RESUMEN
Cancer cells adjust their metabolism to meet energy demands. In particular, glutamine addiction represents a distinctive feature of several types of tumors, including colorectal cancer. In this study, four colorectal cancer cell lines (Caco-2, HCT116, HT29 and SW480) were cultured with or without glutamine. The growth and proliferation rate, colony-forming capacity, apoptosis, cell cycle, redox homeostasis and metabolomic analysis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT), flow cytometry, high-performance liquid chromatography and gas chromatography/mass spectrometry techniques. The results show that glutamine represents an important metabolite for cell growth and that its deprivation reduces the proliferation of colorectal cancer cells. Glutamine depletion induces cell death and cell cycle arrest in the GO/G1 phase by modulating energy metabolism, the amino acid content and antioxidant defenses. Moreover, the combined glutamine starvation with the glycolysis inhibitor 2-deoxy-D-glucose exerted a stronger cytotoxic effect. This study offers a strong rationale for targeting glutamine metabolism alone or in combination with glucose metabolism to achieve a therapeutic benefit in the treatment of colon cancer.
RESUMEN
Epithelial barrier alteration is a central event in the pathogenesis of inflammatory bowel diseases. Lipopolysaccharide, correlated to the pathogenesis of such pathologies, has been demonstrated to cause altered membrane permeability, through the disruption and/or relocation of tight junction proteins, following redox-sensitive mitogen-activated protein kinases (MAPKs) modulation. Pterostilbene and its metabolite pinostilbene are natural stilbenoids which may reach relevant concentrations at intestinal level, together with their glucuronide and sulfate metabolites. The aim of our study was to evaluate the ability of these compounds to inhibit lipopolysaccharide-induced toxic effects on intestinal cell monolayer integrity and to explore the mechanism of action. Caco-2 cells, differentiated as enterocytes, were treated with lipopolysaccharide following pretreatment with the phenolic compounds at 1 µM physiological concentration. Caco-2 monolayer's permeability was monitored with time, measuring the transepithelial electrical resistance. Tight junction proteins were assessed by western blotting and immunofluorescence in lipopolysaccharide-treated cells, in relation to MAPK p38 and ERK1/2 activation. Pretreatment with all the phenolic compounds significantly slowed lipopolysaccharide-induced transepithelial electrical resistance decrease, preserved tight junction proteins levels and reduced MAPKs phosphorylation. The reported findings indicate that pterostilbene and its metabolites may counteract lipopolysaccharide-induced alteration of epithelial permeability, one of the initial events in the intestinal inflammatory process.
Asunto(s)
Mucosa Intestinal/efectos de los fármacos , Lipopolisacáridos/toxicidad , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Estilbenos/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Células CACO-2 , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Permeabilidad/efectos de los fármacos , Estilbenos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Mesenchymal stromal (stem) cells (MSCs) are increasingly demonstrated to ameliorate experimentally induced lung injuries through disease-specific anti-inflammatory actions, thus suggesting that different in vivo inflammatory environments can influence MSC actions. To determine the effects of different representative inflammatory lung conditions, human bone marrow-derived MSCs (hMSCs) were exposed to in vitro culture conditions from bronchoalveolar lavage fluid (BALF) samples obtained from patients with either the acute respiratory distress syndrome (ARDS) or with other lung diseases including acute respiratory exacerbations of cystic fibrosis (CF) (non-ARDS). hMSCs were subsequently assessed for time- and BALF concentration-dependent effects on mRNA expression of selected pro- and anti-inflammatory mediators, and for overall patterns of gene and mRNA expression. Both common and disease-specific patterns were observed in gene expression of different hMSC mediators, notably interleukin (IL)-6. Conditioned media obtained from non-ARDS BALF-exposed hMSCs was more effective in promoting an anti-inflammatory phenotype in monocytes than was conditioned media from ARDS BALF-exposed hMSCs. Neutralizing IL-6 in the conditioned media promoted generation of anti-inflammatory monocyte phenotype. This proof of concept study suggest that different lung inflammatory environments potentially can alter hMSC behaviors. Further identification of these interactions and the driving mechanisms may influence clinical use of MSCs for treating lung diseases.
