Hepatic Synaptotagmin 1 is involved in the remodelling of liver plasma- membrane lipid composition and gene expression in male Apoe-deficient mice consuming a Western diet.

BACKGROUND AND AIMS: The molecular mechanisms by which the liver develops steatotic disease still remain unclear. Previous studies using nutritional and genetic models of hepatic steatosis in mice showed that liver synaptotagmin 1 (Syt1) expression was associated with lipid droplet area. Hepatic Syt1 overexpression was used as a tool to explore its effect on hepatic and plasma lipids. METHODS AND RESULTS: To find out a cause-effect, hepatic mouse Syt1 mRNA was cloned into a vector driving hepatocyte-specific expression and administered by hydrodynamic injection to male Apoe-deficient mice fed on a Western diet, the latter as a model of rapid spontaneous steatosis development. Hepatic microsomal, large vesicle, lysosomal and plasma membrane fractions were enriched in SYT1 protein following gene overexpression. In these conditions, very low density lipoprotein esterified cholesterol increased. Likewise, the transgene caused an alteration in lipid droplet surface and a positive correlation between Syt1 expression and hepatic total cholesterol content. A lipidomic approach evidenced a decrease in lysophosphatidylcholine, phosphatidylcholine and triglycerides in isolated plasma membrane fraction. Expressions of genes involved in biosynthesis of bile acids, fatty acid metabolism, lipoprotein dynamics and vesicular transport were modified by the increased SYT1 expression. CONCLUSIONS: These results indicate that this protein is involved in hepatic management of lipids and in the regulation of genes involved in lipid metabolism.

Diagnosis of genetic amyloidosis through the analysis of transthyretin gene mutation using high-resolution melting.

Transthyretin amyloidosis can be either the wild-type (ATTR-wt) or the hereditary form (ATTR-m) with autosomal dominant inheritance. ATTR seems to be an underdiagnosed disease, despite now being recognized as one of the most frequent causes of heart failure (HF) with preserved ejection fraction. The confirmation of diagnosis includes a genetic analysis as a critical step to distinguish between ATTR-wt and hereditary amyloidosis. The present study aimed to evaluate the potential application of High-Resolution Melting (HRM) analysis for identifying gene mutations in patients with suspected ATTR-m. We have adapted and validated the use of HRM for TTR mutations. We, therefore, sequenced the TTR gene and used HRM in a group of 134 patients suspected of suffering from amyloidosis. Seven patients were diagnosed with mutations in the TTR gene (p.Glu74Gln, heterozygous p.Val142Ile, and homozygous p.Val142Ile). HRM is capable of clearly detecting these TTR mutations, including the heterozygous and homozygous variants. The results show a 100% correlation between the HRM study and TTR sequencing. These results support future studies of applying HRM analysis as a diagnostic approach for ATTR-m, mainly for epidemiological studies.

Several phosphate transport processes are present in vascular smooth muscle cells.

We have studied inorganic phosphate (Pi) handling in rat aortic vascular smooth muscle cells (VSMC) using 32P-radiotracer assays. Our results have revealed a complex set of mechanisms consisting of 1) well-known PiT1/PiT2-mediated sodium-dependent Pi transport; 2) Slc20-unrelated sodium-dependent Pi transport that is sensitive to the stilbene derivatives 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS); 3) a sodium-independent Pi uptake system that is competitively inhibited by sulfate, bicarbonate, and arsenate and is weakly inhibited by DIDS, SITS, and phosphonoformate; and 4) an exit pathway from the cell that is partially chloride dependent and unrelated to the known anion-exchangers expressed in VSMC. The inhibitions of sodium-independent Pi transport by sulfate and of sodium-dependent transport by SITS were studied in greater detail. The maximal inhibition by sulfate was similar to that of Pi itself, with a very high inhibition constant (212 mM). SITS only partially inhibited sodium-dependent Pi transport, but the Ki was very low (14 µM). Nevertheless, SITS and DIDS did not inhibit Pi transport in Xenopus laevis oocytes expressing PiT1 or PiT2. Both the sodium-dependent and sodium-independent transport systems were highly dependent on VSMC confluence and on the differentiation state, but they were not modified by incubating VSMC for 7 days with 2 mM Pi under nonprecipitating conditions. This work not only shows that the Pi handling by cells is highly complex but also that the transport systems are shared with other ions such as bicarbonate or sulfate.NEW & NOTEWORTHY In addition to the inorganic phosphate (Pi) transporters PiT1 and PiT2, rat vascular smooth muscle cells show a sodium-dependent Pi transport system that is inhibited by DIDS and SITS. A sodium-independent Pi uptake system of high affinity is also expressed, which is inhibited by sulfate, bicarbonate, and arsenate. The exit of excess Pi is through an exchange with extracellular chloride. Whereas the metabolic effects of the inhibitors, if any, cannot be discarded, kinetic analysis during initial velocity suggests competitive inhibition.

