Binding of Low Density Lipoprotein to Platelet Apolipoprotein E Receptor 2' results in phosphorylation of p38MAPK

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Endothelial Cell and Platelet Bioenergetics: Effect of Glucose and Nutrient Composition

It has been suggested that cells that are independent of insulin for glucose uptake, when exposed to high glucose or other nutrient concentrations, manifest enhanced mitochondrial substrate oxidation with consequent enhanced potential and generation of reactive oxygen species (ROS); a paradigm that could predispose to vascular complications of diabetes. Here we exposed bovine aortic endothelial (BAE) cells and human platelets to variable glucose and fatty acid concentrations. We then examined oxygen consumption and acidification rates using recently available technology in the form of an extracellular oxygen and proton flux analyzer. Acute or overnight exposure of confluent BAE cells to glucose concentrations from 5.5 to 25 mM did not enhance or change the rate of oxygen consumption (OCR) under basal conditions, during ATP synthesis, or under uncoupled conditions. Glucose also did not alter OCR in sub-confluent cells, in cells exposed to low serum, or in cells treated with added pyruvate. Likewise, overnight exposure to fatty acids of varying saturation had no such effects. Overnight exposure of BAE cells to low glucose concentration decreased maximal uncoupled respiration, but not basal or ATP related oxygen consumption. Labeled glucose oxidation to CO2 increased, but only marginally after high glucose exposure while oleate oxidation to CO2 decreased. Overnight exposure to linolenic acid, but not oleic or linoleic acid increased extracellular acidification consistent with enhanced glycolytic metabolism. We were unable to detect an increase in production of reactive oxygen species (ROS) from BAE cells exposed to high medium glucose. Like BAE cells, exposure of human platelets to glucose did not increase oxygen consumption. As opposed to BAE cells, platelet mitochondria demonstrate less respiratory reserve capacity (beyond that needed for basal metabolism). Our data do not support the concept that exposure to high glucose or fatty acids accelerates mitochondrial oxidative metabolism in endothelial cells or platelets

Development of platelet inhibition by cAMP during megakaryocytopoiesis.

Prostacyclin is a potent inhibitor of agonist-induced Ca2+ increases in platelets, but in the megakaryocytic cell line MEG-01 this inhibition is absent. Using human megakaryocytic cell lines representing different stages in megakaryocyte (Mk) maturation as well as stem cells and immature and mature megakaryocytes, we show that the inhibition by prostacyclin develops at a late maturation stage shortly before platelets are formed. This late appearance is not caused by insufficient cAMP formation or absent protein kinase A (PKA) activity in immature cells. Instead, the appearance of Ca2+ inhibition by prostacyclin is accompanied by a sharp increase in the expression of the catalytic subunit of PKA (PKA-C) but not by changes in the expression of the PKA-regulatory subunits Ialpha/beta, IIalpha, and IIbeta. Overexpression of PKA-C in the megakaryocytic cell line CHRF-288-11 potentiates the Ca2+ inhibition by prostacyclin. Thus, up-regulation of PKA-C appears to be a key step in the development of Ca2+ inhibition by prostacyclin in platelets

Plasma ectonucleotidases prevent desensitization of purinergic receptors in stored platelets: importance for platelet activity during thrombus formation

