Mercedes Heras

Unsaturated fatty acids and their oxidation products stimulate CD36 gene expression in human macrophages

Fatty acids (FA) have been implicated in the control of expression of several atherosclerosis-related genes. Similarly, the CD36 receptor has recently been shown to play an important role in atherosclerosis and other pathologies. The aim of the present study was to evaluate the direct effect of FA and their oxidation products (aldehydes), on the expression of CD36 in both THP-1 macrophages and human monocyte-derived macrophages (HMDM). The FA tested included the saturated FA (SFA) lauric, myristic, palmitic and stearic acid; the monounsaturated FA oleic acid; and the unsaturated FA (UFA) linoleic, arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Aldehydes used were malondialdehyde (MDA), hexanal, 2,4-decadienal (DDE) and 4-hydroxynonenal (HNE). CD36 expression was measured by RT-PCR, Western blot and immunofluorescence. Incubation of THP-1 macrophages for 24 h with non-cytotoxic concentrations of UFA significantly increased CD36 mRNA expression. By contrast, exposure of THP-1 macrophages to SFA did not affect the levels of CD36 mRNA. Among all UFAs tested, EPA and DHA were the strongest inducers of CD36 mRNA levels, followed by oleic and linoleic acid. Incubation of HMDM with either oleic or linoleic acid significantly increased steady-state CD36 mRNA in a dose-dependent manner. Consistent with the increase of CD36 mRNA expression, incubation of THP-1 macrophages with oleic and linoleic acid for 24 h markedly increased CD36 protein expression. Treatment of THP-1 macrophages with MDA or hexanal for 24 h significantly increased CD36 mRNA expression in a dose dependent manner. In contrast, DDE and HNE significantly decreased this parameter. The data provide evidence for a direct regulatory effect of UFA on CD36 gene expression and support a role for aldehydes in the regulation of CD36 expression by FA.

Soluble fibre (Plantago ovata husk) reduces plasma low-density lipoprotein (LDL) cholesterol, triglycerides, insulin, oxidised LDL and systolic blood pressure in hypercholesterolaemic patients: A randomised trial

UNLABELLED The objective was to evaluate whether the soluble fibre Plantago ovata (Po)-husk improves cardiovascular disease (CVD) risk biomarkers including low-density lipoprotein cholesterol (LDL-C). METHODS In a multi-centred, double-blind, placebo-controlled, parallel, randomised trial conducted in primary care-clinics in Spain, France and Holland, mild-moderate hypercholesterolaemic patients (age range: 43-67 years) received 14 g/d of Po-husk (n=126) or placebo (microcrystalline-cellulose 14 g/d; n=128) in a low saturated fat diet for 8 weeks. Subsequently, if LDL-C remained > or = 3.35 mmol/L [130 mg/dL], participants proceeded with the fibre plus simvastatin (20mg/d) for further 8 weeks. Lipid profile, blood pressure (BP), insulin, oxidised LDL and some gene polymorphisms involved in CVD risk were measured. RESULTS Relative to placebo, Po-husk reduced plasma LDL-C by -6% (P<0.0002), total cholesterol (TC) by -6%, triglycerides (TG) by -21.6%, apolipoprotein (Apo) B-100 by -6.7%, oxidised LDL by a mean of -6.82 U/L (95%CI: 3.15-10.48), insulin by -4.68 pmol/L (95%CI: 0.68-8.67) and systolic BP by -4.0mm Hg (95%CI; 1.2-6.7) (P<0.05). The TG-lowering effect in the Po-husk group was magnified by variants in plasminogen-activator-inhibitor (PAI-1; rs1799768) and fatty acid-binding protein (FABP-2; rs1799883) genes. At 16 weeks, the intra-group action of simvastatin (20mg/d) added to Po-husk or placebo was a similar LDL-C reduction. CONCLUSIONS Po-husk, apart from lowering LDL-C, also reduced TG, TG related to certain gene variants, TC, Apo B-100, oxLDL, insulin-resistance and systolic BP in mild-moderate hypercholesterolaemic individuals. Thus, the target patients to receive Po-husk would be those who present a cluster of various CVD risk factors, such as metabolic syndrome.

