oxLDL Concentrations Research Articles

Urolithin A attenuates ox-LDL-induced endothelial dysfunction partly by modulating microRNA-27 and ERK/PPAR-γ pathway.

Endothelial dysfunction and inflammation are both common events occurring during the development of atherosclerosis. Previous studies have shown that urolithins, the intestinal microflora metabolites of ellagitannin, exhibit anti-inflammation and antioxidative properties. This study aims to investigate the protective effect of urolithin A (UA) on ox-LDL-induced (where ox-LDL is oxidized low-density lipoprotein) endothelial dysfunction and possible modes of action. Human artery endothelial cells were incubated with 50 μg/mL ox-LDL and various concentrations of UA for 24 h. UA improved the productions of nitric oxide and endothelial nitric oxide synthase in a dose-dependent manner. UA markedly reduced the expressions of ICAM-1 (intercellular adhesion molecule 1) and MCP-1 (monocyte chemotactic protein 1) and further attenuated THP-1 (human acute monocytic leukemia cell line) cell adhesion. In addition, UA suppressed expressions of tumor necrosis factor α, interleukin 6, and endothelin 1, and increased PPAR-γ (peroxisome proliferators activated receptor gamma) mRNA expression. Moreover, UA decreased miR-27 expression, and overexpression of miR-27 by adding pre-miR-27 abolished the ability of UA to improve ox-LDL-induced PPAR-γ decrease. Furthermore, UA significantly downregulated phosphorylated ERK1/2 (where ERK is extracellular signal-regulated kinase) while decreasing interleukin 6 level and elevating PPAR-γ. UA could alleviate endothelial dysfunction induced by ox-LDL partially through modulating miR-27 expression and ERK/PPAR-γ pathway.

Inflammatory and Lipid-Associated Markers of Cardiovascular Diseases in Children with First Exacerbation of Inflammatory Bowel Disease.

BackgroundAdult patients with inflammatory bowel disease (IBD) are at increased risk of early atherosclerosis and atherosclerosis-driven cardiovascular diseases. However, data on the development of early, subclinical atherosclerosis in children with IBD are scarce. The aim of this study was to assess selected biomarkers of atherosclerosis in children with IBD.Material/MethodsThe study group comprised 30 children with first exacerbation of IBD. Twenty healthy children were enrolled into the control group. Total cholesterol, triglycerides, low-density lipoproteins (LDL), high-density lipoproteins (HDL), lipoprotein (a) (Lp(a)), interleukin 6 (Il-6), high sensitivity C-reactive protein (hs-CRP), and oxidized LDL (ox LDL) were determined.ResultsThere were no significant differences in lipids profiles in IBD children and controls. Mean IL-6 level (8.996 pg/ml) was significantly higher in the IBD group compared to controls (3.502 pg/ml). Mean hs-CRP concentration was significantly higher in IBD children than in controls (7.648 and 1.290 μg/ml, respectively). In the IBD group, mean ox-LDL concentration (144.837 ng/ml) was lower than in controls (162.352 ng/ml), but the difference was non-significant (P=0.4). Mean Lp(a) serum level was higher in patients with IBD (19.418 mg/dl) than in controls (10.970 mg/dl), but it was also non-significant.ConclusionsNo significant differences were found in biomarkers of atherosclerosis in children with IBD compared to controls. Elevated IL-6 and hs-CRP level are well-established inflammatory markers. Further studies are needed to fully determine cardiovascular risk factors in IBD children.

Bacillus Subtilis-Fermented Red Bean (Red Bean Natto) Reduces Hyperlipidemia Levels in Hamsters Fed an Atherogenic Diet

