Direct observations of the kinetics of migrating T-cells suggest active retention by endothelial cells with continual bidirectional migration.

The kinetics and regulatory mechanisms of T-cell migration through endothelium have not been fully defined. In experimental filter-based assays in vitro, transmigration of lymphocytes takes hours, compared to minutes in vivo. We cultured endothelial cell (EC) monolayers on filters, solid substrates or collagen gels, and treated them with tumour necrosis factor-α (TNF), interferon-γ (IFN), or both, prior to analysis of lymphocyte migration in the presence or absence of flow. Peripheral blood lymphocytes (PBL), CD4+ cells or CD8+ cells, took many hours to migrate through EC-filter constructs for all cytokine treatments. However, direct microscopic observations of EC-filters which had been mounted in a flow chamber showed that PBL crossed the endothelial monolayer in minutes and were highly motile in the subendothelial space. Migration through EC was also observed on clear plastic, with or without flow. After brief settling without flow, PBL and isolated CD3+ or CD4+ cells all crossed EC in minutes, but the numbers of migrated cells varied little with time. Close observation revealed that lymphocytes continuously migrated back and forth across endothelium. Under flow, migration kinetics and the proportions migrating back and forth were little altered. On collagen gels, PBL again crossed EC in minutes and migrated back and forth, but showed little penetration of the gel over hours.In contrast, neutrophils migrated efficiently through EC and into gels. These observations suggest a novel model for lymphoid migration, in which endothelial cells support migration but retain lymphocytes (as opposed to neutrophils), and additional signal(s) are required for onward migration

Analysis of the effects of stromal cells on the migration of lymphocytes into and through inflamed tissue using 3-D culture models

AbstractStromal cells may regulate the recruitment and behaviour of leukocytes during an inflammatory response, potentially through interaction with the endothelial cells (EC) and the leukocytes themselves. Here we describe new in vitro methodologies to characterise the effects of stromal cells on the migration of lymphocytes through endothelium and its underlying matrix. Three-dimensional tissue-like constructs were created in which EC were cultured above a stromal layer incorporating fibroblasts either as a monolayer on a porous filter or dispersed within a matrix of collagen type 1. A major advantage of these constructs is that they enable each step in leukocyte migration to be analysed in sequence (migration through EC and then stroma), as would occur in vivo. Migrated cells can also be retrieved from the constructs to identify which subsets traffic more effectively and how their functional responses evolve during migration. We found that culture of EC with dermal fibroblasts promoted lymphocyte transendothelial migration but not onward transit through matrix. A critical factor influencing the effect of fibroblasts on recruitment proved to be their proximity to the EC, with direct contact tending to disrupt migration. Comparison of the different approaches indicates that choice of an appropriate 3-D model enables the steps in lymphocyte entry into tissue to be studied in sequence, the regulatory mechanism to be dissected, and the effects of changes in stroma to be investigated

Endothelial dysfunction in COVID-19: a position paper of the ESC Working Group for Atherosclerosis and Vascular Biology, and the ESC Council of Basic Cardiovascular Science

The COVID-19 pandemic is an unprecedented healthcare emergency causing mortality and illness across the world. Although primarily affecting the lungs, the SARS-CoV-2 virus also affects the cardiovascular system. In addition to cardiac effects, e.g. myocarditis, arrhythmias, and myocardial damage, the vasculature is affected in COVID-19, both directly by the SARS-CoV-2 virus, and indirectly as a result of a systemic inflammatory cytokine storm. This includes the role of the vascular endothelium in the recruitment of inflammatory leucocytes where they contribute to tissue damage and cytokine release, which are key drivers of acute respiratory distress syndrome (ARDS), in disseminated intravascular coagulation, and cardiovascular complications in COVID-19. There is also evidence linking endothelial cells (ECs) to SARS-CoV-2 infection including: (i) the expression and function of its receptor angiotensin-converting enzyme 2 (ACE2) in the vasculature; (ii) the prevalence of a Kawasaki disease-like syndrome (vasculitis) in COVID-19; and (iii) evidence of EC infection with SARS-CoV-2 in patients with fatal COVID-19. Here, the Working Group on Atherosclerosis and Vascular Biology together with the Council of Basic Cardiovascular Science of the European Society of Cardiology provide a Position Statement on the importance of the endothelium in the underlying pathophysiology behind the clinical presentation in COVID-19 and identify key questions for future research to address. We propose that endothelial biomarkers and tests of function (e.g. flow-mediated dilatation) should be evaluated for their usefulness in the risk stratification of COVID-19 patients. A better understanding of the effects of SARS-CoV-2 on endothelial biology in both the micro- and macrovasculature is required, and endothelial function testing should be considered in the follow-up of convalescent COVID-19 patients for early detection of long-term cardiovascular complications

