The role of microscopy in understanding atherosclerotic lysosomal lipid metabolism.

Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with modified lipoproteins suggests this abnormal metabolism occurs in two steps. Initially, hydrolysis of lipoprotein cholesteryl esters occurs normally, but the resultant free cholesterol cannot exit the lysosome. Further lysosomal cholesterol accumulation inhibits hydrolysis, producing a mixture of cholesterol and cholesteryl esters within swollen lysosomes. Various lipoprotein modifications can produce this lysosomal engorgement in vitro and it remains to be seen which modifications are most important in vivo.     

Analysis of lipoproteins with analytical capillary isotachophoresis

An analytical free flow capillary isotachophoresis procedure, with a discontinuous electrolyte system, for the detailed analysis of lipoproteins in human body fluids has been developed. The technique is based on prestaining whole serum lipoproteins with a lipophilic dye before separation. Human serum lipoproteins are separated into 14 well-characterized subfractions according to their electrophoretic mobility. High density lipoproteins (fraction 1 to 6) are separated into three major subpopulations, the fast migrating high density lipoprotein (HDL) subpopulation, containing mainly apo AI and phosphatidylcholine, the subpopulation with intermediate mobility, consisting of particles rich in apo AII, apo E, and C apolipoproteins, and the slowly migrating HDL subfraction, containing mainly particles rich in apo AI, apo AIV, and lecithin: cholesterol acyltransferase (LCAT) activity. The apo B containing lipoproteins (fraction 7 to 14) can be subdivided into four major functional groups. The first represents chylomicron derived particles and large triglyceride-rich very low density lipoproteins (VLDL). The second group consists of small VLDL and intermediate density lipoprotein (IDL) particles, anf the third and fourth group represent the low density lipoproteins. The isotachophoretic analysis of human serum samples obtained from patients with hyperlipoproteinemias is compatible with the classification according to the Frederickson phenotypes and reflects the respective biochemical abnormalities. Furthermore, several genetic disorders of lipid and lipoprotein metabolism like HDL deficiency syndromes, familial LCAT deficiency, Fish eye disease, hypobetalipoproteinemia and abetalipoproteinemia can be well characterized by analytical capillary iso tachophoresis. In addition to patient analysis we investigated the influence of lipid lowering drugs on the lipoprotein subfraction distribution during therapy with analytical capillary isotachophoresis.     

Confirmation that SNPs in the high mobility group‐A2 gene (HMGA2) are associated with adult height in the Japanese population; wide‐ranging population survey of height‐related SNPs in HMGA2

Adult height is a highly heritable trait in that multiple genes are involved. Recent genome‐wide association studies have identified a novel single‐nucleotide polymorphism (SNP) rs1042725 in the high mobility group‐A2 gene (HMGA2) and shown it to be associated with human height in Caucasian populations. We performed a replication study to examine the associations between SNPs in HMGA2 and adult height in the Japanese population based on autopsy cases. Although we could not confirm a significant association between rs1042725 in HMGA2 and adult height, another SNP, rs7968902, in the gene achieved significance for its association in the same populations, and the effect was the same as that documented previously. These findings permit us to conclude that the SNPs in HMGA2 are common variants influencing human height across different populations. Moreover, a worldwide population study of these SNPs using 14 different populations including Asians, Africans and Caucasians demonstrated that both haplotypes and genotypes for three height‐related SNPs (rs1042725, rs7968682 and rs7968902) in HMGA2 were distributed in an ethnicity‐dependent manner. This information will be useful for clarifying the genetic basis of human height.     

Polymorphisms in genes involved in innate immunity and susceptibility to benzene-induced hematotoxicity

Benzene, a recognized hematotoxicant and carcinogen, can damage the human immune system. We studied the association between single nucleotide polymorphisms (SNPs) in genes involved in innate immunity and benzene hematotoxicity in a cross-sectional study of workers exposed to benzene (250 workers and 140 controls). A total of 1,236 tag SNPs in 149 gene regions of six pathways were included in the analysis. Six gene regions were significant for their association with white blood cell (WBC) counts (MBP, VCAM1, ALOX5, MPO, RAC2, and CRP) based on gene-region (P<0.05) and SNP analyses (FDR<0.05). VCAM1 rs3176867, ALOX5 rs7099684, and MPO rs2071409 were the three most significant SNPs. They showed similar effects on WBC subtypes, especially granulocytes, lymphocytes, and monocytes. A 3-SNP block in ALOXE3 (rs7215658, rs9892383, and rs3027208) showed a global association (omnibus P = 0.0008) with WBCs even though the three SNPs were not significant individually. Our study suggests that polymorphisms in innate immunity genes may play a role in benzene-induced hematotoxicity; however, independent replication is necessary.     

