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Preeclampsia is a severe pregnancy complication characterized by hypertension and may cause maternal morbidity and mortality. A better understanding of the essential genes involved in preeclampsia pathophysiology is urgently needed. This study investigated the function and molecular mechanisms of pumilio RNA binding family member 1 (PUM1) in extravillous trophoblast cells (EVTs). The interaction between protein and mRNA was verified by RNA pull-down assays, RNA immunoprecipitation assays, and luciferase reporter assays. The mRNA and protein levels of the genes involved were determined by RT-qPCR and western blot assays, respectively. Our results demonstrated that PUM1 could bind to the 3'-untranslated region of low-density lipoprotein receptor-related protein 6 (LRP6) mRNA, resulting in reduced expression of LRP6 mRNA and protein. Repression of PUM1 resulted in enhanced colony formation, cell proliferation, migration, and invasion of EVTs. The PUM1-depletion-mediated promotion effects on EVTs could be abrogated by LRP6 knockdown. PUM1 regulates the growth and mobility of EVTs by modulating LRP6 expression. Developing strategies to balance PUM1 and LRP6 levels may be beneficial for the management of preeclampsia patients. Key words: preeclampsia, PUM1, LRP6, cell proliferation, cell migration and invasion. La prééclampsie est une complication grave de la grossesse caractérisée par l'hypertension et qui peut entraîner une morbidité et une mortalité chez la mère. Il est urgent de mieux comprendre les gènes essentiels impliqués dans la physiopathologie de la prééclampsie. Cette étude visait à étudier la fonction et les mécanismes moléculaires de PUM1 (pumilio RNA binding family member 1) dans les cellules trophoblastiques extravillaires (TEV). L'interaction entre la protéine et l'ARNm a été vérifiée par un test de précipitation d'affinité avec l'ARN, un test d'immunoprécipitation d'ARN et un test à l'aide du rapporteur luciférase. Les niveaux d'ARNm et de protéines des gènes impliqués ont été déterminés respectivement par RT-qPCR et par buvardage Western. Les résultats obtenus par les auteurs ont démontré que PUM1 pouvait se lier à la région 3'-non traduite de l'ARNm de LRP6 (low density lipoprotein receptor-related protein 6), ce qui conduisait à une réduction de l'expression de l'ARNm et de la protéine LRP6. La répression de PUM1 conduisait à une augmentation de la formation de colonies, de la prolifération cellulaire, de la migration et de l'invasion des TEV. Les effets stimulateurs sur les TEV médiés par la suppression de PUM1 pouvaient être abrogés par le knockdown de LRP6. PUM1 régule la croissance et la mobilité des cellules TEV en modulant LRP6. Le développement de stratégies visant à équilibrer les niveaux de PUM1 et de LRP6 pourrait être bénéfique pour la prise en charge des patientes atteintes de prééclampsie. [Traduit par la Rédaction] Mots-clés : prééclampsie, PUM1, LRP6, prolifération cellulaire, migration et invasion cellulaire., Introduction Preeclampsia is a particular type of hypertensive disorder of pregnancy that can cause preterm birth, perinatal death, and maternal morbidity and mortality (Adams et al. 2014; Lei et al. [...]

Hypertriglyceridemia is an independent risk factor for cardiovascular disease, and plasma triglycerides (TGs) correlate strongly with plasma apolipoprotein C-III (ApoC-III) levels. Antisense oligonucleotides (ASOs) for ApoC-III reduce plasma TGs in primates and mice, but the underlying mechanism of action remains controversial. We determined that a murine-specific ApoC-III-targeting ASO reduces fasting TG levels through a mechanism that is dependent on low-density lipoprotein receptors (LDLRs) and LDLR-related protein 1 (LRP1). ApoC-III ASO treatment lowered plasma TGs in mice lacking lipoprotein lipase (LPL), hepatic heparan sulfate proteoglycan (HSPG) receptors, LDLR, or LRP1 and in animals with combined deletion of the genes encoding HSPG receptors and LDLRs or LRP1. However, the ApoC-III ASO did not lower TG levels in mice lacking both LDLR and LRP1. LDLR and LRP1 were also required for ApoC-III ASO-induced reduction of plasma TGs in mice fed a high-fat diet, in postprandial clearance studies, and when ApoC-III-rich or ApoC-III-depleted lipoproteins were injected into mice. ASO reduction of ApoC-III had no effect on VLDL secretion, heparin-induced TG reduction, or uptake of lipids into heart and skeletal muscle. Our data indicate that ApoC-III inhibits turnover of TG-rich lipoproteins primarily through a hepatic clearance mechanism mediated by the LDLR/LRP1 axis., Introduction Cardiovascular diseases (CVDs) are the leading cause of death in Western societies and are predicted to become a prominent problem worldwide (1). Most current therapeutic interventions are aimed at [...]

