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The exposure and health implications of exhaled aerosol particles from tobacco products remain a critical area of concern in public health. This research aimed to characterize the cytotoxicity of exhaled aerosol particles from conventional cigarettes (CC) and heated tobacco products (HTP) using a novel "Cells on Particles" in vitro exposure model. The research uniquely captures the physical and chemical characteristics of aerosols by depositing them onto fibrous matrixes, enabling a more accurate representation of exposure conditions. New insights were provided into the differences between CC and HTP in terms of particle size distributions, cell viability, metabolic activity, and the expression of genes related to xenobiotic metabolism and oxidative stress. This approach marks a significant advancement in the field by offering a more direct and representative method to evaluate the potential health hazards of tobacco aerosol particles.
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The presented research introduces the "Cells-on-Particles" integrated aerosol sampling and cytotoxicity testing in vitro platform, which allows for the direct assessment of the biological effects of captured aerosol particles on a selected cell type without the need for extraction or resuspension steps. By utilizing particles with unaltered chemical and physical properties, the method enables simple and fast screening of biological effects on specific cell types, making it a promising tool for assessing the cytotoxicity of particulate matter in ambient and occupational air. Platforms fabricated from cellulose acetate (CA) and poly[ε]caprolactone (PCL) were proven to be biocompatible and promoted the attachment and growth of the human bronchial epithelial cell line BEAS-2B. The PCL platforms were exposed to simulated occupational aerosols of silver, copper, and graphene oxide nanoparticles. Each nanoparticle type exhibited different and dose-dependent cytotoxic effects on cells, evidenced by reduced cell viability and distinct, particle type-dependent gene expression patterns. Notably, copper nanoparticles were identified as the most cytotoxic, and graphene oxide the least. Comparing the "Cells-on-Particles" and submerged exposure ("Particles-on-Cells") testing strategies, BEAS-2B cells responded to selected nanoparticles in a comparable manner, suggesting the developed testing system could be proposed for further evaluation with more complex environmental aerosols. Despite limitations, including particle agglomeration and the need for more replicates to address variability, the "Cells-on-Particles" platform enables effective detection of toxicity induced by relatively low levels of nanoparticles, demonstrating good sensitivity and a relatively simpler procedure compared to standard 2D cell exposure methods.
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BACKGROUND: An increasing number of studies investigate various human microbiotas and their roles in the development of diseases, maintenance of health states, and balanced signaling towards the brain. Current data demonstrate that the nasal microbiota contains a unique and highly variable array of commensal bacteria and opportunistic pathogens. However, we need to understand how to harness current knowledge, enrich nasal microbiota with beneficial microorganisms, and prevent pathogenic developments. RESULTS: In this study, we have obtained nasal, nasopharyngeal, and bronchoalveolar lavage fluid samples from healthy volunteers and patients suffering from chronic respiratory tract diseases for full-length 16 S rRNA sequencing analysis using Oxford Nanopore Technologies. Demographic and clinical data were collected simultaneously. The microbiome analysis of 97 people from Lithuania suffering from chronic inflammatory respiratory tract disease and healthy volunteers revealed that the human nasal microbiome represents the microbiome of the upper airways well. CONCLUSIONS: The nasal microbiota of patients was enriched with opportunistic pathogens, which could be used as indicators of respiratory tract conditions. In addition, we observed that a healthy human nasal microbiome contained several plant- and bee-associated species, suggesting the possibility of enriching human nasal microbiota via such exposures when needed. These candidate probiotics should be investigated for their modulating effects on airway and lung epithelia, immunogenic properties, neurotransmitter content, and roles in maintaining respiratory health and nose-brain interrelationships.
