Role of P-selectin in platelet sequestration in pulmonary capillaries during endotoxemia

Background: There is growing evidence that platelets accumulate in the lung and contribute to the pathogenesis of acute lung injury during endotoxemia. The aims of the present study were to localize platelet sequestration in the pulmonary microcirculation and to investigate the role of P-selectin as a molecular mechanism of platelet endothelial cell interaction. Methods: We used in vivo fluorescence microscopy to quantify the kinetics of fluorescently labeled erythrocytes and platelets in alveolar capillary networks in rabbit lungs. Results: Six hours after onset of endotoxin infusion we observed a massive rolling along and firm adherence of platelets to lung capillary endothelial cells whereas under control conditions no platelet sequestration was detected. P-selectin was expressed on the surface of separated platelets which were incubated with endotoxin and in lung tissue. Pretreatment of platelets with fucoidin, a P-selectin antagonist, significantly attenuated the endotoxin-induced platelet rolling and adherence. In contrast, intravenous infusion of fucoidin in endotoxin-treated rabbits did not inhibit platelet sequestration in pulmonary capillaries. Conclusion: We conclude that platelets accumulate in alveolar capillaries following endotoxemia. P-selectin expressed on the surface of platelets seems to play an important role in mediating this platelet-endothelial cell interaction. Copyright (c) 2006 S. Karger AG, Basel

Cisplatin-induced emesis: systematic review and meta-analysis of the ferret model and the effects of 5-HT3 receptor antagonists

PURPOSE: The ferret cisplatin emesis model has been used for ~30 years and enabled identification of clinically used anti-emetics. We provide an objective assessment of this model including efficacy of 5-HT(3) receptor antagonists to assess its translational validity. METHODS: A systematic review identified available evidence and was used to perform meta-analyses. RESULTS: Of 182 potentially relevant publications, 115 reported cisplatin-induced emesis in ferrets and 68 were included in the analysis. The majority (n = 53) used a 10 mg kg(−1) dose to induce acute emesis, which peaked after 2 h. More recent studies (n = 11) also used 5 mg kg(−1), which induced a biphasic response peaking at 12 h and 48 h. Overall, 5-HT(3) receptor antagonists reduced cisplatin (5 mg kg(−1)) emesis by 68% (45–91%) during the acute phase (day 1) and by 67% (48–86%) and 53% (38–68%, all P < 0.001), during the delayed phase (days 2, 3). In an analysis focused on the acute phase, the efficacy of ondansetron was dependent on the dosage and observation period but not on the dose of cisplatin. CONCLUSION: Our analysis enabled novel findings to be extracted from the literature including factors which may impact on the applicability of preclinical results to humans. It reveals that the efficacy of ondansetron is similar against low and high doses of cisplatin. Additionally, we showed that 5-HT(3) receptor antagonists have a similar efficacy during acute and delayed emesis, which provides a novel insight into the pharmacology of delayed emesis in the ferret

Myocardial Autophagy after Severe Burn in Rats

Autophagy plays a major role in myocardial ischemia and hypoxia injury. The present study investigated the effects of autophagy on cardiac dysfunction in rats after severe burn.Protein expression of the autophagy markers LC3 and Beclin 1 were determined at 0, 1, 3, 6, and 12 h post-burn in Sprague Dawley rats subjected to 30% total body surface area 3rd degree burns. Autophagic, apoptotic, and oncotic cell death were evaluated in the myocardium at each time point by immunofluorescence. Changes of cardiac function were measured in a Langendorff model of isolated heart at 6 h post-burn, and the autophagic response was measured following activation by Rapamycin and inhibition by 3-methyladenine (3-MA). The angiotensin converting enzyme inhibitor enalaprilat, the angiotensin receptor I blocker losartan, and the reactive oxygen species inhibitor diphenylene iodonium (DPI) were also applied to the ex vivo heart model to examine the roles of these factors in post-burn cardiac function.Autophagic cell death was first observed in the myocardium at 3 h post-burn, occurring in 0.008 ± 0.001% of total cardiomyocytes, and continued to increase to a level of 0.022 ± 0.005% by 12 h post-burn. No autophagic cell death was observed in control hearts. Compared with apoptosis, autophagic cell death occurred earlier and in larger quantities. Rapamycin enhanced autophagy and decreased cardiac function in isolated hearts 6 h post-burn, while 3-MA exerted the opposite response. Enalaprilat, losartan, and DPI all inhibited autophagy and enhanced heart function.Myocardial autophagy is enhanced in severe burns and autophagic cell death occurred early at 3 h post-burn, which may contribute to post-burn cardiac dysfunction. Angiotensin II and reactive oxygen species may play important roles in this process by regulating cell signaling transduction

