биохимия атеросклероза

310 Sudesh Vasdev and Vicki Gill

functional changes [44, 52–55]. These conjugates are also known as AGEs and some have been identified and linked to complications of diabetes [52, 55, 56]. Aldehyde conjugates/ AGEs have also been shown to act on receptors of AGEs (RAGEs) and various scavenger receptors to influence protein function and expression [57–59]. Several aldehyde conjugates/AGEs including carboxymethyl-lysine, carboxyethyl-lysine, argpyrimidine, and glycoalde- hyde-pyridine, have been identified, and have been implicated in hypertension and atherosclerosis [27, 60–63].

Under normal physiological conditions, methylglyoxal is formed but 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 [49]. It has been suggested that aldehyde conjugates/AGEs formed under these conditions contribute to the regulation of normal tissue remodeling [64]. However, when there is an excess of methylglyoxal and other aldehydes, more conjugates/AGEs are formed leading to altered structure and function of tissue proteins. High levels of methylglyoxal also lead to a depletion of cysteine and glutathione resulting in increased oxidative stress.

Oxidative stress exists when there is an imbalance between the level of reactive oxygen species (ROS) and the body’s antioxidant capacity. ROS include single electron oxidants such as superoxide radicals (O2−) and hydroxyl radical (.OH), and the two-electron oxidants hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and peroxynitrite [65]. Under normal conditions, ROS play a role in several processes; for example, they act as signaling molecules in regulating VSMC contraction–relaxation and VSMC growth [66]. However, excess ROS have the capacity to exert toxic effects. They can lead to a further formation of aldehydes including malondialdehyde and hydroxynonenal, through lipid peroxidation [49, 66–71]. These lipid-derived aldehydes have been shown to react with cysteine or lysine residues of proteins to form a type of AGE known as advanced lipoxidation end products (ALEs) [50, 72–74]. The myeloperoxidase system produces HOCl, which has been shown to form the AGE, glycoaldehyde pyridine, found in atherosclerotic lesions [27]. ROS also oxidize sugars, proteins, and nucleic acids leading to membrane dysfunction, tissue remodeling, enzyme inhibition, and alteration of gene expression [66, 75]. Levels of ROS are controlled by antioxidants or antioxidant enzymes, which neutralize or scavenge them. The antioxidant enzyme glutathione reductase acts to ensure that reduced glutathione is available to neutralize O2-. Another antioxidant enzyme, glutathione peroxidase, scavenges H2O2, preventing its reduction to .OH [66]. Endogenous antioxidants such as glutathione, and antioxidant nutrients like vitamin C and vitamin E, lipoic acid, and coenzyme Q10, also function to control oxidative stress [21]. There is strong evidence that oxidative stress contributes to the progression of essential hypertension and the development of its complications including atherosclerosis [5, 66, 75–79]. For the purpose of uniformity, we will use the term AGEs throughout this chapter to denote aldehyde conjugates, AGEs, and ALEs.

Causative Role of AGEs in Hypertension and

Atherosclerosis

We have shown that methylglyoxal given in the diet to Wistar-Kyoto (WKY) rats increased tissue AGEs and caused hypertension [80] (Fig. 14.3). Levels of tissue methylglyoxal and AGEs are higher in spontaneously hypertensive rats and sugar-induced hypertensive rats [22, 23, 62, 81, 82]. Although research on AGEs in human essential hypertension is scant, in preeclampsia, a hypertensive condition of pregnancy, RAGE expression was increased in vascular tissue [83]. There is strong evidence in human diabetes, another insulin resistance state, that AGEs are responsible for cellular protein modifications, which contribute to diabetic complications [52, 55, 56, 84–86]. AGE-mediated cross-links in collagen and elastin, also contribute to arterial stiffening, hindering vessel elasticity, and exacerbating hypertension [87]. Treatments which lower AGEs also lower the blood pressure [23, 81, 82, 88].

