Ox-LDL increases OX40L in endothelial cells through a LOX-1-dependent mechanism

Oxidative low-density lipoprotein (Ox-LDL) is a key risk factor for the development of atherosclerosis, and it can stimulate the expression of a variety of inflammatory signals. As a new and highly sensitive inflammation index, OX40L may be a key to understanding the mechanisms that regulate interactions between cells within the vessel wall and inflammatory mediators during the development of atherosclerosis. To investigate whether Ox-LDL regulates OX40L expression through an oxidized LDL-1 receptor (LOX-1)-mediated mechanism, we investigated the effect of different concentrations of Ox-LDL (50, 100, 150 µg/mL) on endothelial cell proliferation and apoptosis. Stimulation with Ox-LDL increased OX40L protein 1.44-fold and mRNA 4.0-fold in endothelial cells, and these effects were inhibited by blocking LOX-1. These results indicate that LOX-1 plays an important role in the chronic inflammatory process in blood vessel walls. Inhibiting LOX-1 may reduce blood vessel inflammation and provide a therapeutic option to limit atherosclerosis progression.

Loss of insulin synthesis and beta cell survival induced by oxidized LDL require activation of reticulum endoplasmic stress: a mechanism countered by HDL

Elevated circulating concentrations in modified LDL-cholesterol particles (e.g. oxidised LDL) and low levels in HDL increase not only the risk for diabetic patients to develop cardiovascular diseases but also may contribute to development and progression of diabetes by directly having adverse effects on β-cells. Chronic exposure of β-cells to 2 mM human oxidised LDL-cholesterol (oxLDL) increases the rate of apoptosis, reduce insulin biosynthesis and the secretory capacity of the cells in response to nutrients. In line with the protective role, HDL efficiently antagonised the harmful effects of ox- LDL, suggesting that low levels of HDL would be inefficient to protect β-cells against oxLDL attack in patients. Activation of endoplasmic reticulum (ER) stress is pointed out to contribute to β-cell dysfunction elicited by environmental stressors. In this study we investigated whether activation of ER stress is required for oxLDL to mediate detrimental effects on β-cells and we tested the potential antagonist properties of HDL: The mouse MIN6 insulin-secreting cells were cultured with 2 mM of LDL-cholesterol preparation (native or in vitro oxidized) in the presence or absence of 1 mM of HDL-cholesterol or the ER stress inhibitor 4-phenylbutyrate (4-PBA): Prolonged exposure of MIN6 cells to 2 mM oxLDL-cholesterol for 48 hours led to an increase in expression of ER stress markers such as ATF4, CHOP and p58 and stimulated the splicing of XBP-1 whereas, induction of these markers was not observable in the cells cultured with native LDL. Treatment of the cells with the 4-PBA chemical chaperone molecule efficiently blocked activation of the ER stress markers induced by oxLDL. The latter mediates β-cell dysfunction and apoptosis by diminishing the expression of islet brain 1 (IB1) and Bcl2. The levels of these two proteins were preserved in the cells that were co-treated with oxLDL and the 4-PBA. Consistent with this result we found that blockade of ER stress activation alleviated the loss of insulin synthesis and abolished apoptosis evoked by oxLDL. However incubation of the cells with 4-PBA did not prevent impairment of insulin secretion elicited by oxLDL, indicating that ER stress is not responsible for the oxLDL-mediated defect of insulin secretion. Co-incubation of the cells with HDL mimicked the effects of 4-PBA on the expression of IB1 and Blc2 and thereby counteracted oxLDL attacks on insulin synthesis and cell survivals. We found that HDL efficiently inhibited activation of the ER stress mediated by oxLDL: These data highlight the contribution of the ER stress in the defects of insulin synthesis and cell survivals induced by oxLDL and emphasize the potent role of HDL to counter activation of the oxLDL-mediated ER-stress activation:

Oxidized LDL and physical fitness in healthy young men: associations with body composition, smoking, metabolic syndrome and androgen status

