Showing papers in "Experimental Lung Research in 2013"
TL;DR: The data clearly indicate that the upregulation of miR146a suppresses inflammatory mediators in LPS induced-ALI model, and miR-146a may be therapeutically targeted as a mean to repress inflammatory response following ALI.
Abstract: Despite the critical role of microRNA in inflammatory response, little is known about its function in inflammation-induced Acute Lung Injury (ALI)/Acute Respiratory Distress Syndrome (ARDS). To investigate the potential role of microRNA146a (miR-146a) in ALI, we used lipopolysaccharide (LPS)-induced ALI rat model. Our data revealed that LPS-induced lung injury in rats resulted in significant upregulation of proinflammatory cytokine tumor necrosis factor-alpha (TNF-α), IL-6, IL-1β, and miR-146a expression. LPS treatment also leads to higher expression of miR-146a as well as increase in secretion of TNF-α, IL-6, and IL-1β in alveolar macrophage (AM) NR8383 cells in a time-dependent manner. Manipulation with miR146a mimic significantly suppressed LPS-mediated TNF-α, IL-6, and IL-1β induction in NR8383 cells by repressing expression of IRAK-1 and TRAF-6. These data clearly indicate that the upregulation of miR146a suppresses inflammatory mediators in LPS induced-ALI model. Therefore, miR-146a may be therapeutically targeted as a mean to repress inflammatory response following ALI.
132 citations
TL;DR: This paper attempts to describe the cellular and molecular mechanisms governing the differentiation of goblet cells in pulmonary diseases, a prerequisite for the development of new therapeutic agents.
Abstract: The mucociliary system, consisting of mucus-secreting goblet cells and ciliated cells, generates a constant overturning layer of protective mucus that lines the airway epithelium. Mucus hypersecretion and the pathophysiological changes associated are hallmarks of many pulmonary diseases including asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Excessive mucus production leads to airway obstruction and, because there is currently no effective treatment, contributes to morbidity and mortality of many patients. Goblet cell differentiation and mucus production are subject to extensive control. An emerging concept is that not all goblet cells are phenotypically identical suggesting that specific molecular pathways orchestrate mucin overproduction. This paper attempts to describe the cellular and molecular mechanisms governing the differentiation of goblet cells in pulmonary diseases, a prerequisite for the development of new therapeutic agents.
66 citations
TL;DR: The intravenous infusion of MSCs, the common mode of delivery, is followed by their entrapment in lung vasculature before MSCS reach to other organ systems thus indicating the feasible and promising approach of M SCs therapy for lung diseases.
Abstract: Lung disorders such as asthma, acute respiratory distress syndrome (ARDS), chronic obstructive lung disease (COPD), and interstitial lung disease (ILD) show a few common threads of pathogenic mechanisms: inflammation, aberrant immune activity, infection, and fibrosis. Currently no modes of effective treatment are available for ILD or emphysema. Being anti-inflammatory, immunomodulatory, and regenerative in nature, the administration of mesenchymal stem cells (MSCs) has shown the capacity to control immune dysfunction and inflammation in the lung. The intravenous infusion of MSCs, the common mode of delivery, is followed by their entrapment in lung vasculature before MSCs reach to other organ systems thus indicating the feasible and promising approach of MSCs therapy for lung diseases. In this review, we discuss the mechanistic basis for MSCs therapy for asthma, ARDS, COPD, and ILD.
61 citations
TL;DR: Most Chinese lung adenocarcinoma patients harbor driver gene mutations, among which ALK rearrangements were more common in Hunan patients than in previously reported populations.
Abstract: Introduction: Mutation analysis of cancer driver genes is helpful for determining an optimal treatment strategy. We evaluated mutations in four driver genes, namely epidermal growth factor receptor (EGFR), Kirsten ras oncogene (KRAS), c-MET, and echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK), in Chinese lung adenocarcinoma patients from Hunan Province. Methods: We enrolled 110 lung adenocarcinoma patients in a single institution. EGFR and KRAS mutations were examined by direct sequencing, the EML4-ALK fusion gene was analyzed by fluorescence in situ hybridization, and c-MET amplification and c-Met protein expression were detected by quantitative PCR and immunohistochemistry, respectively. Results: EGFR and KRAS mutations were observed in 52.7% (58/110) and 3.6% (4/106) of patients, respectively. c-MET amplification was detected in 5.5% (6/110) of patients. In addition, 30% (33/110) of the cases expressed c-Met protein, including all of the patients harboring...
53 citations
TL;DR: The results suggest that NUAK1 plays an important role in NSCLC cell migration and invasion.