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Bacterias , Microbiota , ARN Ribosómico 16S , Humanos , Femenino , Masculino , ARN Ribosómico 16S/genética , Persona de Mediana Edad , Adulto , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Enfermedad Crónica , Líquido del Lavado Bronquioalveolar/microbiología , Nasofaringe/microbiología , Enfermedades Respiratorias/microbiología , Lituania , Nariz/microbiología , Anciano , Adulto Joven , Cavidad Nasal/microbiología , Análisis de Secuencia de ADN/métodos , Voluntarios SanosRESUMEN
This is the first study reporting the presence of airborne nano-sized plastic particles in the bronchoalveolar lavage fluid (BALF) samples of patients undergoing diagnostic bronchoscopy. The results represent the plastic pollution content in the lower airways of the residents of Northern Europe. Airborne micro- and nanoplastic particles (MP/NPs) are widely dispersed worldwide and intrude on human organisms to various extents, with the respiratory tract being the first line of exposure. The amounts of inhaled MP/NPs, their fate in the human respiratory tract, and the effects on the health of human airways and other exposed organs remain largely unknown. In this clinical study, human BALF samples were assessed by means of optical and transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy (TEM-EDX). Results show that MP/NPs levels vary in the interval of 0.14-12.8 particles per 100 ml of BALF and are present in all samples tested, mainly in a fragmented form. External pollution by MP/NPs was excluded by carefully choosing methodology and equipment. This finding is a timely addition of valuable information and stimulates further research into the biological effects of inhaled MP/NPs.
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Publications addressing air pollution-induced human respiratory microbiome shifts are reviewed in this article. The healthy respiratory microbiota is characterized by a low density of bacteria, fungi and viruses with high diversity, and usually consists of Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, Fusobacteria, viruses and fungi. The air's microbiome is highly dependent on air pollution levels and is directly reflected within the human respiratory microbiome. In addition, pollutants indirectly modify the local environment in human respiratory organs by reducing antioxidant capacity, misbalancing proteolysis and modulating inflammation, all of which regulate local microbiomes. Improving air quality leads to more diverse and healthy microbiomes of the local air and, subsequently, residents' airways.
The community of bacteria, viruses and fungi in the human body, known as the microbiome, plays an important role in human health. These communities vary in different locations in the body, for example in the gut, airways and skin. The microbiome within our airways is affected by air pollution because pollutants cause changes in the microbiome that may result in illness. In this article we review the available information on the effect of air pollution on the airway microbiome. We conclude that improving air quality is important to promoting healthy microbiomes and general human health.
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Contaminación del Aire , Microbiota , Humanos , Contaminación del Aire/efectos adversos , Contaminación del Aire/análisis , Sistema Respiratorio/microbiología , Bacterias/genética , InflamaciónRESUMEN
The evolving field of the microbiome and microbiota has become a popular research topic. The human microbiome is defined as a new organ and is considered a living community of commensal, symbiotic and pathogenic microorganisms within a certain body space. The term 'microbiome' is used to define the entire genome of the microbiota. Bacteria, archaea, fungi, algae and small protists are all members of the microbiota, followed by phages, viruses, plasmids and mobile genetic elements. The composition, heterogeneity and dynamics of microbiomes in time and space, their stability and resistance, essential characteristics and key participants, as well as interactions within the microbiome and with the host, are crucial lines of investigation for the development of successful future diagnostics and therapies. Standardization of microbiome studies and harmonized comparable methodologies are required for the transfer of knowledge from fundamental science into the clinic. Human health is dependent on microbiomes and achieved by nurturing beneficial resident microorganisms and their interplay with the host. The present study reviewed scientific knowledge on the major components of the human respiratory microbiome, i.e. bacteria, viruses and fungi, their symbiotic and parasitic roles, and, also, major diseases of the human respiratory tract and their microbial etiology. Bidirectional relationships regulate microbial ecosystems and host susceptibility. Moreover, environmental insults render host tissues and microbiota disease-prone. The human respiratory microbiome reflects the ambient air microbiome. By understanding the human respiratory microbiome, potential therapeutic strategies may be proposed.
