Brain inflammation plays a critical role in the pathophysiology of brain diseases of high prevalence and economic impact, such as major depression, schizophrenia, post traumatic stress disorder, Parkinson’s and Alzheimer’s disease and traumatic brain injury. Our results demonstrate that systemic administration of the centrally acting Angiotensin II AT1 receptor blocker candesartan to normotensive rats decreases the acute brain inflammatory response to administration of the bacterial endotoxin lipopolysaccharide, a model of brain inflammation. The broad anti-inflammatory effects of candesartan were seen across the entire inflammatory cascade, including decreased production and release to the circulation of centrally acting pro-inflammatory cytokines, repression of nuclear transcription factors activation in the brain, reduction of gene expression of brain pro-inflammatory cytokines, cytokine and prostanoid receptors, adhesion molecules, pro-inflammatory inducible enzymes, and reduced microglia activation. These effects are widespread, occurring not only in well-known brain target areas for circulating pro-inflammatory factors and lipopolysaccharide, i.e. hypothalamic paraventricular nucleus and the subfornical organ, but also in the prefrontal cortex, hippocampus and amygdala. Candesartan reduced the associated anorexic effects, and ameliorated associated body weight loss and anxiety. Direct anti-inflammatory effects of candesartan were also documented in cultured rat microglia, cerebellar granule cells and cerebral microvascular endothelial cells. AT1 receptor blockers are widely used in the treatment of hypertension and stroke, and their anti-inflammatory effects contribute to reduce renal and cardiac failure. Our results indicate that these compounds may offer a novel and safe therapeutic approach for the treatment of brain disorders.

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BackgroundFreezing is promising for extended platelet (PLT) storage for transfusion. 6% DMSO cryopreserved PLTs (CPPs) are currently in clinical development. CPPs contain significant amount of platelet membrane vesicles (PMVs). PLT-membrane changes and PMV release in CPP are poorly understood, and haemostatic effects of CPP PMVs are not fully elucidated. This study aims to investigate PLT-membrane alterations in CPPs and provide comprehensive characterization of CPP PMVs, and their contribution to procoagulant activity (PCA) of CPPs.MethodsCPPs and corresponding liquid-stored PLTs (LSPs) were characterized by flow cytometry (FC), fluorescence polarization (FP), nanoparticle tracking analysis (NTA), electron microscopy (SEM, TEM), atomic force microscopy (AFM) and thrombin-generation (TG) test.ResultsSEM and TEM revealed disintegration and vesiculation of the PLT-plasma membrane and loss of intracellular organization in 60% PLTs in CPPs. FP demonstrated that 6% DMSO alone and with freezing–thawing caused marked increase in PLT-membrane fluidity. The FC counts of annexin V-binding PMVs and CD41a+ PMVs were 68- and 56-folds higher, respectively, in CPPs than in LSPs. The AFM and NTA size distribution of PMVs in CPPs indicated a peak diameter of 100 nm, corresponding to exosome-size vesicles. TG-based PCA of CPPs was 2- and 9-folds higher per PLT and per volume, respectively, compared to LSPs. Differential centrifugation showed that CPP supernatant contributed 26% to CPP TG-PCA, mostly by the exosome-size PMVs and their TG-PCA was phosphatidylserine dependent.ConclusionsMajor portion of CPPs does not show activation phenotype but exhibits grape-like membrane disintegration with significant increase of membrane fluidity induced by 6% DMSO alone and further aggravated by freezing–thawing process. DMSO cryopreservation of PLTs is associated with the release of PMVs and marked increase of TG-PCA, as compared to LSPs. Exosome-size PMVs have significant contribution to PCA of CPPs.

Objective Angiotensin II type 1 receptor (AT1) blockers (ARBs) reduce the bacterial endotoxin lipopolysaccharide (LPS)-induced innate immune response in human circulating monocytes expressing few AT1. To clarify the mechanisms of anti-inflammatory effects of ARBs with different peroxisome proliferator-activated receptor-γ (PPARγ)-activating potencies, we focused our study on telmisartan, an ARB with the highest PPARγ-stimulating activity. Methods Human circulating monocytes and monocytic THP-1 (human acute monocytic leukemia cell line) cells were exposed to 50 ng/ml LPS with or without pre-incubation with telmisartan. AT1 mRNA and protein expressions were determined by real-time PCR and membrane receptor binding assay, respectively. The expression of pro-inflammatory factors was determined by real-time PCR, western blot analysis and ELISA. PPARγ activation was measured by electrophoretic mobility shift assay and its role was determined by pharmacological inhibition and PPARγ gene silencing. Results In human monocytes, telmisartan significantly attenuated the LPS-induced expression of pro-inflammatory factors, the release of pro-inflammatory cytokines and prostaglandin E2, nuclear factor-κB activation and reactive oxygen species formation. In THP-1 cells, telmisartan significantly reduced LPS-induced tumor necrosis factor-α, inhibitor of κB-α, monocyte chemotactic protein-1 (MCP-1) and lectin-like oxidized low-density lipoprotein receptor-1 gene expression and MCP-1-directed migration. Telmisartan also stimulated the expression of the PPARγ target genes cluster of differentiation 36 and ATP-binding cassette subfamily G member 1 in monocytes. The anti-inflammatory effects of telmisartan were prevented by both PPARγ antagonism and PPARγ gene silencing. Anti-inflammatory effects of ARBs correlated with their PPARγ agonist potency. Conclusion Our observations demonstrate that in human monocytes, ARBs inhibit the LPS-induced pro-inflammatory response to a major extent through the PPARγ activation pathway and may be beneficial for the treatment of cardiovascular and metabolic disorders in which inflammation plays a major role.