The purpose of this study was to investigate the effects of 50% red beans ethanol natto extracts fermented by Bacillus subtilis on hyperlipidemia in hamsters. Forty male hamsters were randomly assigned to normal control, high-fat control, and high-fat with RBNE (red beans natto extracts) (200 and 1000 mg/kg BW) groups. RBNE treatment for 8 weeks significantly decreased the body and visceral fat weight in hamsters. Compared to the high-fat diet group, the plasma total cholesterol (TC), total triacylglycerol (TAG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and oxidized LDL (oxLDL) concentrations as well as atherogenic index of plasma AIP [Log(TAG/HDL-C)] were significantly lower in the RBNE-fed group. The consumption of RBNE significantly reduced the liver contents of TC, esterified cholesterol (CE) and alanine aminotransferase (ALT), and increased a regulator of lipid metabolism: peroxisome proliferator-activated receptor alpha (PPAR-alpha). Furthermore, the circulating level of irisin was decreased. Practical Application The oral administration of Bacillus subtilis-fermented red bean natto extract (RBNE), a popular Asian dessert, effectively moderated the metabolism of lipids and positively affected the blood and liver profiles of hamsters fed with a high-cholesterol and high-fat diet. Importantly, RBNE significantly suppressed the increase in visceral fat mass. Additionally, RBNE appears to protect the liver and alleviate the magnitude of fatty liver development. RBNE also upregulated PPAR-alpha protein expression in the liver, suggesting that supplementation may improve lipid metabolism in part through the beta-oxidation of lipids and promotion of lipid secretion. Furthermore, RBNE may ameliorate hyperlipidemia involving irisin, a newly identified hormone, in hamsters fed a cholesterol- and lipid-enriched diet. In conclusion, RBNE with high hypocholesterolemic activities may be helpful for patients with atherosclerosis.

Overexpression of LOXIN Protects Endothelial Progenitor Cells From Apoptosis Induced by Oxidized Low Density Lipoprotein.

Human endothelial progenitor cells (hEPC) are adult stem cells located in the bone marrow and peripheral blood. Studies have indicated that hEPC play an important role in the recovery and repair of injured endothelium, however, their quantity and functional capacity is reduced in several diseases including hypercholesterolemia. Recently, it has been demonstrated that hEPC express lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and its activation by oxidized low-density lipoprotein (ox-LDL) induces cellular dysfunction and apoptosis. This study aimed to investigate whether overexpression of LOXIN, a truncated isoform of LOX-1 that acts as a dominant negative, plays a protective role against ox-LDL-induced apoptosis in hEPC. Human endothelial progenitor cells exposed to ox-LDL showed a significant increase in LOX-1 expression, and apoptosis began at ox-LDL concentrations above 50 μg/mL. All hEPC apoptosed at 200 μg/mL ox-LDL. High LOXIN expression was generated using adenoviral systems in hEPC and SiHa cells transduced with 100 colony-forming units per cell. Transduced LOXIN localized to the plasma membrane and blocked ox-LDL uptake mediated by LOX-1. Overexpression of LOXIN protected hEPC from ox-LDL-induced apoptosis, and therefore maybe a novel way of improving hEPC function and quantity. These results suggest that adenoviral vectors of LOXIN may provide a possible treatment for diseases related to ox-LDL and vascular endothelium dysfunction, including atherosclerosis.

The neutral sphingomyelinase-2 is involved in angiogenic signaling triggered by oxidized LDL

Capillaries of the external part of the normal arterial wall constitute the vasa vasorum network. In atherosclerotic lesions, neovascularization occurs in areas of intimal hyperplasia where it may promote plaque expansion, and intraplaque hemorrhage. Oxidized LDL that are present in atherosclerotic areas activate various angiogenic signaling pathways, including reactive oxygen species and the sphingosine kinase/sphingosine-1-phosphate pathway. We aimed to investigate whether oxidized LDL-induced angiogenesis requires neutral sphingomyelinase-2 activation and the neutral sphingomyelinase-2/sphingosine kinase-1 pathway.The role of neutral sphingomyelinase-2 in angiogenic signaling was investigated in Human Microvascular Endothelial Cells (HMEC-1) forming capillary tube on Matrigel and in vivo in the Matrigel plug assay in C57BL/6 mice and in the chicken chorioallantoic membrane model.Low concentration of human oxidized LDL elicits HMEC-1 capillary tube formation and neutral sphingomyelinase-2 activation, which were blocked by neutral sphingomyelinase-2 inhibitors, GW4869 and specific siRNA. This angiogenic effect was mimicked by low concentration of C6-Ceramide and was inhibited by sphingosine kinase-1 inhibitors. Upstream of neutral sphingomyelinase-2, oxidized LDL-induced activation required LOX-1, reactive oxygen species generation by NADPH oxidase and p38-MAPK activation. Inhibition of sphingosine kinase-1 blocked the angiogenic response and triggered HMEC-1 apoptosis.Low concentration of oxidized LDL was angiogenic in vivo, both in the Matrigel plug assay in mice and in the chorioallantoic membrane model, and was blocked by GW4869.In conclusion, low oxLDL concentration triggers sprouting angiogenesis that involves ROS-induced activation of the neutral sphingomyelinase-2/sphingosine kinase-1 pathway, and is effectively inhibited by GW4869.

Reduction of Oxidative Stress in Chronic Kidney Disease Does Not Increase Circulating α-Klotho Concentrations.

