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Targeting cancer cells that are highly dependent on the nicotinamide adenine dinucleotide (NAD+) metabolite is a promising therapeutic strategy. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme catalyzing NAD + production. Despite the high efficacy of several developed NAMPT inhibitors (i.e., FK866 (APO866)) in preclinical studies, their clinical activity was proven to be limited. Here, we report the synthesis of new NAMPT Inhibitors, JJ08, FEI191 and FEI199, which exhibit a broad anticancer activity in vitro. Results show that these compounds are potent NAMPT inhibitors that deplete NAD + and NADP(H) after 24 h of drug treatment, followed by an increase in reactive oxygen species (ROS) accumulation. The latter event leads to ATP loss and mitochondrial depolarization with induction of apoptosis and necrosis. Supplementation with exogenous NAD + precursors or catalase (ROS scavenger) abrogates the cell death induced by the new compounds. Finally, in vivo administration of the new NAMPT inhibitors in a mouse xenograft model of human Burkitt lymphoma delays tumor growth and significantly prolongs mouse survival. The most promising results are collected with JJ08, which completely eradicates tumor growth. Collectively, our findings demonstrate the efficient anticancer activity of the new NAMPT inhibitor JJ08 and highlight a strong interest for further evaluation of this compound in hematological malignancies.

Rationale: How endothelial cells (ECs) migrate and form an immature vascular plexus has been extensively studied. Yet, mechanisms underlying vascular remodeling remain poorly established. A better understanding of these processes may lead to the design of novel therapeutic strategies complementary to current angiogenesis inhibitors. Objective: Starting from our previous observations that PP2A (protein phosphatase 2) regulates the HIF (hypoxia-inducible factor)/PHD-2 (prolyl hydroxylase 2)-constituted oxygen machinery, we hypothesized that this axis could play an important role during blood vessel formation, tissue perfusion, and oxygen restoration. Methods and Results: We show that the PP2A regulatory subunit B55α is at the crossroad between vessel pruning and vessel maturation. Blood vessels with high B55α counter cell stress conditions and thrive for stabilization and maturation. When B55α is inhibited, ECs cannot cope with cell stress and undergo apoptosis, leading to massive pruning of nascent blood vessels. Mechanistically, we found that the B55α/PP2A complex restrains PHD-2 activity, promoting EC survival in a HIF-dependent manner, and furthermore dephosphorylates p38, altogether protecting ECs against cell stress occurring, for example, during the onset of blood flow. In tumors, EC-specific B55α deficiency induces pruning of immature-like tumor blood vessels resulting in delayed tumor growth and metastasis, without affecting nonpathological vessels. Consistently, systemic administration of a pan-PP2A inhibitor disrupts vascular network formation and tumor progression in vivo without additional effects on B55α-deficient vessels. Conclusions: Our data underline a unique role of the B55α/PP2A phosphatase complex in vessel remodeling and suggest the use of PP2A-inhibitors as potent antiangiogenic drugs targeting specifically nascent blood vessels with a mode-of-action complementary to VEGF-R (vascular endothelial growth factor receptor)-targeted therapies. Graphical Abstract: A graphical abstract is available for this article.

Interleukin (IL)-6 is an important mediator of neurovascular dysfunction, neurodegeneration and/or neuroinflammation. We previously reported that brain pericytes released higher levels of IL-6 than did glial cells (astrocytes and microglia) in response to tumor necrosis factor (TNF)-?. Moreover, pericytes stimulated with TNF-? enhanced activation of BV-2 microglia. In this study, we investigated the mechanisms of TNF-? mediated induction of IL-6 release from brain pericytes and astrocytes and whether pericyte-derived IL-6 would facilitate activation of BV-2 microglia. Using rat brain pericyte and astrocyte primary cultures and pharmacological inhibitors, we found that, TNF-? induced the highest levels of IL-6 release from pericytes by activating the inhibitor kappa B (I?B)-nuclear factor kappa-light-chain-enhancer of activated B cells (NF?B) and Janus family of tyrosine kinase (JAK)-signal transducer and activator of transcription (STAT)3 pathways. STAT3 contributed to TNF-? induced nuclear translocation of phospho-NF?B in pericytes. TNF-?-induced IL-6 release in astrocytes was mediated by NF?B but not by STAT3. The presence of pericytes amplified TNF-?-induced iNOS mRNA expression in BV-2 microglia. This effect was blocked by a neutralizing antibody for IL-6. These findings indicated that crosstalk between the I?B-NF?B and JAK-STAT3 pathways is a pericyte specific mechanism, not occurring in astrocytes, for TNF-?-induced IL-6 release. IL-6 derived from pericytes enhanced microglial activation. Our findings increase understanding of the role of pericyte-microglia crosstalk in the brain under neuroinflammatory conditions and suggest a potentially attractive therapeutic target for brain inflammation. © 2018 Elsevier B.V.

Background Gap junction channels made of Connexin37 (Cx37) are expressed by aortic endothelial and smooth muscle cells of hypertensive mice, as well as by the renin‐secreting cells of kidneys. Methods and Results To decipher whether Cx37 has any role in hypertension, angiotensin II (Ang II ) was infused in normotensive wild‐type and Cx37‐deficient mice (Cx37−/−). After 2 to 4 weeks, the resulting increase in blood pressure was lower in Cx37−/− than in wild‐type mice, suggesting an alteration in the Ang II response. To investigate this possibility, mice were submitted to a 2‐kidney, 1‐clip procedure, a renin‐dependent model of hypertension. Two weeks after this clipping, Cx37−/− mice were less hypertensive than wild‐type mice and, 2 weeks later, their blood pressure had returned to control values, in spite of abnormally high plasma renin levels. In contrast, Cx37−/− and wild‐type mice that received N ‐nitro‐ l ‐arginine‐methyl‐ester, a renin‐independent model of hypertension, featured a similar and sustained increase in blood pressure. The data indicate that loss of Cx37 selectively altered the Ang II ‐dependent pathways. Consistent with this conclusion, aortas of Cx37−/− mice featured an increased basal expression of the Ang II type 2 receptors ( AT 2R), and increased transcripts levels of downstream signaling proteins, such as Cnksr1 and Ptpn6 ( SHP ‐1). Accordingly, the response of Cx37−/− mice aortas to an ex vivo Ang II exposure was altered, since phosphorylation levels of several proteins of the Ang II pathway ( MLC 2, ERK , and AKT ) remained unchanged. Conclusions These findings provide evidence that Cx37 selectively influences Ang II signaling, mostly via a modulation of the expression of the Ang II type 2 receptor.

Cholesterol metabolism is subject to complex transcriptional and nontranscriptional regulation. Herein, the role of ubiquitylation is emerging as an important post-translational modification that regulates cholesterol synthesis and uptake. Similar to other post-translational modifications, ubiquitylation is reversible in a process dependent on activity of deubiquitylating enzymes (DUBs). Yet whether these play a role in cholesterol metabolism is largely unknown. As a first step to test this possibility, we used pharmacological inhibition of cellular DUB activity. Short term (2 h) inhibition of DUBs resulted in accumulation of high molecular weight ubiquitylated proteins. This was accompanied by a dramatic decrease in abundance of the LDLR and attenuated LDL uptake into hepatic cells. Importantly, this occurred in the absence of changes in the mRNA levels of the LDLR or other SREBP2-regulated genes, in line with this phenotype being a post-transcriptional event. Mechanistically, we identify transcriptional induction of the E3 ubiquitin ligase IDOL in human and rodent cells as the underlying cause for ubiquitylation-dependent lysosomal degradation of the LDLR following DUB inhibition. In contrast to the established transcriptional regulation of IDOL by the sterol-responsive liver X receptor (LXR) transcription factors, induction of IDOL by DUB inhibition is LXR-independent and occurs in Lxrαβ(-/-) MEFs. Consistent with the role of DUBs in transcriptional regulation, we identified a 70-bp region in the proximal promoter of IDOL, distinct from that containing the LXR-responsive element, which mediates the response to DUB inhibition. In conclusion, we identify a sterol-independent mechanism to regulate IDOL expression and IDOL-mediated lipoprotein receptor degradation.