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Dilated cardiomyopathy (DCM) is the most common type of nonischemic cardiomyopathy characterized by left ventricular or biventricular dilation and impaired contraction leading to heart failure and even patients' death. Therefore, it is important to search for new cardiac tissue regenerating tools. Human mesenchymal stem/stromal cells (hmMSCs) were isolated from post-surgery healthy and DCM myocardial biopsies and their differentiation to the cardiomyogenic direction has been investigated in vitro. Dilated hmMSCs were slightly bigger in size, grew slower, but had almost the same levels of MSC-typical surface markers as healthy hmMSCs. Histone deacetylase (HDAC) activity in dilated hmMSCs was 1.5-fold higher than in healthy ones, which was suppressed by class I and II HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) showing activation of cardiomyogenic differentiation-related genes alpha-cardiac actin (ACTC1) and cardiac troponin T (TNNT2). Both types of hmMSCs cultivated on collagen I hydrogels with hyaluronic acid (HA) or 2-methacryloyloxyethyl phosphorylcholine (MPC) and exposed to SAHA significantly downregulated focal adhesion kinase (PTK2) and activated ACTC1 and TNNT2. Longitudinal cultivation of dilated hmMSC also upregulated alpha-cardiac actin. Thus, HDAC inhibitor SAHA, in combination with collagen I-based hydrogels, can tilt the dilated myocardium hmMSC toward cardiomyogenic direction in vitro with further possible therapeutic application in vivo.
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Biomimética , Cardiomiopatía Dilatada/patología , Diferenciación Celular , Células Madre Mesenquimatosas/patología , Miocitos Cardíacos/citología , Vorinostat/farmacología , Anciano , Cardiomiopatía Dilatada/inducido químicamente , Estudios de Casos y Controles , Proliferación Celular , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Persona de Mediana Edad , Miocitos Cardíacos/efectos de los fármacos , RegeneraciónRESUMEN
Humanity faces an increasing impact of air pollution worldwide, including threats to human health. Air pollutants prompt and promote chronic inflammation, tumourigenesis, autoimmune and other destructive processes in the human body. Post-translational modification of proteins, for example citrullination, results from damaging attacks of pollutants, including smoking, air pollution and others, rendering host tissues immunogenic. Citrullinated proteins and citrullinating enzymes, deiminases, are more prevalent in patients with COPD and correlate with ongoing inflammation and oxidative stress. In this study, we installed an in-house-designed diesel exhaust delivery and cannabidiol vaporization system where mice were exposed to relevant, urban traffic-related levels of diesel exhaust for 14 days and assessed integrity of alveolar tissue, gene expression shifts and changes in protein content in the lungs and other tissues of exposed mice. Systemic presence of modified proteins was also tested. The protective effect of phytocannabinoids was investigated as well. Data obtained in our study show subacute effects of diesel exhaust on mouse lung integrity and protein content. Emphysematous changes are documented in exposed mouse lungs. In parallel, increased levels of citrulline were detected in the alveolar lung tissue and peripheral blood of exposed mice. Pre-treatment with vaporized cannabidiol ameliorated some damaging effects. Results reported hereby provide new insights into subacute lung tissue changes that follow diesel exhaust exposure and suggest possible dietary and/or other therapeutic interventions for maintaining lung health and healthy ageing.
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Contaminantes Atmosféricos/toxicidad , Citrulinación/efectos de los fármacos , Lesión Pulmonar/inducido químicamente , Emisiones de Vehículos/toxicidad , Administración por Inhalación , Animales , Cannabinoides/administración & dosificación , Cannabis/química , Modelos Animales de Enfermedad , Humanos , Lesión Pulmonar/diagnóstico , Lesión Pulmonar/prevención & control , Masculino , Ratones , Nebulizadores y Vaporizadores , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/patología , Resultado del TratamientoRESUMEN
According to World Health Organisation (WHO) air pollution increases the risk of cardiovascular disorders, respiratory diseases, including COPD, lung cancer and acute respiratory infections, neuro-degenerative and other diseases. It is also known that various phytochemicals may mitigate such risks. This study tested if phytochemicals mangiferin (MNG) and Z-ligustilide (Z-LG) may protect PAH-exposed human lung bronchial epithelial cells (BEAS-2B). Organic PAH extract was obtained from the urban fine PM with high benzo(a)pyrene content collected in Eastern European mid-sized city during winter heating season. Cell proliferation traits and levels of intracellular oxidative stress were examined. Effect of MNG (0.5 µg/mL) alone or in combination with PAH on bronchial epithelium wound healing was evaluated. Both phytochemicals were also evaluated for their antioxidant properties in acellular system. Treatment with MNG produced strong cytoprotective effect on PAH-exposed cells (p < 0.01) while Z-LG (0.5 µg/mL) exhibited strong negative effect on cell proliferation in untreated and PAH-exposed cells (p < 0.001). MNG, being many times stronger antioxidant than Z-LG in chemical in vitro assays (p < 0.0001), was also able to decrease PAH-induced oxidative stress in the cell cultures (p < 0.05). Consequently MNG ameliorates oxidative stress, speeds up wound healing process and restores proliferation rate in PAH-exposed bronchial epithelium. Such protective effects of MNG in air pollution affected airway epithelium stimulate further research on this promising phytochemical.
