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Adaptive immunity relies on the efficient recruitment of T cells from the blood into peripheral tissues. However, the current understanding of factor(s) coordinating these events is incomplete. Previous studies on galectin-9 (Gal-9), have proposed a functionally significant role for this lectin in mediating leukocyte adhesion and transmigration. However, very little is known about its function in T cell migration. Here, we have investigated the role of the Gal-9 on the migration behaviour of both human primary CD4+ and CD8+ T cells. Our data indicate that Gal-9 supports both CD4+ and CD8+ T cell adhesion and transmigration in a glycan dependent manner, inducing L-selectin shedding and upregulation of LFA-1 and CXCR4 expression. Additionally, when immobilized, Gal-9 promoted capture and firm adhesion of T cells under flow, in a glycan and integrin-dependent manner. Using an in vivo model, dorsal air pouch, we found that Gal-9 deficient mice display impaired leukocyte trafficking, with a reduction in pro-inflammatory cytokines/chemokines generated locally. Furthermore, we also demonstrate that Gal-9 inhibits the chemotactic function of CXCL12 through direct binding. In conclusion, our study characterises, for the first time, the capture, adhesion, and migration behaviour of CD4+ and CD8+ T cells to immobilised /endothelial presented Gal-9, under static and physiological flow conditions. We also demonstrate the differential binding characteristics of Gal-9 to T cell subtypes, which could be of potential therapeutic significance, particularly in the treatment of inflammatory-based diseases, given Gal-9 ability to promote apoptosis in pathogenic T cell subsets.

Background Large-scale screening for atrial fibrillation (AF) requires reliable methods to identify at-risk populations. Using an experimental semi-quantitative biomarker assay, B-type natriuretic peptide (BNP) and fibroblast growth factor 23 (FGF23) were recently identified as the most suitable biomarkers for detecting AF in combination with simple morphometric parameters (age, sex, and body mass index [BMI]). In this study, we validated the AF model using standardised, high-throughput, high-sensitivity biomarker assays. Methods and findings For this study, 1,625 consecutive patients with either (1) diagnosed AF or (2) sinus rhythm with CHA2DS2-VASc score of 2 or more were recruited from a large teaching hospital in Birmingham, West Midlands, UK, between September 2014 and February 2018. Seven-day ambulatory ECG monitoring excluded silent AF. Patients with tachyarrhythmias apart from AF and incomplete cases were excluded. AF was diagnosed according to current clinical guidelines and confirmed by ECG. We developed a high-throughput, high-sensitivity assay for FGF23, quantified plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) and FGF23, and compared results to the previously used multibiomarker research assay. Data were fitted to the previously derived model, adjusting for differences in measurement platforms and known confounders (heart failure and chronic kidney disease). In 1,084 patients (46% with AF; median [Q1, Q3] age 70 [60, 78] years, median [Q1, Q3] BMI 28.8 [25.1, 32.8] kg/m2, 59% males), patients with AF had higher concentrations of NT-proBNP (median [Q1, Q3] per 100 pg/ml: with AF 12.00 [4.19, 30.15], without AF 4.25 [1.17, 15.70]; p < 0.001) and FGF23 (median [Q1, Q3] per 100 pg/ml: with AF 1.93 [1.30, 4.16], without AF 1.55 [1.04, 2.62]; p < 0.001). Univariate associations remained after adjusting for heart failure and estimated glomerular filtration rate, known confounders of NT-proBNP and FGF23. The fitted model yielded a C-statistic of 0.688 (95% CI 0.656, 0.719), almost identical to that of the derived model (C-statistic 0.691; 95% CI 0.638, 0.744). The key limitation is that this validation was performed in a cohort that is very similar demographically to the one used in model development, calling for further external validation. Conclusions Age, sex, and BMI combined with elevated NT-proBNP and elevated FGF23, quantified on a high-throughput platform, reliably identify patients with AF. Trial registration Registry IRAS ID 97753 Health Research Authority (HRA), United Kingdom, Winnie Chua and colleagues identify and validate biomarkers for atrial fibrillation, Author summary Why was this study done? Atrial fibrillation (AF) is the most commonly sustained arrhythmia in the world, markedly increasing the risk of stroke, heart failure, and cardiovascular death. AF episodes can be transient and go undetected by ECG for a long time, leading to untreated AF and avoidable complications such as stroke. Blood biomarkers are an easy and affordable way to enable screening for undiagnosed, silent AF. An earlier study using a dual-antibody-based research assay identified a simple model, consisting of age, sex, body mass index, and 2 biomarkers, FGF23 and NT-proBNP, that identified patients with AF. What did the researchers do and find? We measured 2 biomarkers, NT-proBNP and FGF23, using high-throughput, high-sensitivity assays that are ready for clinical use. The FGF23 assay was developed for this study. We incorporated these biomarkers into a model including age, sex, and body mass index, which was derived from previous work where research assays were used to identify biomarkers to detect patients with AF. We demonstrated that the model using the standardised assays performed consistently to identify patients with AF. What do these findings mean? Elevated concentrations of FGF23 and NT-proBNP, in addition to older age, female sex, and high body mass index, identify patients who are likely to have AF. In the future, point-of-care testing can be easily performed using these biomarkers in combination with 3 simple pieces of information (age, sex, and body mass index) to target intensive screening, for example, using ECG monitoring.

