Thrombin Generation Is Associated With Extracellular Vesicle and Leukocyte Lipid Membranes in Atherosclerotic Cardiovascular Disease.

BACKGROUND: Clotting, leading to thrombosis, requires interactions of coagulation factors with the membrane aminophospholipids (aPLs) phosphatidylserine and phosphatidylethanolamine. Atherosclerotic cardiovascular disease (ASCVD) is associated with elevated thrombotic risk, which is not fully preventable using current therapies. Currently, the contribution of aPL to thrombotic risk in ASCVD is not known. Here, the aPL composition of circulating membranes in ASCVD of varying severity will be characterized along with the contribution of external facing aPL to plasma thrombin generation in patient samples. METHODS: Thrombin generation was measured using a purified factor assay on platelet, leukocyte, and extracellular vesicles (EVs) from patients with acute coronary syndrome (n=24), stable coronary artery disease (n=18), and positive risk factor (n=23) and compared with healthy controls (n=24). aPL composition of resting/activated platelet and leukocytes and EV membranes was determined using lipidomics. RESULTS: External facing aPLs were detected on EVs, platelets, and leukocytes, elevating significantly following cell activation. Thrombin generation was higher on the surface of EVs from patients with acute coronary syndrome than healthy controls, along with increased circulating EV counts. Thrombin generation correlated significantly with externalized EV phosphatidylserine, plasma EV counts, and total EV membrane surface area. In contrast, aPL levels and thrombin generation from leukocytes and platelets were not impacted by disease, although circulating leukocyte counts were higher in patients. CONCLUSIONS: The aPL membrane of EV supports an elevated level of thrombin generation in patient plasma in ASCVD. Leukocytes may also play a role although the platelet membrane did not seem to contribute. Targeting EV formation/clearance and developing strategies to prevent the aPL surface of EV interacting with coagulation factors represents a novel antithrombotic target in ASCVD.

Dietary n-3 polyunsaturated fatty acids alter the number, fatty acid profile and coagulatory activity of circulating and platelet-derived extracellular vesicles: a randomized, controlled crossover trial.

BACKGROUND: Extracellular vesicles (EVs) are proposed to play a role in the development of cardiovascular diseases (CVDs) and are considered emerging markers of CVDs. n-3 PUFAs are abundant in oily fish and fish oil and are reported to reduce CVD risk, but there has been little research to date examining the effects of n-3 PUFAs on the generation and function of EVs. OBJECTIVES: We aimed to investigate the effects of fish oil supplementation on the number, generation, and function of EVs in subjects with moderate risk of CVDs. METHODS: A total of 40 participants with moderate risk of CVDs were supplemented with capsules containing either fish oil (1.9 g/d n-3 PUFAs) or control oil (high-oleic safflower oil) for 12 wk in a randomized, double-blind, placebo-controlled crossover intervention study. The effects of fish oil supplementation on conventional CVD and thrombogenic risk markers were measured, along with the number and fatty acid composition of circulating and platelet-derived EVs (PDEVs). PDEV proteome profiles were evaluated, and their impact on coagulation was assessed using assays including fibrin clot formation, thrombin generation, fibrinolysis, and ex vivo thrombus formation. RESULTS: n-3 PUFAs decreased the numbers of circulating EVs by 27%, doubled their n-3 PUFA content, and reduced their capacity to support thrombin generation by >20% in subjects at moderate risk of CVDs. EVs derived from n-3 PUFA-enriched platelets in vitro also resulted in lower thrombin generation, but did not alter thrombus formation in a whole blood ex vivo assay. CONCLUSIONS: Dietary n-3 PUFAs alter the number, composition, and function of EVs, reducing their coagulatory activity. This study provides clear evidence that EVs support thrombin generation and that this EV-dependent thrombin generation is reduced by n-3 PUFAs, which has implications for prevention and treatment of thrombosis. CLINICAL TRIAL REGISTRY: This trial was registered at clinicaltrials.gov as NCT03203512.

Phosphatidylthreonine is a procoagulant lipid detected in human blood and elevated in coronary artery disease.