Abstract: Novel (nua) kinase family 1 (NUAK1) is a member of the human adenosine monophosphate (AMP)-activated protein kinase family that has been identified as a key tumor cell survival factor. In the present study, we investigated the role of NUAK1 in the migration and invasion of human nonsmall cell lung cancer (NSCLC) cells. Immunohistochemistry staining showed that the expression of NUAK1 correlated with the differentiation and stage of the carcinoma, as well as with lymph node metastasis. Inhibition of NUAK1 expression by small interference RNA severely impaired migration and invasion in A549 cells. In addition, we found that the knockdown of NUAK1 suppressed the expression of MMP-2 and MMP-9 and the activation of NF-kB, which can regulate the transcription of MMP-2 and MMP-9. Correspondingly, NUAK1 knockdown reduced lung metastasis in a xenograft mouse model of NSCLC. Taken together, our results suggest that NUAK1 plays an important role in NSCLC cell migration and invasion.
50 citations
TL;DR: Intraperitoneal injection of CSE produced emphysema, pulmonary parenchymal apoptosis, and injury of cardiac and skeletal muscles in mice, and all pathobiologically relevant mechanisms in this model are shared with the COPD patients.
Abstract: Background: Chronic obstructive pulmonary disease (COPD) is a chronic, progressive, airway disease. In order to recognize mechanisms of COPD, various types of COPD animal models have been established, and the pathogenesis are different. The present study was designed to establish a COPD animal model by intraperitoneal injection of cigarette smoke extract (CSE) in BALB/C mice. Methods: Mice were injected intraperitoneally with PBS/CSE and sacrificed at day 28. Pulmonary function, pathology of lung tissue, morphology of hearts and skeletal muscle, leukocytes count and antioxidant activity of bronchoalveolar lavage fluid (BALF), pulmonary parenchymal apoptosis index (AI), expression of cleaved caspase-3, expression of MMP-2 and MMP-9 mRNA, and activity of MMP-2 and MMP-9 in lung tissue were measured. Results: Intraperitoneal injection of CSE induced pulmonary parenchymal destruction, pulmonary function reduction, leukocytes count, injury of cardiac and peripheral muscles, and increased pulmonary pare...
41 citations
TL;DR: It is indicated that a HFD inhibits airway eosinophilia while altering levels of PAI-1 and PGE2 in response to CRA in mice, which could lead to the development of lung fibrosis even in the absence of allergen exposure.
Abstract: Obesity is an important risk factor for asthma but the mechanistic basis for this association is not well understood. In the current study, the impact of obesity on lung inflammatory responses after allergen exposure was investigated. C57BL/6 mice maintained on a high-fat diet (HFD) or a normal diet (ND) after weaning were sensitized and challenged with cockroach allergen (CRA). Airway inflammation was assessed based on inflammatory cell recruitment, measurement of lung Th1-Th2 cytokines, chemokines, eicosanoids, and other proinflammatory mediators as well as airway hyperresponsiveness (AHR). CRA-challenged mice fed a HFD exhibited significantly decreased allergen-induced airway eosinophilia along with reduced lung IL-5, IL-13, LTC4, CCL11, and CCL2 levels as well as reduced mucus secretion and smooth muscle mass compared to ND fed mice. However, allergen-challenged HFD fed mice demonstrated significantly increased PAI-1 and reduced PGE2 levels in the lung relative to corresponding ND fed mice. Interestingly, saline-exposed HFD fed mice demonstrated elevated baseline levels of TGF-β1, arginase-1, hypoxia-inducible factor-1α, and lung collagen expression associated with decreased lung function compared to corresponding ND fed mice. These studies indicate that a HFD inhibits airway eosinophilia while altering levels of PAI-1 and PGE2 in response to CRA in mice. Further, a HFD can lead to the development of lung fibrosis even in the absence of allergen exposure which could be due to innate elevated levels of specific profibrotic factors, potentially affecting lung function during asthma.
36 citations
TL;DR: In this article, different types of circulating EMPs with distinct time profiles were detectable during exacerbations. And they hypothesized that the released EMP subtypes correlated with differences in the inflammatory stimuli and the endothelial cell type.
Abstract: Circulating endothelial microparticles (EMPs) are membrane vesicles that are shed into the blood stream from activated or apoptotic endothelial cells. We previously reported that circulating EMP numbers significantly increased in stable chronic obstructive pulmonary disease (COPD) patients and during exacerbation compared with healthy control subjects. However, different types of circulating EMPs with distinct time profiles were detectable during exacerbations. We hypothesized that the released EMP subtypes correlated with differences in the inflammatory stimuli and the endothelial cell type. We compared the EMP subtypes from human aortic endothelial cells (Aortic ECs) and human lung microvascular endothelial cells (Pulmonary microvascular ECs) released in response to various stimuli, including proinflammatory cytokines (TNFα), oxidative stress (H2O2), and cigarette smoke extracts (CSE) in vitro. We defined circulating EMPs by the expression of endothelial antigens: CD144(+) MPs (VE-cadherin EMPs), CD31(+)/CD41(-) MPs (PECAM EMPs), CD62E(+) MPs (E-selectin EMPs), and CD146(+) MPs (MCAM EMPs). E-selectin EMPs were released from both pulmonary microvascular and aortic ECs in response to TNFα but not to H2O2 or CSE stimulation. The amount of MCAM EMPs released from pulmonary microvascular ECs differed significantly between the cells stimulated with H2O2 and those stimulated with CSE. VE-cadherin EMPs were only released from aortic ECs, whereas PECAM EMPs were released exclusively from pulmonary microvascular ECs. The EMP subtypes released differ in vitro among TNFα, H2O2, and CSE stimulation as well as between pulmonary microvascular and aortic ECs. The differences in circulating EMP subtypes may reflect a condition or site of endothelial injury and may serve as markers for endothelial damage in COPD patients.
