Ca 2+ entry via Orai1 store-operated Ca 2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific ‘partner proteins’ and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca 2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca 2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca 2+ signaling and von Willebrand factor release in response to inflammatory stimuli.

AimsAdequate microcirculatory perfusion, and not just opening of occluded arteries, is critical to salvage heart tissue following myocardial infarction. However, the degree of microvascular perfusion taking place is not known, limited primarily by an inability to directly image coronary microcirculation in a beating heart in vivo. Haematopoietic stem/progenitor cells (HSPCs) offer a potential therapy but little is known about their homing dynamics at a cellular level and whether they protect coronary microvessels. This study used intravital microscopy to image the anaesthetized mouse beating heart microcirculation following stabilization.Methods and resultsA 3D-printed stabilizer was attached to the ischaemia–reperfusion injured (IRI) beating heart. The kinetics of neutrophil, platelet and HSPC recruitment, as well as functional capillary density (FCD), was imaged post-reperfusion. Laser speckle contrast imaging (LSCI) was used for the first time to monitor ventricular blood flow in beating hearts. Sustained hyperaemic responses were measured throughout reperfusion, initially indicating adequate flow resumption. Intravital microscopy confirmed large vessel perfusion but demonstrated poor transmission of flow to downstream coronary microvessels. Significant neutrophil adhesion and microthrombus formation occurred within capillaries with the latter occluding them, resulting in patchy perfusion and reduced FCD. Interestingly, ‘patrolling’ neutrophils were also observed in capillaries. Haematopoietic stem/progenitor cells readily trafficked through the heart but local retention was poor. Despite this, remarkable anti-thromboinflammatory effects were observed, consequently improving microvascular perfusion.ConclusionWe present a novel approach for imaging multiple microcirculatory perturbations in the beating heart with LSCI assessment of blood flow. Despite deceptive hyperaemic responses, increased microcirculatory flow heterogeneity was seen, with non-perfused areas interspersed with perfused areas. Microthrombi, rather than neutrophils, appeared to be the major causative factor. We further applied this technique to demonstrate local stem cell presence is not a pre-requisite to confer vasculoprotection. This is the first detailed in vivo characterization of coronary microcirculatory responses post-reperfusion injury.

infarct size was observed when WT-MI hearts were compared to their PMCA1Ht counterparts 1 week post-surgery. Interestingly, whilst both WT and PMCA1Ht MI-treated mice showed a significant deterioration in cardiac function 1 week post-surgery there were differences seen in cardiac structure between the two groups. For example, echocardiography revealed that the WT-MI hearts were significantly more hypertrophic when compared to their PMCA1Ht counterparts shown by a significant increase in the LV diameters alongside a significant difference in the normalised heart weight to tibia length ratios. ECG revealed significantly longer QT intervals and considerably more extra-systolic events among the WT-MI mice. Whilst histological analysis of cardiomyocyte size showed a significantly exacerbated hypertrophic response in the remote regions of WT-MI mice compared to PMCA1 Ht -MI mice. Conclusion Heterozygous deletion of PMCA1 might serve a protective role in the heart following both moderate and severe MI. The protective mechanism most likely develops in the early post-operative phase, as an MI of similar extent is associated with higher mortality rates in WT mice. Future work will aim to elucidate the mechanism producing this phenotype mainly focusing on the potential role that PMCA1 might serve in the process. Introduction Lipids have key roles in CVD but heritability studies focus on few species bar lipoproteins. Lipids play key roles in cell signalling, immunity, inflammation, vasodilation and cell death. Although not encoded, their activities are tightly linked to DNA-encoded entities (e.g. enzymes and other proteins) and those with a strong genetic influence (high heritability) may identify novel pathways in CVD. Purpose: Analysing eicosanoid, endocannabinoid and sphingolipid profiles in 250 British Caucasian families with GWAS data will identify particularly heritable lipid biomarkers for the discovery of causative genetic variation of CVD. Methods An array of 79 eicosanoids and related species, 33 endocannabinoids and congeners, 63 ceramides and related species, from 204 plasma samples (31 families of 1+ individuals with hypertension) were extracted and analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Quality control was accessed and estimates of heritability of each lipid species was estimated using QTDT software. The concentration of each lipid species were assessed for normal distribution, outliers, and adjusted for age and sex. The resulting lipid concentrations will be analysed using FaST-LMM software for associations with 557,124 SNPs. Results 19 species of eicosanoids were identified (mean concentrations 19 pg/ml -7600 pg/ml); the species at highest abundance in plasma (HODEs) are derivatives of the omega-6 fatty acid linoleic acid. 8 species passed quality control assessments and 3 species were estimated to be significantly heritable (9-OxoODE 24%, 12-HETE 44%, 12(13)-EPOME 78%). 15 congeners of endocannabinoids were identified (mean concentrations 20 pg/ml -4000 pg/ml); the...

No abstract

the DCB group (figure 2). Gene expression analysis of CD14 + leucocytes showed that IL18 had decreased expression at two weeks, CXCR4 and IL1β decreased at two months, while pentraxin 3 increased at two weeks and two months. In terms of humoral biomarkers, hsTnI remains elevated up to two weeks post PCI while IL6 and TNFa remain elevated till two months post PCI. Conclusion Intermediate monocytes, a highly proatherogenic monocyte subset, increase significantly two months following elective, uncomplicated PCI. They remain significantly elevated in the DES group but not in the DCB group suggesting that the PCI strategy could be one of the ways to modulate the inflammatory response post PCI and improve patient outcomes.