Identifying early pathogenic events during vascular calcification in uremic rats.

Vascular calcification in chronic kidney disease is a very complex process traditionally explained in multifactorial terms. Here we sought to clarify relevance of the diverse agents acting on vascular calcification in uremic rats and distinguish between initiating and complicating factors. After 5/6 nephrectomy, rats were fed a 1.2% phosphorus diet and analyzed at different time points. The earliest changes observed in the aortic wall were noticed 11 weeks after nephrectomy: increased Wnt inhibitor Dkk1 mRNA expression and tissue non-specific alkaline phosphatase (TNAP) expression and activity. First deposits of aortic calcium were observed after 12 weeks in areas of TNAP expression. Increased mRNA expressions of Runx2, BMP2, Pit1, Pit2, HOXA10, PHOSPHO1, Fetuin-A, ANKH, OPN, Klotho, cathepsin S, MMP2, and ENPP1 were also found after TNAP changes. Increased plasma concentrations of activin A and FGF23 were observed already at 11 weeks post-nephrectomy, while plasma PTH and phosphorus only increased after 20 weeks. Plasma pyrophosphate decreased after 20 weeks, but aortic pyrophosphate was not modified, nor was the aortic expression of MGP, Msx2, several carbonic anhydrases, osteoprotegerin, parathyroid hormone receptor-1, annexins II and V, and CD39. Thus, increased TNAP and Dkk1 expression in the aorta precedes initial calcium deposition, and this increase is only preceded by elevations in circulating FGF23 and activin A. The expression of other agents involved in vascular calcification only changes at later stages of chronic kidney disease, in a complex branching pattern that requires further clarification.

Postprandial changes in high density lipoproteins in rats subjected to gavage administration of virgin olive oil.

BACKGROUND AND AIMS: The present study was designed to verify the influence of acute fat loading on high density lipoprotein (HDL) composition, and the involvement of liver and different segments of small intestine in the changes observed. METHODS AND RESULTS: To address these issues, rats were administered a bolus of 5-ml of extra-virgin olive oil and sacrificed 4 and 8 hours after feeding. In these animals, lipoproteins were analyzed and gene expressions of apolipoprotein and HDL enzymes were assessed in duodenum, jejunum, ileum and liver. Using this experimental design, total plasma and HDL phospholipids increased at the 8-hour-time-point due to increased sphingomyelin content. An increase in apolipoprotein A4 was also observed mainly in lipid-poor HDL. Increased expression of intestinal Apoa1, Apoa4 and Sgms1 mRNA was accompanied by hepatic decreases in the first two genes in liver. Hepatic expression of Abcg1, Apoa1bp, Apoa2, Apoe, Ptlp, Pon1 and Scarb1 decreased significantly following fat gavage, while no changes were observed for Abca1, Lcat or Pla2g7. Significant associations were also noted for hepatic expression of apolipoproteins and Pon1. Manipulation of postprandial triglycerides using an inhibitor of microsomal transfer protein -CP-346086- or of lipoprotein lipase -tyloxapol- did not influence hepatic expression of Apoa1 or Apoa4 mRNA. CONCLUSION: All these data indicate that dietary fat modifies the phospholipid composition of rat HDL, suggesting a mechanism of down-regulation of hepatic HDL when intestine is the main source of those particles and a coordinated regulation of hepatic components of these lipoproteins at the mRNA level, independently of plasma postprandial triglycerides.

In comparison with palm oil, dietary nut supplementation delays the progression of atherosclerotic lesions in female apoE-deficient mice.

