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Department of Clinical Nutrition, Sun Yat-sen University, Guangzhou 510080, China
2To whom correspondence should be addressed. E-mail: whling{at}gzsums.edu.cn.
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONLITERATURE CITED |
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B and formation of nitrotyrosine in the aorta were greater in rats with mild hyperhomocysteinemia than in control rats. Serum levels of malonyldialdehyde (MDA) were higher in mildly hyperhomocysteinemic rats than in control rats. These results suggest that the oxidative stress resulting from elevated plasma homocysteine stimulates the activation of nuclear factor B, and consequently increases the expression of the inflammatory factors in vivo. Such an effect may contribute to atherogenesis by enhancing the inflammatory response of the vascular endothelium.
KEY WORDS: • atherosclerosis • homocysteine • inflammatory factors • nuclear factor B • oxidative stress
Atherosclerosis (AS)3 is a complex multifactorial process and a chronic low-level inflammatory disorder resulting from an excessive inflammatory response to various forms of injurious stimuli to the artery wall. Experimentally confirmed risk factors for cardiovascular disease do not fully account for its widespread prevalence (1). For example, classic risk factors including family history, hypercholesterolemia, male gender, physical inactivity, obesity, and smoking account for only 50 to 70% of the actual risk of cardiovascular disease (2). Therefore, the potential contributions of other variables have come under scrutiny.
Homocysteine (Hcy), a sulfur-containing amino acid, is an intermediate metabolite of the essential amino acid methionine. Hyperhomocysteinemia (HHcy), which refers to total plasma Hcy above 15 µmol/L, is caused by an abnormality in either an enzyme (cystathionine ß-synthetase or temperature-sensitive methylenetetrahydrofolate reductase) or a cofactor (folate, vitamin B-12, or vitamin B-6) required for Hcy metabolism. Moderate HHcy (15 to 30 µmol/L) is most commonly caused by vitamin-B deficiency, especially deficiencies in folic acid, vitamin B-6, and vitamin B-12 (3). Genetic factors, certain drugs, and renal impairment may also contribute (3).
Hyperhomocysteinemia is now regarded as one of the important risk factors for cardiovascular and cerebral vascular disorders (4). Elevated plasma Hcy levels occur in a large proportion of patients with coronary artery disease (5). Several plausible mechanisms for Hcy-induced AS have been proposed, including endothelial dysfunction (6), impaired flow-mediated vasodilation (7), increased proliferation of vascular smooth muscle cells (8), and enhanced coagulability (9).
An inflammatory lesion, consisting only of monocyte-derived macrophages and T lymphocytes, is considered the earliest stage of atherosclerotic lesion (10). The inflammatory molecules, including monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and E-selectin, have been proposed as key elements in this inflammatory response. A few recent in vitro studies indicate that Hcy affects the expression of some inflammatory factors. Studies report that Hcy increases the expression of MCP-1 in both human aortic endothelial cells (11) and monocytes (12). Chronic exposure to Hcy also increases the production of ICAM-1 in endothelial cells (13). These results suggest that the atherogenic effects of Hcy result at least in part from its inflammatory effects.
Most of the studies that have elucidated the mechanism of the adverse effects of Hcy on the vascular endothelium used in vitro experiments and short-term in vivo experiments with HHcy (9,14–16), but the molecular pathogenesis underlying the effects of high Hcy levels is not yet fully defined (11,14). Wang et al. (17) reported that rats fed a high-methionine diet (17 g/kg) for 4 wk developed HHcy, and that HHcy stimulated the endothelial expression of MCP-1, VCAM-1, and E-selectin, causing increased adhesion of monocytes to the aortic endothelium and facilitating the inflammatory response in the early stages of AS. Because the progression of AS is a chronic and long-term process, short-term studies of HHcy cannot elucidate the atherosclerotic changes in the vascular endothelium that are related to HHcy. A study of the effects of long-term HHcy on the aortic inflammatory response is needed to clarify the role of HHcy in the progression of AS. Therefore, the present study tested the hypothesis that long-term elevation of plasma Hcy levels induced by the ingestion of a diet either enriched in L-methionine or deficient in folate could cause an inflammatory response in the aorta in rats.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONLITERATURE CITED |
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Plasma Hcy.