Prolyl oligopeptidase (PREP) is a serine protease that has been studied particularly in the context of neurode-generative diseases for decades but its physiological function has remained unclear. We have previously found that PREP negatively regulates beclinl-mediated macroautophagy (autophagy), and that PREP inhibition by a small-molecule inhibitor induces clearance of protein aggregates in Parkinson's disease models. Since autophagy induction has been suggested as a potential therapy for several diseases, we wanted to further characterize how PREP regulates autophagy. We measured the levels of various kinases and proteins regulating beclin1-autophagy in HEK-293 and SH-SY5Y cell cultures after PREP inhibition, PREP deletion, and PREP overexpression and restoration, and verified the results in vivo by using PREP knock-out and wild-type mouse tissue where PREP was restored or overexpressed, respectively. We found that PREP regulates autophagy by interacting with protein phosphatase 2A (PP2A) and its endogenous inhibitor, protein phosphatase methylesterase 1 (PME1), and activator (protein phosphatase 2 phosphatase activator, PTPA), thus adjusting its activity and the levels of PP2A in the intracellular pool. PREP inhibition and deletion increased PP2A activity, leading to activation of deathassociated protein kinase 1 (DAPK1), beclin1 phosphorylation and induced autophagy while PREP overexpression reduced this. Lowered activity of PP2A is connected to several neurodegenerative disorders and cancers, and PP2A activators would have enormous potential as drug therapy but development of such compounds has been a challenge. The concept of PREP inhibition has been proved safe, and therefore, our study supports the further development of PREP inhibitors as PP2A activators.

In endothelial cells, agonist-induced Ca(2+) entry takes place via the store-operated Ca(2+) entry pathway and/or via channel(s) gated by second messengers. As cell stimulation leads to both a partial Ca(2+) store depletion as well as the production of second messengers, these two pathways are problematic to distinguish. We showed that passive endoplasmic reticulum (ER) depletion by thapsigargin or cell stimulation by histamine activated a similar Ca(2+)-release-activated Ca(2+) current (CRAC)-like current when 10 mM Ba(2+)/2 mM Ca(2+) was present in the extracellular solution. Importantly, during voltage clamp recordings, histamine stimulation largely depleted the ER Ca(2+) store, explaining the activation of a CRAC-like current (due to store depletion) upon histamine in Ba(2+) medium. On the contrary, in the presence of 10 mM Ca(2+), the ER Ca(2+) content remained elevated, and histamine induced an outward rectifying current that was inhibited by Ni(2+) and KB-R7943, two blockers of the Na(+)/Ca(2+) exchanger (NCX). Both blockers also reduced histamine-induced cytosolic Ca(2+) elevation. In addition, removing extracellular Na(+) increased the current amplitude which is in line with a current supported by the NCX. These data are consistent with the involvement of the NCX working in reverse mode (Na(+) out/Ca(2+) in) during agonist-induced Ca(2+) entry in endothelial cells.

AIMS: Atherosclerosis is the leading cause of death in Western societies and a chronic inflammatory disease. However, the key mediators linking recruitment of inflammatory cells to atherogenesis remain poorly defined. Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme, which plays a role in acute inflammatory diseases. METHODS AND RESULTS: In order to test the role of PARP in atherogenesis, we applied chronic pharmacological PARP inhibition or genetic PARP1 deletion in atherosclerosis-prone apolipoprotein E-deficient mice and measured plaque formation, adhesion molecules, and features of plaque vulnerability. After 12 weeks of high-cholesterol diet, plaque formation in male apolipoprotein E-deficient mice was decreased by chronic inhibition of enzymatic PARP activity or genetic deletion of PARP1 by 46 or 51%, respectively (P < 0.05, n >or= 9). PARP inhibition or PARP1 deletion reduced PARP activity and diminished expression of inducible nitric oxide synthase, vascular cell adhesion molecule-1, and P- and E-selectin. Furthermore, chronic PARP inhibition reduced plaque macrophage (CD68) and T-cell infiltration (CD3), increased fibrous cap thickness, and decreased necrotic core size and cell death (P < 0.05, n >or= 6). CONCLUSION: Our data provide pharmacological and genetic evidence that endogenous PARP1 is required for atherogenesis in vivo by increasing adhesion molecules with endothelial activation, enhancing inflammation, and inducing features of plaque vulnerability. Thus, inhibition of PARP1 may represent a promising therapeutic target in atherosclerosis.

SummaryPharmacological treatments targeting CXC chemokines and the associated neutrophil activation and recruitment into atherosclerotic plaques hold promise for treating cardiovascular disorders. Therefore, we investigated whether FK866, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with anti-inflammatory properties that we recently found to reduce neutrophil recruitment into the ischaemic myocardium, would exert beneficial effects in a mouse atherosclerosis model. Atherosclerotic plaque formation was induced by carotid cast implantation in ApoE-/- mice that were fed with a Western-type diet. FK866 or vehicle were administrated intraperitoneally from week 8 until week 11 of the diet. Treatment with FK866 reduced neutrophil infiltration and MMP-9 content and increased collagen levels in atherosclerotic plaques compared to vehicle. No effect on other histological parameters, including intraplaque lipids or macrophages, was observed. These findings were associated with a reduction in both systemic and intraplaque CXCL1 levels in FK866-treated mice. In vitro, FK866 did not affect MMP-9 release by neutrophils, but it strongly reduced CXCL1 production by endothelial cells which, in the in vivo model, were identified as a main CXCL1 source at the plaque level. CXCL1 synthesis inhibition by FK866 appears to reflect interference with nuclear factor-κB signalling as shown by reduced p65 nuclear levels in endothelial cells pre-treated with FK866. In conclusion, pharmacological inhibition of NAMPT activity mitigates inflammation in atherosclerotic plaques by reducing CXCL1-mediated activities on neutrophils. These results support further assessments of NAMPT inhibitors for the potential prevention of plaque vulnerability.

Background: Early cytokine dysregulation upon infection with highly pathogenic avian influenza viruses (HPAIV) is a major determinant of viral pathogenicity. Results: p38 MAPK controls HPAIV-induced gene expression by regulating interferon synthesis and subsequently interferon signaling, whereas its inhibition protects mice from lethal infection. Conclusion: p38 MAPK is crucial for the induction of hypercytokinemia upon infection. Significance: Targeting p38 MAPK is a promising approach for antiviral intervention., Highly pathogenic avian influenza viruses (HPAIV) induce severe inflammation in poultry and men. One characteristic of HPAIV infections is the induction of a cytokine burst that strongly contributes to viral pathogenicity. This cell-intrinsic hypercytokinemia seems to involve hyperinduction of p38 mitogen-activated protein kinase. Here we investigate the role of p38 MAPK signaling in the antiviral response against HPAIV in mice as well as in human endothelial cells, the latter being a primary source of cytokines during systemic infections. Global gene expression profiling of HPAIV-infected endothelial cells in the presence of the p38-specific inhibitor SB 202190 revealed that inhibition of p38 MAPK leads to reduced expression of IFNβ and other cytokines after H5N1 and H7N7 infection. More than 90% of all virus-induced genes were either partially or fully dependent on p38 signaling. Moreover, promoter analysis confirmed a direct impact of p38 on the IFNβ promoter activity. Furthermore, upon treatment with IFN or conditioned media from HPAIV-infected cells, p38 controls interferon-stimulated gene expression by coregulating STAT1 by phosphorylation at serine 727. In vivo inhibition of p38 MAPK greatly diminishes virus-induced cytokine expression concomitant with reduced viral titers, thereby protecting mice from lethal infection. These observations show that p38 MAPK acts on two levels of the antiviral IFN response. Initially the kinase regulates IFN induction and, at a later stage, p38 controls IFN signaling and thereby expression of IFN-stimulated genes. Thus, inhibition of MAP kinase p38 may be an antiviral strategy that protects mice from lethal influenza by suppressing excessive cytokine expression.

