Carbocysteine Modifies Circulating miR-21, IL-8, sRAGE, and fAGEs Levels in Mild Acute Exacerbated COPD Patients: A Pilot Study.

Patients with Chronic Obstructive Pulmonary Disease (COPD) periodically experience acute exacerbation (AECOPD). Carbocysteine represents a valid add on therapy in COPD by exerting antioxidant and anti-inflammatory activities. The in vivo effects of carbocysteine on inflammatory markers are not yet fully understood. The aims of this study were to assess: (i) miR-21, IL-8, soluble Receptor for Advanced Glycation End Products (sRAGE), and fluorescent Advanced Glycation End Products (fAGEs) in control subjects (n = 9), stable (n = 9), and AECOPD patients (n = 24); and (ii) whether carbocysteine modifies these markers and the functional parameters in mild AECOPD patients. Mild AECOPD patients received or not carbocysteine along with background inhalation therapy for 20 days. At the onset and at the end of the observation period, the following parameters were evaluated: FEV1, FEF25-75%, CAT questionnaire; miR-21 by Real Time PCR; IL-8 and sRAGE by ELISA; and fAGEs by spectro-fluorescence method. COPD patients showed higher levels of miR-21, IL-8, fAGEs and lower levels of sRAGE compared to that of controls. miR-21 inversely correlated with FEV1. IL-8 and fAGEs were significantly different in stable and exacerbated COPD patients. Carbocysteine improved symptoms, FEV1 and FEF25-75%, increased sRAGE, and reduced miR-21, IL-8, and fAGEs in mild AECOPD patients. The present study provides compelling evidence that carbocysteine may help to manage mild AECOPD by downregulating some parameters of systemic inflammation.

Cigarette smoke upregulates Notch-1 signaling pathway and promotes lung adenocarcinoma progression.

Notch-1 pathway plays an important role in lung carcinoma, stem cell regulation, cellular communication, growth and differentiation. Cigarette smoke is involved in the regulation of Notch signaling. However, current data regarding the impact of cigarette smoke on the Notch pathway in lung cancer progression are limited. The present study aimed to explore whether cigarette smoke exposure altered Notch-1 pathway in ex-vivo (surgical samples of lung parenchyma from non-smoker and smoker patients with lung adenocarcinoma) and in vitro (adenocarcinoma A549 cell line) approaches. The expression of Notch-1, Jagged-1 and CD133 in surgical samples was evaluated by immunohistochemistry. A549 were exposed to cigarette smoke extracts (2.5 % and 5 % CSE for 6, 24 and 48 h) and the expression of Notch-1, Jagged-1 and Hes-1 was evaluated by Real-Time PCR and Western Blot (nuclear fractions). Expression and localization of Notch-1, Hes-1, CD133 and ABCG2 were assessed by immunofluorescence. The expression of survivin and Ki-67 was assessed by flow cytometry following CSE exposure and inhibition of Notch-1 signaling. Smokers lung parenchyma exhibited higher expression of Notch-1. CSE exposure increased Notch-1 and Hes-1 gene and nuclear protein expression in A549. Immunofluorescence confirmed higher expression of nuclear Hes-1 in CSE-stimulated A549 cells. CSE increased both survivin and Ki-67 expression and this effect was reverted by inhibition of the Notch-1 pathway. In conclusion, these data show that cigarette smoke may promote adenocarcinoma progression by activating the Notch-1 pathway thus supporting its role as hallmark of lung cancer progression and as a new target for lung cancer treatment.

Rbm24 displays dynamic functions required for myogenic differentiation during muscle regeneration.

Skeletal muscle has a remarkable capacity of regeneration after injury, but the regulatory network underlying this repair process remains elusive. RNA-binding proteins play key roles in the post-transcriptional regulation of gene expression and the maintenance of tissue homeostasis and plasticity. Rbm24 regulates myogenic differentiation during early development, but its implication in adult muscle is poorly understood. Here we show that it exerts multiple functions in muscle regeneration. Consistent with its dynamic subcellular localization during embryonic muscle development, Rbm24 also displays cytoplasm to nucleus translocation during C2C12 myoblast differentiation. In adult mice, Rbm24 mRNA is enriched in slow-twitch muscles along with myogenin mRNA. The protein displays nuclear localization in both slow and fast myofibers. Upon injury, Rbm24 is rapidly upregulated in regenerating myofibers and accumulates in the myonucleus of nascent myofibers. Through satellite cell transplantation, we demonstrate that Rbm24 functions sequentially to regulate myogenic differentiation and muscle regeneration. It is required for myogenin expression at early stages of muscle injury and for muscle-specific pre-mRNA alternative splicing at late stages of regeneration. These results identify Rbm24 as a multifaceted regulator of myoblast differentiation. They provide insights into the molecular pathway orchestrating the expression of myogenic factors and muscle functional proteins during regeneration.

