, vitamins such as vitamins C and E, and other reducing compounds such as lipoic acid, glutathione and cysteine nonenzymatically reduce radicals, including O₂⁻ and ·OH. Vitamins themselves become oxidized as a result of this antioxidant action, and are regenerated into their reduced forms by antioxidants of higher electronegativity (Figure 1 and Table 1). For example, vitamin E radical (oxidized) is regenerated to vitamin E (reduced) by vitamin C (ascorbate) or coenzyme Q10 (CoQ10) (reduced). These antioxidants are, in turn, regenerated by dihydrolipoic acid (10). When there is a decreased antioxidant capacity or an increased generation of ROS, the result is an imbalance referred to as oxidative stress. The pathogenic consequence of oxidative stress is oxidative damage, which has been implicated in the development and progression of hypertension and atherosclerosis.

Figure 1).

Regeneration of antioxidants in the body. Oxygen free radicals are quenched by antioxidants, which themselves become oxidized. The oxidized antioxidant is regenerated to its original reduced form by another antioxidant of higher electronegativity. ROO⁻ Free radical; ROOH Nonradical

TABLE 1.

Redox potentials of antioxidants in mammalian oxidation systems (redox pairs are given at increasing electronegativity and antioxidant activity)

Redox pair	E′o (volts)
Oxygen/water	+0.82
Vitamin E oxidized/reduced	+0.37
Coenzyme Q10 oxidized/reduced	+0.10
Vitamin C oxidized/reduced	+0.08
Cystine/cysteine	−0.22
Glutathione oxidized/reduced	−0.24
Lipoate oxidized/reduced	−0.29
NAD+/NADH and NADP+/NADPH	−0.32
H+/H2	−0.42

E′_o Standard redox potential at pH 7.0

HYPERTENSION AND ATHEROSCLEROSIS

Hypertension affects more than 600 million people worldwide and results in 13% of total deaths globally (11). Essential hypertension is characterized by endothelial dysfunction with alterations in NO bioavailability and calcium handling, VSMC proliferation, thickening of the vessel walls, and increased peripheral vascular resistance and blood pressure (12,13). Individuals with hypertension are at an increased risk for atherosclerotic diseases such as stroke, and ischemic heart and kidney diseases.

Heart disease and stroke, two of the common manifestations of atherosclerosis, are leading causes of death in adults in developed countries, and cardiovascular disease is responsible for one-third of all deaths globally (14). Atherosclerosis is an inflammatory condition of the blood vessels (15) in which increased permeability of the endothelium promotes entry of low density lipoprotein (LDL) into the intima, where it becomes oxidized. Oxidized LDL stimulates the expression of adhesion molecules and inflammatory cytokines, resulting in platelet aggregation and monocyte adhesion to the endothelium. The monocytes transmigrate to the subendothelial space, where they differentiate into macrophages. Modified LDL stimulates the scavenger receptors of macrophages, causing internalization and degradation of LDL. In time, these cells are transformed into foam cells, which, along with other cellular debris, evolve into atherosclerotic plaques (1,15–17). Oxidized LDL also stimulates VSMC migration and proliferation, which also contribute to intimal thickening and plaque formation. Although atherosclerotic lesions generally occur at junctions of large- and medium-sized vessels, they may also arise throughout the vasculature (17,18). Through stenosis or embolytic occlusion (via plaque eruption), lesions within the coronary, cerebral, peripheral or renal vessels result in the clinical manifestations of angina, myocardial infarction (MI), stroke, peripheral arterial disease or renal failure (18). Extensive endothelial involvement and plaque formation may also lead to loss of vessel elasticity, contributing to hypertension.

Hypertension is considered to be a risk factor for atherosclerosis, because increased blood pressure itself can contribute to vascular injury, making vessels more susceptible to inflammation (2). However, studies show that lowering blood pressure alone does not completely eliminate the risk of cardiovascular disease (19). Hypertension and atherosclerosis share similar risk factors, and both are characterized by a modified vascular structure and function (20). These cardiovascular conditions are also associated with insulin resistance and oxidative stress (1–4,21–24).

