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Purpose: The triglyceride (TG) content in the human heart is increased in insulin resistance-related diseases. Plasma lipoproteins are source of lipids for the heart. Electronegative LDL [LDL(-)] is a modified subfraction of LDL which is elevated in different cardiometabolic diseases wih a high cardiovascular component. Our purpose was to assess the effect of LDL(-) on the intracellular neutral lipid accumulation in cardiomyocytes under normoxic and hypoxic conditions.Methods: LDL(-) was isolated from total LDL by anion-exchange chromatography. HL-1 cardiomyocytes were exposed to increasing concentrations of either native LDL (nLDL) or LDL(-) under normoxia (21% O2) or hypoxia (1% O2). Intracellular cholesteryl ester (CE) and TG content was analyzed by TLC after cellular lipid extraction. The distribution of LDL-induced intracellular lipid accumulation was assessed by confocal microscopy experiments in HL-1 cells exposed to DiI-labeled nLDL and DiI-labeled LDL(-). Gene expression and protein level determination was carried out by real time PCR and western blot analysis, respectively.Results: LDL(-), differently to native LDL, induced a strong intracellular TG accumulation in HL-1 cardiomyocytes. The intracellular TG accumulation induced by the exposition to LDL(-) was strongly enhanced under hypoxic conditions. Likewise nLDL, endocytosed DiI-LDL(-) was detected into lipid droplets homogeneously distributed around the nucleus of cells under normoxic conditions. Conversely, under hypoxic conditions, the distribution of internalized lipids from DiI-LDL(-) was spread widely into the whole cytoplasm. Furthermore, this abnormal distribution of intracellular lipids was associated with a reduction in the expression of perilipin 5, a key protein involved in the intracellular lipid storage and metabolism, and an exacerbated downregulatory effect of hypoxia on the gene expression of carnitine palmitoyltransferase I, an enzyme essential for mitochondrial fatty acid β-oxidation. These array of changes were associated with increased cellular levels of the reactive oxygen production (ROS) production in HL-1 cardiomyocytes exposed to LDL(-) under hypoxia.Conclusions: LDL (-), by promoting an altered and detrimental lipid intracellular storage and metabolism, may contribute to exacerbate the effects of ischemia. Therefore, our results highlight a potential relevant and novel mechanism underlying cardiometabolic complications of diabetes. [ABSTRACT FROM PUBLISHER]

Patients with type 1 diabetes (T1D) have a greater risk of cardiovascular disease. Proconvertase subtilisin-kexin 9 (PCSK9) is involved in the atherosclerosis process. This study aimed to determine the relationship between PCSK9 levels and epicardial adipose tissue (EAT) volume and cardiometabolic variables in patients with T1D. This was an observational cross-sectional study including 73 patients with T1D. Clinical, biochemical and imaging data were collected. We divided the patients into two groups according to their glycemic control and the EAT index (iEAT) percentile. We performed a correlation analysis between the collected variables and PCSK9 levels; subsequently, we performed a multiple regression analysis with the significant parameters. The mean age was 47.6 ± 8.5 years, 58.9% were men, and the BMI was 26.9 ± 4.6 kg/m 2 . A total of 31.5%, 49.3% and 34.2% of patients had hypertension, dyslipidemia and smoking habit, respectively. The PCSK9 concentration was 0.37 ± 0.12 mg/L, which was greater in patients with worse glycemic control (HbA1c > 7.5%), dyslipidemia and high EAT volume (iEAT > 75th percentile). The PCSK9 concentration was positively correlated with age (r = 0.259; p = 0.027), HbA1c (r = 0.300; p = 0.011), insulin dose (r = 0.275; p = 0.020), VLDL-C level (r = 0.331; p = 0.004), TG level (r = 0.328; p = 0.005), and iEAT (r = 0.438; p < 0.001). Multiple regression analysis revealed that 25% of the PCSK9 variability was explained by iEAT and HbA1c (p < 0.05). The PCSK9 concentration is associated with metabolic syndrome parameters, poor glycemic control and increased EAT volume in patients with T1D., (© 2024. The Author(s).)