Background: Blood biomarkers related to AF could be useful to detect silent AF and to develop stratified strategies for AF prevention. Previous studies identified markers that predict incident AF. However, it is difficult to differentiate whether biomarkers relate to underlying cardiovascular diseases, are generated by the atria in response to an AF episode, or both. We therefore measured a panel of blood biomarkers in patients without overt CVD with and without AF to investigate the association between biomarkers and atrial fibrillation (AF) in patients without overt cardiovascular disease (CVD).Methods: Blood samples - drawn remote from an AF episode - of 60 patients with AF but without overt forms of CVD (idiopathic AF; iAF) were compared to 120 matched patients with sinus rhythm only. A novel antibody-based method for quantification of blood biomarkers (OlinkProseek Multiplex Cardiovascular) was used to compare 92 biomarkers between the two groups.Results: N-terminal pro-B-type natriuretic peptide (NT-proBNP), Cathepsin L1, Endothelial cell-specific molecule 1, Cancer Antigen-125 (CA-125), Heat shock 27kDa protein, Galanin peptides, Proteinase-activated receptor 1, Stem cell factor, and CD40-ligand were all higher in iAF patients than in SR controls. Both NT-proBNP (OR1.55(1.07-2.25);p=0.022) and CA-125 (OR1.68(1.07-2.64);p=0.026) were independently associated with iAF.Conclusions: This exploratory study, investigating over 90 cardiovascular blood biomarkers in patients without known CVD, identified one established biomarker for paroxysmal AF, NT-proBNP, and a novel marker, CA-125. CA-125 - previously unrelated to paroxysmal AF in an otherwise healthy population - may thus be a potential indicator of remote paroxysms of AF.

The (n-3) PUFA, DHA, is widely thought to posses the ability to modulate the inflammatory response. However, its modes of interaction with inflammatory cells are poorly understood. In particular, there are limited data on the interactions of DHA with vascular endothelium, the cells that regulate the traffic of leukocytes from the blood into inflamed tissue. Using human umbilical vein endothelial cells (EC) cultured in a flow-based adhesion assay and activated with TNFα, we tested whether supplementing human umbilical vein EC with physiologically achievable concentrations of DHA would inhibit the recruitment of flowing neutrophils. DHA caused a dose-dependent reduction in neutrophil recruitment to the EC surface, although cells that became adherent were activated and could migrate across the human umbilical vein EC monolayer normally. Using EPA as an alternative supplement had no effect on the levels of neutrophil adhesion in this assay. Analysis of adhesion receptor expression by qPCR demonstrated that DHA did not alter the transcriptional activity of human umbilical vein EC. However, DHA did significantly reduce E-selectin expression at the human umbilical vein EC surface without altering the total cellular pool of this adhesion receptor. Thus, we have identified a novel mechanism by which DHA alters the trafficking of leukocytes during inflammation and demonstrate that this involves disruption of intracellular transport mechanisms used to present adhesion molecules on the surface of cytokine-stimulated EC.

We adapted a monocyte:endothelial cell co-culture model to investigate the pro-inflammatory potential of monocytes from patients with peripheral arterial disease (PAD). Isolated monocytes were cultured with human umbilical vein endothelial cells (HUVEC) for 24h, after which the ability of the HUVEC to recruit flowing neutrophils was tested. Development of a usable protocol required comparisons of primary HUVEC with cells that had been passaged and/or frozen and thawed, evaluation of optimal culture media and comparison of monocytes from freshly drawn and stored blood. We found, for instance, that expansion of HUVEC was assisted by inclusion of hydrocortisone, but this agent was withdrawn before the test phase because it reduced responses of HUVEC. Using the optimal practical protocol, we found great variation in the ability of monocytes from different donors to cause neutrophil adhesion. Slightly more ( approximately 20%) monocytes from patients with PAD adhered to HUVEC than monocytes from healthy controls, and the monocytes from PAD patients induced approximately 70% greater subsequent adhesion of neutrophils. Thus, we developed a functional model of inflammatory potential usable in clinically-related studies and found that patients with PAD had circulating monocytes with greater than normal ability to activate endothelial cells.