INSULIN RESISTANCE AND THE FORMATION OF ADVANCED GLYCATION END PRODUCTS

We suggest that the key etiological factor in hypertension and atherosclerosis is insulin resistance (Figure 2). Insulin resistance can arise from genetic and lifestyle factors. It is characterized by an inadequate glucose uptake in peripheral tissues at a given concentration of plasma insulin, and involves an impairment of the nonoxidative (glycolytic) pathways of intracellular glucose metabolism (21). Under normal physiological conditions, glucose is metabolized via the glycolytic pathway to glyceraldehyde-3-phosphate, which is converted to 1,3-diphosphoglycerate by the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), with further metabolism to pyruvate. Any factor that affects GAPDH, whether through inhibition or downregulation, has an impact on the rate of glucose metabolism. GAPDH activity is upregulated by insulin (25). In insulin-resistant states, altered insulin function may downregulate GAPDH activity, slowing glucose metabolism through the glycolytic pathway, and thus increasing glucose metabolism via the polyol pathway. This may result in a buildup of glyceraldehyde-3-phosphate, leading to an increase in the highly reactive aldehyde methylglyoxal (26,27). Methylglyoxal itself has been shown to inhibit GAPDH, which can result in further abnormalities in glucose metabolism (28). Methylglyoxal also induces aldose reductase, an enzyme known to stimulate glucose flux through the polyol pathway, with further formation of methylglyoxal (29). Insulin resistance is associated with dyslipidemia (22,24), and elevated levels of LDL without the mitigating antioxidant effect of high levels of high density lipoprotein (HDL) may also contribute to an increase in reactive aldehydes (30). Typical North American diets, which are high in fructose- and fat-enriched processed food products, may also contribute to this altered glucose and lipid metabolism, leading to an increase in reactive aldehyde levels.

Figure 2).

Insulin resistance, due to genetic and lifestyle factors, leads to altered glucose and lipid metabolism with an increase in the formation of reactive aldehydes. These excess aldehydes form advanced glycation end products (AGEs) that lead to oxidative stress. AGEs and oxidative stress are responsible for structural and functional damage to lipids and proteins, which contribute to the progression of hypertension and atherosclerosis. NO Nitric oxide; VSMC Vascular smooth muscle cell. Modified with permission from reference 160

Advanced glycation end products (AGEs) are formed when aldehydes react nonenzymatically with the free sulfhydryl (-SH) and amino groups of protein amino acids, including cysteine, arginine and lysine (26,31,32). This direct modification of protein structure results in functional changes (28,33,34). AGE-modified proteins also stimulate receptors of AGEs and various scavenger receptors to influence protein function and expression (35,36). Several specific AGEs, including carboxymethyl-lysine, carboxyethyl-lysine, argpyrimidine and glycoaldehyde-pyridine, have been identified, and have been implicated in the pathology of hypertension and atherosclerosis (16,37–41).

OXIDATIVE STRESS – WHERE DOES IT COME FROM?

Effect of AGEs

Oxidative stress is controlled, in part, by the antioxidant enzymes GPx and GRed (9). These enzymes have -SH and amino groups at their catalytic sites (42,43). Direct modification of these groups by aldehydes to form AGEs or reaction with AGE-modified proteins can inhibit the activity of these enzymes, resulting in increased oxidative stress (28,43–45). Because methylglyoxal is kept at a low level through catabolism via the glutathione-dependent glyoxalase enzyme system or by binding to cysteine and being excreted in bile and urine (26), an excess of this aldehyde may lead to depletion of cysteine and glutathione, in turn causing a decrease in antioxidant capacity. The activation of AGE receptors by AGE results in an NADPH oxidase-mediated increase in intracellular reactive oxygen intermediates, extracellular H₂O₂ and cell surface expression of vascular cell adhesion molecule-1 in human endothelial cells (35). This suggests an ROS-mediated action of AGEs on genetic expression.

AGE-modified bovine serum albumin increases O₂⁻ production in human platelets in vitro (46), and increases intracellular calcium levels and oxidative stress in rat mesangial cells (47). In VSMCs in vitro, methylglyoxal induces significant generation of O₂⁻ and ONOO⁻ (48). Cultured VSMCs from spontaneously hypertensive rats (SHRs) show an increase in levels of methylglyoxal, AGEs and oxidative stress, and a decrease in GSH levels, and GPx and GRed activity (44). SHRs with elevated plasma methylglyoxal show an increase in aortic AGEs and oxidative stress, and a decrease in GSH levels and antioxidant enzyme activity (41). ROS inhibit GAPDH, which may exacerbate insulin resistance, causing a further increase in the level of aldehydes (49,50).