Background: 18 F-fluorodeoxyglucose positron emission tomography ( 18 F-FDG PET) identifies carotid plaque inflammation and predicts stroke recurrence., Aim: Our aim was to evaluate the performance of soluble low-density lipoprotein receptor-related protein 1 (sLRP1) as an indicator of carotid plaque inflammation., Methods: A prospective study was conducted among adult patients with recent (< 7 days) anterior circulation ischemic stroke and at least one atherosclerotic plaque in the ipsilateral internal carotid artery. Patients underwent an early (< 15 days from inclusion) 18 F-FDG PET, and the maximum standardized uptake value (SUVmax) within the plaque was measured. sLRP1 levels were measured in plasma samples by ELISA. The association of sLRP1 with SUVmax was assessed using bivariate and multivariable linear regression analyses. Hazard ratios (HR) were estimated with Cox regression to evaluate the association between circulating sLRP1 and stroke recurrence., Results: The study was conducted with 64 participants, of which 57.8% had ≥ 50% carotid stenosis. The multivariable linear and logistic regression analyses showed that sLRP1 was independently associated with (i) SUVmax within the plaque (β = 0.159, 95% CI 0.062-0.257, p = 0.002) and (ii) a probability of presenting SUVmax ≥ 2.85 g/mL (OR = 1.31, 95% CI 1.00-1.01, p = 0.046), respectively. Participants with stroke recurrence showed higher sLRP1 levels at baseline [6447 ng/mL (4897-11163) vs. 3713 ng/mL (2793-4730); p = 0.018]., Conclusions: sLRP1 was independently associated with carotid plaque inflammation as measured by 18 F-FDG PET in patients with recent ischemic stroke and carotid atherosclerosis., (© 2023. The Author(s).)

This chapter provides details on the methodologies currently used to monitor macrophage cholesterol efflux in vivo in mice. The general principles and techniques described herein can be applied to evaluate the effect of different experimental pathophysiological conditions or the efficacy of different therapeutic strategies on the modulation of in vivo cholesterol efflux to plasma acceptors and the rate of reverse transport of unesterified cholesterol from macrophages to feces in mice., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This chapter provides details on a simple and reproducible method used to determine the capacity of murine HDL to prevent the oxidation of LDL . The principle of the method is based on the rearrangement of double bonds of polyunsaturated fatty acids that occurs during the oxidation of human LDL , which generates a sigmoidal curve. The shape and length of the curve is modified in the presence of HDL , and such modifications are easily quantifiable by measuring the absorbance of conjugated dienes at 234 nm. The general technique described herein may be applied to evaluate the effect of HDL obtained from different experimental murine models of atherosclerosis., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

In this study, we aim to explore the effects on lipids of integrase strand transfer inhibitors (INSTIs) in naïve and switch randomised controlled trials, and compare them with protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). We reviewed phase 3/4 randomised clinical trials in the Cochrane and PubMed databases that compare an INSTI with a boosted PI, an NNRTI, or another INSTI plus one or two nucleoside/nucleotide reverse transcriptase inhibitors (NtRTIs) in naïve patients and switching strategies in HIV-infected patients. We reported the baseline plasma concentration of total cholesterol (TC), low and high-density lipoprotein cholesterol (LDL-c, HDL-c), triglycerides (TG), and the TC/HDL-c ratio, as well as the change at weeks 48 and 96, when available. In naïve HIV-infected patients, raltegravir (RAL) and dolutegravir (DTG) have a more favourable lipid profile compared with NNRTI and boosted PI. Elvitegravir (EVG/c) has a superior lipid profile compared with efavirenz and is similar to that observed with ritonavir-boosted atazanavir except in TG, which increases less with EVG/c. In naïve patients, RAL, DTG, and bictegravir (BIC) produce a similar, slight increase in lipids. In switching trials, the regimen change based on a boosted PI or efavirenz to RAL, DTG, or BIC is associated with clinically significant decreases in lipids that are minor when the change is executed on EVG/c. No changes were observed in lipids by switching trials between INSTIs. In summary, RAL, DTG, and BIC have superior lipid profiles compared with boosted-PI, efavirenz, and EVG/c, in studies conducted in naïve participants, and they are associated with a clinically significant decrease in lipoproteins by switching studies.

