effect of hyperhomocysteinemia on cardiovascular risk factors and initiation of atherosclerosis in...

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logy 574 (2007) 49–60www.elsevier.com/locate/ejphar

European Journal of Pharmaco

Effect of hyperhomocysteinemia on cardiovascular risk factors and initiationof atherosclerosis in Wistar rats

Meenakshi Sharma ⁎, Santosh Kumar Rai, Manisha Tiwari ⁎, Ramesh Chandra

Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110 007, India

Received 20 April 2007; received in revised form 22 June 2007; accepted 4 July 2007Available online 21 July 2007

Abstract

Hyperhomocysteinemia is considered an independent risk factor for atherosclerosis. The present study was designed to assess the effect ofhigh level of serum homocysteine on other cardiovascular risk factors and markers in rats and to study its mode of action in initiatingatherosclerosis. To address this issue, four different doses of methionine (0.1 g/kg, 0.25 g/kg, 0.5 g/kg, 1 g/kg) were orally administered to fourgroups (Group II, III, IV, V respectively) of rats (6 rats in each group) for a period of 8 weeks to get different level of homocysteine in serum.Group I was administered with saline and served as control. Our results revealed that the level of Total cholesterol, Triglyceride, and Oxidizedlow-density lipoproteins increased significantly with the increase in the level of serum homocysteine. The levels of Resistin, C-reactive proteinand cysteinyl–leukotrienes were found to be significantly high in Group IV (Pb0.001 vs Group I) and Group V (Pb0.001 vs Group I) at8 weeks. Total antioxidant capacity and nitrite/nitrate level in serum showed negative correlation with the increased dose of methionine. ThemRNA expression and the enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase significantly increased only inlivers of rats of Group V. Furthermore, high mRNA expression of P2 receptors and caveolin were found in aorta of rats administered with highdose of methionine (Group IV and V at 8 weeks). Data obtained from in-vitro effect of homocysteine on isolated aortic arch also showedinduction in P2 receptors and caveolin with the increase in the concentration of homocysteine. These findings collectively suggest thathyperhomocysteinemia initiates atherosclerosis by modulating the cholesterol biosynthesis and by significantly inducing the level of othercardiovascular risk factors and markers, which play important role in initiating atherosclerosis.© 2007 Elsevier B.V. All rights reserved.

Keywords: Hyperhomocysteinemia; Atherosclerosis; HMG-CoA reductase; P2 receptors; Caveolin

1. Introduction

Elevated plasma concentration of homocysteine, an interme-diate compound derived by de-methylation of methionine, hasemerged as an independent risk factor in the development ofatherosclerosis. Up to 40% of patients diagnosed with prematurecoronary artery cerebrovascular or peripheral vascular disease arereported to have hyperhomocysteinemia. (Refsum et al., 2001).

Every 3 μmol/l plasma increase in homocysteine levelcontributes to a 10% increased risk of stroke (Malinow et al.,1995). The studies by Hofmann and Zhou represent a majoradvance in the understanding of the pathophysiology ofhyperhomocysteinemia (Hofmann et al., 2001; Zhou et al.,

⁎ Corresponding authors. Tel.: +91 11 27666272.E-mail addresses: [email protected] (M. Sharma),

[email protected] (M. Tiwari).

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.07.022

2001). The mechanism by which hyperhomocysteinemiaincreases risk for adverse cardiovascular outcomes, is not fullyunderstood. There is however, an abundant of evidence to suggestthat Hyperhomocysteinemia produces functional abnormalities ofblood vessels and impair the endothelium-dependent vasomotorresponses (Lentz, 2001).

So far, many studies have been performed, describing themechanism of action of homocysteine in initiating atheroscle-rosis by administering methionine to rats, but the correlation ofthe Hyperhomocysteinemia with other cardiovascular riskfactors and markers has never been studied. This particularstudy was designed to study somemajor facets of homocysteine-induced atherosclerosis and its correlation with the other majorcardiovascular risk factors and markers. Varying doses ofmethionine were administered to Wistar rats and subsequentlythe homocysteine levels and lipid profile were assayed in theserum of treated rats. To analyse the effect of methionine

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http://dx.doi.org/10.1016/j.ejphar.2007.07.022

50 M. Sharma et al. / European Journal of Pharmacology 574 (2007) 49–60

treatment on cholesterol biosynthesis, the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in cholesterol biosynthesis, was investigatedwith the assessment of mRNA and protein expression in liver.Further, the total nitrates and nitrites, which are stable nitricoxide metabolites were measured in the serum along with totalantioxidant capacity to evaluate the status of oxidative stress inrats administered with methionine.

Biomarkers that integrate metabolic and inflammatory signalsare attractive candidates for defining the risk of atheroscleroticcardiovascular disease (Reilly et al., 2005). High levels of resistin(a plasma protein) (Reilly et al., 2005), C-reactive protein (Hanet al., 2004) and cysteinyl–leukotrienes (Spanbroek et al., 2003)are important markers of inflammation and are predictive ofcoronary atherosclerosis in humans. We aimed to investigatewhether Hyperhomocysteinemia induces any effect on the levelof these immunological cardiovascular markers.

Receptors for extracellular nucleotides, the P2 receptors, havebeen recognized as fundamental modulators of cardiovasculareffects such as positive inotropic effects in the heart, plateletaggregation, and release of endothelial factors and growthstimulation of vascular smooth muscle cells, vasomotor effectsand blood pressure regulation (Virgilio and Solini., 2002). Wetried to analyse whether Hyperhomocysteinemia was able to alterthe mRNA expression of P2X and P2Y receptors and hence havean influence on the initiation of atherosclerosis in rats.

Scientists have recently shown that along with various otherfactors, the protein caveolin plays an important role in theregulation of the cardiovascular system. Caveolin is the principalstructural protein of caveolae and is involved in signal trans-duction and lipid transport. A number of findings have suggestedthat the expression of caveolin represents a mechanism by whichfree cholesterol efflux can bemodulated in response to changes incellular cholesterol content. Alteration in caveolin abundanceor subcellular location may lead endothelial cells to favorone mode of regulation over the other and thereby alter thesubtle equilibrium governing nitric oxide production in these cells(Gargalovic and Dory, 2003). Therefore, in the present study weexamined whether Hyperhomocysteinemia also produces someeffect on the mRNA expression of caveolin and disturbs theequilibrium of endothelium ultimately leads to atherosclerosis.

An in-vitro aorta organ culture assay was performed to studythe effect of different concentration of homocysteine on themRNA expression of caveolin and P2 receptors for thecorroborative purposes in the isolated aorta.