Aminophospholipids (aPL) such as phosphatidylserine are essential for supporting the activity of coagulation factors, circulating platelets, and blood cells. Phosphatidylthreonine (PT) is an aminophospholipid previously reported in eukaryotic parasites and animal cell cultures, but not yet in human tissues. Here, we evaluated whether PT is present in blood cells and characterized its ability to support coagulation. Several PT molecular species were detected in human blood, washed platelets, extracellular vesicles, and isolated leukocytes from healthy volunteers using liquid chromatography-tandem mass spectrometry. The ability of PT to support coagulation was demonstrated in vitro using biochemical and biophysical assays. In liposomes, PT supported prothrombinase activity in the presence and absence of phosphatidylserine. PT nanodiscs strongly bound FVa and lactadherin (nM affinity) but poorly bound prothrombin and FX, suggesting that PT supports prothrombinase through recruitment of FVa. PT liposomes bearing tissue factor poorly generated thrombin in platelet poor plasma, indicating that PT poorly supports extrinsic tenase activity. On platelet activation, PT is externalized and partially metabolized. Last, PT was significantly higher in platelets and extracellular vesicle from patients with coronary artery disease than in healthy controls. In summary, PT is present in human blood, binds FVa and lactadherin, supports coagulation in vitro through FVa binding, and is elevated in atherosclerotic vascular disease. Our studies reveal a new phospholipid subclass, that contributes to the procoagulant membrane, and may support thrombosis in patients at elevated risk.

Circulating Extracellular Vesicles Are Strongly Associated With Cardiovascular Risk Markers.

Background: Extracellular vesicles (EVs) are submicron membrane-bound vesicles released from various cells, which are emerging as a potential novel biomarker in cardiovascular diseases (CVDs) due to their procoagulatory and prothrombotic properties. However, there is little information about the relationships between circulating EVs and conventional and thrombogenic risk markers of CVDs. Objective: To investigate the relationships between circulating EVs, conventional cardiovascular risk markers and thrombogenic markers in subjects with moderate risk of CVDs. Design: Subjects (n = 40) aged 40-70 years with moderate risk of CVDs were recruited and assessed for body mass index, blood pressure and plasma lipid profile, as well as platelet aggregation, clot formation, thrombin generation and fibrinolysis. Numbers of circulating EVs were assessed by Nanoparticle Tracking Analysis and flow cytometry. A range of assays were used to assess the procoagulatory activity of plasma and circulating EVs. Results: Circulating EV numbers were positively associated with body mass index, blood pressure, plasma triacylglycerol concentration and overall CVD risk. Higher circulating EV numbers were also associated with increased thrombin generation and enhanced clot formation, and EVs isolated from subjects with moderate CVD risk promoted thrombin generation ex vivo. Higher numbers of endothelial-derived EVs were associated with a greater tendency for clot lysis. Plasma triacylglycerol concentration and diastolic blood pressure independently predicted circulating EV numbers, and EV numbers independently predicted aspects of thrombin generation and clot formation and 10-year CVD risk. Conclusion: Circulating EVs were strongly associated with both conventional and thrombogenic risk markers of CVDs, and also with overall CVD risk, highlighting a potentially important role for EVs in CVDs.

Gamma-Linolenic and Pinolenic Acids Exert Anti-Inflammatory Effects in Cultured Human Endothelial Cells Through Their Elongation Products.

SCOPE: Omega-3 fatty acids (FAs) from oily fish reduce cardiovascular disease. This may be partly due to modulation of endothelial cell (EC) inflammation. Fish stocks are declining and there is a need for sustainable alternative FAs. Gamma-linolenic acid (GLA) and pinolenic acid (PLA) are plant-derived FAs, which can fulfil this role. METHODS AND RESULTS: EA.hy926 cells are exposed GLA and PLA prior to stimulation with tumor necrosis factor (TNF)-α. GLA and PLA are incorporated into ECs, resulting in increases in long-chain derivatives produced by elongase 5, dihomo-gamma-linolenic acid (DGLA), and eicosatrienoic acid (ETA). Both GLA and PLA (50 µm) decrease production of soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein 1 (MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES). However, decreases in these mediators are not seen after pre-treatment with GLA or PLA in elongase 5 silenced EA.hy926 cells. DGLA and ETA (10 µm) decrease EC production of sICAM-1, MCP-1, RANTES, and IL-6. All FAs reduce adhesion of THP-1 monocytes to EA.hy926 cells. Both PLA (50 µm) and ETA (10 µm) decrease NFκBp65 phosphorylation. CONCLUSION: These effects suggest potential for GLA, PLA and their long-chain derivatives, DGLA and ETA, as sustainable anti-inflammatory alternatives to fish-derived FAs.