29 citations
TL;DR: BS can cause emphysema and chronic bronchitis similar to that caused by CS, which is accompanied by systemic oxidative stress and inflammation.
Abstract: Background: Epidemiological research and meta-analyses of published data have shown that biomass smoke (BS) is a risk factor for chronic obstructive pulmonary disease (COPD). However, the link between BS and COPD lacks experimental confirmation. Objectives: To verify whether BS can induce pathologic changes and systemic oxidative stress, which may be relevant to the development of emphysema and chronic bronchitis in rats. Methods: Rats were exposed to BS, cigarette smoke (CS), or clean air (sham) for 14 weeks. During the exposure, the O2, SO2, and CO levels were monitored. Pathological changes in the lungs, systemic oxidative stress, and inflammation biomarkers, together with GSTM1 and GSTP1 mRNA expression in the lung were measured. The glutamate–cysteine ligase catalytic subunit (GCLC) protein expression in the lung was measured using immunohistochemistry and western blotting. Results: The O2, CO, and SO2 levels were 20.31 ± 0.03%, 981.72 ± 64.76, and 2.59 ± 0.26 mg/m3 for the BS group, respecti...
28 citations
TL;DR: Autophagy may be a scathing factor responsible for ALI induced by seawater, and autophagy must be understood to develop new methods for prevention and treatment of seawater-induced ALI.
Abstract: Seawater drowning can lead to acute lung injury (ALI). However, the molecular and cellular mechanisms underlying this phenomenon remain elusive. The overall aim of this study is to clarify the role of autophagy in seawater-induced ALI, by which we can further understand the molecular mechanism and develop new methods for prevention and treatment of seawater-induced ALI. In this study, electron microscopy, western blot analysis, and RT-PCR were used to detect autophagy in lung tissues. Moreover, arterial blood gas analysis, lung weight coefficient, TNF-α, IL-8 in bronchoalveolar fluid (BALF), histopathology were used to detect the lung injury of seawater exposure. An inhibitor of autophagy (3-Methyladenine, 3-MA) was injected intraperitoneally before seawater exposure to further explore the role of autophagy in ALI. Electron microscopy revealed increasing autophagosomes in alveolar epithelial cell in seawater group compared with the control. The transcription and expression levels (mRNA and protein levels) of the LC3 II significantly increased in lung tissue of seawater group compared with those in control group. Furthermore, the alterations of autophage were basically consistent with the changes in arterial blood gas, lung weight coefficient, TNF-α, IL-8 in BALF and morphologic findings. In addition, inhibition of autophagy by 3-MA partly ameliorated seawater-induced ALI, as indicated by reduced lung weight coefficient and TNF-α in BALF, as well as increased PaO2. In conclusion, seawater aspiration triggered autophagy, and autophagy may be a scathing factor responsible for ALI induced by seawater.
25 citations
TL;DR: Findings suggest that normal regulation of MUC5AC and Muc5B production by HBE cells is TGF-β isoform-specific and that TGF -β2 downregulates both MUC 5AC andMUC5B.
Abstract: Introduction: Mucus hypersecretion is a major contributor to asthma pathology and occurs as part of a spectrum of structural changes termed airway wall remodeling. Transforming growth factor (TGF)-β is proposed to play a key role in regulating airway matrix remodeling although less is known about the specific action of TGF-β isoforms in regulating mucus production. Methods: Primary human bronchial epithelial (HBE) cells cultured at air–liquid interface were treated with exogenous TGF-β1, TGF-β2, and/or a Th2 cytokine, interleukin (IL)-13. Expression and production of respiratory mucins, MUC5AC and MUC5B, were analyzed by real-time PCR, agarose gel electrophoresis, and western blotting. A murine-transformed Clara cell line (mtCC1-2) transfected with a luciferase reporter driven by the Muc5ac promoter containing Smad4 site-mutated cis sequences was used to determine whether exogenous TGF-β2 affects Muc5ac promoter function. Results: Surprisingly, TGF-β1 showed no measurable effect on MUC5AC or MUC5B...
TL;DR: TRPV1 channel was involved in the regulation of proliferation and apoptosis in asthmatic ASMC and was found a new target to treat airway remodeling in asthma.
