Intramicroparticle nitrogen dioxide is a bubble nucleation site leading to decompression-induced neutrophil activation and vascular injury

2013 ◽  
Vol 114 (5) ◽  
pp. 550-558 ◽  
Author(s):  
Stephen R. Thom ◽  
Ming Yang ◽  
Veena M. Bhopale ◽  
Tatyana N. Milovanova ◽  
Marina Bogush ◽  
...  
Keyword(s):  
Nitrogen Dioxide ◽  
Ex Vivo ◽  
Ambient Pressure ◽  
Annexin V ◽  
Nucleation Site ◽  
Bubble Nucleation ◽  
Neutrophil Activation ◽  
Inert Gases ◽  
Wild Type ◽  
Inos Inhibitor

Inert gases diffuse into tissues in proportion to ambient pressure, and when pressure is reduced, gas efflux forms bubbles due to the presence of gas cavitation nuclei that are predicted based on theory but have never been characterized. Decompression stress triggers elevations in number and diameter of circulating annexin V-coated microparticles (MPs) derived from vascular cells. Here we show that ∼10% MPs from wild-type (WT) but not inflammatory nitric oxide synthase-2 (iNOS) knockout (KO) mice increase in size when exposed to elevated air pressure ex vivo. This response is abrogated by a preceding exposure to hydrostatic pressure, demonstrating the presence of a preformed gas phase. These MPs have lower density than most particles, 10-fold enrichment in iNOS, and generate commensurately more reactive nitrogen species (RNS). Surprisingly, RNS only slowly diffuse from within MPs unless particles are subjected to osmotic stress or membrane cholesterol is removed. WT mice treated with iNOS inhibitor and KO mice exhibit less decompression-induced neutrophil activation and vascular leak. Contrary to injecting naïve mice with MPs from wild-type decompressed mice, injecting KO MPs triggers fewer proinflammatory events. We conclude that nitrogen dioxide is a nascent gas nucleation site synthesized in some MPs and is responsible for initiating postdecompression inflammatory injuries.

scholarly journals NTPDase1 Modulates Smooth Muscle Contraction in Mice Bladder by Regulating Nucleotide Receptor Activation Distinctly in Male and Female

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 147
Author(s):  
Romuald Brice Babou Kammoe ◽  
Gilles Kauffenstein ◽  
Julie Pelletier ◽  
Bernard Robaye ◽  
Jean Sévigny
Keyword(s):  
Smooth Muscle ◽  
Ex Vivo ◽  
Smooth Muscle Contraction ◽  
Receptor Activation ◽  
Nucleoside Triphosphate ◽  
Nucleotide Receptors ◽  
Nerve Terminals ◽  
Wild Type ◽  
Nucleoside Triphosphate Diphosphohydrolase ◽  
Bladder Contraction

Nucleotides released by smooth muscle cells (SMCs) and by innervating nerve terminals activate specific P2 receptors and modulate bladder contraction. We hypothesized that cell surface enzymes regulate SMC contraction in mice bladder by controlling the concentration of nucleotides. We showed by immunohistochemistry, enzymatic histochemistry, and biochemical activities that nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and ecto-5′-nucleotidase were the major ectonucleotidases expressed by SMCs in the bladder. RT-qPCR revealed that, among the nucleotide receptors, there was higher expression of P2X1, P2Y1, and P2Y6 receptors. Ex vivo, nucleotides induced a more potent contraction of bladder strips isolated from NTPDase1 deficient (Entpd1−/−) mice compared to wild type controls. The strongest responses were obtained with uridine 5′-triphosphate (UTP) and uridine 5′-diphosphate (UDP), suggesting the involvement of P2Y6 receptors, which was confirmed with P2ry6−/− bladder strips. Interestingly, this response was reduced in female bladders. Our results also suggest the participation of P2X1, P2Y2 and/or P2Y4, and P2Y12 in these contractions. A reduced response to the thromboxane analogue U46619 was also observed in wild type, Entpd1−/−, and P2ry6−/− female bladders showing another difference due to sex. In summary, NTPDase1 modulates the activation of nucleotide receptors in mouse bladder SMCs, and contractions induced by P2Y6 receptor activation were weaker in female bladders.