Comparative anti-inflammatory effects of plant- and marine-derived omega-3 fatty acids explored in an endothelial cell line.

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) lower risk of cardiovascular disease. The primary source of EPA and DHA is fatty fish. Plant-derived alpha linolenic acid (ALA) and stearidonic acid (SDA) could provide sustainable land-based alternatives, but their functionality is underexplored. Omega-3 fatty acids (n-3 FAs) may influence atherogenic processes through changing endothelial cell (EC) function and lowering inflammation. This study compared effects of marine- and plant-derived n-3 FAs on EC inflammatory responses. EA.hy926 cells were exposed to ALA, SDA, EPA or DHA prior to stimulation with tumor necrosis factor (TNF)-α. All FAs were shown to be incorporated into ECs in a dose-dependent manner. SDA (50 µM) decreased both production and cell-surface expression of intercellular adhesion molecule (ICAM)-1; however EPA and DHA resulted in greater reduction of ICAM-1 production and expression. EPA and DHA also significantly lowered production of monocyte chemoattractant protein 1, interleukin (IL)-6 and IL-8. ALA, SDA and DHA (50 µM) all reduced adhesion of THP-1 monocytes to EA.hy926 cells. DHA significantly decreased nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)p105 gene expression and phosphorylated NFκBp65 protein. Both EPA and DHA (50 µM) significantly decreased cyclooxygenase (COX)-2 protein. Thus, both marine-derived n-3 FAs, particularly DHA, had potent anti-inflammatory effects in this EC model. Of the plant-derived n-3 FAs, SDA showed the greatest inhibition of inflammation. Although neither ALA nor SDA reproduced the anti-inflammatory effects of EPA and DHA in this model, there is some potential for SDA to be a sustainable anti-inflammatory alternative to the marine n-3 FAs.

Impact of a (poly)phenol-rich extract from the brown algae Ascophyllum nodosum on DNA damage and antioxidant activity in an overweight or obese population: a randomized controlled trial.

Background: Epidemiologic evidence suggests that a diet rich in (poly)phenols has beneficial effects on many chronic diseases. Brown seaweed is a rich source of (poly)phenols. Objective: The aim of this study was to investigate the bioavailability and effect of a brown seaweed (Ascophyllum nodosum) (poly)phenol extract on DNA damage, oxidative stress, and inflammation in vivo. Design: A randomized, double-blind, placebo-controlled crossover trial was conducted in 80 participants aged 30-65 y with a body mass index (in kg/m2) ≥25. The participants consumed either a 400-mg capsule containing 100 mg seaweed (poly)phenol and 300 mg maltodextrin or a 400-mg maltodextrin placebo control capsule daily for an 8-wk period. Bioactivity was assessed with a panel of blood-based markers including lymphocyte DNA damage, plasma oxidant capacity, C-reactive protein (CRP), and inflammatory cytokines. To explore the bioavailability of seaweed phenolics, an untargeted metabolomics analysis of urine and plasma samples after seaweed consumption was determined by ultra-high-performance liquid chromatography-high-resolution mass spectrometry. Results: Consumption of the seaweed (poly)phenols resulted in a modest decrease in DNA damage but only in a subset of the total population who were obese. There were no significant changes in CRP, antioxidant status, or inflammatory cytokines. We identified phlorotannin metabolites that are considered potential biomarkers of seaweed consumption including pyrogallol/phloroglucinol-sulfate, hydroxytrifurahol A-glucuronide, dioxinodehydroeckol-glucuronide, diphlorethol sulfates, C-O-C dimer of phloroglucinol sulfate, and C-O-C dimer of phloroglucinol. Conclusions: To the best of our knowledge, this work represents the first comprehensive study investigating the bioactivity and bioavailability of seaweed (poly)phenolics in human participants. We identified several potential biomarkers of seaweed consumption. Intriguingly, the modest improvements in DNA damage were observed only in the obese subset of the total population. The subgroup analysis should be considered exploratory because it was not preplanned; therefore, it was not powered adequately. Elucidation of the biology underpinning this observation will require participant stratification according to weight in future studies. This trial was registered at clinicaltrials.gov as NCT02295878.

Omega-3 fatty acids and leukocyte-endothelium adhesion: Novel anti-atherosclerotic actions.

