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While IκB-kinase-ε (IKKε) induces immunomodulatory genes following viral stimuli, its up-regulation by inflammatory cytokines remains under-explored. Since airway epithelial cells respond to airborne insults and potentiate inflammation, IKKε expression was characterized in pulmonary epithelial cell lines (A549, BEAS-2B) and primary human bronchial epithelial cells grown as submersion or differentiated air-liquid interface cultures. IKKε expression was up-regulated by the pro-inflammatory cytokines, interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNFα). Thus, mechanistic interrogations in A549 cells were used to demonstrate the NF-κB dependence of cytokine-induced IKKε. Furthermore, chromatin immunoprecipitation in A549 and BEAS-2B cells revealed robust recruitment of the NF-κB subunit, p65, to one 5' and two intronic regions within the IKKε locus (IKBKE). In addition, IL-1ß and TNFα induced strong RNA polymerase 2 recruitment to the 5' region, the first intron, and the transcription start site. Stable transfection of the p65-binding regions into A549 cells revealed IL-1ß- and TNFα-inducible reporter activity that required NF-κB, but was not repressed by glucocorticoid. While critical NF-κB motifs were identified in the 5' and downstream intronic regions, the first intronic region did not contain functional NF-κB motifs. Thus, IL-1ß- and TNFα-induced IKKε expression involves three NF-κB-binding regions, containing multiple functional NF-κB motifs, and potentially other mechanisms of p65 binding through non-classical NF-κB binding motifs. By enhancing IKKε expression, IL-1ß may prime, or potentiate, responses to alternative stimuli, as modelled by IKKε phosphorylation induced by phorbol 12-myristate 13-acetate. However, since IKKε expression was only partially repressed by glucocorticoid, IKKε-dependent responses could contribute to glucocorticoid-resistant disease.
Assuntos
Células Epiteliais , Quinase I-kappa B , Humanos , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células A549 , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/genética , Interleucina-1beta/farmacologia , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , NF-kappa B/metabolismo , NF-kappa B/genética , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Pulmão/metabolismo , Pulmão/citologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/citologia , Regulação da Expressão Gênica/efeitos dos fármacosRESUMO
Chronic stress is epidemiologically correlated with physical and psychiatric disorders. Whereas many animal models of chronic stress induce symptoms of psychopathology, repeated homotypic stressors to moderate intensity stimuli typically reduce stress-related responses with fewer, if any, pathological symptoms. Recent results indicate that the rostral posterior hypothalamic (rPH) region is a significant component of the brain circuitry underlying response reductions (habituation) associated with repeated homotypic stress. To test whether posterior hypothalamic transcriptional regulation associates with the neuroendocrine modifications induced by repeated homotypic stress, RNA-seq was performed in the rPH dissected from adult male rats that experienced either no stress, 1, 3, or 7 stressful loud noise exposures. Plasma samples displayed reliable increases of corticosterone in all stressed groups, with the smallest increase in the group exposed to 7 loud noises, indicating significant habituation compared to the other stressed groups. While few or no differentially expressed genes were detected 24-h after one or three loud noise exposures, relatively large numbers of transcripts were differentially expressed between the group exposed to 7 loud noises when compared to the control or 3-stress groups, respectively, which correlated with the corticosterone response habituation observed. Gene ontology analyses indicated multiple significant functional terms related to neuron differentiation, neural membrane potential, pre- and post-synaptic elements, chemical synaptic transmission, vesicles, axon guidance and projection, glutamatergic and GABAergic neurotransmission. Some of the differentially expressed genes (Myt1l, Zmat4, Dlx6, Csrnp3) encode transcription factors that were independently predicted by transcription factor enrichment analysis to target other differentially regulated genes in this study. A similar experiment employing in situ hybridization histochemical analysis in additional animals validated the direction of change of the 5 transcripts investigated (Camk4, Gabrb2, Gad1, Grin2a and Slc32a) with a high level of temporal and regional specificity for the rPH. In aggregate, the results suggest that distinct patterns of gene regulation are obtained in response to a repeated homotypic stress regimen; they also point to a significant reorganization of the rPH region that may critically contribute to the phenotypic modifications associated with repeated homotypic stress habituation.