Through lipid peroxidation, ROS can also cause secondary production of aldehydes such as malondialdehyde and hydroxynonenal (30,51,52). These lipid-derived aldehydes form a type of AGE known as advanced lipoxidation end products (31). Thus, a cycle is created in which ROS and AGEs perpetuate a pro-oxidant state. Taken together, these reports indicate that AGEs can increase oxidative stress by both decreasing antioxidant capacity and increasing the production of ROS (45).

Effects of lifestyle and environmental factors

Oxidative stress may occur when dietary intake of antioxidants does not keep pace with antioxidant requirements or when appropriate combinations of nutrients are not included in the diet. If the relative amounts of antioxidants are disproportionate, inefficient regeneration of oxidized vitamins to their reduced form with a possible accumulation of vitamin radicals may occur. Hypertensive patients and those with cardiovascular disease show low plasma levels of vitamins E and C (53–58). High-salt and high-fat diets, implicated in hypertension and atherosclerosis, respectively, have also been shown to increase oxidative stress (11,14,59,60). Both smoking and high ethanol intake, which are lifestyle risk factors associated with hypertension and atherosclerosis, increase oxidative stress (11,14,61,62).

Oxidative stress appears to contribute to the progression of hypertension and atherosclerosis (1–4). ROS modification of proteins and lipids has the capacity to produce changes that are characteristic of these two vascular disorders. The mechanism of this oxidative damage is discussed next.

MECHANISM OF OXIDATIVE DAMAGE

Proteins, lipids and DNA are cellular targets for oxidation, which leads to alterations in their structure and function (Table 2 and Figure 3). ROS can cause these alterations in the following ways that lead to pathogenic changes: first, oxidation of critical amino acid residues, such as cysteine residues, may alter enzyme activity where -SH groups are in the catalytic domain (49,63–65) or may affect transcriptional activities if they are within the binding site of transcription factors (66,67); second, intra- or intermolecular conformational changes, such as the formation of disulfide bridges between or within proteins, may result in an alteration of protein activity or function (65,68); third, metal-catalyzed oxidative reactions may cause modifications to membrane proteins and lipids, leading to degradation processes (30); fourth, peroxidation reactions may lead to degradation of membrane lipids with loss of membrane integrity, and these reactions also release aldehydes that modify membrane proteins, possibly altering ion channel or receptor function (1,30,69); and fifth, overstimulation of ROS-mediated signalling pathways may result in gene transcription activities that regulate the expression of inflammatory factors, increased vascular cell proliferation and apoptosis (4,70).

TABLE 2.

Effect of oxidative stress in hypertension and atherosclerosis

Affected component	Action of ROS	Effect of oxidative damage (references)
Glutathione peroxidase and glutathione reductase	Inhibition of enzyme activity	A decrease in ability to degrade or neutralize ROS, resulting in an increase in oxidative stress. This leads to lipid peroxidation and formation of secondary aldehydes, setting up a cycle of ROS and AGEs (30,41,44,71)
Glyceraldehyde-3-phosphate dehydrogenase	Inhibition of enzyme activity	Exacerbation of insulin resistance, resulting in an increase in aldehydes and AGEs, and a perpetuation of oxidative stress (26,49,50)
Endothelial nitric oxide synthase	Inhibition of enzyme activity and alteration causing uncoupling of enzyme	Decreased production of NO resulting in an increase in vasoconstriction, blood pressure, platelet aggregation, VSMC proliferation and activation of monocyte adhesion molecules (7,8,13,75,76)
Endothelial NO	Degradation into peroxynitrite and peroxynitrous acid	Decreased bioavailability of NO and an increase in reactive nitrogen species, resulting in an increase in blood pressure, inflammatory processes and nitrosative damage (3,64,77,78)
Membrane NADPH oxidase	Activation of enzyme activity	Increase in oxidative stress at the surface of the endothelial membrane, possibly leading to membrane modification that contributes to atherosclerotic processes (6,81,82,84,92)
Protein tyrosine phosphatase	Inhibition of enzyme activity resulting in an increase in protein tyrosine kinase	Abnormal intracellular cell signal transduction, resulting in VSMC proliferation, apoptosis, contraction and inflammation (65,70,79)
Angiotensin-converting enzyme	Activation of enzyme activity	Increase in production of angiotensin II, resulting in vasoconstriction and increased blood pressure, hypertrophy, proliferation and apoptosis, and activation of NADPH oxidase with an increase in oxidative stress (6,79–83)
Calcium channels and calcium regulatory enzymes and proteins	Modification of vascular calcium channels,	Increased free cytosolic calcium, either from an increased influx or
	inhibition of Ca2+-ATPase, and alteration of transport activating proteins	release from intracellular stores, resulting in an increase in oxidative stress, peripheral vascular resistance and blood pressure, and stimulation of cellular proliferation, apoptosis and inflammation (63,64,86–89)
LDL	Oxidative modification to protein and lipid components of LDL	Oxidized LDL stimulates VSMC proliferation, platelet aggregation and release of inflammatory cytokines. It also stimulates scavenger receptors, increasing uptake by macrophages forming foam cells that contribute to atherosclerotic plaque (3,15,17,91)
HDL	Modification of paraoxonase	Decreases HDL’s ability to protect LDL from oxidation (97,98,102)
Structural lipids and proteins	Modification to lipid and/or proteins of structural membranes	Alters integrity of endothelial membrane, making it vulnerable to atherosclerotic processes (1,30,69)