Cardiovascular disease (CVD) is a major cause of mortality in type 1 diabetes mellitus (T1DM) patients, and cardiovascular risk (CVR) remains high even in T1DM patients with good metabolic control. The underlying mechanisms remain poorly understood and known risk factors seem to operate differently in T1DM and type 2 diabetes mellitus (T2DM) patients. However, evidence of cardiovascular risk assessment and management in T1DM patients often is extrapolated from studies on T2DM patients or the general population. In this review, we examine the existing literature about the prevalence of clinical and subclinical CVD, as well as current knowledge about potential risk factors involved in the development and progression of atherosclerosis in T1DM patients. We also discuss current approaches to the stratification and therapeutic management of CVR in T1DM patients. Chronic hyperglycemia plays an important role, but it is likely that other potential factors are involved in increased atherosclerosis and CVD in T1DM patients. Evidence on the estimation of 10-year and lifetime risk of CVD, as well as the efficiency and age at which current cardiovascular medications should be initiated in young T1DM patients, is very limited and clearly insufficient to establish evidence-based therapeutic approaches to CVD management.

Rationale: The HDL (high-density lipoprotein)-mediated stimulation of cellular cholesterol efflux initiates macrophage-specific reverse cholesterol transport (m-RCT), which ends in the fecal excretion of macrophage-derived unesterified cholesterol (UC). Early studies established that LDL (low-density lipoprotein) particles could act as efficient intermediate acceptors of cellular-derived UC, thereby preventing the saturation of HDL particles and facilitating their cholesterol efflux capacity. However, the capacity of LDL to act as a plasma cholesterol reservoir and its potential impact in supporting the m-RCT pathway in vivo both remain unknown., Objective: We investigated LDL contributions to the m-RCT pathway in hypercholesterolemic mice., Methods and Results: Macrophage cholesterol efflux induced in vitro by LDL added to the culture media either alone or together with HDL or ex vivo by plasma derived from subjects with familial hypercholesterolemia was assessed. In vivo, m-RCT was evaluated in mouse models of hypercholesterolemia that were naturally deficient in CETP (cholesteryl ester transfer protein) and fed a Western-type diet. LDL induced the efflux of radiolabeled UC from cultured macrophages, and, in the simultaneous presence of HDL, a rapid transfer of the radiolabeled UC from HDL to LDL occurred. However, LDL did not exert a synergistic effect on HDL cholesterol efflux capacity in the familial hypercholesterolemia plasma. The m-RCT rates of the LDLr (LDL receptor)-KO (knockout), LDLr-KO/APOB100, and PCSK9 (proprotein convertase subtilisin/kexin type 9)-overexpressing mice were all significantly reduced relative to the wild-type mice. In contrast, m-RCT remained unchanged in HAPOB100 Tg (human APOB100 transgenic) mice with fully functional LDLr, despite increased levels of plasma APO (apolipoprotein)-B-containing lipoproteins., Conclusions: Hepatic LDLr plays a critical role in the flow of macrophage-derived UC to feces, while the plasma increase of APOB-containing lipoproteins is unable to stimulate m-RCT. The results indicate that, besides the major HDL-dependent m-RCT pathway via SR-BI (scavenger receptor class B type 1) to the liver, a CETP-independent m-RCT path exists, in which LDL mediates the transfer of cholesterol from macrophages to feces. Graphical Abstract: A graphical abstract is available for this article.