Recent epidemiological studies have provided evidence supporting the potential benefits of antioxidants in coronary prevention. We have investigated the effects of vitamin E on platelets, monocytes and endothelial cells in vitro. Pre-incubation of platelets with vitamin E inhibited subsequent thrombin- (P 0.05, n = 8); nor was the adhesion of unstimulated platelets significantly affected (P > 0.05, n = 8). Pre-incubation of monocytes with vitamin E inhibited their subsequent adhesion to plastic (P < 0.05, n = 9), and was also associated with an 18% reduction in adhesion to EA.hy 926 endothelial cells (n = 8), although this failed to reach statistical significance. Pre-incubation of the endothelial cells with vitamin E also significantly reduced subsequent mononuclear cell adhesion by 56% (P < 0.05, n = 3).

Mononuclear cells and platelets are intimately involved in the pathogenesis and complications of cardiovascular disease. Platelet activation has been reported in hypertension, though the activation-state of monocytes has received less attention. In this study the adhesiveness of monocytes and platelets was assessed and any relationship between the adhesive properties of these cellular elements and plasma levels of soluble adhesion molecules and blood pressure parameters determined. Fifty six elderly volunteers, of whom 32 were classified hypertensive (daytime SBPor = 135 mmHg), underwent 24 h blood pressure monitoring, assessment of monocyte and platelet adhesion and measurement of the plasma soluble adhesion molecules ICAM-1, L-selectin, E-selectin and vWF. In the elderly hypertensive subjects, monocyte adhesion to collagen coated (P0.05) and tissue culture plastic microwells (P0.05) was significantly elevated and circulating levels of soluble ICAM-1 (P0.01) and soluble E-selectin (P0.05) were significantly raised compared to their normotensive counterparts. A significant correlation was found to exist between monocyte adhesion to collagen and daytime pulse pressure (r = 0.39, P0.01) and also between plasma levels of soluble E-selectin and clinic DBP (r = 0.40, P0.001). The increased monocyte adhesion witnessed in hypertensive subjects and with increasing pulse pressure may contribute to the increased risk of cardiovascular disease in hypertension. Whether this increased adhesiveness is a property of the monocytes. or reflects endothelial cell activation, remains to be determined.

Introduction Atrial fibrillation (AF) affects 2% of the population and is associated with cardiovascular disease and increased stroke and mortality rates. The myocardium releases proteins (SCF, VEGF-D and BNPs). Detection of such markers in blood could be used to differentiate specific types of AF, or to guide screening for silent, undiagnosed AF. Aim To identify plasma proteins discerning between patients with and without AF. Methods We studied blood from consecutive patients undergoing CT coronary angiography at Maastricht Medical center. We only enrolled patients without a history of stroke, hypertension, diabetes or heart failure. Using unique DNA-coupled paired antibodies and qPCR, we simultaneously analysed 92 plasma proteins. CCL21 protein was measured by ELISA (n = 240). Stats: Mann-Whitney test, mean normalized protein expression (NPX) +/-SEM. Results 176 patients (paroxysmal AF=50, sinus rhythm (SR) controls=126) were analysed. Mean age was 54 years in both groups. N— terminal fragment proB-type natriuretic peptide (NT-proBNP) protein was higher in patients with than in those without AF (6.23 ± 0.62 vs 4.72 ± 0.33, p = 0.012, n = 176). We also performed an exploratory analysis only in patients without signs of coronary artery disease on CTCA (AF=27, SR=80). In this subgroup, NT-proBNP (6.478 ± 0.8442 vs 4.554 ± 0.4122, p = 0.023*), BNP (1.111 ± 0.083 vs 0.9713 ± 0.029, p = 0.0175*), Stem Cell Factor (SCF, 162.8 ± 7.860 vs 140.4 ± 5.127 p = 0.0097**) and VEGF-D (47.44 ± 2.708 vs 41.38 ± 1.694 p = 0.0389*) were higher in the 27 patients with AF. Conclusion While NT-proBNP is mostly known as a marker for heart failure, NT-proBNP appears as a potential blood marker for AF in patients without history of stroke, hypertension, diabetes or heart failure. Further validation of these initial, hypothesis-generating results seems warranted.

