RESUMO
Rationale: There are controversial reports on applications of mesenchymal stromal cells (MSCs) in patients with acute respiratory distress syndrome (ARDS). Objectives: We hypothesized that lung microenvironment was the main determinant of beneficial versus detrimental effects of MSCs during ARDS. Methods: Lung proteome was profiled in three models of injury induced by acid instillation and/or mechanical ventilation in mice. Human gene of glutathione peroxidase-1 was delivered before MSC administration; or MSCs carrying human gene of IL-10 or hepatocyte growth factor were administered after lung injury. An inhibitory cocktail against IL-6, fibronectin, and oxidative stress was used in in vitro studies using human small airway epithelial cells and human MSCs after exposure to plasma of patients with ARDS. Measurements and Main Results: Distinct proteomic profiles were observed in three lung injury models. Administration of MSCs protected lung from ventilator-induced injury, whereas it worsened acid-primed lung injuries associated with fibrotic development in lung environment that had high levels of IL-6 and fibronectin along with low antioxidant capacity. Correction of microenvironment with glutathione peroxidase-1, or treatment with MSCs carrying human gene of IL-10 or hepatocyte growth factor after acid-primed injury, reversed the detrimental effects of native MSCs. Proteomic profiles obtained in the mouse models were also similarly observed in human ARDS. Treatment with the inhibitory cocktail in samples of patients with ARDS retained protective effects of MSCs in small airway epithelial cells. Conclusions: MSCs can be beneficial or detrimental depending on microenvironment at the time of administration. Identification of potential beneficiaries seems to be crucial to guide MSC therapy in ARDS.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Proteômica , Síndrome do Desconforto Respiratório/fisiopatologia , Síndrome do Desconforto Respiratório/cirurgia , Animais , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
OBJECTIVE: Neutrophil infiltration and release of the abundant human neutrophil peptides (HNP) are a common clinical feature in critically ill patients. We tested a hypothesis that different cell types respond to HNP differently in lung microenvironment that may influence the host responses. METHODS: Plasma concentrations of HNP were measured in healthy volunteers and patients with sepsis. Cells including the bacteria P. aeruginosa, human lung epithelial cells and mesenchymal stromal cells (MSCs) were exposed to various concentrations of HNP. Bacterial killing, epithelial cell inflammation, MSC adhesion and behaviours were examined after HNP stimulation. RESULTS: Incubation of P. aeruginosa or stimulation of human lung epithelial cells with HNP resulted in bacterial killing or IL-8 production at a dose of 50 µg/mL, while MSC adhesion and alternations of secretome profiles took place after HNP stimulation at a dose of 10 µg/mL. The secretome profile changes were characterized by increased release of the IL-6 family members such as C-reactive protein (CRP), leukemia inhibitory factor (LIF) and interleukin (IL-11), and first apoptosis signal (FAS) and platelet-derived growth factor-AA as compared to a vehicle control group. CONCLUSION: Stimulation of MSCs with HNP resulted in changes of secretome profiles at 5-fold lower concentration than that required for bacterial killing and lung epithelial inflammation. This undisclosed risk factor of HNP in lung environment should be taken into consideration when MSCs are applied as cell therapy in inflammatory lung diseases.
RESUMO
Farber disease (FD) is a debilitating lysosomal storage disorder (LSD) caused by a deficiency of acid ceramidase (ACDase) activity due to mutations in the gene ASAH1. Patients with ACDase deficiency may develop a spectrum of clinical phenotypes. Severe cases of FD are frequently associated with neurological involvement, failure to thrive, and respiratory complications. Mice homozygous ( Asah1P361R/P361R) for an orthologous patient mutation in Asah1 recapitulate human FD. In this study, we show significant impairment in lung function, including low compliance and increased airway resistance in a mouse model of ACDase deficiency. Impaired lung mechanics in Farber mice resulted in decreased blood oxygenation and increased red blood cell production. Inflammatory cells were recruited to both perivascular and peribronchial areas of the lung. We observed large vacuolated foamy histiocytes that were full of storage material. An increase in vascular permeability led to protein leakage, edema, and impacted surfactant homeostasis in the lungs of Asah1P361R/P361R mice. Bronchial alveolar lavage fluid (BALF) extraction and analysis revealed accumulation of a highly turbid lipoprotein-like substance that was composed in part of surfactants, phospholipids, and ceramides. The phospholipid composition of BALF from Asah1P361R/P361R mice was severely altered, with an increase in both phosphatidylethanolamine (PE) and sphingomyelin (SM). Ceramides were also found at significantly higher levels in both BALF and lung tissue from Asah1P361R/P361R mice when compared with levels from wild-type animals. We demonstrate that a deficiency in ACDase leads to sphingolipid and phospholipid imbalance, chronic lung injury caused by significant inflammation, and increased vascular permeability, leading to impaired lung function.
