Hisayo Sameshima

Edaravone attenuates cerebral ischemic injury by suppressing aquaporin-4

Aquaporin-4 (AQP4) plays a role in the generation of post-ischemic edema. Pharmacological modulation of AQP4 function may thus provide a novel therapeutic strategy for the treatment of stroke, tumor-associated edema, epilepsy, traumatic brain injury, and other disorders of the central nervous system (CNS) associated with altered brain water balance. Edaravone, a free radical scavenger, is used for the treatment of acute ischemic stroke (AIS) in Japan. In this study, edaravone significantly reduced the infarct area and improved the neurological deficit scores at 24h after reperfusion in a rat transient focal ischemia model. Furthermore, edaravone markedly reduced AQP4 immunoreactivity and protein levels in the cerebral infarct area. In light of observations that edaravone specifically inhibited AQP4 in a rat transient focal ischemia model, we propose that edaravone might reduce cerebral edema through the inhibition of AQP4 expression following cerebral infarction.

A microchip flow-chamber system for quantitative assessment of the platelet thrombus formation process.

As the pathogenesis of arterial thrombosis often includes platelet thrombus formation (PTF), antiplatelet agents are commonly used for the prevention of thromboembolic events. Here, using a novel microchip flow-chamber system we developed to quantitatively analyze the PTF process, we evaluated the pharmacological efficacies of antiplatelet agents under different arterial shear rates. Hirudin-anticoagulated whole blood was perfused over a collagen-coated microchip at shear rates of 1000, 1500, and 2000s(-1), and PTF in the absence and presence of various antiplatelet agents was observed microscopically and quantified by measuring flow-pressure changes. The onset of PTF was measured as T(10) (time to reach 10 kPa), and AUC(10) (area under the flow pressure curve for the first 10 min) was calculated to quantify the overall stability of the formed thrombus. Aspirin and AR-C66096 (P2Y(12)-antagonist) at high concentrations (50 μM and 1000 nM, respectively) prolonged T(10) only modestly (AR-C66096>aspirin), but effectively decreased AUC(10), resulting in unstable PTF at all examined shear rates. With dual inhibition using both aspirin (25 μM) and ARC-66096 (250 nM), AUC(10) was drastically reduced. Nearly complete suppression of AUC(10) was also observed with abciximab (2 μg ml(-1)) and beraprost (PGI(2)-analog; 4 nM). Although OS-1 (GPIbα-antagonist; 100 nM) prevented complete capillary occlusion, significant amounts of microscopic thrombi were observed on the collagen surface. In contrast to abciximab and beraprost, OS-1 differentially affected PTF under higher shear conditions. Our novel analytical system is capable of distinguishing the pharmacological effects of various antiplatelet agents under physiological shear rates, suggesting that this system may aid in the determination of the appropriate type and dose of antiplatelet agent in the clinical setting.

Analysing responses to aspirin and clopidogrel by measuring platelet thrombus formation under arterial flow conditions

High residual platelet aggregability and circulating platelet-monocyte aggregates in patients administered aspirin and clopidogrel are associated with ischaemic vascular events. To determine the relevance of these factors with residual thrombogenicity, we measured platelet thrombus formation using a microchip-based flow-chamber system in cardiac patients receiving aspirin and/or clopidogrel, and evaluated its correlation with agonist-inducible platelet aggregation and platelet-monocyte aggregates. Platelet thrombus formation was analysed by measuring flow pressure changes due to the occlusion of micro-capillaries and was quantified by calculating AUC 10 (area under the flow pressure curve. The growth and stability of platelet thrombi that formed inside microchips at shear rates of 1000, 1500, and 2000 s⁻¹ were markedly reduced in patients receiving aspirin and/or thienopyridine compared to healthy controls (n=33). AUC 10 values of aspirin therapy patients (n=20) were significantly lower and higher than those of healthy controls and dual antiplatelet therapy patients (n=19), respectively, and showed relatively good correlations with collagen-induced platelet aggregation and platelet-monocyte aggregates at 1000 and 1500 s⁻¹ (rs >0.59, p<0.01). In contrast, AUC 10 in dual antiplatelet therapy patients was significantly correlated with ADP-induced platelet aggregation at all examined shear rates (rs >0.59, p<0.01), but did not correlate with collagen-induced aggregation. Aspirin monotherapy patients with high residual platelet thrombogenicity also exhibited significant elevations in both collagen-induced platelet aggregation and platelet-monocyte aggregates. Our results, although preliminary, suggest that residual platelet thrombogenicity in aspirin-treated patients is associated with either collagen-induced platelet aggregation or circulating platelet-monocyte aggregates, but it is predominantly dependent on ADP-induced platelet aggregation in patients receiving dual antiplatelet therapy.

Minocycline attenuates both OGD-induced HMGB1 release and HMGB1-induced cell death in ischemic neuronal injury in PC12 cells.