There is increasing evidence of a causative role for AGEs in atherosclerosis either through a hypertension-mediated increase in oxidative stress, and/or directly via stimulation of various inflammatory processes. AGEs have the capacity to adversely influence the function and expression of many of the body’s proteins including calcium channels, metabolic, and antioxidant enzymes, receptors, and structural proteins. In the following section, we will discuss these adverse effects and their possible contribution to hypertension and atherosclerosis (Table 14.2).

AGEs and Increased Oxidative Stress

In addition to depleting glutathione, methylglyoxal, and AGEs may affect antioxidant enzyme activity. The antioxidant enzymes, glutathione peroxidase and glutathione reductase contain SH and NH2 groups at their active sites [89, 90]. Formation of AGEs at these sites may result in inhibition of these enzymes. In rat VSMCs in culture, methylglyoxal inhibited these enzymes, leading to oxidative stress, low levels of reduced glutathione and increased levels of oxidized glutathione [22]. Vascular ROS are produced in endothelial, adventitial, and VSMCs by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [66]. AGEs have been shown to activate RAGEs resulting in a NADPH oxidase-mediated increase in intracellular reactive oxygen intermediates and extracellular H2O2 [59]. AGEmodified bovine serum albumin increased O2− production in human platelets in vitro [91]. As discussed above, increased oxidative stress promotes further formation of aldehydes and thus AGEs. This creates a cycle where AGEs and ROS perpetuate a prooxidant state, which contributes to the development and progress of vascular disease.

Methylglyoxal

FIGURE 14.3. (a) The line graph shows the effect of N-acetylcysteine (NAC) on systolic blood pressure in methylglyoxal treated Wistar-Kyoto (WKY) rats. Starting at 7 weeks of age, WKY rats were divided into three groups of six animals each. Animals in the WKY-Control group were given normal diet and normal drinking water; WKYMethylglyoxal, regular diet, and methylglyoxal in drinking water; WKYMethylglyoxal+NAC, 1.5% NAC in diet and methylglyoxal in drinking water for the next 18 weeks. Methylglyoxal in drinking water was given at a concentration of 0.2% during weeks 0–5; 0.4%, weeks 6–10; and 0.8%, weeks 11–18. Values are mean of six animals in each group for each week. Standard deviation of mean was not more than 6 mm Hg in all cases. (b) The bar graph shows the effect of NAC on platelet [Ca2+]i, kidney aldehyde conjugates and circulating nitric oxide in methylglyoxal treated WKY rats. Experimental groups and treatment period were the same as in Figure 14.3a. Values are expressed as a percentage of the control group values at the completion of the study, age 25 weeks.

314 Sudesh Vasdev and Vicki Gill

AGEs and Increased Cytosolic Free Calcium

Vascular calcium channels are dependent on SH groups for normal function [49, 92] and their alteration can lead to increased cytosolic free calcium [Ca2+]i, abnormal contractile activity, and increased peripheral vascular resistance. AGEs impaired type 2 ryanodine receptor calcium release channels (calcium receptors which regulate cardiac contractility) during chronic diabetes [93]. ROS have been shown to increase intracellular calcium in VSMC and endothelial cells [66, 94]. Oxidized LDL was shown to enhance vasoconstriction likely via increased calcium influx into VSMCs [95]. AGEmodified human serum albumin (AGE-HSA) increased ROS and intracellular calcium in neonatal mesengial cells [96]. AGE-HSA also acts via RAGEs to produce ROS and increase the release of calcium from intracellular stores in human neutrophils [97]. In essential, human hypertensives and hypertensive animals, cytosolic [Ca2+]i levels in VSMCs are elevated [98, 99]. We have shown elevated vascular tissue AGEs and increased platelet [Ca2+]i in spontaneously hypertensive rats, a genetic model of hypertension, and in methylglyoxal and fructose-treated WKY rats, dietary models of hypertension [80–82] (Fig. 14.3). In addition to its role in vascular contractility and blood pressure homeostasis, calcium is a major determinant of platelet activation and aggregation, and increased levels may also contribute to enhanced atherogenic conditions [100].