Epidemiological studies have demonstrated the benefits of nut consumption on cardiovascular risk factors and CHD, attributed to their fatty acid profile, rich in unsaturated fatty acids, and also to other nutrients. The effect of nuts on atherosclerotic lesions was studied in female and male apoE-knockout mice fed a diet supplemented with 3 % (w/w) mixed nuts (mix: almonds, hazelnuts and walnuts in a proportion of 0.25:0·25:0.50, respectively), and compared with mice receiving an isoenergetic diet of similar fat content provided as palm oil. After 12 weeks, plasma lipid parameters and aortic lesions were measured. Males receiving nuts had lower plasma cholesterol than the palm oil group, and both sex groups had lower plasma non-HDL-cholesterol and lower content of reactive oxygen species in LDL than mice receiving the palm oil diet, the latter decrease being more pronounced in females than in males. Females consuming the nut diet showed a smaller aortic lesion area than those consuming palm oil, whereas no differences were observed in males. In females, hepatic paraoxonase 2 (Pon2) mRNA increased, and no change was observed in prenylcysteine oxidase 1 (Pcyox1) expression after the consumption of the nut-containing diet. In addition, aortic atherosclerotic lesions correlated directly with total plasma cholesterol and inversely with hepatic Pon2 expression. The results suggest that the beneficial effect of nut intake in female apoE-deficient mice may be attributed to reduced non-HDL-cholesterol levels and enhanced PON2 antioxidant activity.

Cystathionine ß-synthase deficiency causes infertility by impairing decidualization and gene expression networks in uterus implantation sites.

Hyperhomocysteinemia has been reported in human reproduction as a risk factor for early pregnancy loss, preeclampsia, and congenital birth defects like spina bifida. Female infertility was also observed in cystathionine beta synthase-deficient mice (Cbs-KO) as an animal model for severe hyperhomocysteinemia. The aim for the present research was to elucidate the time-point of pregnancy loss and to pinpoint gene and cellular changes involved in the underlying pathological mechanism. By mating 90-day-old wild-type and Cbs-KO female mice with their homologous male partners, we found that pregnancy loss in Cbs-KO occurred between the 8th and 12th gestation day during placenta formation. DNA microarrays were carried out on uterus from implantation and interimplantation samples obtained on day 8. The results allowed us to select genes potentially involved in embryo death; these were individually confirmed by RT-qPCR, and their expressions were also followed throughout pregnancy. We found that changes in expression of Calb1, Ttr, Expi, Inmt, Spink3, Rpgrip1, Krt15, Mt-4, Gzmc, Gzmb, Tdo2, and Afp were important for pregnancy success, since a different regulation in Cbs-KO mice was found. Also, differences in relationships among selected genes were observed, indicating a dysregulation of these genes in Cbs-KO females. In conclusion, our data provide more information on the gene expression cascade and its timely regulated process required for a successful pregnancy. In addition, we unveil new potential avenues to explore further investigations in pregnancy loss.

Proteomics and gene expression analyses of mitochondria from squalene-treated apoE-deficient mice identify short-chain specific acyl-CoA dehydrogenase changes associated with fatty liver amelioration.

Squalene, a hydrocarbon involved in cholesterol biosynthesis, is an abundant component in virgin olive oil. Previous studies showed that its administration decreased atherosclerosis and steatosis in male apoE knock-out mice. To study the effect of squalene on mitochondrial proteins in fatty liver, 1 g/kg/day of this isoprenoid was administered to those mice. After 10 weeks, hepatic fat was assessed and protein extracts from mitochondria enriched fractions from control and squalene-treated animals were analyzed by 2D-DIGE. Spots exhibiting significant differences were identified by MS analysis. Squalene administration modified the expression of eighteen proteins involved in different metabolic processes, 12 associated with hepatic fat content. Methionine adenosyltransferase I alpha (Mat1a) and short-chain specific acyl-CoA dehydrogenase (Acads) showed significant increased and decreased transcripts, respectively, consistent with their protein changes. These mRNAs were also studied in wild-type mice receiving squalene, where Mat1a was found increased and Acads decreased. However, this mRNA was significantly increased in the absence of apolipoprotein E. These results suggest that squalene action may be executed through a complex regulation of mitochondrial protein expression, including changes in Mat1a and Acads levels. Indeed, Mat1a is a target of squalene administration while Acads reflects the anti-steatotic properties of squalene.

Postprandial transcriptome associated with virgin olive oil intake in rat liver.