Background The recreational abuse of anabolic steroids is an emerging global health concern and may disrupt cardiac electrophysiology. Every fifth man joining a gym in the UK uses anabolic steroids for performance enhancement. Based on molecular signaling analyses and considering the volume-loading effects of testosterone, individuals with vulnerable desmosomal (cell-cell contact) proteins may be at increased risk of anabolic steroid induced cardiac electrical remodeling. We therefore studied the impact of chronic anabolic steroid exposure on left atrial (LA) electrophysiology in wildtype (WT) and plakoglobin (Plako; or gamma-catenin, a key desmosomal protein) deficient mice using dihydro-testosterone (DHT-a stable derivative of testosterone). Methods Young adult WT and Plako +/- male mice, bred on 129/sv background were fitted with subcutaneous osmotic mini-pumps containing either DHT (62.5mg/ml) in ethanol, or ethanol alone (Placebo), for 6 weeks. Following treatment, we examined transmembrane action potentials, and generated high spatial resolution activation maps in the Di-4-Anepps loaded LA using the Hamamatsu ORCA flash 4. Significance was taken as P Results DHT minipump exposure lead to supraphysiological DHT plasma levels and increased the ventricular mass:tibial length ratio of both genotypes (WT Placebo 79 ± 2 Plako +/- Placebo 78 ± 2 WT DHT 86 ± 3 Plako +/- DHT 92 ± 3 mg/cm, P +/- LA mass:tibial length ratio (WT Placebo 2.4 ± 0.1 Plako +/- Placebo 2.3 ± 0.1 WT DHT 2.6 ± 0.2 Plako +/- DHT 3.2 ± 0.2mg/cm, P +/- Placebo 0.14 ± 0.006 WT DHT 0.16 ± 0.004, Plako +/- DHT 0.24 ± 0.04 mm 2 , P DHT reduced action potential amplitude in Plako +/- LA, but not WT LA (WT Placebo 82 ± 2 Plako+/- Placebo 80 ± 2 WT DHT 80 ± 2 Plako+/- DHT 71 ± 2mV, P +/- DHT 82 ± 5 V/s, P +/- LA following DHT exposure (WT Placebo 40 ± 0.3 Plako +/- Placebo 40 ± 0.9 WT DHT 37 ± 1.3 Plako +/- DHT 36 ± 1.4 cm/s, P Conclusion Reduced cardiac plakoglobin expression increased susceptibility to steroid-induced left atrial hypertrophy, reduced action potential amplitude and lead to significant conduction slowing. These differences are likely to provide an enhanced substrate for atrial arrhythmogenesis. Our results suggest a potentially important interaction between reduced mechanical cell-cell contacts and anabolic steroid use.

Background. Neutrophil recruitment into glomerular tissues and reduced capillary wall integrity has been implicated in the development of vasculitic glomerulonephritis (VGN). This study investigated the stages and mechanisms through which neutrophil serine proteases (SPs), proteinase 3 (PR3) or elastase contribute to endothelial dysfunction. Methods. Protease-induced damage to endothelium and adhesion molecule upregulation was measured by viability assays and ELISA. Neutrophil/platelet adhesion to human glomerular and umbilical vein endothelium was assessed using in vitro adhesion assays. Results. PR3 and elastase (1 µg/mL, 2 h) significantly induced neutrophil adhesion to endothelial cells (EnC) whilst PR3 also enhanced platelet–EnC interactions. This neutrophil adhesion was associated with enhanced P-selectin expression and required CXCL8 receptor involvement, and could be inhibited by blocking the P-selectin ligand PSGL1. SPs induced damage in a time- and dose-dependent fashion, decreasing cell monolayer integrity followed by cell membrane integrity, inducing caspase-3 activation and p21 cleavage. However, SPs caused significant EnC damage with increasing concentrations and prolonged exposures. Conclusion. Neutrophil SPs induce a pro-adhesive phenotype in glomerular endothelium primarily by inducing neutrophil and platelet adhesion that transits to dysfunction after high/prolonged exposures. Dysregulated release of these enzymes within glomeruli may contribute to injury during diseases such as VGN.

Neutrophil proteases, proteinase-3 (PR3) and elastase play key roles in glomerular endothelial cell (GEC) injury during glomerulonephritis. Endothelial protease-activated receptors (PARs) are potential serine protease targets in glomerulonephritis. We investigated whether PAR1/2 are required for alterations in GEC phenotype that are mediated by PR3 or elastase during active glomerulonephritis. Endothelial PARs were assessed by flow cytometry. Thrombin, trypsin and agonist peptides for PAR1 and PAR2, TFLLR-NH(2) and SLIGKV-NH(2,) respectively, were used to assess alterations in PAR activation induced by PR3 or elastase. Endothelial von Willebrand Factor (vWF)release and calcium signaling were used as PAR activation markers. Both PR3 and elastase induced endothelial vWF release, with elastase inducing the highest response. PAR1 peptide induced GEC vWF release to the same extent as PR3. However, knockdown of PARs by small interfering RNA showed that neither PAR1 nor PAR2 activation caused PR3 or elastase-mediated vWF release. Both proteases interacted with and disarmed surface GEC PAR1, but there was no detectable interaction with cellular PAR2. Neither protease induced a calcium response in GEC. Therefore, PAR signaling and serine protease-induced alterations in endothelial function modulate glomerular inflammation via parallel but independent pathways.

