Search

Lysophosphatidylcholine (LPC), a major component of oxidized low-density lipoprotein, is associated with atherosclerosis, obesity, stroke, and cancer. However, the direction and mechanism of this relationship remains unclear. In this study, we conducted RNA profiling in THP-1 derived macrophages treated with LPC and uncovered a relationship between LPC and the cholesterol biosynthesis pathway. Principal component analysis (PCA) of RNA profiling showed that untreated THP-1 cells and those treated with 10, 20, or 40 µM LPC were distinctly distributed. Functional annotation revealed that LPC affected the expression of genes involved in cytokine-cytokine receptor interaction, TNF signaling, and MAPK signaling. Interestingly, LPC also altered the expression of 11 genes involved in cholesterol synthesis such as those in terpenoid backbone biosynthesis and steroid biosynthesis pathways. This increased gene expression occurred in a dose-dependent manner in response to LPC treatment. Especially, LPC with saturated acyl groups enhanced the expression of these genes compared to LPC with unsaturated acyl groups, and similar results were shown in response to saturated and unsaturated free fatty acids. Our findings demonstrate that LPCs with saturated acyl groups induce the expression of genes involved in cholesterol biosynthesis and may have implications for cholesterol related diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Patients with small-cell lung cancer (SCLC) patients demonstrate varied survival outcomes. Previous studies have reported that lipoproteins are associated with prognosis in various cancers; however, the role of low-density lipoprotein (LDL) and low-density lipoprotein- cholesterol (LDLR) in patients with SCLC has not been studied., Methods: In this study, the impact of LDL and LDLR on the prognosis of SCLC patients was evaluated. A total of 601 patients with SCLC were retrospectively evaluated, in which 198 patients had adequate tissues for immunohistochemistry, and serum LDL and LDLR expression levels at baseline were tested. X-tile tool, and univariate and multivariate Cox analysis were used to assess the association between LDL, LDLR and overall survival (OS)., Results: Univariate analysis demonstrated that a lower LDL level was significantly associated with superior OS (P = 0.037). Similarly, LDLR also significantly predicted OS (P = 0.003). Multivariate Cox analyses confirmed that lower LDL and LDLR expression was independent prognostic factors associated with longer OS (P = 0.019 and P = 0.027, respectively)., Conclusions: This study showed that both LDL and LDLR are prognostic indexes for survival in patients with SCLC. Patients with high LDL or LDLR expression level may benefit from treatment that modulates lipoprotein combined with platinum-based chemotherapy.

Background: Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous plaque in the arteries. Its etiology is very complicated and its risk factors primarily include genetic defects, smoking, hyperlipidemia, hypertension, lack of exercise, and infection. Recent studies suggest that fine particulate matter (PM2.5) air pollution may also contribute to the development of atherosclerosis., Scope of Review: The present review integrates current experimental evidence with mechanistic pathways whereby PM2.5 exposure can promote the development of atherosclerosis., Major Conclusions: PM2.5-mediated enhancement of atherosclerosis is likely due to its pro-oxidant and pro-inflammatory effects, involving multiple organs, different cell types, and various molecular mediators., General Significance: Studies about the effects of PM2.5inhalation on atherosclerosis may yield a better understanding of the link between air pollution and major cardiovascular diseases, and provide useful information for policy makers to determine acceptable levels of PM2.5 air quality. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The paper proposes a pathophysiologic framework to explain the well-established epidemiological association between exposure to ambient air particle pollution and premature cardiovascular mortality, and offers insights into public health solutions that extend beyond regulatory environmental protections to actions that can be taken by individuals, public health officials, healthcare professionals, city and regional planners, local and state governmental officials and all those who possess the capacity to improve cardiovascular health within the population. The foundation of the framework rests on the contribution of traditional cardiovascular risk factors acting alone and in concert with long-term exposures to air pollutants to create a conditional susceptibility for clinical vascular events, such as myocardial ischemia and infarction; stroke and lethal ventricular arrhythmias. The conceptual framework focuses on the fact that short-term exposures to ambient air particulate matter (PM) are associated with vascular thrombosis (acute coronary syndrome, stroke, deep venous thrombosis, and pulmonary embolism) and electrical dysfunction (ventricular arrhythmia); and that individuals having prevalent heart disease are at greatest risk. Moreover, exposure is concomitant with changes in autonomic nervous system balance, systemic inflammation, and prothrombotic/anti-thrombotic and profibrinolytic-antifibrinolytic balance. Thus, a comprehensive solution to the problem of premature mortality triggered by air pollutant exposure will require compliance with regulations to control ambient air particle pollution levels, minimize exposures to air pollutants, as well as a concerted effort to decrease the number of people at-risk for serious clinical cardiovascular events triggered by air pollutant exposure by improving the overall state of cardiovascular health in the population. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu., (Published by Elsevier B.V.)

Aims: Electronegative LDL (LDL(-)) is a plasma LDL subfraction that induces cytokine release in monocytes through toll-like receptor 4 (TLR4) activation. However, the intracellular pathways induced by LDL(-) downstream TLR4 activation are unknown. We aimed to identify the pathways activated by LDL(-) leading to cytokine release in monocytes., Methods and Results: We determined LDL(-)-induced activation of several intracellular kinases in protein extracts from monocytes using a multikinase ELISA array. LDL(-) induced higher p38 mitogen-activated protein kinase (MAPK) phosphorylation than native LDL. This was corroborated by a specific cell-based assay and it was dependent on TLR4 and phosphoinositide 3-kinase (PI3k)/Akt pathway. P38 MAPK activation was involved in cytokine release promoted by LDL(-). A specific ELISA showed that LDL(-) activated cAMP response-element binding (CREB) in a p38 MAPK dependent manner. P38 MAPK was also involved in the nuclear factor kappa-B (NF-kB) and activating protein-1 (AP-1) activation by LDL(-). We found that NF-kB, AP-1 and CREB inhibitors decreased LDL(-)-induced cytokine release, mainly on MCP1, IL6 and IL10 release, respectively., Conclusions: LDL(-) promotes p38 MAPK phosphorylation through TLR4 and PI3k/Akt pathways. Phosphorylation of p38 MAPK is involved in NF-kB, AP-1 and CREB activation, leading to LDL(-)-induced cytokine release in monocytes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Valproic acid (VPA) is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-based in vitro model. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA-) induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG) synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36), low-density lipoprotein receptor-related protein 1 (Lrp1), diacylglycerol acyltransferase 2 (Dgat2), and perilipin 2 (Plin2) were increased, that of carnitine palmitoyltransferase I a (Cpt1a) was not affected, and those of acetyl-Co A carboxylase α (Acca) and fatty acid synthase (Fasn) were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptor γ (PPARγ) nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation.