Assuntos
Ceramidase Ácida/fisiologia , Modelos Animais de Doenças , Lesão Pulmonar/etiologia , Pulmão/patologia , Animais , Líquido da Lavagem Broncoalveolar , Ceramidas/metabolismo , Homozigoto , Pulmão/metabolismo , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Camundongos , Camundongos Knockout , Fenótipo , Fosfolipídeos/metabolismo , Testes de Função RespiratóriaRESUMO
Bacterial colonization of epithelial surfaces and subsequent transmigration across the mucosal barrier are essential for the development of infection. We hypothesized that the methyl-accepting proteins (MCPs), known as chemoreceptors expressed on Escherichia coli (E. coli) bacterial surface, play an important role in mediating bacterial transmigration. We demonstrated a direct interaction between human interleukin-8 (IL-8) and Tsr receptor, a major MCP chemoreceptor. Stimulation of human lung epithelial cell monolayer with IL-8 resulted in increased E. coli adhesion and transmigration of the native strain (RP437) and a strain expressing only Tsr (UU2373), as compared to a strain (UU2599) with Tsr truncation. The augmented E. coli adhesion and migration was associated with a higher expression of carcinoembryonic antigen-related cell adhesion molecule 6 and production of inflammatory cytokines/chemokines, and a lower expression of the tight junction protein claudin-1 and the plasma membrane protein caveolin-1 in lung epithelial cells. An increased E. coli colonization and pulmonary cytokine production induced by the RP437 and UU2373 strains was attenuated in mice challenged with the UU2599 strain. Our results suggest a critical role of the E. coli Tsr chemoreceptor in mediating bacterial colonization and transmigration across human lung epithelium during development of pulmonary infections.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Escherichia coli/imunologia , Escherichia coli/metabolismo , Interleucina-8/metabolismo , Pulmão/patologia , Proteínas de Membrana/metabolismo , Mucosa Respiratória/metabolismo , Antígenos CD/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/genética , Caveolina 1/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Claudina-1/metabolismo , Citocinas/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas Ligadas por GPI/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Proteínas de Membrana/genética , Organismos Geneticamente Modificados , Mucosa Respiratória/patologia , Deleção de Sequência/genética , Migração Transendotelial e TransepitelialRESUMO
STUDY DESIGN: An in vitro and in vivo evaluation of intervertebral disc (IVD)-derived stem/progenitor cells. OBJECTIVE: To determine the chondrogenic, adipogenic, osteogenic, and neurogenic differentiation capacity of disc-derived stem/progenitor cells in vitro and neurogenic differentiation in vivo. SUMMARY OF BACKGROUND DATA: Tissue repair strategies require a source of appropriate cells that could be used to replace dead or damaged cells and tissues such as stem cells. Here we examined the potential use of IVD-derived stem cells in regenerative medicine approaches and neural repair. METHODS: Nonchondrodystrophic canine IVD nucleus pulposus (NP) cells were used to generate stem/progenitor cells (NP progenitor cells [NPPCs]) and the NPPCs were differentiated in vitro into chondrogenic, adipogenic, and neurogenic lineages and in vivo into the neurogenic lineage. NPPCs were compared with bone marrow-derived mesenchymal (stromal) stem cells in terms of the expression of stemness genes. The expression of the neural crest marker protein 0 and the Brachyury gene were evaluated in NP cells and NPPCs. RESULTS: NPPCs contain stem/progenitor cells and express "stemness" genes such as Sox2, Oct3/4, Nanog, CD133, Nestin, and neural cell adhesion molecule but differ from mesenchymal (stromal) stem cells in the higher expression of the Nanog gene by NPPCs. NPPCs do not express protein 0 or the Brachyury gene both of which are expressed by the totality of IVD NP cells. The percentage of NPPCs within the IVD is 1% of the total as derived by colony-forming assay. NPPCs are capable of differentiating along chondrogenic, adipogenic, and neurogenic lineages in vitro and into oligodendrocyte, neuron, and astroglial specific precursor cells in vivo within the compact myelin-deficient shiverer mouse. CONCLUSION: We propose that the IVD NP represents a regenerative niche suggesting that the IVD could represent a readily accessible source of precursor cells for neural repair and regeneration.