Nicotinamide adenine dinucleotide (NAD+) biosynthesis from nicotinamide is used by mammalian cells to replenish their NAD+ stores and to avoid unwanted nicotinamide accumulation. Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the key enzyme in this biosynthetic pathway, almost invariably leads to intracellular NAD+ depletion and, when protracted, to ATP shortage and cell demise. Cancer cells and activated immune cells express high levels of NAMPT and are highly susceptible to NAMPT inhibitors, as shown by the activity of these agents in models of malignant and inflammatory disorders. As the spectrum of conditions which could benefit from pharmacological NAMPT inhibition becomes broader, the mechanisms accounting for their activity are also eventually becoming apparent, including the induction of autophagy and the impairment of Ca2+--and NF-κB-dependent signaling. Here, we discuss the rationales for exploiting NAMPT inhibitors in cancer and inflammatory diseases and provide an overview of the preclinical and clinical studies in which these agents have been evaluated.

BACKGROUND: Vitreoretinal disorders, including proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR) and exudative age-related macular degeneration (AMD), are a major cause of visual impairment worldwide and can lead to blindness when untreated. Loss of blood-retinal barrier (BRB) integrity associated with vitreoretinal fibrin deposition, inflammation, fibrosis and neovascularization contribute to the pathophysiological processes in these disorders. Retinal pigment epithelial (RPE) cells are well recognized to contribute to vitreoretinal inflammation/fibrosis and are likely to encounter contact with coagulation factor upon loss of BRB integrity. METHODS: An extensive study was performed in which we examined the effect of factor Xa and thrombin on the production of a broad panel of cytokines/chemokines and growth factors by RPE cells. For this purpose we used the ARPE-19 cell line as well as primary RPE cells, a glass slide based array that allows simultaneous detection of 120 cytokines/chemokines and growth factors, ELISA and real-time-quantitative PCR. The involved signaling cascade was examined using specific inhibitors for protease activated receptor (PAR)1, PAR2 and nuclear factor kappa-B (NF-κB). RESULTS: Factor Xa and thrombin regulated the production of cytokines and growth factors (including GM-CSF, IL-6, IL-8, MCP-3, PDGF-AA, PDGF-BB, TIMP-1 and TGF-α) that fit well in the pathobiology of vitreoretinal disease. Blocking studies revealed that the effects were mediated via PAR1 induced NF-κB activation. CONCLUSIONS: Our findings suggest that factor Xa and thrombin can drive vitreoretinal inflammation and fibrosis and should be considered as treatment targets in vitreoretinal disorders such as PVR, PDR and AMD.

Objective— Antioxidative drugs continue to be developed for the treatment of atherosclerosis. Apocynin is an nicotinamide adenine dinucleotide phosphate oxidase inhibitor with anti-inflammatory properties. We used contrast-enhanced ultrasound molecular imaging to assess whether short-term apocynin therapy in atherosclerosis reduces vascular oxidative stress and endothelial activation Approach and Results— Genetically modified mice with early atherosclerosis were studied at baseline and after 7 days of therapy with apocynin (4 mg/kg per day IP) or saline. Contrast-enhanced ultrasound molecular imaging of the aorta was performed with microbubbles targeted to vascular cell adhesion molecule 1 (VCAM-1; MB V ), to platelet glycoprotein Ibα (MB Pl ), and control microbubbles (MB Ctr ). Aortic vascular cell adhesion molecule 1 was measured using Western blot. Aortic reactive oxygen species generation was measured using a lucigenin assay. Hydroethidine oxidation was used to assess aortic superoxide generation. Baseline signal for MB V (1.3±0.3 AU) and MB Pl (1.5±0.5 AU) was higher than for MB Ctr (0.5±0.2 AU; P P V : 0.3±0.1; MB Pl : 0.4±0.1; MB Ctr : 0.3±0.2 AU; P =0.6 between agents). Apocynin reduced aortic vascular cell adhesion molecule 1 expression by 50% ( P Conclusions— Short-term treatment with apocynin in atherosclerosis reduces endothelial cell adhesion molecule expression. This change in endothelial phenotype can be detected by molecular imaging before any measurable decrease in macrophage content and is not associated with a detectable change in oxidative burden.

Objective— Hyperuricemia is common in patients with metabolic syndrome. We investigated the role of xanthine oxidoreductase (XOR) in atherosclerosis development, and the effects of the XOR inhibitor allopurinol on this process. Methods and Results— Oral administration of allopurinol to ApoE knockout mice markedly ameliorated lipid accumulation and calcification in the aorta and aortic root. In addition, allopurinol treatment or siRNA-mediated gene knockdown of XOR suppressed transformation of J774.1 murine macrophage cells, treated with acetylated LDL or very low density lipoprotein (VLDL) into foam cells. This inhibitory effect of allopurinol was also observed in primary cultured human macrophages. In contrast, overexpression of XOR promoted transformation of J774.1 cells into foam cells. Interestingly, SR-A1, SR-B1, SR-B II, and VLDL receptors in J774.1 cells were reduced by XOR knockdown, and increased by XOR overexpression. Conversely, expressions of ABCA1 and ABCG1 were increased by XOR knockdown and suppressed by XOR overexpression. Finally, productions of inflammatory cytokines accompanied by foam cell formation were also reduced by allopurinol administration. Conclusion— These results strongly suggest XOR activity and/or its expression level to contribute to macrophage foam cell formation. Thus, XOR inhibitors may be useful for preventing atherosclerosis.

Anabolic androgenic steroids (AAS) are testosterone derivatives used either clinically, in elite sports, or for body shaping with the goal to increase muscle size and strength. Clinically developed compounds and nonclinically tested designer steroids often marketed as food supplements are widely used. Despite the considerable evidence for various adverse effects of AAS use, the underlying molecular mechanisms are insufficiently understood. Here, we investigated whether some AAS, as a result of a lack of target selectivity, might inhibit 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2)-dependent inactivation of glucocorticoids. Using recombinant human 11β-HSD2, we observed inhibitory effects for several AAS. Whereas oxymetholone, oxymesterone, danazol, and testosterone showed medium inhibitory potential, fluoxymesterone was a potent inhibitor of human 11β-HSD2 (half-maximal inhibitory concentration [IC(50)] of 60-100nM in cell lysates; IC(50) of 160nM in intact SW-620, and 530nM in MCF-7 cells). Measurements with rat kidney microsomes and lysates of cells expressing recombinant mouse 11β-HSD2 revealed much weaker inhibition by the AAS tested, indicating that the adverse effects of AAS-dependent 11β-HSD2 inhibition cannot be investigated in rats and mice. Furthermore, we provide evidence that fluoxymesterone is metabolized to 11-oxofluoxymesterone by human 11β-HSD2. Structural modeling revealed similar binding modes for fluoxymesterone and cortisol, supporting a competitive mode of inhibition of 11β-HSD2-dependent cortisol oxidation by this AAS. No direct modulation of mineralocorticoid receptor (MR) function was observed. Thus, 11β-HSD2 inhibition by fluoxymesterone may cause cortisol-induced MR activation, thereby leading to electrolyte disturbances and contributing to the development of hypertension and cardiovascular disease.