Associations between DRDs and schizophrenia in a Korean population: multi-stage association analyses

The dysregulation of the dopaminergic system has been implicated in the pathophysiology of major psychosis, including schizophrenia, with dopamine receptor genes (DRDs) presently targeted as the most promising candidate genes. We investigated DRD1-5 for association with schizophrenia using a multi-stage approach in a Korean sample. One hundred forty-two SNPs in DRD1-5 were selected from the dbSNP, and the associations of each SNP were then screened and typed by MALDI-TOF mass spectrometry using pooled DNA samples from 150 patients with major psychosis and 150 controls. Each of the suggested SNPs was then genotyped and tested for an association within the individual samples comprising each pool. Finally, the positively associated SNPs were genotyped in an extended sample of 270 patients with schizophrenia and 350 controls. Among the 142 SNPs, 88 (62%) SNPs in our Korean population were polymorphic. At the pooling stage, 10 SNPs (DRD1: 2, DRD2: 3, and DRD4: 5) were identified (P<0.05). SNPs rs1799914 of DRD1 (P=0.046) and rs752306 of DRD4 (P=0.017) had significantly different allele frequencies in the individually genotyped samples comprising the pool. In the final stage, with the extended sample, the suggestive association of DRD4 with rs752306 was lost, but the association of DRD1 with rs1799914 gained greater significance (P=0.017). In these large-scale multi-stage analyses, we were able to find a possible association between DRD1 and schizophrenia. These findings suggested the potential contribution of a multi-step strategy for finding genes related to schizophrenia.     

Synthesis,biological activity and toxicity of chromium(III) metformin complex as potential insulin-mimetic agent in C57BL/6 mice

As an oral hypoglycemic agent, metformin (Met) has become a best-selling inexpensive drug worldwide. In this thesis, [Cr(metformin)₃] (CrMet) complex was synthesized and characterized by elemental analysis (EA), electrospray ionization mass spectrometry (ESI-MS), nuclear magnetic resonance (NMR), infrared (IR), UV-visible (UV-vis), and molar conductivity. Meanwhile, the molecule structure of CrMet complex was optimized using Gaussian 09. Considering the therapeutic effect of Met and Met/Cr(III) complex on type 2 diabetes mellitus (T2DM), the biological activities of CrMet in streptozocin (STZ)-induced diabetic mice were evaluated in detail from the aspects of fasting blood glucose (FBG), fasting serum insulin (FINS), triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) levels. These results indicated that CrMet had beneficial function on blood glucose (BG) and lipid metabolism for diabetes. Additionally, the results of cytotoxicity and toxicity experiments showed that CrMet had no damage to cells and relatively high safety in mice. It maybe a potential candidate as a therapeutic agent in T2DM.     

Sequence-based analysis of 5′UTR and coding regions of CASP3 in terms of miRSNPs and SNPs in targetting miRNAs