Metabolic consequences for mice lacking Endosialin: LC-MS/MS-based metabolic phenotyping of serum from C56Bl/6J Control and CD248 knock-out mice

IntroductionThe Endosialin/CD248/TEM1 protein is expressed in adipose tissue and its expression increases with obesity. Recently, genetic deletion of CD248 has been shown to protect mice against atherosclerosis on a high fat diet.ObjectivesWe investigated the effect of high fat diet feeding on visceral fat pads and circulating lipid profiles in CD248 knockout mice compared to controls.MethodsFrom 10 weeks old, CD248-/- and +/+ mice were fed either chow (normal) diet or a high fat diet for 13 weeks. After 13 weeks the metabolic profiles and relative quantities of circulating lipid species were assessed using ultra high performance liquid chromatography-quadrupole time-of flight mass spectrometry (UHPLC-MS) with high resolution accurate mass (HRAM) capability.ResultsWe demonstrate a specific reduction in the size of the perirenal fat pad in CD248-/- mice compared to CD248+/+, despite similar food intake. More strikingly, we identify significant, diet-dependent differences in the serum metabolic phenotypes of CD248 null compared to age and sex-matched wildtype control mice. Generalised protection from HFD-induced lipid accumulation was observed in CD248 null mice compared to wildtype, with particular reduction noted in the lysophosphatidylcholines, phosphatidylcholines, cholesterol and carnitine.ConclusionsOverall these results show a clear and protective metabolic consequence of CD248 deletion in mice, implicating CD248 in lipid metabolism or trafficking and opening new avenues for further investigation using anti-CD248 targeting agents

The role of platelets in the recruitment of leukocytes during vascular disease

Besides their role in the formation of thrombus during haemostasis, it is becoming clear that platelets contribute to a number of other processes within the vasculature. Indeed, the integrated function of the thrombotic and inflammatory systems, which results in platelet-mediated recruitment of leukocytes, is now considered to be of great importance in the propagation, progression and pathogenesis of atherosclerotic disease of the arteries. There are three scenarios by which platelets can interact with leukocytes: (1) during haemostasis, when platelets adhere to and are activated on sub-endothelial matrix proteins exposed by vascular damage and then recruit leukocytes to a growing thrombus. (2) Platelets adhere to and are activated on stimulated endothelial cells and then bridge blood borne leukocytes to the vessel wall and. (3) Adhesion between platelets and leukocytes occurs in the blood leading to formation of heterotypic aggregates prior to contact with endothelial cells. In the following review we will not discuss leukocyte recruitment during haemostasis, as this represents a physiological response to tissue trauma that can progress, at least in its early stages, in the absence of inflammation. Rather we will deal with scenarios 2 and 3, as these pathways of platelet–leukocyte interactions are important during inflammation and in chronic inflammatory diseases such as atherosclerosis. Indeed, these interactions mean that leukocytes possess means of adhesion to the vessel wall under conditions that may not normally be permissive of leukocyte–endothelial cell adhesion, meaning that the disease process may be able to bypass the regulatory pathways which would ordinarily moderate the inflammatory response

Longer lifespan in male mice treated with a weakly estrogenic agonist, an antioxidant, an α-glucosidase inhibitor or a Nrf2-inducer