Background: In the past, oxidized low density lipoprotein (ox-LDL) has been associated with an unbeneficial lipid profile. This atherogenic lipid profile increases the risk of atherosclerotic cardiovascular diseases. Physical fitness has substantial effect on serum lipoprotein concentration as well as body composition and humoral responses, however interrelationships between ox-LDL and physical fitness have not been widely examined in a nationally representative sample. Aims: This thesis evaluates how cardiorespiratory and muscular fitness associate with ox-LDL lipids and how the other known risk factors of atherosclerosis might alter these associations. Subjects and Methods: The study cohort consisted of 846 healthy young males (mean age 25.1, SD 4.6) who were gathered by voluntary nationwide recruitment. Each participant conducted a series of physical fitness tests (cardiorespiratory and muscular fitness) and answered a detailed questionnaire that included lifestyle habits (i.e. smoking and leisuretime physical activity). Venous blood samples including ox-LDL and serum lipids were also collected. Results: Higher levels of ox-LDL were found in overweight and obese men, however, high cardiorespiratory fitness seemed to protect the overweight from high levels of ox-LDL. Young men who smoked and had poor cardiorespiratory or muscular fitness possessed a higher concentration of ox-LDL lipids when compared to comparable levels of cardiorespiratory or muscular fitness non-smoking young men. Metabolic syndrome was associated with increased levels of ox-LDL and high levels of ox-LDL combined with poor cardiorespiratory and abdominal muscle fitness seems to predict metabolic syndrome in young men. Also, participants with poor cardiorespiratory fitness and low levels of testosterone had higher levels of ox-LDL when compared to participants with high cardiorespiratory fitness / low testosterone as well as those with poor cardiorespiratory fitness / high testosterone. Conclusions: Good cardiorespiratory and muscular fitness protects young men from increased levels of ox-LDL lipids. This association was discovered in young men who were categorized as being overweight, smokers, metabolic syndrome or with low levels of testosterone. Being fit seems to prevent higher levels of ox-LDL, even in young healthy

Genistein reverses changes of the proteome induced by oxidized-LDL in EA center dot hy 926 human endothelial cells

Endothelial cells are primary targets for pro-atherosclerotic stressors such as oxidized LDL (ox-LDL). The isoflavone genistein, on the other hand, is suggested to prevent a variety of processes underlying atherosclerosis and cardiovascular diseases. By analyzing the proteome of EA(.)hy 926 endothelial cells, here we show, that genistein reverses the ox-LDL-induced changes of the steady-state levels of several proteins involved in atherosclerosis. These alterations caused by genistein are functionally linked to the inhibition of ox-LDL induced apoptosis.

Proteome analysis for identification of target proteins of genistein in primary human endothelial cells stressed with oxidized LDL or homocysteine

Background Epidemiological studies suggest that soy consumption contributes to the prevention of coronary heart disease. The proposed anti-atherogenic effects of soy appear to be carried by the soy isoflavones with genistein as the most abundant compound. Aim of the study To identify proteins or pathways by which genistein might exert its protective activities on atherosclerosis, we analyzed the proteomic response of primary human umbilical vein endothelial cells ( HUVEC) that were exposed to the pro-atherosclerotic stressors homocysteine or oxidized low-density lipoprotein (ox-LDL). Methods HUVEC were incubated with physiological concentrations of homocysteine or ox-LDL in the absence and presence of genistein at concentrations that can be reached in human plasma by a diet rich in soy products (2.5 muM) or by pharmacological intervention ( 25 muM). Proteins from HUVEC were separated by two-dimensional polyacrylamide gel electrophoresis and those that showed altered expression level upon genistein treatment were identified by peptide mass fingerprints derived from tryptic digests of the protein spots. Results Several proteins were found to be differentially affected by genistein. The most interesting proteins that were potently decreased by homocysteine treatment were annexin V and lamin A. Annexin V is an antithrombotic molecule and mutations in nuclear lamin A have been found to result in perturbations of plasma lipids associated with hypertension. Genistein at low and high concentrations reversed the stressor-induced decrease of these anti-atherogenic proteins. Ox-LDL treatment of HUVEC resulted in an increase in ubiquitin conjugating enzyme 12, a protein involved in foam cell formation. Treatment with genistein at both doses reversed this effect. Conclusions Proteome analysis allows the identification of potential interactions of dietary components in the molecular process of atherosclerosis and consequently provides a powerful tool to define biomarkers of response.