Mimetic peptides are potential therapeutic agents for atherosclerosis. d-[ 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 ]apolipoprotein (apo) J (D-[ 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 ]apoJ) is a 10-residue peptide that is predicted to form a class G* amphipathic helix 6 from apoJ; it shows anti-inflammatory and anti-atherogenic properties. In the present study, we analyzed the effect of d-[ 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 ]apoJ in low-density lipoprotein receptor knockout mice(LDLR-KO) on the development of atherosclerosis and lipoprotein function. Fifteen-week-old female LDLR-KO mice fed an atherogenic Western-type diet were treated for eight weeks with d-[ 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 ]apoJ peptide, a scrambled peptide, or vehicle. Peptides were administered subcutaneously three days per week (200 µg in 100 µL of saline). After euthanasia, blood and hearts were collected and the aortic arch was analyzed for the presence of atherosclerotic lesions. Lipoproteins were isolated and their composition and functionality were studied. The extent of atherosclerotic lesions was 43% lower with d-[ 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 ]apoJ treatment than with the vehicle or scramble. The lipid profile was similar between groups, but the high-density lipoprotein (HDL) of d-[ 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 ]apoJ-treated mice had a higher antioxidant capacity and increased ability to promote cholesterol efflux than the control group. In addition, low-density lipoprotein (LDL) from d-[ 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 ]apoJ-treated mice was more resistant to induced aggregation and presented lower electronegativity than in mice treated with d-[ 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 ]apoJ. Our results demonstrate that the d-[ 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 ]apoJ peptide prevents the extent of atherosclerotic lesions, which could be partially explained by the improvement of lipoprotein functionality.

Electronegative low-density lipoprotein (LDL(-)) is a minor LDL subfraction that is present in blood with inflammatory and apoptotic effects. We aimed to evaluate the role of sphingolipids ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) in the LDL(-)-induced effect on monocytes. Total LDL was subfractioned into native LDL and LDL(-) by anion-exchange chromatography and their sphingolipid content evaluated by mass spectrometry. LDL subfractions were incubated with monocytes in the presence or absence of enzyme inhibitors: chlorpromazine (CPZ), d-erythro-2-( N -myristoyl amino)-1-phenyl-1-propanol (MAPP), and N , N -dimethylsphingosine (DMS), which inhibit Cer, Sph, and S1P generation, respectively. After incubation, we evaluated cytokine release by enzyme-linked immunosorbent assay (ELISA) and apoptosis by flow cytometry. LDL(-) had an increased content in Cer and Sph compared to LDL(+). LDL(-)-induced cytokine release from cultured monocytes was inhibited by CPZ and MAPP, whereas DMS had no effect. LDL(-) promoted monocyte apoptosis, which was inhibited by CPZ, but increased with the addition of DMS. LDL enriched with Sph increased cytokine release in monocytes, and when enriched with Cer, reproduced both the apoptotic and inflammatory effects of LDL(-). These observations indicate that Cer content contributes to the inflammatory and apoptotic effects of LDL(-) on monocytes, whereas Sph plays a more important role in LDL(-)-induced inflammation, and S1P counteracts apoptosis., Competing Interests: The authors declare that they have no conflicts of interest