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Military Deployment to Southwest Asia and Afghanistan and exposure to toxic airborne particulates have been associated with an increased risk of developing respiratory disease, collectively termed deployment-related respiratory diseases (DRRDs). Our knowledge about how particulates mediate respiratory disease is limited, precluding the appropriate recognition or management. Central to this limitation is the lack of understanding of how exposures translate into dysregulated cell identity with dysregulated transcriptional programs. The small airway epithelium is involved in both the pathobiology of DRRD and fine particulate matter deposition. To characterize small airway epithelial cell epigenetic and transcriptional responses to Afghan desert particulate matter (APM) and investigate the functional interactions of transcription factors that mediate these responses, we applied two genomics assays, the assay for transposase accessible chromatin with sequencing (ATAC-seq) and Precision Run-on sequencing (PRO-seq). We identified activity changes in a series of transcriptional pathways as candidate regulators of susceptibility to subsequent insults, including signal-dependent pathways, such as loss of cytochrome P450 or P53/P63, and lineage-determining transcription factors, such as GRHL2 loss or TEAD3 activation. We further demonstrated that TEAD3 activation was unique to APM exposure despite similar inflammatory responses when compared with wood smoke particle exposure and that P53/P63 program loss was uniquely positioned at the intersection of signal-dependent and lineage-determining transcriptional programs. Our results establish the utility of an integrated genomics approach in characterizing responses to exposures and identifying genomic targets for the advanced investigation of the pathogenesis of DRRD.
Assuntos
Células Epiteliais Alveolares , Material Particulado , Fatores de Transcrição , Afeganistão , Células Epiteliais Alveolares/metabolismo , Cromatina/metabolismo , Epigênese Genética , Genômica/métodos , Destacamento Militar , Material Particulado/toxicidade , Doenças Respiratórias/epidemiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transposases/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Rationale: Common genetic variants have been associated with idiopathic pulmonary fibrosis (IPF). Objectives: To determine functional relevance of the 10 IPF-associated common genetic variants we previously identified. Methods: We performed expression quantitative trait loci (eQTL) and methylation quantitative trait loci (mQTL) mapping, followed by co-localization of eQTL and mQTL with genetic association signals and functional validation by luciferase reporter assays. Illumina multi-ethnic genotyping arrays, mRNA sequencing, and Illumina 850k methylation arrays were performed on lung tissue of participants with IPF (234 RNA and 345 DNA samples) and non-diseased controls (188 RNA and 202 DNA samples). Measurements and Main Results: Focusing on genetic variants within 10 IPF-associated genetic loci, we identified 27 eQTLs in controls and 24 eQTLs in cases (false-discovery-rate-adjusted P < 0.05). Among these signals, we identified associations of lead variants rs35705950 with expression of MUC5B and rs2076295 with expression of DSP in both cases and controls. mQTL analysis identified CpGs in gene bodies of MUC5B (cg17589883) and DSP (cg08964675) associated with the lead variants in these two loci. We also demonstrated strong co-localization of eQTL/mQTL and genetic signal in MUC5B (rs35705950) and DSP (rs2076295). Functional validation of the mQTL in MUC5B using luciferase reporter assays demonstrates that the CpG resides within a putative internal repressor element. Conclusions: We have established a relationship of the common IPF genetic risk variants rs35705950 and rs2076295 with respective changes in MUC5B and DSP expression and methylation. These results provide additional evidence that both MUC5B and DSP are involved in the etiology of IPF.