The concentration of total plasma Hcy was analyzed by HPLC (19). Briefly, 100 µL of plasma was added to 10 µL of 0.345 mol/L tri-n-butylphosphine (Sigma) in dimethyl formamide (Sigma). Then, 100 µL of 0.5 mol/L perchloric acid containing 0.5 mmol/L EDTA-Na2 was added. The mixture was centrifuged at 4000 x g for 10 min. To 30 µL of the supernatant, a mixture consisting of 15 µL of 0.5 mol/L NaOH and 30 µL of 7-fluorbenzo-2-oxa-1,3-diazle-4-sulfonic acid (SBD-F; Sigma) [4.25 mmol/L in 0.1 mol/L borate buffer (pH 9.5) containing 2 mmol/L of EDTA] was added. A 10-µL sample of the supernatant was analyzed by HPLC, using an HP 1050LC series liquid chromatograph and workstation and an F1046 fluorescence detector (excitation 385 nm, emission 515 nm; Hewlett-Packard). Separation was carried out using a reversed-phase column (C18 BDS, 150 x 4.6 mm; Hypersil). Analysis was performed under isocratic conditions (40 mmol/L ammonium formate and 30 mmol/L ammonium nitrate buffer with 5% acetonitrile, pH3.5) at a flow rate of 1 mL/min for 15 min. The concentration of plasma Hcy was calculated using Hcy as an external standard. The retention time for Hcy was 
3 min.
Serum folate. Serum folate concentration was analyzed at the Sun Yat-sen University Radioimmunoassay Center, using a commercially available radioimmunoassay kit (folate radioimmunoassay kit from Radioimmunoassay Center of Peking) (20).
Serum nitrite. Serum nitrite concentration was measured as an indicator of NO production, using the Griess reaction (9). A 100-µL sample of serum was mixed with the same volume of Griess reagent (58 mmol/L sulfanilamide in 0.5 mol/L phosphoric acid and 3.85 mmol/L naphthylethylenediamine dihydrochloride in water). The absorbance at 550 nm was assayed using sodium nitrite as the calibration standard.
Aorta nitric oxide synthase (NOS) activity. Each abdominal aorta segment (100 g/L) was homogenized in a solution containing 0.01 mol/L Tris-HCl, 0.1 mmol/L EDTA-Na2, 0.01 mol/L sucrose, and 0.137 mol/L NaCl (pH 7.4). The homogenate was centrifuged at 4000 x g for 15 min at 4°C, and the supernatant was used to assay the activity of NOS. The protein concentration was analyzed using the Bradford method. Under NOS catalysis, NO is produced from L-arginine, and the coloring agent is produced when NO combines with nucleophilic substances. The absorbance at 530 nm was determined to calculate the activity of NOS. The assay was performed using a commercially available kit (NOS activity determination kit, Jiancheng Biotechnology) (21). The activity of NOS was expressed as enzyme activity units/mg protein.
Immunohistochemistry of nitrotyrosine. Peroxynitrite production was assayed by the presence of the stable end product of its interaction with tyrosine residues, nitrotyrosine (22). Paraffin-embedded aortic sections were deparaffinized and incubated with 0.882 mol/L H2O2 for 10 min to quench the endogenous peroxides. The sections were incubated with a blocking solution of nonimmune goat serum for 15 min at room temperature, then with mouse antirat nitrotyrosine monoclonal antibody (Cayman) diluted 1:200 with PBS for antibody dilution for 1 h at 37°C. After washing with PBS, a biotinylated rabbit antimouse secondary antibody solution (Santa Cruz Biotechnology) was added, and the sections were incubated for 20 min at 37°C. Sections were then washed and incubated with horseradish peroxidase-conjugated streptavidin for 15 min at 37°C, stained with diaminobenzidine (DAB), and counterstained with hematoxylin. The presence of nitrotyrosine was detected by DAB staining. The specificity of the immunolabeling was evaluated by omitting the primary antibody in control experiments. Imaging analysis was used as an objective and semiquantitative measure of the presence of nitrotyrosine in the aortic sections (23).