Apoptosis is described as a mechanism of cell death occurring after adequate cellular harm. Deregulation of apoptosis occurs in many human conditions such as autoimmune disorders, ischemic damage, neurodegenerative diseases and different cancer types. Information relating miRNAs to cancer is increasing. miRNAs can affect development of cancer via many different pathways, including apoptosis. Polymorphisms in miRNA genes or miRNA target sites (miRSNPs) can change miRNA activity. Although polymorphisms in miRNA genes are very uncommon, SNPs in miRNA-binding sites of target genes are quite common. Many researches have revealed that SNPs in miRNA target sites improve or decrease the efficacy of the interaction between miRNAs and their target genes. Our aim was to specify miRSNPs on CASP3 gene (caspase-3) and SNPs in miRNA genes targeting 5′UTR and coding exons of CASP3, and evaluate the effect of these miRSNPs and SNPs of miRNA genes with respect to apoptosis. We detected 141 different miRNA binding sites (126 different miRNAs) and 7 different SNPs in binding sites of miRNA in 5′UTR and CDS of CASP3 gene. Intriguingly, miR-339-3p’s binding site on CASP3 has a SNP (rs35372903, G/A) on CASP3 5′UTR and its genomic sequence has a SNP (rs565188493, G/A) at the same nucleotide with rs35372903. Also, miR-339-3p has two other SNPs (rs373011663, C/T rs72631820, A/G) of which the first is positioned at the binding site. Here, miRSNP (rs35372903) at CASP3 5′UTR and SNP (rs565188493) at miR-339-3p genomic sequence cross-matches at the same site of binding region. Besides, miR-339-3p targets many apoptosis related genes (ZNF346, TAOK2, PIM2, HIP1, BBC3, TNFRSF25, CLCF1, IHPK2, NOL3) although it had no apoptosis related interaction proven before. This means that miR-339-3p may also have a critical effect on apoptosis via different pathways other than caspase-3. Hence, we can deduce that this is the first study demonstrating a powerful association between miR-339-3p and apoptosis upon computational analysis.     

Global analysis of single nucleotide polymorphisms in the exons of human deoxyribonuclease I-like 1 and 2 genes

Several SNPs in the deoxyribonuclease I-like 1 (DNase 1L1) and DNase 1L2 were investigated. In the present study, the genotype distributions of three synonymous SNPs (V59V, rs1050095; P67P, rs1130929; A277A, rs17849495) in the DNase 1L1 gene and four non-synonymous SNPs, V122I (rs34952165), Q170H (rs6643670), and D227A (rs5987256) in the DNase 1L1 gene, as well as D197A (rs62621282) in the DNase 1L2 gene were investigated in 13 populations. In all the populations, no variation was found in four SNPs (V59V, Q170H, D227A, and A277A) in DNASE1L1 or in D197A in DNASE1L2. As for V122I, only the German population showed a low degree of polymorphism. The SNP V122I in DNASE1L1 was monoallelic for the G-allele in all of the Asian and African populations examined, with no polymorphism being evident. Since the A-allele in SNP V122I was distributed in only the Caucasian populations, not in the other ethnic groups, it was confirmed that the A-allele in SNP V122I was Caucasian-specific. On the other hand, only P67P in DNASE1L1 was polymorphic among three synonymous SNPs. The effect of nucleotide substitution corresponding to polymorphic SNP P67P on DNase 1L1 activity was examined: the corresponding nucleotide substitution in polymorphic SNP P67P has little effect on the DNase activity.     

Genetic association of the EGR2 gene with bipolar disorder in Korea

The early growth response gene 2 (EGR2) is located at chromosome 10q21, one of the susceptibility loci in bipolar disorder (BD). EGR2 is involved in cognitive function, myelination, and signal transduction related to neuregulin-ErbB receptor, Bcl-2 family proteins, and brain-derived neurotrophic factor. This study investigated the genetic association of the EGR2 gene with BD and schizophrenia (SPR) in Korea. In 946 subjects (350 healthy controls, 352 patients with BD, and 244 with SPR), nine single nucleotide polymorphisms (SNPs) in the EGR2 gene region were genotyped. Five SNPs showed nominally significant allelic associations with BD (rs2295814, rs61865882, rs10995315, rs2297488, and rs2297489), and the positive associations of all except rs2297488 remained significant after multiple testing correction. Linkage disequilibrium structure analysis revealed two haplotype blocks. Among the common identified haplotypes (frequency > 5%), 'T-G-A-C-T (block 1)' and 'A-A-G-C (block 2)' haplotypes were over-represented, while 'C-G-G-T-T (block 1)' haplotype was under-represented in BD. In contrast, no significant associations were found with SPR. Although an extended analysis with a larger sample size or independent replication is required, these findings suggest a genetic association of EGR2 with BD. Combined with a plausible biological function of EGR2, the EGR2 gene is a possible susceptibility gene in BD.     