Background: Saliva contains a variety of substances and could be functionally equivalent to serum in reflecting the physiological state of the body, including metabolic variations. Salivary samples are non-invasive, safe, and easier to handle than serum. Oxidized LDL cholesterol (oxLDL) is an additional cardiovascular risk factor playing an important role in atheromatous plaque formation; overweight/obese subjects present an increase in oxLDL concentrations. The aims of the study were to assess oxLDL salivary levels, if detectable, and to verify their possible correlation with serum in overweight/obese subjects., Methods: Thirty-five consecutive overweight/obese subjects and 10 normal weight controls were enrolled. Serum and salivary oxLDL levels were measured by a commercial enzyme-linked-immunosorbent assay (ELISA method)., Results: oxLDL levels were detectable in salivary samples and correlated (P = 0.001) with serum levels. Overweight/obese subjects showed serum and salivary oxLDL levels higher than controls (P = 0.000 and P = 0.022, respectively)., Conclusions: Our study showed the presence of oxLDL in salivary samples and highlighted a correlation between salivary oxLDL levels and their counterpart in serum. Moreover, salivary oxLDL levels were higher in overweight/obese subjects than in controls. Therefore, a salivary sample could be functionally equivalent to serum in monitoring cardiovascular risk in overweight/obese subjects., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Diabetic (DM) patients have exacerbated atherosclerosis and high CVD burden. Changes in lipid metabolism, lipoprotein structure, and dysfunctional HDL are characteristics of diabetes. Our aim was to investigate whether serum ApoA-I, the main protein in HDL, was biochemically modified in DM patients. By using proteomic technologies, we have identified a 26 kDa ApoA-I form in serum. MS analysis revealed this 26 kDa form as a novel truncated variant lacking amino acids 1-38, ApoA-IΔ(1-38). DM patients show a 2-fold increase in ApoA-IΔ(1-38) over nondiabetic individuals. ApoA-IΔ(1-38) is found in LDL, but not in VLDL or HDL, with an increase in LDL3 and LDL4 subfractions. To identify candidate mechanisms of ApoA-I truncation, we investigated potentially involved enzymes by in silico data mining, and tested the most probable molecule in an established animal model of diabetes. We have found increased hepatic cathepsin D activity as one of the potential proteases involved in ApoA-I truncation. Cathepsin D-cleaved ApoA-I exhibited increased LDL binding affinity and decreased antioxidant activity against LDL oxidation. In conclusion, we show for the first time: a) presence of a novel truncated ApoA-I form, ApoA-IΔ(1-38), in human serum; b) ApoA-IΔ(1-38) is transported by LDL; c) ApoA-IΔ(1-38) is increased in dense LDL fractions of DM patients; and d) cathepsin D-ApoA-I truncation may lead to ApoA-IΔ(1-38) binding to LDLs, increasing their susceptibility to oxidation and contributing to the high cardiovascular risk of DM patients., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

There is a growing body of evidence that excess lipids, hypoxic stress and other inflammatory signals can stimulate endoplasmic reticulum (ER) stress in metabolic diseases. However, the pathophysiological importance and the underlying mechanisms of this phenomenon remain unknown. The current study investigated that 50 ng/ml oxidized LDL promoted unfolded protein response (UPR) and ER stress in J774A1 murine macrophages, which was blocked by extracts (PPE) of purple Perilla frutescens, a plant of the mint family Lamiaceae. The ER stressor tunicamycin was employed as a positive control. Treating 1-10 µg/ml oxidized LDL for 24 h elicited lipotoxic apoptosis in macrophages with obvious nuclear condensation and DNA fragmentation, which was inhibited by PPE. Tunicamycin and oxidized LDL activated and induced the UPR components of activating transcription factor 6 and ER resident chaperone BiP/Grp78 in temporal manners and such effects were blocked by ≥5 µg/ml PPE. In addition, PPE suppressed the enhanced mRNA transcription and splicing of X-box binding protein 1 (XBP1) by tunicamycin and oxidized LDL. The protein induction and nuclear translocation of XBP1 were deterred in PPE-treated macrophages under ER stress. The induction of ATP-binding cassette transporter A1 (ABCA1), scavenger receptor-B1 (SR-B1) and intracellular adhesion molecule-1 (ICAM-1) was abolished by the ER stressor in activated macrophages. The protein induction of ABCA1 and ICAM1 but not SR-B1 was retrieved by adding 10 µg/ml PPE to cells. These results demonstrate that PPE inhibited lipotoxic apoptosis and demoted the induction and activation of UPR components in macrophages. PPE restored normal proteostasis in activated macrophages oxidized LDL. Therefore, PPE was a potent agent antagonizing macrophage ER stress due to lipotoxic signals associated with atherosclerosis.

Background: Increased levels of the most electronegative type of LDL, L5, have been observed in the plasma of patients with metabolic syndrome (MetS) and ST-segment elevation myocardial infarction and can induce endothelial dysfunction. Because men have a higher predisposition to developing coronary artery disease than do premenopausal women, we hypothesized that LDL electronegativity is increased in men and promotes endothelial damage., Methods: L5 levels were compared between middle-aged men and age-matched, premenopausal women with or without MetS. We further studied the effects of gender-influenced LDL electronegativity on aortic cellular senescence and DNA damage in leptin receptor-deficient (db/db) mice by using senescence-associated-β-galactosidase and γH2AX staining, respectively. We also studied the protective effects of 17β-estradiol and genistein against electronegative LDL-induced senescence in cultured bovine aortic endothelial cells (BAECs)., Results: L5 levels were higher in MetS patients than in healthy subjects (P < 0.001), particularly in men (P = 0.001). LDL isolated from male db/db mice was more electronegative than that from male or female wild-type mice. In addition, LDL from male db/db mice contained abundantly more apolipoprotein CIII and induced more BAEC senescence than did female db/db or wild-type LDL. In the aortas of db/db mice but not wild-type mice, we observed cellular senescence and DNA damage, and the effect was more significant in male than in female db/db mice. Pretreatment with 17β-estradiol or genistein inhibited BAEC senescence induced by male or female db/db LDL and downregulated the expression of lectin-like oxidized LDL receptor-1 and tumor necrosis factor-alpha protein., Conclusion: The gender dichotomy of LDL-induced cardiovascular damage may underlie the increased propensity to coronary artery disease in men.

LDL (low-density lipoprotein) is subjected to pro-atherogenic modifications in the circulation. A novel uraemia-independent mechanism of carbamylation of lipoproteins mediated by MPO (myeloperoxidase) has recently been reported. We have investigated whether carbamylation of LDL was increased in patients with Type 2 diabetes without renal impairment and the role of MPO. cLDL (carbamylated LDL) and MPO were measured by ELISA in a cross-sectional study of 198 patients and 174 non-diabetic controls. The impact of lowering MPO on plasma cLDL was determined by assaying cLDL and MPO in archived samples from a previous randomized open-label parallel group study comparing rosiglitazone (n=20) and sulfonylurea (n=24). Both plasma cLDL (P<0.05) and MPO levels (P<0.01) were higher in patients with Type 2 diabetes than controls in the cross-sectional study. Plasma cLDL correlated with MPO (r=0.42 and P<0.01) in subjects with diabetes, and plasma MPO was an independent determinant of plasma cLDL even after adjusting for age, gender, BMI (body mass index), apoB (apolipoprotein B), urea and HbA1c (glycated haemoglobin). In the randomized trial, rosiglitazone significantly lowered MPO (P<0.01) and cLDL (P<0.05), whereas no changes were observed in the sulfonylurea group despite a similar reduction in HbA1c. The magnitude of reduction in plasma cLDL correlated with changes in MPO, but not with HbA1c in the rosiglitazone group, suggesting that lowering MPO reduced plasma cLDL. Plasma cLDL is increased in patients with Type 2 diabetes even in the absence of renal impairment and carbamylation of LDL in these subjects is mainly mediated by MPO and not by urea.

The development of immediate and delayed long-term resistance to hypoxia during a course of intermittent normobaric hypoxia (15 daily sessions of alternating exposure to 10% O2 and atmospheric air for 1 h) correlated with biphasic expression of HIF-1α in neocortex of hypoxia-intolerant rats, which suggests involvement of this protein factor not only in the formation of long-term adaptation, but also in triggering immediate adaptation to hypoxia. Both processes develop under conditions promoting down-regulation of oxidative modification of LDL and increasing tolerance of biological membranes to hypoxia in the absence of activation of the free radical processes, which therefore do not trigger HIF-1α expression under these conditions. Neither cytokines nor NO are the inducers of immediate adaptation, and they are not related to HIF-1α expression during the early post-hypoxic period. In contrast, long-term adaptation in response to the course of intermittent normobaric hypoxia develops against the background of enhanced NO production, activation of pro- and anti-inflammatory factors, and expression of VEGF, the marker of angiogenesis. Therefore, all these factors can promote activation of transcription processes required to form the long-term adaptation.

