Sex differences in murine cardiac pathophysiology with hyperoxia exposure.

Hyperoxia (>90% oxygen) is commonly implemented in mechanically ventilated patients. Reports suggest that hyperoxia is directly associated with in-hospital mortality in ventilated patients. Certain studies also show that mortality in women undergoing mechanical ventilation is significantly higher than that in men. Additionally, females are predisposed to certain cardiac electrophysiological risks, including QTc prolongation. In this study, we assessed the impact of hyperoxia in male and female mice (C57BL/6J) at age 8-10 weeks. On completion of either hyperoxia or normoxia exposures, physical, hemodynamic, biochemical, functional, electrophysiological, and molecular assessments were conducted. Hyperoxia-exposed mice lost a significant amount of body mass, compared with normoxia controls, in both sexes. However, while both genders developed brady-arrhythmia after hyperoxia exposure, female mice exhibited significantly reduced heart rates compared with males, with significantly elevated RR intervals. Additionally, 50% mortality was observed in females, whereas no mortality was reported in males. Furthermore, unlike in male mice, we observed no hypertrophy upon hyperoxia exposure in female mice. We reported that both hyperoxia-treated male and female mice exhibit significant hyperdynamic left ventricular ejection fraction, which is marked by % ejection fraction > 70 compared with the normoxia controls. We also noted significant reductions in stroke volume and cardiac output in both mice with hyperoxia. Surface ECG also demonstrated that hyperoxia exposure significantly augments RR, PR, QRS, QTc, and JT intervals in both sexes. Molecular analysis of left ventricular tissue demonstrated dysregulation of potassium ion channels in hyperoxia-treated males and females. In summary, we determined that sex differences are present with 72 hr hyperoxia exposure.

Loss of phosphatase and tensin homolog (PTEN) induces leptin-mediated leptin gene expression: feed-forward loop operating in the lung.

Elevated levels of systemic and pulmonary leptin are associated with diseases related to lung injury and lung cancer. However, the role of leptin in lung biology and pathology, including the mechanism of leptin gene expression in the pathogenesis of lung diseases, including lung cancer, remains elusive. Here, using conditional deletion of tumor suppressor gene Pten in the lung epithelium in vivo in transgenic mice and human PTEN-null lung epithelial cells, we identify the leptin-driven feed-forward signaling loop in the lung epithelial cells. Leptin-mediated leptin/leptin-receptor gene expression likely amplifies leptin signaling that may contribute to the pathogenesis and severity of lung diseases, resulting in poor clinical outcomes. Loss of Pten in the lung epithelial cells in vivo activated adipokine signaling and induced leptin synthesis as ascertained by genome-wide mRNA profiling and pathway analysis. Leptin gene transcription was mediated by binding of transcription factors NRF-1 and CCAAT/enhancer-binding protein δ (C/EBP) to the proximal promoter regions and STAT3 to the distal promoter regions as revealed by leptin promoter-mutation, chromatin immunoprecipitation, and gain- and loss-of-function studies in lung epithelial cells. Leptin treatment induced expression of the leptin/leptin receptor in the lung epithelial cells via activation of MEK/ERK, PI3K/AKT/mammalian target of rapamycin (mTOR), and JAK2/STAT3 signaling pathways. Expression of constitutively active MEK-1, AKT, and STAT3 proteins increased expression, and treatment with MEK, PI3K, AKT, and mTOR inhibitors decreased LEP expression, indicating that leptin via MAPK/ERK1/2, PI3K/AKT/mTOR, and JAK2/STAT3 pathways, in turn, further induces its own gene expression. Thus, targeted inhibition of the leptin-mediated feed-forward loop provides a novel rationale for pharmacotherapy of disease associated with lung injury and remodeling, including lung cancer.

Smoke extract impairs adenosine wound healing: implications of smoke-generated reactive oxygen species.