Collectively, in the present investigation our aim was toconfirm the finding that hyperhomocysteinemia upregulates thecholesterol biosynthesis and also tried to investigate the effectof high serum homocysteine level on the level of other stimuliinvolved in initiation of atherosclerosis.

2. Methods

2.1. Animals and experimental treatment

All procedures and experimental protocols were approvedby the Institutional Ethical Committee of the Dr. B. R.

Ambedkar Center for Biomedical Research, University ofDelhi. The animal house of the institute is registered forBreeding and Experiments on animals with Committee for thePurpose of Control and Supervision of Experiments onAnimals (CPCSEA), Animal Welfare Division, and Govern-ment of India. Adult male rats of Wistar strain weighing 100–120 g were used in the investigation. Animals were maintainedin an air-conditioned room with, free access to food and water.The room was maintained at 25±2 °C with natural daytime andno light after 1900 h, until morning. Rats were divided into fivegroups of six animals each. The animals were fed with GoldMohar rat feed supplied by Brooke Bond India Ltd. Rats wereassigned to five groups: Group I, Saline fed rats; Group II, III,IV and V were Orally administered with 0.1 g/kg, 0.25 g/kg,0.5 g/kg, 1 g/kg dose of methionine(dissolved in salinesolution) respectively. Blood samples were collected fromretro-orbital plexus for measurement of serum homocysteine,total cholesterol, triglyceride, high density lipoprotein (HDL),resistin, C-reactive protein and cysteinyl–leukotrienes at time 0and after 4 and 8 weeks. The rats were fed their respective dietsfor 8 weeks. Weights of the rats were recorded at 0, 4 and8 weeks.

2.2. Tissue preparation

After a 12 h overnight starvation, animals were anaesthetizedat about 1000 h to minimize the diurnal variation duringcholesterol synthesis after overnight starvation (Jurevics et al.,2000). After dissection, the liver and thoracic aortae werecollected, rinsed in ice-cold solution and aliquoted forpreparation of total RNA extraction. Hepatic microsomeswere prepared by ultracentrifugation. Liver samples werefrozen at −80 °C for further biochemical use (Bligh and Dyer,1959).

2.3. Preparation of aortic tissue for histological examination

Quantification of the aortic fatty streak area was carried out bythe method of Kowala et al. (1993). Briefly, the aortic arches wereexcised, cleaned, fixed with 10% buffered formalin, immersed inOil Red O and photographed under a light microscope (Nikon-Eclipse CFI 60) for observation of orange lipid plaques. Thefollowing morphological criteria were considered: grade0; scale 0(no damage), grade 1; scale + (mild), grade 2; scale ++ (moderate),and grade 3; scale +++ (severe). The final observation was themean of readings taken by three different investigators tominimizethe human error.

2.4. Total homocysteine estimation

Total homocysteine estimation was performed in serum ofmethionine treated rats using the High Performance LiquidChromatography sodium borohydride/monobromobimane(NaBH4/mBrB method used NaBH4 for reduction and mBrBfor derivatization) method, essentially according to the methodof Ubbink et al with homocysteine as an external standard(Ubbink et al., 1991).

Table 1Sequences of PCR primers

Gene Sequence 5′-3′ Productlength(bp)

HMG-CoA reductase FP TGC TGC TTT GGC TGT ATG TC 230RP TGA GCG TGA ACA AGAACC AG

GAPDH FP TTC ACC ACC ATG GAG AAG GC 237RP GGC ATG GAC TGT GGT CAT GA

Caveolin-2 FP CAG TCATGG CTC AGT TGC AT 198RP CTT CAT TGC GGG TAT CCT GT

P2X FP TGC AAA GGG AGG GTG TAG TC 215RP GGC ACC ATC AAG TGG ATC TT

P2Y FP TAG CAG GCC AGT AAG GCT GT 206RP GCT TGG GTG GTATGT GGA GT

FP; forward primer.RP; reverse primer.

51M. Sharma et al. / European Journal of Pharmacology 574 (2007) 49–60

2.5. Measurement of lipid profile

To assess the serum lipid profile of the rats administered withdifferent doses of methionine, the levels of total Cholesterol,triglycerides, oxidized Low density lipoprotein and Highdensity lipoprotein were determined using the method ofZlatkis et al. (1953); Vanhandel and Zilversmit (1957); Ahotupaet al. (1996); Grove (1979) respectively.

2.6. Activity of HMG-CoA reductase

The activity of HMG-CoA reductase in rats administeredwith methionine was determined by two methods, one by themethod of Venugopala and Ramakrishanan et al. (1975).Briefly, the ratio of absorbance of 3-hydroxy-3-methylglutarylCoA and mevalonate was measured in a UV spectrophotometerat 540 nm after treating the 10% liver homogenate (i.e. 1 g ofliver/10 ml of saline arsenate sol.) with hydroxylaminehydrochloride reagent (in water) and alkali hydroxylaminehydrochloride reagent (in sodium hydroxide), respectivelyfollowed by the addition of ferric chloride. The ratio ofHMG-CoA/Mevalonate was taken as an index of HMG-CoAreductase activity.

Second, the activity of HMG-CoA reductase in microsomeswas determined by the method of Hulcher and Oleson (1973).Briefly, measured the released coenzyme A (SH) during thereduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate.Coenzyme A was measured in the presence of dithiothreitol,required for activity, by reaction with 5,5′-dithiobis(2-nitro-benzoic acid). The absorbance due to the coenzyme A4,5′-dithiobis(2-nitrobenzoic acid) reaction is determined by extrap-olating the linear (dithiol) absorbance-time curve to the time ofaddition of the reagent. After subtraction of control absorbance(deletion of NADPH), the concentration of CoA-SH wascalculated from molar extinction coefficient (1.36× l04) at412 nm.

2.7. Measurement of total nitrates and nitrites

Nitric oxide (NO) released in the serum of methioninetreated rats was determined by measuring accumulation ofnitrates and nitrites, as described by Tracey et al. (1995). Nitricoxide reacts with oxygen to form nitrites and nitrates. Griessreagent was used to measure nitrite. First converted all nitrateto nitrite with bacterial nitrate reductase, and then this nitrite(representing total nitrite plus nitrate) was measured with theGriess reagent.

2.8. Total antioxidant capacity

Total antioxidant capacity in serum of rats treated withmethionine, was assessed by method of Koracevic et al. (2001).Briefly, the assay measured the capacity of the serum/plasma toinhibit the production of thiobarbituric acid reactive substances(TBARS) from sodium benzoate under the influence of the freeoxygen radicals derived from Fenton's reaction. A solution of1 mmol/l uric acid was used as standard.