BACKGROUND/OBJECTIVES: Elevated oxidized low-density lipoprotein (OxLDL) may promote inflammation, and is associated with increased risk of atherosclerotic coronary heart disease and worsening complications of diabetes mellitus. The primary objective of this study was to evaluate the efficacy of chitin-glucan (CG), alone and in combination with a potentially anti- inflammatory olive oil (OO) extract, for reducing OxLDL in subjects with borderline to high LDL cholesterol (LDL-C) levels. SUBJECTS/METHODS: This 6-week, randomized, double-blind, placebo-controlled study of a novel, insoluble fiber derived from the Aspergillus niger mycelium, CG, evaluated 130 subjects free of diabetes mellitus with fasting LDL-C 3.37-4.92 mmol/l and glucose [less than or equal to] 6.94 mmol/l. Participants were randomly assigned to receive CG (4.5 g/day; n = 33), CG (1.5 g/day; n = 32), CG (1.5 g/day) plus OO extract (135 mg/day; n = 30), or matching placebo (n = 35). RESULTS: Administration of 4.5 g/day CG for 6 weeks significantly reduced OxLDL compared with placebo (P = 0.035). At the end of study, CG was associated with lower LDL-C levels relative to placebo, although this difference was statistically significant only for the CG 1.5 g/day group (P = 0.019). CG did not significantly affect high-density lipoprotein cholesterol, triglycerides, glucose, insulin or F2-isoprostane levels. Adverse events did not substantively differ between treatments and placebo. CONCLUSIONS: In this 6-week study, CG (4.5 g/day) reduced OxLDL, an effect that might affect the risk for atherosclerosis. European Journal of Clinical Nutrition (2013) 67, 2-7; doi: 10.1038/ejcn.2012.121; published online 5 September 2012 Keywords: atherosclerosis; cardiovascular disease; chitin-glucan; fiber; LDL cholesterol; oxidized LDL, INTRODUCTION Cardiovascular diseases (CVDs), especially atherosclerotic coronary heart disease (CHD) and stroke, are the leading causes of death globally. Low-density lipoprotein cholesterol (LDL-C) level is the primary lipid treatment target [...]

Background/Objectives: The importance of both low-density lipoprotein cholesterol (LDLc) size and the apolipoprotein E (Apo E) in the atherogenic process is known, but there is little information with regard to the effect of phytosterols (PS) on these parameters. The aim of this study was to evaluate the influence of PS on lipid profile and LDLc size according to Apo E genotype. Subjects/Methods: This was a randomized parallel trial employing 75 mild-hypercholesterolemic subjects and consisting of two 3-month intervention phases. After 3 months of receiving a standard healthy diet, subjects were divided into two intervention groups: a diet group (n = 34) and a diet + PS group (n = 41) that received 2g/day of PS. Total cholesterol (TC), triacylglycerols, LDLc, high-density lipoprotein cholesterol (HDLc), non-HDLc, Apo A-I and B-100, LDLc size and Apo E genotype were determined. Results: Patients receiving PS exhibited a significant decrease in TC (5.1%), LDLc (8.1%), non-HDLc (7.4%) and Apo B-100/Apo A-I ratio (7.7%), but these effects did not depend on Apo E genotype. No significant changes were found in lipid profile according to Apo E genotype when patients following dietary recommendations were considered as a whole population or separately. No variations in LDLc size were observed in any of the intervention groups. Conclusion: The results of this study show that Apo E genotype does not have an impact on the lipid response to PS as a cholesterol-lowering agent in mild-hypercholesterolemic patients. Furthermore, the evidence obtained confirms that LDLc particle size is not modified when PS are added to a standard healthy diet. European Journal of Clinical Nutrition (2011) 65, 255-261; doi: 10.1038/ejcn.2010.241; published online 3 November 2010 Keywords: phytosterols; apolipoprotein E; LDLc diameter; hyperlipidemia., Introduction Phytosterols (PS) are known to reduce serum low-density lipoprotein cholesterol (LDLc) levels, and so food products containing these plant compounds are widely used as dietary therapy to reduce plasma [...]

Recent genome-wide association studies have identified a genetic locus at human chromosome 8q24 as having minor alleles associated with lower levels of plasma triglyceride (TG) and LDL cholesterol (LDL-C), higher levels of HDL-C, as well as decreased risk for myocardial infarction. This locus contains only one annotated gene, tribbles homolog 1 (TRIB1), which has not previously been implicated in lipoprotein metabolism. Here we demonstrate a role for Trib1 as a regulator of lipoprotein metabolism in mice. Hepatic- specific overexpression of Trib1 reduced levels of plasma TG and cholesterol by reducing VLDL production; conversely, Trib1-knockout mice showed elevated levels of plasma TG and cholesterol due to increased VLDL production. Hepatic Trib1 expression was inversely associated with the expression of key lipogenic genes and measures of lipogenesis. Thus, we provide functional evidence for what we believe to be a novel gene regulating hepatic lipogenesis and VLDL production in mice that influences plasma lipids and risk for myocardial infarction in humans., Introduction Aberrant plasma lipoprotein concentrations of cholesterol, including LDL cholesterol (LDL-C) and HDL-C, and of triglycerides (TG) are heritable risk factors for atherosclerotic cardiovascular disease, the major cause of death [...]