The CKD-associated decline in soluble α-Klotho levels is considered detrimental. Some in vitro and in vivo animal studies have shown that anti-oxidant therapy can upregulate the expression of α-Klotho in the kidney. We examined the effect of anti-oxidant therapy on α-Klotho concentrations in a clinical cohort with mild tot moderate chronic kidney disease (CKD). We performed a post-hoc analysis of a prospective randomized trial involving 62 patients with mild to moderate CKD (the ATIC study), all using an angiotensin-converting enzyme inhibitor (ACEi) or angiotensin receptor blocker (ARB) for 12 months. On top of that, the intervention group received anti-oxidative therapy consisting of the combination of pravastatin (40 mg/d) and vitamin E (α-tocopherol acetate, 300 mg/d) while the placebo was not treated with anti-oxidants. α-Klotho concentrations were measured at baseline and after 12 months of anti-oxidant therapy. Data were analysed using T-tests and Generalized Estimating Equations, adjusting for potential confounders such as vitamin D, parathyroid hormone, fibroblast-growth-factor 23 (FGF23) and eGFR. The cohort existed of 62 patients with an eGFR (MDRD) of 35 ± 14 ml/min/1.72m2, 34 were male and mean age was 53.0 ± 12.5 years old. Anti-oxidative therapy did successfully reduce oxLDL and LDL concentrations (P <0.001). α-Klotho concentrations did not change in patients receiving either anti-oxidative therapy (476.9 ± 124.3 to 492.7 ± 126.3 pg/mL, P = 0.23) nor in those receiving placebo 483.2 ± 142.5 to 489.6 ± 120.3 pg/mL, P = 0.62). Changes in α-Klotho concentrations were not different between both groups (p = 0.62). No evidence was found that anti-oxidative therapy affected α-Klotho concentrations in patients with mild-moderate CKD.

The relationship between concentrations of magnesium and oxidized low-density lipoprotein and Beta2-microglobulin in the serum of patients on the end-stage of renal disease.

The end-stage of renal disease is associated with increased oxidative stress and oxidative modification of low-density lipoproteins (LDLs). Beta2 microglobulin (beta2M) is accumulated in the serum of dialysis patients. Magnesium (Mg) plays a protective role in the development of oxidative stress in healthy subjects. We studied the relationship between concentrations of magnesium and oxidized LDL (ox-LDL) and beta2M in the serum of patients on the end stage of renal disease. In 96 patients on on-line- predilution hemodiafiltration, beta2M and intact parathormone were measured by radioimmunoassays. High-sensitivity C-reactive protein (hsCRP) and ox-LDL were measured using ΕLISA. Serum bicarbonate levels were measured in the blood gas analyser gas machine. We performed logistic regression analysis models to investigate Mg as an important independent predictor of elevated ox-LDL and high beta2M serum concentrations, after adjustment to traditional and specific for dialysis patients' factors. We observed a positive correlation of Mg with ox-LDL (r = 0.383, P = 0.001), but the association of Mg with beta2M, hsCRP, and serum bicarbonate levels was significantly inverse (r = -0.252, P = 0.01, r = -0.292, P = 0.004, and r = -0.282, P = 0.04 respectively). The built logistic-regression analysis showed that Mg act as a significant independent factor for the elevated ox-LDL and beta2M serum concentrations adjusting to traditional and specific factors for these patients. We observed a positive relationship between magnesium and acidosis status- related ox-LDL concentrations, but the inverse association between magnesium and beta2M serum concentrations in hemodialysis patients.

Oxidized LDL: As a risk factor for cardiovascular disease in renal transplantation.

The mortality rate of chronic kidney disease (CKD) patients that have undergone renal replacement therapy is very high due to cardiovascular diseases (CVD). Some studies have indicated that cyclosporine A, a drug used to prevent transplant rejection, is associated with bone loss following transplantation. Furthermore, it has an oxidative effect on circulating lipids. Its prooxidant effect on cell membranes causes calcium release. This study aimed to examine whether or not renal transplantation result in improvement in oxidative stress and to assess the association between oxidized LDL (ox-LDL) and some variables in the prediction of CVD risk in Renal Transplantation (RT) patients that were compared with the control group. A total number of 30 CKD patients were recruited to evaluate time dependent changes in biomarker of OS before and after RT. The ox-LDL, lipid metabolism parameters, CsA, creatinine, calcium and phosphate were assessed both before RT, 10 days and 6 months after RT in comparison with the control group (n = 30). Over 6 months, ox-LDL concentration changed from 79.7 ± 9.7 to 72 ± 7 mU/mL (p < 0.009). calcium phosphate level was positively correlated with the concentration of ox-LDL (R = 0.467, p = 0.011) and cyclosporine (R = 0.419, p = 0.024) 6 months after transplantation. The findings indicated that restoring renal function by transplantation, improves uremia induced oxidative stress. calcium phosphate product, as an independent risk factor for CVD, correlates with ox-LDL before RT and 6 months after RT. Calcium phosphate product correlates with cyclosporine in the RT group, too.