Catechol-O-methyltransferase (COMT) is an important enzyme in the metabolism of dopamine and disturbance in dopamine function is proposed to be central to the pathogenesis of schizophrenia. Clinical epidemiological studies have indicated cannabis use to confer a 2-fold increase in risk for subsequent onset of psychosis, with adolescent-onset use conveying even higher risk. There is evidence that a high activity COMT polymorphism moderates the effects of adolescent exposure to cannabis on risk for adult psychosis. In this paper we compared the effect of chronic adolescent exposure to the cannabinoid WIN 55212 on sensorimotor gating, behaviours related to the negative symptoms of schizophrenia, anxiety- and stress-related behaviours, as well as ex-vivo brain dopamine and serotonin levels, in COMT KO vs. wild-type (WT) mice. Additionally, we examined the effect of pretreatment with the COMT inhibitor tolcapone on acute effects of this cannabinoid on sensorimotor gating in C57BL/6 mice. COMT KO mice were shown to be more vulnerable than WT to the disruptive effects of adolescent cannabinoid treatment on prepulse inhibition (PPI). Acute pharmacological inhibition of COMT in C57BL/6 mice also modified acute cannabinoid effects on startle reactivity, as well as PPI, indicating that chronic and acute loss of COMT can produce dissociable effects on the behavioural effects of cannabinoids. COMT KO mice also demonstrated differential effects of adolescent cannabinoid administration on sociability and anxiety-related behaviour, both confirming and extending earlier reports of COMT×cannabinoid effects on the expression of schizophrenia-related endophenotypes.

SummaryDecreased β cell mass and function are hallmarks of type 2 diabetes. Here we identified, through a siRNA screen, beta site amyloid precursor protein cleaving enzyme 2 (Bace2) as the sheddase of the proproliferative plasma membrane protein Tmem27 in murine and human β cells. Mice with functionally inactive Bace2 and insulin-resistant mice treated with a newly identified Bace2 inhibitor both display augmented β cell mass and improved control of glucose homeostasis due to increased insulin levels. These results implicate Bace2 in the control of β cell maintenance and provide a rational strategy to inhibit this protease for the expansion of functional pancreatic β cell mass.

Chronic hepatitis C virus (HCV) infection is characterized by increased rates of apoptotic hepatocytes and activated caspases have been shown in HCV-infected patients. GS-9450, a novel caspase-inhibitor has demonstrated hepatoprotective activity in fibrosis/apoptosis animal models. This study evaluated the effects of GS-9450 on peripheral T-cell apoptosis in chronic HCV-infected patients. As sub study of the GS-US-227-0102, a double-blind, placebo-controlled phase 2a trial evaluating the safety and tolerability of GS-9450, apoptosis of peripheral CD4+ and CD8+ T-cells was measured using activated caspase-3, activated caspase-8 and CD95 (Fas). Blood samples were drawn at baseline, day 14 after therapy and at 5 weeks off-treatment follow-up in the first cohort of 10 mg. In contrast to the placebo-treated patients, GS-9450 caused a median of 46% decrease in ALT-values from baseline to day 14 in all treated patients (median of 118-64 U/l) rising again to a median of 140 U/l (19%) at 5 weeks off-treatment follow-up. In GS9450-treated patients, during treatment and follow-up, percentages of activated caspase-3+ and caspase-8 expression tended to decrease, in contrast to placebo-treated patients. Interestingly, compared to healthy controls, higher percentages of caspase-3 and caspase-8 positive CD4+ and CD8+ T-cells were demonstrated in HCV-infected patients at baseline. Decreased ALT-values were observed in all HCV-infected patients during treatment with low dose of the caspase-inhibitor GS-9450 accompanied by a lower expression of caspase-3 and -8 on peripheral T-cells. Furthermore, at baseline percentages of activated caspase-3, activated caspase-8 and CD95+ T-cells were higher in chronic HCV-infected patients compared to healthy controls.

Rationale : Although Nox5 (Nox2 homolog) has been identified in the vasculature, its regulation and functional significance remain unclear. Objectives : We sought to test whether vasoactive agents regulate Nox5 through Ca 2+ /calmodulin-dependent processes and whether Ca 2+ -sensitive Nox5, associated with Rac-1, generates superoxide (O 2 ·− ) and activates growth and inflammatory responses via mitogen-activated protein kinases in human endothelial cells (ECs). Methods and Results : Cultured ECs, exposed to angiotensin II (Ang II) and endothelin (ET)-1 in the absence and presence of diltiazem (Ca 2+ channel blocker), calmidazolium (calmodulin inhibitor), and EHT1864 (Rac-1 inhibitor), were studied. Nox5 was downregulated with small interfering RNA. Ang II and ET-1 increased Nox5 expression (mRNA and protein). Effects were inhibited by actinomycin D and cycloheximide and blunted by diltiazem, calmidazolium and low extracellular Ca 2+ ([Ca 2+ ] e ). Ang II and ET-1 activated NADPH oxidase, an effect blocked by low [Ca 2+ ] e , but not by EHT1864. Nox5 knockdown abrogated agonist-stimulated O 2 ·− production and inhibited phosphorylation of extracellular signal-regulated kinase (ERK)1/2, but not p38 MAPK (mitogen-activated protein kinase) or SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase). Nox5 small interfering RNA blunted Ang II-induced, but not ET-1-induced, upregulation of proliferating-cell nuclear antigen and vascular cell adhesion molecule-1, important in growth and inflammation. Conclusions : Human ECs possess functionally active Nox5, regulated by Ang II and ET-1 through Ca 2+ /calmodulin-dependent, Rac-1-independent mechanisms. Nox5 activation by Ang II and ET-1 induces ROS generation and ERK1/2 phosphorylation. Nox5 is involved in ERK1/2-regulated growth and inflammatory signaling by Ang II but not by ET-1. We elucidate novel mechanisms whereby vasoactive peptides regulate Nox5 in human ECs and demonstrate differential Nox5-mediated functional responses by Ang II and ET-1. Such phenomena link Ca 2+ /calmodulin to Nox5 signaling, potentially important in the regulation of endothelial function by Ang II and ET-1.

Extracellular matrix has a beneficial impact on β-cell spreading and function, but the underlying signaling pathways have yet to be fully elucidated. In other cell types, Rho, a well-characterized member of the family of Rho GTPases, and its effector Rho-associated kinase (ROCK), play an important role as downstream mediators of outside in signaling from extracellular matrix. Therefore, a possible role of the Rho-ROCK pathway in β-cell spreading, actin cytoskeleton dynamics, and function was investigated. Rho was inhibited using a new cell-permeable version of C3 transferase, whereas the activity of ROCK was repressed using the specific ROCK inhibitors H-1152 and Y-27632. Inhibition of Rho and of ROCK increased spreading and improved both short-term and prolonged glucose-stimulated insulin secretion but had no impact on basal secretion. Inhibition of this pathway led to a depolymerization of the actin cytoskeleton. Furthermore, the impact of the inhibition of ROCK on stimulated insulin secretion was acute and reversible, suggesting that rapid signaling such as phosphorylation is involved. Finally, quantification of the activity of RhoA indicated that the extracellular matrix represses RhoA activity. Overall these results show for the first time that the Rho-ROCK signaling pathway contributes to the stabilization of the actin cytoskeleton and inhibits glucose-stimulated insulin secretion in primary pancreatic β-cells. Furthermore, they indicate that inhibition of this pathway might be one of the mechanisms by which the extracellular matrix exerts its beneficial effects on pancreatic β-cell function.