Evaluation of Mode I Fracture Toughness Assisted by the Numerical Determination of K-Resistance

The fracture toughness of a rock often varies depending on the specimen shape and the loading type used to measure it. To investigate the mode I fracture toughness using semi-circular bend (SCB) specimens, we experimentally studied the fracture toughness using SCB and chevron bend (CB) specimens, the latter being one of the specimens used extensively as an International Society for Rock Mechanics (ISRM) suggested method, for comparison. The mode I fracture toughness measured using SCB specimens is lower than both the level I and level II fracture toughness values measured using CB specimens. A numerical study based on discontinuum mechanics was conducted using a two-dimensional distinct element method (DEM) for evaluating crack propagation in the SCB specimen during loading. The numerical results indicate subcritical crack growth as well as sudden crack propagation when the load reaches the maximum. A K-resistance curve is drawn using the crack extension and the load at the point of evaluation. The fracture toughness evaluated by the K-resistance curve is in agreement with the level II fracture toughness measured using CB specimens. Therefore, the SCB specimen yields an improved value for fracture toughness when the increase of K-resistance with stable crack propagation is considered

Morphine activation of mu opioid receptors causes disinhibition of neurons in the ventral tegmental area mediated by β-arrestin2 and c-Src

Abstract The tyrosine kinase, c-Src, participates in mu opioid receptor (MOP) mediated inhibition in sensory neurons in which β-arrestin2 (β-arr2) is implicated in its recruitment. Mice lacking β-arr2 exhibit increased sensitivity to morphine reinforcement; however, whether β-arr2 and/or c-Src participate in the actions of opioids in neurons within the reward pathway is unknown. It is also unclear whether morphine acts exclusively through MOPs, or involves delta opioid receptors (DOPs). We examined the involvement of MOPs, DOPs, β-arr2 and c-Src in the inhibition by morphine of GABAergic inhibitory postsynaptic currents (IPSCs) recorded from neurons in the mouse ventral tegmental area. Morphine inhibited spontaneous IPSC frequency, mainly through MOPs, with only a negligible effect remaining in MOP−/− neurons. However, a reduction in the inhibition by morphine for DOP−/− c.f. WT neurons and a DPDPE-induced decrease of IPSC frequency revealed a role for DOPs. The application of the c-Src inhibitor, PP2, to WT neurons also reduced inhibition by morphine, while the inactive PP3, and the MEK inhibitor, SL327, had no effect. Inhibition of IPSC frequency by morphine was also reduced in β-arr2−/− neurons in which PP2 caused no further reduction. These data suggest that inhibition of IPSCs by morphine involves a β-arr2/c-Src mediated mechanism

Cigarette Smoke-Related Hydroquinone Dysregulates MCP-1, VEGF and PEDF Expression in Retinal Pigment Epithelium in Vitro and in Vivo

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly population. Debris (termed drusen) below the retinal pigment epithelium (RPE) have been recognized as a risk factor for dry AMD and its progression to wet AMD, which is characterized by choroidal neovascularization (CNV). The underlying mechanism of how drusen might elicit CNV remains undefined. Cigarette smoking, oxidative damage to the RPE and inflammation are postulated to be involved in the pathophysiology of the disease. To better understand the cellular mechanism(s) linking oxidative stress and inflammation to AMD, we examined the expression of pro-inflammatory monocyte chemoattractant protein-1 (MCP-1), pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial derived factor (PEDF) in RPE from smoker patients with AMD. We also evaluated the effects of hydroquinone (HQ), a major pro-oxidant in cigarette smoke on MCP-1, VEGF and PEDF expression in cultured ARPE-19 cells and RPE/choroids from C57BL/6 mice.MCP-1, VEGF and PEDF expression was examined by real-time PCR, Western blot, and ELISA. Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls. Both MCP-1 mRNA and protein were downregulated in ARPE-19 cells and RPE/choroids from C57BL/6 mice after 5 days and 3 weeks of exposure to HQ-induced oxidative injury. VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio. Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo.We propose that impaired RPE-derived MCP-1-mediated scavenging macrophages recruitment and phagocytosis might lead to incomplete clearance of proinflammatory debris and infiltration of proangiogenic macrophages which along with increased VEGF/PEDF ratio favoring angiogenesis might promote drusen accumulation and progression to CNV in smoker patients with dry AMD

Rotavirus Rearranged Genomic RNA Segments Are Preferentially Packaged into Viruses Despite Not Conferring Selective Growth Advantage to Viruses