Background and objectives Polymorphisms close to the PITX2 gene on chromosome 4q25 are associated with incident and recurrent atrial fibrillation (AF). Carriers of variant rs10033464 respond relatively well to antiarrhythmic drug therapy with sodium channel blockers. To test whether altered atrial PITX2 mRNA expression alters the electrophysiological effect of sodium channel blockers, we studied the effect of flecainide (Flec) in hearts of mice with heterozygous Pitx2c gene deletion, a model for reduced Pitx2 mRNA expression and AF susceptibility. Methods We assessed the effect of 1µM Flec on left atrial (LA) monophasic, transmembrane and optical action potentials in Pitx2c +/– mice and their wild type (WT) littermates. We measured conduction velocity (CV), action potential duration (APD), AP amplitude (APA), maximum upstroke velocity (dV/dt max ) and resting membrane potential (RMP) during atrial pacing at 80–120 ms fixed-rate cycle lengths (CL); and effective refractory periods (ERP) with arrhythmia inducibility during programmed (S2) stimulation. Post-repolarisation refractoriness (PRR) was defined as the difference between ERP and APD90. We assessed whether the electrophysiological changes associated with reduced Pitx2c mRNA expression alters the response to sodium current (I Na ) block using the Courtemanche et al . human atrial model. Taqman low density array analysis was also used to measure changes in 20 ion channel, and related, genes of interest. Results Flec abolished arrhythmias in Pitx2c +/– (6/18 base vs 0/15 Flec, p +/– atria compared with WT (Pitx2c +/– 14 ± 2 ms, n = 13 vs WT 7 ± 2 ms, n = 10; p +/– 21 ± 3 ms, n = 6 vs WT 6 ± 4 ms, n = 6, p +/– atria had a slightly more positive resting membrane potential (WT –70 ± 0.7 mV, n = 30 cells vs PITX2c +/– –68 ± 0.7 mV, n = 32 cells, p +/– 73 ± 1.3 mV, n = 32). Flec had no effect on the RMP but reduced CV, APA and dV/dt max in both genotypes to a similar extent. Hearts with PRR were not susceptible to arrhythmias (0/17). As with the isolated hearts, I Na block prolonged PRR in the Pitx2c deficiency simulation (500 ms CL: reference model 30ms; Pitx2c deficiency model 42 ms) and had no differential effects of I Na on RMP, CV, APD, or dV/dt max . The human atrial model thus qualitatively reproduced the experimental findings. mRNA expression of Kcnk5 and Kcna6 was altered in Pitx2c +/– left atria. Conclusion Flecainide suppresses atrial arrhythmias in mice with reduced Pitx2 mRNA expression. We propose that this genotype-specific antiarrhythmic effect can be explained by a more positive resting membrane potential and higher post-repolarisation refractoriness. These data suggest that flecainide may be effective for rhythm control in carriers of genetic variants on chromosome 4q25.

Although platelets do not ordinarily bind to endothelial cells (EC), pathological interactions between platelets and arterial EC may contribute to the propagation of atheroma. Previously, in an in vitro model of atherogenesis, where leukocyte adhesion to EC cocultured with smooth muscle cells was greatly enhanced, we also observed attachment of platelets to the EC layer. Developing this system to specifically model platelet adhesion, we show that EC cocultured with smooth muscle cells can bind platelets in a process that is dependent on EC activation by tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β1. Recapitulating the model using EC alone, we found that a combination of TGF-β1and TNF-α promoted high levels of platelet adhesion compared with either agent used in isolation. Platelet adhesion was inhibited by antibodies against GPIb-IX-V or αIIbβ3integrin, indicating that both receptors are required for stable adhesion. Platelet activation during interaction with the EC was also essential, as treatment with prostacyclin or theophylline abolished stable adhesion. Confocal microscopy of the surface of EC activated with TNF-α and TGF-β1revealed an extensive matrix of von Willebrand factor that was able to support the adhesion of flowing platelets at wall shear rates below 400 s−1. Thus, we have demonstrated a novel route of EC activation which is relevant to the atherosclerotic microenvironment. EC activated in this manner would therefore be capable of recruiting platelets in the low-shear environments that commonly exist at points of atheroma formation.