Asunto(s)
Antiinflamatorios/farmacología , Líquido del Lavado Bronquioalveolar/química , Medios de Cultivo Condicionados/farmacología , Fibrosis Quística/terapia , Células Madre Mesenquimatosas/citología , Neumonía/terapia , Síndrome de Dificultad Respiratoria/terapia , Fibrosis Quística/inmunología , Fibrosis Quística/patología , Humanos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Neumonía/inmunología , Neumonía/patología , Síndrome de Dificultad Respiratoria/inmunología , Síndrome de Dificultad Respiratoria/patologíaRESUMEN
The emerging role of the diet in the incidence of intestinal inflammatory diseases has stimulated research on the influence of eating habits with pro-inflammatory properties in inducing epithelial barrier disturbance. Cholesterol oxidation products, namely oxysterols, have been shown to promote and sustain oxidative/inflammatory reactions in human digestive tract. This work investigated in an in vitro model the potential ability of a combination of dietary oxysterols representative of a hyper-cholesterol diet to induce the loss of intestinal epithelial layer integrity. The components of the experimental mixture were the main oxysterols stemming from heat-induced cholesterol auto-oxidation, namely 7-ketocholesterol, 5α,6α-and 5ß,6ß-epoxycholesterol, 7α- and 7ß-hydroxycholesterol. These compounds added to monolayers of differentiated CaCo-2 cells in combination or singularly, caused a time-dependent induction of matrix metalloproteinases (MMP)-2 and -9, also known as gelatinases. The hyperactivation of MMP-2 and -9 was found to be associated with decreased levels of the tight junctions zonula occludens-1 (ZO-1), occludin and Junction Adhesion Molecule-A (JAM-A). Together with such a protein loss, particularly evident for ZO-1, a net perturbation of spatial localization of the three tight junctions was observed. Cell monolayer pre-treatment with the selective inhibitor of MMPs ARP100 or polyphenol (-)-epicathechin, previously shown to inhibit NADPH oxidase in the same model system, demonstrated that the decrease of the three tight junction proteins was mainly a consequence of MMPs induction, which was in turn dependent on the pro-oxidant property of the oxysterols investigated. Although further investigation on oxysterols intestinal layer damage mechanism is to be carried on, the consequent - but incomplete - prevention of oxysterols-dependent TJs alteration due to MMPs inhibition, avoided the loss of scaffold protein ZO-1, with possible significant recovery of intestinal monolayer integrity.
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Colesterol/análogos & derivados , Hidroxicolesteroles/farmacología , Cetocolesteroles/farmacología , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Uniones Estrechas/efectos de los fármacos , Células CACO-2 , Catequina/farmacología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Colesterol/farmacología , Colesterol en la Dieta/metabolismo , Colesterol en la Dieta/farmacología , Impedancia Eléctrica , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Peroxidación de Lípido , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ocludina/genética , Ocludina/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Uniones Estrechas/metabolismo , Uniones Estrechas/ultraestructura , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
BACKGROUND: A novel potential approach for lung transplantation could be to utilize xenogeneic decellularized pig lung scaffolds that are recellularized with human lung cells. However, pig tissues express several immunogenic proteins, notably galactosylated cell surface glycoproteins resulting from alpha 1,3 galactosyltransferase (α-gal) activity, that could conceivably prevent effective use. Use of lungs from α-gal knock out (α-gal KO) pigs presents a potential alternative and thus comparative de- and recellularization of wild-type and α-gal KO pig lungs was assessed. METHODS: Decellularized lungs were compared by histologic, immunohistochemical, and mass spectrometric techniques. Recellularization was assessed following compartmental inoculation of human lung bronchial epithelial cells, human lung fibroblasts, human bone marrow-derived mesenchymal stromal cells (all via airway inoculation), and human pulmonary vascular endothelial cells (CBF) (vascular inoculation). RESULTS: No obvious differences in histologic structure was observed but an approximate 25% difference in retention of residual proteins was determined between decellularized wild-type and α-gal KO pig lungs, including retention of α-galactosylated epitopes in acellular wild-type pig lungs. However, robust initial recellularization and subsequent growth and proliferation was observed for all cell types with no obvious differences between cells seeded into wild-type versus α-gal KO lungs. CONCLUSION: These proof of concept studies demonstrate that decellularized wild-type and α-gal KO pig lungs can be comparably decellularized and comparably support initial growth of human lung cells, despite some differences in retained proteins. α-Gal KO pig lungs are a suitable platform for further studies of xenogeneic lung regeneration.
