биохимия атеросклероза

Introduction

Hyperhomocysteinemia, a condition of elevated blood levels of homocysteine (Hcy), is regarded as one of the common and independent risk factors for atherosclerosis [1–12]. Atherosclerosis is the principal contributor to the pathogenesis of myocardial and cerebral infarctions, which are the leading causes of mortality and morbidity in many countries. Abnormal elevations of Hcy levels, up to 100–250 µM in the blood, have been reported in patients with hyperhomocysteinemia [2, 5, 11]. Many factors may regulate plasma levels of Hcy. For example, severe hyperhomocysteinemia seen in children is usually the result of rare homozygous deficiency of enzymes necessary for Hcy metabolism [11]. Moderate increases in blood Hcy levels occur more frequently and are often found in patients with heterozygous enzyme deficiency, folate or pyridoxine (vitamin B6) deficiency, impaired renal function, as well as in elderly people and in postmenopausal women [6, 7, 11].

Metabolism of Homocysteine

Hcy is a sulfur-containing amino acid formed during the conversion of methionine to cysteine. Plasma Hcy is found primarily in three molecular forms, namely Hcy, disulfide Hcy, and the mixed disulfide Hcy–cysteine [7, 11]. Majority of Hcy molecules in the circulation are in the oxidized and the protein-bound form. Reduced or free Hcy (nonprotein-bound form) constitutes about 1% of the total Hcy level in the blood. The normal range of total Hcy (sum of all forms of Hcy) in adults is 5–15 µM, with a mean level of 10 µM. Hyperhomocysteinemia refers to the total plasma Hcy level above 15 µM nuclear factor-κB (NF-κB) [6, 7, 11]. The cellular homeostasis of Hcy is tightly regulated under normal conditions [1]. Hcy can be metabolized by two major pathways: (1) the transulfuration pathway to form cysteine, which requires vitamin B6 as a cofactor and (2) the remethylation pathway to form methionine, which requires methyltetrahydrofolate as a cosubstrate and vitamin B12 as a cofactor (Fig. 17.1). Factors that perturb steps in Hcy metabolic pathways can cause an increase in its cellular levels and lead to hyperhomocysteinemia (plasma Hcy level higher than 15 µM). Abnormal elevations of Hcy levels up to 100–250 µM in the blood have been reported in patients with hyperhomocysteinemia [8]. Severe hyperhomocysteinemia seen in children is usually the result of a rare homozygous deficiency of enzymes necessary for Hcy catabolism. Moderate increases in blood Hcy levels occur frequently and are often seen in patients with heterozygous enzyme deficiency, impaired kidney function, deficiency of folate, vitamin B6, or vitamin B12, in elderly people, etc. [2, 5, 11, 13]. There is an inverse correlation between plasma levels of Hcy and kidney function [13].

Cysteine

FIGURE 17.1. Homocysteine metabolic pathways. The remethylation pathway and the transsulfuration pathway are the two major pathways for homocysteine metabolism. Reactions involved in these two pathways are catalyzed by the following enzymes: (1) methionine adenosyltransferase; (2) S-adenosylmethionine-dependent methyltransferase; (3) S-adenosyl homocysteine hydrolase; (4) betaine-homocysteine methyltransferase; (5) serine hydroxymethyltransferase; (6) 5,10-methylenetetrahydrofolate reductase; (7) 5-methyltetrahydrofolate-homocysteine methyltransferase; (8) cystathionine β-synthase; and (9) γ-cystathionase.

Hyperhomocysteinemia and Cardiovascular Disorders

Although the precise molecular mechanisms responsible for the pathogenicity of hyperhomocysteinemia remain uncertain, several potential mechanisms have been proposed. These include endothelial dysfunction [10, 14, 15], increased proliferation of smooth muscle cells [16–18], enhanced coagulability [18], and increased cholesterol biosynthesis in hepatocytes [19–21]. Endothelial injury and dysfunction are considered to be one of the leading mechanisms contributing to atherogenesis. Upon injury, endothelial cells are capable of producing various cytokines and growth factors that in turn participate in the development of atherosclerotic lesions.

