Two Secreted Proteoglycans, Activators of Urothelial Cell-Cell Adhesion, Negatively Contribute to Bladder Cancer Initiation and Progression.

Osteomodulin (OMD) and proline/arginine-rich end leucine repeat protein (PRELP) are secreted extracellular matrix proteins belonging to the small leucine-rich proteoglycans family. We found that OMD and PRELP were specifically expressed in umbrella cells in bladder epithelia, and their expression levels were dramatically downregulated in all bladder cancers from very early stages and various epithelial cancers. Our in vitro studies including gene expression profiling using bladder cancer cell lines revealed that OMD or PRELP application suppressed the cancer progression by inhibiting TGF-ß and EGF pathways, which reversed epithelial-mesenchymal transition (EMT), activated cell-cell adhesion, and inhibited various oncogenic pathways. Furthermore, the overexpression of OMD in bladder cancer cells strongly inhibited the anchorage-independent growth and tumorigenicity in mouse xenograft studies. On the other hand, we found that in the bladder epithelia, the knockout mice of OMD and/or PRELP gene caused partial EMT and a loss of tight junctions of the umbrella cells and resulted in formation of a bladder carcinoma in situ-like structure by spontaneous breakdowns of the umbrella cell layer. Furthermore, the ontological analysis of the expression profiling of an OMD knockout mouse bladder demonstrated very high similarity with those obtained from human bladder cancers. Our data indicate that OMD and PRELP are endogenous inhibitors of cancer initiation and progression by controlling EMT. OMD and/or PRELP may have potential for the treatment of bladder cancer.

Risk factors for excessive tidal volumes delivered during intraoperative mechanical ventilation, a retrospective study.

BACKGROUND: Lung Protective Ventilation (LPV) refers to a combination of measures aimed at reducing ventilator-associated lung injury. This includes: delivering tidal volumes of 6-8 ml/kg of ideal body weight, use of positive end expiratory pressure and recruitment maneuvers. With Postoperative Pulmonary Complications (PPCs) contributing towards significant morbidity and mortality following surgery, evidence indicates that effective use of LPV measures intraoperatively has been associated with reduced rates of PPCs. METHODS: We conducted a post-hoc analysis using data from a recent clinical audit on departmental ventilation practices. Potential risk factors for excessive tidal volume ventilation were assessed using univariable and multivariable regression models. RESULTS: Obesity and gender are independently associated with risk of excessive ventilation. In contrast, the urgency and length of surgery, the choice of airway devices and the mode of ventilation were not associated with excessive ventilation. CONCLUSION: There is an association between female gender, obesity and excessive tidal volume ventilation. This may be addressed through formal, protocolized intraoperative ventilation setting.

Risk factors for admission at three urban emergency departments in England: a cross-sectional analysis of attendances over 1 month.

OBJECTIVE: To investigate factors associated with unscheduled admission following presentation to emergency departments (EDs) at three hospitals in England. DESIGN AND SETTING: Cross-sectional analysis of attendance data for patients from three urban EDs in England: a large teaching hospital and major trauma centre (site 1) and two district general hospitals (sites 2 and 3). Variables included patient age, gender, ethnicity, deprivation score, arrival date and time, arrival by ambulance or otherwise, a variety of ED workload measures, inpatient bed occupancy rates and admission outcome. Coding inconsistencies in routine ED data used for this study meant that diagnosis could not be included. OUTCOME MEASURE: The primary outcome for the study was unscheduled admission. PARTICIPANTS: All adults aged 16 and older attending the three inner London EDs in December 2013. Data on 19 734 unique patient attendances were gathered. RESULTS: Outcome data were available for 19 721 attendances (>99%), of whom 6263 (32%) were admitted to hospital. Site 1 was set as the baseline site for analysis of admission risk. Risk of admission was significantly greater at sites 2 and 3 (adjusted OR (AOR) relative to site 1 for site 2 was 1.89, 95% CI 1.74 to 2.05, p<0.001) and for patients of black or black British ethnicity (AOR 1.29, 1.16 to 1.44, p<0.001). Deprivation was strongly associated with admission. Analysis of departmental and hospital-wide workload pressures gave conflicting results, but proximity to the "4-hour target" (a rule that limits patient stays in EDs to 4 hours in the National Health Service in England) emerged as a strong driver for admission in this analysis (AOR 3.61, 95% CI 3.30 to 3.95, p<0.001). CONCLUSION: This study found statistically significant variations in odds of admission between hospital sites when adjusting for various patient demographic and presentation factors, suggesting important variations in ED-level and clinician-level behaviour relating to admission decisions. The 4-hour target is a strong driver for emergency admission.