Atherosclerosis is the most common cause of cardiovascular diseases in the world. Although the development of atherosclerosis appears to be the result of multiple maladaptive pathways, a particularly important factor in the pathogenesis of atherosclerosis is oxidized low-density lipoprotein (ox-LDL), which contributes to endothelial damage. Data from our laboratory and others show that follistatin-related protein (FRP), which is expressed in the vasculature, has cardioprotective effects, suggesting that loss of FRP protection might play a role in the development of atherosclerosis. In the present study, we determined whether FRP overexpression protects against endothelial cell (EC) damage, an intermediate end point for atherosclerosis. We bred apoE-knockout ([apoE.sup.-/-]) mice that were [FRP.sup.+] transgenic (they overexpressed FRP). We compared them with control mice (their littermates). Human umbilical vein endothelial cells (HUVECs) were isolated and treated with ox-LDL and recombinant FRP. FRP-induced signal transduction and Bcl-2 mRNA and protein stability were analyzed. After 16 wk, [apoE.sup.-/-] [FRP.sup.+] mice had significantly fewer apoptotic ECs than controls. In vitro experiments showed that the effect of FRP on EC apoptosis was mediated by upregulation of expression of the antiapoptotic protein Bcl-2. In HUVECs, FRP upregulated Bcl-2 transcription via a PI3K-Akt-NF-[kappa]B pathway. We conclude that FRP overexpression maintains EC viability by preventing apoptosis via Bcl-2 upregulation. FRP may be a novel therapeutic target for the prevention and treatment of vascular EC injury and of atherosclerosis. follistatin-related protein; oxidized low-density lipoprotein; nuclear factor-[kappa]B; apolipoprotein E; Bcl-2 doi: 10.1152/ajpendo.00005.2010.

Oxidatively modified low-density lipoprotein (OxLDL) is a contributing factor of endothelial dysfunction, an early cellular event during atherogenesis. In endothelial cells, OxLDL has been shown to stimulate proinflammatory responses, increase lipid accumulation, and induce the expression of adhesion and extracellular matrix degrading molecules. The primary receptor for OxLDL on endothelial cells has been identified as a member of the scavenger receptor family called lectin-like OxLDL receptor-1 (LOX-1). A number of studies on LOX-1 have implicated its role in multiple cardiovascular diseases including atherosclerosis. To better understand the molecular mechanisms underlying the role of LOX-1 in endothelial cells, we identified interacting proteins in an affinity-purified LOX-1 receptor complex from human aortic endothelial HAECT cells by mass spectrometry. Two molecules involved in Rho signaling pathway, ARHGEF1 and ROCK2, were identified, and their associations with LOX-1 were confirmed in reciprocal immunoprecipitation studies. Particularly, ROCK2 was found to dynamically associate with LOX-1 in the presence of OxLDL. In addition, OxLDL treatment stimulated ROCK2 catalytic activity, and ROCK2 inhibition attenuated NF-[kappa]B activation and IL-8 production resulting from OxLDL activation of LOX-1. In summary, a functional proteomics approach has enabled us to identify novel LOX-1 interactors that potentially contribute to the cellular and signaling functions of LOX-1. affinity proteomics; Rho-associated, coiled-coil containing protein kinase 2; Rho guanine nucleotide exchange factor 1; interleukin-8 doi: 10.1152/ajpcell.00483.2009.

A chiral porphyrazine (pz), [H.sub.2][pz(trans-[A.sub.2][B.sub.2])] (247), has been prepared that exhibits preferential in vivo accumulation in the cells of tumors. Pz 247 exhibits near-infrared (NIR) emission with [lambda] > 700 nm in the required wavelength range for maximum tissue penetration. When MDA-MB-231 breast tumor cells are treated with 247, the agent shows strong intracellular fluorescence with an emission maximum, 704 nm, which indicates that it localizes within a hydrophobic microenvironment. Pz 247 is shown to associate with the lipophilic core of LDL and undergo cellular entry primarily through receptor-mediated endocytosis accumulating in lysosomes. Preliminary in vivo studies show that 247 exhibits preferential accumulation and retention in the cells of MDA-MB231 tumors subcutaneously implanted in mice, thereby enabling NIR optical imaging with excellent contrast between tumor and surrounding tissue. The intensity of fluorescence from 247 within the tumor increases over time up to 48 h after injection presumably due to the sequestration of circulating 247/LDL complex by the tumor tissue. As the need for cholesterol, and thus LDL, is elevated in highly proliferative tumor cells over nontumorigenic cells, 247 has potential application for all such tumors. low-density lipoprotein | molecular imaging | near-IR fluorescence | porphyrinoid | tumor imaging doi/ 10.1073/pnas.0912811107