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4-Butirolactona/análogos & derivados , Material Particulado/toxicidad , Xantonas/farmacología , 4-Butirolactona/farmacología , Contaminantes Atmosféricos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Humanos , Estrés Oxidativo/efectos de los fármacos , Material Particulado/química , Fitoquímicos/farmacología , Mucosa Respiratoria/citologíaRESUMEN
Atmospheric particulate matter (PM) constitutes the major part of urban air pollution and is a heterogeneous mixture of solid and liquid particles of different origin, size, and chemistry. Human exposure to PM in urban areas poses considerable and significant adverse effects on the respiratory system and human health in general. Major contributors to PM content are combustion-related sources such as diesel vehicles, household, and industrial heating. PM is composed of thousands of different high molecular weight organic compounds, including poly-aromatic hydrocarbons (PAHs). The aim of this study was to clarify the cytotoxic effects of the extract of actual urban PM1 with high benzo[a]pyrene (BaP) content collected in Eastern European mid-sized city during winter heating season on human bronchial epithelial cells (BEAS-2B). Decreased cell viability, alteration of cell layer integrity, increased apoptosis, and oxidative stress were observed during the 3-day exposure to the PM extract. In addition, following PM exposure pro-inflammatory cytokine expression was upregulated at gene and protein levels. Morphology and motility changes, i.e., decreased cells' ability to cover scratch area, were also documented. We report here that the extract of urban PM1 may induce bronchial epithelium changes and render it pro-inflammatory and compromised within 3 days.
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Contaminantes Atmosféricos/toxicidad , Material Particulado/toxicidad , Contaminantes Atmosféricos/análisis , Contaminación del Aire/análisis , Ciudades , Células Epiteliales/efectos de los fármacos , Humanos , Industrias , Estrés Oxidativo/efectos de los fármacos , Material Particulado/análisis , Estaciones del Año , Pruebas de ToxicidadRESUMEN
Studies of lung diseases in vitro often rely on flat, plastic-based monocultures, due to short lifespan of primary cells, complicated anatomy, lack of explants, etc. We hereby present a native 3D model with cues for repopulating epithelial cells. Abilities of mesenchymal stem cells (MSC) to modulate bacterial lipopolysaccharide (LPS) and cigarette smoke-induced injury to pulmonary epithelium were tested in our model. Post-mortem human lung tissue was sliced, cut and decellularized. Resulting matrix pads were reseeded with pulmonary epithelium (A549 line). Markers of the layer integrity and certain secreted proteins in the presence of cigarette smoke extract (CSE) and LPS were assessed via Western blot, ELISA and RT-PCR assays. In parallel, the effects of MSC paracrine factors on exposed epithelial cells were also investigated at gene and protein levels. When cultured on native 3D matrix, A549 cells obtain dual, type I- and II-like morphology. Exposure to CSE and LPS leads to downregulation of several epithelial proteins and suppressed proliferation rate. MSC medium added to the model restores proliferation rate and some of the epithelial proteins, i.e. e-cadherin and beta-catenin. CSE also increases secretion of pro-inflammatory cytokines by epithelial cells and upregulates transcription factor NFκB. Some of these effects might be counteracted by MSC in our model. We introduce repopulated decellularized lung matrix that highly resembles in vivo situation and is convenient for studies of disease pathogenesis, cytotoxicology and for exploring therapeutic strategies in the human lung context in vitro. MSC paracrine products have produced protecting effects in our model.