Asunto(s)
Células Epiteliales/citología , Fibroblastos/citología , Galactosiltransferasas/fisiología , Pulmón/citología , Células Madre Mesenquimatosas/citología , Regeneración/fisiología , Animales , Animales Modificados Genéticamente , Proliferación Celular , Células Epiteliales/enzimología , Matriz Extracelular/enzimología , Fibroblastos/enzimología , Humanos , Pulmón/enzimología , Células Madre Mesenquimatosas/enzimología , PorcinosRESUMEN
UNLABELLED: Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that mediate most of the effects elicited by the thyroid hormone, 3,5,3'-L-triiodothyronine (T3). TRs have been implicated in tumorigenesis, although it is unclear whether they act as oncogenes or tumor suppressors, and at which stage of tumorigenesis their dysregulation occurs. Using the resistant-hepatocyte rat model (R-H model), we found down-regulation of TRß1 and TRα1 and their target genes in early preneoplastic lesions and hepatocellular carcinoma (HCCs), suggesting that a hypothyroid status favors the onset and progression of preneoplastic lesions to HCC. Notably, TRß1 and, to a lesser extent, TRα1 down-regulation was observed only in preneoplastic lesions positive for the progenitor cell marker, cytokeratin-19 (Krt-19) and characterized by a higher proliferative activity, compared to the Krt-19 negative ones. TRß1 down-regulation was observed also in the vast majority of the analyzed human HCCs, compared to the matched peritumorous liver or to normal liver. Hyperthyroidism induced by T3 treatment caused up-regulation of TRß1 and of its target genes in Krt-19(+) preneoplastic rat lesions and was associated with nodule regression. In HCC, TRß1 down-regulation was not the result of hypermethylation of its promoter, but was associated with an increased expression of TRß1-targeting microRNAs ([miR]-27a, -181a, and -204). An inverse correlation between TRß1 and miR-181a was also found in human cirrhotic peritumoral tissue, compared to normal liver. CONCLUSION: Down-regulation of TRs, especially TRß1, is an early and relevant event in liver cancer development and is species and etiology independent. The results also suggest that a hypothyroid status of preneoplastic lesions may contribute to their progression to HCC and that the reversion of this condition may represent a possible therapeutic goal to interfere with the development of this tumor.
Asunto(s)
Carcinoma Hepatocelular/etiología , Hipotiroidismo/complicaciones , Neoplasias Hepáticas Experimentales/etiología , Lesiones Precancerosas/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Carcinogénesis , Proliferación Celular , Islas de CpG , Femenino , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Hipotiroidismo/metabolismo , Cirrosis Hepática/metabolismo , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , Ratas Endogámicas F344 , Receptores de Hormona Tiroidea/genéticaRESUMEN
Systemic administration of mesenchymal stromal cells (MSCs) suppresses airway inflammation and methacholine-induced airway hyper-responsiveness (AHR) in mouse models of T helper cell (Th) type 2-mediated eosinophilic allergic airway inflammation (AAI); however, the efficacy of MSCs in mouse models of severe Th17-mediated neutrophilic AAI has not yet been demonstrated. We assessed MSC effects in a mouse model of mixed Th2/Th17 AAI produced by mucosal exposure to Aspergillus fumigatus hyphal extract (AHE). Following sensitization produced by oropharyngeal AHE administration, systemic (tail vein) administration of syngeneic MSCs on the first day of challenge significantly reduced acute AHR predominantly through reduction of Th17-mediated airway inflammation. In parallel experiments, MSCs also mitigated AHR when administered during recurrent challenge 10 weeks after initial sensitization and challenge through reduction in systemic Th17-mediated inflammation. Investigation into potential mechanistic actions of MSCs in this model demonstrated that although T regulatory cells were increased in all AHE-treated mice, MSC administration did not alter T regulatory cell numbers in either the acute or recurrent model. Differential induction of interleukin-17a secretion was observed in ex vivo restimulation of mediastinal lymph node mixed-cell cytokine analyses. Although the mechanisms by which MSCs act to decrease inflammation and AHR in this model are not yet fully elucidated, decrease in Th17-mediated airway inflammation appears to play a significant role. These results provide a basis for further investigations of MSC administration as a potential therapeutic approach for severe refractory neutrophilic asthma.