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Background: This study was undertaken to examine putative mechanisms of calcium independent signal transduction pathway of cell swelling-induced insulin secretion. Methods: The role of phospholipase A₂, G proteins, and soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) in insulin secretion induced by 30% hypotonic medium was studied using isolated rat pancreatic islets. Results: In contrast to glucose stimulation, osmotically induced insulin secretion from pancreatic islets was not inhibited by 10 µmol/l bromoenol lactone, an iPLA₂ (Ca²⁺ independent phospholipase) inhibitor. Similarly, preincubation of islets for 20 hours with 25 µg/ml mycophenolic acid to inhibit GTP synthesis fully abolished glucose-induced insulin secretion but was without effect on hypotonicity stimulated insulin release. Glucose-induced insulin secretion was prevented by preincubation with 20 nmol/l tetanus toxin (TeTx), a metalloprotease inactivating soluble SNARE. Cell swelling-induced insulin secretion was inhibited by TeTx in the presence of calcium ions but not in calcium depleted medium. The presence of N-ethylmaleimide (NEM, 5 mmol/l, another inhibitor of SNARE proteins) in the medium resulted in high basal insulin secretion and lacking response to glucose stimulation. In contrast, high basal insulin secretion from NEM treated islets further increased after hypotonic stimulation. Conclusion: G proteins and iPLA₂ – putative mediators of Ca²⁺ independent signaling pathway participate in glucose but not in hypotonicity-induced insulin secretion. Hypotonicity-induced insulin secretion is sensitive to clostridial neurotoxin TeTx but is resistant to NEM.

Secretion of insulin could be stimulated by several ways. Comparison of glucose-and swelling-induced mechanisms in pancreatic islets revealed the involvement of a novel signal transduction pathway with specific features of osmotically stimulated peptide hormone release including Ca 2+ independence and resistance to noradrenalin (NA) inhibition. Cell swelling can be induced by hypotonicity or small permeant molecules (e.g. ethanol, urea). Our experiments were aimed to compare the effect of these permeants on insulin secretion from natural system -freshly isolated pancreatic islets and rat insulinoma cell lines INS-1 and INS-1E. As expected glucose and both permeants (80 mM ethanol and urea in isosmotic medium) induced insulin release from islets and NA did not inhibit permeant-induced secretion. Although ethanol and urea induced similar swelling of tumor cells, they produced opposite effect on insulin secretion; while exposure to ethanol led to stimulation of insulin secretion, exposure to urea led to suppression in both types of neoplastic cells. Surprisingly, stimulating effect of ethanol was completely suppressed by NA in both tumor cell lines. Ethanol in hyperosmotic medium failed to stimulate and even inhibited insulin release from both tumor cell lines in present study indicating thus involvement of an osmotic component. In conclusion, the opposite effect of ethanol and urea on insulin secretion from insulinoma cells and sensitivity of ethanol stimulation to NA indicate utilization of different cellular signaling pathways in tumor cells as compared to natural β-cells. Participation of permeant effect in the mechanism of ethanol stimulation remains to be clarified.

Alcohol causes reactive hypoglycemia by attenuating the release of counter regulatory hormones, redistribution of pancreatic blood flow and direct stimulation of insulin secretion. Objective of this study was characterization of ethanol-induced insulin secretion. Signaling of ethanol- and glucose-induced insulin release from INS-1 and INS-1E cells was compared. Both cell lines responded similarly to all experimental interventions. In contrast to glucose, ethanol-induced insulin secretion was not hindered in calcium depleted medium or by addition of 10 μM BAPTA/AM (intracellular chelator). Inhibitor of protein kinase C Bisindolylmaleimide (3 μM) abolished glucose- but not ethanol-induced insulin secretion. Tetanus toxin (20 nM), inhibitor of SNARE proteins complex formation, blocked ethanol-induced insulin secretion. Both 5 mM N-ethylamaleimide and 10 μM ZnCl₂ (inhibitor of protein tyrosine phosphatases), which block disassembly of SNARE complexes and their further participation in exocytosis, increased basal insulin secretion. In contrast to glucose, already high insulin secretion was further increased after ethanol stimulation in either treatment. Conclusion: Signaling of ethanol-induced insulin secretion from INS-1 and INS-1E cell lines bypasses calcium and PKC involving steps, is sensitive to tetanus toxin but resistant to N-ethymaleimide and ZnCl₂. An extra pool of secretory vesicles not available for glucose is exploited for exocytosis after ethanol stimulation.