Circulating oxidized low-density lipoprotein (LDL) is associated with risk factors of the metabolic syndrome and LDL size in clinically healthy 58-year-old men (AIR study).

OBJECTIVES Hypothetically the atherogenic effect of the metabolic syndrome may be mediated through the increased occurrence of small LDL-particles which are easily modified to atherogenic oxidized LDL (ox-LDL). The aim of this study was to test this concept by examining the association between circulating ox-LDL, LDL-particle size, and the metabolic syndrome. DESIGN AND RESULTS A population-based sample of clinically healthy 58-year-old men (n = 391) was recruited. Ox-LDL was measured by ELISA (specific monoclonal antibody, mAb-4E6) and LDL-particle size by gradient gel electrophoresis. The results showed that ox-LDL significantly correlated to factors constituting the metabolic syndrome; triglycerides (r = 0.43), plasma insulin (r = 0.20), body mass index (r = 0.20), waist-to-hip ratio (r = 0.21) and HDL (r = -0.24); (P < 0.001). Ox-LDL correlated also to LDL-particle size (r = -0.42), Apo-B (r = 0.70), LDL (r = 0.65); (P < 0.001) and, furthermore, with Apo A-1 (r = -0.13) and heart rate (r = 0.13); (P < 0.01). CONCLUSION The metabolic syndrome was accompanied by high plasma ox-LDL concentrations compared with those without the syndrome. Ox-LDL levels were associated with most of the risk factors constituting the metabolic syndrome and was, in addition related to small LDL-particle size. To our knowledge the present study is the first one to demonstrate that circulating ox-LDL levels are associated with small LDL-particle size in a population representative sample of clinically healthy middle-aged men. The high degree of intercorrelation amongst several factors makes it difficult to clarify the independent role of any specific factor.

Oxidised LDL-lipids alter redox ratio, lipid raft formation and increase amyloid beta production by SHSY-5Y cells

Elevated cholesterol in mid-life has been associated with increased risk of dementia in later life. We have previously shown that low density lipoprotein (LDL) is more oxidised in the plasma of dementia patients although total cholesterol levels remained unchanged [1]. We have investigated the hypothesis that amyloid beta production and neurodegeneration can be driven by oxidised lipids derived from LDL following the loss of blood brain barrier integrity with ageing. Therefore, we have investigated amyloid beta formation in SHSY5Y cells treated with LDL, minimally modified (ox) LDL, and lipids extracted from both forms of LDL. LDL-treated SHSY-5Y cell viability was not significantly decreased with up to 8 μg LDL/2 × 104 cells compared to untreated cells. However, 8 μg oxLDL protein/2 × 104 cells decreased the cell viability significantly by 33.7% (P < 0.05). A more significant decrease in cell viability was observed when treating cells with extracted lipids from 8 μg of LDL (by 32.7%; P < 0.01) and oxLDL (by 41%; P < 0.01). In parallel, the ratio of reduced to oxidised GSH was decreased; GSH concentrations were significantly decreased following treatment with 0.8 μg/ml oxLD-L (7.35 ± 0.58;P < 0.01), 1.6 μg/ml (5.27 ± 0.23; P < 0.001) and 4 μg/ml (5.31 ± 0.31; P < 0.001). This decrease in redox potential was associated with an increase acid sphingomyelinase activity and lipid raft formation which could be inhibited by desipramine; SHSY5Y cells treated with oxLDL, and lipids from LDL and oxLDL for 16 h showed significantly increased acid sphingomyelinase activity (5.32 ± 0.35; P < 0.05, 5.21 ± 0.6; P < 0.05, and 5.58 ± 0.44; P < 0.01, respectively) compared to control cells (2.96 ± 0.34). As amyloid beta production is driven by the activity of beta secretase and its association with lipid rafts, we investigated whether lipids from ox-LDL can influence amyloid beta by SHSY-5Y cells in the presence of oxLDL. Using ELISA and Western blot, we confirmed that secretion of amyloid beta oligomers is increased by SHSY-5Y cells in the presence of oxLDL lipids. These data suggest a mechanism whereby LDL, and more significantly oxLDL lipids, can drive amyloid beta production and cytotoxicity in neuronal cells. [1] Li L, Willets RS, Polidori MC, Stahl W, Nelles G, Sies H, Griffiths HR. Oxidative LDL modification is increased in vascular dementia and is inversely associated with cognitive performance. Free Radic Res. 2010 Mar; 44(3): 241–8.