International audience; Two distinct native alpha-bungarotoxin (alpha-Bgt)-insensitive nicotinic acetylcholine receptors (nAChRs), named nAChR1 and nAChR2, were identified in the cockroach Periplaneta americana dorsal unpaired median (DUM) neurons. They differed in their electrophysiological, pharmacological properties and intracellular regulation pathways. nAChR2 being an atypical nicotinic receptor closed upon agonist application and its current-voltage relationship resulted from a reduction in potassium conductance. In this study, using whole-cell patch-clamp technique, we demonstrated that calcium modulated nAChR2-mediated nicotine response. Under 0.5 microM alpha-Bgt and 20 mM d-tubocurarine, the nicotine-induced inward current amplitude was strongly reduced in the presence of intracellularly applied BAPTA or bath application of calcium-free solution. In addition, using cadmium chloride, we showed that nicotine response was modulated by extracellular calcium through plasma membrane calcium channels. Moreover, extracellular application of caffeine and thapsigargin reduced nAChR2-mediated response. Together these experiments revealed a complex calcium-dependent regulation of nAChR2.

The members of the NOX/DUOX family of NADPH oxidases mediate such physiologic functions as host defense, cell signaling, and thyroid hormone biosynthesis through the generation of reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide. Moreover, ROS are involved in a broad range of fundamental biochemical and cellular processes, and data accumulated in recent years indicate that the NOX enzymes comprise one of the most important biological sources of ROS. Given the high biochemical reactivity of ROS, it is not surprising that they have been implicated in a wide variety of pathologies and diseases. Prominent among the settings that feature ROS-mediated tissue injury are disorders associated with inflammation, aging, and progressive degenerative changes in cells and organ systems, and it appears that essentially no organ system is exempt. Among the disorders currently believed to be mediated at least in part by NOX-derived ROS are hypertension, aortic aneurysm, myocardial infarction (and other ischemia-reperfusion disorders), pulmonary fibrosis and hypertension, amyotropic lateral sclerosis, Alzheimer's disease, Parkinson's disease, ischemic stroke, diabetic nephropathy, and renal cell carcinoma. Several small-molecule and peptide inhibitors of the NOX enzymes have been useful in experimental studies, but issues of specificity, potency, and toxicity militate against any of the existing published compounds as candidates for drug development. Given the broad array of disease targets documented in recent work, the time is here for vigorous efforts to develop clinically useful inhibitors of the NOX enzymes. As most (though not all) NOX-related diseases appear to be mediated by a single member of the NOX family, agents with isoform specificity will be preferred, although broadly active NOX inhibitors may prove to be useful in some settings.

Early evidence demonstrates that exogenous nitric oxide (NO) and the NO produced by inducible nitric oxide synthase (iNOS) can induce insulin resistance. Here, we investigated whether this insulin resistance, mediated by S-nitrosation of proteins involved in early steps of the insulin signal transduction pathway, could be reversed by acute physical exercise. Rats on a high-fat diet were subjected to swimming for two 3 h-long bouts, separated by a 45 min rest period. Two or 16 h after the exercise protocol the rats were killed and proteins from the insulin signalling pathway were analysed by immunoprecipitation and immunoblotting. We demonstrated that a high-fat diet led to an increase in the iNOS protein level and S-nitrosation of insulin receptor beta (IR beta), insulin receptor substrate 1 (IRS1) and Akt. Interestingly, an acute bout of exercise reduced iNOS expression and S-nitrosation of proteins involved in the early steps of insulin action, and improved insulin sensitivity in diet-induced obesity rats. Furthermore, administration of GSNO (NO donor) prevents this improvement in insulin action and the use of an inhibitor of iNOS (L-N6-(1-iminoethyl)lysine; L-NIL) simulates the effects of exercise on insulin action, insulin signalling and S-nitrosation of IR beta, IRS1 and Akt. In summary, a single bout of exercise reverses insulin sensitivity in diet-induced obese rats by improving the insulin signalling pathway, in parallel with a decrease in iNOS expression and in the S-nitrosation of IR/IRS1/Akt. The decrease in iNOS protein expression in the muscle of diet-induced obese rats after an acute bout of exercise was accompanied by an increase in AMP-activated protein kinase (AMPK) activity. These results provide new insights into the mechanism by which exercise restores insulin sensitivity.

Strong evidence suggests that phospholipase Cγ1 (PLCγ1) is a suitable target to counteract tumourigenesis and metastasis dissemination. We recently identified a novel signalling pathway required for PLCγ1 activation which involves formation of a protein complex with 3-phosphoinositide-dependent protein kinase 1 (PDK1). In an effort to define novel strategies to inhibit PLCγ1-dependent signals we tested here whether a newly identified and highly specific PDK1 inhibitor, 2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5), could affect PDK1/PLCγ1 interaction and impair PLCγ1-dependent cellular functions in cancer cells. Here, we demonstrate that 2-O-Bn-InsP5 interacts specifically with the pleckstrin homology domain of PDK1 and impairs formation of a PDK1/PLCγ1 complex. 2-O-Bn-InsP5 is able to inhibit the epidermal growth factor-induced PLCγ1 phosphorylation and activity, ultimately resulting in impaired cancer cell migration and invasion. Importantly, we report that 2-O-Bn-InsP5 inhibits cancer cell dissemination in zebrafish xenotransplants. This work demonstrates that the PDK1/PLCγ1 complex is a potential therapeutic target to prevent metastasis and it identifies 2-O-Bn-InsP5 as a leading compound for development of anti-metastatic drugs.

Background: The development of collateral circulation is proposed as an inherent compensatory mechanism to restore impaired blood perfusion after ischemia, at least in the penumbra. We have studied the dynamic macro- and microcirculation after ischemia-reperfusion in the juvenile rat brain and evaluated the impact of neuronal nitric oxide synthase (nNOS) inhibition on the collateral flow. Methods: Fourteen-day-old (P14) rats were subjected to ischemia-reperfusion and treated with either PBS or 7-nitroindazole (7-NI, an nNOS inhibitor, 25 mg/kg). Arterial blood flow (BF) was measured using 2D-color-coded pulsed ultrasound imaging. Laser speckle contrast (LSC) imaging and sidestream dark-field videomicroscopy were used to measure cortical and microvascular BF, respectively. Results: In basal conditions, 7-NI reduced BF in the internal carotids (by ∼25%) and cortical (by ∼30%) BF, as compared to PBS. During ischemia, the increased mean BF velocity in the basilar trunk after both PBS and 7-NI demonstrated the establishment of collateral support and patency. Upon re-flow, BF immediately recovered to basal values in the internal carotid arteries under both conditions. The 7-NI group showed increased collateral flow in the penumbral tissue during early re-flow compared to PBS, as shown with both LSC imaging and side-stream dark-field videomicroscopy. The proportion of perfused capillaries was significantly increased under 7-NI as compared to PBS when given before ischemia (67.0 ± 3.9 vs. 46.8 ± 8.8, p < 0.01). Perfused capillaries (63.1 ± 17.7 vs. 81.1 ± 20.7, p < 0.001) and the BF index (2.4 ± 0.6 vs. 1.3 ± 0.1, p < 0.001) significantly increased under 7-NI given at the re-flow onset. Conclusions: Collateral support in the penumbra is initiated during ischemia, and may be increased during early re-flow by neuronal NOS inhibition (given in pre- and post-treatment), which may preserve brain tissue in juvenile rats.

Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease., Author Summary Neutrophil infiltration is characteristic of immune-induced pathology in tuberculosis but mechanisms whereby neutrophils cause tissue destruction are not fully understood. In this study, we show that neutrophils secrete the collagenase MMP-8 in response to direct infection with Mycobacterium tuberculosis and via cellular networks. MMP-8 is up-regulated in respiratory samples from TB patients, driving matrix destruction associated with neutrophil activation and reflects disease severity. Neutrophils are present adjacent to the wall of TB cavities in human histology specimens. The metabolic pathway AMP-activated protein kinase (AMPK) regulates neutrophil MMP-8 secretion with data supported by studies in human neutrophils from AMPK-deficient patients. Host-directed therapy against neutrophil MMP-8 may reduce innate-immune mediated tissue damage in TB.