scholarly journals Cooperative Blockade of CK2 and ATM Kinases Drives Apoptosis in VHL-Deficient Renal Carcinoma Cells through ROS Overproduction

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 576
Author(s):  
Sofia Giacosa ◽  
Catherine Pillet ◽  
Irinka Séraudie ◽  
Laurent Guyon ◽  
Yann Wallez ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Renal Carcinoma ◽  
Kinase Inhibitors ◽  
Ex Vivo ◽  
Cancer Therapeutics ◽  
Carcinoma Cells ◽  
Wild Type ◽  
Cell Renal Cell Carcinoma ◽  
Von Hippel Lindau ◽  
Renal Carcinoma Cells

Kinase-targeted agents demonstrate antitumor activity in advanced metastatic clear cell renal cell carcinoma (ccRCC), which remains largely incurable. Integration of genomic approaches through small-molecules and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. The 786-O cell line represents a model for most ccRCC that have a loss of functional pVHL (von Hippel-Lindau). A multiplexed assay was used to study the cellular fitness of a panel of engineered ccRCC isogenic 786-O VHL− cell lines in response to a collection of targeted cancer therapeutics including kinase inhibitors, allowing the interrogation of over 2880 drug–gene pairs. Among diverse patterns of drug sensitivities, investigation of the mechanistic effect of one selected drug combination on tumor spheroids and ex vivo renal tumor slice cultures showed that VHL-defective ccRCC cells were more vulnerable to the combined inhibition of the CK2 and ATM kinases than wild-type VHL cells. Importantly, we found that HIF-2α acts as a key mediator that potentiates the response to combined CK2/ATM inhibition by triggering ROS-dependent apoptosis. Importantly, our findings reveal a selective killing of VHL-deficient renal carcinoma cells and provide a rationale for a mechanism-based use of combined CK2/ATM inhibitors for improved patient care in metastatic VHL-ccRCC.

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

scholarly journals Pharmacological ablation of astrocytes reduces Aβ degradation and synaptic connectivity in an ex vivo model of Alzheimer’s disease

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Nicola Davis ◽  
Bibiana C. Mota ◽  
Larissa Stead ◽  
Emily O. C. Palmer ◽  
Laura Lombardero ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Culture Media ◽  
Ex Vivo ◽  
Wild Type ◽  
Insulin Degrading Enzyme ◽  
Ex Vivo Model ◽  
Model Of Alzheimer’S Disease ◽  
Aβ Clearance ◽  
5Xfad Mice

Abstract Background Astrocytes provide a vital support to neurons in normal and pathological conditions. In Alzheimer’s disease (AD) brains, reactive astrocytes have been found surrounding amyloid plaques, forming an astrocytic scar. However, their role and potential mechanisms whereby they affect neuroinflammation, amyloid pathology, and synaptic density in AD remain unclear. Methods To explore the role of astrocytes on Aβ pathology and neuroinflammatory markers, we pharmacologically ablated them in organotypic brain culture slices (OBCSs) from 5XFAD mouse model of AD and wild-type (WT) littermates with the selective astrocytic toxin L-alpha-aminoadipate (L-AAA). To examine the effects on synaptic circuitry, we measured dendritic spine number and size in OBCSs from Thy-1-GFP transgenic mice incubated with synthetic Aβ42 or double transgenics Thy-1-GFP/5XFAD mice treated with LAAA or vehicle for 24 h. Results Treatment of OBCSs with L-AAA resulted in an increased expression of pro-inflammatory cytokine IL-6 in conditioned media of WTs and 5XFAD slices, associated with changes in microglia morphology but not in density. The profile of inflammatory markers following astrocytic loss was different in WT and transgenic cultures, showing reductions in inflammatory mediators produced in astrocytes only in WT sections. In addition, pharmacological ablation of astrocytes led to an increase in Aβ levels in homogenates of OBCS from 5XFAD mice compared with vehicle controls, with reduced enzymatic degradation of Aβ due to lower neprilysin and insulin-degrading enzyme (IDE) expression. Furthermore, OBSCs from wild-type mice treated with L-AAA and synthetic amyloid presented 56% higher levels of Aβ in culture media compared to sections treated with Aβ alone, concomitant with reduced expression of IDE in culture medium, suggesting that astrocytes contribute to Aβ clearance and degradation. Quantification of hippocampal dendritic spines revealed a reduction in their density following L-AAA treatment in all groups analyzed. In addition, pharmacological ablation of astrocytes resulted in a decrease in spine size in 5XFAD OBCSs but not in OBCSs from WT treated with synthetic Aβ compared to vehicle control. Conclusions Astrocytes play a protective role in AD by aiding Aβ clearance and supporting synaptic plasticity.