Serum MCP-1. Serum MCP-1 concentration was analyzed using a Rat MCP-1 detector kit (Pierce-Endogen). The analysis was conducted according to the manual provided with the kit (24). Briefly, diluted (1:25) serum samples were added to a 96-well plate precoated with anti-MCP-1 antibody. After incubating for 1 h at room temperature, the plate was washed 3 times. Biotinylated antibody reagent was added to each well, and the plate was again incubated for 1 h. Then streptavidin-horseradish peroxidase (HRP) solution was added to each well, and the plate was incubated for 30 min at room temperature. After washing, tetramethyl benzidine (TMB) substrate solution was added, and the enzymatic color reaction was developed in darkness at room temperature for 30 min without covering the plate. The reaction was terminated by adding the stop solution. Absorbance was detected at 450 nm, using a Titertek ELISA reader (Bio-TEK Instruments), and the results were expressed as absorbance at 450 nm.
ICAM-1 protein. Thoracic aorta tissue stored at -80°C was homogenized in ice-cold buffer as described by Jung et al. (25). The homogenates of aorta tissue were centrifuged at 12,000 x g for 10 min, and the supernatant was collected and stored at -20°C for analysis. The total protein concentration was analyzed using the Bradford method. Proteins from each study group (60 µg/lane) were mixed with sample buffer [60 mmol/L Tris-HCl, 25% glycerol, 0.087 mol/L sodium dodecylsulfate (SDS), 14.4 mmol/L ß-mercaptoethanol, and 1.49 mmol/L bromophenol blue] at a 4:1 ratio(v/v) of sample to buffer and heated at 95°C for 5 min. Electrophoresis was carried out on 8% polyacrylamide minigels (Bio-Rad Laboratories). Protein was electrophoretically transferred from the gel to a nitrocellulose membrane. The membranes were blocked with Tris-buffer saline with Tween (TBST; 0.15 mol/L sodium chloride, 20 mmol/L Tris-HCl, and 1 g/L Tween; pH 7.0) containing 50 g/L of nonfat milk, then incubated with a mouse antirat ICAM-1 monoclonal antibody (Santa Cruz Biotechnology) diluted 1:200 with TBST. Immunoreactive bands were detected using the enhanced chemiluminescence method with an enhanced chemiluminescence kit (Santa Cruz Biotechnology). The membranes in this assay were also stripped (26) via 30 min of incubation with a stripping buffer containing 62.5 mmol/L Tris base (pH 6.8), 100 mmol/L ß-mercaptoethanol, and 0.087 mol/L SDS at 55°C, and were then blocked with TBST solution containing 50 g/L of nonfat milk at room temperature. The stripped membranes were then probed at 4°C overnight with anti-ß-actin antibody diluted 1:500 with TBST, and were then processed as ICAM-1.
Aorta nuclear factor B (NF-B) p65 protein.
Pure nuclear proteins were extracted according to the method of Basheer et al. (27) with minor modifications. In brief, the frozen aortic tissue (the ascending aorta and aortic arch segment) was homogenized in 4 x tissue volume of cold buffer A [0.5 mol/L sucrose, 10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl, 10% glycerol, 1 mmol/L EDTA, and 1 mmol/L dithiothreitol (DTT)] with the addition of protease inhibitors, 1 mmol/L phenyl-methylsulfonyl fluoride and 0.308 µmol/L aprotinin and 0.0047 mmol/L leupeptin (final concentrations). Homogenates were transferred into Eppendorf tubes, and the cells were allowed to swell on ice for 15 min. Nonidet-P40 was added to the tubes for 0.6% final concentration. The mixture was centrifuged at 2000 x g for 10 min at 4°C. The pellet was resuspended in 1 x tissue volume of cold buffer C [20 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 0.3 mol/L NaCl, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, and 20% glycerol], the protease inhibitors were added as described above, and the samples were incubated for 30 min. After centrifuging at 20,000 x g for 15 min at 4°C, the supernatant, containing mainly nuclear protein, was stored at -80°C. Protein concentrations were analyzed using the Bradford method. The procedure for assaying NF-B p65 concentration by Western blotting was similar to that for ICAM-1. The amount of nuclear proteins from each study group was 60 µg/lane. Rabbit antirat NF-B p65 polyclonal antibody (Santa Cruz Biotechnology) was diluted 1:200 before incubation.