In his letter, Dr. Pick criticizes our use of relative values when representing the NOX2 inhibitory action of a novel small molecule (GSK2795039) in a semi-recombinant NOX2 membrane assay. To address this concern, we performed additional experiments using the superoxide inhibitable assays cytochrome C and water soluble tetrazolium salt (WST-1) reduction. In this letter, we document turnover values between 80 and 100 mol O2(•-)/s/mol cytochrome b558 in our semi-recombinant assay and confirmed that GSK2795039 inhibits the NOX2 isoform in the submicromolar range. Antioxid. Redox Signal. 23, 1251-1253.

Luteolin and quercetin are widely distributed plant flavonoids that possess a variety of chemical and biological activities, including free-radical scavenging and antioxidant activity. Recently, both flavonoids have been reported to inhibit DNA topoisomerases I and II (topo I and topo II), a property that, together with their ability to induce DNA and chromosome damage, has made them candidate anticancer compounds. In the present study, we confirmed that both compounds are topo II inhibitors by conducting a comparative study of their effect on topo II activity from Chinese hamster ovary AA8 cells. Because interference with the function of topo II to resolve DNA entanglement at the end of replication results in chromosome malsegregation at mitosis, we investigated whether luteolin and quercetin are effective in inducing endoreduplication in AA8 cells. Concentrations of luteolin and quercetin that inhibited topo II catalytic activity resulted in extraordinarily high yields of metaphases showing diplochromosomes. Given the established relationship of polyploidy with tumor development via aneuploidy and genetic instability, these results question the usefulness of luteolin and quercetin in cancer therapy.

Drugs of abuse induce long-lasting changes in neural circuits that may underlie core components of addiction. Here we focus on glutamatergic synapses onto dopamine (DA) neurons of the ventral tegmental area (VTA). Using an 'ex vivo' approach in mice, we show that a single injection of cocaine caused strong rectification and conferred sensitivity to the polyamine Joro spider toxin (JST) of AMPAR-mediated excitatory postsynaptic currents (AMPAR EPSCs), indicating the recruitment of receptors that lack GluR2. This qualitative change in transmission was paralleled by an increase in the AMPAR:NMDAR ratio and was prevented by interfering with the protein interacting with C kinase-1 (PICK1) in vivo. Activation of metabotropic glutamate receptors (mGluR1s) by intraperitoneal injection of a positive modulator depotentiated synapses and abolished rectification in slices of cocaine-treated mice, revealing a mechanism to reverse cocaine-induced synaptic plasticity in vivo.

The growth of both normal and transformed epithelial cells of the female reproductive system is stimulated by estrogens, mainly through the activation of estrogen receptor α (ERα), which is a ligand-regulated transcription factor. The selective ER modulator tamoxifen (TAM) has been widely used as an ER antagonist in breast tumor; however, long-term treatment is associated with an increased risk of endometrial cancer. To provide new insights into the potential mechanisms involved in the agonistic activity exerted by TAM in the uterus, we evaluated the potential of 4-hydroxytamoxifen (OHT), the active metabolite of TAM, to transactivate wild-type ERα and its splice variant expressed in Ishikawa and HEC1A endometrial tumor cells, respectively. OHT was able to antagonize only the activation of ERα by 17β-estradiol (E2) in Ishikawa cells, whereas it up-regulated c-fos expression in a rapid manner similar to E2 and independently of ERα in both cell lines. This stimulation occurred through the G protein-coupled receptor named GPR30 and required Src-related and epidermal growth factor receptor tyrosine kinase activities, along with the activation of both ERK1/2 and phosphatidylinositol 3-kinase/AKT pathways. Most importantly, OHT, like E2, stimulated the proliferation of Ishikawa as well as HEC1A cells. Transfecting a GPR30 antisense expression vector in both endometrial cancer cell lines, OHT was no longer able to induce growth effects, whereas the proliferative response to E2 was completely abrogated only in HEC1A cells. Furthermore, in the presence of the inhibitors of MAPK and phosphatidylinositol 3-kinase pathways, PD 98059 and wortmannin, respectively, E2 and OHT did not elicit growth stimulation. Our data demonstrate a new mode of action of E2 and OHT in endometrial cancer cells, contributing to a better understanding of the molecular mechanisms involved in their uterine agonistic activity.

Nicotine, a component of tobacco, is highly addictive but possesses beneficial properties such as cognitive improvements and memory maintenance. Involved in these processes is the neuronal nicotinic acetylcholine receptor (nAChR) α7, whose activation triggers depolarization, intracellular signaling cascades, and synaptic plasticity underlying addiction and cognition. It is therefore important to investigate intracellular mechanisms by which a cell regulates α7 nAChR activity. We have examined the role of phosphorylation by combining molecular biology, biochemistry, and electrophysiology in SH-SY5Y neuroblastoma cells,Xenopusoocytes, rat hippocampal interneurons, and neurons from the supraoptic nucleus, and we found tyrosine phosphorylation of α7 nAChRs. Tyrosine kinase inhibition by genistein decreased α7 nAChR phosphorylation but strongly increased acetylcholine-evoked currents, whereas tyrosine phosphatase inhibition by pervanadate produced opposite effects. Src-family kinases (SFKs) directly interacted with the cytoplasmic loop of α7 nAChRs and phosphorylated the receptors at the plasma membrane. SFK inhibition by PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656 (2,3-dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide) increased α7 nAChR-mediated responses, whereas expression of active Src reduced α7 nAChR activity. Mutant α7 nAChRs lacking cytoplasmic loop tyrosine residues because of alanine replacement of Tyr-386 and Tyr-442 were more active than wild-type receptors and insensitive to kinase or phosphatase inhibition. Because the amount of surface α7 receptors was not affected by kinase or phosphatase inhibitors, these data show that functional properties of α7 nAChRs depend on the tyrosine phosphorylation status of the receptor and are the result of a balance between SFKs and tyrosine phosphatases. These findings reveal novel regulatory mechanisms that may help to understand nicotinic receptor-dependent plasticity, addiction, and pathology.

In addition to promoting insulin secretion, an increase in cytosolic Ca2+triggered by glucose has been shown to be crucial for spreading of β-cells attached on extracellular matrix (804G matrix). Calpains are Ca2+-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca2+-induced insulin secretion and spreading of rat pancreatic β-cells. The results indicate calpain dependency of β-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibitors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox-iranecarboxylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secretory response after 24 h. In addition, incubation with calpeptin did not affect insulin secretory response to mastoparan that acts on exocytosis downstream of intracellular calcium [Ca2+]i. Finally, calpeptin was shown to affect the [Ca2+]iresponse to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary pancreatic β-cells. It is therefore suggested that calpain could be a mediator of Ca2+-induced-insulin secretion and β-cell spreading.

Induction of long-term potentiation and application of short periods of anoxia/hypoglycemia result in the growth of dendritic filopodia and formation of new spines. Here we investigated whether these conditions also affected the morphology of presynaptic structures. Using confocal imaging of DiI-labeled axons, electron microscopy, and stereological analyses, we show that short anoxia/hypoglycemia and theta burst stimulation induced rapid, calcium-dependent growth of presynaptic filopodia-like protrusions and remodeling of presynaptic varicosities. Three-dimensional reconstruction of axonal outgrowths revealed that, within 30 min, they made contacts and triggered the formation of a postsynaptic density on the target cell. Interestingly, these axonal filopodia first established synapses with the dendritic shaft and later mostly with spines. They also contributed to the formation of multi-innervated spines. Because these presynaptic growth mechanisms depended on NMDA receptor activation, we investigated whether a diffusing messenger could be involved. We found that blockade of nitric oxide synthase prevented these changes, and conversely, a nitric oxide donor could reproduce them. A model is presented that proposes that activation of NMDA receptors and subsequent release of nitric oxide could trigger the growth of presynaptic filopodia, which, in turn, play an active role in synaptogenesis and spine formation.