Liver has been proposed as a gatekeeper that regulates postprandial lipemia and a potential target for regulation by acute intake of virgin olive oil. To characterize the hepatic gene expression response to a fat gavage, male rats were fed a bolus of 5 ml of extra-virgin olive oil and the hepatic mRNA expression analyzed 4 hours later using DNA microarrays. To provide an initial screening of candidate genes, only twenty one with remarkably modified expression between both conditions (signal log2 ratio > 2.5 or < -2.5) were considered and confirmed by quantitative real time PCR. Those that presented biological significance were also analyzed 8 hours after the experimental approach. Hepatic A2m Slc13a5 and Nrep mRNA expressions were found significantly changed in both studied conditions and showed the highest significant associations with postprandial plasma triglycerides and lack of association with basal triglyceridemia. These results highlight new gene regulation in liver by postprandial triglyceridemia and will help to understand the complex human pathology providing the involvement of hepatic proteins and new strategies to cope with postprandial metabolism.

Cysteinemia, rather than homocysteinemia, is associated with plasma apolipoprotein A-I levels in hyperhomocysteinemia: lipid metabolism in cystathionine beta-synthase deficiency.

OBJECTIVE: Genetic and dietary hyperhomocysteinemia has been found to decrease high density lipoproteins (HDL) and their apolipoprotein A1 (APOA1). To test the hypothesis that the presence of cysteine could normalize HDL levels in hyperhomocysteinemic cystathionine beta-synthase (Cbs)-deficient mice and that the inclusion of glycine would block this effect. METHODS: Lipids and HDL cholesterol were studied in Cbs-deficient mice and wild-type animals fed a low-methionine diet supplemented with cysteine and glycine and in Cbs-deficient mice on the same diet supplemented only with cysteine. RESULTS: Triglyceride and homocysteine levels were significantly decreased and increased, respectively in Cbs-deficient mice irrespective of treatment. However, plasma cholesterol, glucose and APOA1 were significantly decreased in homozygous Cbs-deficient mice when they received the cysteine and glycine-enriched beverage. This group of mice also showed decreased mRNA levels and increased hepatic content of APOA1 protein, the latter increase was observed in endothelial cells. A significant, inverse relationship was observed between plasma and hepatic APOA1 concentrations while a positive one was found between plasma levels of cysteine and APOA1. CONCLUSION: These data suggest an altered hepatic management of APOA1 and that cysteine may be involved in the control of this apolipoprotein at this level. Overall these findings represent a new aspect of dietary regulation of HDL at the hepatic transendothelial transport.

Microarray analysis of hepatic gene expression identifies new genes involved in steatotic liver.

UNLABELLED: Trans-10, cis-12-conjugated linoleic acid (CLA)-enriched diets promote fatty liver in mice, while cis-9, trans-11-CLA ameliorates this effect, suggesting regulation of multiple genes. To test this hypothesis, apoE-deficient mice were fed a Western-type diet enriched with linoleic acid isomers, and their hepatic gene expression was analyzed with DNA microarrays. To provide an initial screening of candidate genes, only 12 with remarkably modified expression between both CLA isomers were considered and confirmed by quantitative RT-PCR. Additionally mRNA expression of 15 genes involved in lipid metabolism was also studied. Ten genes (Fsp27, Aqp4, Cd36, Ly6d, Scd1, Hsd3b5, Syt1, Cyp7b1, and Tff3) showed significant associations among their expressions and the degree of hepatic steatosis. Their involvement was also analyzed in other models of steatosis. In hyperhomocysteinemic mice lacking Cbs gene, only Fsp27, Cd36, Scd1, Syt1, and Hsd3b5 hepatic expressions were associated with steatosis. In apoE-deficient mice consuming olive-enriched diet displaying reduction of the fatty liver, only Fsp27 and Syt1 expressions were found associated. Using this strategy, we have shown that expression of these genes is highly associated with hepatic steatosis in a genetic disease such as Cbs deficiency and in two common situations such as Western diets containing CLA isomers or a Mediterranean-type diet. CONCLUSION: The results highlight new processes involved in lipid handling in liver and will help to understand the complex human pathology providing new proteins and new strategies to cope with hepatic steatosis.

Knowledge of the biological actions of extra virgin olive oil gained from mice lacking apolipoprotein E.