Lipid peroxidation. Lipid peroxdation was quantified by measuring the serum concentration of malonydialdehyde (MDA), a product of lipid peroxidation, as previously described by Huang et al. (28). The reaction agent contained 3 g/L of SDS, 0.1 mol/L HCl, 10 g/L of phosphotungstic acid, and 0.7 g/L of 2-thiobarbituric acid. The sample mixture was incubated for 45 min at 95°C, and the TBARS produced by the reaction were extracted in 2.5 mL of L-butanol. After centrifugation at 1000 x g for 10 min, the fluorescence of the butanol layer was measured using an Hitachi F-3010 fluorescence spectrophotometer (Hitachi) at 555 nm emission and 515 nm excitation. Tetraethoxypropane was used as a standard.
Statistical analysis. Results are presented as means ± SD. Data were analyzed by one-way ANOVA and a post-hoc least significant difference (LSD)-t multiple comparison test. Differences were considered significant at P < 0.05.
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RESULTS |
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Aorta nitrotyrosine formation. Immunostaining of aortic tissue with an antinitrotyrosine antibody showed greater staining in the smooth muscle cells (in media). There was no evidence of nonspecific immunostaining (Fig. 1). Image analysis showed greater staining for nitrotyrosine in the 2 hyperhomocysteinemic groups, compared to the control group (P < 0.05) (Fig. 1).
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B p65 activation.
There were clear protein bands at 65 kDa in the lanes loaded with samples from rats with mild HHcy; however, the band was almost undetectable in samples from the control rats (Fig. 3). These data suggest that elevated plasma Hcy concentration activates NF-B.
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONLITERATURE CITED |
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We found that 2 hyperhomocysteinemic diets significantly increased plasma Hcy concentrations in rats, compared to the control diet. Mild HHcy (plasma Hcy increased to 2–3 times baseline levels) was induced by feeding rats the ME or FD diets. The magnitude of increase in plasma Hcy induced by a diet rich in methionine or deficient in folate differed in several previous reports. Feeding a diet supplemented with methionine (12 g/kg) to rats for 4 wk increased plasma Hcy 2- to 3-fold (28). Feeding a diet deficient in folate for 4 or 8 wk caused a 2-fold increase in plasma Hcy in rats (29,30). However, Wang et al. (17) reported that a high-methionine diet (17 g/kg) for 4 wk increased plasma Hcy levels in rats 3- to 4-fold. Because there is no information available on the effects of long-term feeding (14 wk) of folate-deficient or methionine-enriched diets on plasma Hcy levels in rats, it is hard to compare the present results with those reported elsewhere. We assume that diets high in methionine or deficient in folate affect Hcy metabolism. Rowling et al. (31) reported that high L-methionine (5 to 10 g/kg) supplementation for 10 d upregulated hepatic glycine N-methyltransferase. Long-term feeding of diets rich in methionine or deficient in folate may alter the metabolic pathway of Hcy, such as by upregulating the conversion of Hcy back to methionine or cysteine, leading to the mild increase in plasma Hcy levels reported in the present study. The body weight gain was less in the rats fed the ME and FD diets from wk 13 to 14 of feeding period. This may also partly affect the magnitude of increase in the plasma Hcy levels. However, the mechanism underlying this increase needs further investigation.
Both high plasma Hcy and low serum folate concentrations may be related to the early processes of AS. Low levels of folate are associated with both moderate HHcy and carotid artery stenosis in humans, particularly in elderly populations (32). Because the ME diet caused higher expression of adhesion molecules and positive formation of nitrotyrosine in rats that had high serum Hcy and adequate serum folate levels, it is possible that the vascular inflammatory response was mainly caused by HHcy, not by decreased serum folate. It is also possible that other features of the modified diet (such as methionine enrichment) may be important in altering the vascular function.
Intracellular adhesion molecule-1 is an adhesion molecule, primarily from endothelial cells (ECs), that is thought to be involved in the firm adhesion step in leukocyte infiltration (33). Monocyte chemoattractant protein-1 is a potent chemoattractant protein that stimulates the migration of monocytes into the intima of the arterial walls in the inflammatory response (12). One of the important early features of AS is monocyte infiltration into the injured arterial wall, followed by differentiation into macrophages. These macrophages then take up large amounts of lipids and become foam cells. In vitro cell culture studies have reported that Hcy increased the production of inflammatory factors such as ICAM-1 and MCP-1 (13,34). Wang et al. (17) reported that elevated plasma Hcy levels stimulated the expression of chemokine (MCP-1) and adhesion (VCAM-1), but not ICAM-1 and P-selectin. The authors hypothesized that ICAM-1 might be involved in the later stages of AS, that is, after the stage marked by MCP-1, VCAM-1, and E-selectin in hyperhomocysteinemic rats (16). The present study proved that elevated Hcy levels induce the production of MCP-1 and ICAM-1 in rats fed a high methionine diet for 14 wk, further supporting the hypothesis that ICAM-1 is involved in the later stages of AS.