BACKGROUND: Platelets (PLTs) contain purinergic receptors for ATP (P2X1) and ADP (P2Y1 and P2Y12) that rapidly desensitize upon stimulation with these nucleotides. In vivo, this is antagonized by ectonucleotidases on the surface of endothelial cells and white blood cells (WBCs). The receptor desensitization of ATP- and ADP-induced responses of PLTs stored in plasma without WBCs was investigated. STUDY DESIGN AND METHODS: ATP- and ADP-induced PLT shape change (shear-induced) aggregation and Ca2+ signaling were measured in the presence or absence of plasma. Degradation of nucleotides in plasma was quantified by high-performance liquid chromatography. RESULTS: Washed PLTs became refractory for ATP and ADP in shape change, aggregation, and Ca2+ responses during a 90-minute incubation at 37 degrees C. The PLT responses mediated by P2X1, P2Y1, and P2Y12 receptors gradually reduced or disappeared. When plasma was present, however, the PLTs persistently showed high responses to ATP and ADP. Heat treatment of plasma abolished this effect. Also under conditions of flow and high shear, PLTs in plasma kept high P2X1 activity, mediating aggregate formation. In isolated plasma, not containing WBCs, nucleotides were degraded in the order of ADP/UDP>ATP/UTP. Degradation of ATP was partly inhibited by blocking the ecto-NTPDase CD39, whereas degradation of both ATP and ADP was inhibited by blocking ectopyrophosphatase/phosphodiesterase activity. Part of the nucleotide-degrading activities appeared to be membrane-bound. CONCLUSION: Ectonucleotidases in plasma preserve the functionality of P2X1 and P2Y receptors. Upon PLT storage, these plasma activities are essential to ensure adequate (shear-dependent) formation of aggregates and thrombi

Wave overtopping erosion tests at Groningen sea dyke

In the present report field erosion tests of the inner slope of a sea dyke in the province of Groningen (near Delfzijl) are described for the situation of severe wave overtopping. Three types of tests have been performed: tests at the present grass cover, tests at a reinforced grass cover and tests at a section of bare clay. At the reinforced grass cover section a provisional Smart Grass Reinforcement (SGR) system was installed in May 2006 (Royal Haskoning & Infram, 2005). The test sections were 4 m wide and extended over about 16 m along the inner slope, down from the dyke crest. It should be remarked that the erosion tests focused on the inner slope from the crest to some distance above the level of the service road. Below the service road, the berm slope was artificially strengthened with riprap as to avoid any damage on beforehand. The tests have been carried out under the framework of ComCoast, Work Package 3 (WP3). Additional measurements have been commissioned by the SBW program (Sterkte & Belastingen Waterkeringen or in English: Strength & Loads Water Defences), a research program for improvement of knowledge of present defences. These measurements included: measurement of flow velocities and water depths, infiltration tests, determination of the soil shear strength and an additional erosion test at a bare clay section. The present report is an overall report, presenting a full overview of all test activities, as far as conducted under the responsibility of the consortium. At a later stage, the SBW measurements will be analysed further within the scope of the SBW program. Where applicable, however, some preliminary results are included in this report. For further information, the present report addresses background reports that were written within the framework of ComCoast or SBW. Special reference is made here to the report on the wave overtopping simulator (Infram & Royal Haskoning, 2007), a summary of which has been presented in the present report in Chapter 4.Civil Engineering and Geoscience

Development of Alternative Overtopping-Resistant Sea Defences, Phase 2: Elaboration of Smart Grass Reinforcement Concept

In the present report, a Smart Grass Reinforcement for overtopping resistant sea defences is elaborated on a theoretical basis, within the framework of ComCoast, Work Package 3 (WP3). The smart grass revetment concept aims at strengthening the present grass revetments at the crests and inners slopes of sea embankments. Smart denotes a significant reinforcement of the grass revetment, a high-cost effectiveness, easy installation with minimum disturbance of the existing grass revetment, hidden presence and thus invisible, and durable. In addition, the system should have no environmental adverse impacts. The Hondsbossche Sea Defence and the North Frysian Sea Defence (grass test sections for studying the effect of maintenance strategies) have been visited in order to get insight in the feasibility of the meanwhile developed ideas in true field situations. Two, potentially feasible, concepts have been identified: 1. Geogrid system, to be placed underneath the temporary lifted (thin-cut) grass layer; 2. Geocell system, to be penetrated through the existing grass revetment, without lifting it. The Geogrid system can be applied at a rather flat surface and good grass cover. The Geocell system can be applied as well at a more uneven surface and a poor grass cover. The proposed application of these systems is new, as far as we know, and some engineering (system, equipment and installation) still needs to be done. At this point, we believe that this will lead to a successful and highly cost-effective application. A cost indication shows that smart application of the Geogrid and Geocell systems will reduce the costs by a factor 10 or more, as compared to raising the crest of the dike. In addition, the risk of applying a smart reinforcement system is much smaller than of regular dike strengthening (raising and extending dikes), as the recovery time of the grass revetment is very short (months at the most) as compared to some years. We recommend the systems to be tested during the anticipated field tests in the ComCoast WP research programme Phase 3.The engineering should preferably be done by the end of this year, in order to be able to apply the smart systems at the field test section by the end of April 2006. This will allow proper field testing in the summer and/or winter of 2006.Civil Engineering and Geoscience