AGEs and Endothelial Dysfunction

Maintaining normal endothelial function is essential to blood pressure homeostasis and vessel integrity. One of the major factors involved in regulation of endothelial function is NO. Endothelium-derived NO is not only a potent vasodilator but also inhibits platelet aggregation, VSMC migration and proliferation, monocyte adhesion, and adhesion molecule expression, thus regulating blood pressure and protecting vascular function [101]. Therefore, abnormalities in NO bioavailability impair endothelial function. This has been demonstrated in both hypertension and atherosclerosis [4, 102].

NO is synthesized from arginine by the thiol-containing endothelial nitric oxide synthase (eNOS) and cofactor tetrahydrobiopterin (BH4), and its production is regulated in part by insulin acting on specific receptors on the cell surface [40, 103–105]. Thus, the ability to form adequate amounts of NO depends on the availability of arginine, active eNOS, and BH4, and appropriate insulin response. Excess methylglyoxal and other aldehydes may limit substrate by forming AGEs with arginine [84]. Methylglyoxal has been shown to react with arginine residues to form several AGEs including argpyrimidine, which has been demonstrated in human serum and kidney [63, 106, 107]. The importance of SH groups in normal catalytic activity of eNOS has been demonstrated [104] and corroborates evidence that alteration of cysteine residues (c184) of human eNOS results in loss of activity [108]. AGEs inhibit

eNOS activity and expression [109–111]. We have shown that methylglyoxal given in the diet to WKY rats increased tissue AGEs, decreased plasma NO, and caused hypertension [80] (Fig. 14.3). NOS activity requires the presence of BH4. It has been suggested that insulin stimulates BH4 synthesis and that this insulin action is impaired in insulin resistant states [102]. Also, oxidation of BH4 by ROS promotes uncoupling of eNOS therefore decreasing production of NO [102, 105]. Superoxide reacts with NO, also reducing bioavailability, forming peroxynitrite. Peroxynitrite and peroxynitrous acid are powerful and cytotoxic oxidants, also referred to as reactive nitrogen species, which may cause further damage to vascular tissue [112].

AGEs and Atherosclerotic Lesion Formation

Shear stress caused by elevated blood pressure results in endothelial injury enhancing formation of atherosclerotic plaques, particularly at junctions of vessels where turbulence is greatest. Subsequent oxidative stress will also contribute to this atherogenic environment. Oxidized LDL is recognized as a key component in atherosclerotic lesions and hypertensive subjects exhibit an enhanced susceptibility to LDL oxidation [13, 113]. AGEs may contribute to atherosclerosis in various ways. AGE-LDL caused cholesterol and cholesterol ester accumulation in macrophages in vitro [25] and has been identified in the cytoplasm of foam cells and extracellularly in the core of atherosclerotic lesions in humans and animals [14, 26, 27]. AGE-LDL and AGEmodified protein are ligands for scavenger receptors. Binding to these receptors leads to endocytic uptake of LDL and accumulation in human monocytes–macrophages [57, 114]. AGEs also bind to RAGEs generating a cascade of intracellular signals, which result in inflammatory responses including an increased expression of NF-κB, endothelin-1, vascular cell adhesion molecule-1, insulin-like growth factors, and interleukin-6. These changes result in monocyte recruitment and vascular adhesion, cell proliferation, and vasoconstriction [22, 58, 59, 64, 115, 116]. Endothelial growth factor receptor (EGFR) is involved in the regulation of multiple cellular processes such as cell growth, motility, differentiation, survival, and death. It is a common receptor shared by several growth factors. Methylglyoxal and glyoxal directly inhibit EGFR autophosphorylation and tyrosine kinase activity, by forming AGEs with the free NH2 group of EGFR protein thus impairing downstream signaling [117]. ROS may also affect this signaling pathway by oxidation of the cysteine residue of tyrosine phosphatase, thus influencing receptor protein tyrosine kinases including EGFR [66]. This AGE-induced alteration in signaling may play a role in the cellular changes associated with hypertension and atherosclerosis.

AGEs may contribute to platelet aggregation and thrombus formation. AGEs increased O2− production and aggregation in human platelets in vitro [91]. Alterations due to AGEs reduce NO bioavailability resulting in an increase in platelet aggregation. It has been suggested that AGEs stimulate

316 Sudesh Vasdev and Vicki Gill

externalization of phosphatidylserine, which activates clotting factors leading to platelet adhesion [118].