Th17/Treg cell expression in children with primary nephritic syndrome and the effects of ox-LDL on Th17/Treg cells.

To investigate the role of T-helper cells/Treg (Th17/Treg) and morbidity factors related to primary nephritic syndrome (PNS) in children, as well as the influence of ox-low density lipoprotein (ox-LDL) on Th17/Treg expression in children with PNS. To clarify the pathogenesis of PNS in children, 50 children with PNS treated in our hospital were enrolled in the study group. Additionally, 20 healthy children who came to our hospital for physical examination during the same period were enrolled in the control group. Th17 and Treg cells in children belonging to the two groups were detected by flow cytometry; the numbers of Th17/Treg cells in peripheral blood mononuclear cells at different concentrations of ox-LDL were detected simultaneously. Ox-LDL can affect the number of Th17/Treg cells in peripheral blood mononuclear cells, and both cell types decreased with increasing concentration of ox-LDL, with the numbers being significantly lower in the control group. However, the decrease in the number of Th17 cells was statistically insignificant (P > 0.05), whereas the decrease in Treg cells was more obvious and statistically significant (P < 0.05). The effect of ox-LDL the number of Treg cells was stronger than that on Th17 cells. We concluded that the imbalance of Th17/Treg cells influenced by high and low ox-LDL concentrations in children with PNS might be the immunological basis of the disease.

Oxidized Low-Density Lipoprotein (ox-LDL) to LDL Ratio (ox-LDL/LDL) and ox-LDL to High-Density Lipoprotein Ratio (ox-LDL/HDL).

Oxidized low-density lipoprotein (ox-LDL) plays a principal role in diabetes complications, though ox-LDL concentrations and its functions are dependents of other oxidative and anti-oxidative particles. The oxLDL to low-density lipoprotein ratio (ox-LDL/LDL) and ox-LDL to high-density lipoprotein ratio (ox-LDL/HDL) as new lipid biomarkers may serve as a good estimation of oxidation and anti-oxidation in type 2 diabetes mellitus. The aim of this study was first to examine ox-LDL/LDL and ox-LDL/HDL levels in patients with type 2 diabetes mellitus compared to a control group and evaluate their diagnostic accuracies, and, second, to investigate the correlation of these two ratios with triglyceride and HDL levels. This study was included 144 patients admitted to the diabetes clinic of a University general hospital and 41 healthy individuals as controls. Levels of LDL, HDL, triglyceride (TG) ox-LDL, and malondialdehyde (MDA) were measured for all patients. Levels of ox-LDL/LDL and ox-LDL/HDL were calculated. Diagnostic accuracies were determined by receiver-operating characteristic curve analysis by measuring the area under the curve. Ox-LDL, ox-LDL/LDL, and ox-LDL/HDL were significantly higher in patients compared to the control group after adjustment for age, gender, and BMI (p = 0.000). The area under the curve for diagnosing diabetes was 0.946 for ox-LDL/HDL, 0.918 for ox-LDL/LDL, and 0.832 for ox-LDL. Ox-LDL/HDL was positively correlated with MDA (r = 0.210, p = 0.011). Ox-LDL/LDL was negatively correlated with HDL (r = -0.2 p = 0.016) and positively correlated with TG (r = 0.45 p = 0.000). Ox-LDL/HDL and TG had significant positive correlation (r = 0.173 p = 0.037). The ox-LDL/LDL level was significantly higher in patients with coronary heart disease (CHD) or hypertension (HTN) compared to those without (p = 0.042). Our findings revealed that ox-LDL/LDL and ox-LDL/HDL are potent biomarkers of type 2 diabetes, which apparently reflect the association between lipids in the state of oxidative stress.