Human plasma low-density lipoproteins (LDL), a risk factor for cardiovascular disease, transfer cholesterol from plasma to liver cells via the LDL receptor (LDLr). Here, we report the structures of LDL and its complex with the LDL receptor extracellular domain (LDL. LDLr) at extracellular pH determined by cryoEM. Difference imaging between LDL x LDLr and LDL localizes the site of LDLr bound to its ligand. The structural features revealed from the cryoEM map lead to a juxtaposed stacking model of cholesteryl esters (CEs). High density in the outer shell identifies protein-rich regions that can be accounted for by a single apolipoprotein (apo B-100, 500 kDa) leading to a model for the distribution of its [alpha]-helix and [beta]-sheet rich domains across the surface. The structural relationship between the apo B-100 and CEs appears to dictate the structural stability and function of normal LDL. apolipoprotein B-100 | cholesteryl ester | electron cryomicroscopy | LDL receptor doi/10.1073/pnas.0908004107

Plasminogen activator inhibitor-1 (PAI-1) is implicated in thrombogenesis, inflammation, and extracellular matrix remodeling. Previous studies indicated that oxidized low-density lipoprotein (LDL) stimulated the generation of PAI-1 in vascular endothelial cells (EC). The present study demonstrated that LDL oxidized by copper, iron, or 3-morpholinosydnonimine increased the expression of NADPH oxidase (NOX) 2, PAI-1, and heat shock factor-1 (HSF1) in human umbilical vein EC or coronary artery EC compared with LDL or vehicle. Diphenyleneiodonium, a NOX inhibitor, prevented the increases of the expression of HSF1 and PAI-1 in EC induced by oxidized LDLs. Small-interference RNA (siRNA) for [p22.sup.phox], an essential subunit of NOX, prevented oxidized LDL-induced expression of NOX2, HSF1, and PAI-1 in EC. HSF1 siRNA inhibited oxidized LDL-induced expression of PAI-1 and HSF1, but not NOX2, in EC. The binding of HSF1 to PAI-1 promoter and the activity of PAI-1 promoter in EC were enhanced by oxidized LDL. Butylated hydroxytulene, a potent antioxidant, inhibited oxidized LDL-induced release of hydrogen peroxide ([H.sub.2][O.sub.2]) and the expression of NOX2, HSF1, and PAI-1 in EC. Treatment with [H.sub.2][O.sub.2] increased the abundance of NOX2, HSF1, and PAI-1 in EC. The results of the present study indicate that oxidized LDL-induced expression of NOX may lead to the elevated release of reactive oxygen species, the activation of HSF1, and the enhancement of the transcription of PAI-1 gene in cultured vascular EC. reduced nicotinamide adenine dinucleotide phosphatase; oxidized low-density lipoprotein

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels [approximately equal to]2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in [LDLR.sup.-/-] mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia. antibody | LDL-C | LDLR | PCSK9 | hypercholesterolemia

Proprotein convertase subtilisin/kexin type 9 (PCSK9) posttranslationally promotes the degradation of the low-density lipoprotein receptor (LDLr) in hepatocytes and increases plasma LDL cholesterol. It is not clear, however, whether PCSK9 plays a role in the small intestine. Here, we characterized the patterns of variations of PCSK9 and LDLr in fully differentiated Caco-2/15 cells as a function of various potential effectors. Cholesterol (100 [micro]M) solubilized in albumin or micelles significantly downregulated PCSK9 gene (30%, P < 0.05) and protein expression (50%, P < 0.05), surprisingly in concert with a decrease in LDLr protein levels (45%, P < 0.05). Cells treated with 25-hydroxycholesterol (50 [micro]M) also displayed significant reduction in PCSK9 gene (37%, P < 0.01) and protein (75% P < 0.001) expression, whereas LDLr showed a decrease at the gene (30%, P < 0.05) and protein (57%, P < 0.01) levels, respectively. The amounts of PCSK9 mRNA and protein in Caco-2/15 cells were associated to the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein-2 (SREBP-2) that can transcriptionally activate PCSK9 via sterol-regulatory elements located in its proximal promoter region. On the other hand, depletion of cholesterol content by hydroxypropyl-[beta]-cyclodextrin upregulated PCSK9 transcripts (20%, P < 0.05) and protein mass (540%, P < 0.001), in parallel with SREBP-2 protein levels. The addition of bile acids (BA) taurocholate and deoxycholate to the apical culture medium lowered PCSK9 gene expression (25%, P < 0.01) and raised PCSK9 protein expression (30%, P < 0.01), respectively, probably via the modulation of farnesoid X receptor. Furthermore, unconjugated and conjugated BA exhibited different effects on PCSK9 and LDLr. Altogether, these data indicate that intestinal PCSK9 is highly modulated by sterols and emphasize the distinct effects of BA species. enterocyte; PCSK9; cholesterol; bile acids; farnesoid X receptor; SREBP-2