Abstract: Background: Hyperplasia of airway smooth muscle cells (ASMC) is a major contributor to airway remodeling in asthma. Transient receptor potential vanilloid 1 (TRPV1) is an important channel to mediate Ca2+ influx. This study explores the expression of TRPV1 channel and its effect on the proliferation and apoptosis in rat ASMC, in order to find a new target to treat airway remodeling in asthma. Methods: Rats were sensitized and challenged with ovalbumin to replicate asthmatic models. Proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry. Reverse transcriptase-polymerase chain reaction, immunocytochemistry, and Western blot were used to detect the mRNA and protein expression of TRPV1 channel. Intracellular calcium ([Ca2+]i) was detected using confocal fluorescence Ca2+ imaging. [3H] thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were used to observe the DNA synthesis and proliferation. TUNEL assay was used to detect the ap...
TL;DR: The results provided the evidence of a cross-race susceptibility of the lung cancer locus and clarified the association that rs2736100 of the TERT gene conferred the highest significant risk of lung cancer.
Abstract: Lung cancer is a leading cause of cancer-related deaths throughout the world. Recent genome-wide association studies and consecutive validation supported that the 5p15.33 region containing telomerase reverse transcriptase gene (TERT) and cleft lip and palate transmembrane protein 1-like (CLPTM1L) gene showed significant association with lung cancer in multiple populations. Here we studied a large Chinese Han cohort consisting of 1759 cases and 1804 controls. In the 1st stage (784 cases versus 782 controls) we genotyped 13 tag SNPs within 5p15.33 region to further investigate the association. After the 2nd stage validation (975 cases versus 1022 controls), the study clarified the association that rs2736100 of the TERT gene conferred the highest significant risk of lung cancer (P = 4 × 10−3 in the 1st stage association, P = 4 × 10−4 in the 2nd stage validation, and P = 1 × 10−5, odds ratio = 1.24 in the combined population). The results provided the evidence of a cross-race susceptibility of the lun...
TL;DR: The present study indicates that the consumption of a single cigarette may alter lung mechanics and FENO production among young smokers, and further research is needed to assess the mechanisms and washout period after which these parameters return to normal.
Abstract: Introduction: Smoking is known to have a long-term impact on lung function; however, the acute physiological response of smoking a single cigarette and the influential role of pack years and cigarettes per day on pulmonary indices remains an area of interest, especially among young smokers. Methods: 50 naive smokers (ages: 18–26, 24 males: mean pack years 3.8) participated in this experimental study. Respiratory resistance (R), reactance (X ), and impedance (Z ) were assessed through impulse oscillometry. The participants’ fraction of exhaled nitric oxide (FENO) was measured. All tests were performed immediately before and after smoking one single cigarette. Results: Smoking a single cigarette was found to immediately increase airway impedance (Z 5 Hz) by 0.024kPa/(L/s) (P = .002), airway resistance at R 5 Hz, R 10 Hz, and R 20 Hz by 0.024kPa/(L/s)(P < .001), 0.016kPa/(L/s)(P = .019), and 0.023kPa/(L/s) (P = .007), respectively, after adjusting for BMI, age, gender, and pack years. FENO concentrat...
TL;DR: The interaction between rhinovirus and PM2.5 significantly impairs airway function in asthma, and a larger sample size study is suggested to investigate these observations.
Abstract: Background: Elevated levels of particulate matter PM2.5 and rhinovirus infection have been known to exacerbate asthma. However, the combined effect of rhinovirus infection and high PM2.5 has not been investigated. Purpose: To investigate the effect of PM2.5 and concomitant rhinovirus infection on airway function in asthma in an area with high PM2.5 concentration. Methods: Asthmatics and their matched controls were monitored for lung function, exhaled nitric oxide (eNO) and respiratory symptoms on days with varying levels of PM2.5. As the study was a repeated measure design, repeated clinical findings, and laboratory data were used in the mixed model analysis. Results: Wheezing and dyspnea in asthmatics were worsened with increasing ambient PM2.5. Increasing PM2.5 decreased FEV1% predicted (–0.51, –0.79 to –0.23) and FEF25–75% predicted (–0.66, –1.07 to –0.24) in subjects with asthma (all P < .01). Rhino viral infection reduced FEF25–75% predicted in subjects with asthma (–11.7, –20 to –2.9). The r...
TL;DR: The results suggest that the high methylation statuses of p16, RASSF1A, or FHIT genes were associated with a significantly increased risk of lung cancer; the risk of Lung cancer increased as the methylation status increased.