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Chronic obstructive pulmonary disease (COPD), a major cause of death and morbidity worldwide, is characterized by expiratory airflow limitation that is not fully reversible, deregulated chronic inflammation, and emphysematous destruction of the lungs. Despite the fact that COPD is a steadily growing global healthcare problem, the conventional therapies remain palliative, and regenerative approaches for disease management are not available yet. We aim to provide an overview of key reviews, experimental, and clinical studies addressing lung emphysema development and repair mechanisms published in the past decade. Novel aspects discussed herein include integral revision of the literature focused on lung microflora changes in COPD, autoimmune component of the disease, and environmental risk factors other than cigarette smoke. The time span of studies on COPD, including emphysema, chronic bronchitis, and asthmatic bronchitis, covers almost 200 years, and several crucial mechanisms of COPD pathogenesis are described and studied. However, we still lack the holistic understanding of COPD development and the exact picture of the time-course and interplay of the events during stable, exacerbated, corticosteroid-treated COPD states, and transitions in-between. Several generally recognized mechanisms will be discussed shortly herein, ie, unregulated inflammation, proteolysis/antiproteolysis imbalance, and destroyed repair mechanisms, while novel topics such as deviated microbiota, air pollutants-related damage, and autoimmune process within the lung tissue will be discussed more extensively. Considerable influx of new data from the clinic, in vivo and in vitro studies stimulate to search for novel concise explanation and holistic understanding of COPD nowadays.
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Remodelación de las Vías Aéreas (Respiratorias) , Pulmón/fisiopatología , Enfermedad Pulmonar Obstructiva Crónica/etiología , Enfisema Pulmonar/etiología , Regeneración , Remodelación de las Vías Aéreas (Respiratorias)/efectos de los fármacos , Animales , Autoinmunidad , Disbiosis , Ambiente , Epigénesis Genética , Predisposición Genética a la Enfermedad , Humanos , Mediadores de Inflamación/metabolismo , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/microbiología , Microbiota , Estrés Oxidativo , Pronóstico , Proteolisis , Enfermedad Pulmonar Obstructiva Crónica/genética , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/microbiología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Enfermedad Pulmonar Obstructiva Crónica/terapia , Enfisema Pulmonar/inmunología , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/microbiología , Enfisema Pulmonar/fisiopatología , Enfisema Pulmonar/terapia , Especies Reactivas de Oxígeno/metabolismo , Regeneración/efectos de los fármacos , Factores de Riesgo , Transducción de SeñalRESUMEN
Stem cells isolated from human adult tissues represent a promising source for neural differentiation studies in vitro. We have isolated and characterized stem cells from human exfoliated deciduous teeth (SHEDs). These originate from the neural crest and therefore particularly suitable for induction of neural differentiation. We here established a novel three-stage protocol for neural differentiation of SHEDs cells. After adaptation to a serum-free and neurogenic environment, SHEDs were induced to differentiate. This resulted in the formation of stellate or bipolar round-shaped neuron-like cells with subpopulations expressing markers of sensory neurons (Brn3a, peripherin) and glia (myelin basic protein). Commercial PCR array analyses addressed the expression profiles of genes related to neurogenesis and cAMP/calcium signalling. We found distinct evidence for the upregulation of genes regulating the specification of sensory (MAF), sympathetic (midkine, pleitrophin) and dopaminergic (tyrosine hydroxylase, Nurr1) neurons and the differentiation and support of myelinating and non-myelinating Schwann cells (Krox24, Krox20, apolipoprotein E). Moreover, for genes controlling major developmental signalling pathways, there was upregulation of BMP (TGF ß-3, BMP2) and Notch (Notch 2, DLL1, HES1, HEY1, HEY2) in the differentiating SHEDs. SHEDs treated according to our new differentiation protocol gave rise to mixed neuronal/glial cell cultures, which opens new possibilities for in vitro studies of neuronal and glial specification and broadens the potential for the employment of such cells in experimental models and future treatment strategies.
