Samantha Tull

Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step in Neutrophil Recruitment

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.

Cellular Pathology of Atherosclerosis. Smooth Muscle Cells Promote Adhesion of Platelets to Cocultured Endothelial Cells

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-β1 and 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 αIIbβ3 integrin, 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-β1 revealed 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.

Docosahexaenoic Acid Inhibits the Adhesion of Flowing Neutrophils to Cytokine Stimulated Human Umbilical Vein Endothelial Cells

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.

PITX2 Modulates Atrial Membrane Potential and the Antiarrhythmic Effects of Sodium-Channel Blockers

Background Antiarrhythmic drugs are widely used to treat patients with atrial fibrillation (AF), but the mechanisms conveying their variable effectiveness are not known. Recent data suggested that paired like homeodomain-2 transcription factor (PITX2) might play an important role in regulating gene expression and electrical function of the adult left atrium (LA). Objectives After determining LA PITX2 expression in AF patients requiring rhythm control therapy, the authors assessed the effects of Pitx2c on LA electrophysiology and the effect of antiarrhythmic drugs. Methods LA PITX2 messenger ribonucleic acid (mRNA) levels were measured in 95 patients undergoing thoracoscopic AF ablation. The effects of flecainide, a sodium (Na+)-channel blocker, and d,l-sotalol, a potassium channel blocker, were studied in littermate mice with normal and reduced Pitx2c mRNA by electrophysiological study, optical mapping, and patch clamp studies. PITX2-dependent mechanisms of antiarrhythmic drug action were studied in human embryonic kidney (HEK) cells expressing human Na channels and by modeling human action potentials. Results Flecainide 1 μmol/l was more effective in suppressing atrial arrhythmias in atria with reduced Pitx2c mRNA levels (Pitx2c+/–). Resting membrane potential was more depolarized in Pitx2c+/– atria, and TWIK-related acid-sensitive K+ channel 2 (TASK-2) gene and protein expression were decreased. This resulted in enhanced post-repolarization refractoriness and more effective Na-channel inhibition. Defined holding potentials eliminated differences in flecainide’s effects between wild-type and Pitx2c+/– atrial cardiomyocytes. More positive holding potentials replicated the increased effectiveness of flecainide in blocking human Nav1.5 channels in HEK293 cells. Computer modeling reproduced an enhanced effectiveness of Na-channel block when resting membrane potential was slightly depolarized. Conclusions PITX2 mRNA modulates atrial resting membrane potential and thereby alters the effectiveness of Na-channel blockers. PITX2 and ion channels regulating the resting membrane potential may provide novel targets for antiarrhythmic drug development and companion therapeutics in AF.

PR3 and Elastase Alter PAR1 Signaling and Trigger vWF Release via a Calcium-Independent Mechanism from Glomerular Endothelial Cells

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-NH2 and SLIGKV-NH2, 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.

Neutrophil serine proteases mediate inflammatory cell recruitment by glomerular endothelium and progression towards dysfunction

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 PSGL-1. 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.

A Regional Reduction in Ito and IKACh in the Murine Posterior Left Atrial Myocardium Is Associated with Action Potential Prolongation and Increased Ectopic Activity

Background The left atrial posterior wall (LAPW) is potentially an important area for the development and maintenance of atrial fibrillation. We assessed whether there are regional electrical differences throughout the murine left atrial myocardium that could underlie regional differences in arrhythmia susceptibility. Methods We used high-resolution optical mapping and sharp microelectrode recordings to quantify regional differences in electrical activation and repolarisation within the intact, superfused murine left atrium and quantified regional ion channel mRNA expression by Taqman Low Density Array. We also performed selected cellular electrophysiology experiments to validate regional differences in ion channel function. Results Spontaneous ectopic activity was observed during sustained 1Hz pacing in 10/19 intact LA and this was abolished following resection of LAPW (0/19 resected LA, P<0.001). The source of the ectopic activity was the LAPW myocardium, distinct from the pulmonary vein sleeve and LAA, determined by optical mapping. Overall, LAPW action potentials (APs) were ca. 40% longer than the LAA and this region displayed more APD heterogeneity. mRNA expression of Kcna4, Kcnj3 and Kcnj5 was lower in the LAPW myocardium than in the LAA. Cardiomyocytes isolated from the LAPW had decreased Ito and a reduced IKACh current density at both positive and negative test potentials. Conclusions The murine LAPW myocardium has a different electrical phenotype and ion channel mRNA expression profile compared with other regions of the LA, and this is associated with increased ectopic activity. If similar regional electrical differences are present in the human LA, then the LAPW may be a potential future target for treatment of atrial fibrillation.

217 Differences in Blood Biomarker Composition Between Paroxysmal AF and Sinus Rhythm Patients, Without Heart Failure

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.Abstract 217 Figure 1 Plasma NT-pro-BNP

169 PITX2C deficiency augments the anti-arrhythmic properties of flecainide: results in a mouse model and validation in a human atrium simulation study

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/dtmax) 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 (INa) 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 < 0.05) but not in WT atria (3/15 base vs 3/12 Flec). Flec doubled the increase in ERP in Pitx2c+/– atria compared with WT (Pitx2c+/– 14 ± 2 ms, n = 13 vs WT 7 ± 2 ms, n = 10; p < 0.05), and tripled post-repolarisation refractoriness (PRR; Pitx2c+/– 21 ± 3 ms, n = 6 vs WT 6 ± 4 ms, n = 6, p < 0.01). Pitx2c+/– 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 < 0.05) and lower APA (WT 78 ± 1.2mV, n = 30; Pitx2c+/– 73 ± 1.3 mV, n = 32). Flec had no effect on the RMP but reduced CV, APA and dV/dtmax in both genotypes to a similar extent. Hearts with PRR were not susceptible to arrhythmias (0/17). As with the isolated hearts, INa 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 INa on RMP, CV, APD, or dV/dtmax. 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.