Abstract: Background: Lung cancer is the leading cause of cancer-related deaths worldwide due mainly to late diagnosis and poor prognosis. Aberrant promoter methylation is an important mechanism for silencing of tumor suppressor genes during carcinogenesis and a promising tool for the development of molecular biomarkers. Methods: We evaluated the p16, RASSF1A, and FHIT genes promoter methylation status in peripheral blood DNA between 200 lung cancer patients and 200 normal controls by using SYBR green-based quantitative methylation-specific PCR (qMSP). Results: There were statistically significant differences in the methylation status of p16, RASSF1A, and FHIT between the cancer cases and controls (p16: P = .008, RASSF1A: P = .038, FHIT: P = .002). When the subjects were categorized into quartiles based on the genes methylation status, the risk of lung cancer was found to increase as methylation status increased (p16: Ptrend = .002, RASSF1A: Ptrend = .014, FHIT: Ptrend = .001). When the median of methylatio...
TL;DR: 5-HT inhibited PASMC apoptosis by activating the 5-HT2A receptor through the pERK1/2 and PDK pathways, which is involved, at least in part, in pulmonary artery remolding.
Abstract: Decreased pulmonary artery smooth muscle cell (PASMC) apoptosis play a key role in pulmonary artery remodeling during pulmonary artery hypertension (PAH), but the mechanisms involved are unclear. Serotonin (5-HT) inhibits apoptosis in many pathologic processes by activating the 5-HT2A receptor. Therefore, we hypothesized that 5-HT may be the promoter of decreased apoptosis in PAH through the 5-HT2A receptor. We found that inhibition of the 5-HT2A receptor prevented the increase in pulmonary artery pressure and pulmonary artery remodeling in rats stimulated by monocrotaline. This effect was accompanied by increased apoptosis in the pulmonary artery. Cultured PASMCs stimulated with 5-HT showed a decrease in apoptosis with increased phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), pyruvate dehydrogenase kinase (PDK), and mitochondrial transmembrane potential. These effects were markedly prevented by a 5-HT2A receptor inhibitor, an ERK1/2 activation inhibitor peptide I, or a PDK inhibitor. In conclusion, 5-HT inhibited PASMC apoptosis by activating the 5-HT2A receptor through the pERK1/2 and PDK pathways.5-HT decreasing apoptosis through 5-HT2A receptor is involved, at least in part, in pulmonary artery remolding.
TL;DR: Findings indicated the anti-KL-6 antibody may potentially be developed as a useful inhibitor of pulmonary fibrosis.
Abstract: Background: Elevated levels of KL-6 are reported in the serum and/or bronchoalveolar lavage fluid (BALF) of patients with interstitial lung disease (ILD) and are useful to estimate the severity and prognosis of the disease. However, whether the anti-KL-6 antibody could attenuate pulmonary fibrosis remains unclear. Objectives: This study aims to investigate the therapeutic effects and mechanisms of anti-KL-6 antibody on bleomycin-induced pulmonary fibrosis. Methods: A mouse model of pulmonary fibrosis was established by intratracheal injection of bleomycin (5 mg/kg). Mouse received anti-KL-6 antibody (20 ug/day, once a day) from day 7 to 21 after bleomycin injection. The effects of anti-KL-6 antibody were evaluated by pathological examination, measuring hydroxyproline measurements in lung tissues, leukocyte counts in BALF and the expression of collagen type I and type III using qRT-PCR. The expression of profibrotic cytokine (transforming growth factor-β1, TGF-β1), antifibrotic cytokine (hepatocyte...
TL;DR: The interaction between c-Kit and sKitL in bone marrow EPCs, a critical step in endothelial repair, is negatively affected in a CSE-induced emphysema model.
Abstract: Background: Smoking causes lung endothelial cell apoptosis and emphysema. Derived from bone marrow, circulating endothelial progenitor cells (EPCs) maintain vascular integrity by replacing and repairing damaged endothelial cells. Smoking influences the number of circulating EPCs. Recruitment of EPCs from bone marrow to peripheral blood depends on the interaction of c-Kit/soluble c-Kit ligand (sKitL). We hypothesized that smoking might influence c-Kit(+) EPCs/sKitL interaction in bone marrow in the development of smoking-related emphysema. In this study, we used a cigarette smoke extract (CSE)-induced emphysema model. Methods: Mice were injected intraperitoneally with PBS/CSE and sacrificed at day 28. Lung function and pathology of lung tissue were measured to characterize the model. Expressions of c-Kit in the lung tissue were assayed. Bone marrow cells were isolated by red blood cell lysis. EPCs/c-Kit(+) EPCs in nonred blood cells were analyzed by flow cytometry. Expressions of KitL and MMP-9, an...
TL;DR: In this article, the effects of thrombin, an important mediator of interstitial lung fibrosis, on EMT in A549 human alveolar epithelial cells were investigated.