Sulfate-based lipids (SL) have been proposed as players in inflammation, immunity and infection. In spite of the many biochemical processes linked to SL, analysis on this class of lipids has only focused on specific SL sub-classes in individual fluids or cells leaving a range of additional SL in other biological samples unaccounted for. This study describes the mass spectrometry screening of SL in lipid extracts of human fluids (saliva, plasma, urine, seminal fluid) and primary human cells (RBC, neutrophils, fibroblasts and skin epidermal) using targeted precursor ion scanning (PIS) approach. The PIS 97 mass spectra reveal a wide diversity of SL including steroid sulfates, sulfoglycolipids and other unidentified SL, as well as metabolites such as taurines, sulfated polyphenols and hypurate conjugates. Semi-quantification of SL revealed that plasma exhibited the highest content of SL whereas seminal fluid and epithelial cells contained the highest sulphur to phosphorous (S/P) ratio. The complexity of biofluids and cells sulfateome presented in this study highlight the importance of expanding the panel of synthetic sulfate-based lipid standards. Also, the heterogenous distribution of SL provides evidence for the interplay of sulfotransferases/sulfatases, opening new avenues for biomarker discovery in oral health, cardiovascular, fertility and dermatology research areas., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Aggregated LDL is the first ligand reported to interact with the cluster II CR9 domain of low-density lipoprotein receptor-related protein 1 (LRP1). In particular, the C-terminal half of domain CR9, comprising the region Gly 1127 -Cys 1140 exclusively recognizes aggregated LDL and it is crucial for aggregated LDL binding. Our aim was to study the effect of the sequence Gly 1127 -Cys 1140 (named peptide LP3 and its retro-enantio version, named peptide DP3) on the structural characteristics of sphingomyelinase- (SMase) and phospholipase 2 (PLA 2 )-modified LDL particles. Turbidimetry, gel filtration chromatography (GFC) and transmission electronic microscopy (TEM) analysis showed that LP3 and DP3 peptides strongly inhibited SMase- and PLA 2 -induced LDL aggregation. Nondenaturing polyacrylamide gradient gel electrophoresis (GGE), agarose gel electrophoresis and high-performance thin-layer chromatography (HPTLC) indicated that LP3 and DP3 prevented SMase-induced alterations in LDL particle size, electric charge and phospholipid content, respectively, but not those induced by PLA 2 . Western blot analysis showed that LP3 and DP3 counteracted changes in ApoB-100 conformation induced by the two enzymes. LDL proteomics (LDL trypsin digestion followed by mass spectroscopy) and computational modeling methods evidenced that peptides preserve ApoB-100 conformation due to their electrostatic interactions with a basic region of ApoB-100. These results demonstrate that LRP1-derived peptides are protective against LDL aggregation, even in conditions of extreme lipolysis, through their capacity to bind to ApoB-100 regions critical for ApoB-100 conformational preservation. These results suggests that these LRP1(CR9) derived peptides could be promising tools to prevent LDL aggregation induced by the main proteolytic enzymes acting in the arterial intima., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Epicardial adipose tissue (EAT) constitutes a novel parameter for cardiometabolic risk assessment and a target for therapy. Here, we evaluated for the first time the plasma microRNA (miRNA) profile as a source of biomarkers for epicardial fat volume (EFV). miRNAs were profiled in plasma samples from 180 patients whose EFV was quantified using multidetector computed tomography. In the screening study, 54 deregulated miRNAs were identified in patients with high EFV levels (highest tertile) compared with matched patients with low EFV levels (lowest tertile). After filtering, 12 miRNAs were selected for subsequent validation. In the validation study, miR-15b-3p, miR-22-3p, miR-148a-3p miR-148b-3p and miR-590-5p were directly associated with EFV, even after adjustment for confounding factors ( p value < 0.05 for all models). The addition of miRNA combinations to a model based on clinical variables improved the discrimination (area under the receiver-operating-characteristic curve (AUC) from 0.721 to 0.787). miRNAs correctly reclassified a significant proportion of patients with an integrated discrimination improvement (IDI) index of 0.101 and a net reclassification improvement (NRI) index of 0.650. Decision tree models used miRNA combinations to improve their classification accuracy. These results were reproduced using two proposed clinical cutoffs for epicardial fat burden. Internal validation corroborated the robustness of the models. In conclusion, plasma miRNAs constitute novel biomarkers of epicardial fat burden.