AGEs Advanced glycation end products; HDL High density lipoprotein; LDL Low density lipoprotein; NO Nitric oxide; ROS Reactive oxygen species; VSMC Vascular smooth muscle cell

Figure 3).

Advanced glycation end products (AGEs) inhibit antioxidant enzymes, which results in increased oxidative stress (inset). Reactive oxygen species (ROS) alter the structure and function of cellular proteins, including cell membranes, calcium channels and enzymes such as endothelial nitric oxide synthase (eNOS), which results in a decrease in nitric oxide (NO) and enothelial dysfunction, an increase in cytokines, inflammation, platelet aggregation, altered calcium handling and vascular smooth muscle cell (VSMC) proliferation. AGEs and ROS also modify low density lipoprotein (LDL), increasing uptake by macrophages and contributing to the formation of plaque. Modified with permission from reference 160

Modification of enzymes

Numerous enzymes are involved in cellular regulatory functions, and oxidative modification of these enzymes may result in adverse effects contributing to the development and progression of hypertension and atherosclerosis (Figure 3). As discussed previously, GPx and GRed are two key antioxidant enzymes that protect cells from oxidative damage. These enzymes themselves have been shown to be susceptible to oxidative modification with loss of activity (71). This would result in a reduction in antioxidant defence and a perpetuation of oxidative stress. In both in vitro and in vivo studies of SHRs, which are models of human essential hypertension, there are increased ROS levels in vascular tissue, with a decrease in GSH levels and antioxidant enzyme activity (41,44). Increased ROS levels and decreased antioxidant capacity are documented in essential hypertensive humans, from juvenile to elderly people (56,72–74).

As previously stated, the glycolytic enzyme GAPDH may play a key role in insulin resistance. Oxidative modification of this enzyme may exacerbate this condition, creating a biochemical loop that perpetuates an oxidant environment. O₂⁻-induced peptide and protein peroxides decrease free thiols of GAPDH and inhibit its activity (49). Similarly, incubation of GAPDH with the lipid peroxide product 4-hydroxy-2-nonenal (an aldehyde) decreases enzyme activity and triggers enzyme degradation (50).

Endothelial NOS (eNOS) is an important enzyme involved in regulating vascular tone. It acts on the substrate arginine to produce the vasodilatory molecule NO. eNOS is an SH-dependent enzyme with a cysteine residue (C184) at its active site. Alterations to this residue result in loss of catalytic activity (75). Oxidation of eNOS cofactor tetrahydrobiopterin causes the uncoupling of eNOS, with a decreased formation of NO and an increase in O₂⁻ production (7,8,76). NO is not only a potent vasodilator, but it also inhibits platelet aggregation, VSMC migration and proliferation, monocyte adhesion and adhesion molecule expression (13). ROS act directly on NO to produce the potent radicals ONOO⁻ and peroxynitric acid (3), which have been implicated in tissue injury and protein dysfunction (64,77,78). Decreasing NO bioavailability, either through a decrease in eNOS or NO, while at the same time producing ROS, leads to the endothelial dysfunction (76,77) that is seen in hypertension and atherosclerosis (76–78).