Adenosine concentrations are elevated in the lungs of patients with asthma and chronic obstructive pulmonary disease, where it balances between tissue repair and excessive airway remodeling. We previously demonstrated that the activation of the adenosine A2A receptor promotes epithelial wound closure. However, the mechanism by which adenosine-mediated wound healing occurs after cigarette smoke exposure has not been investigated. The present study investigates whether cigarette smoke exposure alters adenosine-mediated reparative properties via its ability to induce a shift in the oxidant/antioxidant balance. Using an in vitro wounding model, bronchial epithelial cells were exposed to 5% cigarette smoke extract, were wounded, and were then stimulated with either 10 µM adenosine or the specific A2A receptor agonist, 5'-(N-cyclopropyl)-carboxamido-adenosine (CPCA; 10 µM), and assessed for wound closure. In a subset of experiments, bronchial epithelial cells were infected with adenovirus vectors encoding human superoxide dismutase and/or catalase or control vector. In the presence of 5% smoke extract, significant delay was evident in both adenosine-mediated and CPCA-mediated wound closure. However, cells pretreated with N-acetylcysteine (NAC), a nonspecific antioxidant, reversed smoke extract-mediated inhibition. We found that cells overexpressing mitochondrial catalase repealed the smoke extract inhibition of CPCA-stimulated wound closure, whereas superoxide dismutase overexpression exerted no effect. Kinase experiments revealed that smoke extract significantly reduced the A2A-mediated activation of cyclic adenosine monophosphate-dependent protein kinase. However, pretreatment with NAC reversed this effect. In conclusion, our data suggest that cigarette smoke exposure impairs A2A-stimulated wound repair via a reactive oxygen species-dependent mechanism, thereby providing a better understanding of adenosine signaling that may direct the development of pharmacological tools for the treatment of chronic inflammatory lung disorders.

NLRP3 deletion protects from hyperoxia-induced acute lung injury.

Inspiration of a high concentration of oxygen, a therapy for acute lung injury (ALI), could unexpectedly lead to reactive oxygen species (ROS) production and hyperoxia-induced acute lung injury (HALI). Nucleotide-binding domain and leucine-rich repeat PYD-containing protein 3 (NLRP3) senses the ROS, triggering inflammasome activation and interleukin-1ß (IL-1ß) production and secretion. However, the role of NLRP3 inflammasome in HALI is unclear. The main aim of this study is to determine the effect of NLRP3 gene deletion on inflammatory response and lung epithelial cell death. Wild-type (WT) and NLRP3(-/-) mice were exposed to 100% O2 for 48-72 h. Bronchoalveolar lavage fluid and lung tissues were examined for proinflammatory cytokine production and lung inflammation. Hyperoxia-induced lung pathological score was suppressed in NLRP3(-/-) mice compared with WT mice. Hyperoxia-induced recruitment of inflammatory cells and elevation of IL-1ß, TNFα, macrophage inflammatory protein-2, and monocyte chemoattractant protein-1 were attenuated in NLRP3(-/-) mice. NLRP3 deletion decreased lung epithelial cell death and caspase-3 levels and a suppressed NF-κB levels compared with WT controls. Taken together, this research demonstrates for the first time that NLRP3-deficient mice have suppressed inflammatory response and blunted lung epithelial cell apoptosis to HALI.

Co-exposure to cigarette smoke and alcohol decreases airway epithelial cell cilia beating in a protein kinase Cε-dependent manner.

Alcohol use disorders are associated with increased lung infections and exacerbations of chronic lung diseases. Whereas the effects of cigarette smoke are well recognized, the interplay of smoke and alcohol in modulating lung diseases is not clear. Because innate lung defense is mechanically maintained by airway cilia action and protein kinase C (PKC)-activating agents slow ciliary beat frequency (CBF), we hypothesized that the combination of smoke and alcohol would decrease CBF in a PKC-dependent manner. Primary ciliated bronchial epithelial cells were exposed to 5% cigarette smoke extract plus100 mmol/L ethanol for up to 24 hours and assayed for CBF and PKCε. Smoke and alcohol co-exposure activated PKCε by 1 hour and decreased both CBF and total number of beating cilia by 6 hours. A specific activator of PKCε, DCP-LA, slowed CBF after maximal PKCε activation. Interestingly, activation of PKCε by smoke and alcohol was only observed in ciliated cells, not basal bronchial epithelium. In precision-cut mouse lung slices treated with smoke and alcohol, PKCε activation preceded CBF slowing. Correspondingly, increased PKCε activity and cilia slowing were only observed in mice co-exposed to smoke and alcohol, regardless of the sequence of the combination exposure. No decreases in CBF were observed in PKCε knockout mice co-exposed to smoke and alcohol. These data identify PKCε as a key regulator of cilia slowing in response to combined smoke and alcohol-induced lung injury.