Objective: Electronegative LDL (LDL(-)) is involved in atherosclerosis through the activation of the TLR4/CD14 inflammatory pathway in monocytes. Matrix metalloproteinases (MMP) and their inhibitors (tissue inhibitors of metalloproteinase [TIMP]) are also crucially involved in atherosclerosis, but their modulation by LDL(-) has never been investigated. The aim of this study was to examine the ability of LDL(-) to release MMPs and TIMPs in human monocytes and to determine whether sulodexide (SDX), a glycosaminoglycan-based drug, was able to affect their secretion., Approach and Results: Native LDL (LDL(+)) and LDL(-) separated by anion-exchange chromatography were added to THP1-CD14 monocytes in the presence or absence of SDX for 24 h. A panel of 9 MMPs and 4 TIMPs was analyzed in cell supernatants with multiplex immunoassays. The gelatinolytic activity of MMP-9 was assessed by gelatin zymography. LDL(-) stimulated the release of MMP-9 (13-fold) and TIMP-1 (4-fold) in THP1-CD14 monocytes, as well as the gelatinolytic activity of MMP-9. Co-incubation of monocytes with LDL(-) and SDX for 24 h significantly reduced both the release of MMP-9 and TIMP-1 and gelatinase activity. In THP1 cells not expressing CD14, no effect of LDL(-) on MMP-9 or TIMP-1 release was observed. The uptake of DiI-labeled LDL(-) was higher than that of DiI-LDL(+) in THP1-CD14 but not in THP1 cells. This increase was inhibited by SDX. Experiments in microtiter wells coated with SDX demonstrated a specific interaction of LDL(-) with SDX., Conclusions: LDL(-) induced the release of MMP-9 and TIMP-1 in monocytes through CD14. SDX affects the ability of LDL(-) to promote TIMP-1 and MMP-9 release by its interaction with LDL(-)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cardiovascular disease of atherosclerotic origin is the main cause of death in diabetes mellitus and metabolic syndrome. One of the mechanisms involved in such increased risk is the high incidence of lipoprotein modification in these pathologies. Increased glycosylation, oxidative stress or high non-esterified fatty acid levels in blood, among other factors, promote the modification and subsequent alteration of the properties of lipoproteins. Since the modification of low-density lipoprotein (LDL) is the triggering factor in the development of atherosclerosis, considerable research has been focused on the quantification of modified LDLs in blood to be used as biomarkers of cardiovascular risk. The present review deals with the main molecular mechanisms involved in the modification of LDL in diabetes and metabolic syndrome and briefly describe the atherogenic effects that these modified LDLs exert on the arterial wall. The possibility of using the high levels of modified LDLs or their immunocomplexes as a predictive tool for cardiovascular risk in diabetes-related pathologies is also discussed.

Background and Aims: Type 2 diabetic patients have an increased proportion of electronegative low-density lipoprotein (LDL(-)), an inflammatory LDL subfraction present in blood, and dysfunctional high-density lipoprotein (HDL). We aimed at examining the inflammatory effect of LDL(-) on monocytes and the counteracting effect of HDL in the context of type 2 diabetes., Methods: This was a cross-sectional study in which the population comprised 3 groups (n = 12 in each group): type 2 diabetic patients with good glycaemic control (GC-T2DM patients), type 2 diabetic patients with poor glycaemic control (PC-T2DM), and a control group. Total LDL, HDL, and monocytes were isolated from plasma of these subjects. LDL(-) was isolated from total LDL by anion-exchange chromatography. LDL(-) from the three groups of subjects was added to monocytes in the presence or absence of HDL, and cytokines released by monocytes were quantified by ELISA., Results: LDL(-) proportion and plasma inflammatory markers were increased in PC-T2DM patients. LDL(-) from PC-T2DM patients induced the highest IL1β, IL6, and IL10 release in monocytes compared to LDL(-) from GC-T2DM and healthy subjects, and presented the highest content of non-esterified fatty acids (NEFA). In turn, HDL from PC-T2DM patients showed the lowest ability to inhibit LDL(-)-induced cytokine release in parallel to an impaired ability to decrease NEFA content in LDL(-)., Conclusions: Our findings show an imbalance in the pro- and anti-inflammatory effects of lipoproteins from T2DM patients, particularly in PC-T2DM., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.)

Background: Electronegative LDL (LDL(−)), a modified LDL fraction found in blood, induces the release of inflammatory mediators in endothelial cells and leukocytes. However, the inflammatory pathways activated by LDL(−) have not been fully defined. We aim to study whether LDL(−) induced release of the first-wave proinflammatory IL-1β in monocytes and monocyte-derived macrophages (MDM) and the mechanisms involved., Methods: LDL(−) was isolated from total LDL by anion exchange chromatography. Monocytes and MDM were isolated from healthy donors and stimulated with LDL(+) and LDL(−) (100 mg apoB/L)., Results: In monocytes, LDL(−) promoted IL-1β release in a time-dependent manner, obtaining at 20 h-incubation the double of IL-1β release induced by LDL(−) than by native LDL. LDL(−)-induced IL-1β release involved activation of the CD14-TLR4 receptor complex. LDL(−) induced priming, the first step of IL-1β release, since it increased the transcription of pro-IL-1β (8-fold) and NLRP3 (3-fold) compared to native LDL. Several findings show that LDL(−) induced inflammasome activation, the second step necessary for IL-1β release. Preincubation of monocytes with K+ channel inhibitors decreased LDL(−)-induced IL-1β release. LDL(−) induced formation of the NLRP3-ASC complex. LDL(−) triggered 2-fold caspase-1 activation compared to native LDL and IL-1β release was strongly diminished in the presence of the caspase-1 inhibitor Z-YVAD. In MDM, LDL(−) promoted IL-1β release, which was also associated with caspase-1 activation., Conclusions: LDL(−) promotes release of biologically active IL-1β in monocytes and MDM by induction of the two steps involved: priming and NLRP3 inflammasome activation., Significance: By IL-1β release, LDL(−) could regulate inflammation in atherosclerosis.