Hyperhomocysteinemia and Endothelial Function

It has been proposed that Hcy-caused endothelial injury may be due to oxidative stress, attenuation of nitric oxide-mediated vasodilatation, and disturbances in the antithrombotic activities of the endothelium [20–28]. This topic was recently reviewed by Austin et al. [10]. Several animal models with hyperhomocysteinemia have been developed in monkeys [26], apoE-null mice [27], cystathionine β-synthase (CBS)-deficient mice [23], and high-methion- ine fed rats [28]. In diet-induced moderate hyperhomocysteinemic monkeys, Lentz et al. [26] noticed increased platelet-mediated vasoconstriction, impaired endothelium-dependent vasodilatation, and decreased thrombo- modulin-dependent activation of protein C, when compared to that of monkeys fed a normal diet. In the apoE-null mice with dietary-induced hyperhomocysteinemia, Hofmann et al. [27] reported a twofold increase in the aortic root lesion size. These mice also had significantly elevated levels of vascular cell adhesion molecule-1 (VCAM-1) and tumor necrosis factor-α (TNF-α). An impaired endothelium-dependent vasodilatation function, likely due to diminished nitric oxide bioactivity was observed in the CBSdeficient mice, which had impaired Hcy metabolism [23]. These studies indicate that Hcy may enhance vascular inflammation and endothelial dysfunction in animals that are prone to the development of atherosclerosis. In our recent study, hyperhomocysteinemia was induced in rats that were fed a high-methionine diet for 4 weeks [28]. In this animal model, serum Hcy level was 4–5-fold higher than that of control rats. In aortic rings isolated from hyperhomocysteinemic rats, endothelium-dependent vessel relaxation was impaired while endothelium-independent vessel relaxation was not affected. This study clearly demonstrates that in the absence of other risk factors, hyperhomocysteinemia alone is able to cause endothelial dysfunction. Taken together, results obtained from various animal models indicate that Hcy, at pathological concentrations, can act synergistically with other risk factors as well as act independently causing endothelial dysfunction.

Oxidative Stress and Activation of NF-κB

Many risk factors causing atherosclerosis share a common feature of generating intracellular oxidative stress [15, 29–34]. It has been suggested that generation of reactive oxygen species (ROS) is responsible for Hcy-induced cell injury [15, 29, 33, 34]. ROS are often generated in cells as by-products in many metabolic and signal transduction pathways [32]. Under normal conditions, small amount of ROS generated inside cells can be scavenged by cellular antioxidant defense mechanism. For example, superoxide anions generated can be scavenged by superoxide dismutase to form oxygen and hydrogen peroxide, the latter is decomposed by catalase and glutathione

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peroxidase [22, 35–37]. However, ROS scavengers would no longer be sufficient for their removal when ROS are above a certain level. As a result, ROS accumulate inside cells and oxidative stress occurs. The major ROS include superoxide anion, hydrogen peroxide, hydroxyl radical, and peroxynitrite, which are formed during the sequential reduction of oxygen. Overproduction of these free radicals can induce oxidation of DNA, proteins, and lipids resulting in mutations, loss of enzymatic activities, and alteration of lipid functions. The initial product of ROS is superoxide anion. Many in vitro studies demonstrated that Hcy was able to stimulate intracellular superoxide anion generations in vascular cells as well as in other types of cells [10, 38–40]. We observed that Hcy, at higher concentrations (50–200 µM), caused an increase in intracellular superoxide anion levels in vascular endothelial cells [38], in vascular smooth muscle cells [41], and in monocytes/macrophages [39, 40]. As a consequence, there was a marked increase in the expression of inflammatory genes such as monocyte chemoattractant protein-1 (MCP-1), chemokine receptor (CCR2), and inducible nitric oxide synthase (iNOS) in these cells [39–41]. Pretreatment of cells with cell-permeable polyethylene glycol-bound superoxide dismutase, a known superoxide scavenger, reversed Hcy-induced elevation of superoxide anion levels in endothelial cells, vascular smooth muscle cells, and monocytes/macrophages. The polyethylene glycol-bound superoxide dismutase treatment also abolished Hcy-induced mRNA expression of MCP-1, CCR2, and iNOS. These results suggest that oxidative stress contributes to inflammatory responses elicited by Hcy in vascular cells.

The expression of inflammatory factors is mainly controlled by transcription factors such as NF-κB. NF-κB plays an important role in the expression of inflammatory factors in the vasculature [42–44]. Oxidative stress can activate this transcription factor leading to upregulating the expression of its target genes [39, 41–45]. Our laboratory reported that Hcy treatment could activate NF-κB in vascular cells as well as in macrophages via superoxide anion generation [39]. In resting cells, NF-κB is normally present in the cytoplasm in an inactive form associated with an inhibitory protein (IκB) [45–49]. IκB-α is one of the best-characterized forms of IκB proteins. Upon stimulation, there is a rapid phosphorylation of IκB-α and subsequent degradation of IκB-α by the proteasome, leading to the release of NF-κB. After dissociation from IκB, the activated NF-κB is translocated into the nucleus where it binds to the κB binding motifs in the promoters or enhancers of the genes encoding cytokines. Activated NF-κB (found in the nucleus) has been detected in macrophages, endothelial cells, and smooth muscle cells of human atherosclerotic lesions [43, 44, 46]. In contrast, little or no activation of NF-κB is found in normal arterial walls. In diet-induced hyperhomocysteinemic rat, we observed a marked elevation of superoxide anion level and the presence of activated NF-κB in the aorta while little superoxide anion and activated NF-κB were detected in the control aorta [38]. Several lines of evidence indicated that NF-κB was activated in endothelial cells upon Hcy treatment. First, nuclear translocation of NF-κB occurred in endothelial cells