Alcohol increases the permeability of airway epithelial tight junctions in Beas-2B and NHBE cells.

BACKGROUND: Tight junctions form a continuous belt-like structure between cells and act to regulate paracellular signaling. Protein kinase C (PKC) has been shown to regulate tight junction assembly and disassembly and is activated by alcohol. Previous research has shown that alcohol increases the permeability of tight junctions in lung alveolar cells. However, little is known about alcohol's effect on tight junctions in epithelium of the conducting airways. We hypothesized that long-term alcohol exposure reduces zonula occluden-1 (ZO-1) and claudin-1 localization at the cell membrane and increases permeability through a PKC-dependent mechanism. METHODS: To test this hypothesis, we exposed normal human bronchial epithelial (NHBE) cells, cells from a human bronchial epithelial transformed cell line (Beas-2B), and Beas-2B expressing a PKCα dominant negative (DN) to alcohol (20, 50, and 100 mM) for up to 48 hours. Immunofluorescence was used to assess changes in ZO-1, claudin-1, claudin-5, and claudin-7 localization. Electric cell-substrate impedance sensing was used to measure the permeability of tight junctions between monolayers of NHBE, Beas-2B, and DN cells. RESULTS: Alcohol increased tight junction permeability in a concentration-dependent manner and decreased ZO-1, claudin-1, claudin-5, and claudin-7 localization at the cell membrane. To determine a possible signaling mechanism, we measured the activity of PKC isoforms (alpha, delta, epsilon, and zeta). PKCα activity significantly increased in Beas-2B cells from 1 to 6 hours of 100 mM alcohol exposure, while PKCζ activity significantly decreased at 1 hour and increased at 3 hours. Inhibiting PKCα with Gö-6976 prevented the alcohol-induced protein changes in both ZO-1 and claudin-1 at the cell membrane. PKCα DN Beas-2B cells were resistant to alcohol-induced protein alterations. CONCLUSIONS: These results suggest that alcohol disrupts ZO-1, claudin-1, claudin-5, and claudin-7 through the activation of PKCα, leading to an alcohol-induced "leakiness" in bronchial epithelial cells. Such alcohol-induced airway-leak state likely contributes to the impaired airway host defenses associated with acute and chronic alcohol ingestion.

The p38 mitogen-activated protein kinases modulate endothelial cell survival and tissue repair.

OBJECTIVE AND DESIGN: This study is designed to investigate the role of p38 MAPK in modulating human pulmonary artery endothelial cells (HPAECs) survival and tissue repair functions. METHODS: HPAECs (passage 8-12) were used for all experiments. Cells were treated with IL-1ß (0.5 or 2 ng/ml) or p38 inhibitor (SB203580 or SB220025, 5 µM each). Cells were also transfected with 50 nM siRNAs. Cell length was measured using ImageJ software. Collagen gel contraction and wound close assay were performed to evaluate tissue repair functions. RESULTS: IL-1ß activated p38 MAPK and induced morphologic change of HPAECs. The p38 inhibitors further augmented IL-1ß-induced cell morphologic change, prevented cell death, and augmented collagen gel contraction. Suppression of p38α, γ, or δ, but not p38ß resulted in cell morphologic alteration, and suppressing any one of p38 isoforms by siRNAs increased cell survival. Suppression of p38α or δ augmented gel contraction. While p38α suppression stimulated cell migration, suppressing the rest of three isoforms inhibit cell migration. Nuclear factor p65-siRNA blocked IL-1ß-induced cell morphologic change, but did not affect p38 inhibitor-induced change. CONCLUSION: These findings suggest that p38 MAPK may negatively modulate tissue repair functions of endothelial cells via p65 independent pathway.

The Arf6/PIP5K pathway activates IKACh in cigarette smoke mediated atrial fibrillation.