Antioxidants in the Treatment of Hypertension

and Atherosclerosis

We have presented evidence that insulin resistance leading to increased formation of AGEs and oxidative stress contributes to the etiology of essential hypertension and subsequent development of atherosclerosis. It is likely that by attenuating insulin resistance and decreasing levels of AGEs and ROS it should be possible to prevent or treat these diseases (Fig. 14.4). The protective effect of individual antioxidants and antioxidant combinations has been investigated in humans and animals. Supplementation with antioxidants including vitamin C, E, or B6, lipoic acid and coenzyme Q10 has been shown to lower blood pressure in animal models and humans with essential hypertension [21]. However, evidence supporting their efficacy in the treatment of atherosclerosis is somewhat less conclusive [119]. This may have less to do with the potential of these compounds to achieve antiatherosclerotic effects and more to do with factors such as the nature of the atherosclerotic lesion and degree of damage already incurred, and which antioxidants were used and in what combinations.

Cardiovascular research usually entails monitoring the effect of dietary intervention on the occurrence of hypertensive or atherosclerotic events such as myocardial infarction, stroke, and kidney failure. If subjects have been

Improve glucose metabolism and quench free radicals

Increase glutathione and antioxidant activity

Decrease aldehydes and AGE and ROS formation

Normalize cytosolic [ca2+]i and nitric oxide

Prevent hypertension and atherosclerosis

FIGURE 14.4. Mechanism of action of antioxidants for the prevention of hypertension and atherosclerosis

hypertensive for a number of years and atherosclerosis is well established, antioxidant therapy may prevent further damage from AGEs and ROS but may not be able to completely restore vasculature to normal. The nature of atherosclerotic plaque itself may not be conducive to physical infiltration or repair by antioxidants. Tissue proteins already altered by AGEs will require time to regenerate and some tissues have long turnover times.

Typically, studies of cardiovascular disease use either high doses of single antioxidants, for example vitamin E, or combinations including β-carotene or vitamin C. Many of these studies have given variable results. Recent discussions, which suggest that vitamin E supplementation may be detrimental to cardiovascular health, may be an example of this [120]. Most of these studies do not consider that antioxidants in their oxidized form have the capacity to act as radicals and that it is necessary to maintain an adequate antioxidant balance such that these radicals are regenerated back into their reduced states. Antioxidants have been shown to regenerate each other from their oxidized to reduced forms [121–123] (Fig. 14.5). For example, vitamin E radical (oxidized) is regenerated to vitamin E (reduced) by vitamin C (ascorbate) or coenzyme Q10 (reduced). These antioxidants are in turn regenerated by dihydrolipoic acid. Thus, 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 radical. When choosing antioxidant

Dehydroascorbate

ROOH

Glutathione

(oxidized)

Glutathione

(reduced)

Lipoic

acid

FIGURE 14.5. Regeneration of antioxidants in the body. Oxygen free radicals are quenched by antioxidants which themselves are oxidized. The oxidized antioxidant is regenerated to its original reduced form by another antioxidant of higher electronegativity. ROO−/ROOH (Free radical/non-radical); NAD+/NADH (Nicotinamide adenine dinucleotide oxidized/reduced).

318 Sudesh Vasdev and Vicki Gill

combinations for cardiovascular studies, it may be as important to consider how these antioxidants work with each other, as it is to contemplate their mechanism of action with regards to the disease state. Studies using combinations of standard or moderate doses of combinations of antioxidants may show better efficacy in the treatment of hypertension and atherosclerosis. Choosing an antioxidant with high electronegativity (i.e., more reducing power) such as lipoic acid as one of the component antioxidants may ensure that radicals created from those antioxidants of lower electronegativity will be regenerated (Table 14.3). Using a combination of antioxidants, which are effective against both oneand two-electron radicals, should confer protection against a broader spectrum of radical agents. Lipoic acid, as well as cysteine, also improve glucose metabolism and in addition have the capacity to bind aldehydes directly preventing AGE formation and subsequent increase in ROS, making these antioxidants a more versatile treatment [124].