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Current cell therapy protocols require considerable numbers of mesenchymal stromal cells (MSCs), which can be obtained only by in vitro expansion. The most important issue is a choice of optimal growth supplements for cell culture. Ideally, these should be of known composition, free of animal components and allow production of large homogenic populations of MSCs in a considerably short period of time. Since this standard has not been achieved to date, we aimed to assess the molecular responses of MSCs to different growth supplements commonly in use. MSCs were isolated from breast or abdominal adipose tissue and plated into DMEM supplemented with one of four different sera: fetal calf serum (FCS), pretested fetal calf serum (FCS-Sp), human allogeneic serum (HS) or artificial serum substitute (AS). MSCs cultivated with different serum supplements demonstrated distinct morphologies, high adipogenic and osteogenic differentiation potential and expressed characteristic antigens. Using real-time PCR, we found a large increase in PPARγ and Msx2 gene expression in both lines of proliferating MSCs cultivated with AS. We found that MSCs cultivated in the presence of different sera had similar global proteomic expression patterns, but comparisons of identified proteins revealed most differences in the MSCs cultivated with AS. Our results indicate that MSCs cultivated in the presence of FCS and HS display similar growth, differentiation, immunophenotypic and proteomic properties, while AS induces more profound changes in the physiology of MSCs, suggesting that further fundamental studies should be done before its introduction into clinical practice.
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Tejido Adiposo/citología , Células Madre Mesenquimatosas/citología , Suero/metabolismo , Adipogénesis/efectos de los fármacos , Adipogénesis/genética , Adulto , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Electroforesis en Gel Bidimensional , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunofenotipificación , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Osteopontina/genética , Osteopontina/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , ProteómicaRESUMEN
AIM: Intrinsic tissue regeneration mechanisms are still not fully understood. The destruction/reconstruction processes are usually in fine balance; however, this can be easily destroyed, for example in the environment of chronic inflammation. One of the major proteins present at the inflammatory sites is the multifunctional protein alpha-1-antitrypsin (AAT). In this study, potential therapeutic effects of this major human antiprotease on progenitor cells are assessed. MATERIALS & METHODS: Stromal cells from human exfoliated deciduous teeth (SHEDs) were used, which are similar to the mesenchymal stromal cells isolated from other tissues. SHEDs were cultivated in the presence of subphysiological, physiological and inflammatory concentrations of AAT, and their proliferation and motility traits were assayed. Some intracellular signaling pathways, AAT internalization by SHEDs and their matrix metalloprotease profile were studied in parallel. RESULTS: Physiologic and inflammatory concentrations of AAT significantly increased the cell proliferation rate, induced phosphorylation of several key protein kinases and increased the amount of secreted active gelatinases. Moreover, cells exposed to physiologic and inflammatory levels of AAT were able to invade and migrate more efficiently. Subphysiologic AAT levels did not change cell behavior significantly. CONCLUSION: AAT at physiologic and inflammatory concentrations positively modulates the proliferation and motility of SHEDs in vitro. These results suggest the importance of AAT in the maintenance and regulation of tissue progenitor cells in vivo.