2.9. C-reactive protein, resistin and cysteinyl–leukotrieneestimation

To analyse the effect of homocysteine on cardiovascularmarkers the serum level of C-reactive protein, resistin andcysteinyl–leukotriene were estimated. C-reactive protein(CRP)was analysed by High Sensitive C-reactive Protein EnzymeImmunoassay test kit (hsCRP ELISA) (Diagnostic AutomationInc.USA) according to the kit manufacturer's instructions. Serumresistin was assessed using the murine Enzyme Immunoassay testkit from Cayman Chemical company (Ann Arbor, MI, USA)according to the kit manufacturer's instructions. Serum cystei-nyl–leukotriene was measured using the Enzyme Immunoassaytest kit from Cayman Chemical company (Ann Arbor, MI, USA)according to the kit manufacturer's instructions.

2.10. Incubation of aortic ring

To evaluate the in-vitro effect of homocysteine on isolatedaorta, rats were killed by cervical dislocation. The aortas(thoracic and abdominal) were excised and cleaned of alladherent tissues in ice-cold phosphate buffered saline (pH 7.4)under sterile conditions. The isolated aortas were placed in ice-cold Na+ free choline-containing Krebs solution, washed threetimes in phosphate buffered saline, and then cut into segments∼15 mm long, using a scalpel. Each segment was blotted dryand weighed. The segments were incubated in culture dishescontaining Krebs buffer (pH 7.2) in an atmosphere of 5%CO2

and 95% O2 at 37 °C. After incubation for 30 min, Differentconcentrations of Homocysteine (1 nmol/l, 1 μmol/l and1 mmol/l) was administered (Yang et al., 2006).

2.11. In vitro NO production in the aortic ring

After 3 h incubation, the homocysteine incubated culturemedium of each dish was collected in separate microcentrifugetubes. The medium of each tube was subsequently dried and theresulting pellets were redissolved with 100 μL of distilled water.Nitrate/nitrite levels were measured with use of Aspergillusnitrate reductase and Greiss reagent and the absorbance weredetermined at 540 nm on spectrophotometry. The concentrations

Table 2Body weight gain in rats fed the respective diets for 8 weeks

Body weightgain(g)

Group I Group II Group III Group IV Group V

After 4 weeks 14±1.50 15±2.0 11±2.5 13±1.0 11±1.5After 8 weeks 40±2.34 54.1±2.56 45.3±3.0 43.5±3.5 46.4 ±2.89

Values are mean±SE of six rats in each group.


were calculated from a standard curve derived from solutions ofNANO3 (5–100 μmol/l) (Yang et al., 2006).

2.12. RNA isolation

To assess the mRNA expression of HMG-CoA reductase inliver and mRNA expression of P2 receptors and caveolin inaorta, the total RNA was isolated from the liver and aorta withTrizol reagent according to the manufacturer's instructions. Inin-vitro study after incubation in the presence and absence ofdifferent concentration of Homocysteine for 8 h, total RNA inaortas was also prepared by in situ lysis with Trizol reagent(Dragin et al., 2006).

2.13. Reverse transcription and polymerase chain reaction

2 μg of total RNA (2 μl) was reverse transcribed into cDNAusing RevertAid™ first strand cDNA synthesis kit (Fermentas).Primers for HMG-CoA reductase, P2X, P2Y, Caveolin andGlyceraldehyde-3-phosphate dehydrogenase (GAPDH) werepurchased from Prolab Marketing, India (Table 1). PCR wasperformed in 20 μl reaction mixture containing 10× PCR buffer,

Fig. 1. Sequential changes in serum homocysteine (⁎Pb0.05, ⁎⁎Pb0.01, ⁎⁎⁎Pblipoproteins and triglyceride levels (⁎Pb0.05, ⁎⁎Pb0.01, ⁎⁎⁎Pb0.001, compared to

2 mM MgCl2, 0.2 mM of each dNTP, 0.5 μm of each primer,0.625 U of Taq polymerase and nuclease free water. 1 μl ofcDNA was used as a template in each PCR. Data was nor-malized to the GAPDH signal. The amplicons were resolved in2% agarose gel containing ethidium bromide (1 μg/ml). Theintensity of bands were analysed on Alpha Imager™2200(Alpha Innotech Corporation, USA). Each experiment wasperformed in duplicate (Baba et al., 2006).

2.14. Immunoblot analysis

The expression of HMG-CoA reductase in liver of methioninetreated rats was estimated by western blot analysis. The proteincontent of microsomes was measured using Lowry's method(Lowry et al., 1951). Proteins were resolved on sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) using7.5% gel and transferred onto polyvinylidene difluroide mem-branes (PVDF). Western blot was performed using antibodiesdirected against HMG-CoA reductase. The Western Blot wasgenerated from three replicate runs. Relative amount of proteinloaded and transferred onto the blot were assessed by CoomassieBlue staining and quantified by Alpha Imager™2200 (AlphaInnotech Corporation, USA). Integrated peaks densities wereexpressed as arbitrary units relative to protein content.

2.15. Densitometric and statistical analysis

The intensities of bands obtained from western blots and RT-PCRwere estimated with Alpha Imager™2200 (Alpha InnotechCorporation, USA). The values were expressed as mean±S.E.

0.001, compared to time 0 in respective Groups), cholesterol, high densitysaline fed rats) of experimental Groups. Results are expressed as mean±SEM.


The significance of difference betweenmeans of two groups wasobtained with one-way analysis of variance (ANOVA), Tukey-Kramer multiple comparisons test, using Graph pad Prism 3.0computer software. Pb0.05 was considered to be statisticallysignificant.

3. Results

3.1. Body weight

The changes in the bodyweights of the rats of the experimentalgroups are shown in Table 2. There was progressive increasein the body weight of all groups. No significant change in bodyweight was found in animals administered with different con-centrations of methionine.

3.2. Level of total homocysteine in serum

The serum total homocysteine levels showed a significantincrease in Group V (1 g/kg of methionine) at weeks 4compared with time 0 but remained practically unchanged inother groups (Fig. 1). Groups II, III and IV showed significantincrease only at 8 weeks as compared to homocystiene level attime 0.

Fig. 2. Effect of different doses of methionine on the sequential changes in level of oand nitrates level, the level of resistin (⁎Pb0.05, ⁎⁎Pb0.01, ⁎⁎⁎Pb0.001, compa⁎⁎Pb0.01, ⁎⁎⁎Pb0.001, compared to time 0 in respective groups) in the experimen

3.3. Lipid profile

The levels of serum cholesterol remained unchanged ingroups II and III at week 4, but increased remarkably in groupIV and V as compared to saline fed rats (Pb0.01, Pb0.001respectively) (Fig. 1). After 8 weeks the levels of cholesterolincreased by ∼16.2%, ∼45.4%, ∼84.7% and ∼107% in groupII, III, IV and V as compared to saline fed rats.