Oxidative stress may contribute to the deleterious effects of Hcy. Homocysteine induces endothelial cell injury through an oxidant-mediated mechanism (35). Oxidative stress can stimulate the activation of NF-B, which plays a pivotal role in the regulation of many genes involved in the inflammatory response (36). Several lines of evidence indicate that NF-B may have an important role in AS by regulating or signaling the inflammation reaction (37,38). Various genes whose products are putatively involved in the atherosclerotic process are regulated by NF-B, such as VCAM-1, MCP-1, and so forth. In the present study, both oxidative stress (measured as serum MDA concentration) and the aortic nuclear protein NF-B p65 concentration were elevated in the groups with mild HHcy, indicating that the oxidative stress–NF-B pathway promotes the expression of MCP-1 and ICAM-1 in hyperhomocysteinemic rats.
Early AS processes may also be directly or indirectly caused by the disturbance of NO metabolism in rats with HHcy. Nitric oxide has antiadhesive properties that influence the interaction between leukocytes and endothelial cells, perhaps by interfering with the migration of monocytes and lymphocytes at the atherosclerotic site (39). Nitric oxide also contributes to the pathogenesis of inflammatory disorders (40), and it is induced in response to a variety of inflammatory cytokines, many of which are produced in the progression of AS. The reaction of NO with superoxide to form peroxynitrite is a major mechanism of endothelial dysfunction associated with the production of NO in vivo (41). Peroxynitrite has been hypothesized to be involved in the pathology of wide range of diseases, including AS (42). Nitrotyrosine, a stable marker of the production of reactive nitrogen-associated oxidants, is generated when peroxynitrite is added to tyrosine itself or to proteins containing tyrosine residues (42).
Earlier studies in vitro showed that Hcy treatment of vessel rings or endothelial cells reduces the biological activity of NO. The mechanism of this reduction is related to the generation of hydrogen peroxide and the impairment of the antioxidative ability of ECs to eliminate H2O2 (30,43). In the present study, the production of NO did not differ among the control and hyperhomocysteinemic groups, but oxidative stress (measured as serum MDA concentration) increased in the groups with HHcy. The elevated plasma Hcy level caused the production of reactive oxygen species, which interacted with NO to form peroxynitrite, as indicated by the overproduction of nitrotyrosine, a stable biomarker of peroxynitrite formation. The subsequent loss of NO bioavailability may be linked to endothelial inflammation and thus promote the development of AS. The increased formation of nitrotyrosine in the aortic tissue of rats with HHcy suggests that HHcy reduces the bioavailability of NO by increasing the production of peroxynitrite or nitrotyrosine.
In summary, mild HHcy induced by the ME or FD diet was positively related to the elevation of oxidative stress as indicated by serum MDA concentration and aortic nitrotyrosine formation, which in turn stimulated NF-B activation. Consequently, we concluded that HHcy contributes to the early stages of the development of AS by increasing the expression of vascular inflammatory molecules (such as MCP-1 and ICAM-1) and decreasing the availability of NO. Further studies are required to evaluate whether long-term HHcy induced by FD or ME diets cause histological atherosclerotic lesions of the aortic tissue in addition to the changes in vascular inflammatory molecules.
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FOOTNOTES |
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3 Abbreviations used: AS, atherosclerosis; DTT, dithiothreitol; ECs, endothelial cells; FD, folate-deficient; Hcy, homocysteine; HHcy, hyperhomocysteinemia; ICAM-1, intracellular adhesion molecule-1; MCP-1, monocyte chemoattractant protein-1; MDA, malonyldialdehyde; ME, methionine-enriched; NOS, nitric oxide synthase; SDS, sodium dodecylsulfate; TBST, Tris-buffer saline with Tween; VCAM-1, vascular cell adhesion molecule-1.
Manuscript received 9 December 3003. Initial review completed 29 December 2003. Revision accepted 16 January 2004.
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LITERATURE CITED |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONLITERATURE CITED |
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