Asunto(s)
Aspergilosis Broncopulmonar Alérgica/inmunología , Aspergilosis Broncopulmonar Alérgica/terapia , Aspergillus fumigatus/inmunología , Células Madre Mesenquimatosas/microbiología , Transducción de Señal/inmunología , Células Th17/microbiología , Animales , Aspergilosis Broncopulmonar Alérgica/metabolismo , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Modelos Animales de Enfermedad , Interleucina-17/inmunología , Interleucina-17/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/microbiología , Prueba de Cultivo Mixto de Linfocitos , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/microbiología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/microbiología , Células Th17/inmunología , Células Th17/metabolismoRESUMEN
Acellular whole human lung scaffolds represent a unique opportunity for ex vivo tissue engineering. However, it remains unclear whether lungs from individuals with chronic lung diseases such as chronic obstructive pulmonary disease (COPD) can be appropriately decellularized and recellularized. To assess this, cadaveric human lungs from normal (non-smoking) patients and from patients with COPD (smoking history) were decellularized and found by histochemical and immunohistochemical staining, electron microscopy, and mass spectrometry to retain characteristic histological architecture and extracellular matrix components (ECM) reflecting either normal or COPD, particularly emphysematous, origin. Inoculation of human bronchial epithelial cells, endothelial progenitor cells, bone marrow-derived mesenchymal stem cells, and lung fibroblasts via airway or vascular routes into small, excised segments of the decellularized lungs demonstrated that normal lung scaffolds robustly supported initial engraftment and growth of each cell type for up to one month. In contrast, despite initial binding, all cell types inoculated into decellularized emphysematous lungs did not survive beyond one week. However, cell attachment and proliferation on solubilized ECM homogenates of decellularized normal and emphysematous lungs coated onto tissue culture plates was comparable and not impaired, suggesting that the 3-dimensional decellularized emphysematous scaffolds may lack the necessary ECM architecture to support sustained cell growth.
Asunto(s)
Enfisema/patología , Pulmón/patología , Línea Celular , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Espectrometría de Masas , Microscopía ElectrónicaRESUMEN
Using a model of postpneumonectomy (PNY) compensatory lung growth in mice, we previously observed an increase in numbers of a putative endogenous distal airway progenitor cell population (CCSP(pos) /pro-SPC(pos) cells located at bronchoalveolar duct junctions [BADJs]), at 3, 7, and 14 days after pneumonectomy, returning to baseline at 28 days post-PNY. As the origin of these cells is poorly understood, we evaluated whether bone marrow cells contributed to the pool of these or other cells during prolonged post-PNY lung regrowth. Naïve and sex-mismatched chimeric mice underwent left PNY and were evaluated at 1, 2, and 3 months for numbers of BADJ CCSP(pos) /pro-SPC(pos) cells and presence of donor-derived marrow cells engrafted as airway or alveolar epithelium. Nonchimeric mice were also examined at 12 months after PNY for numbers of BADJ CCSP(pos) /pro-SPC(pos) cells. Notably, the right accessory lobe (RAL) continued to grow disproportionately over 12 months, a novel finding not previously described. Assessment of lung mechanics demonstrated an increase in lung stiffness following PNY, which significantly diminished over 1 year, but remained elevated relative to 1-year-old naïve controls. However, the number of CCSP(pos) /pro-SPC(pos) BADJ cells ≥1-month following PNY was equivalent to that found in naïve controls even after 12 months of continued RAL growth. Notably, no donor bone marrow-derived cells engrafted as airway or alveolar epithelial cells, including those at the BADJ, up to 3 months after PNY. These studies suggest that lung epithelial cells, including CCSP(pos) /pro-SPC(pos) cells, are not replenished from marrow-derived cells during post-PNY lung growth in mice.