The values of serum HDL remained unchanged in all theexperimental groups after 4 weeks (Fig. 1). After 8 weeks, thelevels of HDL decreased significantly in group V as comparedto control group (Pb0.01).

The values of triglycerides remained unchanged after4 weeks in group II and III and increased significantly ingroup IVand V (Pb0.05, Pb0.01 respectively) as compared tosaline fed rats. After 8 weeks the levels of triglyceride increasedby ∼23.14%, ∼50.7%, ∼79.0% in group III, IV and V respec-tively as compared to saline fed rats (Fig. 1).

The concentrations of Oxidised low density lipoproteinsshowed no significant change in group II and III and increasedsignificantly in group IV and V as compared to saline fed rats(Pb0.05,Pb0.01 respectively). At 8 weeks the levels of Oxidisedlow density lipoproteins increased by ∼45%, ∼71%, ∼85% ingroups III, IV and Vas compared to saline fed rats (Fig. 2).

xidised low density lipoproteins, serum total antioxidant capacity, serum nitritesred to saline fed rats), C-reactive protein and cysteinyl–leukotriens (⁎Pb0.05,tal Groups. Results are expressed as mean±SEM.


3.4. Total antioxidant capacity and nitric oxide levels

The total antioxidant capacity in serum remained unchangedat 4 weeks in all the experimental groups. At 8 weeks totalantioxidant capacity decreased significantly in groups III, IVand V (Pb0.05, Pb0.05, Pb0.01 respectively) as compared tosaline fed rats (Fig. 2). The levels of Nitric oxide (NO) in theserum were shown in Fig. 2. Groups II and III showed no

Fig. 3. Treatment effects on aortic plaque burden. Representative images of oilred O-stained after en face preparation. (A) Aorta of saline fed rats. (B) Aorta ofrats administered with 0.5 g/kg of methionine. (C) Aorta of rats administeredwith 1.0 g/kg of methionine. Note bright red-stained lipids in atheroscleroticplaques were shown by arrows. Magnification is ×100.

change in the level of nitric oxide at 4 weeks, but a significantdecrease was observed in groups IV and Vat 4 weeks (Pb0.05,Pb0.05 respectively). The levels of nitric oxide showed adecrease of ∼57%, ∼67% in groups III and IV respectively ascompared to saline fed rats at week 8. Group V showed nochange in the values of nitric oxide level as compared to valuesat 4 weeks (Fig. 2).

3.5. Serum level of resistin, C-reactive protein andcysteinyl–leukotrienes

The levels of serum resistin in Groups II and III showed nochange at 4 weeks, but group IV and V showed a significantincrease as compared to saline fed rats (Pb0.01, Pb0.01respectively) (Fig. 2). At 8 weeks the level of resistin in group IIshowed no significant change. However, the level of resistinshowed significant increase in group III, IV and V (Pb0.05,Pb0.001, Pb0.001 respectively).

The levels of C-reactive protein remained unchanged inGroups II, III and IV and increased in Groups Vas compared tozero time (Pb0.05). The values were increased by ∼26%,∼31% and∼40% in Group III, IVand Vat 8 weeks as comparedto Group I (saline fed rats) (Fig. 2). The basal levels of cysteinyl–leukotrienes showed no significant difference among theexperimental groups. Groups IV and V showed a significantincrease at 4weeks as compared to zero time (Pb0.05, Pb0.01respectively). The group II showed no significant difference at8 weeks but a significant increase in the levels of cysteinyl–leukotrienes was observed in groups III, IV and V (Pb0.01,Pb0.01, Pb0.001 respectively) as compared to time 0 (Fig. 2).

3.6. Atherosclerotic changes in the aorta

The severity of aortic atherosclerosis as judged by grossgrading of Oil red O staining, Group I and Group II did not showany evidence of atherosclerosis (grade 0; scale 0) (Fig. 3). GroupIII, showed a lower degree of atherosclerosis (grade 1; scale++).A significant area of the Group IV and Group V was coveredwith atherosclerotic plaques (grade 2; scale++) ((Fig. 3B and C).

3.7. mRNA level, protein expression and activity of HMG-CoAreductase in liver

3.7.1. Effect on hepatic HMG-CoA reductase mRNA levelsThe mRNA levels of HMG-CoA reductase in Group I was

comparable to group II. Group III and group IV showed anincrease of ∼15% and ∼31% in mRNA levels of HMG-CoAreductase (Fig. 4B and C), respectively. A significant increaseof ∼65% was observed in group V as compared to group I.

3.7.2. Effect on HMG-CoA reductase protein expressionWestern Blot analysis revealed that HMG-CoA reductase was

not expressed inGroup I (saline fed rats) (Fig. 4D). The expressionof HMG-CoA reductase was found to be minimal in Group II.In Groups III and IV, the expression of this protein increasedof ∼15% and ∼12% respectively. An increase of ∼17% wasobserved in Group V as compared to Group I (Fig. 4E).

Fig. 4. Effect of different doses of methionine on mRNA levels of HMG-CoA reductase (230 bp) and GAPDH (237 bp) in experimental animals by RT-PCR. (A) mRNAlevels of GAPDH (B)mRNA levels of HMG-CoA reductase. Lane (1) saline fed with normal chow diet, lane (2) orally administered with 0.1 g/kg of methionine with normaldiet, lane (3) orally administered with 0.25 g/kg of methionine with normal diet, lane (4) orally administered with 0.5 g/kg of methionine with normal diet, lane (5) orallyadministered with 1 g/kg of methionine with normal diet, lane (M) DNA ladder (100–1000 bp), (C) comparison of mRNA levels of HMG-CoA reductase with respect tocorresponding levels of GAPDH standard. Integrated density values (IDV) of all the groupswere normalizedwith IDVofGAPDH. Lanes 1,2,3,4, and 5 correspond toGroupsI, II, III, IVandVrespectively. Each treatment on ratswas repeated three times and values representmeans±SEobtained fromduplicate assays on every treatment. ⁎Pb0.05 ascompared to Group I, ⁎⁎Pb0.01 as compared to Group I, ⁎⁎⁎Pb0.001 as compared to Group I. (D) Effect of different doses of methionine on hepatic HMG-CoA reductaseprotein expression (98KDa) in experimental animals byWestern Blot analysis. Lane (M)molecular weight marker 117–19KDa). (E) Comparison of effect of different dosesof methionine on hepatic HMG-CoA reductase protein levels. Lanes 1,2,3,4, and 5 correspond Groups I, II, III, IVand V respectively. Each treatment on rats was repeatedthree times and values represent means±SE obtained from duplicate assays on every treatment. ⁎Pb0.01 as compared to Group I, ⁎⁎Pb0.05 as compared to Group I.