Bilirubin is independently associated with oxidized LDL levels in young obese patients

BACKGROUND: Bilirubin can prevent lipid oxidation in vitro, but the association in vivo with oxidized low-density lipoprotein (Ox-LDL) levels has been poorly explored. Our aim is to the association of Ox-LDL with total bilirubin (TB) levels and with variables related with metabolic syndrome and inflammation, in young obese individuals. FINDINGS: 125 obese patients (13.4 years; 53.6% females) were studied. TB, lipid profile including Ox-LDL, markers of glucose metabolism, and levels of C-reactive protein (CRP) and adiponectin were determined. Anthropometric data was also collected. In all patients, Ox-LDL correlated positively with BMI, total cholesterol, LDLc, triglycerides (TG), CRP, glucose, insulin and HOMAIR; while inversely with TB and HDLc/Total cholesterol ratio (P < 0.05 for all). In multiple linear regression analysis, LDLc, TG, HDLc and TB levels were significantly associated with Ox-LDL (standardized Beta: 0.656, 0.293, -0.283, -0.164, respectively; P < 0.01 for all). After removing TG and HDLc from the analysis, HOMAIR was included in the regression model. In this new model, LDLc remained the best predictor of Ox-LDL levels (β = 0.665, P < 0.001), followed by TB (β = -0.202, P = 0.002) and HOMAIR (β = 0.163, P = 0.010). CONCLUSIONS: Lower bilirubin levels may contribute to increased LDL oxidation in obese children and adolescents, predisposing to increased cardiovascular risk.

Influence of a probiotic soy product on fecal microbiota and its association with cardiovascular risk factors in an animal model

Background: Previous work showed that daily ingestion of an aqueous soy extract fermented with Enterococcus faecium CRL 183 and Lactobacillus helveticus 416, supplemented or not with isoflavones, reduced the total cholesterol and non-HDL-cholesterol levels, increased the high-density lipoprotein (HDL) concentration and inhibited the raising of autoantibody against oxidized low-density lipoprotein (ox-LDL Ab) and the development of atherosclerotic lesions. Objective: The aim of this study was to characterize the fecal microbiota in order to investigate the possible correlation between fecal microbiota, serum lipid parameters and atherosclerotic lesion development in rabbits with induced hypercholesterolemia, that ingested the aqueous soy extract fermented with Enterococcus faecium CRL 183 and Lactobacillus helveticus 416. Methods: The rabbits were randomly allocated to five experimental groups (n = 6): control (C), hypercholesterolemic (H), hypercholesterolemic plus unfermented soy product (HUF), hypercholesterolemic plus fermented soy product (HF) and hypercholesterolemic plus isoflavone-supplemented fermented soy product (HIF). Lipid parameters and microbiota composition were analyzed on days 0 and 60 of the treatment and the atherosclerotic lesions were quantified at the end of the experiment. The fecal microbiota was characterized by enumerating the Lactobacillus spp., Bifidobacterium spp., Enterococcus spp., Enterobacteria and Clostridium spp. populations. Results: After 60 days of the experiment, intake of the probiotic soy product was correlated with significant increases (P < 0.05) on Lactobacillus spp., Bifidobacterium spp. and Enterococcus spp. and a decrease in the Enterobacteria population. A strong correlation was observed between microbiota composition and lipid profile. Populations of Enterococcus spp., Lactobacillus spp. and Bifidobacterium spp. were negatively correlated with total cholesterol, non-HDL-cholesterol, autoantibodies against oxidized LDL (ox-LDL Ab) and lesion size. HDL-C levels were positively correlated with Lactobacillus spp., Bifidobacterium spp., and Enterococcus spp. populations. Conclusion: In conclusion, daily ingestion of the probiotic soy product, supplemented or not with isoflavones, may contribute to a beneficial balance of the fecal microbiota and this modulation is associated with an improved cholesterol profile and inhibition of atherosclerotic lesion development.