Therapies targeting insulin resistance, AGEs and oxidative stress should be effective in the treatment of hypertension and atherosclerosis (Fig. 14.4). A first line approach to these vascular disorders would include preventative measures such as participating in moderate physical activity, and eating a well-balanced diet rich in fruits and vegetables, low in salt and sugar, with nuts and lean meats. Healthy lifestyle choices include not smoking and limiting alcohol intake. Since antioxidants including vitamin E, vitamin C, and lipoic acid have been shown to improve glucose metabolism, lower AGEs and oxidative stress, reduce inflammatory response, and prevent hypertension, they will likely prevent subsequent damage due to atherosclerosis [21, 125, 126]. Considering the greater likelihood of benefit than harm, we suggest that an antioxidant combination supplement, as an additive to a well-balanced diet, is appropriate for most adults as a preventative measure. Early screening to evaluate blood pressure, glucose, and lipid abnormalities will also allow timely diagnosis and increase the opportunity to limit tissue damage and halt or slow disease progress (Table 14.1).

TABLE 14.3. Redox potentials of antioxidants in mammalian oxidation systems (Redox pairs given at increasing electronegativity and antioxidant activity).

Conclusion

Hypertension and atherosclerosis share similar risk factor conditions and both involve structural and functional changes of the vasculature. Increased blood pressure contributes to atherosclerosis but it is likely that these two conditions also have a common biochemical causation. We suggest that AGEs, formed as a result of insulin resistance found in these two disorders, may be the common link. AGEs and subsequent oxidative stress have the capability to alter calcium channels, metabolic and antioxidant enzymes, receptors and structural proteins resulting in hypertension and atherosclerosis. We suggest that lifestyle choices and appropriate antioxidant supplements which attenuate insulin resistance, reduce the formation of aldehydes and AGEs and lower oxidative stress will prevent or attenuate these two vascular diseases.

Acknowledgments: We would like to thank the Canadian Institutes of Health Research Regional Partnership Program for their financial support.

References

1.World Hypertension League: Know your blood pressure. http://www. meduohio.edu/org/whl/know.html : 1, 2004.

2.World Health Organization: Global strategy on diet, physical activity and health: Chronic Disease. http:// www.who.int/dietphysicalactivity/publications/ facts/riskfactors/en/index/html :1–2, 2003.

3.Warnock DG, Textor SC: Core curriculum in Nephrology: hypertension. Am J Kidney Dis 44: 369–375, 2004.

4.Taddei S, Virdis D, Ghiadoni L, Salvetti G, Salvetti A: Endothelial dysfunction in hypertension. J Nephrol 13: 205–210, 2000.

5.Portaluppi F, Boari B, Manfredini R: Oxidative stress in essential hypertension. Curr Pharm Des 10: 1695–1698, 2004.

6.Resnick LM: Ionic basis of hypertension, insulin resistance, vascular disease, and related disorders. The mechanism of “syndrome X”. Am J Hypertens 6: 123S–134S, 1993.

7.Oshima T, Young EW: Systemic and cellular calcium metabolism and hypertension. Semin Nephrol 15: 496–503, 1995.

8.American Stroke Association: Heart disease and stroke statistics – 2005 update, http://www.americanheart.org/downloadable/heart/1105390918119HDSStats 2005Update.pdf: 1–63, 2005.

9.Collins R, Peto R, MacMahon S, Hebert P, Fiebach NH, Eberland KA, Godwin J, Qizilbash N, Taylor JO, Hennekens CH: Blood pressure, stroke and coronary heart disease. Part 2, Short-term reductions in blood pressure:overview of randomised drug trials in their epidemiological context. Lancet 335: 827–838, 1990.

10.World Health Organization: World Health Report. Neglected Global Epidemics: Three growing threats. http://www.who.int/whr/2003/chapter6/en/ : 1–5, 2003.

11.Ross R: Atherosclerosis—an inflammatory disease. N Engl J Med 340: 115–126, 1999.