3.7.3. Effect on activity of HMG-CoA reductase (ratio of HMG/mevalonate)

The activity of HMG-CoA reductase was determined bycalculating the ratio of HMG to mevalonate. An increase in the

Table 3Effect on HMG-CoA reductase activity

Groups

Group I G

Ratio of HMG/mevalonate 1.232±0.06 1.Microsomal specific activity of HMG-CoA

reductase (nmol of coenzyme A/mg protein/min.)1.51±0.046 1

Values are mean±SE.nsnot significant.aPb0.05 as compared to Group I.bPb0.01 as compared to Group I.cPb0.001 as compared to GroupI.The experiment has been run in triplicates.

ratio of HMG/mevalonate is an indication of a decline in HMG-CoA reductase activity. HMG-CoA reductase activity wasfound to show no significant change in Group II as compared toGroup I. Groups III, IV and V showed an increase of ∼17%,

roup II Group III Group IV Group V

130±0.01ns 1.02±0.02a 0.987±0.024b 0.763±0.013b

.61±0.043ns 1.78±0.049b 2.03±0.042c 2.2±0.037c

Fig. 5. Effect of different doses ofmethionine onmRNA levels of caveolin-2 (198 bp), P2X receptors (215 bp) and P2Y receptors (206 bp) in experimental animals byRT-PCR. (A)mRNA levels of caveolin-2. (B) Comparison ofmRNA levels of caveolin-2 with respect to corresponding levels of GAPDH standard. (C)mRNA levels of P2X,(D) comparison ofmRNA levels of P2Xwith respect to corresponding levels of GAPDHstandard. (E) mRNA levels of P2Y. (F) Comparison ofmRNA levels of P2Xwithrespect to corresponding levels of GAPDH standard. Lane (1) normal chow diet, lane (2) orally administered with 0.1 g/kg of methionine with normal diet, lane (3) orallyadministered with 0.25 g/kg of methionine with normal diet, lane (4) orally administered with 0.5 g/kg of methionine with normal diet, lane (5) orally administered with1 g/kg of methionine with normal diet, lane (M) DNA ladder 100–1000 bp. Integrated density values IDV of all the Groups were normalized with IDVof GAPDH(Fig. 4A). Lanes 1,2,3,4, and 5 correspond to Groups I, II, III, IV and V respectively. Each treatment on rats was repeated three times and values represent means±SEobtained from duplicate assays on every treatment. ⁎Pb0.05 as compared to Group I, ⁎⁎Pb0.01 as compared to Group I, ⁎⁎⁎Pb0.001 as compared to Group I.


∼20% and ∼38% in the activity of HMG-CoA reductase,respectively (Table 3).

3.7.4. Effect on microsomal specific activity of HMG-CoAreductase

HMG-CoA reductase activity was found to show nosignificant change in Group II as compared to Group I. GroupsIII, IV and V showed an increase of ∼18%, ∼34% and ∼46%in the activity of HMG-CoA reductase, respectively (Table 3).

3.8. mRNA levels of caveolin protein in aorta (in-vivo)

The mRNA expression of Group II was comparable torats fed a normal diet. Groups III and IV showed ∼1 fold

increase in the mRNA levels of caveolin. A ∼2 foldincrease was observed in mRNA levels in Group V (Fig. 5Aand B).

3.9. mRNA levels of P2X and P2Y receptors in aorta (in-vivo)

The mRNA levels of P2X in Groups II, III and IV werecomparable to rats fed a normal diet. Group V showed anincrease of∼43% in the mRNA levels as compared to saline fedrats (Fig. 5C, D). The mRNA expression of P2Y showed nosignificant change in Group II as compared to Group I. ThemRNA expression increased by ∼11%, ∼18% and ∼54% inGroup III, IV and V as compared to Group I, respectively(Fig. 5E and F).

Table 4Effect on nitrite/nitrate (NO level) production in the rat aorta

Control 1 nmol/l Hcy 1 μmol/l Hcy 1 mmol/l Hcy

NO2− (μmol/mgprotein)

8.48±0.59 4.43±0.49a 3.89±0.51a 3.74±0.55a

aPb0.01 as compared to control.The experiment has been run in triplicates.


3.10. Effect on nitric oxide (total nitrate/nitrite level) produc-tion in the isolated rat aorta medium (in-vitro)

In vitro incubation with 10−9, 10−6 and 10−3 mol/l homo-cysteine for 3 h resulted in a concentration dependent decreasein nitric oxide production. Nitrite and nitrate content in mediumwas ∼47%, ∼54% and ∼56% lower, respectively, than that inthe control Group (all Pb0.01) (Table 4).

3.11. Effect of different concentration of homocysteine onmRNA expression of caveolin, P2X and P2Y in the isolated rataorta (in-vitro)

Homocysteine (10−3 mol/l) stimulated the mRNA expres-sion of caveolin, P2X and P2Y in aortic tissue by∼82%,∼79%and ∼51% respectively as compared to control aortic tissue(Fig. 6A–G). A dose dependent increase in mRNA expressionof caveolin, P2X and P2Y was observed upon incubation withdifferent concentration of homocysteine with aortic tissue(Fig. 6H).

4. Discussion

In the present study, we have evaluated the effect of highlevel of homocysteine on the level of other cardiovascular riskfactors and markers in rats. Along with this, we have alsoassessed the finding that hyperhomocysteinemia initiates theatherosclerosis by inducing the cholesterol biosynthesis.Homocysteine concentration in serum was regulated by thedietary intake of methionine. Administration of varying dosesof methionine for 8 weeks led to a dose dependent increase inthe levels of total homocysteine.

A large number of epidemiological studies have confirmedthat an elevation of total plasma homocysteine is prevalent inpatients with stroke, myocardial infarction, peripheral vasculardisease and venous thrombosis (Herrmann and Knapp, 2002).The molecular mechanism by which homocysteine promotesatherosclerosis is not clear.