The growth and development of prostate gland is governed by testosterone. Testosterone helps in maintaining the adipose tissue stores of the body. It is well documented that with advancing age there has been a gradual decline in testosterone levels. Our aim was to study the protective role of daidzein on flutamide-induced androgen deprivation on matrix degrading genes, lipid profile and oxidative stress in Wistar rats. Sub-chronic (60 days) flutamide (30 mg/kg b.wt) administration resulted in marked increase in expressions of matrix degrading genes [matrix metalloproteases 9 and urokinase plasminogen activation receptor]. Additionally, it increased the levels of low density lipoproteins, total cholesterol, triglycerides, and lowered the levels of high density lipoproteins and endogenous antioxidant levels. Oral administration of daidzein (20 and 60 mg/kg b.wt) restituted the levels to normal. Daidzein administration resulted in amelioration of the prostate atrophy, degeneracy and invasiveness induced by flutamide. Our findings suggest that the daidzein may be given as dietary supplement to patients who are on androgen deprivation therapy, to minimize the adverse effects related to it and also retarding susceptibility of patients to cardiovascular diseases.

Considerable attention has focused on cannabidiol (CBD), a major non-psychotropic constituent of fiber-type cannabis plant, and it has been reported to possess diverse biological activities. Although CBD is obtained from non-enzymatic decarboxylation of its parent molecule, cannabidiolic acid (CBDA), several studies have investigated whether CBDA itself is biologically active. In the present report, the author summarizes findings indicating that; 1) CBDA is a selective cyclooxygenase-2 (COX-2) inhibitor, and ii) CBDA possesses an anti-migrative potential for highly invasive cancer cells, apparently through a mechanism involving inhibition of cAMP-dependent protein kinase A, coupled with an activation of the small GTPase, RhoA. Further, the author introduces recent findings on the medicinal chemistry and pharmacology of the CBD derivative, CBD-2',6'-dimethyl ether (CBDD), that exhibits inhibitory activity toward 15-lipoxygenase (15-LOX), an enzyme responsible for the production of oxidized low-density lipoprotein (LDL). These studies establish CBD as both an important experimental tool and as a lead compound for pharmaceutical development. In this review, the author further discusses the potential uses of CBD and its derivatives in future medicines.

Objective: Familial hypercholesterolaemia (FH) is associated with increased risk of premature atherosclerosis. Inflammation is a key event in atherogenesis, and we have previously reported an inflammatory imbalance between tumour necrosis factor (TNF)α and interleukin-10 in children with FH. Based on the potential role of TNF-related molecules in inflammation, we investigated the regulation of other members of the TNF superfamily (TNFSF)/TNF receptor superfamily (TNFRSF) in children and young adults with FH and matched healthy controls., Methods: Expression of TNFSF/TNFRSF genes in peripheral blood mononuclear cells (PBMCs) was quantified in children and young adults with FH prior to (n = 42) and after statin treatment (n = 10) and in controls (n = 25) by quantitative real-time polymerase chain reaction., Results: First we found that, compared with controls, the mRNA levels of OX40L, BAFFR and TRAILR1 were significantly higher, whereas TRAIL and TRAILR3 were significantly lower in children and young adults with FH. Secondly, levels of oxidized low-density lipoprotein (oxLDL) were significantly raised in the FH group, and correlated with the expression of OX40L, BAFFR and TRAILR1. Thirdly, oxLDL increased mRNA levels of BAFFR, TRAILR1 and TRAILR4 in PBMCs ex vivo from individuals with FH. Fourthly, OX40, acting through OX40L, enhanced the oxLDL-induced expression of matrix metalloproteinase-9 in THP-1 monocytes in vitro. Finally, after statin treatment in children with FH (n = 10), mRNA levels of OX40L and TRAILR1 decreased, whereas levels BAFF, TRAIL and TRAILR3 increased., Conclusion: Our findings suggest the involvement of some TNFSF/TNFRSF members and oxLDL in the early stages of atherogenesis; this may potentially contribute to the accelerated rate of atherosclerosis observed in individuals with FH., (© 2012 The Association for the Publication of the Journal of Internal Medicine.)

Problem: To address the question of whether the high levels of oxidative modified low-density lipoproteins (oxLDL) in pregnancy are opposed by an appropriate humoral autoimmune response providing anti-oxLDL autoantibodies in maternal serum of healthy women throughout gestation., Method of Study: Blood was taken from 33 patients at four different time points from early to late gestation and post-partum. OxLDL and anti-oxLDL concentrations were measured by enzyme-linked immunosorbent assays. ANOVA was used for statistical evaluations followed by post hoc test with Bonferoni adjustment., Results: Oxidized Low Density Lipoprotein concentrations increased while anti-oxLDL levels decreased significantly from early to late gestation. OxLDL was strongly positively correlated with LDL concentration and mildly negatively associated with anti-oxLDL levels. Estimating the status of oxidation by calculating oxLDL/LDL ratio revealed decreasing values with ongoing pregnancy. Multivariate analysis showed that anti-oxLDL levels were dependent on gestational age but neither on oxLDL levels nor on the oxLDL/LDL ratio., Conclusions: The results indicate that normal pregnancy is a well-balanced state of oxidative and anti-oxidative processes. However, we could not confirm a dependence of anti-oxLDL autoantibodies on oxLDL concentration. Whether or not the humoral immune system is involved in oxidative defence remains to be elucidated., (© 2012 John Wiley & Sons A/S.)

In chronic inflammatory diseases, cardiovascular disease risk is increased and is the main cause of increased mortality. Oxidized LDL (oxLDL) and scavenger receptors participate in atherogenesis. Using human arterial endothelial cells (HAECs), we evaluated the effect of IL-6 and TNF-α on the expression of scavenger receptors. IL-6 induced expression of SR-A mRNA and TNF-α induced both SR-A and LOX-1 mRNA. Both did induce either CD36 or CD68. To assess the function of scavenger receptors, MCP-1 production by oxLDL from cytokine-pretreated HAEC was examined. In accordance with scavenger receptor expression, oxLDL-induced MCP-1 production was increased in IL-6- or TNF-α-pretreatment. Serum from rheumatoid arthritis patients but not from healthy subjects increased mRNA expressions of SR-A, LOX-1 and CD36 in HAEC. SR-A expression was inhibited by both anti-IL-6 receptor antibody (α-IL-6R Ab) and TNF-α receptor (p75)-Fc (TNFR-Fc) and LOX-1 expression was inhibited by TNFR-Fc. CD36 expression was affected by neither. Serum from rheumatoid arthritis patients augmented oxLDL-induced MCP-1 production. Both α-IL-6R Ab and TNFR-Fc partially inhibited this MCP-1 production. In conclusion, our results strongly support that blocking therapy of IL-6 and TNF-α might be beneficial to reduce atherosclerosis risk in chronic inflammatory diseases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The aim of this study was to explore the influence of simvastatin dosing time, variable compliance and circadian cholesterol production on the reduction of low-density lipoprotein (LDL). A published pharmacokinetic-pharmacodynamic (PKPD) model for simvastatin was identified and evaluated. A model for circadian LDL production was incorporated into the PKPD model. Reduction in LDL from baseline was simulated stochastically from the full model at dose levels of 10, 20, 40 and 80 mg daily for 30 days. Simulated dosing times for each data set were morning (8.00 a.m.), evening (22.00 p.m.), evening with reduced compliance and evening for a hypothetical bioequivalent generic. Differences in LDL reduction from baseline between evening (33-43%) and morning dosing (31-43%) were negligible across a range of doses. Any differences were negated when variable compliance was considered. In addition, differences in simvastatin effect between morning and evening dosing were found to be within the range of LDL concentrations that would be permissible for a bioequivalent generic (at the lower limit) and hence are not likely to be important clinically. The results of this study suggest that taking simvastatin in the evening is not superior to morning dosing., (© 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.)

