Activated Platelets Interfere with Recruitment of Mesenchymal Stem Cells to Apoptotic Cardiac Cells via High Mobility Group Box 1/Toll-like Receptor 4-mediated Down-regulation of Hepatocyte Growth Factor Receptor MET

Recruitment of mesenchymal stem cells (MSC) following cardiac injury, such as myocardial infarction, plays a critical role in tissue repair and may contribute to myocardial recovery. However, the mechanisms that regulate migration of MSC to the site of tissue damage remain elusive. Here, we demonstrate in vitro that activated platelets substantially inhibit recruitment of MSC toward apoptotic cardiac myocytes and fibroblasts. The alarmin high mobility group box 1 (HMGB1) was released by platelets upon activation and mediated inhibition of the cell death-dependent migratory response through Toll-like receptor (TLR)-4 expressed on the MSC. Migration of MSC to apoptotic cardiac myocytes and fibroblasts was driven by hepatocyte growth factor (HGF), and platelet activation was followed by HMGB1/TLR-4-dependent down-regulation of HGF receptor MET on MSC, thereby impairing HGF-driven MSC recruitment. We identify a novel mechanism by which platelets, upon activation, interfere with MSC recruitment to apoptotic cardiac cells, a process that may be of particular relevance for myocardial repair and regeneration.     

Secondary succession in a Swiss mire after a bog burst

Severe natural disturbances can lead to the recovery of the original vegetation or the shift to new vegetation types. While post-disturbance succession is well documented for regularly disturbed ecosystems, little is known about the pathways and rapidity of vegetation dynamics after rare events such as peat mass movements in bogs. We monitored the floristic changes in a mire subject to a bog burst in 1987 for two decades through the repeated sampling of permanent plots. The mean species number per plot increased continuously, while the evenness increased only in the first decade and then slightly decreased. Declining species were mostly mire species, while colonist species were mostly wet meadow species. Species turnover was higher in the first decade after the disturbance, and was also higher in the area of peat erosion than in the area of peat accumulation. Changes in plant species composition indicate a succession towards tall-forb vegetation (Filipendulion), acidic fen vegetation (Caricion fuscae) and swamp willow forest (Salicion). We conclude that the effects of the disturbance are still ongoing, and that the mire’s potential for recovery is therefore difficult to predict.     

The GPVI – Fc Fusion Protein Revacept Reduces Thrombus Formation and Improves Vascular Dysfunction in Atherosclerosis without Any Impact on Bleeding Times

Aims

Glycoprotein VI (GPVI) is a key platelet receptor which mediates plaque-induced platelet activation and consecutive atherothrombosis, but GPVI is also involved in platelet-mediated atheroprogression. Therefore, interference in GPVI-mediated platelet activation has the potential to combine short-term and long-term beneficial effects, specificity and safety especially regarding bleeding complications.

Methods and Results

We investigated the effects of the soluble dimeric GPVI receptor fusion protein, Revacept, an antagonist of collagen-mediated platelet activation, in an animal model of atherosclerosis: twenty week old rabbits, which had been fed on a cholesterol-rich diet for 8 weeks, received Revacept (8 mg/kg) or control twice weekly for 4 weeks. Pharmacokinetics indicated a slight accumulation of the drug in the serum after repeated dosing of Revacept for 3 weeks. A significant improvement of endothelial dysfunction after 0.06 and 0.6 µg/min acetylcholine and a significant decrease of vessel wall thickening were found after Revacept treatment. Accordingly, aortic vessel weight was reduced, and plaque sizes, macrophage and T-cell invasion tended to be reduced in histological evaluations. Bleeding time was determined after tail clipping in mice. Revacept alone or in combination with widely used anti-platelet drugs revealed a high safety margin with no prolongation of bleeding times.

Conclusion

Repeated doses of Revacept led to a significant improvement of endothelial dysfunction and vascular morphology in atherosclerotic rabbits. Furthermore, no influence of Revacept on bleeding time alone or in combinations with various anti-platelet drugs was found in mice. Thus, the inhibition of collagen-mediated platelet interaction with the atherosclerotic endothelium by Revacept exerts beneficial effects on morphology and vascular function in vivo and seems to have a wide therapeutic window without influencing the bleeding time.     