Assuntos
Diferenciação Celular , Disco Intervertebral/citologia , Medicina Regenerativa/métodos , Células-Tronco/citologia , Antígeno AC133 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Antígenos CD/genética , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Cães , Expressão Gênica , Glicoproteínas/genética , Proteínas de Homeodomínio/genética , Proteínas de Filamentos Intermediários/genética , Disco Intervertebral/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Nestina , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Peptídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Células-Tronco/metabolismo , Proteínas com Domínio T/genéticaRESUMO
INTRODUCTION: The relative resistance of non-chondrodystrophic (NCD) canines to degenerative disc disease (DDD) may be due to a combination of anabolic and anti-catabolic factors secreted by notochordal cells within the intervertebral disc (IVD) nucleus pulposus (NP). Factors known to induce DDD include interleukin-1 beta (IL-1ß) and/or Fas-Ligand (Fas-L). Therefore we evaluated the ability of notochordal cell conditioned medium (NCCM) to protect NP cells from IL-1ß and IL-1ß +FasL-mediated cell death and degeneration. METHODS: We cultured bovine NP cells with IL-1ß or IL-1ß+FasL under hypoxic serum-free conditions (3.5% O2) and treated the cells with either serum-free NCCM or basal medium (Advanced DMEM/F-12). We used flow cytometry to evaluate cell death and real-time (RT-)PCR to determine the gene expression of aggrecan, collagen 2, and link protein, mediators of matrix degradation ADAMTS-4 and MMP3, the matrix protection molecule TIMP1, the cluster of differentiation (CD)44 receptor, the inflammatory cytokine IL-6 and Ank. We then determined the expression of specific apoptotic pathways in bovine NP cells by characterizing the expression of activated caspases-3, -8 and -9 in the presence of IL-1ß+FasL when cultured with NCCM, conditioned medium obtained using bovine NP cells (BCCM), and basal medium all supplemented with 2% FBS. RESULTS: NCCM inhibits bovine NP cell death and apoptosis via suppression of activated caspase-9 and caspase-3/7. Furthermore, NCCM protects NP cells from the degradative effects of IL-1ß and IL-1ß+Fas-L by up-regulating the expression of anabolic/matrix protective genes (aggrecan, collagen type 2, CD44, link protein and TIMP-1) and down-regulating matrix degrading genes such as MMP-3. Expression of ADAMTS-4, which encodes a protein for aggrecan remodeling, is increased. NCCM also protects against IL-1+FasL-mediated down-regulation of Ank expression. Furthermore, NP cells treated with NCCM in the presence of IL-1ß+Fas-L down-regulate the expression of IL-6 by almost 50%. BCCM does not mediate cell death/apoptosis in target bovine NP cells. CONCLUSIONS: Notochordal cell-secreted factors suppress NP cell death by inhibition of activated caspase-9 and -3/7 activity and by up-regulating genes contributing anabolic activity and matrix protection of the IVD NP. Harnessing the restorative powers of the notochordal cell could lead to novel cellular and molecular strategies in the treatment of DDD.