Hydrolysis and oxidation of LDL stimulate LDL entrapment in the arterial wall and promote inflammation and atherosclerosis via various mechanisms including lipoprotein fusion and lipid droplet formation. To determine the effects of FFA on these transitions, we hydrolyzed LDL by phospholipase A(2) (PLA(2)), removed FFA by albumin, and analyzed structural stability of the modified lipoproteins. Earlier, we showed that heating induces LDL remodeling, rupture, and coalescence into lipid droplets resembling those found in atherosclerotic lesions. Here, we report how FFA affect these transitions. Circular dichroism showed that mild LDL lipolysis induces partial β-sheet unfolding in apolipoprotein B. Electron microscopy, turbidity, and differential scanning calorimetry showed that mild lipolysis promotes LDL coalescence into lipid droplets. FFA removal by albumin restores LDL stability but not the protein conformation. Consequently, FFA enhance LDL coalescence into lipid droplets. Similar effects of FFA were observed in minimally oxidized LDL, in LDL enriched with exogenous FFA, and in HDL and VLDL. Our results imply that FFA promote lipoprotein coalescence into lipid droplets and explain why LDL oxidation enhances such coalescence in vivo but hampers it in vitro. Such lipid droplet formation potentially contributes to the pro-atherogenic effects of FFA.

Background: Myocardial infarction and colorectal cancer are associated at the population level and in autoptic studies. Glycated apolipoprotein B (apoB) is a risk factor for the development of myocardial infarction. The association of glycated apoB with dysplastic and neoplastic colorectal tissue was investigated., Materials and Methods: Forty-eight consecutive surgical specimens, 26 colorectal adenomas and 22 colorectal carcinomas, retrieved from the archives of the Pathologic Anatomy Department of our institution, were examined. The tissue content of glycated apoB was determined using a monoclonal antibody., Results: Glycated apoB was detected in 27% of the adenomas and 45% of the cancer tissue, but only in 18% of the normal tissue near the cancer site., Conclusion: Glycated apoB is associated with dysplastic and even more so with neoplastic cancer tissue.

15-Lipoxygenase (15-LOX) is one of the key enzymes responsible for the formation of oxidized low-density lipoprotein (ox-LDL), a major causal factor for atherosclerosis. Both enzymatic (15-LOX) and non-enzymatic (Cu(2+)) mechanisms have been proposed for the production of ox-LDL. We have recently reported that cannabidiol-2',6'-dimethyl ether (CBDD) is a selective and potent inhibitor of 15-LOX-catalyzed linoleic acid oxygenation (Takeda et al., Drug Metab. Dispos., 37, 1733-1737 (2009)). In the LDL, linoleic acid is present as cholesteryl linoleate, the major fatty acid esterified to cholesterol, and is susceptible to oxidative modification by 15-LOX or Cu(2+). In this investigation, we examined the efficacy of CBDD on i) 15-LOX-catalyzed oxygenation of cholesteryl linoleate, and ii) ox-LDL formation catalyzed by 15-LOX versus Cu(2+)-mediated non-enzymatic generation of this important mediator. The results obtained demonstrate that CBDD is a potent and selective inhibitor of ox-LDL formation generated by the 15-LOX pathway. These studies establish CBDD as both an important experimental tool for characterizing 15-LOX-mediated ox-LDL formation, and as a potentially useful therapeutic agent for treatment of atherosclerosis.

Background: Here, we aim to identify defects of apolipoprotein (apo) B lipoprotein metabolism that characterize hypertriglyceridemia, focusing on apoC-III and apoE., Methods and Results: We studied the transport of plasma apoB within 21 distinct subfractions as separated by anti-apoC-III and anti-apoE immunoaffinity chromatography and ultracentrifugation in 9 patients with moderate hypertriglyceridemia and 12 normotriglyceridemic control subjects. Hypertriglyceridemia was characterized by a 3-fold higher liver secretion of very low-density lipoprotein (VLDL) that had apoC-III but not apoE and a 50% lower secretion of VLDL with both apoC-III and apoE (both P<0.05). This shift in VLDL secretion pattern from apoE to apoC-III resulted in significantly reduced clearance of light VLDL (-39%; P<0.05), compatible with the antagonizing effects of apoC-III on apoE-induced clearance of triglyceride-rich lipoproteins. In addition, rate constants for clearance were reduced for apoE-containing triglyceride-rich lipoproteins in hypertriglyceridemia, associated with increased apoC-III contents of these particles. LDL distribution shifted from light and medium LDL to dense LDL in hypertriglyceridemia through a quartet of kinetic perturbations: increased flux from apoC-III-containing triglyceride-rich lipoproteins, a shift in liver LDL secretion pattern from light to dense LDL, an increased conversion rate from light and medium LDL to dense LDL, and retarded catabolism of dense LDL., Conclusions: These results support a central role for apoC-III in metabolic defects leading to hypertriglyceridemia. Triglyceride-rich lipoprotein metabolism shifts from an apoE-dominated system in normotriglyceridemic participants characterized by rapid clearance from circulation of VLDL to an apoC-III-dominated system in hypertriglyceridemic patients characterized by reduced clearance of triglyceride-rich lipoproteins and the formation of the dense LDL phenotype.

The antioxidant activities of the thymoquinone-rich fraction (TQRF) extracted from Nigella sativa and its bioactive compound, thymoquinone (TQ), in rats with induced hypercholesterolemia were investigated. Rats were fed a semipurified diet supplemented with 1% (w/w) cholesterol and were treated with TQRF and TQ at dosages ranging from 0.5 to 1.5 g/kg and 20 to 100 mg/kg body wt, respectively, for 8 weeks. The hydroxyl radical (OH(.))-scavenging activity of plasma samples collected from experimental rats was measured by electron spin resonance. The GenomeLab Genetic Analysis System was used to study the molecular mechanism that mediates the antioxidative properties of TQRF and TQ. Plasma total cholesterol and low-density-lipoprotein cholesterol levels were significantly decreased in the TQRF- and TQ-treated rats compared to untreated rats. Feeding rats a 1% cholesterol diet for 8 weeks resulted in a significant decrease in plasma antioxidant capacity, as measured by the capacity to scavenge hydroxyl radicals. However, rats treated with TQRF and TQ at various doses showed significant inhibitory activity toward the formation of OH(.) compared to untreated rats. Upon examination of liver RNA expression levels, treatment with TQRF and TQ caused the up-regulation of the superoxide dismutase 1 (SOD1), catalase, and glutathione peroxidase 2 (GPX) genes compared to untreated rats (P<0.05). In support of this, liver antioxidant enzyme levels, including SOD1 and GPX, were also apparently increased in the TQRF- and TQ-treated rats compared to untreated rats (P<0.05). In conclusion, TQRF and TQ effectively improved the plasma and liver antioxidant capacity and enhanced the expression of liver antioxidant genes of hypercholesterolemic rats., ((c) 2009 Elsevier Inc. All rights reserved.)

Chemical characterization and acute and sub-acute toxicity study of Trikatu, a generic herbal formulation of Indian system of medicine, was carried out in Charles Foster (CF) rats for safety profiling. In acute toxicity experiment, Trikatu at 2,000 mg/kg body weight once orally was well tolerated by the experimental animals (both male and female) and no changes were observed in mortality, morbidity, gross pathology, gain in weight, vital organ weight, hematological (total white blood cells (WBC) and red blood cells (RBC) count), biochemical parameters such as serum creatinine, serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), serum lipid profile and tissue biochemical parameters such as reduced glutathione and malonaldehyde content as oxidative stress markers. In sub-acute experiment, Trikatu was administered at 5, 50 and 300 mg/kg body weight once daily for 28 days in female CF rats, and non-significant changes were found in most of the parameters studied such as acute experiment except significant increase in low density lipoprotein (LDL) cholesterol level at 50 and 300 mg/kg body weight, decrease in high density lipoprotein (HDL) cholesterol level at 300 mg/kg body weight, increase in SGPT activity at 50 mg/kg body weight and decrease in WBC count at 300 mg/kg body weight on 28(th) day post treatment.