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Pulmón/fisiología , Neumonectomía/métodos , Mecánica Respiratoria/fisiología , Células Madre/fisiología , Animales , Pulmón/citología , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mecánica Respiratoria/genética , Células Madre/citología , Células Madre/metabolismoRESUMEN
Despite growing interest on the potential use of de-cellularized whole lungs as 3-dimensional scaffolds for ex vivo lung tissue generation, optimal processing including sterilization and storage conditions, are not well defined. Further, it is unclear whether lungs need to be obtained immediately or may be usable even if harvested several days post-mortem, a situation mimicking potential procurement of human lungs from autopsy. We therefore assessed effects of delayed necropsy, prolonged storage (3 and 6 months), and of two commonly utilized sterilization approaches: irradiation or final rinse with peracetic acid, on architecture and extracellular matrix (ECM) protein characteristics of de-cellularized mouse lungs. These different approaches resulted in significant differences in both histologic appearance and in retention of ECM and intracellular proteins as assessed by immunohistochemistry and mass spectrometry. Despite these differences, binding and proliferation of bone marrow-derived mesenchymal stromal cells (MSCs) over a one month period following intratracheal inoculation was similar between experimental conditions. In contrast, significant differences occurred with C10 mouse lung epithelial cells between the different conditions. Therefore, delayed necropsy, duration of scaffold storage, sterilization approach, and cell type used for re-cellularization may significantly impact the usefulness of this biological scaffold-based model of ex vivo lung tissue regeneration.
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Pulmón/citología , Preservación de Órganos , Esterilización , Animales , Proliferación Celular , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Pulmón/anatomía & histología , Pulmón/metabolismo , Masculino , Espectrometría de Masas , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Use of de-cellularized cadaveric lungs as 3-dimensional scaffolds for ex vivo lung tissue generation offers a new potential therapeutic approach for clinical lung transplantation. However, it is likely that some of the available cadaveric human lungs may be from older donors or from donors with previously existing structural lung diseases such as emphysema or pulmonary fibrosis. It is not known whether these lungs will be suitable for either de-cellularization or re-cellularization. To investigate this, we assessed the effects of advanced age, representative emphysematous and fibrotic injuries, and the combination of advanced age and emphysematous injury and found significant differences both in histologic appearance and in the retention of extracellular matrix (ECM) and other proteins, as assessed by immunohistochemistry and mass spectrometry, between the different conditions. However, despite these differences, binding, retention and growth of bone marrow-derived mesenchymal stromal cells (MSCs) over a 1-month period following intratracheal inoculation were similar between the different experimental conditions. In contrast, significant differences occurred in the growth of C10 mouse lung epithelial cells between the different conditions. Therefore, age, lung injury, and the cell type used for re-cellularization may significantly impact the usefulness of de-cellularized whole lungs for ex vivo lung tissue regeneration.