High mobility group box-1 (HMGB1), a non-histone DNA-binding protein, is massively released into the extracellular space from neuronal cells after ischemic insult and exacerbates brain tissue damage in rats. Minocycline is a semisynthetic second-generation tetracycline antibiotic which has recently been shown to be a promising neuroprotective agent. In this study, we found that minocycline inhibited HMGB1 release in oxygen-glucose deprivation (OGD)-treated PC12 cells and triggered the activation of p38mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK1/2). The ERK kinase (MEK)1/2 inhibitor U-0126 and p38MAPK inhibitor SB203580 blocked HMGB1 release in response to OGD. Furthermore, HMGB1 triggered cell death in a dose-dependent fashion. Minocycline significantly rescued HMGB1-induced cell death in a dose-dependent manner. In light of recent observations as well as the good safety profile of minocycline in humans, we propose that minocycline might play a potent neuroprotective role through the inhibition of HMGB1-induced neuronal cell death in cerebral infarction.

Nucleophosmin may act as an alarmin: implications for severe sepsis.

NPM is a major nucleolar multifunctional protein involved in ribosome biogenesis, centrosome duplication, cell‐cycle progression, apoptosis, cell differentiation, and sensing cellular stress. Alarmins are endogenous molecules released from activated cells and/or dying cells, which activate the immune system and cause severe damage to cells and tissue organs. In the present work, stimulation of cells with the alarmin‐inducible molecule endotoxin, for 16 h, resulted in NPM release into the culture supernatants of RAW264.7 cells, a murine macrophage cell line. Extracellular NPM was detected in the ascites of the CLP model. NPM was translocated into the cytoplasm from the nucleus in LPS ‐stimulated RAW264.7 cells; furthermore, NPM was detected in the cytosols of infiltrated macrophages in the CLP model. rNPM induced release of proinflammatory cytokines, TNF‐α, IL‐6, and MCP‐1, from RAW264.7 cells and increased the expression level of ICAM‐1 in HUVECs. NPM induced the phosphorylation of MAPKs in RAW264.7 cells. Our data indicate that NPM may have potent biological activities that contribute to systemic inflammation. Further investigations of the role of NPM may lead to new therapies for patients with septic shock or other inflammatory diseases.

Comparative evaluation of direct thrombin and factor Xa inhibitors with antiplatelet agents under flow and static conditions: an in vitro flow chamber model.

Dabigatran and rivaroxaban are novel oral anticoagulants that specifically inhibit thrombin and factor Xa, respectively. The aim of this study is to elucidate antithrombotic properties of these anticoagulant agents under arterial and venous shear conditions. Whole blood samples treated with dabigatran or rivaroxaban at 250, 500, and 1000 nM, with/without aspirin and AR-C66096, a P2Y12 antagonist, were perfused over a microchip coated with collagen and tissue thromboplastin at shear rates of 240 and 600 s−1. Fibrin-rich platelet thrombus formation was quantified by monitoring flow pressure changes. Dabigatran at higher concentrations (500 and 1000 nM) potently inhibited thrombus formation at both shear rates, whereas 1000 nM of rivaroxaban delayed, but did not completely inhibit, thrombus formation. Dual antiplatelet agents weakly suppressed thrombus formation at both shear rates, but intensified the anticoagulant effects of dabigatran and rivaroxaban. The anticoagulant effects of dabigatran and rivaroxaban were also evaluated under static conditions using thrombin generation (TG) assay. In platelet-poor plasma, dabigatran at 250 and 500 nM efficiently prolonged the lag time (LT) and moderately reduce peak height (PH) of TG, whereas rivaroxaban at 250 nM efficiently prolonged LT and reduced PH of TG. In platelet-rich plasma, however, both anticoagulants efficiently delayed LT and reduced PH of TG. Our results suggest that dabigatran and rivaroxaban may exert distinct antithrombotic effects under flow conditions, particularly in combination with dual antiplatelet therapy.

Antithrombotic effects of PAR1 and PAR4 antagonists evaluated under flow and static conditions