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Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidores de Serina Proteinasa/farmacología , Células del Estroma/efectos de los fármacos , Diente Primario/citología , Diente Primario/efectos de los fármacos , alfa 1-Antitripsina/farmacología , Western Blotting , Diferenciación Celular/efectos de los fármacos , Niño , Femenino , Citometría de Flujo , Gelatinasas/metabolismo , Humanos , Técnicas para Inmunoenzimas , Células del Estroma/citologíaRESUMEN
Cigarette smoke (CS)-induced activation of proteases such as matrix metalloproteinases (MMPs) contributes to lung alveolar destruction due to cell death. The aim of this study was to determine whether MMPs are produced in pulmonary artery endothelial cells (PAECs) and whether CS activation of MMPs is associated with apoptosis. Cultured PAECs were exposed to CS and subjected to assessments of apoptosis and MMPs. Western blotting and in situ zymography were performed to localize gelatinolytic activity and to identify enzymes. CS-induced apoptosis, i.e., enhanced annexin V binding and cleaved poly-ADP-ribose-polymerase (PARP), correlated with increased degradation of gelatin, a substrate of MMPs. The levels of pro-MMP-2 and active MMP-2 were increased in cytosolic and nuclear fractions isolated from CS-exposed cells. MMP-2 protein colocalized with gelatinolytic activity in the nucleus of CS-exposed cells undergoing apoptosis. These observations support the notion that MMP-2 contributes to CS-induced gelatinase activity, which localizes in the nuclear region primarily and correlates with annexin V binding and PARP cleavage. This suggests a novel function of MMP-2 in the degradation of the nuclear matrix in CS-induced endothelial apoptosis.
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Apoptosis/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/metabolismo , Arteria Pulmonar/efectos de los fármacos , Animales , Anexina A5/metabolismo , Núcleo Celular/enzimología , Núcleo Celular/patología , Células Cultivadas , Endotelio Vascular/enzimología , Endotelio Vascular/patología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Arteria Pulmonar/enzimología , Arteria Pulmonar/patología , Humo/efectos adversos , Fumar/efectos adversos , PorcinosRESUMEN
BACKGROUND: Deficiency of the antiprotease alpha-1-antitrypsin (AAT) and exposure to cigarette smoke (CS) contribute to the development of early onset emphysema. CS-induced apoptosis of alveolar cells including endothelial cells plays critical role in the lung destruction. AAT deficiency is associated with increased lung tissue destruction as well. We hypothesize that AAT protects lung alveoli from noxious environmental stimuli such as CS-induced apoptosis. METHODS: Porcine pulmonary artery endothelial cells (PAEC) were exposed to CS in the presence or absence of AAT (20 microM). AAT internalization and markers for apoptosis were assessed by confocal microscopy. Flow cytometry was performed in parallel to quantify the number of AAT-loaded and apoptotic cells. RESULTS: We demonstrated that exogenous AAT accumulated in PAEC and protected cells from CS-induced apoptosis. AAT-loaded CS-exposed cells exhibited increased amounts of chaperone HSP-70 in their cytosol and less apoptosis inducing factor in their nuclei compared to AAT-untreated, CS-exposed cells. CONCLUSIONS: Our results suggest that AAT is taken up by endothelial cells via two mechanisms and that intracellular AAT may have a protective role in CS-induced endothelial apoptosis. This may open new insights into the field of endothelial serpins as agents capable of protecting the vasculature from environment-derived noxious substances.
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Apoptosis/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Arteria Pulmonar/efectos de los fármacos , Fumar/efectos adversos , Inhibidores de Tripsina/farmacología , alfa 1-Antitripsina/farmacología , Animales , Caveolinas/metabolismo , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Proteínas HSP70 de Choque Térmico/metabolismo , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , PorcinosRESUMEN
BACKGROUND: Alpha1-antitrypsin (AAT) is one of the major serine proteinase inhibitors controlling proteinases in many biological pathways. There is increasing evidence that AAT is able to exert other than antiproteolytic effects. To further examine this question we compared how various doses of the native (inhibitory) and the polymerised (non-inhibitory) molecular form of AAT affect pro-inflammatory responses in human monocytes, in vitro. Human monocytes isolated from different donors were exposed to the native or polymerised form of AAT at concentrations of 0.01, 0.02, 0.05, 0.1, 0.5 and 1 mg/ml for 18 h, and analysed to determine the release of cytokines and to detect the activity of NF-kappaB. RESULTS: We found that native and polymerised AAT at lower concentrations, such as 0.1 mg/ml, enhance expression of TNFalpha (10.9- and 4.8-fold, p < 0.001), IL-6 (22.8- and 23.4-fold, p < 0.001), IL-8 (2.4- and 5.5-fold, p < 0.001) and MCP-1 (8.3- and 7.7-fold, p < 0.001), respectively, compared to buffer exposed cells or cells treated with higher doses of AAT (0.5 and 1 mg/ml). In parallel to increased cytokine levels, low concentrations of either conformation of AAT (0.02-0.1 mg/ml) induced NF-kappaB p50 activation, while 1 mg/ml of either conformation of AAT suppressed the activity of NF-kappaB, compared to controls. CONCLUSIONS: The observations reported here provide further support for a central role of AAT in inflammation, both as a regulator of proteinase activity, and as a signalling molecule for the expression of pro-inflammatory molecules. This latter role is dependent on the concentration of AAT, rather than on its proteinase inhibitory activity.