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Envejecimiento/patología , Lesión Pulmonar/patología , Pulmón/patología , Enfisema Pulmonar/patología , Animales , Apoptosis , Bleomicina , Caspasa 3/metabolismo , Proliferación Celular , Células Epiteliales/metabolismo , Células Epiteliales/patología , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/metabolismo , Fibrosis , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Lesión Pulmonar/complicaciones , Masculino , Espectrometría de Masas , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Elastasa Pancreática , Enfisema Pulmonar/complicacionesRESUMEN
UNLABELLED: Human hepatocellular carcinoma (HCC) is a heterogeneous disease of distinct clinical subgroups. A principal source of tumor heterogeneity may be cell type of origin, which in liver includes hepatocyte or adult stem/progenitor cells. To address this issue, we investigated the molecular mechanisms underlying the fate of the enzyme-altered preneoplastic lesions in the resistant hepatocyte (RH) model. Sixty samples classified as focal lesions, adenoma, and early and advanced HCCs were microdissected after morphological and immunohistochemical evaluation and subjected to global gene expression profiling. The analysis of progression of the persistent glutathione S-transferase (GSTP)(+) focal lesions to fully developed HCC showed that approximately 50% of persistent nodules and all HCCs expressed cytokeratin 19 (CK19), whereas 14% of remodeling nodules were CK19(+). Unsupervised hierarchical clustering of the expression profiles also grouped the samples according to CK19 expression. Furthermore, supervised analysis using the differentially expressed genes in each cluster combined with gene connectivity tools identified 1308 unique genes and a predominance of the AP-1/JUN network in the CK19(+) lesions. In contrast, the CK19-negative cluster exhibited only limited molecular changes (156 differentially expressed genes versus normal liver) consistent with remodeling toward differentiated phenotype. Finally, comparative functional genomics showed a stringent clustering of CK19(+) early lesions and advanced HCCs with human HCCs characterized by poor prognosis. Furthermore, the CK19-associated gene expression signature accurately predicted patient survival (P < 0.009) and tumor recurrence (P < 0.006). CONCLUSION: Our data establish CK19 as a prognostic marker of early neoplastic lesions and strongly suggest the progenitor derivation of HCC in the rat RH model. The capacity of CK19-associated gene signatures to stratify HCC patients according to clinical prognosis indicates the usefulness of the RH model for studies of stem/progenitor-derived HCC.
Asunto(s)
Carcinoma Hepatocelular/etiología , Neoplasias Hepáticas/etiología , Células Madre/patología , Animales , Carcinoma Hepatocelular/patología , Glutatión Transferasa/análisis , Inmunohistoquímica , Queratina-19/análisis , Queratina-19/genética , Neoplasias Hepáticas/patología , Masculino , Proteína Quinasa 14 Activada por Mitógenos/análisis , Proteína Quinasa 14 Activada por Mitógenos/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Modelos de Riesgos Proporcionales , Ratas , Ratas Endogámicas F344RESUMEN
RATIONALE: Recent studies have suggested that both embryonic stem cells and adult bone marrow stem cells can participate in the regeneration and repair of diseased adult organs, including the lungs. However, the extent of airway epithelial remodeling with adult marrow stem cells is low, and there are no available in vivo data with embryonic stem cells. Human umbilical cord blood contains both hematopoietic and nonhematopoietic stem cells, which have been used clinically as an alternative to bone marrow transplantation for hematologic malignancies and other diseases. OBJECTIVES: We hypothesized that human umbilical cord blood stem cells might be an effective alternative to adult bone marrow and embryonic stem cells for regeneration and repair of injured airway epithelium. METHODS: Human cord blood was obtained from normal deliveries at the University of Vermont. Cultured plastic adherent cells were characterized as mesenchymal stem cells (MSCs) by flow cytometry and differentiation assays. Cord blood-derived MSCs (CB-MSCs) were cultured in specialized airway growth media or with specific growth factors, including keratinocyte growth factor and retinoic acid. mRNA and protein expression were analyzed with PCR and immunofluorescent staining. CB-MSCs were systematically administered to immunotolerant, nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice. Lungs were analyzed for presence of human cells. MEASUREMENTS AND MAIN RESULTS: When cultured in specialized airway growth media or with specific growth factors, CB-MSCs differentially expressed Clara cell secretory protein (CCSP), cystic fibrosis transmembrane conductance regulator (CFTR), surfactant protein C, and thyroid transcription factor-1 mRNA, and CCSP and CFTR protein. Furthermore, CB-MSCs were easily transduced with recombinant lentiviral vectors to express human CFTR. After systemic administration to immunotolerant, NOD-SCID, mice, rare cells were found in the airway epithelium that had acquired cytokeratin and human CFTR expression. CONCLUSIONS: CB-MSCs appear to be comparable to MSCs obtained from adult bone marrow in ability to express phenotypic markers of airway epithelium and to participate in airway remodeling in vivo.