Fatty acid-binding protein 4 impairs the insulin-dependent nitric oxide pathway in vascular endothelial cells.

BackgroundRecent studies have shown that fatty acid-binding protein 4 (FABP4) plasma levels are associated with impaired endothelial function in type 2 diabetes (T2D). In this work, we analysed the effect of FABP4 on the insulin-mediated nitric oxide (NO) production by endothelial cells in vitro.MethodsIn human umbilical vascular endothelial cells (HUVECs), we measured the effects of FABP4 on the insulin-mediated endothelial nitric oxide synthase (eNOS) expression and activation and on NO production. We also explored the impact of exogenous FABP4 on the insulin-signalling pathway (insulin receptor substrate 1 (IRS1) and Akt).ResultsWe found that eNOS expression and activation and NO production are significantly inhibited by exogenous FABP4 in HUVECs. FABP4 induced an alteration of the insulin-mediated eNOS pathway by inhibiting IRS1 and Akt activation. These results suggest that FABP4 induces endothelial dysfunction by inhibiting the activation of the insulin-signalling pathway resulting in decreased eNOS activation and NO production.ConclusionThese findings provide a mechanistic linkage between FABP4 and impaired endothelial function in diabetes, which leads to an increased cardiovascular risk.

Fatty acid-binding protein 4 is associated with endothelial dysfunction in patients with type 2 diabetes

OBJECTIVE Adipocyte fatty acid-binding protein (FABP4) plasma levels are higher in type 2 diabetes (T2D). Endothelial dysfunction is also common in T2D. We have investigated the relationship between circulating FABP4 levels and endothelial function in diabetic patients. METHODS In 257 patients (105 diabetic and 152 non-diabetic) at increased risk of cardiovascular disease, we measured circulating FABP4, reactive hyperemia index (RHI) by peripheral artery tonometry, intima-media thickness, and biomarkers of inflammation, oxidation and endothelial function. RESULTS In T2D subjects, FABP4 was negatively associated with endothelial function, as measured by RHI (r=-0.226, P=0.05). In a stepwise multivariate linear regression model, FABP4 was a predictor of RHI in T2D patients (P=0.04). CONCLUSION Circulating levels of FABP4 are inversely associated with endothelial function in T2D patients, as measured by RHI. We suggest a direct effect of plasma FABP4 on the vascular endothelium in those with T2D.

Oxidized Lipoproteins Including HDL and Their Lipid Peroxidation Products Inhibit TNF-α Secretion by THP-1 Human Macrophages

It has been established that oxidized LDL (ox-LDL) modifies cytokine secretion by macrophages, for example, by reducing tumor necrosis factor alpha (TNF-(alpha) m-RNA. However, little is known about the effects of oxidized high density lipoprotein (ox-HDL). This study reports the effects of ox-HDL subfractions 2 and 3 (ox-HDL2, ox-HDL3) compared with that of ox-LDL and some products of oxidation (hydroperoxides and aldehydes) on the secretion of TNF-alpha from THP-1 human monocytes derived macrophages in vitro. HDL2, HDL3 and LDL were oxidized with 10 microM Cu++ for 12 h and/or 24 h. Native and oxidized HDL and LDL were incubated for 24 h with macrophages with or without LPS (10 ng/ml) after which TNF-alpha secretion was measured in the culture medium. Lipid hydroperoxides and apolar aldehydes were also incubated with the cells for 2 h following which the medium was replaced and TNF-alpha secretion measured after a further 22 h of incubation. An inhibition of TNF-alpha by ox-HDL2 (p < .05), ox-HDL3 (p < .05) and ox-LDL (p < .05) from THP-1 macrophages was observed in the presence and absence of LPS. This inhibition remained the same after incubation with ox-HDL 12 h and 24 h. Hydroperoxides of linoleic acid did not modify TNF-alpha secretion by cells while five out of eight aldehydes analyzed (2,4-heptadienal, hexanal, 2-nonenal, 2-octenal, 2,4-decadienal) inhibited TNF-alpha secretion (p < .05). These findings demonstrate that ox-HDL, and some of its lipid peroxidation products, plays a role in the modulation of the inflammatory response by macrophages as previously observed for ox-LDL.