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Monocitos/inmunología , alfa 1-Antitripsina/farmacología , Biopolímeros/química , Biopolímeros/farmacología , Células Cultivadas , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Humanos , Peso Molecular , Monocitos/efectos de los fármacos , FN-kappa B/metabolismo , alfa 1-Antitripsina/químicaRESUMEN
The finding that alphal-antitrypsin (AAT) deficiency, PiZZ, a well-established genetic risk factor for COPD, is related to high levels of circulating AAT polymers, prompted us to measure serum levels of such polymers and selected markers of inflammation in age- and gender-matched patients with stable COPD and control subjects with and without severe AAT deficiency, and to assess their relationship with each other and with the genetic AAT-variant. We found that COPD individuals (n= 20), independent of AAT-variant, had significantly higher serum levels of AAT and its polymers, MMP-9, sICAM-1, VEGF and sE-selectin than controls (n=30). Subjects with PiZZ COPD (n= 10) showed significantly elevated serum levels of AAT-polymers, sE-selectin and sICAM-1, while patients with PiMM COPD (n= 10) showed higher levels of MMP-9, VEGF, IL-8 and MCP-1 than controls. By using factor analysis we were able to split the analysed biomarkers into two independent components: the first containing MMP-9, MCP-1, IL-8 and VEGF and the second-AAT and its polymers and sE-selectin. The result from the binomial logistic regression showed that 95.2 percent of the control individuals and 94.7 percent of the COPD patients can be correctly classified on the basis of the measured serum biomarkers. These observations highlight the importance of the finding sets of biomolecules, which could offer new strategies for the diagnosis of COPD and may have value for monitoring progression of COPD.
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Biomarcadores/sangre , Enfermedad Pulmonar Obstructiva Crónica/sangre , Moléculas de Adhesión Celular/sangre , Estudios Transversales , Citocinas/sangre , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nefelometría y Turbidimetría , Pronóstico , Enfermedad Pulmonar Obstructiva Crónica/genética , alfa 1-Antitripsina/sangreRESUMEN
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by incompletely reversible airflow obstruction associated with inflammation in which monocytes/macrophages are the predominant inflammatory cells. The only known genetic factor related to COPD is inherited PiZZ deficiency of alpha1-antitrypsin (AAT), an inhibitor of serine proteases. METHODS: We investigated the basal and LPS-stimulated release of pro-inflammatory molecules from blood monocytes isolated from age and gender matched healthy (n = 30) and COPD (n = 20) individuals with and without AAT deficiency. RESULTS: After 18 h of cell culture the basal release of MMP-9 was 2.5-fold, p < 0.02 greater, whereas IL-8 was 1.8-fold (p < 0.01) lower from COPD patient monocytes than from controls. LPS-stimulated release of IL-6 and MCP-1 was greater from COPD patient's monocytes relative to controls, while activation of control cells resulted in enhanced secretion of ICAM-1 and MMP-9 compared to COPD patients. Independent of disease status, monocytes from PiZZ AAT carriers released less TNFalpha (by 2.3-fold, p < 0.03). CONCLUSIONS: The basal and LPS-stimulated secretion of specific pro-inflammatory molecules from circulating monocytes differs between healthy and COPD subjects. These findings may be valuable for further studies on the mechanisms involved in recruitment and activation of inflammatory cells in COPD.