Asunto(s)
Trasplante de Células Madre de Sangre del Cordón Umbilical , Fibrosis Quística/cirugía , Células Madre Mesenquimatosas , Regeneración , Mucosa Respiratoria/fisiología , Animales , Técnicas de Cultivo de Célula , Fibrosis Quística/patología , Humanos , Queratinas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Transducción Genética/métodosRESUMEN
The effect of lung irradiation on subsequent inflammatory or fibrotic lung injuries remains poorly understood. We postulated that irradiation and bone marrow transplantation might impact the development and progression of lung remodeling resulting from asbestos inhalation. Our objective was to determine whether irradiation and bone marrow transplantation affected inflammation and fibrosis associated with inhaled asbestos exposure. Inflammation, cytokine production, and fibrosis were assessed in lungs of naïve and sex-mismatched chimeric mice exposed to asbestos for 3, 9, or 40 days. Potential engraftment of donor-derived cells in recipient lungs was examined by fluorescence in situ hybridization and immunohistochemistry. Compared with asbestos-exposed naïve (nonchimeric) mice, chimeric mice exposed to asbestos for 3, 9, or 40 days demonstrated significant abrogation of acute increases in asbestos-associated inflammatory mediators and fibrosis. Donor-derived cells trafficked to lung but did not significantly engraft as phenotypic lung cells. Irradiation and bone marrow transplantation alters inflammatory and fibrotic responses to asbestos, likely through modulation of soluble inflammatory mediators.
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Amianto/toxicidad , Asbestosis/metabolismo , Trasplante de Médula Ósea , Citocinas/biosíntesis , Rayos gamma/efectos adversos , Mediadores de Inflamación/metabolismo , Neumonía/metabolismo , Fibrosis Pulmonar/metabolismo , Quimera por Trasplante/metabolismo , Irradiación Corporal Total , Animales , Asbestosis/patología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de la radiación , Femenino , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Neumonía/etiología , Neumonía/patología , Fibrosis Pulmonar/etiología , Fibrosis Pulmonar/patología , Factores de Tiempo , Trasplante HomólogoRESUMEN
Chronic pulmonary inflammation and infection are the leading causes of morbidity and mortality in cystic fibrosis (CF). While the effect of mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) on airways remains controversial, some groups have demonstrated increases in Na(+) and Cl(-) in CF airway surface liquid compared to normal airways. We investigated the consequences of NaCl on pro-inflammatory chemokine and cytokine production by macrophages. Stimulation of mouse macrophages with increasing amounts of NaCl induced macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-alpha (TNF-alpha) production. Further, co-incubation of macrophages with NaCl in the presence of either lipopolysaccharide (LPS) or TNF-alpha synergistically increased MIP-2 production. Both the NaCl and NaCl plus LPS responses were partially dependent on endogenous production and autocrine signaling by TNF-alpha. To investigate the role of CFTR in MIP-2 production, we compared the responses of wild-type and DeltaF508 CF mouse macrophages to NaCl and LPS. The responses of macrophages from both strains were indistinguishable. In addition, CFTR mRNA was not expressed in macrophages. Taken together, these findings suggest that NaCl stimulates MIP-2 production by macrophages through a mechanism that is partially dependent on TNF-alpha but independent of macrophage CFTR expression.
Asunto(s)
Quimiocinas/biosíntesis , Macrófagos/metabolismo , Cloruro de Sodio/metabolismo , Animales , Células Cultivadas , Quimiocina CXCL2 , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
RATIONALE: Recent literature suggests that adult bone marrow-derived cells can localize to lung and acquire immunophenotypic characteristics of lung epithelial cells. We speculated this might be a potential therapeutic approach for correcting defective lung epithelium in cystic fibrosis. OBJECTIVE: To determine whether adult bone marrow-derived cells containing normal cystic fibrosis transmembrane conductance regulator protein (CFTR) could repopulate lung epithelium in transgenic mice deficient in that protein. METHODS: Stromal marrow cells or total marrow obtained from adult male wild-type mice were transplanted into adult female Cftr knockout mice. To increase marrow cell recruitment naphthalene was used to induce airway epithelial injury in recipient mice. MEASUREMENTS AND MAIN RESULTS: At 1 wk, 1 mo, and 3 mo after transplantation, Cftr mRNA was detected in lung homogenates of recipient mice by reverse transcription-polymerase chain reaction. Cftr mRNA was not found in either donor marrow cells or mature circulating leukocytes. In situ examination of recipient mouse lungs demonstrated rare (0.025%) chimeric airway epithelial cells, some of which (0.01%) expressed CFTR protein. Naphthalene-induced airway remodeling nonsignificantly increased the number of chimeric airway epithelial cells expressing Cftr. CONCLUSIONS: These results demonstrate that adult marrow cells can be recruited to airway epithelium and induced to express Cftr in mice otherwise lacking this protein. However, the number of observed chimeric epithelial cells is small and new strategies for enhancing airway epithelial remodeling by adult bone marrow-derived cells will be necessary for correction of defective CFTR-dependent chloride transport.