Development of Alternative Overtopping-Resistant Sea Defences: Proposal for concepts

The present proposal deals with a presentation of three potentially feasible concepts for strengthening of the sea defences as to allow for increased wave overtopping, within the framework of ComCoast. In addition, for each concept an indicative proposal is given for a further theoretical study (Phase 2) and an indicative approach is shown for further testing. This proposal is denoted Phase 1: generation of alternative concepts. For the present proposal and the activities involved, as well as possible future assignments within Phase 2, a consortium has been formed between Royal Haskoning and INFRAM (Royal Haskoning, being the leading party), the two partners that also participated in the Inventory Study that preceded this proposal. We think that this combination brings about the outstanding expertise from both firms, which we hope is reflected in the present proposal. The three alternative concepts focus on different types of innovative reinforcement measures, i.e.: reduction of the wave overtopping flow attack, strengthening of the present grass revetments and strengthening of the subsoil. After an introductory chapter, Chapter 1, the relevant failure phenomena are indicated in Chapter 2. The Concepts A (low hedges), B (application of innovative grass reinforcement) and C (application of Smartsoils techniques) developed to cope with these phenomena are presented in Appendices A, B and C and are summarized and discussed briefly in Chapter 3. Chapter 4 presents the indicative set-up for the further theoretical studies of Phase 2 for each concept, whereas in Chapter 5 the approach for testing of the concepts is indicated. The Concepts A and B are highly promising solutions, and we think that they only need limited elaboration and research. The costs are very competitive as compared to the costs for traditional raising of a dike crest (=Reference). The savings can be so big, that further in-depth research on optimization seems anyhow to be useful. Concept C is still highly experimental for application on sea defences and clayey soils. Nevertheless, it would be an interesting option to further explore The charm of these Concepts is that they can also be combined in an economical way (A and B together being still much more economical as the Reference) as to cope with increasing Sea Level Rise in future in a flexible way).Civil Engineering and Geoscience

Platelet Inhibition by Insulin Is Absent in Type 2 Diabetes Mellitus

Objective—ADP-induced P2y12 signaling is crucial for formation and stabilization of an arterial thrombus. We demonstrated recently in platelets from healthy subjects that insulin interferes with Ca2+ increases induced by ADP-P2y1 contact through blockade of the G-protein Gi, and thereby with P2y12-mediated suppression of cAMP

Cyclic AMP raises intracellular Ca(2+) in human megakaryocytes independent of protein kinase A.

The immature megakaryoblastic cell line MEG-01 responds to iloprost with an increase in cytosolic Ca(2+) and cAMP. The Ca(2+) response is almost absent in CHRF-288-11 cells, but cAMP formation is preserved in this more mature megakaryoblastic cell line. Also, in human hematopoietic stem cells, iloprost induces a Ca(2+) response and cAMP formation. The Ca(2+) response is downregulated during megakaryocytopoiesis, but cAMP formation remains unchanged. The Ca(2+) increase may be caused by cAMP-mediated inhibition of Ca(2+) sequestration, because it is (1) independent of Ca(2+) entry; (2) mimicked by forskolin, an activator of adenylyl cyclase, and isobutylmethylxanthine, an inhibitor of phosphodiesterases; and (3) preserved in the presence of inhibitors of protein kinase A and inositol-1,4,5-triphosphate receptors. The small GTPase Rap1 has been implicated in the control of Ca(2+) sequestration. Indeed, Rap1 activation parallels the iloprost- and forskolin-induced Ca(2+) increase and is unaffected by the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid-AM. These findings reveal a novel mechanism for elevating cytosolic Ca(2+) by cAMP, possibly via GTP-Rap1