INTRODUCTION Thrombin-mediated activation of human platelets involves the G-protein-coupled protease-activated receptors PAR1 and PAR4. Inhibition of PAR1 and/or PAR4 is thought to modulate platelet activation and subsequent procoagulant reactions. However, the antithrombotic effects of PAR1 and PAR4 antagonism have not been fully elucidated, particularly under flow conditions. MATERIALS AND METHODS A microchip-based flow chamber system was used to evaluate the influence of SCH79797 (PAR1 antagonist) and YD-3 (PAR4 antagonist) on thrombus formation mediated by collagen and tissue thromboplastin at shear rates simulating those experienced in small- to medium-sized arteries (600s(-1)) and large arteries and small veins (240s(-1)). RESULTS At a shear rate of 600s(-1), SCH79797 (10μM) efficiently reduced fibrin-rich platelet thrombi and significantly delayed occlusion of the flow chamber capillary (1.44 fold of control; P<0.001). The inhibitory activity of SCH79797 was diminished at 240s(-1). YD-3 (20μM) had no significant effect at either shear rate. The antithrombotic effects of SCH79797 were significantly augmented when combined with aspirin and AR-C66096 (P2Y12 antagonist), but not with YD-3. In contrast, no significant inhibition of tissue factor-induced clot formation under static conditions was observed in blood treated with SCH79797 and YD-3, although thrombin generation in platelet-rich plasma was weakly delayed by these antagonists. CONCLUSIONS Our results suggest that the antithrombotic activities of PAR1 and/or PAR4 antagonism is influenced by shear conditions as well as by combined platelet inhibition with aspirin and a P2Y12-antagonist.

Attenuation of LPS-induced iNOS expression by 1,5-anhydro-D-fructose

1,5-anhydro-d-fructose (1,5-AF), a monosaccharide formed from starch and glycogen, exhibits antioxidant and antibacterial activity, and inhibits cytokine release by attenuating NF-kappaB activation in LPS-stimulated mice. The present study examined whether 1,5-AF inhibits lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) in vitro and in vivo. We found that 1,5-AF significantly blocked the production of NO, and protein and mRNA expression of iNOS, and up-regulated IL-10 production in vitro. We also investigated the effects of 1,5-AF on acute lung inflammation in C57BL/6J mice. We found that protein and mRNA expression of iNOS in lung tissues were inhibited by 1,5-AF pretreatment. In addition, the serum level of IL-10 was upregulated by 1,5-AF. Collectively, the iNOS transcriptional and translational inhibitory effects of 1,5-AF seem to be prolonged and enhanced by the production of IL-10. These results suggest that 1,5-AF could be a useful adjunct in the treatment of acute lung inflammation.

1,5-Anhydro-d-fructose attenuates lipopolysaccharide-induced cytokine release via suppression of NF-κB p65 phosphorylation

Lipopolysaccharide (LPS) stimulates macrophages by activating NF-kappaB, which contributes to the release of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. 1,5-Anhydro-D-fructose (1,5-AF), a monosaccharide formed from starch and glycogen, exhibits anti-oxidant activity and enhances insulin secretion. This study examined the effects of 1,5-AF on LPS-induced inflammatory reactions and elucidated its molecular mechanisms. Before LPS challenge, mice were pretreated with 1,5-AF (38.5 mg/kg). We found that 1,5-AF pretreatment attenuated cytokine release into the serum, including TNF-alpha, IL-6 and macrophage chemoattractant protein (MCP)-1. Furthermore, pretreatment with 1,5-AF (500 microg/ml) attenuated cytokine release, and 1,5-AF directly inhibited the nuclear translocalization of the NF-kappaB p65 subunit in LPS-stimulated murine macrophage-like RAW264.7 cells. This inhibition was responsible for decreased LPS-induced phosphorylation on Ser536 of the NF-kappaB p65 subunit, which is a posttranslational modification involved in the non-canonical pathway. Collectively, these findings indicate that the anti-inflammatory activity of 1,5-AF occurs via inactivation of NF-kappaB.

Edaravone, a Synthetic Free Radical Scavenger, Enhances Alteplase-Mediated Thrombolysis

The combination of alteplase, a recombinant tissue plasminogen activator, and edaravone, an antioxidant, reportedly enhances recanalization after acute ischemic stroke. We examined the influence of edaravone on the thrombolytic efficacy of alteplase by measuring thrombolysis using a newly developed microchip-based flow-chamber assay. Rat models of embolic cerebral ischemia were treated with either alteplase or alteplase-edaravone combination therapy. The combination therapy significantly reduced the infarct volume and improved neurological deficits. Human blood samples from healthy volunteers were exposed to edaravone, alteplase, or a combination of alteplase and edaravone or hydrogen peroxide. Whole blood was perfused over a collagen- and thromboplastin-coated microchip; capillary occlusion was monitored with a video microscope and flow-pressure sensor. The area under the curve (extent of thrombogenesis or thrombolysis) at 30 minutes was 69.9% lower in the edaravone-alteplase- than alteplase-treated group. The thrombolytic effect of alteplase was significantly attenuated in the presence of hydrogen peroxide, suggesting that oxidative stress might hinder thrombolysis. D-dimers were measured to evaluate these effects in human platelet-poor plasma samples. Although hydrogen peroxide significantly decreased the elevation of D-dimers by alteplase, edaravone significantly inhibited the decrease. Edaravone enhances alteplase-mediated thrombolysis, likely by preventing oxidative stress, which inhibits fibrinolysis by alteplase in thrombi.