Associations between CD36 gene polymorphisms and susceptibility to coronary artery heart disease

Associations between polymorphisms of the CD36 gene and susceptibility to coronary artery heart disease (CHD) are not clear. We assessed allele frequencies and genotype distributions of CD36 gene polymorphisms in 112 CHD patients and 129 control patients using semi-quantitative polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Additionally, we detected CD36 mRNA expression by real-time quantitative PCR, and we quantified plasma levels of oxidized low-density lipoprotein (ox-LDL) using an enzyme-linked immunosorbent assay (ELISA). There were no significant differences between the two groups (P>0.05) in allele frequencies of rs1761667 or in genotype distribution and allele frequencies of rs3173798. The genotype distribution of rs1761667 significantly differed between CHD patients and controls (P=0.034), with a significantly higher frequency of the AG genotype in the CHD group compared to the control group (P=0.011). The plasma levels of ox-LDL in patients with the AG genotype were remarkably higher than those with the GG and AA genotypes (P=0.010). In a randomized sample taken from patients in the two groups, the CD36 mRNA expression of the CHD patients was higher than that of the controls. In CHD patients, the CD36 mRNA expression in AG genotype patients was remarkably higher than in those with an AA genotype (P=0.005). After adjusted logistic regression analysis, the AG genotype of rs1761667 was associated with an increased risk of CHD (OR=2.337, 95% CI=1.336-4.087, P=0.003). In conclusion, the rs1761667 polymorphism may be closely associated with developing CHD in the Chongqing Han population of China, and an AG genotype may be a genetic susceptibility factor for CHD.

Convergencia de vías de señalización en un modelo de apoptosis

La sepsis es un evento inflamatorio generalizado del organismo inducido por un daño causado generalmente por un agente infeccioso. El patógeno más frecuentemente asociado con esta entidad es el Staphylococcus aureus, responsable de la inducción de apoptosis en células endoteliales debida a la producción de ceramida. Se ha descrito el efecto protector de la proteína C activada (PCA) en sepsis y su relación con la disminución de la apoptosis de las células endoteliales. En este trabajo se analizó la activación de las quinasas AKT, ASK1, SAPK/JNK y p38 en un modelo de apoptosis endotelial usando las técnicas de Western Blotting y ELISA. Las células endoteliales (EA.hy926), se trataron con C2-ceramida (130μM) en presencia de inhibidores químicos de cada una de estas quinasas y PCA. La supervivencia de las células en presencia de inhibidores químicos y PCA fue evaluada por medio de ensayos de activación de las caspasas 3, 7 y 9, que verificaban la muerte celular por apoptosis. Los resultados evidencian que la ceramida reduce la activación de AKT y aumenta la activación de las quinasas ASK, SAPK/JNK y p38, en tanto que PCA ejerce el efecto contrario. Adicionalmente se encontró que la tiorredoxina incrementa la activación/fosforilación de AKT, mientras que la quinasa p38 induce la defosforilación de AKT.

Effect of oxidized low-density lipoprotein on differential gene expression in primary human endothelial cells

Oxidative modification of low-density lipoprotein (LDL) plays an important role in the initiation and progression of atherosclerosis. It has been proposed that the biological action of oxidized LDL (ox-LDL) may be partially attributed to its effect on a shift of the pattern of gene expression in endothelial cells. To examine the transcriptional response to ox-LDL, we applied cDNA array technology to cultured primary human endothelial cells challenged with oxidized human LDL. A twofold or greater difference in the expression of a particular gene was considered a significant difference in transcript abundance. Seventy-eight of the 588 genes analyzed were differentially expressed in response to the treatment. Ox-LDL significantly affected the expression of genes encoding for transcription factors, cell receptors, growth factors, adhesion molecules, extracellular matrix proteins, and enzymes involved in cholesterol metabolism. The alteration of the expression pattern of several genes was substantiated post hoc using RT-PCR. The experimental strategy identified several novel ox-LDL-sensitive genes associated with a "response to injury" providing a conceptual background to be utilized for future studies addressing the molecular basis of the early stages of atherogenesis.