Endothelial cells (ECs) play a role in the optimal function of blood vessels. When endothelial function becomes dysregulated, the risk of developing atherosclerosis increases. Specifically, upregulation of adhesion molecule expression on ECs promotes the movement of leukocytes, particularly monocytes, into the vessel wall. Here, monocytes differentiate into macrophages and may become foam cells, contributing to the initiation and progression of an atherosclerotic plaque. The ability of omega-3 (n-3) polyunsaturated fatty acids (PUFAs) to influence the expression of adhesion molecules by ECs and to modulate leukocyte-endothelial adhesion has been studied in cell culture using various types of ECs, in animal feeding studies and in human trials; the latter have tended to evaluate soluble forms of adhesion molecules that circulate in the bloodstream. These studies indicate that n-3 PUFAs (both eicosapentaenoic acid and docosahexaenoic acid) can decrease the expression of key adhesion molecules, such as vascular cell adhesion molecule 1, by ECs and that this results in decreased adhesive interactions between leukocytes and ECs. These findings suggest that n-3 PUFAs may lower leukocyte infiltration into the vascular wall, which could contribute to reduced atherosclerosis and lowered risk of cardiovascular disease.

Replacement of dietary saturated fat with unsaturated fats increases numbers of circulating endothelial progenitor cells and decreases numbers of microparticles: findings from the randomized, controlled Dietary Intervention and VAScular function (DIVAS) study.

Background: Endothelial progenitor cells (EPCs) and microparticles are emerging as novel markers of cardiovascular disease (CVD) risk, which could potentially be modified by dietary fat. We have previously shown that replacing dietary saturated fatty acids (SFAs) with monounsaturated or n-6 (ω-6) polyunsaturated fatty acids (MUFAs or PUFAs, respectively) improved lipid biomarkers, blood pressure, and markers of endothelial activation, but their effects on circulating EPCs and microparticles are unclear. Objective: The Dietary Intervention and VAScular function (DIVAS) Study investigated the replacement of 9.5-9.6% of total energy (%TE) contributed by SFAs with MUFAs or n-6 PUFAs for 16 wk on EPC and microparticle numbers in United Kingdom adults with moderate CVD risk. Design: In this randomized, controlled, single-blind, parallel-group dietary intervention, men and women aged 21-60 y (n = 190) with moderate CVD risk (≥50% above the population mean) consumed 1 of three 16-wk isoenergetic diets. Target compositions for total fat, SFAs, MUFAs, and n-6 PUFAs (%TE) were as follows: SFA-rich diet (36:17:11:4; n = 64), MUFA-rich diet (36:9:19:4; n = 62), and n-6 PUFA-rich diet (36:9:13:10; n = 66). Circulating EPC, endothelial microparticle (EMP), and platelet microparticle (PMP) numbers were analyzed by flow cytometry. Dietary intake, vascular function, and other cardiometabolic risk factors were determined at baseline. Results: Relative to the SFA-rich diet, MUFA- and n-6 PUFA-rich diets decreased EMP (-47.3%, -44.9%) respectively and PMP (-36.8%, -39.1%) numbers (overall diet effects, P < 0.01). The MUFA-rich diet increased EPC numbers (+28.4%; P = 0.023). Additional analyses that used stepwise regression models identified the augmentation index (measuring arterial stiffness determined by pulse-wave analysis) as an independent predictor of baseline EPC and microparticle numbers. Conclusions: Replacement of 9.5-9.6%TE dietary SFAs with MUFAs increased EPC numbers, and replacement with either MUFAs or n-6 PUFAs decreased microparticle numbers, suggesting beneficial effects on endothelial repair and maintenance. Further studies are warranted to determine the mechanisms underlying the favorable effects on EPC and microparticle numbers after SFA replacement. This trial was registered at www.clinicaltrials.gov as NCT01478958.

Age-Related Changes in the Natural Killer Cell Response to Seasonal Influenza Vaccination Are Not Influenced by a Synbiotic: a Randomised Controlled Trial.