Assuntos
Apoptose/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Disco Intervertebral/efeitos dos fármacos , Notocorda/metabolismo , Proteínas ADAM/genética , Proteína ADAMTS4 , Agrecanas/genética , Animais , Caspases/metabolismo , Bovinos , Hipóxia Celular , Células Cultivadas , Colágeno Tipo II/genética , Meios de Cultivo Condicionados/metabolismo , Cães , Proteínas da Matriz Extracelular/genética , Proteína Ligante Fas/farmacologia , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Receptores de Hialuronatos/genética , Interleucina-1beta/farmacologia , Interleucina-6/genética , Disco Intervertebral/citologia , Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/prevenção & controle , Masculino , Metaloproteinase 3 da Matriz/genética , Notocorda/citologia , Pró-Colágeno N-Endopeptidase/genética , Proteoglicanas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-1/genéticaRESUMO
Both Epac and PKA are effectors of the second messenger cAMP. Utilizing an exchange protein directly activated by cAMP (Epac) pathway-specific cAMP analog (ESCA), we previously reported that Epac signaling regulates proglucagon gene (gcg) expression in the glucagon-like peptide-1 (GLP-1)-producing intestinal endocrine L-cell lines GLUTag and STC-1. We now show that Epac-2 is also expressed in glucagon-producing pancreatic alpha-cell lines, including PKA-deficient InR1-G9 cells, and that ESCA stimulates gcg promoter and mRNA expression in the InR1-G9 cells. Using a dominant-negative Epac-2 expression plasmid (Epac-2DN), we found that Epac inhibition attenuated forskolin-stimulated gcg promoter expression in the PKA-active STC-1 cell line and blocked forskolin-stimulated gcg promoter expression in the InR1-G9 cells. Consistently, ESCA was shown to stimulate glucagon and GLP-1 production in the InR1-G9 and GLUTag cell lines, respectively. Surprisingly, ESCA treatment did not show a notable stimulation of glucagon or GLP-1 secretion from these two cell lines. This is in contrast to its ability to stimulate insulin secretion from the pancreatic INS-1 beta-cell line. Our findings suggest that Epac is selectively involved in peptide hormone secretion in pancreatic and intestinal endocrine cells and that distinct signaling cascades are involved in stimulating production vs. secretion of glucagon and GLP-1 in response to cAMP elevation.
Assuntos
AMP Cíclico/metabolismo , Células Enteroendócrinas/metabolismo , Células Secretoras de Glucagon/metabolismo , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Proglucagon/biossíntese , Northern Blotting , Western Blotting , Linhagem Celular , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/biossíntese , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Imuno-Histoquímica , Proglucagon/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , TransfecçãoRESUMO
Although proglucagon gene expression and the synthesis of proglucagon encoded peptide hormones could be activated by protein kinase A (PKA) activators such as forskolin/3-isobutyl-1-methylxanthine (IBMX) and cholera toxin, whether the activation is entirely attributed to PKA has not been previously examined. We found that forskolin/IBMX also activate ERK1/2 phosphorylation in intestinal and pancreatic proglucagon-producing cell lines. The MEK inhibitors PD98059 and U0126 were found to repress the expression of proglucagon promoter as well as endogenous proglucagon mRNA in two intestinal proglucagon-producing cell lines and to block the stimulatory effect of forskolin/IBMX on proglucagon mRNA expression. The repressive effect of the PKA-specific inhibitors H-89 and KT-5720, however, was either not observable or much less potent. Forskolin could activate ERK1/2 phosphorylation and proglucagon gene transcription on its own, whereas forskolin plus IBMX are required to effectively activate the PKA pathway in the proglucagon-producing cells. Exchange protein directly activated by cyclic AMP 2 (Epac2, or cAMP-binding guanine nucleotide exchange factor-2) was found to be expressed in gut and pancreatic proglucagon-producing cell lines, whereas the Epac-pathway-specific cAMP analog, 8-pMeOPT-2'O-Me-cAMP, effectively stimulated ERK1/2 phosphorylation as well as proglucagon mRNA expression. We therefore suggest that cAMP at least partially regulates proglucagon gene expression via the Epac-Ras/Rap-Raf-MEK-ERK signaling pathway.