Background: HIV-infected patients develop multiple metabolic abnormalities including insulin resistance, lipodystrophy and dyslipidemia. Although progression of these disorders has been associated with the use of various protease inhibitors and other antiretroviral drugs, HIV-infected individuals who have not received these treatments also develop lipid abnormalities albeit to a lesser extent. How HIV alters lipid metabolism in an infected cell and what molecular changes are affected through protein interaction pathways are not well-understood., Results: Since many genetic, epigenetic, dietary and other factors influence lipid metabolism in vivo, we have chosen to study genome-wide changes in the proteomes of a human T-cell line before and after HIV infection in order to circumvent computational problems associated with multiple variables. Four separate experiments were conducted including one that compared 14 different time points over a period of >3 months. By subtractive analyses of protein profiles overtime, several hundred differentially expressed proteins were identified in HIV-infected cells by mass spectrometry and each protein was scrutinized for its biological functions by using various bioinformatics programs. Herein, we report 18 HIV-modulated proteins and their interaction pathways that enhance fatty acid synthesis, increase low density lipoproteins (triglycerides), dysregulate lipid transport, oxidize lipids, and alter cellular lipid metabolism., Conclusions: We conclude that HIV replication alone (i.e. without any influence of antiviral drugs, or other human genetic factors), can induce novel cellular enzymes and proteins that are significantly associated with biologically relevant processes involved in lipid synthesis, transport and metabolism (p = <0.0002-0.01). Translational and clinical studies on the newly discovered proteins may now shed light on how some of these proteins may be useful for early diagnosis of individuals who might be at high risk for developing lipid-related disorders. The target proteins could then be used for future studies in the development of inhibitors for preventing lipid-metabolic anomalies. This is the first direct evidence that HIV-modulates production of proteins that are significantly involved in disrupting the normal lipid-metabolic pathways.

Objective: Cholesteryl ester transfer protein (CETP) inhibition with torcetrapib not only increases high-density lipoprotein cholesterol levels but also significantly reduces plasma triglyceride, low-density lipoprotein (LDL) cholesterol, and apolipoprotein B (apoB) levels. The goal of the present study was to define the kinetic mechanism(s) by which CETP inhibition reduces levels of apoB-containing lipoproteins., Methods and Results: Nineteen subjects, 9 of whom were pretreated with 20 mg atorvastatin, received placebo for 4 weeks, followed by 120 mg torcetrapib once daily for 4 weeks. Six subjects in the nonatorvastatin group received 120 mg torcetrapib twice daily for an additional 4 weeks. After each phase, subjects underwent a primed-constant infusion of deuterated leucine to endogenously label newly synthesized apoB to determine very low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL) and LDL apoB100 production, and fractional catabolic rates (FCRs). Once-daily 120 mg torcetrapib significantly reduced VLDL, IDL, and LDL apoB100 pool sizes by enhancing the FCR of apoB100 within each fraction. On a background of atorvastatin, 120 mg torcetrapib significantly reduced VLDL, IDL, and LDL apoB100 pool sizes. The reduction in VLDL apoB100 was associated with an enhanced apoB100 FCR, whereas the decreases in IDL and LDL apoB100 were associated with reduced apoB100 production., Conclusions: These data indicate that when used alone, torcetrapib reduces VLDL, IDL, and LDL apoB100 levels primarily by increasing the rate of apoB100 clearance. In contrast, when added to atorvastatin treatment, torcetrapib reduces apoB100 levels mainly by enhancing VLDL apoB100 clearance and reducing production of IDL and LDL apoB100.

Objective: To investigate the effects of oxidized low-density lipoprotein receptor 1 (LOX-1) on secretion of adhesive molecules mediated by ox-LDL in human umbilical endothelial cells (HUVECs)., Methods: HUVECs with different concentration of ox-LDL (0, 10, 20, 50, 100 microg/ml) were incubated for 24 h, or HUVECs were pretreated with 250 microg/ml poly (I) or 250 microg/ml carrageenan for 2 h and then incubated with 50 microg/ml ox-LDL for another 24 h. Expression of LOX-1 was determined by realtime RT-PCR and Western blot. mRNA and protein of ICAM-1, VCAM-1 and E-selectin were examined by RT-PCR and Western blot respectively., Results: Incubation of HUVECs with ox-LDL (10-100 microg/ml) enhanced the expressions of LOX-1, ICAM-1 and E-selectin in a concentration-dependent manner (P < 0.01). On the contrary, ox-LDL did not affect the expression of VCAM-1 by HUVECs. The expression of LOX-1, ICAM-1 and E-selectin induced by ox-LDL were reduced in HUVECs pretreated with 250 microg/ml poly (I) or 250 microg/ml carrageenan for 2 h and then incubated with 50 microg/ml ox-LDL for 24 h. This showed that both poly (I) and carrageenan obviously decreased the expression of LOX-1, ICAM-1 and E-selectin induced by ox-LDL., Conclusion: ox-LDL may upregulate the expression of LOX-1, ICAM-1 and E-selectin, and LOX-1 blocker may partly inhibit this upregulation. The results suggest that the expression of inflammatory molecules induced by ox-LDL in HUVECs is mediated by LOX-1.

Objective: To investigate for the presence of oxidized low-density lipoprotein (ox-LDL) and lectin-like oxidized LDL receptor 1 (LOX-1) in cartilage specimens from rheumatoid arthritis (RA) joints and to determine whether the interaction of ox-LDL with LOX-1 can induce matrix metalloproteinase 3 (MMP-3) in articular cartilage explant culture., Methods: Human articular cartilage specimens obtained from patients with RA, osteoarthritis (OA), and femoral neck fractures were examined for LOX-1 and ox-LDL by confocal fluorescence microscopy. The association between ox-LDL and LOX-1 was evaluated by immunofluorescence analysis. Articular cartilage specimens from patients with femoral neck fractures were incubated with ox-LDL, with or without preincubation with neutralizing anti-LOX-1 antibody. MMP-3 synthesis by chondrocytes in explant cartilage was evaluated by immunofluorescence, and protein secretion into conditioned medium was monitored by immunoblotting and enzyme-linked immunosorbent assay., Results: The majority of the RA chondrocytes stained positively with both anti-LOX-1 and anti-ox-LDL antibodies; however, no positive cells were found in OA and normal cartilage specimens. Anti-LOX-1 antibody suppressed the binding of DiI-labeled ox-LDL to chondrocytes in explant culture, suggesting that the interaction was mediated by LOX-1. In contrast to native LDL, ox-LDL induced MMP-3 synthesis by articular chondrocytes in association with the induction of LOX-1, which resulted in enhanced secretion of MMP-3 into the culture medium. Anti-LOX-1 antibody reversed ox-LDL-stimulated MMP-3 synthesis to control levels., Conclusion: Ox-LDL, principally mediated by LOX-1, enhanced MMP-3 production in articular chondrocytes. Increased accumulation of ox-LDL with elevated expression of LOX-1 in RA cartilage indicates a specific role of the receptor-ligand interaction in cartilage pathology in RA.

The GALAXY Programme is a comprehensive global research initiative that will address several important unanswered questions in statin research and investigate the impact of rosuvastatin on cardiovascular risk reduction and patient outcomes. Studies already completed demonstrate that rosuvastatin provides greater reductions in low-density lipoprotein cholesterol (LDL-C) than other statins, enabling more patients to achieve LDL-C treatment goals. Additionally, rosuvastatin provides beneficial effects on other components of the atherogenic lipid profile. Ongoing studies will evaluate whether these effects translate into beneficial effects on atherosclerosis and significant reductions in cardiovascular events. Important information will also be provided on the role of statins in less well studied groups, including patients with heart failure, end stage renal disease, and individuals without elevated LDL-C but at heightened vascular risk as a result of increased systemic inflammation. Ultimately, the GALAXY Programme will provide clinical data that will enable physicians to make more effective statin treatment decisions, which will lead to improved patient care and cardiovascular outcomes.