Natural killer (NK) cells are an important component of the immune response to influenza infection, but are subject to alteration during aging, which may play a role in impaired response to infection and vaccination in older people. Enhancement of NK cell activity could, therefore, present a means to improve the immune response to vaccination in older subjects, and pre- and probiotics offer an opportunity to modulate antiviral defenses via alteration of the gut microbiota. This study investigated the effect of a novel probiotic, Bifidobacterium longum bv. infantis CCUG 52486, combined with a prebiotic, gluco-oligosaccharide (B. longum + Gl-OS), on the NK cell response to seasonal influenza vaccination in young and older subjects in a double-blind, randomized controlled trial. There were significant effects of aging on NK cell phenotype, the most notable of which were an increase in CD56dim cells, mainly reflected in the CD16+ subset, a decrease in CD56bright cells, mainly reflected in the CD16- subset, and greater expression of the immunosenescence marker, CD57, on NK cell subsets. However, these changes only partially translated to differences in NK cell activity, observed as trends toward reduced NK cell activity in older subjects when analyzed on a per cell basis. Influenza vaccination increased the proportion of CD56bright cells and decreased the proportion of CD56dim cells, in young, but not older subjects. Although NK cell activity in response to vaccination was not significantly different between the young and older subjects, low post-vaccination NK cell activity was associated with poor seroconversion in only the older subjects. There was no influence of the synbiotic on NK cell phenotype or activity, either before or after influenza vaccination. In conclusion, aging is associated with marked alteration of the phenotype of the NK cell population and there was evidence of an impaired NK cell response to influenza vaccination in older subjects. The effects of aging on NK cell phenotype and activity could not be offset by B. longum + Gl-OS. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT01066377.

Effects of pelargonidin-3-O-glucoside and its metabolites on lipopolysaccharide-stimulated cytokine production by THP-1 monocytes and macrophages.

Epidemiological evidence suggests cardioprotective effects of anthocyanin consumption. This study examined the predominant strawberry anthocyanin, pelargonidin-3-O-glucoside (Pg-3-glc), and three of its plasma metabolites (protocatechuic acid [PCA], 4-hydroxybenzoic acid, and phloroglucinaldehyde [PGA]) for effects on the production of selected cytokines by lipopolysaccharide-stimulated THP-1 monocytes and macrophages. Concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, IL-8 and IL-10 were determined using a cytometric bead array kit. PCA at 0.31, 1.25 and 20 µM and PGA at 5 and 20 µM decreased the concentration of IL-6 in the monocyte cultures, but there were no effects on TNF-α, IL-1ß, IL-8 and IL-10 and there were no effects of the other compounds. In the macrophage cultures, PGA at 20 µM decreased the concentrations of IL-6 and IL-10, but there was no effect on TNF-α, IL-1ß and IL-8 and there were no effects of the other compounds. In conclusion, while the effects of PGA were only observed at the higher, supraphysiological concentration and are thus considered of limited physiological relevance overall, the anti-inflammatory properties of PCA were observed at both the lower, physiologically relevant, and the higher concentrations; however, effects were modest and limited to IL-6 and monocytes. These preliminary data suggest potential for physiologically attainable PCA concentrations to modulate IL-6 production by monocytes.

Impact of ageing and a synbiotic on the immune response to seasonal influenza vaccination; a randomised controlled trial.

BACKGROUND & AIMS: Ageing increases risk of respiratory infections and impairs the response to influenza vaccination. Pre- and pro-biotics offer an opportunity to modulate anti-viral defenses and the response to vaccination via alteration of the gut microbiota. This study investigated the effect of a novel probiotic, Bifidobacterium longum bv. infantis CCUG 52486, combined with a prebiotic, gluco-oligosaccharide, on the B and T cell response to seasonal influenza vaccination in young and older subjects . METHODS: In a double-blind, randomized controlled trial, 58 young (18-35 y) and 54 older (60-85 y) subjects were supplemented with the synbiotic for 8 weeks. At 4 weeks they were administered with a seasonal influenza vaccine. B and T cell phenotype and responsiveness to in vitro re-stimulation with the vaccine were assessed at baseline, 4, 6 and 8 weeks. RESULTS: B and T cell profiles differed markedly between young and older subjects. Vaccination increased numbers of memory, IgA+ memory, IgG+ memory and total IgG+ B cells in young subjects, but failed to do so in older subjects and did not significantly alter T cell subsets. Seroconversion to the H1N1 subunit in the older subjects was associated with higher post-vaccination numbers of plasma B cells, but seroconversion was less consistently associated with T cell phenotype. B and T cell subsets from both young and older subjects demonstrated a strong antigen-specific recall challenge, and although not influenced by age, responsiveness to the recall challenge was associated with seroconversion. In older subjects, CMV seropositivity was associated with a significantly lower recall response to the vaccine, but the synbiotic did not affect the responsiveness of B or T cells to re-stimulation with influenza vaccine. CONCLUSIONS: Antigen-specific B and T cell activation following an in vitro recall challenge with the influenza vaccine was influenced by CMV seropositivity, but not by a synbiotic. Registered under ClinicalTrials.gov Identifier no. NCT01066377.