Circulating Lipoprotein Concentrations Correlate with Total but Not Free Heme in Different Sickle Cell Disease Genotypes

In chronic hemolytic states, hemoglobin is released in the bloodstream and free heme is formed upon hemoglobin oxidation. Free heme has been shown to trigger vaso-occlusion in animal models of sickle cell disease (SCD) by activating endothelial cells through toll-like receptor 4 (TLR4). Serum levels of glycoproteins specialized in clearing hemoglobin and heme, such as haptoglobin and hemopexin, are typically depleted in SCD plasma. High-density (HDL) and low-density lipoproteins (LDL) can also bind heme and work as the first line of defense against heme toxicity. Heme can react with LDL and generate oxidized LDL (ox-LDL), another TLR4 ligand.We aimed to investigate the importance of lipoproteins as heme scavengers by comparing SCD patients with homozygous sickle cell anemia (SS) patients and double heterozygous hemoglobin SC disease (SC) patients, who present with higher and lower hemolytic rates, respectively.Eighty-three patients (aged 18-68 years old, 44 female) participated in this study: 43 SS (12 receiving hydroxyurea - HU) and 40 SC. Exclusion criteria were pregnancy, blood transfusion, pain crisis, or use of lipid lowering drugs in the past 3 months. Peripheral venous blood samples were collected in tubes with EDTA for complete blood counts and without antiocoagulant for serum separation after centrifugation at 500g for 20 min. Serum lactate dehydrogenase, total cholesterol and HDL were measured by conventional enzymatic-colorimetric methods on a Roche Hitachi Modular automation system (Roche, Germany). LDL was calculated by Friedewald equation. Serum oxidized LDL (ox-LDL), total and free heme were determined with commercially available ELISA assays (Mercodia Oxidized LDL ELISA, Heme QuantiChrom Assay, Bioassay Systems, USA). Statistical analysis was performed with SPSS v.22.0.LDH and reticulocyte count were significantly higher in SS patients than SC, and hemoglobin was significantly lower (P =0.001). Total heme was significantly higher in SS patients taking HU (SSHU) (90 ± 59 mM) compared with SC patients (66 ± 18 mM), P =0.01. Total cholesterol was higher in SC than in SS patients, 141 ± 28 vs. 107 ± 20 mg/dL, respectively, P =0.01). We found no significant differences between groups regarding LDL (P =0.08), HDL (P =0.10), ox-LDL (P =0.52). Total heme correlated negatively with total cholesterol (r=-0.33, P =0.037) and HDL (r=-0.49, P =0.001) in the whole population, regardless of genotype or treatment with HU. Surprisingly, free heme did not differ between SS, SSHU, or SC groups of patients (P=0.19), and did not correlate with cholesterol levels.The negative correlation between total heme and total cholesterol supports that lipoproteins contribute to defend the body against heme toxicity. Despite HDL, LDL, and ox-LDL not being significantly different among the groups, the stronger negative correlation between total heme and HDL suggests that this cholesterol fraction may be more important in heme scavenging. We hypothesize that more intense hemolysis may lead to the production of heme capable of oxidizing lipoproteins, which can then be rapidly cleared by macrophages, explaining hypocholesterolemia in SS patients. Similar concentrations of free heme and ox-LDL in all groups suggests that, in steady state, their concentrations are kept relatively constant regardless of hemolytic rate. Since circulating concentrations of these TLR4 ligands did not differ between SC and SS patients in our study, this might indicate that the contribution of hemolysis to endothelial dysfunction and to differences in severity and in the variety of complications between these genotypes may be better explained by TLR4 activation by bound heme rather than by activation by free heme or ox-LDL.Financial support: FAPESP/CAPES/CNPq DisclosuresNo relevant conflicts of interest to declare.

Asymmetric dimethylarginine and homocysteine in exudative age-related macular degeneration

Introduction: The oxidative stress has been proposed as an important case of exudative age-related macular degeneration (E-ARMD). The aim of the present study was to investigate homocysteine (Hcy), asymmetric dimethylarginine (ADMA) and oxidized low-density lipoprotein cholesterol (Ox-LDL-C) levels, the factors involved in oxidative stress, in the patients with E-ARMD. Methods: In a cross-sectional study, 45 patients with E-ARMD were compared with 45 sex- and age-matched healthy controls. The levels of biochemical factors, Hcy, ADMA, and of Ox-LDL were estimated by standard methods in both study groups. Results: The levels of Hcy (15.4 ± 7.2 vs. 10.7 ± 3.7 μM, P = 0.001), Ox-LDL (52.2 ± 13.8 vs. 37.8 ± 10.8 U/l, P = 0.001), and ADMA (0.84 ± 0.23 vs. 0.71 ± 0.26 μM, P = 0.012) were significantly higher in the patients with E-ARMD than those in the controls. In the patient group, there was a positive and significant correlation between serum Ox-LDL and Hcy concentrations (r = 0.719, P = 0.001), but no correlation was found between serum ADMA and Ox-LDL (r = 0.010, P = 0.900) and also between serum Hcy and ADMA levels (r = -0.070, P = 0.600). Conclusion: The high levels of Hcy as an oxidant agent and ADMA as an endogenous nitric oxide synthase inhibitor can lead to increase Ox-LDL levels, and they may have important roles in oxidative stress, which can be a trigger in E-ARMD.