Abstract: Thrombin activates protease-activated receptor (PAR)-1 and induces a myofibroblast phenotype in normal lung fibroblasts. The origins of myofibroblasts are resident fibroblasts, fibrocytes, and epithelial-mesenchymal transition (EMT). We investigated the effects of thrombin, an important mediator of interstitial lung fibrosis, on EMT in A549 human alveolar epithelial cells. We show that thrombin induced EMT and collagen I secretion through the activation of PAR-1, and PKC and ERK1/2 phosphorylation in A549 cells. These effects were largely prevented by a specific PAR-1 antagonist, short interfering RNA (siRNA) directed against PAR-1, or specific PKCα/β, δ, and e inhibitors. These data indicated that interaction with thrombin and alveolar epithelial cells might directly contribute to the pathogenesis of pulmonary fibrosis through EMT. Targeting PAR-1 on the pulmonary epithelium or specific inhibitors to PKCα/β, δ, and e might stop the fibrotic processes in human idiopathic pulmonary fibrosis by preventing thrombin-induced EMT.
TL;DR: It is found that andrographolide mitigates CSE-induced inflammatory response in A549 cells, largely through inhibition of NF-κB activation via upregulation of miR-218, and thus has preventive benefits in CS-inducedinflammatory lung diseases.
Abstract: Andrographolide is a major bioactive labdane diterpenoid isolated from Andrographis paniculata and has protective effects against cigarette smoke (CS)-induced lung injury. This study was done to determine whether such protective effects were mediated through modulation of microRNA (miR)-218 expression. Therefore, we exposed human alveolar epithelial A549 cells to cigarette smoke extract (CSE) with or without andrographolide pretreatment and measured the level of glutathione, nuclear factor-kappaB (NF-κB) activation, proinflammatory cytokine production, and miR-218 expression. We found that andrographolide pretreatment significantly restored the glutathione level in CSE-exposed A549 cells, coupled with reduced inhibitor κB (IκB)-α phosphorylation and p65 nuclear translocation and interleukin (IL)-8 and IL-6 secretion. The miR-218 expression was significantly upregulated by andrographolide pretreatment. To determine the biological role of miR-218, we overexpressed and downregulated its expression us...
TL;DR: Findings suggest that GSTs are upregulated in the airways of asthmatics in response to increased oxidative stress.
Abstract: Reactive oxygen species (ROS) are known aggravating factors for airway inflammation in asthma. Glutathione S-transferases (GSTs) detoxify ROS and toxic compounds in environmental exposures. However, little is known about the regulation of GST and expression of GST subtypes in asthma. The aim of this study was to evaluate how GSTs are regulated in asthma. We observed total GST activity and expression of GST subtypes in murine asthma models and GST expressions in induced sputum cells of asthmatics. Total GST activity was increased in BAL fluids of OVA-treated murine asthma model. GSTP and GSTA are highly expressed in peribronchiolar mononuclear inflammatory cells and epithelial cells in OVA-treated mice. GSTM are expressed in epithelial cells in both OVA and PBS-treated groups. GSTP1 mRNA expression was increased in the lung of OVA-treated mice compared with PBS-treated mice. GSTA1, GSTM1, and GSTT1 mRNA expressions were not different between both groups. GSTA1 mRNA expression was increased in induced sputum cells of asthmatics compared with healthy controls. GSTP1, GSTM1, and GSTT1 mRNA expressions were not different between asthmatics and healthy controls. In asthmatics, GSTP1 and GSTA1 mRNA expressions were higher in induced sputum cells of asthmatics with PC20 ≤ 4 mg/ml than those with PC20 > 4 mg/ml. GSTM1 and GSTT1 mRNA expressions were not different between two groups. These findings suggest that GSTs are upregulated in the airways of asthmatics in response to increased oxidative stress. GSTP and GSTA are thought to play an important role in protecting the airways of asthmatics compared with GSTM and GSTT.
TL;DR: The goal of this investigation was to develop a fast, repeatable method to individually isolate these low-frequency cell types from BAL and may be of use to statistically correlate low- frequency cell type abundance to the onset and development of BOS.
Abstract: The long-term outcome of lung transplants is poor with 60%-70% of patients developing chronic rejection. Chronic rejection is manifested histologically by obliterative bronchiolitis with bronchiolitis obliterans syndrome (BOS), the clinical surrogate. Recent studies suggest that fibroblasts and epithelial cells present in bronchoalveolar lavage (BAL) may be a clinically relevant biomarker for BOS. The goal of this investigation was to develop a fast, repeatable method to individually isolate these low-frequency cell types. Fibroblasts and epithelial cells were isolated from BAL using attachment methods and the phenotype of the cells confirmed using immunostaining for vimentin (fibroblasts) and epithelial cell adhesion molecule (EpCAM, epithelial cells). Both fibroblasts and epithelial cells were isolated in every sample of BAL processed with the frequency of fibroblasts ranging from 0.03% to 0.48% and epithelial cells ranging from 0.05% to 1.5% of the total sample. Additional studies were performed using cytospins of cells after macrophages were depleted; cells exhibiting characteristics of both fibroblasts and epithelial cells were observed. The frequency of the cells of interest suggests that conventional methods of immunomagnetic isolation will not be effective in isolating these subpopulations. Finally, some of the low-frequency cells isolated via cytospin exhibit characteristics of epithelial to mesenchymal transition (which was not observed in plating incubations), indicating that the epithelial to mesenchymal cell transition fibroblasts may be nonadherent. In future studies, this technique and dataset may be of use to statistically correlate low-frequency cell type abundance to the onset and development of BOS.