The soluble adhesion molecules P-selectin (sP-selectin) and intercellular adhesion molecule-1 (sICAM-1) are derived from platelets and endothelial cells. Circulating concentrations of these soluble adhesion molecules are raised in patients with atherosclerosis and following percutaneous transluminal coronary angioplasty (PTCA). We have investigated the effects of vitamin E supplements (800 IU/day) on circulating plasma ICAM-1 and P-selectin levels pre- and post-PTCA. Patients, randomized to group, were pre-treated with vitamin E or placebo (soybean oil) for 1 month before routine PTCA. Plasma sICAM-1 and sP-selectin were measured by enzyme-linked immunosorbent assay on blood taken immediately pre- and post-PTCA. Total protein and α-tocopherol were measured on the same samples. Plasma α-tocopherol concentrations increased in patients receiving vitamin E: 19·1 (1·5) [mean (standard error of the mean, SEM)] mg/mL post-PTCA versus 13·9 (0·6) mg/mL pre-PTCA ( n=23; P

Several recent studies have indicated the possible beneficial effects of antioxidants, specifically vitamin E, in primary and secondary coronary prevention. These studies suggest that a diet enriched in vitamin E is insufficient to have a significant protective effect, whereas supplements, in excess of 200 international units (IU) per day, are efficacious in preventing coronary disease in both men and women. The mechanisms by which vitamin E may exert its protection are uncertain, but, vitamin E is lipophilic and has been shown to inhibit the oxidative modification of low density lipoprotein (LDL), a process thought to be of crucial importance in atherogenesis. We have also previously shown that α-tocopherol (the biologically most potent isomer of vitamin E) has important direct effects on vascular endothelial and smooth muscle cells. In the present study we have investigated the effects of oral supplements of vitamin E (400 IU per day) on platelet and mononuclear cell function in patients with hypercholesterolaemia. We found that although vitamin E supplementation had no significant effect on mononuclear cell adhesion ex vivo, it had a significant effect on the thrombin-induced platelet aggregation (P

While investigating new mechanisms by which the dietary omega-3 fatty acids regulate inflammation, the authors have identified a new step in the regulation of neutrophil migration across vascular endothelial cells., Inflammation is a physiological response to tissue trauma or infection, but leukocytes, which are the effector cells of the inflammatory process, have powerful tissue remodelling capabilities. Thus, to ensure their precise localisation, passage of leukocytes from the blood into inflamed tissue is tightly regulated. Recruitment of blood borne neutrophils to the tissue stroma occurs during early inflammation. In this process, peptide agonists of the chemokine family are assumed to provide a chemotactic stimulus capable of supporting the migration of neutrophils across vascular endothelial cells, through the basement membrane of the vessel wall, and out into the tissue stroma. Here, we show that, although an initial chemokine stimulus is essential for the recruitment of flowing neutrophils by endothelial cells stimulated with the inflammatory cytokine tumour necrosis factor-α, transit of the endothelial monolayer is regulated by an additional and downstream stimulus. This signal is supplied by the metabolism of the omega-6-polyunsaturated fatty acid (n-6-PUFA), arachidonic acid, into the eicosanoid prostaglandin-D2 (PGD2) by cyclooxygenase (COX) enzymes. This new step in the neutrophil recruitment process was revealed when the dietary n-3-PUFA, eicosapentaenoic acid (EPA), was utilised as an alternative substrate for COX enzymes, leading to the generation of PGD3. This alternative series eicosanoid inhibited the migration of neutrophils across endothelial cells by antagonising the PGD2 receptor. Here, we describe a new step in the neutrophil recruitment process that relies upon a lipid-mediated signal to regulate the migration of neutrophils across endothelial cells. PGD2 signalling is subordinate to the chemokine-mediated activation of neutrophils, but without the sequential delivery of this signal, neutrophils fail to penetrate the endothelial cell monolayer. Importantly, the ability of the dietary n-3-PUFA, EPA, to inhibit this process not only revealed an unsuspected level of regulation in the migration of inflammatory leukocytes, it also contributes to our understanding of the interactions of this bioactive lipid with the inflammatory system. Moreover, it indicates the potential for novel therapeutics that target the inflammatory system with greater affinity and/or specificity than supplementing the diet with n-3-PUFAs., Author Summary Inflammation is a physiological response to tissue trauma or infection. Neutrophils, which circulate in the blood stream, are the first inflammatory cells to be recruited to a site of tissue inflammation. In response to recruitment signals provided by chemotactic peptides called chemokines, neutrophils traverse the endothelial cell lining of blood vessels. This process involves a multistep cascade of neutrophil adhesion and activation events on the endothelial barrier. While investigating the anti-inflammatory functions of the omega-3 fatty acid , EPA, which is found, for instance, in dietary fish oil, we identified an additional unexpected lipid-derived signal that is essential for neutrophil migration across the endothelium. Our experiments show that a chemokine delivered the first signal needed to bind neutrophils firmly to the endothelial surface. However, in order to traverse the endothelium, a subsequent signal delivered by prostaglandin-D2 (PGD2), a lipid derived from the omega-6 fatty acid arachidonic acid, was essential. When EPA, was introduced into the experiment, it was used to form PGD3. This alternative lipid blocked interactions between PGD2 and its receptor on neutrophils, preventing the process of migration across the endothelial barrier. Thus, we reveal a new step in the recruitment of neutrophils during inflammation, and a novel anti-inflammatory mechanism of action of dietary EPA.