Protein tyrosine phosphatases are enzymes that have cysteine residues at their active sites. Their function is to regulate another group of enzymes called protein tyrosine kinases (PTKs) via phosphorylation/dephosphorylation. PTKs are involved in initiating cell signalling pathways that regulate cell growth and inflammatory responses. Oxidative modification of protein tyrosine phosphatase results in its inhibition, with an increase in the activity of PTKs. This leads to abnormal intracellular signal transduction that results in cellular responses characteristic of hypertension and atherosclerosis, including vascular cell proliferation, apoptosis and contraction, and proinflammatory processes (65,70,79).

ROS act on yet another enzyme, angiotensin-converting enzyme (ACE), to increase catalytic activity, resulting in an increase in angiotensin II (AII) production (80). AII is a potent vasoconstrictor implicated in the pathogenesis of hypertension and atherosclerosis. AII binds to its type 1 receptor, which results in an increase in contraction, hypertrophy, proliferation and apoptosis. These abnormalities may be due to activation of receptor and nonreceptor kinases, as described above. AII stimulation of its type 1 receptor also increases the production of O₂⁻ by the enzyme NADPH oxidase (6,79). This enzyme is implicated as a major source of oxidative stress in cardiovascular disease (6,81–83). ROS from this source may also activate intracellular nonreceptor kinases and contribute to abnormal cell signalling (79). NADPH oxidase is also activated by another ROS, H₂O₂, enhancing the production of O₂⁻ and contributing to vascular injury (84). AII-induced oxidative stress (via NADPH oxidase) may be responsible, at least in part, for the decrease in NO bioavailability and the subsequent endothelial dysfunction found in hypertension and atherosclerosis (82,85).

Alteration of cytosolic free calcium in VSMC

Calcium is essential to normal cell homeostasis, regulating contraction in VSMCs, transcription activities and expression of cell growth factors. Its levels within the cytoplasm and intracellular organelles are controlled by various means, including membrane ion channels, transporters and ATPase pumps. ROS have been shown to increase intracellular calcium levels due to either increased transport from outside the cell or release from intracellular stores (Figure 3) (86,87). Vascular membrane calcium channels are dependent on -SH groups for normal function (86), and it has been suggested that oxidation of these groups, resulting in a cross-linkage reaction, keeps the channels in an open state that allows for an increased influx of calcium into the cytosol (68). Oxidative modification of cysteine and lysine of Ca²⁺-ATPase leads to inhibition of this enzyme (63,64,88), causing increased levels of cytosolic free calcium. Additionally, dysfunction of the Ca²⁺-ATPase-activating protein calmodulin may contribute to an increase in intracellular calcium (89).

In rat mesangial cells, AGEs increase oxidative stress and intracellular calcium, causing a cytosol-to-membrane translocation of protein kinase C (beta II), an enzyme isoform associated with proliferation and matrix deposition (47). ROS, in particular O₂⁻, inhibit Ca2+-ATPase of the VSMC sarcoplasmic reticulum (88). This results in the release of calcium from the sarcoplasmic reticulum, which initiates signal transduction processes. H₂O₂ increases cytosolic free calcium and contractile responses in VSMCs of SHRs. This effect appears to be the result of an increased influx of extracellular calcium via upregulated calcium channels (87). Increased cytosolic calcium also triggers an increase in ROS (90), which may result in a perpetuation of oxidative stress and cellular damage. Thus, ROS, by causing dysregulation of membrane calcium transport, lead to an increase in cytosolic free calcium, peripheral vascular resistance, cell proliferation and apoptosis, as well as inflammation and a further increase in oxidative stress.

Oxidation of circulating and structural proteins

ROS oxidize lipid and protein components of LDL. Oxidized LDL initiates inflammatory processes that result in platelet aggregation and attraction of monocytes to vessel walls, apoptosis and proliferation of VSMCs (Figure 3) (3,15,17,91). Physical or biochemical activation of membrane NADPH oxidase may increase ROS concentration in close proximity to the vascular endothelium, increasing the possibility of oxidative damage at this critical site (6,81,82,84,92). This may be a source of the endothelial damage that is thought to precede formation of atherosclerotic lesions. Peroxidation of membrane lipids and oxidation of proteins may alter the integrity of the endothelial lining, allowing migration of the monocytes into the intima, where they differentiate into macrophages (1,69). Oxidized LDLs act as ligands to scavenger receptors of these macrophages, stimulating LDL uptake and the formation of foam cells. Oxidized LDL is a main component in atherosclerotic lesions (3,15), and individuals with cardiovascular disease and hypertension show an increase in plasma very low density lipoprotein and LDL peroxides, with an increase in the susceptibility of LDL to oxidation (93–95). ROS production by NADPH oxidase is increased in hypertensive patients and those with coronary artery disease (81,82).