Cigarette smoking (CS) is a major cause of cardiovascular diseases. Smokers are at a significantly higher risk for developing atrial fibrillation (AF), a dangerous and abnormal heart rhythm. In the US, 15.5% of adults are current smokers, and it is becoming clear that CS is an independent risk factor for AF, but a detailed mechanistic understanding of how CS contributes to the molecular patho-electrophysiology of AF remains elusive. We investigated if CS related AF is in part mediated through a mechanism that depends on the cardiac acetylcholine activated inward rectifier potassium current (IKACh). We tested the hypothesis that CS increases IKACh via phosphatidylinositol 4-phosphate 5-kinase alpha (PIP5K) and ADP ribosylation factor 6 (Arf6) signaling, leading to AF perpetuation. In vivo inducibility of AF was assessed in mice exposed to CS for 8 weeks. AF duration was increased in CS exposed mice, and TertiapinQ, an IKACh blocker prevented AF development in CS exposed mice. In HEK293 cells stably transfected with Kir3.1 and Kir3.4, the molecular correlates of IKACh, CS exposure increased the expression of the Kir3.1 and Kir3.4 proteins at the cell surface, activated Arf6 and increased the IKACh current. Inhibition of PIP5K, or of Kir3.1/Kir3.4 trafficking via Arf6 abrogated the CS effects on IKACh. Cigarette smoke modifies the atrial electrophysiological substrate, leading to arrhythmogenesis, in part, through IKACh activation via an Arf6/PIP5K dependent pathway.

Adenosine activation of A(2B) receptor(s) is essential for stimulated epithelial ciliary motility and clearance.

Mucociliary clearance, vital to lung clearance, is dependent on cilia beat frequency (CBF), coordination of cilia, and the maintenance of periciliary fluid. Adenosine, the metabolic breakdown product of ATP, is an important modulator of ciliary motility. However, the contributions of specific adenosine receptors to key airway ciliary motility processes are unclear. We hypothesized that adenosine modulates ciliary motility via activation of its cell surface receptors (A(1), A(2A), A(2B), or A(3)). To test this hypothesis, mouse tracheal rings (MTRs) excised from wild-type and adenosine receptor knockout mice (A(1), A(2A), A(2B), or A(3), respectively), and bovine ciliated bronchial epithelial cells (BBECs) were stimulated with known cilia activators, isoproterenol (ISO; 10 µM) and/or procaterol (10 µM), in the presence or absence of 5'-(N-ethylcarboxamido) adenosine (NECA), a nonselective adenosine receptor agonist [100 nM (A(1), A(2A), A(3)); 10 µM (A(2B))], and CBF was measured. Cells and MTRs were also stimulated with NECA (100 nM or 10 µM) in the presence and absence of adenosine deaminase inhibitor, erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (10 µM). Both ISO and procaterol stimulated CBF in untreated cells and/or MTRs from both wild-type and adenosine knockout mice by ~3 Hz. Likewise, CBF significantly increased ~2-3 Hz in BBECs and wild-type MTRs stimulated with NECA. MTRs from A(1), A(2A), and A(3) knockout mice stimulated with NECA also demonstrated an increase in CBF. However, NECA failed to stimulate CBF in MTRs from A(2B) knockout mice. To confirm the mechanism by which adenosine modulates CBF, protein kinase activity assays were conducted. The data revealed that NECA-stimulated CBF is mediated by the activation of cAMP-dependent PKA. Collectively, these data indicate that purinergic stimulation of CBF requires A(2B) adenosine receptor activation, likely via a PKA-dependent pathway.

Co-inhibition of CD73 and ADORA2B Improves Long-Term Cigarette Smoke Induced Lung Injury.

Adenosine (ADO) involvement in lung injury depends on the activation of its receptors. The ADO A2A receptor (ADORA2A) and A2B receptor (ADORA2B) are best described to have both tissue-protective and tissue-destructive processes. However, no approach has been effective in delineating the mechanism(s) involved with ADO shifting from its tissue-protective to tissue-destructive properties in chronic airway injury. Using cigarette smoke (CS) as our model of injury, we chronically exposed Nuli-1 cells to 5% CS extract (CSE) for 3 years establishing a long-term CSE exposure model (LTC). We found significant morphological changes, decreased proliferation, and migration resulting in impaired airway wound closure in LTC. Further investigations showed that long-term CSE exposure upregulates CD73 and ADORA2B expression, increases ADO production, inhibits PKC alpha activity and p-ERK signaling pathway. Knocking down ADORA2B and/or CD73 in LTC activates PKC alpha and increases p-ERK signaling. Knocking down both showed better improvement in wound repair than either alone. In vivo experiments also showed that double knockout CD73 and ADORA2B remarkably improved CS-induced lung injury by activating PKC alpha, reducing the inflammatory cell number in bronchoalveolar lavage fluid and the production of inflammatory mediator IL-6, inhibiting the fibrosis-like lesions and decreasing collagen deposition surrounding bronchioles. Collectively, long-term CSE exposure upregulates CD73 expression and increases ADO production, which promotes low affinity ADORA2B activation and subsequent diminution of PKC alpha activity and ERK signaling pathway, and inhibition of airway wound repair. Moreover, the data suggesting ADORA2B and CD73 as potential therapeutic targets may be more efficacious in improving chronic CS lung diseases and impaired wound repair.