The National Institute on Aging Interventions Testing Program (ITP) evaluates agents hypothesized to increase healthy lifespan in genetically heterogeneous mice. Each compound is tested in parallel at three sites, and all results are published. We report the effects of lifelong treatment of mice with four agents not previously tested: Protandim, fish oil, ursodeoxycholic acid (UDCA) and metformin – the latter with and without rapamycin, and two drugs previously examined: 17-α-estradiol and nordihydroguaiaretic acid (NDGA), at doses greater and less than used previously. 17-α-estradiol at a threefold higher dose robustly extended both median and maximal lifespan, but still only in males. The male-specific extension of median lifespan by NDGA was replicated at the original dose, and using doses threefold lower and higher. The effects of NDGA were dose dependent and male specific but without an effect on maximal lifespan. Protandim, a mixture of botanical extracts that activate Nrf2, extended median lifespan in males only. Metformin alone, at a dose of 0.1% in the diet, did not significantly extend lifespan. Metformin (0.1%) combined with rapamycin (14 ppm) robustly extended lifespan, suggestive of an added benefit, based on historical comparison with earlier studies of rapamycin given alone. The α-glucosidase inhibitor, acarbose, at a concentration previously tested (1000 ppm), significantly increased median longevity in males and 90th percentile lifespan in both sexes, even when treatment was started at 16 months. Neither fish oil nor UDCA extended lifespan. These results underscore the reproducibility of ITP longevity studies and illustrate the importance of identifying optimal doses in lifespan studies

Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets

Thrombosis is a common, life-threatening consequence of systemic infection; however, the underlying mechanisms that drive the formation of infection-associated thrombi are poorly understood. Here, using a mouse model of systemic Salmonella Typhimurium infection, we determined that inflammation in tissues triggers thrombosis within vessels via ligation of C-type lectin-like receptor-2 (CLEC-2) on platelets by podoplanin exposed to the vasculature following breaching of the vessel wall. During infection, mice developed thrombi that persisted for weeks within the liver. Bacteria triggered but did not maintain this process, as thrombosis peaked at times when bacteremia was absent and bacteria in tissues were reduced by more than 90% from their peak levels. Thrombus development was triggered by an innate, TLR4-dependent inflammatory cascade that was independent of classical glycoprotein VI-mediated (GPVI-mediated) platelet activation. After infection, IFN-ã release enhanced the number of podoplanin-expressing monocytes and Kupffer cells in the hepatic parenchyma and perivascular sites and absence of TLR4, IFN-ã, or depletion of monocytic-lineage cells or CLEC-2 on platelets markedly inhibited the process. Together, our data indicate that infection-driven thrombosis follows local inflammation and upregulation of podoplanin and platelet activation. The identification of this pathway offers potential therapeutic opportunities to control the devastating consequences of infection-driven thrombosis without increasing the risk of bleeding

Nitrite circumvents platelet resistance to nitric oxide in patients with heart failure preserved ejection fraction and chronic atrial fibrillation

Aims: Heart failure (HF) is a pro-thrombotic state. Both platelet and vascular responses to nitric oxide (NO) donors are impaired in HF patients with reduced ejection fraction (HFrEF) compared to healthy volunteers (HV) due to scavenging of NO, and possibly also reduced activity of the principal NO sensor, soluble guanylate cyclase (sGC), limiting the therapeutic potential of NO donors as anti-aggregatory agents. Previous studies have shown that nitrite inhibits platelet activation presumptively after its reduction to NO, but the mechanism(s) involved remain poorly characterized. Our aim was to compare the effects of nitrite on platelet function in HV vs. HF patients with preserved ejection fraction (HFpEF) and chronic atrial fibrillation (HFpEF-AF), vs. patients with chronic AF without HF, and to assess whether these effects occur independent of the interaction with other formed elements of blood. Methods and Results: Platelet responses to nitrite and the NO donor sodium nitroprusside (SNP) were compared in age-matched HV controls (n = 12), HFpEF-AF patients (n = 29) and chronic AF patients (n = 8). Anti-aggregatory effects of nitrite in the presence of NO scavengers/sGC inhibitor were determined and vasodilator-stimulated phosphoprotein (VASP) phosphorylation was assessed using Western blotting. In HV and chronic AF, both nitrite and SNP inhibited platelet aggregation in a concentration-dependent manner. Inhibition of platelet aggregation by the NO donor SNP was impaired in HFpEF-AF patients compared to healthy and chronic AF individuals, but there was no impairment of the anti-aggregatory effects of nitrite. Nitrite circumvented platelet NO resistance independently of other blood cells by directly activating sGC and phosphorylating VASP. Conclusion: We here show for the first time that HFpEF-AF (but not chronic AF without HF) is associated with marked impairment of platelet NO responses due to sGC dysfunction and nitrite circumvents the “platelet NO resistance” phenomenon in human HFpEF, at least partly, by acting as a direct sGC activator independent of NO