HDL is known to have protective effects in cardiovascular disease (96). Paraoxonase, an antioxidant enzyme closely associated with HDL, may contribute to this effect. It protects both HDL and LDL from oxidation and lipid peroxidation (97,98), and low HDL and paraoxonase are associated with oxidative stress, hypertension and coronary artery disease (99–101). Overexpression of human paraoxonase-1 inhibits atherosclerosis in a murine model (102). Blockage of free -SH groups (cysteine residue) of paraoxonase reduces its ability to protect LDL from oxidation (98), suggesting a role for oxidative modification of this enzyme.

Thus, ROS have the ability to damage the endothelium, oxidize LDL and set off a cascade of aggregatory and inflammatory processes. They also decrease the bioavailability of NO, increase cytosolic free calcium, and cause a series of proliferative and apoptotic events in VSMCs. These alterations are characteristic of hypertension and atherosclerosis. Because oxidation may contribute to many of the vascular changes seen in these two diseases, antioxidants should prevent or attenuate these adverse effects.

MODULATION OF OXIDATIVE STRESS WITH ANTIOXIDANTS

Mechanism of antioxidant action

Antioxidants have direct and indirect actions (Figure 4). Direct action involves agents that have the ability to reduce oxidized substances. They include vitamins such as vitamins E and C, and thiol compounds including cysteine, GSH and dihydrolipoic acid, which are able to directly neutralize ROS and lipid peroxides. Polyphenols, components of fruits and wines, and some antihypertensive and antilipidemic agents, such as ACE inhibitors and statins, also have direct antioxidant activity (103–105).

Figure 4).

Antioxidants work directly to neutralize reactive oxygen species (ROS). They also work indirectly to decrease ROS formation by improving insulin resistance and decreasing aldehyde and advanced glycation end product (AGE) formation. Reduction of oxidative stress prevents oxidative damage to lipids and proteins, attenuating the progression of hypertension and atherosclerosis

When it comes to their reducing potential, not all antioxidants are the same. Due to chemical configurations, some antioxidants are more potent than others (Table 1). Not only are antioxidants with high reducing potential more effective against oxidative stress, as discussed earlier, they also regenerate antioxidants of lesser reducing potential. This is essential to prevent the accumulation of vitamin radicals and maintain normal physiological antioxidant capacity (Figure 1) (10). Some antioxidants also act to spare other antioxidants. Supplementation with lipoic acid or vitamin E increases the levels of the endogenous antioxidant GSH (10,106). Because GSH is a powerful ubiquitous antioxidant, increasing its level boosts total antioxidant capacity.

The water or lipid solubility of antioxidants affects their sites of action. For example, lipoic acid is both lipid and water soluble. It is converted intracellularly to dihydrolipoic acid, which readily moves outside the cell and, therefore, is capable of fending off oxidative stress in both the intra- and extracellular compartments (10). Vitamin E is lipophilic, which makes it ideally suited to protect endothelial membrane lipids from oxidative damage. Because vitamin E is the most abundant antioxidant in LDL, it is essential in maintaining this lipoprotein in a reduced state (3). CoQ10 is also a lipid-soluble antioxidant that is widely distributed in biological membranes. CoQ10 spares vitamin E when both are present in the same liposomal membranes (107) and protects against lipid peroxidation more effectively than vitamin E (108). Vitamin C is a water-soluble antioxidant. Its presence within the plasma and its higher reducing potential enables vitamin C to keep LDL-associated vitamin E in a reduced form (109). It is also able to counteract oxidative stress throughout the vasculature. A suitable balance of antioxidants in vivo is likely important in combating oxidative stress wherever it is generated in the body.