Pelargonidin-3-O-glucoside and its metabolites have modest anti-inflammatory effects in human whole blood cultures.

This study hypothesized that the predominant strawberry anthocyanin, pelargonidin-3-O-glucoside (Pg-3-glc), and 3 of its plasma metabolites (4-hydroxybenzoic acid, protocatechuic acid, and phloroglucinaldehyde [PGA]) would affect phagocytosis, oxidative burst, and the production of selected pro- and anti-inflammatory cytokines in a whole blood culture model. For the assessment of phagocytosis and oxidative burst activity of monocytes and neutrophils, whole blood was preincubated in the presence or absence of the test compounds at concentrations up to 5 µmol/L, followed by analysis of phagocytic and oxidative burst activity using commercially available test kits. For the cytokine analysis, diluted whole blood was stimulated with lipopolysaccharide in the presence or absence of the test compounds at concentrations up to 5 µmol/L. Concentrations of selected cytokines (tumor necrosis factor-α, interleukin [IL]-1ß, IL-6, IL-8, and IL-10) were determined using a cytometric bead array kit. There were no effects of any of the test compounds on phagocytosis of opsonized or nonopsonized Escherichia coli or on oxidative burst activity. Pg-3-glc and PGA at 0.08 µmol/L increased the concentration of IL-10 (P<.01 and P<.001, respectively), but there was no effect on tumor necrosis factor-α, IL-1ß, IL-6, and IL-8, and there were no effects of the other compounds. In conclusion, this study demonstrated a lack of effect of these compounds on the opsonization, engulfment, and subsequent destruction of bacteria. Pg-3-glc and PGA, at physiologically relevant concentrations, had anti-inflammatory properties; however, effects were modest, only observed at the lowest dose tested and limited to IL-10.

Effect of simulated gastrointestinal digestion and fermentation on polyphenolic content and bioactivity of brown seaweed phlorotannin-rich extracts.

SCOPE: Unlike other classes of polyphenols, there is a lack of knowledge regarding brown seaweed phlorotannins and their bioactivity. We investigated the impact of in vitro gastrointestinal digestion and colonic fermentation on the bioactivity of a seaweed phlorotannin extract from Ascophyllum nodosum and its high molecular weight (HMW) and low molecular weight (LMW) fractions. METHODS AND RESULTS: The highest phlorotannin and total polyphenol (TP) concentration was observed in the HMW fraction. Antioxidant capacity broadly followed phlorotannin and TP levels, with HMW having the highest activity. Both gastrointestinal digestion (GID) and colonic fermentation (CF) significantly affected phlorotannin and TP levels, and antioxidant capacity of the extract and fractions. Despite this, in HT-29 cells, all GID extracts significantly inhibit cell growth, whereas CF extracts effectively counteracted H2 O2 induced DNA damage. CONCLUSION: Although phlorotannins, TP levels and antioxidant power of the extracts were strongly reduced after in vitro digestion and fermentation, their anti-genotoxic activity and cell growth inhibitory effect in colon HT-29 cells was maintained and enhanced. HMW was the most effective fraction, indicating that the high molecular weight phlorotannins potentially exert a stronger beneficial effect in the colon.

Farnesoid X Receptor and Liver X Receptor Ligands Initiate Formation of Coated Platelets.

OBJECTIVES: The liver X receptors (LXRs) and farnesoid X receptor (FXR) have been identified in human platelets. Ligands of these receptors have been shown to have nongenomic inhibitory effects on platelet activation by platelet agonists. This, however, seems contradictory with the platelet hyper-reactivity that is associated with several pathological conditions that are associated with increased circulating levels of molecules that are LXR and FXR ligands, such as hyperlipidemia, type 2 diabetes mellitus, and obesity. APPROACH AND RESULTS: We, therefore, investigated whether ligands for the LXR and FXR receptors were capable of priming platelets to the activated state without stimulation by platelet agonists. Treatment of platelets with ligands for LXR and FXR converted platelets to the procoagulant state, with increases in phosphatidylserine exposure, platelet swelling, reduced membrane integrity, depolarization of the mitochondrial membrane, and microparticle release observed. Additionally, platelets also displayed features associated with coated platelets such as P-selectin exposure, fibrinogen binding, fibrin generation that is supported by increased serine protease activity, and inhibition of integrin αIIbß3. LXR and FXR ligand-induced formation of coated platelets was found to be dependent on both reactive oxygen species and intracellular calcium mobilization, and for FXR ligands, this process was found to be dependent on cyclophilin D. CONCLUSIONS: We conclude that treatment with LXR and FXR ligands initiates coated platelet formation, which is thought to support coagulation but results in desensitization to platelet stimuli through inhibition of αIIbß3 consistent with their ability to inhibit platelet function and stable thrombus formation in vivo.