The low incidence of cardiovascular disease in countries bordering the Mediterranean basin, where olive oil is the main source of dietary fat, has stimulated interest in the chemical composition of olive oil and in the production of other oils enriched with its minor components. This review summarizes what has been learned about the effects of different olive oil preparations on the development of atherosclerosis and about the prognostic value of associated plasma variables in the disease from experiments on genetically modified mice that spontaneously develop atherosclerosis. The limitations of this animal model associated with its morphological and physiological differences with humans are minimized by the similarity of the two genomes and by the potential for increased understanding attainable, given that the dietary interventions reported here would have taken 400 years to achieve in humans. As observed in traditional Mediterranean populations, it has been confirmed that extra virgin olive oil is beneficial when consumed judiciously and in a diet that is low in cholesterol due to the relative scarcity of animal products. Furthermore, the use of genomic techniques has led to the identification of new markers of response to olive oil. In conclusion, multidisciplinary research into extra virgin olive oil is expanding our knowledge of the substance's biological properties.

Lipopolysaccharide induces inhibition of galactose intestinal transport in rabbits in vitro.

BACKGROUND/AIMS: Previous studies from our laboratory have revealed impaired intestinal absorption of D-galactose in lipopolysaccharide-treated rabbits. The aim of the present work was to examine the effect of LPS on D-galactose intestinal absorption in vitro. METHODS: D-galactose intestinal transport was assessed employing three techniques: sugar uptake in rings of everted jejunum, transepithelial flux in Ussing-type chambers and transport assays in brush border membrane vesicles. The level of expression of the Na(+)/D-galactose cotransporter (SGLT1) was analyzed by Western blot. RESULTS: LPS decreased the mucosal D-galactose transport in rabbit jejunum but a preexposition to the endotoxin was required. LPS affected the Na(+)-dependent transport system by increasing the apparent Km value without affecting the Vmax. It also decreased the Na(+), K(+)-ATPase activity. However, it did not inhibit neither the uptake of D-galactose by brush border membrane vesicles nor modified the SGLT1 protein levels in the brush border, suggesting an indirect endotoxin effect. This inhibitory effect, was reduced by selective inhibitors of Ca(2+)-calmodulin (W13), protein kinase C (GF 109203X), p38 mitogen-activated protein kinase (SB 203580), c-Jun N-terminal kinase (SP 600125) and mitogen extracellular kinase (U 0126). CONCLUSION: LPS inhibits the mucosal Na(+)-dependent D-galactose intestinal absorption and the Na(+), K(+)-ATPase activity when it is added to the tissue. Intracellular processes related to protein kinases seem to be implicated in the endotoxin effect.

Simvastatin reverses the hypertension of heterozygous mice lacking cystathionine beta-synthase and apolipoprotein A-I.

Double heterozygous mice lacking Apoa1 and Cbs genes show mild hyperhomocysteinemia in combination with hypoalphalipoproteinemia. This situation leads to a moderate hypertension associated with a dysregulation in nitric oxide metabolism. The aim of this study was to investigate the potential beneficial effects of statin treatment in these mice. After 4 weeks of simvastatin administration, plasma parameters; apolipoproteins A-I, A-II and A-IV; lipid profile; and blood pressure were assessed, Western blotting was performed in the aorta of these mice to measure endothelial nitric oxide synthase and caveolin-1 content. The high blood pressure level present in the double heterozygous group was corrected down to that of the wild-type group after simvastatin treatment (124+/-7.7 vs. 109+/-11.2 mmHg, p<0.01). Concomitant with this effect, an increase in nitric oxide levels was observed in these double heterozygous mice receiving simvastatin treatment probably mediated in part by a decrease in caveolin-1 levels. Blood pressure changes appeared to be independent of the arylesterase activity of paraoxonase or the lipid content. Another remarkable result was the significant increase in apoA-IV content in animals receiving simvastatin, an effect considered to be protective for the endothelium. In conclusion, the results of this study demonstrate that the use of simvastatin can improve blood pressure control in mice with elevated homocysteinemia and low levels of apoA-I, and this effect is mediated by mechanisms independent of plasma lipids and related to nitric oxide levels.

Protein kinases, TNF-{alpha}, and proteasome contribute in the inhibition of fructose intestinal transport by sepsis in vivo.

