Platelet PECAM-1 Inhibits Thrombus Formation In Vivo

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells including platelets, and is also on vascular endothelial cells. PECAM-1 possesses adhesive and signalling properties, the latter being mediated by an Immunoreceptor Tyrosine-based Inhibitory Motif present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signalling inhibits the aggregation of platelets. In the present study we have utilised PECAM-1 deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1 deficient mice were larger, formed more rapidly than in control mice and were more stable. Larger thrombi were also formed in control mice transplanted with PECAM-1 deficient bone marrow, in comparison to control-transplanted mice. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1 deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation

Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

© 2018 The Author(s). Background: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Results: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusions: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers

Sex-specific differences in white matter microvascular integrity after ischaemic stroke

Background and purpose Functional outcomes after ischaemic stroke are worse in women, despite adjusting for differences in comorbidities and treatment approaches. White matter microvascular integrity represents one risk factor for poor long-term functional outcomes after ischaemic stroke. The aim of the study is to characterise sex-specific differences in microvascular integrity in individuals with acute ischaemic stroke.Methods A retrospective analysis of subjects with acute ischaemic stroke and brain MRI with diffusion-weighted (DWI) and dynamic-susceptibility contrast-enhanced (DSC) perfusion-weighted imaging obtained within 9 hours of last known well was performed. In the hemisphere contralateral to the acute infarct, normal-appearing white matter (NAWM) microvascular integrity was measured using the K-2 coefficient and apparent diffusion coefficient (ADC) values. Regression analyses for predictors of K-2 coefficient, DWI volume and good outcome (90-day modified Rankin scale (mRS) score <2) were performed.Results 105 men and 79 women met inclusion criteria for analysis. Despite no difference in age, women had increased NAWM K-2 coefficient (1027.4 vs 692.7x10(-6)/s; p=0.006). In women, atrial fibrillation (beta=583.6; p=0.04) and increasing NAWM ADC (beta=4.4; p=0.02) were associated with increased NAWM K-2 coefficient. In multivariable regression analysis, the K-2 coefficient was an independent predictor of DWI volume in women (beta=0.007; p=0.01) but not men.Conclusions In women with acute ischaemic stroke, increased NAWM K-2 coefficient is associated with increased infarct volume and chronic white matter structural integrity. Prospective studies investigating sex-specific differences in white matter microvascular integrity are needed

Effect of Vitamin K Supplementation on Insulin Resistance in Older Men and Women

OBJECTIVE—Vitamin K has a potentially beneficial role in insulin resistance, but evidence is limited in humans. We tested the hypothesis that vitamin K supplementation for 36 months will improve insulin resistance in older men and women

Discovery of 1,3-Diaminobenzenes as Selective Inhibitors of Platelet Activation at the PAR1 Receptor

A high-throughput screen of the NIH-MLSMR compound collection, along with a series of secondary assays to identify potential targets of hit compounds, previously identified a 1,3-diaminobenzene scaffold that targets protease-activated receptor 1 (PAR1). We now report additional structure–activity relationship (SAR) studies that delineate the requirements for activity at PAR1 and identify plasma-stable analogues with nanomolar inhibition of PAR1-mediated platelet activation. Compound 4 was declared as a probe (ML161) with the NIH Molecular Libraries Program. This compound inhibited platelet aggregation induced by a PAR1 peptide agonist or by thrombin but not by several other platelet agonists. Initial studies suggest that ML161 is an allosteric inhibitor of PAR1. These findings may be important for the discovery of antithrombotics with an improved safety profile

Ischaemic strokes in patients with pulmonary arteriovenous malformations and hereditary hemorrhagic telangiectasia: associations with iron deficiency and platelets.

<div><p>Background</p><p>Pulmonary first pass filtration of particles marginally exceeding ∼7 µm (the size of a red blood cell) is used routinely in diagnostics, and allows cellular aggregates forming or entering the circulation in the preceding cardiac cycle to lodge safely in pulmonary capillaries/arterioles. Pulmonary arteriovenous malformations compromise capillary bed filtration, and are commonly associated with ischaemic stroke. Cohorts with CT-scan evident malformations associated with the highest contrast echocardiographic shunt grades are known to be at higher stroke risk. Our goal was to identify within this broad grouping, which patients were at higher risk of stroke.</p><p>Methodology</p><p>497 consecutive patients with CT-proven pulmonary arteriovenous malformations due to hereditary haemorrhagic telangiectasia were studied. Relationships with radiologically-confirmed clinical ischaemic stroke were examined using logistic regression, receiver operating characteristic analyses, and platelet studies.</p><p>Principal Findings</p><p>Sixty-one individuals (12.3%) had acute, non-iatrogenic ischaemic clinical strokes at a median age of 52 (IQR 41–63) years. In crude and age-adjusted logistic regression, stroke risk was associated not with venous thromboemboli or conventional neurovascular risk factors, but with low serum iron (adjusted odds ratio 0.96 [95% confidence intervals 0.92, 1.00]), and more weakly with low oxygen saturations reflecting a larger right-to-left shunt (adjusted OR 0.96 [0.92, 1.01]). For the same pulmonary arteriovenous malformations, the stroke risk would approximately double with serum iron 6 µmol/L compared to mid-normal range (7–27 µmol/L). Platelet studies confirmed overlooked data that iron deficiency is associated with exuberant platelet aggregation to serotonin (5HT), correcting following iron treatment. By MANOVA, adjusting for participant and 5HT, iron or ferritin explained 14% of the variance in log-transformed aggregation-rate (p = 0.039/p = 0.021).</p><p>Significance</p><p>These data suggest that patients with compromised pulmonary capillary filtration due to pulmonary arteriovenous malformations are at increased risk of ischaemic stroke if they are iron deficient, and that mechanisms are likely to include enhanced aggregation of circulating platelets.</p></div

Thymoma calcification: Is it clinically meaningful?