Ageing alters the impact of nutrition on immune function.

Immunosenescence during ageing is a major challenge which weakens the ability of older individuals to respond to infection or vaccination. There has been much interest in dietary strategies to improve immunity in older people, but there is an assumption that modulation of the immune response in older people will be based on the same principles as for younger adults. Recent evidence suggests that ageing fundamentally alters the impact of nutrition on immune function. As a result, interpretation of data from studies investigating the impact of diet on immune function is highly dependent on subject age. Study design is critically important when investigating the efficacy of dietary components, and most studies involving older people include rigorous inclusion/exclusion criteria based on medical history, laboratory tests, general health status and often nutritional status. However, immunological status is rarely accounted for, but can vary significantly, even amongst healthy older people. There are several clear examples of age-related changes in immune cell composition, phenotype and/or function, which can directly alter the outcome of an intervention. This review uses two case studies to illustrate how the effects of n-3 PUFA and probiotics differ markedly in young v. older subjects. Evidence from both suggests that baseline differences in immunosenescence influence the outcome of an intervention, highlighting the need for detailed immunological characterisation of subjects prior to interventions. Finally, future work elucidating alterations in metabolic regulation within cells of the immune system as a result of ageing may be important in understanding the impact of diet on immune function in older people.

Impact of phenolic-rich olive leaf extract on blood pressure, plasma lipids and inflammatory markers: a randomised controlled trial.

PURPOSE: Dietary polyphenols have been demonstrated to favourably modify a number of cardiovascular risk markers such as blood pressure (BP), endothelial function and plasma lipids. We conducted a randomised, double-blind, controlled, crossover trial to investigate the effects of a phenolic-rich olive leaf extract (OLE) on BP and a number of associated vascular and metabolic measures. METHODS: A total of 60 pre-hypertensive [systolic blood pressure (SBP): 121-140 mmHg; diastolic blood pressure (DBP): 81-90 mmHg] males [mean age 45 (±SD 12.7 years, BMI 26.7 (±3.21) kg/m2] consumed either OLE (136 mg oleuropein; 6 mg hydroxytyrosol) or a polyphenol-free control daily for 6 weeks before switching to the alternate arm after a 4-week washout. RESULTS: Daytime [-3.95 (±SD 11.48) mmHg, p = 0.027] and 24-h SBP [-3.33 (±SD 10.81) mmHg, p = 0.045] and daytime and 24-h DBP [-3.00 (±SD 8.54) mmHg, p = 0.025; -2.42 (±SD 7.61) mmHg, p = 0.039] were all significantly lower following OLE intake, relative to the control. Reductions in plasma total cholesterol [-0.32 (±SD 0.70) mmol/L, p = 0.002], LDL cholesterol [-0.19 (±SD 0.56) mmol/L, p = 0.017] and triglycerides [-0.18 (±SD 0.48), p = 0.008] were also induced by OLE compared to control, whilst a reduction in interleukin-8 [-0.63 (±SD 1.13) pg/ml; p = 0.026] was also detected. Other markers of inflammation, vascular function and glucose metabolism were not affected. CONCLUSION: Our data support previous research, suggesting that OLE intake engenders hypotensive and lipid-lowering effects in vivo.

Metabolism and functional effects of plant-derived omega-3 fatty acids in humans.