We observed that level of cholesterol and triglycerideincreased significantly with the increase in the concentrationof homocysteine in serum of rats. The increase in cholesterollevels can be explained by studying the effect of homocysteineon the activity of HMG-CoA reductase, a rate-limiting enzymein cholesterol biosynthesis. From our study, we found thatmethionine increased the mRNA levels and hepatic activity ofHMG-CoA reductase up to ∼65% and ∼46%, respectively inGroup V as compared to saline. These results showedconsistency with the findings of Woo et al. (2005) and Liet al. (2002). Western blot analysis of HMG-CoA reductase

showed negligible change in the protein expression in all groupsof rats, except group V that showed ∼17% induction in theexpression as compared to saline fed rats. In contrast, Li et al.(2002) found that there was continuous increase in expressionof protein of HMG-CoA reductase with increase in level ofhomocysteine. The discrepancy between the mRNA and proteinlevel of HMG-CoA reductase might be due to some othermechanism that warrants further investigation.

Low doses of methionine produced no significant effect onthe High-density lipoprotein levels in serum. Dose of 1 g/kg ofmethionine produced significant decline in the levels of High-density lipoprotein. This inverse relationship of High densitylipoprotein with homocysteine is in agreement with the findingsof Liao et al. (2006), who reported that homocysteine reducedthe level of High density lipoprotein in plasma.

Atherosclerosis is associated with an impairment ofendothelium-dependent relaxations, which represents the re-duced bioavailability of nitric oxide produced from endothelialnitric oxide synthase (Kawashima and Yokohama, 2004). Ourdata showed that rats administered with 0.5 g/kg and 1 g/kg ofmethionine led to a significant decrease in nitric oxide levels inthe serum in a dose dependent manner. Our results are inagreement with those of Zhang et al. (2000) who showed thathigh homocysteine level in serum suppress the nitric oxidereleased from the endothelium. Furthermore, our in-vitro datashowed that incubation of isolated aorta with homocysteine,decreased the nitrates and nitrites level but the dose dependenteffect was not very remarkable. The medium of aortic ringsincubated with 1 mmol/l of homocysteine showed no significantdifference as compared to aortic rings medium incubated with1 nmol/l and 1 μmol/l of homocysteine.

In our study, the pro-oxidant properties of homocysteinewere confirmed by decreased total antioxidant capacity andincreased concentration of oxidized low-density lipoprotein inmethionine treated rats. The mechanism of these changes can beexplained on the basis of the fact that homocysteine is injuriousto vascular endothelial cells and impairs normal cellularfunction. The thiol group of homocysteine readily undergoesautooxidation in plasma to generate reactive oxygen species andinduces cell injury/dysfunction via a mechanism involvingoxidative stress (Tyagi et al., 2005).

In our investigation, we have studied the effect ofhomocysteine on the serum levels of resistin. Resistin belongsto a family of cysteine-rich secretory proteins called resistin-likemolecules or FIZZ (found in inflammatory zones) proteins(Degawa-Yamauchi, 2003), secreted from macrophages inatheroma and promotes atherosclerosis (Jung et al., 2006).High concentrations of resistin were shown to induce vascularendothelial dysfunction and vascular smooth muscle cellproliferation. Our results demonstrated that methionine, espe-cially in higher doses, showed increased serum level of resistin.We can speculate that hyperhomocysteinemia caused atheromaformation in the vessel wall and ultimately led to an increase inthe concentration of resistin in the serum.

C-reactive protein is amarker for inflammation.HighC-reactiveprotein is associated with increased coronary heart diseases;hence, the level of C-reactive protein may ultimately represent an

Fig. 6. The mRNA levels of caveolin-2 (198 bp), P2X receptors (215 bp) and P2Y receptors(206 bp) of isolated aortic rings incubated with different doses ofhomocysteine by RT-PCR (A) mRNA levels of GAPDH (B) mRNA levels of caveolin-2. (C) Comparison of mRNA levels of caveolin-2 with respect to correspondinglevels of GAPDH standard. (D) mRNA levels of P2X, (E) comparison of mRNA levels of P2X with respect to corresponding levels of GAPDH standard. (F) mRNAlevels of P2Y. (G) Comparison of mRNA levels of P2X with respect to corresponding levels of GAPDH standard. Lane (1) normal, lane (2) 1 nM/l of Homocysteine,lane (3) 1 uM/l of homocysteine, lane (4) 1 mM/l of homocysteine, lane (M) DNA ladder (100–1000 bp). Integrated density values (IDV) of all the groups werenormalized with IDVof GAPDH (A). The experiment was repeated three times and values represent means±SE obtained from duplicate assays on every treatment.⁎Pb0.05 as compared to as compared to control, ⁎⁎Pb0.01 as compared to Control, ⁎⁎⁎Pb0.001 as compared to Control.


important therapeutic target in managing coronary arterydisease (Han et al., 2004). It is evident from our studies that levelsof C-reactive protein induced with the increased level ofhomocysteine in serum. Our data showed consistency with anepidemiological data collected by Balogh et al. (2006). In contrast,

the studies of Auer et al. (2001) reported lack of association ofincreased C-reactive protein and total homocysteine in patients ofcardiovascular diseases.

Leukotrienes are autocrine and paracrine eicosanoid lipidmediators derived from arachidonic acid by 5-lipoxygenase.


During cysteinyl–leukotriene interaction, they can stimulateproinflammatory activities such as endothelial cell adherenceand chemokine production by mast cells. An increase in thelevel of cysteinyl–leukotrienes is a marker of cardiovasculardiseases (Spanbroek et al., 2003). Our results showed asignificant increase in the level of cysteinyl–leukotrienes inanimals administered with methionine. No other relevant data isavailable which clarifies the correlation of cysteinyl–leuko-trienes with hyperhomocysteinemia.

During our investigation, we studied the effect of theinteraction of homocystiene with P2 receptors. In fast (shortterm) purinergic signalling, ATP acts as an excitatory cotrans-mitter and produce vasoconstriction via P2X-purinoceptors andcan act via P2Y-purinoceptors and produces vasodilatation(Burnstock, 1990) to maintain the normal vascular tone ofendothelium. In addition to their short-term effects on vasculartone, P2 receptors are also involved in long-term trophic effectson cell growth, proliferation, and death, which have importantimplications for atherosclerosis. The shear stress that occursduring changes in blood flow during development of athero-sclerosis leads to a substantial release of ATP (and UTP) fromendothelial cells, Occupation of P2X7 receptors leads to theproduction of proinflammatory response(Virgilio and Solini,2002). Our data showed that aorta of rats administered with highdose of methionine (1 g/kg) showed significant induction themRNA expression of P2X receptors. Our results are consistentwith the findings of Pulvirenti et al. (2000) that showed highdensity of P2X receptors in rabbit aorta following injury toendothelial cells and cholesterol feeding.