Cigarette smoke extract reduces FOXO3a promoting tumor progression and cell migration in lung cancer.

Lung cancer is the leading cause of cancer death worldwide, and the carcinogens in tobacco smoke play a role in its progression and metastasis. The related molecular events are largely unknown. FOXO3a is a transcription factor considered a tumor suppressor. Its inhibition leads to cell transformation, tumor progression and metastasis. The aim of this study was to investigate, in different types of lung cancer cell lines (A549, COLO 699 N, SK-MES-1), the effects of cigarette smoke on mitochondrial status and cell metabolism and on key pathways involved in tumor progression and cell migration, looking at the role of FOXO3a in these mechanisms. The different lung cancer cells were exposed to cigarette smoke extract (CSE) and TGF-ß1. Reactive oxygen species (ROS), mitochondrial superoxide, intracellular ATP, extracellular lactate, FOXO3a, p21, survivin, epithelial-to-mesenchymal transition (EMT) markers (E-cadherin, SNAIL1), MMP-9 and cellular migration were assessed by flow-cytometry, fluorimetry, western blot analysis, Real-Time PCR and scratch test. Our results showed that exposure to CSE: (i) increased ROS, mitochondrial superoxide, lactate release while reducing intracellular ATP; (ii) decreased FOXO3a and increased survivin and p21 in the cytoplasm; (iii) decreased E-cadherin, increased SNAIL1 and MMP-9 and promoted cell migration like TGF-ß1 did. These effects could be partly explained by downregulation of FOXO3a, as demonstrated by silencing experiments. These data suggest that cigarette smoke induces oxidative stress and mitochondrial damage leading to metabolic reprogramming associated with increased glycolytic flux. This is accompanied with a downregulation of FOXO3a contributing to EMT processes and cell migration therefore promoting tumor progression.

Indexes of Angiogenic Activation in Myocardial Samples of Patients with Advanced Chronic Heart Failure.

Background and objectives: Ischemic and idiopathic heart failure are characterized by reactive cardiac fibrosis and impaired vasculogenesis involving pro-angiogenic factors such as angiogenin, angiopoietin-1 (Ang-1), and angiopoietin-2 (Ang-2), as demonstrated in experimental models of heart failure. However, differences in the molecular pathways between these cardiomyopathies are still unclear. In this short communication, we evaluate and compare the expression of pro-angiogenic molecules in the heart tissue of patients with advanced chronic heart failure (CHF) of ischemic vs. nonischemic etiology. Materials and Methods: We obtained heart tissue at transplantation from left ventricular walls of 16 explanted native hearts affected by either ischemic (ICM) or nonischemic dilated cardiomyopathy (NIDCM). Tissue samples were examined using immunohistochemistry for angiogenic molecules. Results: We found immunopositivity (I-pos) for angiopoietin-1 mainly in the cardiomyocytes, while we observed I-pos for Ang-2 and Tie-2 receptor mainly in endothelial cells. Expression of Procollagen-I (PICP), angiogenin, Ang-1, and Tie-2 receptor was similar in ICM and NIDCM. In contrast, endothelial immunopositivity for Ang-2 was higher in ICM samples than NIDCM (p = 0.03). Conclusions: In our series of CHF heart samples, distribution of Ang-1 and angiogenin was higher in cardiomyocytes while that of Ang-2 was higher in endothelial cells; moreover, Ang-2 expression was higher in ICS than NIDCM. Despite the small series examined, these findings suggest different patterns of angiogenic stimulation in ICM and NIDCM, or at least a more altered endothelial integrity in ICD. Our data may contribute to a better understanding of the angiogenesis signaling pathways in CHF. Further studies should investigate differences in the biochemical processes leading to heart failure.

Bacterial load and inflammatory response in sputum of alpha-1 antitrypsin deficiency patients with COPD.