Asunto(s)
Células de la Médula Ósea/fisiología , Trasplante de Médula Ósea/métodos , Fibrosis Quística/cirugía , Pulmón/patología , Mucosa Respiratoria/citología , Animales , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/biosíntesis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/uso terapéutico , Femenino , Citometría de Flujo/métodos , Expresión Génica/fisiología , Pulmón/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Tiempo , Trasplante HomólogoRESUMEN
BACKGROUND/AIMS: The role of adult bone marrow-derived cells (BMC) in hepatic regeneration is controversial. Both transdifferentiation of BMC as well as fusion with hepatocytes have been suggested in toxin-based and genetic selection models. METHODS: We have developed a transgenic mouse model of immune-mediated hepatitis to clarify the role of BMC in liver regeneration following injury mediated by T cells. RESULTS: Repeated adoptive transfer of transgenic T cells into bone marrow chimeras resulted in multiple waves of hepatitis. Hepatocytes derived from donor bone marrow were identified using a self-protein that does not interfere with hepatocyte function and proliferation in recipient animals. Some cells contained one recipient nucleus and another independent donor bone marrow-derived nucleus, suggesting that cellular fusion plays some role in liver repair after immune hepatitis. However, despite pronounced infiltration by myeloid cells, the frequency of fusion was extremely low. CONCLUSIONS: This study provides a unique, clinically relevant model in which fusion hepatocytes can be purified and characterized by the expression of donor MHC antigen. It demonstrates that although fusion between BMC and hepatocytes occurs under conditions of inflammation that correspond to human disease, its frequency needs to be increased to be of any therapeutic value.
Asunto(s)
Células de la Médula Ósea/inmunología , Hepatitis/inmunología , Hepatocitos/inmunología , Animales , Células de la Médula Ósea/citología , Comunicación Celular/inmunología , Fusión Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Citometría de Flujo , Hepatitis/patología , Hepatocitos/citología , Inmunohistoquímica , Hibridación Fluorescente in Situ , Ratones , Ratones Transgénicos , Linfocitos T/inmunología , Linfocitos T/trasplanteRESUMEN
Adult marrow-derived stem cells can localize to lung and acquire immunophenotypic characteristics of lung epithelial cells. Lung injury increases recruitment of the marrow-derived cells. We speculated that comparing patterns of lung engraftment following different lung injuries would provide insight into potential mechanisms by which marrow-derived cells were recruited to lung. To evaluate this, adult female C57Bl/6 mice irradiated and engrafted with marrow from adult male transgenic GFP mice were exposed to either intranasal inhalation of endotoxin (25 microg/mouse) or 3 days of 25 ppm NO(2) and then compared 1 or 3 months later to transplanted but otherwise uninjured mice. In all cases, the majority of marrow-derived cells recruited to lung were CD45(+) leukocytes. In lungs of transplanted but otherwise uninjured mice, small numbers of CD45(-) donor-derived cells in alveolar septae stained positively for pro-surfactant protein C. Rare donor-derived cells located in the airway epithelium stained positively with cytokeratin. Subsequent exposure of engrafted mice to NO(2) or endotoxin did not significantly increase the number or pattern of donor-derived CD45(-) cells found in recipient lungs. These results suggest that NO(2) or endotoxin lung injury does not result in significant engraftment of marrow-derived cells in lung.