Melatonin Regulates Breast Cancer Progression by the lnc010561/miR-30/FKBP3 Axis.

Melatonin has been recognized to slow breast cancer growth. The molecular mechanisms may involve long non-coding RNAs (lncRNAs). However, little is known on how melatonin affects lncRNA expression and function in breast cancer. We used microarrays to explore the expression profile of mRNAs and lncRNAs in melatonin-treated breast cancer cells. Kyoto encyclopedia of genes and genomes (KEGG) and Reactome pathways analysis were performed to identify the signaling pathways affected by altered expressed mRNAs after melatonin treatment. To explore the functions and mechanisms of the selected differentially expressed mRNA and lncRNA in breast cancer, we performed a series of experiments. We found that FK506-binding protein 3 (FKBP3) and lnc010561 were downregulated in melatonin-treated breast cancer cells. Knockdown of FKBP3 and lnc010561 inhibited breast cancer proliferation and invasion, and induced apoptosis. Also, lnc010561 and FKBP3 functioned as competing endogenous RNAs (ceRNAs) for miR-30. Our findings suggested that melatonin regulated breast cancer progression by the lnc010561/miR-30/FKBP3 axis. Melatonin may, therefore, function as an anticancer strategy for breast cancer.

Influence of Cigarettes and Alcohol on the Severity and Death of COVID-19: A Multicenter Retrospective Study in Wuhan, China.

BACKGROUND: The recent emergence and rapid global spread of coronavirus disease 2019 (COVID-19) is leading to public health crises worldwide. Alcohol consumption and cigarette smoking (CS) are two known risk factors in many diseases including respiratory infections. METHODS: We performed a multi-center study in the four largest hospitals designated for COVID-19 patients in Wuhan. There are totally 1547 patients diagnosed with COVID-19 enrolled in the study, alcohol consumption and CS history were evaluated among these patients. The epidemiology, laboratory findings and outcomes of patients contracted COVID-19 were further studied. RESULTS: Our findings indicated that COVID-19 patients with a history of CS tend to have more severe outcomes than non-smoking patients. However, alcohol consumption did not reveal significant effects on neither development of severe illness nor death rates in COVID-19 patients. CONCLUSION: CS is a risk factor for developing severe illness and increasing mortality during the SARS-CoV-2 infection. We believe that our findings will provide a better understanding on the effects of alcohol intake and CS exposure in COVID-19 patients.

Ethanol blocks adenosine uptake via inhibiting the nucleoside transport system in bronchial epithelial cells.

BACKGROUND: Adenosine uptake into cells by nucleoside transporters plays a significant role in governing extracellular adenosine concentration. Extracellular adenosine is an important signaling molecule that modulates many cellular functions via 4 G-protein-coupled receptor subtypes (A(1), A(2A), A(2B), and A(3)). Previously, we demonstrated that adenosine is critical in maintaining airway homeostasis and airway repair and that airway host defenses are impaired by alcohol. Taken together, we hypothesized that ethanol impairs adenosine uptake via the nucleoside transport system. METHODS: To examine ethanol-induced alteration on adenosine transport, we used a human bronchial epithelial cell line (BEAS-2B). Cells were preincubated for 10 minutes in the presence and absence of varying concentrations of ethanol (EtOH). In addition, some cells were pretreated with S-(4-Nitrobenzyl)-6-thioinosine (100 microM: NBT), a potent adenosine uptake inhibitor. Uptake was then determined by addition of [(3)H]-adenosine at various time intervals. RESULTS: Increasing EtOH concentrations resulted in increasing inhibition of adenosine uptake when measured at 1 minute. Cells pretreated with NBT effectively blocked adenosine uptake. In addition, short-term EtOH revealed increased extracellular adenosine concentration. Conversely, adenosine transport became desensitized in cells exposed to EtOH (100 mM) for 24 hours. To determine the mechanism of EtOH-induced desensitization of adenosine transport, cAMP activity was assessed in response to EtOH. Short-term EtOH exposure (10 minutes) had little or no effect on adenosine-mediated cAMP activation, whereas long-term EtOH exposure (24 hours) blocked adenosine-mediated cAMP activation. Western blot analysis of lysates from unstimulated BEAS-2B cells detected a single 55 kDa band indicating the presence of hENT1 and hENT2, respectively. Real-time RT-PCR of RNA from BEAS-2B revealed transcriptional expression of ENT1 and ENT2. CONCLUSIONS: Collectively, these data reveal that acute exposure of cells to EtOH inhibits adenosine uptake via a nucleoside transporter, and chronic exposure of cells to EtOH desensitizes the adenosine transporter to these inhibitory effects of ethanol. Furthermore, our data suggest that inhibition of adenosine uptake by EtOH leads to an increased extracellular adenosine accumulation, influencing the effect of adenosine at the epithelial cell surface, which may alter airway homeostasis.