Effect of virgin olive oil and thyme phenolic compounds on blood lipid profile: implications of human gut microbiota.

To investigate the effect of virgin olive oil phenolic compounds (PC) alone or in combination with thyme PC on blood lipid profile from hypercholesterolemic humans, and whether the changes generated are related with changes in gut microbiota populations and activities. A randomized, controlled, double-blind, crossover human trial (n=12) was carried out. Participants ingested 25mL/day for 3weeks, preceded by 2-week washout periods, three raw virgin olive oils differing in the concentration and origin of PC: (1) a virgin olive oil(OO) naturally containing 80mgPC/kg, (VOO), (2) a PC-enriched virgin olive oil containing 500mgPC/kg, from OO (FVOO), and (3) a PC-enriched virgin olive oil containing a mixture of 500mgPC/kg from OO and thyme, 1:1 (FVOOT). Blood lipid values and faecal quantitative changes in microbial populations, short chain fatty acids, cholesterol microbial metabolites, bile acids, and phenolic metabolites were analysed. FVOOT decreased seric ox-LDL concentrations compared with pre-FVOOT, and increased numbers of bifidobacteria and the levels of the phenolic metabolite protocatechuic acid compared to VOO (P<0.05). FVOO did not lead to changes in blood lipid profile nor quantitative changes in the microbial populations analysed, but increased the coprostanone compared to FVOOT (P<0.05), and the levels of the faecal hydroxytyrosol and dihydroxyphenylacetic acids, compared with pre-intervention values and to VOO, respectively (P<0.05). The ingestion of a PC-enriched virgin olive oil, containing a mixture of olive oil and thyme PC for 3weeks, decreases blood ox-LDL in hypercholesterolemic humans. This cardio-protective effect could be mediated by the increases in populations of bifidobacteria together with increases in PC microbial metabolites with antioxidant activities.

LOX-1 in macrophage migration in response to ox-LDL and the involvement of calpains

Previous studies have shown that oxidized low-density lipoprotein (ox-LDL) inhibits macrophage migration, but the precise mechanisms remain unclear. Lectin-like ox-LDL receptor-1 (LOX-1) is a scavenger receptor that is expressed in macrophages and binds ox-LDL. Calpains, a family of calcium-dependent proteases, influence several aspects of cell migration. In this study, we investigated the role of LOX-1 in macrophage migration in response to ox-LDL and the involvement of calpains in this process. Peritoneal macrophages from wild type C57BL/6 mice were exposed to different concentrations of ox-LDL (1–20 μg/mL), and expression of LOX-1 and calpain-1 and -2, cell migration and intracellular calcium (Ca2+in) were measured. Our results showed that ox-LDL stimulated LOX-1 and calpain-2 expression, and inhibited calpain-1 expression in a dose- and time-dependent manner. Further, ox-LDL inhibited macrophage migration and increased Ca2+in concentration in macrophages. To further elucidate the role of LOX-1 in ox-LDL-impaired macrophage migration, we isolated peritoneal macrophages from LOX-1 knockout mice, and treated them with ox-LDL. Interestingly, calpain-1 expression was much higher, and calpain-2 expression was lower in LOX-1 knockout macrophages than in wild-type macrophages following exposure to ox-LDL. LOX-1 deletion significantly improved macrophage migration and decreased Ca2+in concentration. These data indicate that LOX-1 is, at least in part, responsible for the inhibitory effect of ox-LDL on macrophage migration and this process involves calpain-1 and -2.

Galectin-3-induced oxidized low-density lipoprotein promotes the phenotypic transformation of vascular smooth muscle cells.