Mammalian cells acquire cholesterol mainly from LDL. LDL enter the endosomes, allowing cholesteryl esters to be hydrolyzed by acid lipase. The hydrolyzed cholesterol (LDL-CHOL) enters the Niemann--Pick type C1 (NPC1)-containing endosomal compartment en route to various destinations. Whether the Golgi is involved in LDL-CHOL transport downstream of the NPC1 compartment has not been demonstrated. Using subcellular fractionation and immunoadsorption to enrich for specific membrane fractions, here we show that, when parental Chinese hamster ovary (CHO) cells are briefly exposed to [sup.3]H-cholesteryl linoleate (CL) labeled-LDL, newly liberated [sup.3]H-LDL-CHOL appears in membranes rich in trans-Golgi network (TGN) long before it becomes available for re-esterification at the endoplasmic reticulum (ER) or for efflux at the plasma membrane. In mutant cells lacking NPC1, the appearance of newly liberated [sup.3]H-LDL-CHOL in the TGN-rich fractions is much reduced. We next report a reconstituted transport system that recapitulates the transport of LDL-CHOL to the TGN and to the ER. The transport system requires ATP and cytosolic factors and depends on functionality of NPC1. We demonstrate that knockdown by RNAi of 3 TGN-specific SNAREs (VAMP4, syntaxin 6, and syntaxin 16) reduces [greater than or equal to] 50% of the LDL-CHOL transport in intact cells and in vitro. These results show that vesicular trafficking is involved in transporting a significant portion of LDL-CHOL from the NPC1-containing endosomal compartment to the TGN before its arrival at the ER. lipid metabolism | membrane trafficking | vesicular transport

A single bout of strenuous endurance exercise reduces fasting plasma triglyceride (TG) concentrations the next day (12-24 h later) by augmenting the efficiency of very low-density lipoprotein (VLDL)-TG removal from the circulation. Although much of the hypotriglyceridemia associated with training is attributed to the last bout of exercise, the relevant changes in VLDL-TG metabolism have never been investigated. We therefore examined basal VLDL-TG kinetics in a group of sedentary young men (n = 7) who underwent 2 mo of supervised high-intensity interval training (3 sessions/wk; running at 60 and 90% of peak oxygen consumption in 4-min intervals for a total of 32 min; gross energy expenditure: 446 [+ or -] 29 kcal) and a nonexercising control group (n = 8). Each subject completed two stable isotope-labeled tracer infusion studies in the postabsorptive state, once before and again after the intervention (~48 h after the last exercise bout in the training group). Peak oxygen consumption increased by ~18% after training (P [less than or equal to] 0.05), whereas body weight and body composition were not altered. Fasting plasma VLDL-TG concentration was reduced after training by ~28% (P [less than or equal to] 0.05), and this was due to reduced hepatic VLDL-TG secretion rate (by ~35%, P [less than or equal to] 0.05) with no changes ( 0.7) in VLDL-TG plasma clearance rate and the mean residence time of VLDL-TG in the circulation. No significant changes in VLDL-TG concentration and kinetics were observed in the nonexercising control group (all P [greater than or equal to] 0.3). We conclude that a short period of high-intensity interval aerobic training lowers the rate of VLDL-TG secretion by the liver in previously sedentary men. This is different from the mechanism underlying the hypotriglyceridemia of acute exercise; however, it remains to be established whether our finding reflects an effect of the longer time lapse from the last exercise bout, an effect specific to the type of exercise performed, or an effect of aerobic training itself. isotope; triacylglycerol; endurance; physical activity; chronic exercise

Fluid shear stress modulates vascular production of endothelial superoxide anion ([[O.sub.2].sup.-]) and nitric oxide (NO). Whether the characteristics of shear stress influence the spatial variations in mitochondrial manganese superoxide dismutase (Mn-SOD) expression in vasculatures is not well defined. We constructed a three-dimensional computational fluid dynamics model simulating spatial variations in shear stress at the arterial bifurcation. In parallel, explants of arterial bifurcations were sectioned from the human left main coronary bifurcation and right coronary arteries for immunohistolocalization of Mn-SOD expression. We demonstrated that Mn-SOD staining was prominent in the pulsatile shear stress (PSS)-exposed and atheroprotective regions, but it was nearly absent in the oscillatory shear stress (OSS)-exposed regions and lateral wall of arterial bifurcation. In cultured bovine aortic endothelial cells, PSS at mean shear stress ([[tau].sub.ave]) of 23 dyn/[cm.sup.2] upregulated Mn-SOD mRNA expression at a higher level than did OSS at [[tau].sub.ave] = 0.02 dyn/[cm.sup.2] [+ or -] 3.0 dyn x [cm.sup.-2] x [s.sup.-1] and at 1 Hz (PSS by 11.3 [+ or -] 0.4-fold vs. OSS by 5.0 [+ or -] 0.5-fold vs. static condition; P < 0.05, n = 4). By liquid chromatography and tandem mass spectrometry, it was found that PSS decreased the extent of low-density lipoprotein (LDL) nitration, whereas OSS increased nitration (P < 0.05, n = 4). In the presence of LDL, treatment with Mn-SOD small interfering RNA increased intracellular nitrotyrosine level (P < 0.5, n = 4), a fingerprint for nitrotyrosine formation. Our findings indicate that shear stress in the atheroprone versus atheroprotective regions regulates spatial variations in mitochondrial Mn-SOD expression with an implication for modulating LDL nitration. superoxide dismutase; superoxide anion; nitric oxide; nitrotyrosine; low-density lipoprotein