A phytosociological classification of Swiss mire vegetation

The mapping and monitoring of Swiss mires has so far relied on a classification system based on expert judgement, which was not supported by a quantitative vegetation analysis and which did not include all wetland vegetation types described in the country. Based on a spatially representative sample of 17,608 relevés from 112 Swiss mires, we address the following questions: (1) How abundant are wetland vegetation types (phytosociological alliances) in Swiss mires? (2) How are they distributed across the country––is there a regional pattern? (3) How clearly are they separated from each other? (4) How clear and reliable is their ecological interpretation? Using published wetland vegetation relevés and lists of diagnostic species for phytosociological units (associations and alliances) established by experts, we developed a numerical method for assigning relevés to units through the calculation of similarity indices. We applied this method to our sample of 17,608 relevés and estimated the total area covered by each vegetation type in Switzerland. We found that vegetation types not included in previous mapping were either rare in Switzerland (partly due to mire drainage) or poorly distinguished from other vegetation units. In an ordination, the Swiss mire vegetation formed a triangular gradient system with the Sphagnion medii, the Caricion davallianae and the Phragmition australis as extreme types. Phytosociological alliances were clearly separated in a subset of 2,265 relevés, which had a strong similarity to one particular association, but poorly separated across all relevés, of which many could not be unequivocally assigned to one association. However, ecological gradients were reflected equally well by the vegetation types in either case. Overall, phytosociological alliances distinguished until now proved suitable schemes to describe and interpret vegetation gradients. Nevertheless, we see the urgent need to establish a data base of Swiss wetland relevés for a more reliable definition of some vegetation units.     

Dynamic adhesion of eryptotic erythrocytes to endothelial cells via CXCL16/SR-PSOX

Suicidal death of erythrocytes, or eryptosis, is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the cell surface. Eryptosis is triggered by increase of cytosolic Ca2+ activity, which may result from treatment with the Ca2+ ionophore ionomycin or from energy depletion by removal of glucose. The present study tested the hypothesis that phosphatidylserine exposure at the erythrocyte surface fosters adherence to endothelial cells of the vascular wall under flow conditions at arterial shear rates and that binding of eryptotic cells to endothelial cells is mediated by the transmembrane CXC chemokine ligand 16 (CXCL16). To this end, human erythrocytes were exposed to energy depletion (for 48 h) or treated with the Ca2+ ionophore ionomycin (1 μM for 30 min). Phosphatidylserine exposure was quantified utilizing annexin-V binding, cell volume was estimated from forward scatter in FACS analysis, and erythrocyte adhesion to human vascular endothelial cells (HUVEC) was determined in a flow chamber model. As a result, both, ionomycin and glucose depletion, triggered eryptosis and enhanced the percentage of erythrocytes adhering to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly blunted in the presence of erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml), of a neutralizing antibody against endothelial CXCL16 (4 μg/ml), and following silencing of endothelial CXCL16 with small interfering RNA. The present observations demonstrate that eryptotic erythrocytes adhere to endothelial cells of the vascular wall in part by interaction of phosphatidylserine exposed at the erythrocyte surface with endothelial CXCL16.     

Gremlin-1 Is an Inhibitor of Macrophage Migration Inhibitory Factor and Attenuates Atherosclerotic Plaque Growth in ApoE?/? Mice