Softness of atherogenic lipoproteins: a comparison of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) using elastic incoherent neutron scattering (EINS)

Apolipoprotein B100 (apoB100)-containing plasma lipoproteins (LDL and VLDL) supply tissues and cells with cholesterol and fat. During lipolytic conversion from VLDL to LDL the size and chemical composition of the particles change, but the apoB100 molecule remains bound to the lipids and regulates the receptor mediated uptake. The molecular physical parameters which control lipoprotein remodeling and enable particle stabilization by apoB100 are largely unknown. Here, we have compared the molecular dynamics and elasticities of VLDL and LDL derived by elastic neutron scattering temperature scans. We have determined thermal motions, dynamical transitions, and molecular fluctuations, which reflect the temperature-dependent motional coupling between lipid and protein. Our results revealed that lipoprotein particles are extremely soft and flexible. We found substantial differences in the molecular resiliences of lipoproteins, especially at higher temperatures. These discrepancies not only can be explained in terms of lipid composition and mobility but also suggest that apoB100 displays different dynamics dependent on the lipoprotein it is bound to. Hence, we suppose that the inherent conformational flexibility of apoB100 permits particle stabilization upon lipid exchange, whereas the dynamic coupling between protein and lipids might be a key determinant for lipoprotein conversion and atherogenicity.     

Hypocholesterolemic Effect of Blackcurrant (Ribes nigrum) Extract in Healthy Female Subjects: A Pilot Study

Blackcurrant extract (BCE) ameliorates dyslipidemia in menopausal model animals and in elderly women at a risk of dyslipidemia. However, it is unknown whether the daily intake of BCE can prevent lipid abnormalities in healthy individuals. Lipids are essential for the body, but they also cause arteriosclerosis. In this noncomparative pilot study, we examined the effects of BCE administered for 29 days on serum lipids in young healthy women. Blood samples were collected before and on days 4 and 29 after BCE intake, and 20 lipoprotein fractions in the serum were separated using a gel-permeation high-performance liquid chromatography method to measure the triacylglycerol and cholesterol levels in lipoproteins. There were no effects on lipids on day 4 of BCE intake, but the total cholesterol level decreased on day 29. Furthermore, the levels of total very-low-density lipoprotein (VLDL) cholesterol, small VLDL cholesterol, and large low-density lipoprotein cholesterol were significantly decreased. These results suggest that the daily intake of BCE has a hypocholesterolemic effect in healthy women, and that it is effective in preventing atherosclerosis.     

A rapid gel electrophoretic chip for serum cholesterol determination

We present a rapid gel electrophoretic chip, composed of 2.5% (w/v) acrylamide and 1% (w/v) agarose gel, for serum cholesterol determination using a photo lithography technique. After optimizations, we determined the lipoprotein concentration of standard serum using a conventional enzyme method. The serum was diluted, stained and loaded for 15 min onto the chip. After loading, the intensities of low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) bands separated at the chip were estimated using an image analyzer. The intensities of these bands corresponded to concentrations obtained from a standard enzyme-based method. The detected LDL-C and HDL-C concentrations were linear up to 146 mg dL(-1) and 53 mg dL(-1) respectively. Finally, we carried out the cholesterol analysis using real biological samples obtained from nine volunteers using our electrophoretic chip. The LDL-C and HDL-C levels detected using our chip correlated well with the results obtained using the conventional enzyme-based method r(2) = 0.98 and r(2) = 0.86 for LDL-C and HDL-C, respectively. Although our sample size is small and confined only to health volunteers, we have demonstrated that this proof-of-concept gel electrophoretic chip can determine lipoproteins, simultaneously.     