Liposcale: a novel advanced lipoprotein test based on 2D diffusion-ordered 1H NMR spectroscopy

Determination of lipoprotein particle size and number using advanced lipoprotein tests (ALTs) is of particular importance to improve cardiovascular risk prediction. Here we present the Liposcale test, a novel ALT based on 2D diffusion-ordered 1H NMR spectroscopy. Our method uses diffusion coefficients to provide a direct measure of the mean particle sizes and numbers. Using 177 plasma samples from healthy individuals and the concentration of ApoB and ApoA from isolated lipoprotein fractions, our test showed a stronger correlation between the NMR-derived lipoprotein particle numbers and apolipoprotein concentrations than the LipoProfile® test commercialized by Liposcience. We also converted LDL particle numbers to ApoB equivalents (milligrams per deciliter) and our test yielded similar values of LDL-ApoB to the LipoProfile® test (absolute mean bias of 8.5 and 7.4 mg/dl, respectively). In addition, our HDL particle number values were more concordant with the calibrated values determined recently using ion mobility. Finally, principal component analysis distinguished type 2 diabetic patients with and without atherogenic dyslipidemia (AD) on a second cohort of 307 subjects characterized using the Liposcale test (area under the curve = 0.88) and showed concordant relationships between variables explaining AD. Altogether, our method provides reproducible and reliable characterization of lipoprotein particles and it is applicable to pathological states such as AD.

Cocoa, Hazelnuts, Sterols and Soluble Fiber Cream Reduces Lipids and Inflammation Biomarkers in Hypertensive Patients: A Randomized Controlled Trial

Background Cocoa, mixed with other food ingredients, intake can have beneficial effects on cardiovascular disease (CVD) biomarkers. We compared the effects of 4 cocoa cream products on some of these biomarkers. Methods and Findings In this multi-centered, randomized, controlled, double-blind, parallel trial, volunteers (n = 113; age range: 43–65 years) who were pre-hypertensive, stage-1 hypertensive and hypercholesterolemic received one of 4 cocoa cream products (13 g/unit; 1 g cocoa/unit, 6 units/d; 465 Kcal/d) added to a low saturated fat diet for 4 weeks. The groups were: A) (n = 28), cocoa cream considered as control; B) (n = 28), cocoa+hazelnut cream (30 g/d hazelnuts); C) (n = 30), cocoa+hazelnuts+phytosterols (2 g/d); and D) (n = 27), cocoa+hazelnuts+phytosterols+soluble fiber (20 g/d) the patented “LMN product”. Primary outcome measures were BP, LDL-c, apolipoprotein B-100 (Apo B), ApoB/ApoA ratio, oxidized LDL (oxLDL) and high-sensitive C-reactive protein (hsCRP) determined at baseline and post-cocoa cream product intake. Statistical analysis used was ANCOVA or mixed models (in case of repeated measurements), with baseline observation included as a covariate. After 4 weeks, compared to product A, product C reduced LDL-c by 11.2%, Apo B by 8.1% and ApoB/ApoA ratio by 7.8% (P = 0.01). LMN decreased LDL-c by 9.2%, Apo B-100 by 8.5%, ApoB/ApoA ratio by 10.5%, hsCRP by 33.4% and oxLDL by 5.9% (P = 0.01). Surprisingly, even “control” product A reduced systolic BP (−7.89 mmHg; 95%CI: −11.45 to −4.3) and diastolic BP (−5.54 mmHg; 95%CI: −7.79 to −3.29). The BP reductions were similar with the other 3 products. Limitations of the study are that the trial period was relatively short and that a better “BP control” product would have been preferable. Conclusion The creams (particularly the LMN) have anti-inflammatory and antioxidant effects in addition to lowering LDL-c, Apo B and ApoB/ApoA ratio. Thus, the soluble fiber effects amplified with sterols (as contained in the cocoa creams) provide new dietary therapeutic perspectives. Trial Registration Clinicaltrials.gov NCT00511420