Positive feedback regulation between gamma-aminobutyric acid type A (GABA(A)) receptor signaling and brain-derived neurotrophic factor (BDNF) release in developing neurons.

During the early development of the nervous system, γ-aminobutyric acid (GABA) type A receptor (GABA(A)R)-mediated signaling parallels the neurotrophin/tropomyosin-related kinase (Trk)-dependent signaling in controlling a number of processes from cell proliferation and migration, via dendritic and axonal outgrowth, to synapse formation and plasticity. Here we present the first evidence that these two signaling systems regulate each other through a complex positive feedback mechanism. We first demonstrate that GABA(A)R activation leads to an increase in the cell surface expression of these receptors in cultured embryonic cerebrocortical neurons, specifically at the stage when this activity causes depolarization of the plasma membrane and Ca(2+) influx through L-type voltage-gated Ca(2+) channels. We further demonstrate that GABA(A)R activity triggers release of the brain-derived neurotrophic factor (BDNF), which, in turn by activating TrkB receptors, mediates the observed increase in cell surface expression of GABA(A)Rs. This BDNF/TrkB-dependent increase in surface levels of GABA(A)Rs requires the activity of phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) and does not involve the extracellular signal-regulated kinase (ERK) 1/2 activity. The increase in GABA(A)R surface levels occurs due to an inhibition of the receptor endocytosis by BDNF, whereas the receptor reinsertion into the plasma membrane remains unaltered. Thus, GABA(A)R activity is a potent regulator of the BDNF release during neuronal development, and at the same time, it is strongly enhanced by the activity of the BDNF/TrkB/PI3K/PKC signaling pathway.

Genetic ablation of retinal pigment epithelial cells reveals the adaptive response of the epithelium and impact on photoreceptors.

The retinal pigment epithelium (RPE) plays a critical role in the maintenance of the outer retina. RPE cell death or dysfunction drives the pathophysiology of many retinal diseases, but the physiological response of the retina to RPE cell loss is poorly understood, mainly because of the absence of suitable experimental models. Here, we generated a transgenic mouse in which an inducible Cre recombinase is expressed exclusively in the RPE under the control of the monocarboxylate transporter 3 gene promoter (RPE(CreER)). This was crossed with a transgenic mouse harboring a diphtheria toxin A (DTA) chain gene rendered transcriptionally silent by a floxed stop sequence. We show that activation of DTA in the double transgenic mouse (RPE(CreER)/DTA) led to 60-80% RPE cell death, with surviving cells maintaining the integrity of the monolayer by increasing their size. Despite the apparent morphological normality of the enlarged RPE cells in the RPE(CreER)/DTA mice, functional analysis revealed significant deficits on electroretinography, and retinal histopathology showed regions of photoreceptor rosetting and degeneration although with retention of a normal vascular network. Our study reveals that whilst the RPE monolayer has a remarkable intrinsic capacity to cope with cellular attrition, specific aspects of RPE multifunctionality essential for photoreceptor survival are compromised. The RPE(CreER)/DTA mouse offers advantages over models that employ chemical or mechanical strategies to kill RPE cells, and should be useful for the development and evaluation of RPE-based therapies, such as stem cell transplantation.

ICAM-1-mediated endothelial nitric oxide synthase activation via calcium and AMP-activated protein kinase is required for transendothelial lymphocyte migration.