after incubation with Hcy for 15–30 min. Second, the results from electrophoretic mobility shift assay demonstrated that Hcy treatment caused a significant increase in the NF-κB/DNA binding activity. Third, results from transient transfection demonstrated an enhanced NF-κB-regulated transcriptional activity in Hcy-treated cells. Further investigation revealed that oxidative stress and subsequent activation of IκB kinases (IKK-α and IKK-β) are essential for Hcy-induced activation of NF-κB in endothelial cells [38]. Such a mechanism may regulate the inflammatory response in the vascular wall during the early stage of atherosclerosis in hyperhomocysteinemia. ROS have been implicated to stimulate IκB-α degradation and NF-κB activation in vascular cells [50]. It was reported that hydrogen peroxide stimulated NF- κB activity via activation of IKK-α and IKK-β, which were kinases that phosphorylate IκB protein, in HeLa cells [51]. Antioxidants were shown to be able to block IκB-α degradation and NF-κB activation [52, 53].

Homocysteine and Chemokine Expression in Vascular

Cells

One of the important features during the early stage of atherogenesis is monocyte adhesion to the injured arterial endothelium followed by their differentiation into macrophages. Macrophages are able to uptake large amounts of lipids, particularly cholesterol from oxidized lipoproteins contributing to lipid accumulation in the atherosclerotic lesion [54–57]. MCP-1 is a potent chemokine that stimulates monocyte migration into the intima of arterial walls [53–57]. The amount of this chemokine appears to be increased in atherosclerotic lesions in human and in experimental animals [28, 54–57]. The expression of MCP-1 and other inflammatory factors (i.e., adhesion molecules) in atherosclerotic lesions can be upregulated by NF-κB. Our recent studies demonstrated that Hcy, at pathological concentrations, stimulated the expression of MCP-1 mRNA in cultured endothelial cells [58, 59], in vascular smooth muscle cells [41], and in macrophages [60]. The experimental results suggest that NF-κB activation and protein kinase signaling pathways may play important roles in Hcy-induced MCP-1 expression. Elevated MCP-1 production by these cells, in turn, stimulated monocyte chemotaxis in vitro [41, 58–60]. Pretreatment of cells with NF-κB inhibitors could alleviate the stimulatory effect of Hcy on MCP-1 expression, supporting the notion that Hcy-stimulated chemokine expression was mediated via NF-κB activation [41, 60]. It is generally believed that endothelial expression of MCP-1 initiates the migration of monocytes into the arterial wall. Based on results obtained from our laboratory [41, 60, 61] as well as from other investigators, we speculate that Hcy-induced endothelial MCP-1 expression may be associated with early development of atherosclerosis by stimulating monocyte migration into the subendothelial space and differentiation into macrophages. On the other hand, MCP-1 produced in smooth muscle cells as

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well as in macrophages may facilitate the recruitment of additional monocytes into the lesion at later stages of atherosclerosis in patients with hyperhomocysteinemia. MCP-1 exerts its action mainly through the interaction with the CCR2 on the surface of monocytes. We observed that Hcy was able to stimulate CCR2 expression in human peripheral blood monocytes as well as in THP-1 cells (derived from human monocytic cell line) [40]. We hypothesize that Hcy-induced MCP-1 expression in vascular cells, together with enhanced CCR2 expression in peripheral blood monocytes, may represent a mechanism for monocyte/macrophage accumulation in the arterial wall during atherogenesis. Indeed, we observed that Hcy-treated monocytes/ macrophages as well as endothelial cells were able to take up oxidized LDL causing intracellular lipid accumulation [61]. We also examined the in vivo effect of Hcy on MCP-1 expression leading to monocyte adhesion to the endothelium [28]. Male Sprague–Dawley rats developed hyperhomocysteinemia after being fed a high-methionine diet (regular chow plus 1.7% methionine, wt/wt) for 4 weeks. In diet-induced hyperhomocysteinemic rats, the number of monocytes present on the surface of the aortic endothelium was significantly elevated when compared to the control rats. There was a significant increase in the expression of MCP-1 protein in the endothelium. Further analysis revealed that the expression of adhesion molecules such as VCAM-1 and E-selectin was also significantly elevated in the aortic endothelium of hyperhomocysteinemic rats. Pretreatment with specific antibodies against MCP-1, VCAM, or E-selectin could block monocyte binding to the aortic endothelium of hyperhomocysteinemic rats. These results indicated that elevation of MCP-1 and adhesion molecules in vascular cells was responsible for enhanced monocyte adhesion to the endothelium. Increased monocyte/macrophage binding and adhesion to the vascular endothelium may represent an early feature of atherosclerotic development in hyperhomocysteinemia [12, 28]. In the same animal model, we also observed that hyperhomocysteinemia was associated with reduced endothelium-dependent vessel relaxation [28]. These findings suggest that hyperhomocysteinemia can act independently in the development of vascular dysfunction. Folic acid supplementation to rats fed a high-methionine diet prevented an elevation of MCP-1, VCAM-1, and E-selectin expression in rat aortic endothelium.