Repetitive organic dust exposure in vitro impairs macrophage differentiation and function.

BACKGROUND: Organic dust exposure in the agricultural industry results in significant airway disease and lung function decrease. Mononuclear phagocytes are key cells that mediate the inflammatory and innate immune response after dust exposure. OBJECTIVE: We sought to investigate the effect of organic dust extract (ODE) from modern swine operations on monocyte-derived macrophage (MDM) phenotype and function. METHODS: Peripheral blood monocytes were obtained by means of elutriation methodology (>99% CD14(+)) and differentiated into macrophages in the presence of GM-CSF (1 week) with and without ODE (0.1%). At 1 week, cells were analyzed by means of flow cytometry for cell-surface marker expression (HLA-DR, CD80, CD86, Toll-like receptor 2, Toll-like receptor 4, mCD14, and CD16), phagocytosis (IgG-opsonized zymosan particles), and intracellular killing of Streptococcus pneumoniae. At 1 week, MDMs were rechallenged with high-dose ODE (1%), LPS, and peptidoglycan (PGN), and cytokine levels (TNF-alpha, IL-6, IL-10, and CXCL8/IL-8) were measured. Comparisons were made to MDMs conditioned with heat-inactivated dust, endotoxin-depleted dust, LPS, and PGN to elucidate ODE-associated factors. RESULTS: Expression of HLA-DR, CD80, and CD86; phagocytosis; and intracellular bacterial killing were significantly decreased with ODE-challenged versus control MDMs. Responses were retained after marked depletion of endotoxin. PGN, LPS, and PGN plus LPS significantly reduced MDM surface marker expression and, except for LPS alone, also reduced phagocytosis. ODE-challenged MDMs had significantly diminished cytokine responses (TNF-alpha, IL-6, and IL-10) after repeat challenge with high-dose ODE. Cross-tolerant cytokine responses were also observed. CONCLUSION: Repetitive organic dust exposure significantly decreases markers of antigen presentation and host defense function in MDMs. Bacterial cell components appear to be driving these impaired responses.

Study on the therapeutic index and synergistic effect of Chitosan-zinc oxide nanomicellar composites for drug-resistant bacterial biofilm inhibition.

The synergistic effectiveness of chitosan with zinc oxide nanomicelles (CZNPs) on broad spectrum of multidrug resistance (MDR) was previously evidenced in our labs, requiring elucidation of the therapeutic index (TI) for safe in vivo use. This in vitro assessment estimated the effective dose (ED50) of micellar CZNPs for eradication of the MDR Enterococcus faecium 1449 model and the corresponding cytotoxic dose (LD50) against rat small intestinal epithelial cells as functions of TI. In order to visually determine the mechanistic effects of micellar CZNPs on bacterial biofilm size reduction, LIVE/DEAD viability assay was used in conjunction with advanced fluorescence imaging and 3D confocal microscopy. Biofilm quantification was performed through the measure of the fluorescence intensity, using the Biotek Synergy Neo2 for calculating the ED50. To generate the LD50, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was implemented. Quantification results revealed, at the same concentration (200 µg/mL), micellar CZNPs had average biofilm reduction of approximately 50.22% at 24 h (ED50 = 199.13 µg/mL, LD50 = 240.20 µg/mL, TI = 1.2062), compared to chitosan (15.66%) and ZnO (13.94%) alone. Conclusively, the ED50 of micellar CZNPs on MDR bacterial biofilms (199.13 µg/mL) as a function of TI reveals a promising nanotherapeutic agent in comparison to either Chitosan or ZnO alone.

PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer.

PTEN phosphorylation at its C-terminal (C-tail) serine/threonine cluster negatively regulates its tumor suppressor function. However, the consequence of such inhibition and its downstream effects in driving lung cancer remain unexplored. Herein, we ascertain the molecular mechanisms by which phosphorylation compromises PTEN function, contributing to lung cancer. Replacement of the serine/threonine residues with alanine generated PTEN-4A, a phosphorylation-deficient PTEN mutant, which suppressed lung cancer cell proliferation and migration. PTEN-4A preferentially localized to the nucleus where it suppressed E2F1-mediated transcription of cell cycle genes. PTEN-4A physically interacted with the transcription factor E2F1 and associated with chromatin at gene promoters with E2F1 DNA-binding sites, a likely mechanism for its transcriptional suppression function. Deletion analysis revealed that the C2 domain of PTEN was indispensable for suppression of E2F1-mediated transcription. Further, we uncovered cancer-associated C2 domain mutant proteins that had lost their ability to suppress E2F1-mediated transcription, supporting the concept that these mutations are oncogenic in patients. Consistent with these findings, we observed increased PTEN phosphorylation and reduced nuclear PTEN levels in lung cancer patient samples establishing phosphorylation as a bona fide inactivation mechanism for PTEN in lung cancer. Thus, use of small molecule inhibitors that hinder PTEN phosphorylation is a plausible approach to activate PTEN function in the treatment of lung cancer. Abbreviations AKT V-Akt Murine Thymoma Viral Oncogene CA Cancer adjacent CDK1 Cyclin dependent kinase 1 CENPC-C Centromere Protein C ChIP Chromatin Immunoprecipitation co-IP Co-immunoprecipitation COSMIC Catalog of Somatic Mutations In Cancer CREB cAMP Responsive Element Binding Protein C-tail Carboxy terminal tail E2F1 E2F Transcription Factor 1 ECIS Electric Cell-substrate Impedance Sensing EGFR Epidermal Growth Factor Receptor GSI Gamma Secretase Inhibitor HDAC1 Histone Deacetylase 1 HP1 Heterochromatin protein 1 KAP1/TRIM28 KRAB-Associated Protein 1/Tripartite Motif Containing 28 MAF1 Repressor of RNA polymerase III transcription MAF1 homolog MCM2 Minichromosome Maintenance Complex Component 2 miRNA micro RNA MTF1 Metal-Regulatory Transcription Factor 1 PARP Poly(ADP-Ribose) Polymerase PD-1 Programmed Cell Death 1 PD-L1 Programmed Cell Death 1 Ligand 1 PI3K Phosphatidylinositol-4,5-Bisphosphate 3-Kinase PLK Polo-like Kinase pPTEN Phosphorylated PTEN PTEN Phosphatase and Tensin Homolog deleted on chromosome ten PTM Post Translational Modification Rad51 RAD51 Recombinase Rad52 RAD52 Recombinase RPA1 Replication protein A SILAC Stable Isotope Labeling with Amino Acids in Cell Culture SRF Serum Response Factor TKI Tyrosine Kinase inhbitors TMA Tissue Microarray TOP2A DNA Topoisomerase 2A.

Adenosine promotion of cellular migration in bronchial epithelial cells is mediated by the activation of cyclic adenosine monophosphate-dependent protein kinase A.