Assuntos
Fibrose Pulmonar Idiopática , Humanos , DNA , Metilação de DNA/genética , Expressão Gênica , Predisposição Genética para Doença/genética , Fibrose Pulmonar Idiopática/genética , Mucina-5B/genética , Locos de Características Quantitativas/genética , RNARESUMO
Resolution of inflammation is an active process that is tightly regulated to achieve repair and tissue homeostasis. In the absence of resolution, persistent inflammation underlies the pathogenesis of chronic lung disease such as chronic obstructive pulmonary disease (COPD) with recurrent exacerbations. Over the course of inflammation, macrophage programming transitions from pro-inflammatory to pro-resolving, which is in part regulated by the nuclear receptor Peroxisome Proliferator-Activated Receptor γ (PPARγ). Our previous work demonstrated an association between Fatty Acid Binding Protein 5 (FABP5) expression and PPARγ activity in peripheral blood mononuclear cells of healthy and COPD patients. However, a role for FABP5 in macrophage programming has not been examined. Here, using a combination of in vitro and in vivo approaches, we demonstrate that FABP5 is necessary for PPARγ activation. In turn, PPARγ acts directly to increase FABP5 expression in primary human alveolar macrophages. We further illustrate that lack of FABP5 expression promotes a pro-inflammatory macrophage programming with increased secretion of pro-inflammatory cytokines and increased chromatin accessibility for pro-inflammatory transcription factors (e.g., NF-κB and MAPK). And finally, real-time cell metabolic analysis using the Seahorse technology shows an inhibition of oxidative phosphorylation in FABP5-deficient macrophages. Taken together, our data indicate that FABP5 and PPARγ reciprocally regulate each other's expression and function, consistent with a novel positive feedback loop between the two factors that mediates macrophage pro-resolving programming. Our studies highlight the importance of defining targets and regulatory mechanisms that control the resolution of inflammation and may serve to inform novel interventional strategies directed towards COPD.
Assuntos
PPAR gama , Doença Pulmonar Obstrutiva Crônica , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamação/metabolismo , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo , PPAR gama/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismoRESUMO
While glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to reduce the expression of many inflammatory genes, repression is not an invariable outcome. Here, we explore synergy occurring between synthetic glucocorticoids (dexamethasone and budesonide) and proinflammatory cytokines (IL1B and TNF) on the expression of the toll-like receptor 2 (TLR2). This effect is observed in epithelial cell lines and both undifferentiated and differentiated primary human bronchial epithelial cells (pHBECs). In A549 cells, IL1B-plus-glucocorticoid-induced TLR2 expression required nuclear factor (NF)-κB and GR. Likewise, in A549 cells, BEAS-2B cells, and pHBECs, chromatin immunoprecipitation identified GR- and NF-κB/p65-binding regions â¼32 kb (R1) and â¼7.3 kb (R2) upstream of the TLR2 gene. Treatment of BEAS-2B cells with TNF or/and dexamethasone followed by global run-on sequencing confirmed transcriptional activity at these regions. Furthermore, cloning R1 or R2 into luciferase reporters revealed transcriptional activation by budesonide or IL1B, respectively, while R1+R2 juxtaposition enabled synergistic activation by IL1B and budesonide. In addition, small-molecule inhibitors and siRNA knockdown showed p38α MAPK to negatively regulate both IL1B-induced TLR2 expression and R1+R2 reporter activity. Finally, agonism of IL1B-plus-dexamethasone-induced TLR2 in A549 cells and pHBECs stimulated NF-κB- and interferon regulatory factor-dependent reporter activity and chemokine release. We conclude that glucocorticoid-plus-cytokine-driven synergy at TLR2 involves GR and NF-κB acting via specific enhancer regions, which combined with the inhibition of p38α MAPK promotes TLR2 expression. Subsequent inflammatory effects that occur following TLR2 agonism may be pertinent in severe neutrophilic asthma or chronic obstructive pulmonary disease, where glucocorticoid-based therapies are less efficacious.