scholarly journals Differential effect of anesthetics on mucociliary clearance in vivo in mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kyle S. Feldman ◽  
Eunwon Kim ◽  
Michael J. Czachowski ◽  
Yijen Wu ◽  
Cecilia W. Lo ◽  
...  
Keyword(s):  
Mucociliary Clearance ◽  
Defense Mechanism ◽  
Ex Vivo ◽  
Beat Frequency ◽  
Ciliary Beat Frequency ◽  
Wild Type ◽  
Sulfur Colloid ◽  
Ct System

AbstractRespiratory mucociliary clearance (MCC) is a key defense mechanism that functions to entrap and transport inhaled pollutants, particulates, and pathogens away from the lungs. Previous work has identified a number of anesthetics to have cilia depressive effects in vitro. Wild-type C57BL/6 J mice received intra-tracheal installation of 99mTc-Sulfur colloid, and were imaged using a dual-modality SPECT/CT system at 0 and 6 h to measure baseline MCC (n = 8). Mice were challenged for one hour with inhalational 1.5% isoflurane, or intraperitoneal ketamine (100 mg/kg)/xylazine (20 mg/kg), ketamine (0.5 mg/kg)/dexmedetomidine (50 mg/kg), fentanyl (0.2 mg/kg)/1.5% isoflurane, propofol (120 mg/Kg), or fentanyl/midazolam/dexmedetomidine (0.025 mg/kg/2.5 mg/kg/0.25 mg/kg) prior to MCC assessment. The baseline MCC was 6.4%, and was significantly reduced to 3.7% (p = 0.04) and 3.0% (p = 0.01) by ketamine/xylazine and ketamine/dexmedetomidine challenge respectively. Importantly, combinations of drugs containing fentanyl, and propofol in isolation did not significantly depress MCC. Although no change in cilia length or percent ciliation was expected, we tried to correlate ex-vivo tracheal cilia ciliary beat frequency and cilia-generated flow velocities with MCC and found no correlation. Our results indicate that anesthetics containing ketamine (ketamine/xylazine and ketamine/dexmedetomidine) significantly depress MCC, while combinations containing fentanyl (fentanyl/isoflurane, fentanyl/midazolam/dexmedetomidine) and propofol do not. Our method for assessing MCC is reproducible and has utility for studying the effects of other drug combinations.

scholarly journals GPR109A mediates the effects of hippuric acid on regulating osteoclastogenesis and bone resorption in mice

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Jin-Ran Chen ◽  
Haijun Zhao ◽  
Umesh D. Wankhade ◽  
Sree V. Chintapalli ◽  
Can Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Resorption ◽  
Bone Mass ◽  
Hippuric Acid ◽  
Ex Vivo ◽  
Marrow Cell ◽  
Osteoclast Differentiation ◽  
Bone Homeostasis ◽  
Wild Type ◽  
Osteoclast Precursors