We observed that rats administered with 0.5 g/kg and 1 g/kgmethionine significantly increased the expression of P2Y(Pb0.001, Pb0.001) in aorta respectively. This upreglation ofP2Y receptors can be postulated by the fact that prolongedstimulation of endothelial cells by haemodinamic stress, orplatelet aggregation on the endothelial surface, may cause a largeand sustained ATP release that via an immunological cascade,stimulates smooth muscle cell and endothelial cell proliferationvia P2Y2 and/or P2Y4 receptors and initiate vascular diseaseslike atherosclerosis (Virgilio and Solini, 2002).

Our subsequent in-vitro studies on isolated rat aorta, whichwasincubated with different concentrations of homocysteine, alsoshowed significant increase in the mRNA expression of P2X andP2Y. These in-vitro results support our in-vivo experimental data.From these results it can be speculated that a high concentration ofhomocysteine can induce stress in the endothelium and then causethe accumulation of ATP in the cells and ultimately increase in theexpression of P2 receptors (P2X and P2Y) in aorta. The dose-dependent effects of methionine/homocysteine on ATP turnoverwere not assessed in this study but would provide an interestingarea for future research.

The increase in P2 receptors mRNA expression showedpositive correlation with the level of cysteinyl–leukotrienes.This assessment showed consistency with the investigation ofBallerini et al. (2005) in which they have suggested a cross talkbetween the purine and leukotriene systems in a possibleautocrine/paracrine control of the microglia-mediated initiationand progression of an inflammatory response.

A number of findings suggested that the expression ofcaveolin represents a mechanism by which free cholesterolefflux can be modulated in response to changes in cellularcholesterol content. Caveolin-2 plays a role in cancer andvascular diseases (Gargalovic and Dory, 2003). Caveolin formsoligomers and associates with cholesterol and sphingolipids incertain areas of the cell membrane of cells, and causes theformation of caveolae. We observed that expression of caveolin-2 increased significantly in rats administered with 0.5 g/kg and1.0 g/kg of methionine (Pb0.001, Pb0.001 respectively) indose dependent manner. The isolated aortic rings incubated with1 mmol/l of homocysteine showed ∼82% increase in themRNA level of caveolin as compared to control.

In conclusion, our studies confirm the fact that the homocysteinemodulates the metabolism of cholesterol by inducing thetranscription of HMG-CoA reductase and the activity of HMG-CoA reductase in liver. Homocysteine also has a significant impacton immunological parameters, as shown by the increased levels ofresistin, C-reactive protein and cysteinyl–leukotrienes, in the serumof methionine treated rats. From our results, we can postulate thatthe P2 receptors and caveolin plays an important role inhomocysteine-induced atherosclerosis. Hence, this study providesa new understanding in pathophysiology of hyperhomocysteine-mia. Further investigations are needed to suggest a mechanism ofaction of the positive correlation of high level of homocysteinewiththe levels of resistin, C-reactive protein, cysteinyl–leukotrienes, P2receptors and caveolin.

Acknowledgements

The authors wish to thanks Prof. Vani Brahmachari for valuablesuggestions. The authors also wish to thank Dr. Rakesh KumarTiwari and Dr. Unnikrishanan for helpful discussions. The authorswish to acknowledge the financial support received from theDepartment of Science and Technology, Delhi, India. The facilitiesprovided by Dr. B. R. Ambedkar Center for Biomedical Research,University of Delhi are gratefully acknowledged.

References

Ahotupa, M., Ruutu, M., Mantyla, E., 1996. Simple methods of quantifyingoxidation products and antioxidant potential of low density lipoproteins.Clin. Chem. 29, 139–144.

Auer, J.W., Berent, R., Eber, B., 2001. Lack of association of increased C-reactiveprotein and total plasma homocysteine. Circulation 104, e164.

Baba, M.I., Kaul, D., Grover, A., 2006. Importance of blood cellular genomicprofile in coronary heart diseases. J. Biomed. Sci. 13, 17–26.

Ballerini, P., Ciccarelli, R., Caciagli, F., Rathbone,M.P., Werstiuk, E.S., Traversa, U.,Buccella, S., Giuliani, P., Jang, S., Nargi, E., Visini, D., Santavenere, C., Di lorio,P., 2005. P2X7 receptor activation in rat brain cultured astrocytes increases thebiosynthetic release of cysteinyl–leukotrienes. Int. J. Immunopathol. Pharmacol.18, 417–430.

Balogh, E., Czuriga, I., Bereczky, Z., Boda, K., Kosezegi, Z., Konya, C.,Csaszar, A., Muszbek, L., Edes, I., Ferdinandy, P., 2006. Increase ofhomocysteine in cardiovascular diseases in Hungary. Orv. Hetil. 147,1685–1690.

Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction andpurification. Can. J. Biochem. Physiol. 37, 911–917.

Burnstock, G., 1990. Dual control of local blood flow by purines. Ann. N.Y.Acad. Sci. 603, 31–44.


Degawa-Yamauchi, M., Bovenkerk, J.E., Juliar, B.E., Waston, W., Kerr, K.,Jones, R., Zhu, Q., Considine, R.V., 2003. Serum resistin (FIZZ3) protein isincreased in obese humans. J. Clin. Endocrinol. Metab. 88, 5452–5455.

Dragin, N., Sandrine, M.S., Clement, A.D., Moranvillier, D.I., Pierre, Jean-YvesC., Peuchant, D.E., 2006. Acute oxidative stress is associated with cellproliferation in the mouse liver. FEBS Lett. 580, 3845–3852.

Gargalovic, P., Dory, L., 2003. Caveolins and macrophage lipid metabolism.J. Lipid Res. 44, 11–21.

Grove, T.H., 1979. Effect of reagent pH on determination of high densitylipoprotein cholesterol by precipitation with sodium phosphotungustate-magnesium. Clin. Chem. 25, 560–564.

Han, K.H., Hong, K.H., Park, J.H., Ko, J., Kang, D.H., Choi, K.J., Hong, M.K.,Park, S.W., Park, S.J., 2004. C-reactive protein promotes monocytechemoattractant protein-1 — mediated chemotaxis through upregulatingCC chemokine receptor 2 expression in human monocytes. Circulation 109,2566–2571.

Herrmann, W., Knapp, J.P., 2002. Hyperhomocysteinemia: a new risk factor fordegenerative diseases. Clin. Lab. 48, 471–481.

Hofmann, M.A., Lalla, E., Lu, Y., Gleason, M.R., Wolf, B.M., Tanji, N., Ferran,L.J., Kohl, B., Rao, V., Kisiel, W., 2001. Hyperhomocysteinemia enhancesvascular inflammation and accelerates atherosclerosis in a murine model.J. Clin. Invest. 107, 675–683.

Hulcher, F.H., Oleson, W.H., 1973. Simplified spectrophotometric assay formicrosomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement ofcoenzyme A. J. Lipid Res. 14, 625–631.