In addition to their direct neutralizing properties, some antioxidants have alternate properties that allow them to reduce oxidative stress indirectly. By addressing the sources of oxidative stress, these agents can prevent the formation of ROS and lipid peroxides. For example, some antioxidants reduce insulin resistance, the defect we believe to be a major cause of oxidative stress, as well as the primary etiological factor in hypertension and atherosclerosis. One such antioxidant, lipoic acid, has strong direct antioxidant potential, but also functions as a cofactor of key mitochondrial enzyme complexes that control glucose oxidation, such as pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase (10). Lipoic acid improves glucose metabolism and insulin resistance in diabetic animal models, type II diabetic patients and hypertensive rat models (110–114). N-acetylcysteine (NAC) improves insulin sensitivity in women with polycystic ovarian syndrome, another insulin-resistant state. This action may be due to a protective effect on insulin receptors (115).

Vitamins E and C also improve the action of insulin. These vitamins increase GSH levels and decrease the oxidized glutathione to GSH ratio, which is suggested to improve the physical state of membranes and related glucose transporter activity (116,117). Additionally, lipoic acid increases the conversion of cystine to cysteine, and NAC is a cysteine analogue that, when given orally, increases tissue cysteine. Cystine/cysteine is a relatively powerful redox couple (Table 1), and cysteine has the capacity to bind reactive aldehydes for excretion. Dihydrolipoic acid (lipoic acid [reduced form]) contains two -SH groups, and may act similarly to cysteine to bind aldehydes directly and promote excretion. Lipoic acid, NAC, and vitamins C and E all increase GSH levels, which aids in methylglyoxal breakdown via the glyoxalase enzyme system (10,106,118,119). These alternate functions of antioxidants decrease insulin resistance, lower levels of aldehydes and AGEs, and may prevent AGE-induced oxidative stress. These latter functions are also substantiated by studies using other AGE-lowering agents, such as pyridoxamine, which demonstrate that lowering levels of AGEs results in a decrease in oxidative stress and oxidative damage (33,112,120).

Some pharmaceutical agents exhibit antioxidant activity in addition to their primary action. Although some thiol-containing ACE inhibitors show direct antioxidant activity (104), nonthiol inhibitors act indirectly. Through their primary antihypertensive activity of inhibiting ACE activity, these inhibitors secondarily decreae AII-stimulated NADPH oxidase-induced oxidative stress (121). Other antihypertensive medications, such as calcium channel blockers, also reduce oxidative stress. This may be an indirect effect due to either a decrease in calcium-induced ROS production in the cytosol or the attenuation of pressure-induced activation of NADPH oxidase secondary to a blood pressure-lowering effect (122,123). Statins, a group of antilipidemic agents, also have direct and indirect antioxidant properties (124).

Modulation of oxidative stress-induced changes in hypertension and atherosclerosis by antioxidants

There is no doubt that oxidative stress is associated with hypertension and atherosclerosis (1–4). We have discussed the ability of ROS to cause oxidative damage, a characteristic of both of these disease conditions. It is logical that the modulation of oxidative stress through the use of antioxidants would ameliorate oxidative damage, and attenuate high blood pressure and the clinical end points of atherosclerosis (angina, MI, stroke and renal artery stenosis).

In vitro and ex vivo studies using antioxidants demonstrate that they can effectively reduce oxidative stress and its damage (10,125–130), but are they able to exert the same beneficial effects in vivo? The protective effect of individual antioxidants and antioxidant combinations has been investigated in humans and animals. Animal studies of spontaneous hypertension, and sugar- and salt-induced hypertension, show that supplementation with antioxidants, including vitamins E and C, lipoic acid, NAC and CoQ10, decreases oxidative stress (112,113,118,131–134), normalizes calcium handling (135–137), decreases insulin resistance (112,113,134), attenuates changes to renal vascular morphology (111,132,135–137), improves renal and endothelial function (131,133,138,139), and decreases blood pressure (112,118,131,133,134,139–142).