Among anterior mediastinal lesions, thymoma is the most common. Thymomas are tumors of thymic epithelial cell origin that are distinguished by inconsistent histological and biologic behavior. Chest imaging studies typically show a round or lobulated tumor in the anterior mediastinum. Calcifications in thymomas are classically punctuate or amorphous, positioned within the lesion. Chest computed tomography (CT) features suggesting higher risk thymoma consist of tumor heterogeneity, vascular involvement, lobulation, pulmonary nodules, lymphadenopathy, and pleural manifestations. Imaging findings have an imperfect ability to predict stage and prognosis for thymoma patients. Our objective is to highlight the clinical implications of thymoma calcifications on the diagnosis, clinical manifestation and prognosis. A pubmed and google search was performed using the following words: thymoma calcification, calcified thymus, mediastinal calcification, anterior mediastinal calcification, and calcified thymoma. After reviewing 370 articles, 32 eligible articles describing thymoma calcifications were found and included in this review. Although the presence of thymus calcifications was more common in patients with invasive thymomas, they were present in significant portion of non-invasive thymomas. The presence of calcifications was not a significant factor in differentiating between benign and malignant thymoma. As a result, the type, location, size or other characteristics of thymus gland calcifications were not relevant features in clinical and radiologic diagnosis of thymoma. The histopathological diagnosis is still the only possible way to confirm the neoplastic nature of thymoma. All types of thymomas should be evaluated and managed independently of the presence of calcifications

Association of a rare variant of the TNFSF13B gene with susceptibility to Rheumatoid Arthritis and Systemic Lupus Erythematosus

Abstract: A rare variant (BAFF-var) of the tumor necrosis factor superfamily 13b (TNFSF13B) gene has been recently associated with multiple sclerosis (MS) and systemic lupus erythematosus (SLE). The aim of this study was to investigate the association between TNFSF13B BAFF-var and susceptibility to rheumatoid arthritis (RA) and replicate that association in SLE. 6,218 RA patients, 2,575 SLE patients and 4,403 healthy controls from three different countries were included in the study. TNFSF13B BAFF-var was genotyped using TaqMan allelic discrimination assay. PLINK software was used for statistical analyses. TNFSF13B BAFF-var was significantly associated with RA (p = 0.015, OR = 1.21, 95% CI = 1.03–1.41) in the Spanish cohort. A trend of association was observed in the Dutch (p = 0.115) and German (p = 0.228) RA cohorts. A meta-analysis of the three RA cohorts included in this study revealed a statistically significant association (p = 0.002, OR = 1.24, 95% CI = 1.10–1.38). In addition, TNFSF13B BAFF-var was significantly associated with SLE in the Spanish (p = 0.001, OR = 1.41, 95% CI = 1.14–1.74) and the German cohorts (p = 0.030, OR = 1.86, 95% CI = 1.05–3.28), with a statistically significant p-value obtained in the meta-analysis (p = 0.0002, OR = 1.46, 95% CI = 1.09–2.32). The results obtained confirm the known association of TNFSF13B BAFF-var with SLE and, for the first time, demonstrate that this variant contributes to susceptibility to RA.This work was supported by the following grants: P12-BIO-1395 from Consejería de Innovación, Ciencia y Tecnología, Junta de Andalucía (Spain), and the Cooperative Research Thematic Network (RETICS) program, RD16/0012/0004 (RIER), from Instituto de Salud Carlos III (ISCIII, Health Ministry, Madrid, Spain). DGS was supported by the Spanish Ministry of Economy and Competitiveness through the program FPI (SAF2015-66761-P)

The 10th Biennial Hatter Cardiovascular Institute workshop: cellular protection—evaluating new directions in the setting of myocardial infarction, ischaemic stroke, and cardio-oncology

Due to its poor capacity for regeneration, the heart is particularly sensitive to the loss of contractile cardiomyocytes. The onslaught of damage caused by ischaemia and reperfusion, occurring during an acute myocardial infarction and the subsequent reperfusion therapy, can wipe out upwards of a billion cardiomyocytes. A similar program of cell death can cause the irreversible loss of neurons in ischaemic stroke. Similar pathways of lethal cell injury can contribute to other pathologies such as left ventricular dysfunction and heart failure caused by cancer therapy. Consequently, strategies designed to protect the heart from lethal cell injury have the potential to be applicable across all three pathologies. The investigators meeting at the 10th Hatter Cardiovascular Institute workshop examined the parallels between ST-segment elevation myocardial infarction (STEMI), ischaemic stroke, and other pathologies that cause the loss of cardiomyocytes including cancer therapeutic cardiotoxicity. They examined the prospects for protection by remote ischaemic conditioning (RIC) in each scenario, and evaluated impasses and novel opportunities for cellular protection, with the future landscape for RIC in the clinical setting to be determined by the outcome of the large ERIC-PPCI/CONDI2 study. It was agreed that the way forward must include measures to improve experimental methodologies, such that they better reflect the clinical scenario and to judiciously select combinations of therapies targeting specific pathways of cellular death and injury