Forty-eight multiparous Holstein cows were fed treatments consisting of either 0, 45, 60, or 75 g/d of a rumen-protected choline (RPC) source in a completely randomized design from 21 d before expected calving to 63 d postpartum to determine whether choline supplementation to the diet would affect hepatic fatty acid and glucose metabolism, key metabolites in plasma, and cow performance. Dry matter intake (DMI), milk yield, body condition score, and body weights (BW) were similar for cows receiving the four treatments. Feeding RPC tended to increase yields of milk fat, 3.5% fat-corrected milk, and total solids. Plasma concentrations of nonesterified fatty acids and beta-hydroxybutyrate were not different among cows fed the four treatments. Concentrations of triglycerides in liver were similar, but concentrations of glycogen in liver increased as cows consumed increasing amounts of RPC. Hepatic capacity for storage of [1-(14)C]palmitate as esterified products within liver slices tended to decrease as the amount of RPC consumed by cows increased; however, effects of treatment on hepatic capacity for oxidation of [1-(14)C]palmitate to CO2 were not significant. These data imply that choline may increase the rate of very low density lipoprotein synthesis and secretion of esterified lipid products from liver. Hepatic capacities for conversion of [1-(14)C] propionate to CO2 and to glucose in liver were similar among cows fed the four treatments. Collectively, these results suggest that hepatic fatty acid metabolism and cow performance are responsive to increasing the supply of choline during the periparturient period.

Lipoprotein(a) is composed of low density lipoprotein and apolipoprotein(a). Apolipoprotein(a) has evolved from plasminogen and contains 10 different plasminogen kringle 4 homologous domains [KIV(1-110)]. Previous studies indicated that lipoprotein(a) non-covalently binds the N-terminal region of lipoprotein B100 and the plasminogen kringle 4 binding plasma protein tetranectin. In this study recombinant KIV(2), KIV(7) and KIV(10) derived from apolipoprotein(a) were produced in E. coli and the binding to tetranectin and low density lipoprotein was examined. Only KIV(10) bound to tetranectin and binding was similar to that of plasminogen kringle 4 to tetranectin. Only KIV(7) bound to LDL. In order to identify the residues responsible for the difference in specificity between KIV(7) and KIV(10), a number of surface-exposed residues located around the lysine binding clefts were exchanged. Ligand binding analysis of these derivatives showed that Y62, and to a minor extent W32 and E56, of KIV(7) are important for LDL binding to KIV(7), whereas R32 and D56 of KIV(10) are required for tetranectin binding of KIV(10).

The aim of the present study was to determine the direct effect of glucose on LDL oxidation, a key step in the development of atherosclerosis. Purified human LDL were incubated with glucose (500 mg/dl) and LDL oxidation was started by adding CuCl(2) to the media. Glucose delayed the vitamin E consumption, but accelerated the formation of conjugated dienes and increased both the formation of thiobarbituric acid reacting substances (TBARS) and LDL electrophoretic mobility. When LDL were incubated with increasing concentrations of glucose and submitted to oxidation, the formation of conjugated dienes, TBARS, and the electrophoretic mobility increased in a concentration-dependent manner. When LDL was enriched with vitamin E, it showed a delay in the formation of conjugated dienes, even in the presence of glucose. To determine whether glucose had any effect on LDL oxidation, once the process was started and vitamin E consumed, LDL were submitted to oxidation and, at different times thereafter, glucose was added into the media. Under these conditions glucose also accelerated the LDL oxidation. In summary, present results show that in LDL submitted to oxidation, glucose delays the early phases of the oxidation, slowing the vitamin E consumption, but it accelerates the rate of LDL oxidation once LDL vitamin E has been consumed; the effect being concentration-dependent.

Myeloperoxidase, in the presence of hydrogen peroxide and nitrite, promotes the lipid peroxidation of low density lipoprotein (LDL); the modified lipoprotein is then capable of being readily endocytosed by macrophages. Since acetaminophen has been shown to inhibit the leukocyte myeloperoxidase antimicrobial system and is, under certain experimental conditions, an antioxidant, the effect of acetaminophen on the myeloperoxidase-hydrogen peroxide-nitrite mediated oxidation of LDL was examined. The content of LDL lipid hydroperoxides after incubation with 50 nM myeloperoxidase, 100 microM nitrite and a hydrogen peroxide generating system for 6 h was reduced by approx. 80% in the presence of 25-250 microM acetaminophen. The production of thiobarbituric acid-reactive substances was also inhibited by acetaminophen to a similar extent. Acetylsalicylic acid (25-100 microM) did not inhibit LDL lipid peroxidation mediated by the myeloperoxidase enzyme system. LDL, treated with myeloperoxidase, hydrogen peroxide and nitrite for 14 h, was metabolized by macrophages to a much greater extent than native LDL. The presence of acetaminophen prevented the modification of LDL; the lipoprotein was metabolized by macrophages to the same extent as was native LDL. These results demonstrate that acetaminophen is a potent inhibitor of the myeloperoxidase-hydrogen peroxide-nitrite mediated modification of LDL.

Background: Cyclosporin A is a lipogenic immunosuppressor that can induce posttransplant hyperlipidaemia. Oxidation of low-density lipoprotein (LDL) has been recognized as a major atherogenic factor. Tacrolimus seems to be less lipogenic with an apparently better cardiovascular profile than CsA., Methods: We have studied the lipidic profile and the oxidation of HDL and LDL in 20 renal transplant patients, 12 male and 8 female, mean age 45 +/- 10 year, who where switched from CsA to tacrolimus due to CsA adverse effects. LDL were determined by ultracentrifugation. Oxidation study before and 6 months after conversion to tacrolimus was performed by adding CuSO4., Results: After conversion, systolic blood pressure (BP) decreased from 154 +/- 21 to 133 +/- 21 mm Hg (p = 0.008), diastolic BP from 97 +/- 13 to 77 +/- 15 mm Hg (p = 0.016), total cholesterol from 6.08 +/- 0.9 to 5.68 +/- 1.1 mmol/l (p = 0.02), LDL-chol from 3.29 +/- 1.01 to 2.96 +/- 0.3 mmol/l (p = 0.04) and apo-B lipoprotein from 1.42 +/- 0.28 to 1.15 +/- 0.34 mg/dl (p = 0.003). The oxidation of LDL improved after conversion: the initial dienic compounds decreased from 95 +/- 20 to 63 +/- 12 umol/g and the final DC from 207 +/- 56 to 107 +/- 35 umol/g. Lag-phase increased from 33 +/- 21 to 45 +/- 17 min (p < 0.05)., Conclusion: Tacrolimus has improved hyperlipidaemia in our cyclosporin previously treated patients and increased the resistance to oxidation of high and low-density lipoproteins.

Objective: To study the effect of vitamin C on preventing oxidative modification of low density lipoprotein cholesterol (LDL) in vitro., Methods: Different levels of vitamin C were added to two different systems of lipid peroxidation of LDL induced by either Cu(2+) or macrophage. The level of VC in systems induced by Cu(2+) were 10, 50, 100 and 200 micromol/L respectively and by macrophage were 50, 100, 200 micromol/L respectively. Vitamin E (200 micromol/L) and VC(0) (0 micromol/L) were served as positive and negative control respectively. The content of TBARS, Ox-LDL, fluorescent compounds, electrophoretic mobility of LDL and the lag-phage of oxidation of LDL were measured., Results: In the oxidative systems induced by Cu(2+), the groups with higher levels of VC (100, 200 micromol/L) reduced the levels of TBARS, Ox-LDL at 3, 6, and 9 hour after adding Cu(2+), but low dose groups (10, 50 micromol/L) had no the effects. In the macrophage systems, higher levels of VC (100, 200 micromol/L) significantly reduced levels of fluorescent compounds and TBARS, and also lowered electrophoretic mobility of LDL and increased the lag-phage of oxidation of LDL., Conclusion: Vitamin C has dual effect on oxidation of LDL. Low dose treatment enhanced oxidation of LDL, but high doses has anti-oxidative effects on LDL.