Insulin-resistant states are commonly associated with both increased circulating levels of tumor necrosis factor (TNF)-[alpha] and hepatic over-production of very low density lipoproteins (VLDL). Here, we provide evidence that increased TNF-[alpha] can directly stimulate the hepatic assembly and secretion of apolipoprotein B (apoB) 100-containing VLD[L.sub.1], using the Syrian golden hamster, an animal model that closely resembles humans in hepatic VLDL-apoB100 metabolism. In vivo TNF-[alpha] infusion for 4 h in chow-fed hamsters induced whole-body insulin resistance on the basis of euglycemic hyperinsulinemic clamp studies. Immunoprecipitation and immunoblotting analysis of livers from TNF-[alpha]-treated hamsters indicated decreased tyrosine phosphorylation of insulin receptor (IR)-[beta], IR substrate-1 (Tyr), Akt ([Ser.sup.473]), p38, ERK1/2, and JNK but increased serine phosphorylation of IRS-1 ([Ser.sup.307]) and Shc. TNF-[alpha] infusion also significantly increased hepatic production of total circulating apoB 100 and VLDL-apoB100 in both fasting and postprandial (fat load) states. Ex vivo experiments, using cultured primary hepatocytes from hamsters, also showed TNF-[alpha]-induced VLDL-apoB 100 oversecretion, an effect that was blocked by TNF receptor 2 antibody. Unexpectedly, TNF-[alpha] decreased the sterol regulatory element-binding protein-1c mass and mRNA levels but significantly increased microsomal triglyceride transfer protein mass and mRNA levels in primary hepatocytes. In summary, these data provide direct evidence that TNF-[alpha] induces whole-body insulin resistance and impairs hepatic insulin signaling accompanied by overproduction of apoBl00-containing VLDL particles, an effect likely mediated via TNF receptor 2. TNF-[alpha]; liver; insulin resistance; lipid; lipoprotein; apoB

Background: Familial hypercholesterolemia (FH) is associated with a high risk of coronary heart disease. Pharmacological treatment and diet are both essential for the management of FH. Foods rich in plant [...]

A novel protein, PA0122, has been identified in Pseudomonas aeruginosa and shown to bind to oxidized low-density lipoprotein (Ox-LDL). The PA0122 gene was recognized based on gene expression pattern differences between two strains of P. aeruginosa isolated from the sputum of an individual with cystic fibrosis (CF). There was an approximately eightfold increase in PA0122 expression in the non-mucoid strain 383, compared to that in the mucoid strain 2192. Quantitative real-time RT-PCR (qRT-PCR) supported PA01 22 transcript expression differences between strains 383 and 2192 and revealed growth-phase dependence, with the highest level of expression at early stationary phase ([OD.sub.600] 1.5). PA0122 encodes a 136 aa 'conserved hypothetical' protein that has similarity to Aspergillus fumigatus Asp-haemolysin, which is an Ox-LDL-binding protein, and possessed a motif that is homologous to the fungal aegerolysin family of proteins. Antibodies produced to purified recombinant PA0122 recognized a 16 kDa protein band in cell lysates as well as in the supernatant fractions of strain 383. The PA0122 protein expression pattern was growth phase-dependent, with maximal production observed at [OD.sub.600] 1.5 that was consistent with the PA01 22 transcript expression profile. Subcellular fractionation studies revealed differences in the localization of PA01 22 between strains 383 and 2192. In 383, PA0122 was observed in the cytoplasm and in membrane fractions. In 2192, PA0122 was found in the cytoplasm but was not detected in membrane fractions. Surface plasmon resonance revealed that recombinant PA0122 binds with high affinity to Ox-LDL and to its major subcomponent, lysophosphatidylcholine, but not to non-oxidized LDL.

Chylomicrons produced by the human gut contain apolipoprotein (apo) B48, whereas very-low-density lipoproteins made by the liver contain apo B100. To study how these molecules function during lipid absorption, we examined the process as it occurs in apobec-1 knockout mice (able to produce only apo B100; KO) and in wild-type mice (of which the normally functioning intestine makes apo B48, WT). Using the lymph fistula model, we studied the process of lipid absorption when animals were intraduodenally infused with a lipid emulsion (4 or 6 [micro]mol/h of triolein). KO mice transported triacylglycerol (TG) as efficiently as WT mice when infused with the lower lipid dose; when infused with 6 [micro]mol/h of triolein, however, KO mice transported significantly less TG to lymph than WT mice, leading to the accumulation of mucosal TG. Interestingly, the size of lipoprotein particles from both KO and WT mice were enlarged to chylomicron-size particles during absorption of the higher dose. These increased-size particles produced by KO mice were not associated with increased apo AIV secretion. However, we found that the gut of the KO mice secreted fewer apo B molecules to lymph (compared with WT), during both fasting and lipid infusion, leading us to conclude that the KO gut produced fewer numbers of TG-rich lipoproteins (including chylomicron) than the wild-type animals. The reduced apo B secretion in KO mice was not related to reduced microsomal triglyceride transfer protein lipid transfer activity. We propose that apo B48 is the preferred protein for the gut to coat chylomicrons to ensure efficient chylomicron formation and lipid absorption. very low-density lipoprotein; lymph