Assuntos
Asma , NF-kappa B , Receptores de Glucocorticoides , Receptor 2 Toll-Like , Proteínas Quinases p38 Ativadas por Mitógeno , Asma/fisiopatologia , Budesonida/farmacologia , Citocinas/metabolismo , Dexametasona/farmacologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Humanos , Pulmão/citologia , Pulmão/metabolismo , NF-kappa B/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The heterogeneity of respirable particulates and compounds complicates our understanding of transcriptional responses to air pollution. Here, we address this by applying precision nuclear run-on sequencing and the assay for transposase-accessible chromatin sequencing to measure nascent transcription and chromatin accessibility in airway epithelial cells after wood smoke particle (WSP) exposure. We used transcription factor enrichment analysis to identify temporally distinct roles for ternary response factor-serum response factor complexes, the aryl hydrocarbon receptor (AHR), and NFκB in regulating transcriptional changes induced by WSP. Transcription of canonical targets of the AHR, such as CYP1A1 and AHRR, was robustly increased after just 30 min of WSP exposure, and we discovered novel AHR-regulated pathways and targets including the DNA methyltransferase, DNMT3L. Transcription of these genes and associated enhancers rapidly returned to near baseline by 120 min after exposure. The kinetics of AHR- and NFκB-regulated responses to WSP were distinguishable based on the timing of both transcriptional responses and chromatin remodeling, with induction of several cytokines implicated in maintaining NFκB-mediated responses through 120 min of exposure. In aggregate, our data establish a direct and primary role for AHR in mediating airway epithelial responses to WSP and identify crosstalk between AHR and NFκB signaling in controlling proinflammatory gene expression. This work also defines an integrated genomics-based strategy for deconvoluting multiplexed transcriptional responses to heterogeneous environmental exposures.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fumaça/efeitos adversos , Transcrição Gênica , Madeira , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Transformada , Montagem e Desmontagem da Cromatina , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , DNA (Citosina-5-)-Metiltransferases/biossíntese , DNA (Citosina-5-)-Metiltransferases/genética , Humanos , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Células NIH 3T3 , Receptores de Hidrocarboneto Arílico/genética , Proteínas Repressoras/genéticaRESUMO
Glucocorticoids are potent anti-inflammatory drugs that are used to treat an extraordinary range of human disease, including COVID-19, underscoring the ongoing importance of understanding their molecular mechanisms. Early studies of GR signaling led to broad acceptance of models in which glucocorticoid receptor (GR) monomers tether repressively to inflammatory transcription factors, thus abrogating inflammatory gene expression. However, newer data challenge this core concept and present an exciting opportunity to reframe our understanding of GR signaling. Here, we present an alternate, two-part model for transcriptional repression by glucocorticoids. First, widespread GR-mediated induction of transcription results in rapid, primary repression of inflammatory gene transcription and associated enhancers through competition-based mechanisms. Second, a subset of GR-induced genes, including targets that are regulated in coordination with inflammatory transcription factors such as NF-κB, exerts secondary repressive effects on inflammatory gene expression. Within this framework, emerging data indicate that the gene set regulated through the cooperative convergence of GR and NF-κB signaling is central to the broad clinical effectiveness of glucocorticoids in terminating inflammation and promoting tissue repair.
Assuntos
Anti-Inflamatórios/uso terapêutico , Tratamento Farmacológico da COVID-19 , Dexametasona/uso terapêutico , Glucocorticoides/uso terapêutico , NF-kappa B/genética , Receptores de Glucocorticoides/genética , Animais , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Regulação da Expressão Gênica , Genômica/métodos , Humanos , Inflamação/prevenção & controle , Modelos Genéticos , NF-kappa B/antagonistas & inibidores , NF-kappa B/imunologia , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/imunologia , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/imunologiaRESUMO
The G/T transversion rs35705950, located approximately 3 kb upstream of the MUC5B start site, is the cardinal risk factor for idiopathic pulmonary fibrosis (IPF). Here, we investigate the function and chromatin structure of this -3 kb region and provide evidence that it functions as a classically defined enhancer subject to epigenetic programming. We use nascent transcript analysis to show that RNA polymerase II loads within 10 bp of the G/T transversion site, definitively establishing enhancer function for the region. By integrating Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis of fresh and cultured human airway epithelial cells with nuclease sensitivity data, we demonstrate that this region is in accessible chromatin that affects the expression of MUC5B. Through applying paired single-nucleus RNA- and ATAC-seq to frozen tissue from IPF lungs, we extend these findings directly to disease, with results indicating that epigenetic programming of the -3 kb enhancer in IPF occurs in both MUC5B-expressing and nonexpressing lineages. In aggregate, our results indicate that the MUC5B-associated variant rs35705950 resides within an enhancer that is subject to epigenetic remodeling and contributes to pathologic misexpression in IPF.