The kidney medulla is physiologically exposed to variations in extracellular osmolality. In response to hypertonic cell shrinkage, cells of the rat kidney medullary thick ascending limb of Henle's loop undergo p38 kinase-dependent regulatory volume increase (RVI). In the present study, we investigated the role of actin cytoskeleton reorganization in this process. Addition of hyperosmotic NaCl or sucrose, which activates MAP kinases and reduces cellular volume, induced a sustained actin polymerization occurring after 10 min and concurrently with RVI. In contrast, hyperosmotic urea, which does not modify MAP kinase activity and cellular volume, did not induce sustained actin polymerization. Fluorescence microscopy revealed that hyperosmotic NaCl and sucrose, but not urea, induced the redistribution of F-actin from a dense cortical ring to a diffuse network of actin bundles. Stabilization of actin filaments by jasplakinolide and inhibition of the generation of new actin filaments by swinholide A prevented RVI, whereas depolymerization of actin filaments by latrunculin B attenuated cell shrinkage and enhanced RVI. These actin-interfering drugs did not alter extracellular regulated kinase and p38 kinase activation under hypertonic conditions. Similar to swinholide A, inhibiting p38 kinase with SB-203580 abolished sustained actin polymerization, actin redistribution, and decreased RVI efficacy. We therefore propose that in rat kidney the medullary thick ascending limb of Henle's loop exposed to extracellular hypertonicity, p38 kinase activation induces depolymerization of the F-actin cortical ring and polymerization of a dense diffuse F-actin network that both contribute to increase RVI efficacy.

Contains fulltext : 184929.pdf (Publisher’s version ) (Closed access) A new assay was developed to measure the N-deacetylase activity of the glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs), which are key enzymes in sulfation of heparan sulfate (HS)/heparin. The assay is based on the recognition of NDST-generated N-unsubstituted glucosamine units in Escherichia coli K5 capsular polysaccharide or in HSs by monoclonal antibody JM-403. Substrate specificity and potential product inhibition of the NDST isoforms 1 and 2 were analyzed by comparing lysates of human 293 kidney cells stably transfected with mouse NDST-1 or -2. We found HSs to be excellent substrates for both NDST enzymes. Both NDST-1 and -2 N-deacetylate heparan sulfate from human aorta ( approximately 0.6 sulfate groups/disaccharide) with comparable high efficiency, apparent Km values of 0.35 and 0.76 microM (calculation based on [HexA]) being lower (representing a higher affinity) than those for K5 polysaccharide (13.3 and 4.7 microM, respectively). Comparison of various HS preparations and the unsulfated K5 polysaccharide as substrates indicate that both NDST-1 and -2 can differentially N-sulfate polysaccharides already modified to some extent by various other enzymes involved in HS/heparin synthesis. Both enzymes were equally inhibited by N-sulfated sequences (>or=6 sugar residues) present in N-sulfated K5, N-deacetylated N-resulfated HS, and heparin. Our primary findings were confirmed in the conventional N-deacetylase assay measuring the release of 3H-acetate of radiolabeled K5 or HS as substrates. We furthermore showed that NDST N-deacetylase activity in crude cell/tissue lysates can be partially blocked by endogenous HS/heparin. We speculate that in HS biosynthesis, some NDST variants initiate HS modification/sulfation reactions, whereas other (or the same) NDST isoforms later on fill in or extend already modified HS sequences.

Systemic as well as hippocampal administration of nNOS inhibitors induces antidepressant-like effects in animal models. However, the mechanisms underlying these effects have not been completely understood. Evidence has suggested that nNOS inhibition increases serotonin signaling in the brain. Moreover, activation of prosencephalic 5HT1A receptors is considered to mediate stress coping and antidepressant effects. On this basis, the aim of this study was to investigate the hypothesis that the antidepressant-like effect induced by nNOS inhibition in the dorsal hippocampus (DH) would involve local serotonergic signaling and 5HT1A receptor activation. Therefore, rats were subjected to the forced swimming test and received microinjections of fluoxetine, NPA (Nω-propyl-L-arginine, selective nNOS inhibitor), WAY100635 (5HT1A antagonist), and vehicle, alone or in combination, into the DH. Exposure to the forced swimming test increased nitric oxide acid levels in the DH. The administration of NPA or fluoxetine in the DH induced dose-dependent antidepressant-like effects. WAY100635 microinjection in the DH did not induce any effect per se, but it counteracted NPA-induced and fluoxetine-induced effects. These results suggest that the antidepressant-like effect induced by the administration of nNOS inhibitors in the DH involves local serotonergic signaling and 5HT1A receptor activation.

International audience; The microphthalmia-associated transcription factor (MITF) is indispensable for the viability of melanocytic cells, is an oncogene in melanoma and has a cell type-specific expression pattern. As the modulation of MITF activity by direct chemical targeting remains a challenge, we assessed a panel of drugs for their ability to downregulate MITF expression or activity by targeting its upstream modulators. We found that the multi-kinase inhibitors midostaurin and sunitinib downregulate MITF protein levels. To identify the target molecules shared by both the drugs in melanocytic cells, a chemical proteomic approach was applied and AMP-activated kinase (AMPK) was identified as the relevant target for the observed phenotype. RNA interference and chemical inhibition of AMPK led to a decrease in MITF protein levels. Reduction of MITF protein levels was the result of proteasomal degradation, which was preceded by enhanced phosphorylation of MITF mediated by ERK. As expected, downregulation of MITF protein levels by AMPK inhibition was associated with decreased viability. Together, these results identify AMPK as an important regulator for the maintenance of MITF protein levels in melanocytic cells.

Item does not contain fulltext We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show tight negative correlation between Parp-1 expression and energy expenditure in heterogeneous mouse populations, indicating that variations in PARP-1 activity have an impact on metabolic homeostasis. Notably, these genetic correlations can be translated into pharmacological applications. Long-term treatment with PARP inhibitors enhances fitness in mice by increasing the abundance of mitochondrial respiratory complexes and boosting mitochondrial respiratory capacity. Furthermore, PARP inhibitors reverse mitochondrial defects in primary myotubes of obese humans and attenuate genetic defects of mitochondrial metabolism in human fibroblasts and C. elegans. Overall, our work validates in worm, mouse, and human models that PARP inhibition may be used to treat both genetic and acquired muscle dysfunction linked to defective mitochondrial function.

Background Myocardial perfusion during adenosine-induced hyperemia is used both in clinical diagnosis of coronary heart disease and for scientific investigations of the myocardial microcirculation. The objective of this study was to clarify whether adenosine-induced hyperemia is dependent on endothelial NO production or is influenced by adrenergic mechanisms. Methods and Results In 12 healthy men, myocardial perfusion was measured with PET in 2 protocols performed in random order, each including 3 perfusion measurements. First, perfusion was measured at rest. Second, either saline or the NO synthase inhibitor N G -nitro- l -arginine methyl ester (L-NAME, 4 mg/kg) was infused, and perfusion during adenosine-induced hyperemia was determined. Last, in both protocols, the α-receptor blocker phentolamine was infused, and perfusion during adenosine-induced hyperemia was determined again. Resting perfusion was similar in the 2 protocols (0.69±0.14 and 0.66±0.18 mL · min −1 · g −1 ). L-NAME increased mean arterial blood pressure by 12±7 mm Hg ( P P −1 · g −1 ) was attenuated by L-NAME (1.50±0.55 mL · min −1 · g −1 , P −1 · g −1 , P =NS). In the presence of L-NAME, however, when the adenosine response was attenuated, phentolamine was able to increase hyperemic perfusion (2.05±0.44 mL · min −1 · g −1 , P Conclusions Inhibition of endogenous NO synthesis attenuates myocardial perfusion during adenosine-induced hyperemia, indicating that coronary vasodilation by adenosine is partly endothelium dependent. α-Adrenergic blockade has no effect on adenosine-induced hyperemia unless NO synthesis is inhibited.