Plasma lipoproteins are a source of lipids for the heart, and the proportion of electronegative low density lipoprotein [LDL(-)] is elevated in cardiometabolic diseases. Perilipin 5 (Plin5) is a crucial protein for lipid droplet management in the heart. Our aim was to assess the effect of LDL(-) on intracellular lipid content and Plin5 levels in cardiomyocytes and to determine whether these effects were influenced by hypoxia. HL-1 cardiomyocytes were exposed to native LDL [LDL(+)], LDL(-), and LDL(+) enriched in non-esterified fatty acids (NEFA) by phospholipase A2 (PLA2)-mediated lipolysis [PLA2-LDL(+)] or by NEFA loading [NEFA-LDL(+)] under normoxia or hypoxia. LDL(-), PLA2-LDL(+) and NEFA-LDL(+) raised the intracellular NEFA and triglyceride (TG) content of normoxic cardiomyocytes. Plin5 was moderately upregulated by LDL(+) but more highly upregulated by LDL(-), PLA2-LDL(+) and NEFA-LDL(+) in normoxic cardiomyocytes. Hypoxia enhanced the effect of LDL(-), PLA2-LDL(+) and NEFA-LDL(+) on intracellular TG and NEFA concentrations but, in contrast, counteracted the upregulatory effect of these LDLs on Plin5. Fluorescence microscopy experiments showed that hypoxic cardiomyocytes exposed to LDL(-), PLA2-LDL(+) and NEFA-LDL(+) have an increased production of reactive oxygen species (ROS). By treating hypoxic cardiomyocytes with WY-14643 (PPARα agonist), Plin5 remained high. In this situation, LDL(-) failed to enhance intracellular NEFA concentration and ROS production. In conclusion, these results show that Plin5 deficiency in hypoxic cardiomyocytes exposed to LDL(-) dramatically increases the levels of unpacked NEFA and ROS., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aims: Electronegative LDL (LDL(-)), a minor modified LDL present in the circulation, induces cytokine release in monocytes. We aimed to determine the role of the receptor CD14 and toll-like receptors 2 and 4 (TLR2, TLR4) in the inflammatory action promoted by LDL(-) in human monocytes., Methods and Results: Monocytes were preincubated with antibodies to neutralize CD14, TLR2 and TLR4. The release of monocyte chemoattractant protein 1 (MCP1), and interleukin 6 and 10 (IL6 and IL10) promoted by LDL(-) was inhibited 70-80% by antiCD14 and antiTLR4, and 15-25% by antiTLR2. The involvement of CD14 and TLR4 was confirmed by gene silencing experiments. The human monocytic THP1 cell line overexpressing CD14 released more cytokines in response to LDL(-) than the same THP1 cell line without expressing CD14. VIPER, a specific inhibitor of the TLR4 signaling pathway, blocked 75-90% the cytokine release promoted by LDL(-). Cell binding experiments showed that monocytes preincubated with neutralizing antibodies presented lesser LDL(-) binding than non-preincubated monocytes The inhibitory capacity was antiCD14>antiTLR4>>antiTLR2. Cell-free experiments performed in CD14-coated microtiter wells confirmed that CD14 was involved in LDL(-) binding. When LDL(-) and lipopolysaccharide (LPS) were added simultaneously to monocytes, cytokine release was similar to that promoted by LDL(-) alone. Binding experiments showed that LDL(-) and LPS competed for binding to monocytes and to CD14 coated-wells., Conclusions: CD14 and TLR4 mediate cytokine release induced by LDL(-) in human monocytes. The cross-competition between LPS and LDL(-) for the same receptors could be a counteracting action of LDL(-) in inflammatory situations., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Electronegative low-density lipoprotein (LDL(-)) is a minor modified LDL subfraction that is present in blood. LDL(-) promotes inflammation and is associated with the development of atherosclerosis. We previously reported that the increase of cytokine release promoted by this lipoprotein subfraction in monocytes is counteracted by high-density lipoprotein (HDL). HDL also inhibits a phospholipase C-like activity (PLC-like) intrinsic to LDL(-). The aim of this work was to assess whether the inhibition of the PLC-like activity by HDL could decrease the content of ceramide (CER) and diacylglycerol (DAG) generated in LDL(-). This knowledge would allow us to establish a relationship between these compounds and the inflammatory activity of LDL(-). LDL(-) incubated at 37 °C for 20 h increased its PLC-like activity and, subsequently, the amount of CER and DAG. We found that incubating LDL(-) with HDL decreased both products in LDL(-). Native LDL was modified by lipolysis with PLC or by incubation with CER-enriched or DAG-enriched liposomes. The increase of CER in native LDL significantly increased cytokine release, whereas the enrichment in DAG did not show these inflammatory properties. These data point to CER, a resultant product of the PLC-like activity, as a major determinant of the inflammatory activity induced by LDL(-) in monocytes.