Enzymatic determination of the major serum lipids with a common indicator reaction

Enzymatic, colorimetric assays for serum cholesterol, choline-containing phospholipids, and triglycerides were developed with the intent of being able to specifically determine these individual lipids after isolation of serum lipoproteins by a technology such as ultracentrifugation that is already developed, electrophoresis that is already available for cholesterol and awaits the other lipid stains, or chemical fractionation. Once the relative amounts of the lipids in each lipoprotein fraction are known, the absolute amounts in each separated fraction can then be calculated using the total serum concentrations. As a secondary purpose, the measurement and arithmetic summation of serum cholesterol, choline-containing phospholipids, and triglycerides concentrations appears to provide a close estimate of the major serum total lipids. This summation technique results in apparent lower serum total lipids values than are obtained by a conventional turbidimetric method. This bias appears to be due to a combination of overestimation by the turbidimetric method and underestimation by the summation technique. However, this summation technique appears to be able to provide more information for specific lipid measurements on chemical, ultracentrifugal, or electrophoretic separation of lipoproteins by revealing which lipid class(es) is/are present in an abnormal concentration. It could also prove useful in providing major total lipid information for nonspecific staining of electrophoretic zones where fat-soluble stains such as Sudan black B are presently used. In the latter case, cholesterol and triglycerides are already determined and only the choline-containing phospholipid assay is additional.     

Electrochemical behaviour of carbon paste electrodes enriched with tin oxide nanoparticles using voltammetry and electrochemical impedance spectroscopy

The effect of the SnO(2) nanoparticles (SNPs) on the behaviour of voltammetric carbon paste electrodes were studied for possible use of this material in biosensor development. The electrochemical behaviour of SNP modified carbon paste electrodes (CPE) was first investigated by using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques. The performance of the SNP modified electrodes were compared to those of unmodified ones and the parameters affecting the response of the modified electrode were optimized. The SNP modified electrodes were then tested for the electrochemical sensing of DNA purine base adenine to explore their further development in biosensor applications.     

Solanum anguivi Lam. Fruits: Their Potential Effects on Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder of glucose homeostasis associated with a status of insulin resistance, impaired insulin signaling, β-cell dysfunction, impaired glucose and lipid metabolism, sub-clinical inflammation, and increased oxidative stress. Consuming fruits and vegetables rich in phytochemicals with potential antidiabetic effects may prevent T2DM and/or support a conservative T2DM treatment while being safer and more affordable for people from low-income countries. Solanum anguivi Lam. fruits (SALF) have been suggested to exhibit antidiabetic properties, potentially due to the presence of various phytochemicals, including saponins, phenolics, alkaloids, ascorbic acid, and flavonoids. For the saponin fraction, antidiabetic effects have already been reported. However, it remains unclear whether this is also true for the other phytochemicals present in SALF. This review article covers information on glucose homeostasis, T2DM pathogenesis, and also the potential antidiabetic effects of phytochemicals present in SALF, including their potential mechanisms of action.     

Analysis of Fatty Acid, and Lipoprotein, Metabolism by GC and HPLC: Effect of Low-Density Lipoprotein Apheresis

 Blood samples from 5 hyperlipidemic patients on chronic treatment with low-density lipoprotein (LDL) – apheresis were analysed for lipids and fatty acids in serum, lipoprotein fractions and erythrocyte membrane by capillary gas chromatography (GC), reversed-phase high-performance liquid chromatography (LC), spectrofluorometry and spectrophotometry. LDL-apheresis has been associated with significant changes of fatty acids metabolism in relation to triglyceride-rich lipoproteins. Oleic acid may exert its hypotriglyceridemic effect via VLDL, IDL, LDL and HDL fractions. Polyunsaturated fatty acids, associated with triglyceride metabolism via IDL or VLDL, are linoleic, gamma-linolenic and docosahexaenoic fatty acids. Received November 25, 1999. Revision September 5, 2000.     

Anion-exchange HPLC separation of five major rabbit lipoproteins using a nonporous diethylaminoethyl-ligated gel with a perchlorate-containing eluent