The accumulation of low-density lipoprotein (LDL) is recognized as one of the main contributors in atherogenesis. Mathematical models have been constructed to simulate mass transport in large arteries and the consequent lipid accumulation in the arterial wall. The objective of this study was to investigate the influences of wall shear stress and transmural pressure on LDL accumulation in the arterial wall by a multilayered, coupled lumen-wall model. The model employs the Navier-Stokes equations and Darcy's Law for fluid dynamics, convection-diffusion-reaction equations for mass balance, and Kedem-Katchalsky equations for interracial coupling. To determine physiologically realistic model parameters, an optimization approach that searches optimal parameters based on experimental data was developed. Two sets of model parameters corresponding to different transmural pressures were found by the optimization approach using experimental data in the literature. Furthermore, a shear-dependent hydraulic conductivity relation reported previously was adopted. The integrated multilayered model was applied to an axisymmetric stenosis simulating an idealized, mildly stenosed coronary artery. The results show that low wall shear stress leads to focal LDL accumulation by weakening the convective clearance effect of transmural flow, whereas high transmural pressure, associated with hypertension, leads to global elevation of LDL concentration in the arterial wall by facilitating the passage of LDL through wall layers. low-desity lipoprotein transport; lipid accumulation; atherosclerosis; hypertension doi:10.1152/ajpheart.01281.2006

Glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to cluster in microdomains enriched in glycosphingolipids and cholesterol and represent a relatively selective marker of lipid rafts. In recent years, several attempts have been made to use fluorescent probes to nondisruptively label these domains in living cells. Here, we have transfected endothelial cells with a GPI-anchored thermotolerant green fluorescent protein (ttGFP) to show colocalization of this fluoroprobe with another marker of lipid rafts, urokinase-type plasminogen activator receptor-1. ttGFP was used to quantify the cell surface area occupied by lipid rafts and to examine the effect of various proatherogenic signals on lipid rafts. Exposure of endothelial cells to asymmetric dimethylarginine and oxidized LDL (oxLDL), as well as oxidant stress, reduced the cell surface area occupied by lipid rafts. Next, the property of ttGFP to undergo a shift in absorbance depending on the clustering of these molecules was utilized to perform proximity imaging (PRIM). PRIM showed that nitric oxide (NO) increased the distance between GPI-anchored ttGFP molecules clustered in lipid-rich microdomains. This 'unclustering' of GPI-anchored ttGFP was not reproduced by prooxidant signals and was due to reduction in membrane-cytoskeletal constraints on the lipid rafts. These findings suggested that two fundamentally different mechanisms modulate lipid rafts: 1) substance regulation of lipid rafts involving modification of cholesterol and sphingolipids and 2) structural regulation of lipid rafts through disruption of membrane-cytoskeletal interactions, switching off the spatial confinement of lipid rafts. nitric oxide; endothelium; urokinase-type plasminogen activator receptor; oxidized low-density lipoprotein

Objective: We studied whether consumption of phenol-rich extra virgin olive oil affects the susceptibility of low density lipoproteins (LDL) to oxidation and other markers of oxidation in humans. Design: Randomized cross-over intervention trial, stratified according to sex, age and energy intake. Setting: Division of Human Nutrition and Epidemiology, Wageningen University, The Netherlands. Subjects: Forty-six healthy men and women completed the study. Intervention: Subjects consumed two diets supplying 69 g per day of extra virgin olive oil either rich or poor in phenols for 3 weeks each. The mean difference in phenol intake between the treatments was 18 mg per day. Vitamin E intake was low during the whole study. Fasting blood samples were taken twice at the end of each period. Results: Resistance of LDL and high density lipoprotein (HDL) to oxidation was not affected by treatment. The mean lag time of copper-induced formation of conjugated dienes was 1.6 min shorter in LDL and 0.4 min longer in HDL after the high phenol diet. Other markers of antioxidant capacity in plasma were also not affected: mean lipid hydroperoxides were 0.07 [micro]mol/l higher, mean malondialdehydes were 0.001 [micro]mol/l higher, mean protein carbonyls were 0.001 nmol/mg protein lower, and the mean ferric reducing ability of plasma (FRAP) was 0.006 mmol/l higher after the high phenol diet. All 95% confidence intervals enclosed zero. Serum cholesterol concentrations were not affected by the treatment. Conclusion: Consumption of 18 mg per day of phenols from extra virgin olive oil for 3 weeks did not affect LDL or HDL oxidation or other markers of antioxidant capacity in fasting plasma samples. Sponsorship: Supported by the International Olive Oil Council and the Foundation for Nutrition and Health Research. Descriptors: phenols; tyrosol; hydroxytyrosol; oleuropein; olive oil; antioxidants; LDL-oxidation; HDLoxidation; lipid hydroperoxides; malondialdehyde; protein carbonyls; FRAP, Introduction The Mediterranean diet, with olive oil as the major fat source, has been shown in epidemiological studies to be associated with a reduced incidence of coronary heart disease (Keys [...]