Background: Airway inflammation may drive the progression of chronic obstructive pulmonary disease (COPD) associated with alpha-1 antitrypsin deficiency (AATD), but the relationship between airway microbiota and inflammation has not been investigated. Methods: We studied 21 non-treated AATD (AATD-noT) patients, 20 AATD-COPD patients under augmentation therapy (AATD-AT), 20 cigarette smoke-associated COPD patients, 20 control healthy smokers (CS) and 21 non-smokers (CON) with normal lung function. We quantified sputum inflammatory cells and inflammatory markers (IL-27, CCL3, CCL5, CXCL8, LTB4, MPO) by ELISA, total bacterial load (16S) and pathogenic bacteria by qRT-PCR. Results: AATD-AT patients were younger but had similar spirometric and DLCO values compared to cigarette smoke-associated COPD, despite a lower burden of smoking history. Compared to cigarette smoke-associated COPD, AATD-noT and AATD-AT patients had lower sputum neutrophil levels (p=0.0446, p=0.0135), total bacterial load (16S) (p=0.0081, p=0.0223), M. catarrhalis (p=0.0115, p=0.0127) and S. pneumoniae (p=0.0013, p=0.0001). Sputum IL-27 was significantly elevated in CS and cigarette smoke-associated COPD. AATD-AT, but not AATD-noT patients, had IL-27 sputum levels (pg/ml) significantly lower than COPD (p=0.0297) and these positively correlated with FEV1% predicted values (r=0.578, p=0.0307). Conclusions: Compared to cigarette smoke-associated COPD, AATD-AT (COPD) patients have a distinct airway inflammatory and microbiological profile. The decreased sputum bacterial load and IL-27 levels in AATD-AT patients suggests that augmentation therapy play a role in these changes.

MiR-21 upregulation increases IL-8 expression and tumorigenesis program in airway epithelial cells exposed to cigarette smoke.

BACKGROUND: Cigarette smoke exposure, increasing Toll-like receptor 4 (TLR4) and reactive oxygen species (ROS), promotes inflammatory responses in airway epithelial cells. Chronic inflammation, microRNA (miRNA), and oxidative stress are associated with cancer development. AIMS: The present study was aimed to explore whether cigarette smoke exposure, altering miR-21 expression, promoted inflammatory responses and tumorigenesis processes in airway epithelial cells. METHODS: Airway normal and cancer epithelial cells (16HBE and A549) were exposed to cigarette smoke extracts (CSE) or with/without agomiR-21, and then it was assessed: a) miR-21 expression; b) signal transducer and activator of transcription 3 (STAT3) nuclear protein expression and ERK1/2 activation; c) IL-8 gene expression and protein release. An antagonist of TLR4 (CLI-095) and the antioxidant flavonoid, apigenin, were also included to evaluate miR-21 expression in CSE exposed cells. RESULTS: It was demonstrated that: a) A549 cells constitutively expressed higher levels of miR-21 and IL-8; b) CSE increased STAT3 nuclear expression in 16HBE; c) in both cell lines, CSE and agomiR-21 increased: miR-21 expression; ERK1/2 activation and IL-8 gene expression and protein release; d) TLR4 inhibition counteracted the effects of CSE on miR-21 in A549; e) apigenin reduced miR-21 and IL-8 gene expression in both cell lines. CONCLUSIONS: Data herein provided identified for the first time new mechanisms supporting the crucial role of cigarette smoke-induced miR-21 expression in the amplification of inflammatory responses and in tumorigenesis processes within the airways.

TGF-ß Signaling Pathways in Different Compartments of the Lower Airways of Patients With Stable COPD.

BACKGROUND: The expression and localization of transforming growth factor-ß (TGF-ß) pathway proteins in different compartments of the lower airways of patients with stable COPD is unclear. We aimed to determine TGF-ß pathway protein expression in patients with stable COPD. METHODS: The expression and localization of TGF-ß pathway components was measured in the bronchial mucosa and peripheral lungs of patients with stable COPD (n = 44), control smokers with normal lung function (n = 24), and control nonsmoking subjects (n = 11) using immunohistochemical analysis. RESULTS: TGF-ß1, TGF-ß3, and connective tissue growth factor expression were significantly decreased in the bronchiolar epithelium, with TGF-ß1 also decreased in alveolar macrophages, in patients with stable COPD compared with control smokers with normal lung function. TGF-ß3 expression was increased in the bronchial lamina propria of both control smokers with normal lung function and smokers with mild/moderate stable COPD compared with control nonsmokers and correlated significantly with pack-years of smoking. However, TGF-ß3+ cells decreased in patients with severe/very severe COPD compared with control smokers. Latent TGF-ß binding protein 1 expression was increased in the bronchial lamina propria in subjects with stable COPD of all severities compared with control smokers with normal lung function. Bone morphogenetic protein and activin membrane-bound inhibitor expression (BAMBI) in the bronchial mucosa was significantly increased in patients with stable COPD of all severities compared with control subjects. No other significant differences were observed between groups for all the other molecules studied in the bronchial mucosa and peripheral lung. CONCLUSIONS: Expression of TGF-ßs and their regulatory proteins is distinct within different lower airway compartments in stable COPD. Selective reduction in TGF-ß1 and enhanced BAMBI expression may be associated with the increase in autoimmunity in COPD.