Oxidized low-density lipoprotein (oxLDL) is involved in the pathological phenotypic transformation of vascular smooth muscle cells in atherosclerosis. Galectin-3 also has an important role in atherosclerosis. However, little is currently known regarding the effects of galectin-3 on the oxLDL-induced phenotypic transformation of vascular smooth muscle cells. In the present study, primary culture human umbilical vascular smooth muscle cells were treated with various oxLDL concentrations (0-50 μg/ml) for 72 h, and phenotypic changes were subsequently recorded. The results of the present study suggested that oxLDL increases the expression levels of galectin-3, and induces the phenotypic transformation of vascular smooth muscle cells. The oxLDL-induced cells exhibited increased expression levels of osteopontin, a smooth muscle synthetic protein, and calponin and α-actin, smooth muscle contractile proteins. The oxLDL-induced changes in cellular phenotype were associated with increased migration, proliferation, and phagocytosis. Concordant with these results, oxLDL-treated smooth muscle cells exhibited activation of canonical Wnt signaling, as determined by an increase in the protein expression levels of β-catenin. Silencing of galectin-3 by small interfering RNA reversed the phenotypic transformation and functional changes observed in the oxLDL-treated cells, suggesting these changes were dependent on the activation of galectin-3. In addition, galectin-3 knockdown decreased the protein expression levels of β-catenin in both the cytoplasm and nucleus; however, the mRNA expression levels of β-catenin remained unchanged. These results suggest that galectin-3 is responsible for the phenotypic transformation of human umbilical vascular smooth muscle cells, and the canonical Wnt/β-catenin signaling pathway may be involved in this process.

Carotenoid consumption is related to lower lipid oxidation and DNA damage in middle-aged men.

The present cross-sectional study assessed the potential relationships of carotenoid intake with lipid and oxidative stress markers in middle-aged men. A total of 296 apparently healthy middle-aged men (mean age 50.5 (SD 5.0) years, BMI 25.8 (SD 3.5) kg/m(2)) were recruited to participate in the study. Dietary intake, anthropometry, blood pressure, lifestyle features, blood and urine biomarkers were assessed using validated procedures. The lipid markers included NEFA, Castelli index, and TAG:HDL ratio; oxidative stress markers included urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), 8-iso-PGF2α and plasma oxidised-LDL (ox-LDL). We observed a significant inverse association (P < 0.05) between NEFA concentrations and consumption of lutein plus zeaxanthin, β-carotene, α-carotene and total carotenoid, while Castelli index was negatively associated with daily intake of lycopene, β-carotene and total carotenoids. Regarding oxidative stress biomarkers, urinary 8-OHdG and ox-LDL concentrations were also inversely associated (P < 0.05) with consumption of lycopene, lutein plus zeaxanthin, β-carotene, α-carotene and total carotenoids, regardless of confounding variables. Moreover, there was a negative association of urinary 8-iso-PGF2α concentration with dietary lutein plus zeaxanthin (β - 0.135, 95% CI - 0.268, - 0.001), β-carotene (β - 0.156, 95% CI - 0.277, - 0.034) and with the sum of all carotenoids (β - 0.189, 95% CI - 0.333, - 0.046). In conclusion, total daily carotenoid intake based on five investigated carotenoid types (β-cryptoxanthin, lycopene, lutein plus zeaxanthin, β-carotene and α-carotene) was inversely associated with relevant lipid and oxidative stress markers in middle-aged men, with emphasis on β-carotene that was negatively associated with five of the six lipid and oxidative stress markers evaluated in the present study.

The biphasic effects of oxidized-low density lipoprotein on the vasculogenic function of endothelial progenitor cells.

Late-outgrowth endothelial progenitor cells (EPCs) are stress-resistant and responsible for reparative functions in the cardiovascular system. Oxidized-LDL (oxLDL) plays a critical role in cardiovascular disease pathogenesis. However, it is largely unknown what the impacts of oxLDL are on late-outgrowth EPCs. This study aimed to investigate the concentration-related effects of oxLDL on EPC functions and related angiogenesis, in vitro and in vivo. In this study, early and late-outgrowth EPCs were generated from circulating human mononuclear cells. oxLDL may regulate EPC vasculogenic function via the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). Lower concentrations (5 μg/mL) of oxLDL can potentiate EPC tube formation in vitro and in vivo by activating eNOS mechanisms, which are mediated by p38 MAPK- and SAPK/JNK-related pathways. Higher concentrations of oxLDL (10-50 μg/mL) impaired EPC function via the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathways and consequent inhibition of eNOS activity, which could be reversed by anti-oxidants (diphenylene iodonium and apocynin) and gp91phox siRNA. In conclusion, oxLDL has concentration-dependent biphasic effects on human late-outgrowth EPC tube formation in vitro and in vivo.