Lipopolysaccharide (LPS) endotoxin is a causative agent of sepsis. The aim of this study was to examine LPS effects on intestinal fructose absorption and to decipher mechanisms. Sepsis was induced by intravenous injection of LPS in rabbits. The ultrastructural study and DNA fragmentation patterns were identical in the intestine of LPS and sham animals. LPS treatment reduced fructose absorption altering both mucosal-to-serosal transepithelial fluxes and uptake into brush border membrane vesicles (BBMVs). Cytochalasin B was ineffective on fructose uptake, indicating that GLUT5, but not GLUT2, transport activity was targeted. GLUT5 protein levels in BBMvs were lower in LPS than in sham-injected rabbits. Thus lower fructose transport resulted from lower levels of GLUT5 protein. LPS treatment decreased GLUT5 levels by proteasome-dependent degradation. Specific inhibitors of PKC, PKA, and MAP kinases (p38MAPK, JNK, MEK1/2) protected fructose uptake from adverse LPS effect. Moreover, a TNF-alpha antagonist blocked LPS action on fructose uptake. We conclude that intestinal fructose transport inhibition by LPS is associated with diminished GLUT5 numbers in the brush border membrane of enterocytes triggered by activation of several interrelated signaling cascades and proteasome degradation.

Microarray analysis of hepatic genes differentially expressed in the presence of the unsaponifiable fraction of olive oil in apolipoprotein E-deficient mice.

The hypothesis that the unsaponifiable fraction of olive oil dramatically influences hepatic gene expression was tested in mice. Two olive oils, obtained from the same olive cultivar but by different technological procedures, were characterized to show that they differed mainly in terms of the composition/quantity of this unsaponifiable fraction. Using DNA microarrays, hepatic gene expression was analysed in apoE-deficient mice fed one of two isoenergetic, isonitrogenous diets containing either 10 % (w/w) olive oil or unsaponifiable fraction-enriched olive oil. To provide an initial screening of potential candidate genes involved in a differential response, only genes with remarkably modified expression (signal log2 ratio >3 or < - 3) were further considered. The eleven genes fulfilling these prerequisites were confirmed by quantitative RT-PCR, and then analysed in apoE-deficient mice with a C57BL/6J genetic background. Orosomucoid and serum amyloid A2 were upregulated (to variable extents depending on the genetic background) in the absence of hepatic steatosis and inflammation. Fabp5 and Mt2 were also strongly upregulated. Several proteases were highly suppressed by the unsaponifiable-enriched olive diet, independent of the genetic background. The findings indicate that change in the expression of these genes is a good marker of the intake of the unsaponifiable fraction of olive oil. The results highlight the important biological effects of the unsaponifiable fraction of olive oil. The term 'monounsaturated fatty acid-enriched oil' no longer appears appropriate for describing all the oils to which it is currently applied since it does not adequately reflect that they have different biological effects.

Accelerated atherosclerosis in apolipoprotein E-deficient mice fed Western diets containing palm oil compared with extra virgin olive oils: a role for small, dense high-density lipoproteins.

To test the hypothesis that extra virgin olive oils from different cultivars added to Western diets might behave differently than palm oil in the development of atherosclerosis, apoE-deficient mice were fed diets containing different cultivars of olive oil for 10 weeks. Female mice were assigned randomly to one of the following five groups: (1-4) fed chow diets supplemented with 0.15% (w/w) cholesterol and 20% (w/w) extra virgin olive oil from the Arbequina, Picual, Cornicabra, or Empeltre cultivars, and (5) fed a chow diet supplemented with 0.15% cholesterol and 20% palm oil. Compared to diets containing palm oil, a Western diet supplemented with one of several varieties of extra virgin olive oil decreased atherosclerosis lesions, reduced plaque size, and decreased macrophage recruitment. Unexpectedly, total plasma paraoxonase activity, apoA-I, plasma triglycerides, and cholesterol played minor roles in the regulation of differential aortic lesion development. Extra virgin olive oil induced a cholesterol-poor, apoA-IV-enriched lipoparticle that has enhanced arylesterase and antioxidant activities, which is closely associated with reductions in atherosclerotic lesions. Given the anti-atherogenic properties of extra virgin olive oil evident in animal models fed a Western diet, clinical trials are needed to establish whether these oils are a safe and effective means of treating atherosclerosis.