HSP60 activity on human bronchial epithelial cells.

HSP60 has been implicated in chronic inflammatory disease pathogenesis, including chronic obstructive pulmonary disease (COPD), but the mechanisms by which this chaperonin would act are poorly understood. A number of studies suggest a role for extracellular HSP60, since it can be secreted from cells and bind Toll-like receptors; however, the effects of this stimulation have never been extensively studied. We investigated the effects (pro- or anti-inflammatory) of HSP60 in human bronchial epithelial cells (16-HBE) alone and in comparison with oxidative, inflammatory, or bacterial challenges. 16-HBE cells were cultured for 1-4 h in the absence or presence of HSP60, H2O2, lipopolysaccharide (LPS), or cytomix. The cell response was evaluated by measuring the expression of IL-8 and IL-10, respectively, pro- and anti-inflammatory cytokines involved in COPD pathogenesis, as well as of pertinent TLR-4 pathway mediators. Stimulation with HSP60 up-regulated IL-8 at mRNA and protein levels and down-regulated IL-10 mRNA and protein. Likewise, CREB1 mRNA was up-regulated. H2O2 and LPS up-regulated IL-8. Experiments with an inhibitor for p38 showed that this mitogen-activated protein kinase could be involved in the HSP60-mediated pro-inflammatory effects. HSP60 showed pro-inflammatory properties in bronchial epithelial cells mediated by activation of TLR-4-related molecules. The results should prompt further studies on more complex ex-vivo or in-vivo models with the aim to elucidate further the role of those molecules in the pathogenesis of COPD.

Nandrolone decanoate interferes with testosterone biosynthesis altering blood-testis barrier components.

The aim of this study was to investigate whether nandrolone decanoate (ND) use affects testosterone production and testicular morphology in a model of trained and sedentary mice. A group of mice underwent endurance training while another set led a sedentary lifestyle and were freely mobile within cages. All experimental groups were treated with either ND or peanut oil at different doses for 6 weeks. Testosterone serum levels were measured via liquid chromatography-mass spectrometry. Western blot analysis and quantitative real-time PCR were utilized to determine gene and protein expression levels of the primary enzymes implicated in testosterone biosynthesis and gene expression levels of the blood-testis barrier (BTB) components. Immunohistochemistry and immunofluorescence were conducted for testicular morphological evaluation. The study demonstrated that moderate to high doses of ND induced a diminished serum testosterone level and altered the expression level of the key steroidogenic enzymes involved in testosterone biosynthesis. At the morphological level, ND induced degradation of the BTB by targeting the tight junction protein-1 (TJP1). ND stimulation deregulated metalloproteinase-9, metalloproteinase-2 (MMP-2) and the tissue inhibitor of MMP-2. Moreover, ND administration resulted in a mislocalization of mucin-1. In conclusion, ND abuse induces a decline in testosterone production that is unable to regulate the internalization and redistribution of TJP1 and may induce the deregulation of other BTB constituents via the inhibition of MMP-2. ND may well be considered as both a potential inducer of male infertility and a potential risk factor to a low endogenous bioavailable testosterone.

Effects of Conjugated Linoleic Acid Associated With Endurance Exercise on Muscle Fibres and Peroxisome Proliferator-Activated Receptor γ Coactivator 1 α Isoforms.

Conjugated linoleic acid (CLA) has been reported to improve muscle hypertrophy, steroidogenesis, physical activity, and endurance capacity in mice, although the molecular mechanisms of its actions are not completely understood. The aim of the present study was to identify whether CLA alters the expression of any of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) isoforms, and to evaluate the possible existence of fibre-type-specific hypertrophy in the gastrocnemius and plantaris muscles. Mice were randomly assigned to one of four groups: placebo sedentary, CLA sedentary, placebo trained, or CLA trained. The CLA groups were gavaged with 35 µl per day of Tonalin® FFA 80 food supplement containing CLA throughout the 6-week experimental period, whereas the placebo groups were gavaged with 35 µl sunflower oil each day. Each administered dose of CLA corresponded to approximately 0.7 g/kg or 0.5%, of the dietary daily intake. Trained groups ran 5 days per week on a Rota-Rod for 6 weeks at increasing speeds and durations. Mice were sacrificed by cervical dislocation and hind limb posterior muscle groups were dissected and used for histological and molecular analyses. Endurance training stimulated mitochondrial biogenesis by PGC1α isoforms (tot, α1, α2, and α3) but CLA supplementation did not stimulate PGC1α isoforms or mitochondrial biogenesis in trained or sedentary mice. In the plantaris muscle, CLA supplementation induced a fibre-type-specific hypertrophy of type IIx muscle fibres, which was associated with increased capillary density and was different from the fibre-type-specific hypertrophy induced by endurance exercise (of types I and IIb muscle fibres). J. Cell. Physiol. 232: 1086-1094, 2017. © 2016 Wiley Periodicals, Inc.