Oxidatively modified LDL (ox-LDL) activates a lectin-like receptor, LOX-1, which results in the expression of adhesion molecules on endothelial surface. We investigated the regulation of the expression of transforming growth factor-beta(1) (TGF-beta(1)) and its receptors by ox-LDL and the functional significance of this interaction with regard to adhesion molecule expression in human coronary artery endothelial cells (HCAECs). Ox-LDL, in a time- and concentration-dependent manner, upregulated the expression of all 3 subtypes (1, 2, and 3 [including endoglin]) of TGF-beta(1) receptors and decreased active TGF-beta(1) synthesis (all P<0.05 versus control and native-LDL-treated cells). Treatment of HCAECs with a monoclonal antibody to LOX-1 attenuated ox-LDL-mediated upregulation of TGF-beta(1) receptors and decrease in TGF-beta(1) synthesis (P<0.05 versus ox-LDL alone). Ox-LDL also enhanced the expression of P-selectin and ICAM-1 as well as monocyte adhesion to HCAECs (P<0.05 versus control untreated cells). Pretreatment with recombinant TGF-beta(1) attenuated the enhanced expression of adhesion molecules and monocyte adhesion to HCAECs (P<0.05 versus ox-LDL alone). Effects of recombinant TGF-beta(1) were blocked by antibody to TGF-beta(1) receptor type 2, but not by antibody to endoglin. Thus ox-LDL, via activation of LOX-1, increases the expression of TGF-beta(1) receptors and decreases TGF-beta(1) synthesis in HCAECs. Recombinant TGF-beta(1), by binding to TGF-beta(1) type 2 receptors, modulates ox-LDL-mediated expression of adhesion molecules and monocyte adhesion to HCAECs.

Transgenic mice overexpressing Cu/Zn superoxide dismutase (hSod1Tg(+/0)) or catalase (hCatTg(+/0)) and knockout mice underexpressing manganese superoxide dismutase (Sod2(+/)(-)) or glutathione peroxidase-1 (Gpx1(-/-)) were used to study the effect of antioxidant enzymes on cell-mediated low density lipoprotein (LDL) oxidation and oxidized LDL (oxLDL)-induced apoptosis. Incubation of LDL with mouse aortic segments or smooth muscle cells (SMCs) resulted in a significant increase in LDL oxidation. However, LDL oxidation was significantly reduced when LDL was incubated with aortic segments and SMCs obtained from hSod1Tg(+/0) and hCatTg(+/0) mice compared with those obtained from wild-type mice. In contrast, LDL oxidation was significantly increased when LDL was incubated with aortic segments and SMCs obtained from Sod2(+/)(-) and Gpx1(-/-) mice. CuSO(4)-oxidized LDL increased DNA fragmentation and caspase activities in the primary cultures of mouse aortic SMCs. However, oxLDL-induced DNA fragmentation and caspase activities were reduced 50% in SMCs obtained from hSod1Tg(+/0) and hCatTg(+/0) mice compared with wild-type control mice. In contrast, oxLDL-induced DNA fragmentation and caspase activities were significantly increased in SMCs obtained from Sod2(+/)(-) and Gpx1(-/-) mice. These findings suggest that overexpression of Cu/Zn superoxide dismutase or catalase reduces cell-mediated LDL oxidation and oxLDL-induced apoptosis, whereas underexpression of manganese superoxide dismutase or glutathione peroxidase-1 increases cell-mediated LDL oxidation and oxLDL-induced apoptosis.

Apolipoprotein (apo) E is a multifunctional protein that can act as a ligand for lipoprotein receptors. The receptor-mediated clearance of the triglyceride-rich lipoproteins (TRL) chylomicrons and VLDL from plasma is, in part, dependent on apo E. Enrichment of VLDL with apo E is thought to enhance receptor-mediated clearance of VLDL resulting in a low rate of conversion of VLDL to LDL. However, the kinetic mechanism controlling the concentration of apo E in VLDL is not known. We conducted kinetic studies on apo E in the TRL fraction (d < 1.006 g/ml) and apo B-100 in the TRL and LDL (d = 1.019-1.063 g/ml) fractions to assess the kinetic determinants of apo E concentration in TRL and to determine the effects that TRL apo E production and clearance rates have on the production rate of LDL apo B-100. Nineteen males between the ages of 24 and 73 underwent a primed-constant infusion with deuterated leucine tracer in the constantly-fed state. Apo B-100 from TRL and LDL, and apo E from TRL were isolated and their tracer incorporation measured by gas chromatography/mass spectrometry. The residence time and production rates of each protein were determined from the kinetic data using the SAAM II modeling program. The residence time and production rate of TRL apo E were about one-half that of TRL apo B-100 (1.8 +/- 1.0 vs. 2.9 +/- 2.1 h and 14.5 +/- 11.0 vs. 27.6 +/- 17.3 mg/kg per day, respectively). The production rate of TRL apo E was weakly correlated with the production rate of TRL apo B-100 (r = 0.424, P = 0.07). Multiple regression analysis showed that the residence time of TRL apo B-100 and the relative TRL apo E production rate (relative to the TRL apo B100 production rate) were negatively associated with LDL apo B-100 production rate, accounting for 68% of its variability. We conclude that (1) the concentration of apo E in TRL is highly correlated to its production rate, suggesting that production rate regulates the TRL apo E concentration, and (2) individuals with a relatively short TRL apo B-100 residence time and those producing TRL with a relatively low apo E content have the highest LDL apo B-100 production rates.

We have determined whether the anti-atherosclerotic effect of a 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitor (fluvastatin) is mediated through nitric oxide (NO) as well as affecting plasma lipids. NO related vascular responses, endothelial nitric oxide synthase (eNOS) mRNA and superoxide anion (O(2)(-)) release were examined in vascular walls of oophorectomized female rabbits fed 0.5% cholesterol chow for 12 weeks with or without fluvastatin (2 mg/kg per day). Serum lipid profile was not different between two groups. NO dependent responses stimulated by acetylcholine and calcium ionophore A23187 and tone related basal NO response induced by N(G)-monomethyl-L-arginine acetate (L-NMA); nitric oxide synthase inhibitor were all improved by fluvastatin treatment. Endothelium independent vasorelaxation induced by nitroglycerin was not different between the two groups of rabbits' arteries. Fluvastatin treatment increased cyclic GMP concentration in aorta of rabbits. eNOS mRNA expression and O(2)(-) release were measured in aorta using competitive reverse transcription-polymerase chain reaction (RT-PCR) and with lucigenin analogue, 2-methyl-3,7-dihydroimidazol [1,2-a]pyrazine-3-one (MCLA) chemiluminescence methods. eNOS mRNA in the endothelial cells of aorta was significantly up-regulated and O(2)(-) production was significantly reduced in fluvastatin treated rabbit aorta. Anti-macrophage staining area, but not anti-smooth muscle cell derived actin stained area in the aorta was also reduced by fluvastatin treatment. Conclusion, fluvastatin, a HMG-CoA reductase inhibitor, retards the initiation of atherosclerosis formation through the improvement of NO bioavailability by both up-regulation of eNOS mRNA and decrease of O(2)(-) production in vascular endothelial cells, and this means that part of the anti-atherosclerotic effect of fluvastatin may be due to nonlipid factors.

Studies in vitro reveal that fluvastatin, an HMG-CoA reductase inhibitor, has a strong DPPH radical scavenging activity and achieves concentration-dependent inhibition of copper- and cell-induced oxidation of low-density lipoprotein (LDL). To further examine the anti-oxidative activity of fluvastatin in vivo, we elucidated the effects of chronic treatment with fluvastatin at a dose insufficient to reduce plasma cholesterol levels (2 mg/kg per day) on vasomotion and vascular oxidative stress in thoracic aortas of 0.5% cholesterol-fed rabbits. After 12 weeks of dietary treatment, aortic segments from rabbits fed cholesterol alone showed impaired endothelium-dependent relaxation responses to acetylcholine and A23187 compared to normal chow-fed rabbits in association with a significant increase in plasma total cholesterol levels. In contrast, although plasma total cholesterol levels were not different from those in control cholesterol-fed rabbits, aortic segments from fluvastatin-treated rabbits showed normal relaxation. Compared with rabbits fed cholesterol alone, fluvastatin treatment decreased susceptibility of LDL to ex vivo copper-induced oxidation, reduced vascular superoxide generation, and atheromatous plaque formation. In conclusion, the potent anti-oxidative properties of fluvastatin in addition to its cholesterol-lowering activity appear to contribute to its anti-atherosclerotic effect in vivo.