Alpha-linolenic acid (ALA) is an essential fatty acid and the substrate for the synthesis of longer-chain, more unsaturated ω-3 fatty acids, eicosapentaenoic acid (EPA), docosapentaenoic acid and docosahexaenoic acid (DHA). EPA and DHA are associated with human health benefits. The primary source of EPA and DHA is seafood. There is a need for sustainable sources of biologically active ω-3 fatty acids. Certain plants contain high concentrations of ALA and stearidonic acid (SDA). Here we review the literature on the metabolism of ALA and SDA in humans, the impact of increased ALA and SDA consumption on concentrations of EPA and DHA in blood and cell lipid pools, and the extent to which ALA and SDA might have health benefits. Although it is generally considered that humans have limited capacity for conversion of ALA to EPA and DHA, sex differences in conversion to DHA have been identified. If conversion of ALA to EPA and DHA is limited, then ALA may have a smaller health benefit than EPA and DHA. SDA is more readily converted to EPA and appears to offer better potential for health improvement than ALA. However, conversion of both ALA and SDA to DHA is limited in most humans.

Effect of a synbiotic on the response to seasonal influenza vaccination is strongly influenced by degree of immunosenescence.

BACKGROUND: Ageing increases risk of respiratory infections and impairs the response to influenza vaccination. Pre- and probiotics offer an opportunity to modulate anti-viral defenses and the response to vaccination via alteration of the gut microbiota. This study investigated the effect of a novel probiotic, Bifidobacterium longum bv. infantis CCUG 52,486, combined with a prebiotic, gluco-oligosaccharide (B. longum + Gl-OS), on the response to seasonal influenza vaccination in young and older subjects in a double-blind, randomized controlled trial, taking into account the influence of immunosenescence markers at baseline. RESULTS: Vaccination resulted in a significant increase in total antibody titres, vaccine-specific IgA, IgM and IgG and seroprotection to all three subunits of the vaccine in both young and older subjects, and in general, the increases in young subjects were greater. There was little effect of the synbiotic, although it tended to reduce seroconversion to the Brisbane subunit of the vaccine and the vaccine-specific IgG response in older subjects. Immunological characterization revealed that older subjects randomized to the synbiotic had a significantly higher number of senescent (CD28(-)CD57(+)) helper T cells at baseline compared with those randomized to the placebo, and they also had significantly higher plasma levels of anti-CMV IgG and a greater tendency for CMV seropositivity. Moreover, higher numbers of CD28(-)CD57(+) helper T cells were associated with failure to seroconvert to Brisbane, strongly suggesting that the subjects randomized to the synbiotic were already at a significant disadvantage in terms of likely ability to respond to the vaccine compared with those randomized to the placebo. CONCLUSIONS: Ageing was associated with marked impairment of the antibody response to influenza vaccination in older subjects and the synbiotic failed to reverse this impairment. However, the older subjects randomized to the synbiotic were at a significant disadvantage due to a greater degree of immunosenscence at baseline compared with those randomized to the placebo. Thus, baseline differences in immunosenescence between the randomized groups are likely to have influenced the outcome of the intervention, highlighting the need for detailed immunological characterization of subjects prior to interventions. TRIAL REGISTRATION: Clinicaltrials.gov NCT01066377.

Gastrointestinal modifications and bioavailability of brown seaweed phlorotannins and effects on inflammatory markers.

Brown seaweeds such as Ascophyllum nodosum are a rich source of phlorotannins (oligomers and polymers of phloroglucinol units), a class of polyphenols that are unique to Phaeophyceae. At present, there is no information on the bioavailability of seaweed polyphenols and limited evidence on their bioactivity in vivo. Consequently, we investigated the gastrointestinal modifications in vitro of seaweed phlorotannins from A. nodosum and their bioavailability and effect on inflammatory markers in healthy participants. In vitro, some phlorotannin oligomers were identified after digestion and colonic fermentation. In addition, seven metabolites corresponding to in vitro-absorbed metabolites were identified. Urine and plasma samples contained a variety of metabolites attributed to both unconjugated and conjugated metabolites (glucuronides and/or sulphates). In both urine and plasma, the majority of the metabolites were found in samples collected at late time points (6-24 h), suggesting colonic metabolism of high-molecular-weight phlorotannins, with three phlorotannin oligomers (hydroxytrifuhalol A, 7-hydroxyeckol, C-O-C dimer of phloroglucinol) identified in urine samples. A significant increase of the cytokine IL-8 was also observed. Our study shows for the first time that seaweed phlorotannins are metabolised and absorbed, predominantly in the large intestine, and there is a large inter-individual variation in their metabolic profile. Three phlorotannin oligomers present in the capsule are excreted in urine. Our study is the first investigation of the metabolism and bioavailability of seaweed phlorotannins and the role of colonic biotransformation. In addition, IL-8 is a possible target for phlorotannin bioactivity.