TL;DR: An adjuvant effect of zymosan was observed when exposures occurred during the sensitization phase in an OVA-induced allergy model in BALB/c mice and a non-linear dose–response relationship was observed in the groups primed with OVA and zymOSan.
Abstract: An association has been observed between indoor mold contamination and lung allergy and asthma. This relationship is not fully understood. 1→3-β-Glucan is the major cell wall component of fungi and a good marker of fungi exposure. The objective was to evaluate the adjuvant effect of zymosan, a crude yeast cell wall preparation of 1→3-β-glucan, during ovalbumin (OVA) sensitization in an allergy model. BALB/c mice were sensitized by pharyngeal aspiration with saline, 50 μg of OVA, or OVA with 1, 10, 50, or 75 μg of zymosan on days 0, 7, and 14. One week after sensitization, each sensitized animal group was challenged with an aspiration dose of 50 μg of OVA once a week for 2 weeks. At 1 day after the last aspiration, bronchoalveolar lavage fluid and blood was collected, and markers of lung allergy and inflammation were assessed. An adjuvant effect of zymosan on OVA allergy during sensitization was observed as indicated by significant elevations in lung eosinophils, serum OVA-specific IgE, and lung IL-5 in the groups sensitized with zymosan and OVA. Pulmonary treatment with zymosan also amplified lung inflammation. Elevations were observed in lung neutrophils, TNF-α, and parameters of lung injury in the groups primed with both zymosan and OVA. In nearly all parameters, a non-linear dose-response relationship was observed in the groups primed with OVA and zymosan. The optimum adjuvant dose of zymosan was 10 μg. This study demonstrated an adjuvant effect of zymosan when exposures occurred during the sensitization phase in an OVA-induced allergy model in BALB/c mice.
TL;DR: Investigation of longitudinal changes of the pulmonary inflammatory process as a result of mechanical stress due to mechanical ventilation in bronchoalveolar lavage found high concentrations of IL-8, TNF-α, MIP-1β, nitrites/nitrates, and inducible nitric oxide synthases (iNOS).
Abstract: The aim of this study was to investigate longitudinal changes of the pulmonary inflammatory process as a result of mechanical stress due to mechanical ventilation. The concentrations of IL-8, TNF-α, MIP-1β, nitrites/nitrates, and inducible nitric oxide synthases (iNOS) were investigated indicate in bronchoalveolar lavage (BAL). Twenty-three piglets were divided into three groups. Group I: animals breathing spontaneously; group II: mechanical ventilation (tidal volume (TV) = 7 mL/kg, PEEP = 5 cmH2O); group III: mechanical ventilation (TV = 15 mL/kg, PEEP = 0 cmH20). Concentrations of BAL nitrites/nitrates from groups II and III increased during the first hour of mechanical ventilation (P = .03 and .02, respectively). The highest expression of iNOS was observed during the first hour in groups II and III. IL-8 concentration increased significantly in groups II and III. Production of TNF-α increased significantly in group III during the second and third hour (P = .01). Concentration of MIP-1β was sign...
TL;DR: This work creates an in situ bronchial cast of the conducting airways in rats that can be subsequently imaged in situ using three-dimensional micro-CT imaging and demonstrates that deformations in airway branch angles resulting from the casting procedure are small and can be reversed through an interactive adjustment of the segmented cast geometry.
Abstract: The use of anatomically accurate, animal-specific airway geometries is important for understanding and modeling the physiology of the respiratory system. One approach for acquiring detailed airway architecture is to create a bronchial cast of the conducting airways. However, typical casting procedures either do not faithfully preserve the in vivo branching angles or produce rigid casts that when removed for imaging are fragile and thus easily damaged. We address these problems by creating an in situ bronchial cast of the conducting airways in rats that can be subsequently imaged in situ using three-dimensional micro-CT imaging. We also demonstrate that deformations in airway branch angles resulting from the casting procedure are small, and that these angle deformations can be reversed through an interactive adjustment of the segmented cast geometry. Animal work was approved by the Institutional Animal Care and Use Committee of Pacific Northwest National Laboratory.
TL;DR: It is demonstrated that exogenous Gal-9 suppressed dsRNA-induced AHR in an acute exacerbation model of chronic asthma in mice, and suggested that recombinant Gal- 9 could be therapeutically effective for preventing acute asthma exacerbation.