Influence of a probiotic soy product on fecal microbiota and its association with cardiovascular risk factors in an animal model

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Beneficial effects of diclofenac therapy on serum lipids, oxidized low-density lipoprotein and antioxidant defenses in rats

To study the effects of diclofenac, a nonselective nonsteroidal anti-inflammatory drug (NSAID), on lipid profile, oxidized low-density-lipoprotein (Ox-LDL), serum antioxidant defenses and markers of oxidative stress, male Wistar rats were divided into two groups (n = 10): (C) receiving intramuscularly a single daily dose of saline (NaCl 0.9%), and (AI) receiving intramuscularly a single daily dose of 10 mg/kg diclofenac sodium (C14H10C12NNaO2). After 28 days diclofenac-treated rats had lower Ox-LDL, apoprotein B (apo-B), apo-B/LDL-cholesterol and lipid hydroperoxide than C. Total antioxidant substances and superoxide dismutase were increased in diclofenac-treated rats, while no significant changes were observed in catalase, glutathione peroxidase and nitric oxide. A perincubation test done to examine the possibility of mechanism-based activation showed that diclofenac had no effect on maximal superoxide dismutase velocity, but significantly reduced the Michaelis-Menten (K-M) constant, indicating that diclofenac induced SOD activation increasing substrate linkage affinity to the enzyme-catalytic site. In conclusion, diclofenac had beneficial effects decreasing Ox-LDL and improving antioxidant defense. It appears that the application of this agent may be feasible and beneficial for serum antioxidant protection, which certainly would bring new insights on dyslipidemia control. (C) 2008 Elsevier B.V. All rights reserved.

Effects of N-acetylcysteine on alcohol abstinence and alcohol-induced adverse effects in rats

Alcoholism is rampant in modern society and some antioxidant compound could perhaps be useful to reduce the damage done by alcohol consumption and abstinence. The present study was undertaken to investigate the association of N-acetylcysteine (NAC) intake, alcoholism, and alcohol abstinence on lipid profile, in vivo low-density lipoprotein (LDL) oxidation, oxidative stress, and antioxidant status in serum and liver of rats. Initially, male Wistar 30 rats were divided into two groups: (C, N = 6) given standard chow and water; (E, N = 24) receiving standard chow and aqueous ethanol solution in semi-voluntary research. After 30 days of ethanol exposure, (E) group was divided into four subgroups (N = 6/group): (E-E) continued drinking 30% ethanol solution; (E-NAC) drinking ethanol solution containing 2 g/L NAC (AB) changed ethanol solution to water; (AB-NAC) changed ethanol to aqueous solution 2 g/L NAC. After 15 days of the E-group division, E-E rats had higher serum alanine transaminase, lower body weight, and surface area, despite higher energy intake than C. E-E rats had also lower feed efficiency, dyslipidemia with enhanced triacyl glycerol, very low-density lipoprotein (VLDL), lipid hydroperoxide (LH) and in vivo oxidized-LDL (ox-LDL). AB, E-NAC, and AB-NAC rats ameliorated serum oxidative stress markers and normalized serum lipids. E-E rats had higher hepatic LH and lower reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio than C, indicating hepatic oxidative stress. AB and E-NAC rats normalized hepatic LH, GSSG, and the GSH/GSSG ratio, compared to E-E. AB-NAC rats had the lowest serum ox-LDL, hepatic LH levels, and the highest GSH reductase activity in hepatic tissue. In conclusion, the present study brought new insights into alcohol consumption, because ethanol exposure enhanced serum in vivo ox-LDL, as well as serum and hepatic oxidative stress. N-acetylcysteine offers promising therapeutic value to inhibit ethanol-induced adverse effects. Ethanol withdrawal had beneficial effects on serum lipids, but was more effective when coupled with NAC supplementation. Ethanol abstinence and NAC intake interact synergistically, improving serum lipids and hepatic antioxidant defenses. (c) 2009 Elsevier B.V. All rights reserved.