Jung, H.S., Ki-Ho, P., Cho, Y.M., Chung, S.S., Cho, H.J., Cho, S.J., Kim, S.J.,Lee, H.J., Park, K.S., 2006. Resistin is secreted from macrophages inatheromas and promotes atherosclerosis. Cardiovasc. Res. 69, 76–85.

Jurevics, H., Hostettler, J., Barrett, C., Morell, P., Toews, A.D., 2000. Diurnaland dietary-induced changes in cholesterol synthesis correlate with levels ofmRNA for HMG-CoA reductase. J. Lipid Res. 41, 1048–1053.

Kawashima, S., Yokoyama, M., 2004. Dysfunction of endothelial nitric oxidesynthase and atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 24, 998–1005.

Koracevic, D., Koracevic, G., Djordjevic, V., Andrejevic, S., Cosic, V., 2001.Methods for the measurement of antioxidant activity in human fluids.J. Clin. Pathol. 54, 356–361.

Kowala, M.C., Mazzucco, C.E., Hartl, K.S., Seiler, S.M., Warr, G.A., Abid, S.,Grove, R.I., 1993. Prostacyclin agonists reduce early atherosclerosis inhyperlipidemic hamsters. Octimibate and BMY 42393 suppress monocytechemotaxis, macrophage cholesteryl ester accumulation, scavenger receptoractivity, and tumor necrosis factor production. Arterioscler. Thromb. Vasc.Biol. 13, 435–444.

Lentz, S.R., 2001. Does homocysteine promote atherosclerosis. Arterioscler.Thromb. Vasc. Biol. 21, 1385–1386.

Li, H., Lewis, A., Brodsky, S., Rieger, R., Iden, C., Goligorsky, S.M., 2002.Homocysteine induces 3-hydroxy-3-methylglutaryl coenzyme a reductase invascular endothelial cells a mechanism for development of atherosclerosis?Circulation 105, 1037–1043.

Liao, D., Tan, H., Hui, R., Li, Z., Jiang, X., Gaubatz, J., Yang, F., Durante, W.,Chan, L., Schafer, A.I., Pownall, H.J., Yang, X., Wang, H., 2006.Hyperhomocysteinemia decreases circulating HDL by inhibiting apoA-Iprotein synthesis and enhancing HDL-C clearance. Circ. Res. 99, 598–606.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Proteinmeasurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275.

Malinow, M.R., Levenson, J., Giral, P., Nieto, F.J., 1995. Rate of blood pressure,uric acid and hemorrheological parametes on plasma homocysteineconcentrations. Atherosclerosis 114, 175–183.

Pulvirenti, T.J., Yin, J.L., Chaufour, X., McLachlan, C., Hambly, B.D., Bennett,M.R., Barden, J.A., 2000. P2X (purinergic) receptor redistribution inrabbit aorta following injury to endothelial cells and cholesterol feeding.J. Neurocytol. 29, 623–631.

Refsum, H., Yajnik, C.S., Gadkari, M., Schneede, J., Vollset, S.E., Örning, L.,Guttormsen, A.B., Joglekar, A., Sayyad, M.G., Ulvik, A., Ueland, P.M.,2001. Hyperhomocysteinemia and elevated methylmalonic acid indicate ahigh prevalence of cobalamin deficiency in Asian Indians. Am. J. Clin. Nutr.74, 233–241.

Reilly, M.P., Lehrke, M., Wolfe, M.L., Rohatgi, A., Lazar, M.A., Rader, D.J.,2005. Resistin is an inflammatory marker of atherosclerosis in humans.Circulation 111, 932–939.

Spanbroek, R., Grabner, R., Lotzer, K., 2003. Expanding expression of the5-lipoxygenase pathway within the arterial wall during human atherogenesis.Proc. Natl. Acad. Sci. U. S. A. 100, 1238–1243.

Tracey, W.R., Tse, J., Carter, G., 1995. Lipopolysaccharides induced changes inplasma nitrite and nitrate concentrations in rats and mice; pharmacologicalevaluation of nitric oxide synthase inhibitors. J. Pharmacol. Exp. Ther. 282,1011–1015.

Tyagi, N., Moshal, S.K., Ovechkin, A.V., Rodriguez, W., Steed, M., Henderson, B.,Roberts, A.M., Joshua, G.I., Tyagi, C.S., 2005. J. Cell. Biochem. 96, 665–671.

Ubbink, J.B., Hayward Vermaak, W.J., Bissbort, S., 1991. Rapid highperformance liquid chromotagraphic assay for total homocysteine levels inhuman serum. J. Chromatogr. 565, 441–446.

Vanhandel, E., Zilversmit, D.B., 1957. Micromethod for the direct determinationof serum triglycerides. J. Lab. Clin. Invest. 50, 152–157.

Venugopala, R.A., Ramakrishanan, S., 1975. Indirect assessment of Hydro-xymethylglutaryl-CoA reductase (NADPH). Clin. Chem. 21, 1523–1525.

Virgilio, F.D., Solini, A., 2002. P2 receptors: new potential players inatherosclerosis. Br. J. Pharmacol. 135, 831–842.

Woo, H.W.C., Siow, L.Y., Pierce, N.G., Choy, C.P., Minuk, Y.G., Mymin, D.,Karmin, O., 2005. Hyperhomocysteinemia induces hepatic cholesterolbiosynthesis and lipid accumulation via activation of transcription factors.Am. J. Physiol. Endocrinol. Metab. 288, 1002–1010.

Yang, J.H., Pan, C.S., Jia, Y.X., Zhang, J., Zhao, Z., Pang, Y.Z., Yang, J., Tang,C.S., Qi, Y.F., 2006. Intermedin1-53 activates L-arginine/nitric oxide synthase/nitric oxide pathway in rat aortas. Biochem. Biophy. Res. Comm. 341,567–572.

Zhang, X., Li, H., Jin, H., Ebin, Z., Brodsky, S., Goligorsky, S.M., 2000. Effectsof homocysteine on endothelial nitric oxide production. Am. J. Physiol.Renal Physiol. 279, F671–F678.

Zhou, J., Moller, J., Danielsen, C.C., Bentzon, J., Ravn, H.B., Austin, R.C.,Falk, E., 2001. Dietary supplementation with methionine and homocysteinepromotes early atherosclerosis but not plaque rupture in ApoE-deficientmice. Arterioscler. Thromb. Vasc. Biol. 21, 1470–1476.

Zlatkis, A., Zak, B., Boyle, A.J., 1953. A new method for the directdetermination of serum cholesterol. J. Lab. Clin. Invest. 41, 486–492.

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