Skeletal muscle Heat shock protein 60 increases after endurance training and induces peroxisome proliferator-activated receptor gamma coactivator 1 α1 expression.

Heat shock protein 60 (Hsp60) is a chaperone localizing in skeletal muscle mitochondria, whose role is poorly understood. In the present study, the levels of Hsp60 in fibres of the entire posterior group of hindlimb muscles (gastrocnemius, soleus, and plantaris) were evaluated in mice after completing a 6-week endurance training program. The correlation between Hsp60 levels and the expression of four isoforms of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α) were investigated only in soleus. Short-term overexpression of hsp60, achieved by in vitro plasmid transfection, was then performed to determine whether this chaperone could have a role in the activation of the expression levels of PGC1α isoforms. The levels of Hsp60 protein were fibre-type specific in the posterior muscles and endurance training increased its content in type I muscle fibers. Concomitantly with the increased levels of Hsp60 released in the blood stream of trained mice, mitochondrial copy number and the expression of three isoforms of PGC1α increased. Overexpressing hsp60 in cultured myoblasts induced only the expression of PGC1 1α, suggesting a correlation between Hsp60 overexpression and PGC1 1 α activation.

Alcoholic Liver Disease: A Mouse Model Reveals Protection by Lactobacillus fermentum.

OBJECTIVES: Alcoholism is one of the most devastating diseases with high incidence, but knowledge of its pathology and treatment is still plagued with gaps mostly because of the inherent limitations of research with patients. We developed an animal model for studying liver histopathology, Hsp (heat-shock protein)-chaperones involvement, and response to treatment. METHODS: The system was standardized using mice to which ethanol was orally administered alone or in combination with Lactobacillus fermentum following a precise schedule over time and applying, at predetermined intervals, a battery of techniques (histology, immunohistochemistry, western blotting, real-time PCR, immunoprecipitation, 3-nitrotyrosine labeling) to assess liver pathology (e.g., steatosis, fibrosis), and Hsp60 and iNOS (inducible form of nitric oxide synthase) gene expression and protein levels, and post-translational modifications. RESULTS: Typical ethanol-induced liver pathology occurred and the effect of the probiotic could be reliably monitored. Steatosis score, iNOS levels, and nitrated proteins (e.g., Hsp60) decreased after probiotic intake. CONCLUSIONS: We describe a mouse model useful for studying liver disease induced by chronic ethanol intake and for testing pertinent therapeutic agents, e.g., probiotics. We tested L. fermentum, which reduced considerably ethanol-induced tissue damage and deleterious post-translational modifications of the chaperone Hsp60. The model is available to test other agents and probiotics with therapeutic potential in alcoholic liver disease.

Effects of Nandrolone Stimulation on Testosterone Biosynthesis in Leydig Cells.

Anabolic androgenic steroids (AAS) are among the drugs most used by athletes for improving physical performance, as well as for aesthetic purposes. A number of papers have showed the side effects of AAS in different organs and tissues. For example, AAS are known to suppress gonadotropin-releasing hormone, luteinizing hormone, and follicle-stimulating hormone. This study investigates the effects of nandrolone on testosterone biosynthesis in Leydig cells using various methods, including mass spectrometry, western blotting, confocal microscopy and quantitative real-time PCR. The results obtained show that testosterone levels increase at a 3.9 µM concentration of nandrolone and return to the basal level a 15.6 µM dose of nandrolone. Nandrolone-induced testosterone increment was associated with upregulation of the steroidogenic acute regulatory protein (StAR) and downregulation of 17a-hydroxylase/17, 20 lyase (CYP17A1). Instead, a 15.6 µM dose of nandrolone induced a down-regulation of CYP17A1. Further in vivo studies based on these data are needed to better understand the relationship between disturbed testosterone homeostasis and reproductive system impairment in male subjects.