Migration of neighboring cells into the injury is important for rapid repair of damaged airway epithelium. We previously reported that activation of the A(2A )receptors (A(2A)ARs) mediates adenosine-stimulated epithelial wound healing, suggesting a role for adenosine in migration. Because A(2A)AR increases cyclic adenosine monophosphate (cAMP) levels in many cells, we hypothesized that cAMP-dependent protein kinase A (PKA) is involved in adenosine-mediated cellular migration. To test this hypothesis, we stimulated a human bronchial epithelial cell line with adenosine and/or A(2A)AR agonist (5'-(N-cyclopropyl)-carboxamido-adenosine [CPCA]) in the presence or absence of adenosine deaminase inhibitor (erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride [EHNA]). Cells treated with adenosine or CPCA demonstrated a concentration-dependent increase in migration. Similar results were observed in the presence and absence of EHNA. To confirm A(2A) involvement, we pretreated the cells for 1 hour with the A(2A) receptor antagonist ZM241385 and then stimulated them with either adenosine or CPCA. To elucidate PKA's role, cells were pretreated for 1 hour with either a PKA inhibitor (KT5720) or a cAMP antagonist analogue (Rp-cAMPS) and then stimulated with adenosine and/or CPCA. Pretreatment with KT5720 or Rp-cAMPS resulted in a significant decrease in adenosine-mediated cellular migration. PKA activity confirmed that bronchial epithelial migration requires cAMP and PKA activity. When cells were wounded and stimulated with CPCA, an increase in PKA activity occurred. Pretreatment for 1 hour with either KT5720 or Rp-cAMPS resulted in a significant decrease in adenosine-mediated PKA activation. These data suggest that adenosine activation of A(2A)AR augments epithelial repair by increasing airway cellular migration by PKA-dependent mechanisms.

Cigarette Smoke Impairs A2A Adenosine Receptor Mediated Wound Repair through Up-regulation of Duox-1 Expression.

Cigarette smoke (CS) exposure and intrinsic factors such as the NADPH oxidases produce high levels of reactive oxygen species (ROS), ensuing inflammatory tissue injury. We previously demonstrated that CS-generated ROS, particularly hydrogen peroxide (H2O2), impaired adenosine stimulated wound repair. We hypothesized that CS exposure modulates expression of Dual oxidase 1 (Duox-1), a NADPH oxidases known to generate H2O2. To test this hypothesis, we used human bronchial epithelial cell line Nuli-1 and C57BL/6 mice. Cells were treated with 5% CS extract (CSE) for various periods of time, and mice were exposed to whole body CS for six weeks. Both CSE and CS treatment induced increased expression of Duox-1, and silencing of Doux-1 improved the rate of cell wound repair induced by CSE treatment. Nuli-1 cells pretreated with thapsigargin but not calcium ionophore exhibited increased Duox-1 mRNA expression. CSE treatment stimulated PKCα activation, which was effectively blocked by pretreatment with diphenylene iodonium, a NADPH oxidase inhibitor. Compared to control, lungs from CS-exposed mice showed a significant increase in PKCα activity and Duox-1 expression. Collectively, the data demonstrated that CS exposure upregulates expression of Duox-1 protein. This further leads to H2O2 production and PKCα activation, inhibiting A2AAR-stimulated wound repair.

Galectin-1 inhibition attenuates profibrotic signaling in hypoxia-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by lung remodeling arising from epithelial injury, aberrant fibroblast growth, and excessive deposition of extracellular matrix. Repeated epithelial injury elicits abnormal wound repair and lung remodeling, often associated with alveolar collapse and edema, leading to focal hypoxia. Here, we demonstrate that hypoxia is a physiological insult that contributes to pulmonary fibrosis (PF) and define its molecular roles in profibrotic activation of lung epithelial cells. Hypoxia increased transcription of profibrotic genes and altered the proteomic signatures of lung epithelial cells. Network analysis of the hypoxic epithelial proteome revealed a crosstalk between transforming growth factor-ß1 and FAK1 (focal adhesion kinase-1) signaling, which regulated transcription of galectin-1, a profibrotic molecule. Galectin-1 physically interacted with and activated FAK1 in lung epithelial cells. We developed a novel model of exacerbated PF wherein hypoxia, as a secondary insult, caused PF in mice injured with subclinical levels of bleomycin. Hypoxia elevated expression of phosphorylated FAK1, galectin-1, and α-smooth muscle actin and reduced caspase-3 activation, suggesting aberrant injury repair. Galectin-1 inhibition caused apoptosis in the lung parenchyma and reduced FAK1 activation, preventing the development of hypoxia-induced PF. Galectin-1 inhibition also attenuated fibrosis-associated lung function decline. Further, galectin-1 transcript levels were increased in the lungs of IPF patients. In summary, we have identified a profibrotic role of galectin-1 in hypoxia signaling driving PF.