Monocyte infiltration and macrophage formation are pivotal steps in atherosclerosis and plaque vulnerability. Gremlin-1/Drm is crucial in embryo-/organogenesis and has been shown to be expressed in the adult organism at sites of arterial injury and to inhibit monocyte migration. The purpose of the present study was to evaluate and characterize the role of Gremlin-1 in atherosclerosis. Here we report that Gremlin-1 is highly expressed primarily by monocytes/macrophages in aortic atherosclerotic lesions of ApoE^−/− mice and is secreted from activated monocytes and during macrophage development in vitro. Gremlin-1 reduces macrophage formation by inhibiting macrophage migration inhibitory factor (MIF), a cytokine critically involved in atherosclerotic plaque progression and vulnerability. Gremlin-1 binds with high affinity to MIF (K_D = 54 nm), as evidenced by surface plasmon resonance analysis and co-immunoprecipitation, and reduces MIF-induced release of TNF-α from macrophages. Treatment of ApoE^−/− mice with a dimeric recombinant fusion protein, _mGremlin1-Fc, but not with equimolar control Fc or inactivated _mGremlin1-Fc, reduced TNF-α expression, the content of monocytes/macrophages of atherosclerotic lesions, and attenuated atheroprogression. The present data disclose that Gremlin-1 is an endogenous antagonist of MIF and define a role for Gremlin-1/MIF interaction in atherosclerosis.     

Extracellular peroxidases of suspension culture cells of spruce (Picea abies): fungal elicitor-induced inactivation

Extracellular peroxidases of suspension cultures of spruce (Picea abies) (L.) (Karst) become inactivated when the cell suspension is elicited with a cell wall preparation of the spruce pathogenic fungus Rhizosphaera kalkhoffii. In contrast, cellular peroxidases are induced under these conditions. Both changes of activity are reflected in the isoenzyme profiles.Inactivation of the extracellular peroxidases is caused by an effector, arising from the cells after contact with the elicitor. Formation of the effector is limited to the beginning of elicitation, showing maximal activity at this period of time. Subsequently it becomes increasingly ineffective, probably due to inactivation. The effector is able to also inactivate commercial (horseradish) peroxidase. Inactivation was not the result of the action of a protease present in the medium.The elicitor exerts two different effects on the spruce cell suspension culture. It induces synthesis of enzymes correlated with lignin synthesis and an accumulation of lignin-like material. It also induces secretion of the negative effector which inactivates extracellular peroxidases.The elicitor-induced inactivation is not specific for peroxidases. Other extracellular enzymes, -glucosidase and acid phosphatase (secreted by the cells into the medium) and -amylase and pectinase (from Aspergillus strains) are also inactivated.     

High Platelet Reactivity in Patients with Acute Coronary Syndromes Undergoing Percutaneous Coronary Intervention: Randomised Controlled Trial Comparing Prasugrel and Clopidogrel

Background

Prasugrel is more effective than clopidogrel in reducing platelet aggregation in acute coronary syndromes. Data available on prasugrel reloading in clopidogrel treated patients with high residual platelet reactivity (HRPR) i.e. poor responders, is limited.

Objectives

To determine the effects of prasugrel loading on platelet function in patients on clopidogrel and high platelet reactivity undergoing percutaneous coronary intervention for acute coronary syndrome (ACS).

Patients

Patients with ACS on clopidogrel who were scheduled for PCI found to have a platelet reactivity ≥40 AUC with the Multiplate Analyzer, i.e. “poor responders” were randomised to prasugrel (60 mg loading and 10 mg maintenance dose) or clopidogrel (600 mg reloading and 150 mg maintenance dose). The primary outcome measure was proportion of patients with platelet reactivity <40 AUC 4 hours after loading with study medication, and also at one hour (secondary outcome). 44 patients were enrolled and the study was terminated early as clopidogrel use decreased sharply due to introduction of newer P2Y12 inhibitors.

Results

At 4 hours after study medication 100% of patients treated with prasugrel compared to 91% of those treated with clopidogrel had platelet reactivity <40 AUC (p = 0.49), while at 1 hour the proportions were 95% and 64% respectively (p = 0.02). Mean platelet reactivity at 4 and 1 hours after study medication in prasugrel and clopidogrel groups respectively were 12 versus 22 (p = 0.005) and 19 versus 34 (p = 0.01) respectively.

Conclusions

Routine platelet function testing identifies patients with high residual platelet reactivity (“poor responders”) on clopidogrel. A strategy of prasugrel rather than clopidogrel reloading results in earlier and more sustained suppression of platelet reactivity. Future trials need to identify if this translates into clinical benefit.

Trial Registration

ClinicalTrials.gov NCT01339026