Electronegative LDL (LDL(-)) is a minor modified LDL subfraction that promotes cytokine release by human mononuclear cells. The aim of the current study was to evaluate changes in gene expression induced by LDL(-) versus native LDL in lymphocytes and monocytes. Therefore, mononuclear cells were incubated with these LDL subfractions and their effects on expression in human whole genome were analyzed by gene array. Differential expression of the genes was quantified by real-time RT-PCR. LDL(-) altered the gene expression pattern, particularly of inflammatory genes. LDL(-) down-regulated CD36 and colony-stimulating factor 1 receptor (CSF1R) genes and up-regulated Fas expression and Fas protein on cellular membrane. LDL(-) seemed to promote the alterations in these genes by activation of NF-kB and inhibition of AP1 and PPARG. In conclusion, LDL(-) induced changes in gene expression in monocytes and lymphocytes. Fas up-regulation suggests a proinflammatory action; however, CSF1R and CD36 down-regulation could decrease monocyte differentiation and activation. Therefore, LDL(-) promoted not only inflammatory effects but also counteracting actions in circulating mononuclear cells.

Physical activity (PA) is associated with a reduced risk of coronary heart disease, and may favorably modify the antioxidant-prooxidant balance. This study assessed the effects of aerobic PA training on antioxidant enzyme activity, oxidized LDL concentration, and LDL resistance to oxidation, as well as the effect of acute PA on antioxidant enzyme activity before and after the training period. Seventeen sedentary healthy young men and women were recruited for 16 weeks of training. The activity of superoxide dismutase in erythrocytes (E-SOD), glutathione peroxidase in whole blood (GSH-Px), and glutathione reductase in plasma (P-GR), and the oxidized LDL concentration and LDL composition, diameter, and resistance to oxidation were determined before and after training. Shortly before and after this training period they also performed a bout of aerobic PA for 30 min. The antioxidant enzyme activity was also determined at 0 min, 30 min, 60 min, 120 min, and 24 h after both bouts of PA. Training induces an increase in GSH-Px (27.7%), P-GR (17.6%), and LDL resistance to oxidation, and a decrease in oxidized LDL (-15.9%). After the bout of PA, an increase in E-SOD and GSH-Px was observed at 0 min, with a posterior decrease in enzyme activity until 30-60 min, and a tendency to recover the basal values at 120 min and 24 h. Training did not modify this global response pattern. Regular PA increases endogenous antioxidant activity and LDL resistance to oxidation, and decreases oxidized LDL concentration; 30 min of aerobic PA decreases P-GR and B-GSH-Px activity in the first 30-60 min with a posterior recovery.