Rabbits are often used as experimental animals in studies of atherosclerosis and hyperlipidemia. Although rabbit lipoproteins can be quantitated by sequential ultracentrifugation, a simpler and more reproducible method is desirable for detailed analyses. The current study describes a method to analyze rabbit lipoproteins in plasma by anion‐exchange high‐performance liquid chromatography using a column filled with nonporous, diethylaminoethyl‐ligated polymers. A solution of NaClO₄ was used to adjust the ionic strength of the eluent. The method required only 15 μL of plasma and analysis was completed in 23 min. Five lipoprotein fractions (high‐density lipoprotein, low‐density lipoprotein, intermediate‐density lipoprotein, very‐low‐density lipoprotein and chylomicrons) were eluted with step‐wise increases in a concentration of NaClO₄. The post‐column eluate was reacted with an enzymatic reagent to determine total cholesterol, and the lipoprotein‐cholesterol fraction was calculated according to relative peak areas in the chromatogram. The within‐day and between‐day assay coefficients of variation for lipoprotein cholesterol levels ranged between 0.436 and 7.143% and between 2.905 and 10.526%, respectively. Administering a high‐fat diet increased lipoprotein‐cholesterol concentrations by 6‐ to 77‐fold. The method described here was nevertheless able to quantitate levels of lipoprotein‐cholesterol in plasma samples from these rabbits. These results indicate that this method may be applied to lipoprotein studies using hyperlipidemic rabbit models. Copyright © 2011 John Wiley & Sons, Ltd.     

An effective and rapid determination by MALDI/TOF/TOF of methionine sulphoxide content of ApoA‐I in type 2 diabetic patients

Increased oxidation of low density lipoprotein (LDL) is characteristic of atherosclerosis. In this frame, high density lipoproteins (HDL) play an important role, being able to remove lipid peroxides (LPOs) and cholesterol from oxidized LDL, so exhibiting a protective role against atherosclerosis. A wide range of reactive compounds lead to the oxidation of methionine (Met) residues with the formation of methionine sulphoxide (MetO) in apolipoprotein A‐I (ApoA‐I). Consequently, the determination of MetO level can give both an evaluation of oxidative stress and the reduced capability of ApoA‐I in LPOs and cholesterol transport. For these reasons, the development of analytical methods able to determine the MetO level is surely of interest, and we report here the results obtained by MALDI mass spectrometry. Copyright © 2013 John Wiley & Sons, Ltd.     

THE ROLE OF LIPOPROTEINS IN THE DISTRIBUTION OF TIN ETIOPURPURIN (SnET2) IN THE TUMOR-BEARING RAT

The role of plasma lipoproteins in the distribution of the photosensitizing agent tin etiopurpurin (SnET2) was examined in male rats bearing the N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide-induced tumor. Treatement with 17α-ethinyl estradiol resulted in the depletion of total plasma cholesterol by > 70% and a corresponding decrease in plasma lipoproteins. To both control and estradiol-treated animals, a therapeutic dose (1.5mg/kg) of SnET2 was administered and biodistribution measured 24 h later. Estradiol treatment was not associated with difference in the distribution of SnET2 to liver, skin or tumor, or in the pattern of affinity of SnET2 to plasma albumin and lipoprotein. These results indicate that a substantial decrease in circulating lipoprotein levels does not alter patterns of SnET2 biodistribution.     

Induction of hepatic inducible nitric oxide synthase by cholesterol in vivo and in vitro

Cholesterol-rich diet impairs endothelial NO synthase (eNOS) and enhances inducible NOS (iNOS) expression. In this study, we investigated effects of cholesterol on iNOS expression in high-fat-fed rat models, HepG2 and RAW264.7 cells. The high-fat diet increased the plasma total cholesterol level 6-7 fold and low-density lipoprotein cholesterol level (LDL-C) approximately 70 fold and slightly increased the level of lipid peroxidation as determined by thiobarbituric acid-reactive substance assay. The high-fat diet also increased plasma nitric oxide (NO) concentrations up to 5 fold, and induced iNOS mRNA expression in liver. The contractile responses of the endothelium-denuded thoracic aortic rings to phenylephrine were significantly damaged in high-fat-fed rats when assessed by organ chamber study. Treatment with estrogen for 4 days failed to reduce iNOS expressions as well as aortic contractility, although it improved lipid profiles. In cultured HepG2 or murine macrophage RAW264.7 cells, 3 days treatment with either 25-hydroxycholesterol or 7-ketocholesterol induced iNOS mRNA expression, as determined by RT-PCR. Our data suggested that the chronic exposure of hepatocytes and macrophage cells to high concentration of cholesterol or oxysterols may induce iNOS expression and subsequent synthesis of NO, which may be important in the pathogenesis of atherosclerosis.