Mesenchymal Stem Cells Exhibit Firm Adhesion, Crawling, Spreading and Transmigration across Aortic Endothelial Cells: Effects of Chemokines and Shear

Mesenchymal stem cells (MSCs) have anti-inflammatory and immunosuppressive properties and may be useful in the therapy of diseases such as arteriosclerosis. MSCs have some ability to traffic into inflamed tissues, however to exploit this therapeutically their migratory mechanisms need to be elucidated. This study examines the interaction of murine MSCs (mMSCs) with, and their migration across, murine aortic endothelial cells (MAECs), and the effects of chemokines and shear stress. The interaction of mMSCs with MAECs was examined under physiological flow conditions. mMSCs showed lack of interaction with MAECs under continuous flow. However, when the flow was stopped (for 10min) and then started, mMSCs adhered and crawled on the endothelial surface, extending fine microvillous processes (filopodia). They then spread extending pseudopodia in multiple directions. CXCL9 significantly enhanced the percentage of mMSCs adhering, crawling and spreading and shear forces markedly stimulated crawling and spreading. CXCL9, CXCL16, CCL20 and CCL25 significantly enhanced transendothelial migration across MAECs. The transmigrated mMSCs had down-regulated receptors CXCR3, CXCR6, CCR6 and CCR9. This study furthers the knowledge of MSC transendothelial migration and the effects of chemokines and shear stress which is of relevance to inflammatory diseases such as arteriosclerosis

Appropriation of GPIb from platelet-derived extracellular vesicles supports monocyte recruitment in systemic inflammation

Interactions between platelets, leukocytes and the vessel wall provide alternative pathological routes of thrombo-inflammatory leukocyte recruitment. We found that when platelets were activated by a range of agonists in whole blood, they shed platelet-derived extracellular vesicles which rapidly and preferentially bound to blood monocytes compared to other leukocytes. Platelet-derived extracellular vesicle binding to monocytes was initiated by P-selectin-dependent adhesion and was stabilised by binding of phosphatidylserine. These interactions resulted in the progressive transfer of the platelet adhesion receptor GPIbα to monocytes. GPIbα+-monocytes tethered and rolled on immobilised von Willebrand Factor or were recruited and activated on endothelial cells treated with TGF-β1 to induce the expression of von Willebrand Factor. In both models monocyte adhesion was ablated by a function-blocking antibody against GPIbα. Monocytes could also bind platelet-derived extracellular vesicle in mouse blood in vitro and in vivo. Intratracheal instillations of diesel nanoparticles, to model chronic pulmonary inflammation, induced accumulation of GPIbα on circulating monocytes. In intravital experiments, GPIbα+-monocytes adhered to the microcirculation of the TGF-β1-stimulated cremaster muscle, while in the ApoE−/− model of atherosclerosis, GPIbα+-monocytes adhered to the carotid arteries. In trauma patients, monocytes bore platelet markers within 1 hour of injury, the levels of which correlated with severity of trauma and resulted in monocyte clearance from the circulation. Thus, we have defined a novel thrombo-inflammatory pathway in which platelet-derived extracellular vesicles transfer a platelet adhesion receptor to monocytes, allowing their recruitment in large and small blood vessels, and which is likely to be pathogenic