Abstract: Background. Viral respiratory infection is the most common cause of acute asthma exacerbation in patients with stable asthma. The replication of most respiratory viruses requires the generation of double-stranded RNA (dsRNA), resulting in the activation of host immune responses. Synthetic dsRNA, polyinosinic–polycytidylic acid (PolyIC), mimics the effects of viruses in various cell types. To evaluate new therapies for mite antigen-induced chronic asthma, we developed an acute exacerbation model of mouse chronic asthma using mite antigen and PolyIC. We also examined the preventive effects of recombinant galectin-9 (Gal-9) on acute asthma exacerbation in this model. Methods. Airway hyperresponsiveness (AHR) was examined to evaluate the exacerbation of chronic asthma. To analyze airway inflammation, the numbers of inflammatory cells and concentrations of cytokines in the bronchoalveolar lavage fluid (BALF) were estimated by flow cytometry and enzyme-linked immunosorbent assay, respectively. Results. ...
TL;DR: The data suggest that TxA2 and histamine participate in AI-AO likely through an IgE mechanism, but IgA and IgG1 seem to be involved.
Abstract: Background: Airway obstruction after antigen challenge is not always observed in patients with allergic asthma, even if they develop hyperresponsiveness. A similar event is observed in our guinea pig model of allergic asthma. Our aim was to study this phenomenon. Methods: Sensitized guinea pigs were challenged with ovalbumin (OVA) 3 times every 10 days. Animals were divided into 2 groups: (1) Guinea pigs exhibiting airway obstruction after antigen challenge (R = responders), and (2) guinea pigs lacking airway obstruction response (NR = nonresponders). After the third antigen challenge, antigen-induced airway hyperresponsiveness (AI-AHR), serum OVA-specific immunoglobulins, bronchoalveolar lavage fluid (BALF) inflammatory cells, histamine, cysteinyl leukotrienes and thromboxane A2 (TxA2) BALF levels, and in vitro tracheal contraction induced by contractile mediators and OVA were evaluated. Results: R group consistently displayed a transient antigen-induced airway obstruction (AI-AO) as well as AI-A...
TL;DR: SFN can antagonize CSE-induced growth arrest of alveolar epithelial cells and IGFBP-3 probably plays an important role in the process.
Abstract: Background: Sulforaphane (SFN) is an excellent antioxidant agent, few of the studies focus on the possible protective role of SFN from cigarette smoke-induced injury on alveolar epithelial cells. Objectives: the aim of the study is to observe the possible protective role of SFN, as well as the function of insulin-like growth factor binding protein-3 (IGFBP-3) in the process. Methods: MTT assay was used to evaluate cell viability after cigarette smoke extract (CSE) and/or SFN exposure, cell cycle was analyzed using flow cytometry, intracellular reactive oxygen species (ROS) level was detected by staining with fluorescent indicator 2′, 7′-dichlorofluorescin diacetate (DCFH-DA), finally both real-time quantitative RT-PCR and western blot were employed to observe mRNA and protein levels of IGFBP-3. Results: SFN could restore the viability of A549 cells, attenuate G1 block of the cell cycle, and significantly reduce the proportion of sub-G1 cells; at the same time, CSE-induced accumulation of intracell...
TL;DR: It is concluded that moderate hypothermia (32°C) of alveolar epithelial A549 cells seems to provide protection against H2O2-induced 24-hour oxidative stress by attenuating cell death and intracellular damage.
Abstract: Reactive oxygen species (ROS) are generally involved in lung inflammation and acute lung injury. We investigated the effects of hypothermia on ROS-induced cell damage in human alveolar type II cells. A549 cells were exposed to H2O2 and cultured at different temperatures, namely, normthermia (37 ◦ C), mild hypothermia (34 ◦ C), or moderate hypothermia (32 ◦ C). Cell damage was measured using various assays. The biochemical studies demonstrated a significant increase in apoptosis and intracellular ROS at 32 ◦ C in uninjured A549 cells. After exposure to H2O2, a marked decrease in cell viability (<50%) was demonstrated, and this was significantly ameliorated upon culture at 32 ◦ C. Significantly intracellular damage was found to affect the 24-hour H 2O2-exposed cells in 37 ◦ C( P < .05), including an increase in apoptosis and necrosis, intracellular ROS, caspase-3 activity, HMGB1 protein expression, and some alterations to the cell cycle. On hypothermic treatment, the 24-hour H2O2-induced caspase-3 activation was significantly suppressed in cells cultured at both 32 ◦ C and 34 ◦ C( P < .05 versus 37 ◦ C). The cell cycle changes in 24-hour H2O2-exposed cells were significantly diminished when the cells were cultured in 32 ◦ C( P < .05 versus 37 ◦ C). However, these intracellular alterations were not seen in 6-hour H2O2-exposed cells. We concluded that moderate hypothermia (32 ◦ C) of alveolar epithelial A549 cells seems to provide protection against H2O2-induced 24-hour oxidative stress by attenuating cell death and intracellular damage. However, moderate hypothermia might cause minor damage to uninjured cells, so the use of hypothermic treatment needs to be judiciously applied.