Hyperhomocysteinemia as a Risk Factor for Other Disorders

Although hyperhomocysteinemia is regarded as an independent risk factor for atherosclerosis, elevation of plasma Hcy levels is often associated with diseases that involve other organs. Hyperhomocysteinemia has been indicated as a potential risk factor for Alzheimer’s disease [62–65], osteoporosis/hip fracture [66, 67], liver dysfunction [11, 14, 21, 68, 69], and kidney injury [11, 14, 70–73].

Homocysteine and Hepatic Lipid Metabolism

A positive correlation between the plasma concentrations of Hcy and cholesterol was observed in patients with hyperhomocysteinemia [11, 68]. Over three decades ago, McCully [14] reported postmortem observation of extensive arteriosclerosis in two children with severe hyperhomocysteinemia and proposed a pathogenic link between elevated blood Hcy levels and atherogenesis. In subsequent studies, a correlation between the plasma levels of Hcy and cholesterol was found in patients [68]. Abnormal lipid metabolism was also found in hyperhomocysteinemic animal models [20, 21]. In hyperhomocysteinemic mice caused by CBS deficiency, there was excessive accumulation of lipid droplets in hepatocytes due to increased endoplasmic reticulum stress [21]. In a recent study, ethanol was shown to induce fatty liver and apoptosis in mice through Hcy-induced endoplasmic reticulum stress [74]. We previously reported that Hcy stimulated the production and secretion of cholesterol in human hepatoma cells (HepG2) via activation of 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase [19]. Subsequent study revealed an activation of HMG-CoA reductase in the liver of hyperhomocysteinemic rat [20]. In dietinduced hyperhomocysteinemic rats, there were accumulation of hepatic lipids and elevation of serum cholesterol contents due to increased HMG-CoA reductase gene expression in the liver. The HMG-CoA reductase is the ratelimiting enzyme, catalyzing the conversion of HMG-CoA to mevalonate, in cholesterol biosynthesis [75, 76]. The activity of this enzyme is regulated by several mechanisms including transcriptional regulation, posttranslational modification, allosteric regulation, and levels of endogenous and exogenous cholesterol [77–79]. Sterol regulatory element binding proteins (SREBPs), a subfamily of basic helix-loop-helix zipper proteins, are important transcription factors regulating lipid homeostasis [80–82]. The SREPB-2 preferentially activates enzymes involved in cholesterol production such as HMG-CoA reductase [80, 81]. Two other transcription factors, namely, cAMP response element binding protein and nuclear factor Y, have been identified as important SREBP-2 coregulators and act synergistically for upregulation of HMG-CoA reductase gene expression [82–85]. In the rat model, further investigation revealed that hyperhomocysteinemia could upregulate HMG-CoA reductase gene expression in the liver via the activation of transcription factors (SREBP- 2, cAMP response element binding protein, nuclear factor Y) in hepatocytes [20]. Abnormal cholesterol metabolism can cause serious complications including hepatic fatty infiltration, which can progress to fibrosis and cirrhosis leading to liver failure [19–21, 86, 87]. There were small lipid droplets accumulated in the liver of hyperhomocysteinemic rat, a condition assembled as fatty liver [20, 21]. We postulated that the ability of Hcy to promote cholesterol biosynthesis in hepatocytes is a mechanism of hyperhomocysteinemia-associated liver pathology. Although hyperhomocysteinemia is regarded as an independent risk factor for the development of atherosclerosis, a high blood level of Hcy is often associated with other risk factors. In the Hordaland Study [68], it was found

organs. Biochemical and molecular mechanisms by which Hcy, at an elevated level, affects various organs remains to be further investigated. The knowledge obtained will provide a scientific basis for prevention and therapeutic intervention of patients with cardiovascular disorders associated with hyperhomocysteinemia or with other risk factors.

Acknowledgment: The work was supported, in part, by Heart and Stroke Foundation of Manitoba, Canadian Institute of Health Research, and Natural Sciences and Engineering Research Council. Grant.M.Hatch is a Canada Research Chair in Molecular Cardiolipin Metabolism.

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