The dietary n-3 PUFA, eicosapentaenoic acid (EPA) is thought to be anti-inflammatory. However, data on its ability to regulate leukocyte recruitment by vascular endothelial cells (EC) is scant. Here, primary human EC were treated with EPA for 24h prior to stimulation with TNF- and assessment of neutrophil recruitment in a flow-based assay. At levels achievable by dietary supplementation, EPA dose-dependently decreased migration of neutrophils through the EC without inhibiting their stable adhesion to the surface, a process requiring initial CXC chemokine derived signals that were unaffected by EPA. Inhibition could be replicated using inhibitors of cycloxygenase (COX-)1 and 2, while in the presence of EPA, neutrophil migration could be reconstituted by addition of arachidonic acid (AA) during the period of cytokine stimulation. These results strongly imply a role for COX generated eicosanoids in neutrophil trans-migration. Treating neutrophils with the prostaglandin-D2 [PGD2] receptor antagonist, BW868C, also dose-dependently inhibited neutrophil migration across TNF stimulated EC. Conversely, perfusion of PGD2 (derived from AA) but not PGD3 (derived from EPA) across neutrophils bound to EPA-treated EC induced them to migrate normally. Thus we describe a new PGD2-dependent step in neutrophil migration acting down stream of chemokine activation which is inhibited by EPA. Funded by the BBSRC and BHF.

Background A programme of monitoring and treatment with low-dose 10 per cent Dextran-40 (20 ml h−1) can successfully prevent thrombotic occlusion of the carotid artery after endarterectomy. This is a clinically effective treatment, over 500 CEAs have been performed with no thrombotic strokes, but little is known about the mechanism by which low-dose Dextran-40 affects platelet-rich thrombus formation. Methods In study 1, platelets from ten patients undergoing CEA were collected in a standardized manner. The response to therapeutic doses of Dextran-40 was assessed in vitro using whole-blood flow cytometric analysis of fibrinogen binding in response to adenosine 5′-diphosphate (ADP) and thrombin. Platelet aggregation in response to ADP and collagen was recorded. In study 2, blood collected from ten normal subjects was incubated with fluorescently labelled Dextran-40 and binding was measured using flow cytometry. In these experiments blood was either first incubated for 10 min with the FITC–Dextran-40 and then activated with thrombin 0·4 units ml−1 or activated with thrombin 0·4 units ml−1 for 10 min before incubation with FITC–Dextran-40. Results In study 1, reincubation of the patients' blood with Dextran-40 increased fibrinogen binding to resting platelets by 352 per cent (P < 0·05) and to ADP-stimulated platelets by 39 per cent (P < 0·05). Dextran-40 also increased fibrinogen binding in response to thrombin although this difference was not significant (P = 0·12), and increased platelet aggregation in response to ADP (48 per cent) and collagen (32 per cent) (P < 0·05 for both). In study 2, fluorescently labelled Dextran-40 bound to the surface of resting platelets from normal subjects in a dose-dependent manner, and caused platelet activation, as measured by increased P-selectin expression. Incubation with Dextran followed by activation caused a 28 per cent increase in Dextran binding; however, if the platelets were activated first and then exposed to Dextran the binding increased 94 per cent (P = 0·001). Conclusion Low-dose Dextran-40 is very effective in preventing thromboembolic complications after CEA. However, in vitro, Dextran-40 has a direct stimulatory effect on platelets. Dextran-40 binds to platelets and shows preferential binding to activated, compared with resting, platelets. Dextran may exert a beneficial antiplatelet/antithrombotic effect in vivo by inhibiting the recruitment of activated platelets to a growing thrombus.