As a gatekeeper of leukocyte trafficking the vasculature fulfills an essential immune function. We have recently shown that paracellular transendothelial lymphocyte migration is controlled by intercellular adhesion molecule 1 (ICAM-1)-mediated vascular endothelial cadherin (VEC) phosphorylation [Turowski et al., J. Cell Sci. 121, 29-37 (2008)]. Here we show that endothelial nitric oxide synthase (eNOS) is a critical regulator of this pathway. ICAM-1 stimulated eNOS by a mechanism that was clearly distinct from that utilized by insulin. In particular, phosphorylation of eNOS on S1177 in response to ICAM-1 activation was regulated by src family protein kinase, rho GTPase, Ca(2+), CaMKK, and AMPK, but not Akt/PI3K. Functional neutralization of any component of this pathway or its downstream effector guanylyl cyclase significantly reduced lymphocyte diapedesis across the endothelial monolayer. In turn, activation of NO signaling promoted lymphocyte transmigration. The eNOS signaling pathway was required for T-cell transmigration across primary rat and human microvascular endothelial cells and also when shear flow was applied, suggesting that this pathway is ubiquitously used. These data reveal a novel and essential role of eNOS in basic immune function and provide a key link in the molecular network governing endothelial cell compliance to diapedesis.

ATP depletion inhibits Ca2+ release, influx and extrusion in pancreatic acinar cells but not pathological Ca2+ responses induced by bile.

Here, we describe novel mechanisms limiting a toxic cytosolic Ca(2+) rise during adenosine 5'-triphosphate (ATP) depletion. We studied the effect of ATP depletion on Ca(2+) signalling in mouse pancreatic acinar cells. Measurements of ATP in isolated cells after adenovirus-mediated expression of firefly luciferase revealed that the cytosolic ATP concentration fell from approximately 1 mM to near zero after treatment with oligomycin plus iodoacetate. ATP depletion resulted in the inhibition of Ca(2+) extrusion, which was accompanied by a remarkably synchronous inhibition of store-operated Ca(2+) influx. Alternative inhibition of Ca(2+) extrusion by carboxyeosin had a much smaller effect on Ca(2+) influx. The coordinated metabolic inhibition of Ca(2+) influx and extrusion suggests the existence of a common ATP-dependent master regulator of both processes. ATP-depletion also suppressed acetylcholine (ACh)-induced Ca(2+) oscillations, which was due to the inhibition of Ca(2+) release from internal stores. This could be particularly important for limiting Ca(2+) toxicity during periods of hypoxia. In contrast, metabolic control of Ca(2+) influx and Ca(2+) release from internal stores spectacularly failed to prevent large toxic Ca(2+) responses induced by bile acids-activators of acute pancreatitis (a frequent and often fatal disease of the exocrine pancreas). The bile acids taurolithocholic acid 3-sulphate (TLC-S), taurochenodeoxycholic acid (TCDC) and taurocholic acid (TC) were used in our experiments. Neither Ca(2+) release from internal stores nor Ca(2+) influx triggered by bile acids were inhibited by ATP depletion, emphasising the danger of these pathological mechanisms.

Bile acids induce calcium signals in mouse pancreatic acinar cells: implications for bile-induced pancreatic pathology.

The effect of the natural bile acid, taurolithocholic acid 3-sulfate (TLC-S), on calcium signalling in pancreatic acinar cells has been investigated. TLC-S induced global calcium oscillations and extended calcium transients as well as calcium signals localised to the secretory granule (apical) region of acinar cells. These calcium signals could still be triggered by TLC-S in a calcium-free external solution. TLC-S-induced calcium signals were not inhibited by atropine, but were abolished by caffeine or by depletion of calcium stores, due to prolonged application of ACh. Global calcium signals, produced by TLC-S application, displayed vectorial apical-to-basal polarity. The signals originated in the apical part and were then propagated to the basal region. Other natural bile acids, taurocholate (TC) and taurodeoxycholate (TDC), were also able to produce local and global calcium oscillations (but at higher concentrations than TLC-S). Bile, which can enter pancreas by reflux, has been implicated in the pathology of acute pancreatitis. The calcium releasing properties of bile acids suggest that calcium toxicity could be an important contributing factor in the bile acid-induced cellular damage.