Rat islet beta-cells spread in response to glucose when attached on the matrix produced by a rat bladder carcinoma cell line (804G). Furthermore, in a mixed population of cells, it has been observed previously that spread cells secrete more insulin acutely in response to glucose, compared with cells that remain rounded. These results suggest bi-directional signaling between the islet beta-cell and the extracellular matrix. In the present study, the role of increased intracellular free Ca2+ concentration [Ca2+]i as an intracellular step linking glucose stimulation and beta-cell spreading (inside-out signaling) was investigated. Purified rat beta-cells were attached to this matrix and incubated under various conditions known to affect [Ca2+]i. The effect of glucose on beta-cell spreading was mimicked by 25 mmol/l KCl (which induces calcium influx) and inhibited by diazoxide (which impairs depolarization and calcium entry) and by the L-type Ca2+ channel blocker SR-7037. When a 24-h incubation at 16.7 glucose was followed by 24 h at 2.8 mmol/l, beta-cells that had first spread regained a round phenotype. In the presence of thapsigargin, spreading progressed throughout the experiment, suggesting that capture of calcium by the endoplasmic reticulum is involved in the reversibility of spreading previously induced by glucose. Spreading was still observed in degranulated beta-cells and in botulinum neurotoxin E-expressing beta-cells when exocytosis was prevented. In summary, the results indicate that increased [Ca2+]i is required for the glucose-induced spreading of beta-cells on 804G matrix and that it is not a consequence of exocytotic processes that follow elevation of [Ca2+]i.

Reference EPFL-ARTICLE-147759doi:10.1152/ajpheart.00709.2006View record in Web of Science Record created on 2010-03-25, modified on 2017-05-12

BACKGROUND: More than 50% of patients with Parkinson's disease develop motor response fluctuations (the "wearing off" phenomenon) after more than five years of levodopa therapy. Inhibition of catechol-O-methyltransferase by tolcapone has been shown to increase levodopa bioavailability and plasma elimination half life, thereby prolonging the efficacy of levodopa. OBJECTIVES: The primary objective was to evaluate the efficacy of tolcapone in reducing "wearing off" in levodopa treated, fluctuating parkinsonian patients. Secondary objectives included assessment of reduction in levodopa requirements, improvement in patients' clinical status, duration of improvements, and tolerability of tolcapone. METHODS: In this multicentre, randomised, double blind, placebo controlled trial, 58 patients received placebo, 60 received 100 mg tolcapone three times daily (tid), and 59 received 200 mg tolcapone tid, in addition to levodopa/benserazide. RESULTS: After three months with 200 mg tolcapone tid, "off" time decreased by 26.2% of the baseline value, "on" time increased by 20.6% (P

Long-term potentiation (LTP), a cellular model of learning and memory, requires calcium-dependent protein kinases. Induction of LTP increased the phosphorus-32 labeling of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)–type glutamate receptors (AMPA-Rs), which mediate rapid excitatory synaptic transmission. This AMPA-R phosphorylation appeared to be catalyzed by Ca 2+ - and calmodulin-dependent protein kinase II (CaM-KII): (i) it correlated with the activation and autophosphorylation of CaM-KII, (ii) it was blocked by the CaM-KII inhibitor KN-62, and (iii) its phosphorus-32 peptide map was the same as that of GluR1 coexpressed with activated CaM-KII in HEK-293 cells. This covalent modulation of AMPA-Rs in LTP provides a postsynaptic molecular mechanism for synaptic plasticity.

Glucagon-like peptide-1 (GLP-1) is a gut-derived incretin hormone that has been shown to improve glucose homeostasis in type 2 diabetes. The biological effects of GLP-1 are mediated by its specific receptor GLP-1R that is expressed in a wide range of tissues, where it is responsible of the extra-pancreatic effects of GLP-1. Since the retinal pigment epithelium (RPE), that forms the outer retinal barrier, has a key role in protecting from diabetic retinopathy (DR), we investigated the potential expression and function of GLP-1R in a RPE cell line. ARPE-19 cells were cultured in DMEM/F12 supplemented with 10% FBS. The expression of GLP-1R was evaluated at both mRNA and protein levels. Then, the activation postreceptor intracellular signal transduction pathways (extracellular signal-regulated kinases 1 and 2 [ERK1/2] and protein kinase B [PKB]) were assessed by western blot in normal cells or silenced for GLP-1R in the presence or absence of 10 nmol/L GLP-1. The potential connections between intracellular signalling pathways triggered by GLP-1 stimulation were performed before incubating cells with kinase pharmacological inhibitors of mitogen-activated protein kinase (MEK)1/2, phosphatydilinositol-3kinase (PI3K), or epidermal growth factor receptor (EGFR). The results showed that GLP1R is expressed at both mRNA and protein level in ARPE-19 cells. Stimulation with GLP-1 strongly activated PKB and ERK1/2 phosphorylation till 40 min of exposure. GLP-1-mediated activation of both kinases was dependent on the upstream activation of PI3K and EGFR. Finally, treatment with GLP-1 did not affect the spontaneous release of VEGF-A from ARPE-19 cells. In conclusion, this paper showed that the presence of functional GLP-1R is expressed in RPE cells. These data might represent the rationale to further investigate the potential direct beneficial effects of GLP-1 treatment against DR.

Objective— Endocannabinoid levels are elevated in human and mouse atherosclerosis, but their causal role is not well understood. Therefore, we studied the involvement of fatty acid amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in atherosclerotic plaque vulnerability. Methods and Results— We assessed atherosclerosis in apolipoprotein E–deficient (ApoE –/– ) and ApoE –/– FAAH –/– mice. Before and after 5, 10, and 15 weeks on high-cholesterol diet, we analyzed weight, serum cholesterol, and endocannabinoid levels, and atherosclerotic lesions in thoracoabdominal aortas and aortic sinuses. Serum levels of FAAH substrates anandamide, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) were 1.4- to 2-fold higher in case of FAAH deficiency. ApoE –/– FAAH –/– mice had smaller plaques with significantly lower content of smooth muscle cells, increased matrix metalloproteinase-9 expression, and neutrophil content. Circulating and bone marrow neutrophil counts were comparable between both genotypes, whereas CXC ligand1 levels were locally elevated in aortas of FAAH-deficient mice. We observed enhanced recruitment of neutrophils, but not monocytes, to large arteries of ApoE –/– mice treated with FAAH inhibitor URB597. Spleens of ApoE –/– FAAH –/– mice had reduced CD4+FoxP3+regulatory T-cell content, and in vitro stimulation of splenocytes revealed significantly elevated interferon-γ and tumor necrosis factor-α production in case of FAAH deficiency. Conclusion— Increased anandamide and related FAAH substrate levels are associated with the development of smaller atherosclerotic plaques with high neutrophil content, accompanied by an increased proinflammatory immune response.

Accumulating evidence has demonstrated that S-nitrosation of proteins plays a critical role in several human diseases. Here, we explored the role of inducible nitric oxide synthase (iNOS) in the S-nitrosation of proteins involved in the early steps of the insulin-signaling pathway and insulin resistance in the skeletal muscle of aged mice. Aging increased iNOS expression and S-nitrosation of major proteins involved in insulin signaling, thereby reducing insulin sensitivity in skeletal muscle. Conversely, aged iNOS-null mice were protected from S-nitrosation–induced insulin resistance. Moreover, pharmacological treatment with an iNOS inhibitor and acute exercise reduced iNOS-induced S-nitrosation and increased insulin sensitivity in the muscle of aged animals. These findings indicate that the insulin resistance observed in aged mice is mainly mediated through the S-nitrosation of the insulin-signaling pathway.