AbstractThe G protein-coupled receptor 109 A (GPR109A) is robustly expressed in osteoclastic precursor macrophages. Previous studies suggested that GPR109A mediates effects of diet-derived phenolic acids such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA) on promoting bone formation. However, the role of GPR109A in metabolic bone homeostasis and osteoclast differentiation has not been investigated. Using densitometric, bone histologic and molecular signaling analytic methods, we uncovered that bone mass and strength were significantly higher in tibia and spine of standard rodent diet weaned 4-week-old and 6-month-old GPR109A gene deletion (GPR109A−/−) mice, compared to their wild type controls. Osteoclast numbers in bone and in ex vivo bone marrow cell cultures were significantly decreased in GPR109A−/− mice compared to wild type controls. In accordance with these data, CTX-1 in bone marrow plasma and gene expression of bone resorption markers (TNFα, TRAP, Cathepsin K) were significantly decreased in GPR109A−/− mice, while on the other hand, P1NP was increased in serum from both male and female GPR109A−/− mice compared to their respective controls. GPR109A deletion led to suppressed Wnt/β-catenin signaling in osteoclast precursors to inhibit osteoclast differentiation and activity. Indeed, HA and 3-3-PPA substantially inhibited RANKL-induced GPR109A expression and Wnt/β-catenin signaling in osteoclast precursors and osteoclast differentiation. Resultantly, HA significantly inhibited bone resorption and increased bone mass in wild type mice, but had no additional effects on bone in GPR109A−/− mice compared with their respective untreated control mice. These results suggest an important role for GPR109A during osteoclast differentiation and bone resorption mediating effects of HA and 3-3-PPA on inhibiting bone resorption during skeletal development.

scholarly journals Akt isoforms differentially regulate neutrophil functions

Blood ◽  
2010 ◽  
Vol 115 (21) ◽  
pp. 4237-4246 ◽  
Author(s):  
Jia Chen ◽  
Haiyang Tang ◽  
Nissim Hay ◽  
Jingsong Xu ◽  
Richard D. Ye
Keyword(s):  
Kinase Inhibitor ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Leading Edge ◽  
Neutrophil Activation ◽  
Enzyme Release ◽  
Wild Type ◽  
Akt Isoforms ◽  
Phosphoinositide 3 Kinase ◽  
O2 Production

In neutrophils, the phosphoinositide 3-kinase/Akt signaling cascade is involved in migration, degranulation, and O2− production. However, it is unclear whether the Akt kinase isoforms have distinct functions in neutrophil activation. Here we report functional differences between the 2 major Akt isoforms in neutrophil activation on the basis of studies in which we used individual Akt1 and Akt2 knockout mice. Akt2−/− neutrophils exhibited decreased cell migration, granule enzyme release, and O2− production compared with wild-type and Akt1−/− neutrophils. Surprisingly, Akt2 deficiency and pharmacologic inhibition of Akt also abrogated phorbol ester-induced O2− production, which was unaffected by treatment with the phosphoinositide 3-kinase inhibitor LY294002. The decreased O2− production in Akt2−/− neutrophils was accompanied by reduced p47phox phosphorylation and its membrane translocation, suggesting that Akt2 is important for the assembly of phagocyte nicotinamide adenine dinucleotide phosphate oxidase. In wild-type neutrophils, Akt2 but not Akt1 translocated to plasma membrane upon chemoattractant stimulation and to the leading edge in polarized neutrophils. In the absence of Akt2, chemoattractant-induced Akt protein phosphorylation was significantly reduced. These results demonstrate a predominant role of Akt2 in regulating neutrophil functions and provide evidence for differential activation of the 2 Akt isoforms in neutrophils.

scholarly journals KCNJ11 knockout morula re-engineered by stem cell diploid aggregation

2008 ◽  
Vol 364 (1514) ◽  
pp. 269-276 ◽  
Author(s):  
Timothy J Nelson ◽  
Almudena Martinez-Fernandez ◽  
Andre Terzic
Keyword(s):  
Metabolic Flux ◽  
Ex Vivo ◽  
Gene Knockout ◽  
Embryonic Stem ◽  
Adaptive Behaviour ◽  
Wild Type ◽  
New Paradigm ◽  
Cell Functions ◽  
Dependent Cell ◽  
Channel Dependent