Sex Differences in the Impact of the Mediterranean Diet on LDL Particle Size Distribution and Oxidation

Sex differences have been previously highlighted in the cardioprotective effects of the Mediterranean diet (MedDiet). The objective of this study was to investigate whether sex differences also exist with regard to LDL particle size distribution and oxidation. Participants were 37 men and 32 premenopausal women (24–53 years) with slightly elevated LDL-C concentrations (3.4–4.9 mmol/L) or total cholesterol/HDL-C ≥5.0. Variables were measured before and after a four-week isoenergetic MedDiet. Sex differences were found in response to the MedDiet for the proportion of medium LDL (255–260 Å) (p for sex-by-time interaction = 0.01) and small, dense LDL (sdLDL; <255 Å) (trend; p for sex-by-time interaction = 0.06), men experiencing an increase in the proportion of medium LDL with a concomitant reduction in the proportion of sdLDL, while an opposite trend was observed in women. A sex difference was also noted for estimated cholesterol concentrations among sdLDL (p for sex-by-time interaction = 0.03), with only men experiencing a reduction in response to the MedDiet. The MedDiet marginally reduced oxidized LDL (oxLDL) concentrations (p = 0.07), with no sex difference. Results suggest that short-termconsumption of the MedDiet leads to a favorable redistribution of LDL subclasses from smaller to larger LDL only in men. These results highlight the importance of considering sex issues in cardiovascular benefits of the MedDiet.

Using image-based flow cytometry to measure monocyte oxidized LDL phagocytosis: A potential risk factor for CVD?

Obesity and cardiovascular disease is a worldwide health concern that has been a major focus in research for several decades. Among these diseases, atherosclerosis is one of the leading causes of death and disability nationwide. Circulating monocytes are believed to be primary cells responsible for foam cell formation. The present report describes a novel method for measuring monocyte oxLDL phagocytosis capacity using image-based flow cytometry. Human venous blood monocytes were incubated with different concentrations of oxLDL for different lengths of time to optimize the assay. High (post-meal) and low (pre-meal) responder samples were generated by feeding human subjects a high-fat (~85% of daily fat allowance), high-calorie (~65% of daily calorie needs) meal. This is a relevant model with respect to obesity and risk of developing atherogenesis. After the functional assay, classic (CD14+/CD16−) and pro-inflammatory (CD14+/CD16+) monocytes were assessed for oxLDL uptake, adhesion molecule expression (CD11b and CD18), and scavenger receptor expression (CD36) using an image-based flow cytometer (FlowSight). The present method represents a novel advance in methods available to detect the propensity of circulating monocytes to become intima foam cells. We found the assay to be most effective at separating high from low responder samples when using a fixed oxLDL concentration (120μL/mL) and incubation length (1-h). In a clinical application, this method demonstrated that consuming a single high-fat meal causes an increase in the proportion of monocyte oxLDL phagocytosis and their adhesion capacity, suggesting a higher propensity to become foam cells.

Oxidized HDL and LDL in adolescents with type 2 diabetes compared to normal weight and obese peers

ObjectiveObesity and type 2 diabetes mellitus (T2DM) are associated with oxidative stress. Oxidative damage of high-density lipoprotein (oxHDL) leads to a dysfunctional molecule, potentially a mediator and/or marker of cardiometabolic disease. We tested the hypothesis that circulating concentration of oxHDL is higher in obese (Ob) or T2DM adolescents compared to normal-weight (NW) peers. MethodsIn 37 NW, 38 Ob, and 42 T2DM adolescents, ages 11–18 y, fasting concentrations of HDL and LDL cholesterol, oxHDL, oxidized low-density lipoprotein (oxLDL), and myeloperoxidase (MPO) were measured. ResultsCompared to the NW group, oxHDL in the Ob group was not different, but was 65% higher (p<0.01) in the T2DM group. Within the T2DM group oxHDL was higher in boys than in girls, but this sex difference was not evident in NW or Ob groups. OxLDL was 23% higher in Ob (p=0.02), and 56% higher in T2DM (p<0.01) versus NW and did not differ between boys and girls. MPO was not different between NW and Ob but was 88% (p<0.02) higher in T2DM compared to NW. Contrary to our hypothesis MPO and insulin resistance (HOMA-IR) were not correlated with oxHDL. OxHDL was positively associated with oxLDL and lean body mass while oxLDL was positively associated with apolipoprotein B, triglycerides, HOMA-IR and trunk fat. ConclusionsThe higher concentrations of oxHDL and oxLDL, along with higher MPO in children with T2DM reflect higher oxidative stress compared with obesity alone and potentially increased cardiovascular disease risk in youth with T2DM.