Cocoa consumption reduces NF-κB activation in peripheral blood mononuclear cells in humans.

BACKGROUND AND AIMS: Epidemiological studies have demonstrated an association between high-polyphenol intake and reduced incidence of atherosclerosis. The healthy effects of cocoa-polyphenols may be due to their antioxidant and anti-inflammatory actions, although the exact mechanisms are unknown and depend on the matrix in which cocoa-polyphenols are delivered. Nuclear factor κB (NF-κB) is a key molecule in the pathophysiology of atherosclerosis involved in the regulation of adhesion molecules(AM) and cytokine expression and its activation is the first step in triggering the inflammatory process. The aim of this study was to evaluate the effect of acute cocoa consumption in different matrices related to the bioavailability of cocoa-polyphenols in NF-κB activation and the expression of AM. METHODS AND RESULTS: Eighteen healthy volunteers randomly received 3 interventions: 40g of cocoa powder with milk (CM), with water (CW), and only milk (M). NF-κB activation in leukocytes and AM (sICAM, sVCAM, E-selectin) were measured before and 6h after each intervention. Consumption of CW significantly decreased NF-κB activation compared to baseline and to CM (P < 0.05, both), did not change after CM intervention, and significantly increased after M intervention (P = 0.014). sICAM-1 concentrations significantly decreased after 6h of CW and CM interventions (P ≤ 0.026; both) and E-selectin only decreased after CW intervention (P = 0.028). No significant changes were observed in sVCAM-1 concentrations. CONCLUSIONS: The anti-inflammatory effect of cocoa intake may depend on the bioavailability of bioactive compounds and may be mediated at least in part by the modulation of NF-κB activation and downstream molecules reinforcing the link between cocoa intake and health.

Aggregated low density lipoprotein induces tissue factor by inhibiting sphingomyelinase activity in human vascular smooth muscle cells.

BACKGROUND: Our previous results demonstrated that aggregated low density lipoprotein (agLDL) induces tissue factor (TF) expression and activation through Rho A translocation in human vascular smooth muscle cells (VSMC). We also previously demonstrated that membrane sphingomyelin (SM) content is higher in agLDL-exposed VSMC than in control cells. The main enzymes regulating cellular SM content are the family of sphingomyelinases (Smases) that hydrolize SM to phosphorylcholine and ceramide (CER). OBJECTIVES: We wished to investigate whether agLDL has the ability to modulate acidic- (A-) and neutral (N-) Smase activity and whether or not this effect is related to the upregulatory effect of agLDL on Rho A translocation and TF activation in human VSMC. METHODS AND RESULTS: By measuring generated [(14)C]-phosphorylcholine, we found that agLDL significantly decreased A-Smase and specially N-Smase activity. Pharmacological Smase inhibitors increased Rho A and TF. Specific loss-of-function of A-Smase or N-Smase 1 (N1-Smase) by siRNA treatment (500 nmol L(-1), 12 hours) dramatically increased membrane Rho A protein levels (5- and 3-fold, respectively). Concomitantly, TF protein expression and TF procoagulant activity were also increased. Inhibition of A-Smase or N-Smase activity by agLDL, siRNA-anti A- or N1-Smase or pharmacological treatment significantly increased the SM content of vascular cells. The inhibition of SM synthesis by fumonisin B(1) (FB(1)) prevented the upregulatory effect of agLDL on TF. CONCLUSIONS: These results demonstrate that inhibition of both A- and N1-Smase might explain the upregulatory effect of agLDL on TF activation, and suggest that this effect is related, at least in part, to membrane SM enrichment.

Tissue factor induction by aggregated LDL depends on LDL receptor-related protein expression (LRP1) and Rho A translocation in human vascular smooth muscle cells.

OBJECTIVE: Low density lipoprotein (LDL) internalized in the vascular wall and modified by binding to extracellular matrix-proteoglycans (ECM) becomes aggregated (agLDL). AgLDL induces tissue factor (TF) expression and activity in human vascular smooth muscle cells (VSMC). TF expression in vascular cells promotes the prothrombotic transformation of the vascular wall. However, the mechanisms by which agLDL induces TF are not known. The aim of this study was to investigate the mechanisms involved in TF activation by extracellular matrix-modified LDL in human VSMC. METHODS AND RESULTS: AgLDL significantly induces TF expression (real time PCR and Western blot analysis) and procoagulant activity (factor Xa generation test) in human VSMC. HMG-CoA reductase inhibition completely prevents agLDL-induced TF expression and partially inhibits agLDL-TF activation. These effects are reverted by geranylgeranyl pyrophosphate (GGPP) but not by farnesyl pyrophosphate (FPP), suggesting the involvement of a geranylated protein in agLDL-TF induction. AgLDL increases Rho A translocation (2-fold) from the cytoplasm to the cell membrane in control but not in simvastatin-treated VSMC. Exoenzyme C3, a specific Rho A inhibitor, completely prevents agLDL-induced TF overexpression and partially agLDL-TF activation. Blocking LRP1, the receptor of agLDL, with anti-LRP1 antibodies or inhibiting LRP1 expression by small interference RNA treatment (siRNA-LRP1) impairs agLDL-induced TF overexpression and activation. CONCLUSIONS: These results demonstrate that TF induction by agLDL depends on LRP1 expression and requires Rho A translocation to the cellular membrane.

Sterol regulatory element-binding protein-2 negatively regulates low density lipoprotein receptor-related protein transcription.

Low density lipoprotein receptor-related protein (LRP1) binds aggregated LDL (agLDL) leading to a high intracellular cholesteryl ester (CE) accumulation. AgLDL up-regulates LRP1 expression concomitantly with an LDL receptor (LDLR) and sterol regulatory element binding protein (SREBP-2) down-regulation. The objectives were to investigate whether SREBP-2 regulates LRP1 transcription and determine the molecular mechanisms involved in the process. Down-regulation of active SREBP-2 by nLDL and agLDL led to LDLR down-regulation and LRP1 up-regulation. Enforced expression of an active form of SREBP-2 (SREBP-2-NT, amino acid residues 1-468) decreased LRP1 expression and LRP1 promoter (WT-LRP1) luciferase activity in a dose-dependent manner. LDL did not exert any significant effect on LRP1 promoter activity when a putative sterol regulatory element (SRE) (5-GTGGGGTGA-3'; +225 to +233) was mutated (SRE-MT-LRP1). SREBP-2 overexpression exerted stronger down-regulatory effects on WT-LRP1 than on SRE-MT-LRP1 promoter activity both in control, nLDL- and agLDL-exposed HeLa cells. Gel mobility shift assays showed that recombinant SREBP-2-NT protein (1-468) binds to a double-stranded LRP1 DNA fragment (215 to 245) containing a wild-type (wt) SRE sequence but not to a mutated SRE (mt) sequence (5-GAATTCGA-3'). Our results demonstrate that LDL stimulates LRP1 transcription and decreases SREBP-2 active form which negatively regulates LRP1 transcription. SRE sequence (+225 to +233) plays a pivotal role for the down-regulatory effect of SREBP-2 on LRP1 promoter activity.