Similarly, in animal models of atherosclerosis, treatment with antioxidants shows beneficial effects, such as a decrease in oxidative stress (127,143,144), a decrease in total cholesterol (145) with suppression of LDL oxidation, inhibition of foam cell formation, attenuation of atherosclerotic lesions (146–148), improvement in cardiac performance (127,149) and a decrease in microvascular changes (150). Because clinical end points are not usually evaluated in experimental studies, little information exists on this aspect of antiatherosclerotic benefit in animals. Contrarily, limitations of human investigations make it difficult to study oxidative stress-induced alterations and assess antioxidant effects at the cellular level. In humans, some biochemical indexes, functional improvements and reductions in clinical end points can be evaluated, and these are the usual methods employed to assess effect. Studies in humans have observed that low plasma levels of antioxidants are associated with increased levels of blood pressure (53,55,56). Most controlled trials of the antihypertensive effects of antioxidants have not measured the existing or post-treatment levels of oxidative stress, nor do they consider the possible antioxidant effect of concomitant antihypertensive medications. Despite these omissions, there is strong evidence that antioxidants exert antihypertensive effects. Because antioxidant compounds may have other mechanisms of antihypertensive action (eg, decreases in insulin resistance and AGEs), it is difficult to estimate how much of the antihypertensive effect is from antioxidant action. Whatever the mechanism, several studies in humans show that antioxidants, including vitamin E, vitamin C, lipoic acid, NAC and CoQ10, lower blood pressure (140,151–154).

On the other hand, the evidence supporting the beneficial effect of antioxidants in the treatment of atherosclerosis in humans is somewhat less conclusive. Low plasma levels of antioxidants correlate with an increased incidence of cardiovascular disease (54,57,58), and some studies have shown that supplementation with antioxidants lowers the risk of total and cardiovascular mortality, and decreases the risk of cardiovascular disease and nonfatal MIs (155–157). However, other studies show no cardiovascular benefit (158,159). This lack of demonstrable benefit may have to do with factors such as the nature of the atherosclerotic lesion and the degree of damage already incurred, or which antioxidants were used and in what combinations. If atherosclerosis is already well established, antioxidant therapy may prevent further damage from ROS, but may not be able to completely restore the vasculature to a normal state. Our earlier discussion highlighted the importance of an appropriate antioxidant balance to ensure adequate regeneration of vitamin radicals to nonradicals. Most cardiovascular studies have used high doses of single vitamins or combinations of two or three vitamins. These antioxidant choices may not have been ideal and may be yet another reason for the inconclusive results in these studies. Recent suggestions that vitamin E supplementation may be detrimental to cardiovascular health may be an example of this. Supplementation with vitamin E in high doses without the benefit of other regenerating antioxidants in the diet could conceivably result in damage from vitamin E radicals. In addition, studies using more than one antioxidant may not have included both lipid- and water-soluble choices, or antioxidants with a wide range of reducing potentials. Because in vivo studies in animals show a benefit in atherosclerosis, it may be a matter of choosing appropriate antioxidants and instituting therapy at an early stage as a preventative measure to more clearly demonstrate a benefit in humans.

SUMMARY

Genetic and dietary factors can lead to insulin resistance and AGE formation, which creates a situation in which ROS production is increased and/or antioxidant capacity is decreased. This results in an imbalance that leads to oxidative stress. ROS have the ability to damage the endothelium, oxidize LDL, and set off a cascade of aggregatory and inflammatory processes. They also decrease the bioavailability of NO, increase cytosolic free calcium, and cause a series of proliferative and apoptotic events in VSMCs. These alterations are characteristic of hypertension and atherosclerosis. There is strong evidence in animal studies that antioxidants modulate oxidative damage, lower blood pressure and attenuate atherosclerosis. Antioxidants show antihypertensive effects in humans, with some success in improving the end points of atherosclerosis. Because some antioxidants must be obtained in the diet (eg, vitamins C and E), the importance of a well-balanced diet that is low in fat and includes an abundance of fruits and vegetables, with adequate amounts of proteins (the Dietary Approaches to Stop Hypertension [DASH] diet), must be emphasized. In conditions in which there is chronic oxidative stress, endogenous antioxidants may become depleted and increased intake may be needed to meet requirements. When considering antioxidant supplementation, it may be as important to consider how antioxidants work with each other as it is to contemplate their mechanism of action with regard to the disease state. Using a supplement that contains sufficient amounts of water- and lipid-soluble vitamins, with a wide range of reducing potentials, may be more effective in the treatment of hypertension and atherosclerosis. It may be more appropriate to supplement with antioxidant combinations that include agents (eg, lipoic acid) that are capable of improving insulin resistance, lowering AGEs and preventing oxidative stress. In cases in which antihypertensive or antilipidemic pharmaceuticals must be prescribed, it may be reasonable to choose agents with antioxidant properties.