Alpha-Tocopherol and BAY 11-7082 Reduce Vascular Cell Adhesion Molecule in Human Aortic Endothelial Cells

Background: In endothelial dysfunction, vascular cell adhesion molecule-1 (VCAM-1), E-selectin and intercellular adhesion molecule-1 (ICAM-1) expression (collectively termed cell adhesion molecules; CAMs) increase at sites of atherosclerosis and are stimulated by proinflammatory cytokines such as tumor necrosis factor-α (TNF-α). Methods: We evaluated the effect of alpha-tocopherol (AT; 10–150 µM) and BAY 11-7082 (BAY; 0.1 or 1 µM) on CAMs mRNA expression as well as their protein in soluble release form (sCAMs) in human aortic endothelial cells (HAECs) activated by TNF-α (1 or 10 ng/ml). Also, we determined the extent of lymphocyte adhesion to activated HAECs. Results: BAY reduced VCAM-1, E-selectin and ICAM-1 mRNA expression by 30, 30 and 10%, respectively. Furthermore, protein reduction of sVCAM-1 by 70%, sE-selectin by 51% and sICAM-1 by 25% compared to HAECs stimulated by TNF-α was observed (p < 0.05). AT (50, 75 and 150 µM) decreased VCAM-1 mRNA expression by 30% and sVCAM-1 protein by 33% compared to HAECs stimulated by TNF-α (p < 0.05). TNF-α-activated HAEC adhesion to human Jurkat T lymphocytes was higher compared to nonactivated HAECs (p < 0.05). BAY (2 and 5 µM) reduced this lymphocyte adhesion (p < 0.05). Conclusion: BAY reduces all the CAMs studied as well as cell adhesion, while AT selectively inhibits VCAM-1; both induce endothelial dysfunction improvement.

2,4-Decadienal downregulates TNF-α gene expression in THP-1 human macrophages

Abstract Oxidized lipoproteins inhibit TNF-α secretion by human THP-1 macrophages due, at least in part, to aldehydes derived from the oxidation of polyunsaturated fatty acids. This study extends these findings by investigating the effect of three aldehydes (2,4-decadienal (2,4-DDE), hexanal and 4-hydroxynonenal (4-HNE)) on TNF-α and IL-1β mRNA expression. The 2,4-DDE and 4-HNE showed considerable biological activity which induced cytotoxicity on THP-1 macrophages at concentration of 50 μM. Hexanal, on the other hand, had a lower cytotoxic capacity and concentration of 1000 μM was needed for the effect to be observed. Exposure of THP-1 macrophages to aldehydes for 24 h inhibited TNF-α mRNA expression but increased or did not affect IL-1β mRNA levels. The inhibitory action of 2,4-DDE was dose dependent and began at 5 μM (46%, P P

Cytotoxic effects of the lipid peroxidation product 2,4-decadienal in vascular smooth muscle cells

It is well known that oxidized LDL can be cytotoxic to smooth muscle cells (SMC) and then contribute to the progression of atherosclerosis. Nevertheless, which oxidized compound and which mechanism are involved in cell death is still under study. In this work we have studied the role of two representative apolar aldehydes (hexanal and 2,4-decadienal (2,4-DDE)), derived from polyunsaturated fatty acids oxidation, on human SMC cytotoxicity. Cell cytotoxicity was assessed by means of lactate deshydrogenase (LDH) release, cell morphology and DNA fragmentation. Results showed that hexanal up to 50 microM for 24 h was not cytotoxic to cells. However, 2,4-DDE at 50 microM for 24 h induced a 48% LDH leakage. The observed cytotoxic effect of 2,4-DDE was not due to a programmed cell death as no DNA ladder was detected. After aldehydes exposition a decreased expression of p53 and c-myc mRNA, genes involved in cell death regulation, was also demonstrated by RT-PCR. These observations suggest that 2,4-DDE may be the molecular cause of lipid oxidation cytotoxicity to human vascular SMC. By inducing cell necrosis in advanced stages, lipid oxidation may contribute to the cell debris core which is growing in the atherosclerotic lesion leading to a weakened and unstable plaque.