Familial defective apolipoprotein (apo) B-100 (FDB) is a frequent cause of hypercholesterolemia. Hypercholesterolemia in homozygous FDB is less severe than in homozygotes for familial hypercholesterolemia. Recently, we showed decreased low density lipoprotein (LDL) apoB-100 fractional catabolism and decreased production of LDL due to an enhanced removal of apoE-containing precursors in a patient with homozygous FDB. The effects of defective apoB-100 on high density lipoprotein (HDL) metabolism are unknown. We studied HDL apoA-I metabolism in this FDB patient and in 6 control subjects by using (2)H(3)-L-leucine as a tracer. ApoA-I levels were normal in all study subjects. However, the fractional catabolic rate and the production rate of apoA-I were increased, by 79% and 70%, respectively, in FDB; the fractional catabolic rate of apoA-I in FDB was 0.34 day(-1) compared with 0.19+/-0.03 day(-1) in normal controls. The production rate of apoA-I in FDB was 18.4 mg. kg(-1). d(-1) compared with 10.8+/-2.3 mg. kg(-1). d(-1) in controls. Thus, we have shown for the first time that defective apoB-100 may influence HDL kinetics. The increase in total HDL turnover might enhance reverse cholesterol transport and could contribute to the seemingly benign clinical course of FDB compared with that of familial hypercholesterolemia.

The literature relating lipid and lipoprotein oxidation to atherosclerosis has expanded enormously in recent years. Papers on the "oxidative modification hypothesis" of atherogenesis have ranged from the most basic studies of the chemistry and enzymology of LDL oxidation, through studies of the biological effects of oxidized LDL on cultured cells, and on to in vivo studies of the effects of antioxidants on atherosclerosis in animals and humans. The data in support of this theory are mounting but many key questions remain unanswered. For example, while it is generally agreed that LDL undergoes oxidation and that oxidized LDL is present in arterial lesions, it is still not known how and where LDL gets oxidized in vivo nor which of its many biological effects demonstrable in vitro are relevant to atherogenesis in vivo. This brief review is not intended to be comprehensive but rather to offer a perspective and a context for this Forum. We discuss the strengths and weaknesses of each line of evidence, try to identify areas in which further research is needed, assess the relevance of the hypothesis to the human disease, and point to some of the potential targets for therapy.

Chlamydia pneumoniae is an obligate intracellular prokaryotic human pathogen responsible for a significant portion of atypical pneumonia and associated with a variety of chronic sequelae, the most significant of which is atherosclerosis. The organism is endowed with several attributes that may contribute to the development of atherosclerotic lesions or promote tissue damage at the site of an existing lesion. Two key events that are directly involved in the atherogenic process include the development of foam cells from macrophages and the oxidation of lipoproteins at the site of lesion development. The former process allows for deposition of cholesterol-containing low-density lipoprotein (LDL) and the latter can contribute directly to tissue damage locally. We have hypothesized that C pneumoniae may interact with mononuclear phagocytes in ways that are consistent with the view that this organism contributes to atherosclerotic lesion development. We have demonstrated that the presence of C pneumoniae causes macrophage foam cell formation and lipid oxidation with murine and human cells cocultured in the presence of LDL. In addition, we have provided evidence that implicates 2 putative chlamydial virulence factors in the development of these pathologic processes. Chlamydial lipopolysaccharide has been shown to cause macrophages to develop into foam cells in the presence of LDL, and the 60-kDa chlamydial heat shock protein (cHsp60), a known pathogenesis-inducing protein, has been found to contribute to oxidation of LDL in the presence of macrophages. Work is currently underway to define mechanisms involved in these processes and to further refine the putative role of C pneumoniae in atherogenesis and atherosclerotic lesion development.

To establish a role of the 12-lipoxygenase on the generation of oxidized low density lipoprotein (LDL) in macrophages that leads to foam cell formation in atherosclerosis, we overexpressed 12-lipoxygenases in a macrophage-like cell line, J774A.1, that does not show intrinsic enzyme activity. When the 12-lipoxygenase-expressing cells were incubated with 400 microg.mL-1 LDL in Dulbecco's modified Eagle's medium at 37 degrees C for 12 h, LDL oxidation, as determined by thiobarbituric acid reactive substance, was markedly increased compared with the mock-transfected cells. Oxygenated products in the modified LDL were examined by HPLC before and after alkaline hydrolysis. Most of the oxygenated derivatives were of an esterified form, and the major product was identified as 13S-hydroxyoctadeca-9Z,11E-dienoic acid. These results clearly demonstrate that esterified fatty acids in LDL are oxygenated by the 12-lipoxygenases expressed in the J774A.1 cells. Furthermore, the oxidized LDL generated by intracellular 12-lipoxygenases was recognized by a scavenger receptor as assessed by macrophage degradation assay.

Cellular apoB in primary rat hepatocyte cultures was pulse-labeled with [(35)S]methionine for 1 h. Cells were then chased with excess unlabeled methionine for periods of up to 16 h in the presence or absence of BMS-200150, an inhibitor of microsomal triglyceride transfer protein (MTP). The secretion of apoB-48-VLDL was more sensitive to MTP inhibition than was apoB-100-VLDL. Inhibition of MTP had no inhibitory effect on the secretion of denser particles (apoB-48 HDL and apoB-100 HDL). BMS-200150 delayed the net removal of newly synthesized apoB-48 and apoB-100 from the microsomal and Golgi membranes, but not from the corresponding lumenal compartments. Only minor proportions of the microsomal lumen apoB-48 and apoB-100 (12-16% and 17-19%, respectively) were present as VLDL irrespective of whether MTP was inactivated or not. The HDL fraction contained most of the lumenal apoB-48 (67-73%) and a somewhat smaller proportion of apoB-100 (44-47%). The remainder of the lumenal apoB was associated with the IDL/LDL fraction. These proportions were unaffected by MTP inactivation. Excess labeled apoB which accumulated in the membranes in the presence of BMS-200150 was degraded. Inhibition of MTP prevented the removal of pre-synthesized triacylglycerol (TAG) from the hepatocytes as apoB-VLDL. Under these conditions intracellular TAG accumulated mainly in the cell cytosol, but also, to a lesser extent, in the microsomal membranes. The results suggest that inactivation of MTP inhibits a pathway of VLDL assembly which does not involve the bulk lumenal compartments of the microsomes. Suppression of this pathway ultimately prevents the net transfer of cytosolic TAG into mature apoB-VLDL.

Some observations have suggested that the extracellular group IIa phospholipase A2 (sPLA2), previously implicated in chronic inflammatory conditions such as arthritis, may contribute to atherosclerosis. We have examined this hypothesis by studying transgenic mice expressing the human enzyme. Compared with nontransgenic littermates, the transgenic mice exhibited dramatically increased atherosclerotic lesions when maintained on a high-fat, high-cholesterol diet. Surprisingly, the transgenic mice also exhibited significant atherosclerotic lesions when maintained on a low-fat chow diet. Immunohistochemical staining indicated that sPLA2 was present in the atherosclerotic lesions of the transgenic mice. On both chow and atherogenic diets, the transgenic mice exhibited decreased levels of HDLs and slightly increased levels of LDLs compared with nontransgenic littermates. These data indicate that group IIa sPLA2 may promote atherogenesis, in part, through its effects on lipoprotein levels. These data also provide a possible mechanism for the observation that there is an increased incidence of coronary artery disease in many chronic inflammatory diseases.