KCNJ11 -encoded Kir6.2 assembles with ATP-binding cassette sulphonylurea receptors to generate ATP-sensitive K + (K ATP ) channel complexes. Expressed in tissues with dynamic metabolic flux, these evolutionarily conserved yet structurally and functionally unique heteromultimers serve as high-fidelity rheostats that adjust membrane potential-dependent cell functions to match energetic demand. Genetic defects in channel subunits disrupt the cellular homeostatic response to environmental stress, compromising organ tolerance in the adult. As maladaptation characterizes malignant K ATP channelopathies, establishment of platforms to examine progression of K ATP channel-dependent adaptive behaviour is warranted. Chimeras provide a powerful tool to assay the contribution of genetic variance to stress intolerance during prenatal or post-natal development. Here, KCNJ11 K ATP channel gene knockout↔wild-type chimeras were engineered through diploid aggregation. Integration of wild-type embryonic stem cells into zona pellucida-denuded morula derived from knockout embryos achieved varying degrees of incorporation of stress-tolerant tissue within the K ATP channel-deficient background. Despite the stress-vulnerable phenotype of the knockout, ex vivo derived mosaic blastocysts tolerated intrauterine transfer and implantation, followed by full-term embryonic development in pseudopregnant surrogates to produce live chimeric offspring. The development of adult chimerism from the knockout↔wild-type mosaic embryo offers thereby a new paradigm to probe the ecogenetic control of the K ATP channel-dependent stress response.

Abstract 292: The Role of the ATF6 Branch of the ER Stress Response in the Endocrine Heart

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Erik A Blackwood ◽  
Christopher C Glembotski
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Knockout Mice ◽  
Ex Vivo ◽  
Proteolytic Cleavage ◽  
Isolated Perfused Heart ◽  
Wild Type ◽  
Atrial Myocytes ◽  
Perfused Heart ◽  
Er Stress Response

Rationale: Atrial natriuretic peptide (ANP) is stored in the heart in large dense core granules of atrial myocytes as a biologically inactive precursor, pro-ANP. Hemodynamic stress and atrial stretch stimulate coordinate secretion and proteolytic cleavage of pro-ANP to its bioactive form, ANP, which promotes renal salt excretion and vasodilation, which, together contribute to decreasing blood pressure. While the ATF6 branch of the ER stress response has been studied in ventricular tissue mouse models of myocardial ischemia and pathological hypertrophy, roles for ATF6 and ER stress on the endocrine function of atrial myocytes have not been studied. Objective/Methods: To address this gap in our knowledge, we knocked down ATF6 in primary cultured neonatal rat atrial myocytes (NRAMs) using a chemical inhibitor of the proteolytic cleavage site enabling ATF6 activation and siRNA and measured ANP expression and secretion basally and in response to alpha- adrenergic agonist stimulation using phenylephrine. We also compared the ANP secretion from wild- type mice and ATF6 knockout mice in an ex vivo Langendorff model of the isolated perfused heart. Results: ATF6 knockdown in NRAMs significantly impaired basal and phenylephrine-stimulated ANP secretion. ATF6 knockout mice displayed lower levels of ANP in atrial tissue at baseline as well as after phenylephrine treatment. Similarly, in the ex vivo isolated perfused heart model, less ANP was detected in effluent of ATF6 knockout hearts compared to wild-type hearts. Conclusions: The ATF6 branch of the ER stress response is necessary for efficient co-secretional processing of pro-ANP to ANP and for agonist-stimulated ANP secretion from atrial myocytes. As ANP is secreted in a regulated manner in response to a stimulus and pro-ANP is synthesized and packaged through the classical secretory pathway, we posit that ATF6 is required for adequate expression, folding, trafficking, processing and secretion of biologically active ANP from the endocrine heart.

Abstract P283: Parkin Mediates Mitophagy in Cardioprotection

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Allen M Andres ◽  
Chengqun Huang ◽  
Eric P Ratliff ◽  
Genaro Hernandez ◽  
Pamela Lee ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Wild Type ◽  
Simulated Ischemia ◽  
Selective Targeting ◽  
Cardioprotective Effects ◽  
Mitochondrial Turnover ◽  
First Time

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

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