Objective: The objective of this trial was to compare the effect on the susceptibility of plasma Low Density Lipoprotein (LDL) to oxidative modifications of consumption of two oleic rich diets, prepared with two different plant oils, virgin olive oil (OL)(1) and refined high monounsaturated fatty acids (MUFA sunflower oil (SU)), with the susceptibility of plasma LDL to oxidation after an National Cholesterol Education Program step 1 (NCEP-I) phase diet. Design: A randomized crossover design. Subjects and interventions: Twenty-two healthy normolipidemic young males consumed an NCEP-I diet for a 4-week period. Subjects were then assigned to two diets each of 4-weeks duration. Group one was placed on an olive oil enriched diet (40% fat, 22% MUFA) followed by a 4-week period of a MUFA diet enriched in sunflower oil (40% fat, 22% MUFA). In group two, the order of the diets was reversed. Results: Both MUFA diets induced a decrease in saturated (14:0, 16:0, and 18:0) and an increase in monounsaturated and polyunsaturated n-6 (18:2, 20:3, and 20:5) plasma LDL-phospholipid fatty acids, compared to the NCEP-I diet (P < 0.01). No significant differences in lag times were observed between the olive oil and the NCEP-I diet periods. However there was a greater inhibition time (P < 0.001) when subjects consumed the MUFA rich sunflower oil diet compared to the NCEP-I diet. These differences were probably related to the relative enrichment of plasma LDL particles in [alpha]-tocopherol due to the high vitamin E content of the MUFA-rich sunflower oil. Indeed, the [alpha]-tocopherol content was positively correlated with lag time (r=0.338; P < 0.008). Conclusion: Our findings suggest that changes in plasma LDL [alpha]-tocopherol content with practical solid-food diets can decrease its susceptibility to oxidation. Sponsorship: This work has been supported by grants from the Investigaciones de la Seguridad Social (FIS 92/0182, to Francisco Perez Jimenez); and from Koype Co, Andujar, Jaen, Spain. Descriptors: oxidized low density lipoproteins; [alpha]-tocopherol; phospholipid; monounsaturated fatty acids; low fat diets; fatty acids, Introduction Coronary heart disease (CHD) due to atherosclerosis remains the most prevalent cause of death and disability in Europe and North America. The dramatic geographic differences in the incidence of [...]

Alternate day fasting (ADF) with a low-fat (LF) diet increases low-density lipoprotein (LDL) particle size. Whether these beneficial effects can be reproduced by a high-fat (HF) ADF diet is unclear. This study compared an ADF-HF to an ADF-LF diet on plasma lipids, LDL size and high-density lipoprotein (HDL) size. Thirty-five obese subjects were randomized to an ADF-HF or ADF-LF diet for 10 weeks. Body weight decreased (P < 0.0001) by 4.3 ± 1.0 kg (4.8 ± 1.1%) and 3.7 ± 0.7 kg (4.2 ± 0.8%) in the ADF-HF and ADF-LF group, respectively. LDL cholesterol was reduced (P < 0.0001) by 19 ± 8 mg/dl (18 ± 5%) by ADF-HF and 28 ± 7 mg/dl (25 ± 3%) by ADF-LF. LDL particle size increased (P < 0.005) by 3 ± 1 Å in both groups. The proportion of small LDL particles decreased (P < 0.005) by 8 ± 2% and 11 ± 3% in the ADF-HF and ADF-LF groups, respectively. HDL cholesterol and HDL size remained unchanged. Thus, our results suggest that the ADF-HF diet is equally as effective as the ADF-LF diet in improving LDL particle size and distribution. European Journal of Clinical Nutrition (2013) 67, 783-785; doi: 10.1038/ejcn.2013.83; published online 24 April 2013 Keywords: alternate day fasting; calorie restriction; dietary fat; weight loss; LDL particle size; HDL particle size, INTRODUCTION Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particle size are important predictors of cardiovascular events and progression. (1) Small LDL particles are detrimental to vascular health due to an [...]

Byline: Medine. Cure, Ahmet. Tufekci, Erkan. Cure, Serkan. Kirbas, Sabri. Ogullar, Aynur. Kirbas, Huseyin. Unal, Suleyman. Yuce, Sevim. Cakmak Objectives: Small dense (sd) low-density lipoprotein (LDL), tumor necrosis factor (TNF) [...]

CHO cell lines coexpressing betaCTF and wild-type or mutant presenilin 1/2 were generated and treated with DAPT, to understand more about the cleavage mechanism by gamma-secretase. In all cell lines treated with DAPT, as the levels of Abeta40 decreased, Abeta43 and Abeta46 accumulated.