Background Aspirin therapy fails to prevent a number of postprocedural thrombotic events, yet it still remains the standard antiplatelet regimen in most vascular surgical centres. After carotid endarterectomy (CEA), thrombosis of the endarterectomized vessel is preceded by increasing numbers of microemboli that can be detected with transcranial Doppler (TCD). The number and rate of emboli is highly predictive of thrombotic stroke. It was hypothesized that a preoperative test of platelet function might identify the mechanism(s) underlying post-CEA thrombosis. Methods Blood was taken from 120 patients using a standardized phlebotomy technique. Platelet fibrinogen binding was measured by whole blood flow cytometry, in unstimulated samples, and in response to adenosine 5′-diphosphate (ADP) (10−5−10−7 mol l−1) and thrombin (0·02–0·16 units ml−1). Platelet aggregation was measured using ADP (4–20 × 10−7 mol l−1). The ability of aspirin to inhibit platelets was assessed by the aggregation induced by arachidonic acid. For the first 3 h after operation, the number of emboli occurring was quantified using TCD. Results Of the 120 patients studied, 110 were monitored by TCD. These were divided into patients with more than 25 postoperative emboli (n = 22) and those with fewer than 25 emboli (n = 88). The degree of platelet inhibition induced by aspirin was not significantly different between the two groups (P = 0·89). However, platelets from the group with high rates of embolization bound 58 per cent more fibrinogen on flow cytometry in response to stimulation with a physiological dose of ADP (10−7 mol l−1) (P = 0·006). Aggregation of platelets from this group was also increased in response to ADP (35 per cent) relative to the group with few emboli (P = 0·001). ADP also induced more rapid aggregation in the patients with more than 25 emboli (P = 0·04). There was no difference in the activity of resting platelets (P = 0·4) or platelets stimulated by thrombin (P = 0·43), between the two groups of patients. Conclusion These data suggest that it is the platelet response to ADP which is important in arterial thrombotic complications rather than products of the cyclo-oxygenase pathway. This observation could have significant therapeutic implications for other vascular or interventional procedures in which the endothelium is disrupted.

Vitamin E, the major lipid soluble plasma antioxidant, has been reported to be reduced in patients with coronary atherosclerosis. We have measured the levels of plasma alpha-tocopherol (the predominant form of plasma vitamin E) in 128 patients with different reported degrees of angina. Patients with mild to moderate angina (grades I or II (CSS score)) (n = 64), and patients with severe angina (grades III and IV) (n = 64) were recruited from Cardiology Clinics in the U. K. Healthy controls (n = 33) and patients with hyperlipidaemia (n = 28) were also recruited. The groups of patients with angina did not differ significantly for mean age (58 +/- 1.0 years vs. 59 +/- 1.0 years, respectively); sex distribution (the M:F ratio was 48 : 16 and 46 : 18 for the respective groups); or prevalence of smoking (12% vs. 9%), or hypertension (19% vs. 33%). Total cholesterol levels were higher in the group with severe angina (5.9 +/- 0.16 mmol/l vs. 5.3 +/- 0.13 mmol/l P < 0.05). Absolute levels of plasma vitamin E were not significantly different between the angina subgroups (12.9 +/- 0.40 mg/l for the mild-moderate angina group vs. 12.5 +/- 0.51 mg/l for the severely affected group), but were positively correlated with plasma cholesterol concentrations in each case (P < 0.001). The ratio between plasma vitamin E: total cholesterol was significantly lower in the patients with severe angina (mean 2.20 +/- 0.09 mg/mmol) vs. a mean value of 2.46 +/- 0. 08 mg/m mol in the mildly affected group (P < 0.05). The plasma vitamin E: total cholesterol ratio in patients with severe angina was also significantly lower (P < 0.05) compared to either healthy controls with comparable total cholesterol levels (n = 33), or hypercholesterolaemic subjects (n = 28) without symptomatic coronary disease (mean ratios were 2.69 +/- 0.40 mg/mmol and 2.74 +/- 0.68 mg/mmol, respectively). Vitamin E has previously been demonstrated to protect endothelial function in the presence of hypercholesterolaemia, possibly by preserving nitric oxide bio-activity. It also inhibits LDL oxidation. Hence, a high plasma vitamin E: total cholesterol ratio may be associated with an amelioration of angina.

Background and purposePostoperative microemboli in patients undergoing carotid endarterectomy are a significant risk factor for stroke. These emboli can be detected by intraoperative transcranial Doppler monitoring. They are not linked to technical error and are variable between patients. As it is known that platelets play a key role in arterial thrombosis, it was hypothesized that a patient's risk of postoperative carotid thrombosis was linked to the individual's platelet response to physiologic agonists.MethodsBlood samples from 120 patients undergoing carotid endarterectomy were analyzed before surgery. Platelet aggregation was measured in response to adenosine diphosphate (ADP) (0.5 to 4 μmol/L), collagen (10 to 50 mg/mL), and arachidonic acid (3 or 6 μmol/L), and fibrinogen binding to GPIIb-IIIa was measured by whole blood flow cytometry in response to ADP (0.1 to 10 μmol/L) and thrombin (0.02 to 0.16 μ/mL). Patients underwent intraoperative transcranial Doppler monitoring for 3 hours after surgery, and platelet functional data of those who had >25 emboli in this period (n = 22) were compared with the data of those with 25 emboli, as measured both by aggregometry (P = .0012) and by flow cytometry (P < .0001). Platelet aggregation with collagen was also significantly higher in this group (P = .0018), but the response to thrombin was not statistically different in the two groups. In addition, there was no difference in the response to arachidonic acid between the groups.ConclusionThe platelet response to ADP may be linked to clinical outcome, and thus, specific ADP receptor inhibitors may be appropriate for this group of patients.