The rotavirus (RV) genome consists of 11 double-stranded RNA segments. Sometimes, partial sequence duplication of an RNA segment leads to a rearranged RNA segment. To specify the impact of rearrangement, the replication efficiencies of human RV with rearranged segments 7, 11 or both were compared to these of the homologous human wild-type RV (wt-RV) and of the bovine wt-RV strain RF. As judged by viral growth curves, rotaviruses with a rearranged genome (r-RV) had no selective growth advantage over the homologous wt-RV. In contrast, r-RV were selected over wt-RV during competitive experiments (i.e mixed infections between r-RV and wt-RV followed by serial passages in cell culture). Moreover, when competitive experiments were performed between a human r-RV and the bovine wt-RV strain RF, which had a clear growth advantage, rearranged segments 7, 11 or both always segregated in viral progenies even when performing mixed infections at an MOI ratio of 1 r-RV to 100 wt-RV. Lastly, bovine reassortant viruses that had inherited a rearranged segment 7 from human r-RV were generated. Although substitution of wt by rearranged segment 7 did not result in any growth advantage, the rearranged segment was selected in the viral progenies resulting from mixed infections by bovine reassortant r-RV and wt-RV, even for an MOI ratio of 1 r-RV to 107 wt-RV. Lack of selective growth advantage of r-RV over wt-RV in cell culture suggests a mechanism of preferential packaging of the rearranged segments over their standard counterparts in the viral progeny

2-Deoxy-D-Glucose Treatment of Endothelial Cells Induces Autophagy by Reactive Oxygen Species-Mediated Activation of the AMP-Activated Protein Kinase

Autophagy is a cellular self-digestion process activated in response to stresses such as energy deprivation and oxidative stress. However, the mechanisms by which energy deprivation and oxidative stress trigger autophagy remain undefined. Here, we report that activation of AMP-activated protein kinase (AMPK) by mitochondria-derived reactive oxygen species (ROS) is required for autophagy in cultured endothelial cells. AMPK activity, ROS levels, and the markers of autophagy were monitored in confluent bovine aortic endothelial cells (BAEC) treated with the glycolysis blocker 2-deoxy-D-glucose (2-DG). Treatment of BAEC with 2-DG (5 mM) for 24 hours or with low concentrations of H2O2 (100 µM) induced autophagy, including increased conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II, accumulation of GFP-tagged LC3 positive intracellular vacuoles, and increased fusion of autophagosomes with lysosomes. 2-DG-treatment also induced AMPK phosphorylation, which was blocked by either co-administration of two potent anti-oxidants (Tempol and N-Acetyl-L-cysteine) or overexpression of superoxide dismutase 1 or catalase in BAEC. Further, 2-DG-induced autophagy in BAEC was blocked by overexpressing catalase or siRNA-mediated knockdown of AMPK. Finally, pretreatment of BAEC with 2-DG increased endothelial cell viability after exposure to hypoxic stress. Thus, AMPK is required for ROS-triggered autophagy in endothelial cells, which increases endothelial cell survival in response to cell stress

Olmesartan/amlodipine vs olmesartan/hydrochlorothiazide in hypertensive patients with metabolic syndrome: the OLAS study

We studied the effects of treatment with olmesartan/amlodipine and olmesartan/hydrochlorothiazide on inflammatory and metabolic parameters (including new-onset diabetes as a secondary endpoint) in non-diabetic hypertensive patients with metabolic syndrome (MetS). A total of 120 patients with MetS and stage I and II hypertension were randomized to olmesartan 20 mg/amlodipine 5 mg or olmesartan 20 mg/hydrochlorothiazide 12.5 mg. If target systolic blood pressure (<140 mm Hg) was not reached, doses were doubled after 13 weeks; doxazosin 4 mg was added after 26 weeks, and doubled after 39 weeks; follow-up ended at 78 weeks. At each visit, blood pressure (BP), fasting plasma glucose, insulin, adiponectin, tumour necrosis factor-α, C-reactive protein (CRP), intercellular adhesion molecule-1, vascular cell adhesion molecule-1, interleukins-1β, -6 and -8, and albuminuria were measured; BP was similarly reduced in both groups; 80% of patients reached target BP. Reductions in albuminuria were also similar (50%). Only olmesartan/amlodipine reduced the insulin resistance index (24%, P<0.01), increased plasma adiponectin (16%, P<0.05) and significantly reduced all of the inflammation markers studied, except CRP, which showed a similar reduction in each group. The risk of new-onset diabetes was significantly lower with olmesartan/amlodipine (P=0.02). Both olmesartan-based combinations were effective, but the amlodipine combination resulted in metabolic and anti-inflammatory effects that may have advantages over the hydrochlorothiazide combination