Assuntos
Fibrose Pulmonar Idiopática/genética , Mucina-5B/genética , Células A549 , Sítios de Ligação/genética , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Epigênese Genética , Mutação com Ganho de Função , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Modelos Genéticos , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-ets/metabolismo , RNA Polimerase II/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcriptomes in different cell types. However, some genes, including Krüppel-like factor 9 (KLF9), a putative transcriptional repressor, demonstrate conserved responses. We show that glucocorticoids induce KLF9 expression in the human airways in vivo and in differentiated human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI). In A549 and BEAS-2B pulmonary epithelial cells, glucocorticoids induce KLF9 expression with similar kinetics to primary HBE cells in submersion culture. A549 and BEAS-2B ChIP-seq data reveal four common glucocorticoid-induced GR binding sites (GBSs). Two GBSs mapped to the 5'-proximal region relative to KLF9 transcription start site (TSS) and two occurred at distal sites. These were all confirmed in primary HBE cells. Global run-on (GRO) sequencing indicated robust enhancer RNA (eRNA) production from three of these GBSs in BEAS-2B cells. This was confirmed in A549 cells, plus submersion, and ALI culture of HBE cells. Cloning each GBS into luciferase reporters revealed glucocorticoid-induced activity requiring a glucocorticoid response element (GRE) within each distal GBS. While the proximal GBSs drove modest reporter induction by glucocorticoids, this region exhibited basal eRNA production, RNA polymerase II enrichment, and looping to the TSS, plausibly underlying constitutive KLF9 expression. Post glucocorticoid treatment, interactions between distal and proximal GBSs and the TSS correlated with KLF9 induction. CBP/P300 silencing reduced proximal GBS activity, but negligibly affected KLF9 expression. Overall, a model for glucocorticoid-mediated regulation of KLF9 involving multiple GBSs is depicted. This work unequivocally demonstrates that mechanistic insights gained from cell lines can translate to physiologically relevant systems.
Assuntos
Dexametasona/farmacologia , Genômica , Glucocorticoides/farmacologia , Fatores de Transcrição Kruppel-Like/biossíntese , Pulmão/efeitos dos fármacos , Células A549 , Elementos Facilitadores Genéticos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Ligação Proteica , RNA Mensageiro/genética , Receptores de Glucocorticoides/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
BACKGROUND: Ventricular arrhythmias (VA) are a common cause of sudden death after myocardial infarction (MI). Therefore, developing new therapeutic methods for the prevention and treatment of VA is of prime importance. METHODS: Human bone marrow derived CD271+ mesenchymal stem cells (MSC) were tested for their antiarrhythmic effect. This was done through the development of a novel mouse model using an immunocompromised Rag2-/- γc-/- mouse strain subjected to myocardial "infarction-reinfarction". The mice underwent a first ischemia-reperfusion through the left anterior descending (LAD) artery closure for 45 minutes with a subsequent second permanent LAD ligation after seven days from the first infarct. RESULTS: This mouse model induced various types of VA detected with continuous electrocardiogram (ECG) monitoring via implanted telemetry device. The immediate intramyocardial delivery of CD271+ MSC after the first MI significantly reduced VA induced after the second MI. CONCLUSIONS: In addition to the clinical relevance, more closely reflecting patients who suffer from severe ischemic heart disease and related arrhythmias, our new mouse model bearing reinfarction warrants the time required for stem cell engraftment and for the first time enables us to analyze and verify significant antiarrhythmic effects of human CD271+ stem cells in vivo.
Assuntos
Adapaleno/imunologia , Antiarrítmicos/uso terapêutico , Modelos Animais de Doenças , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Infarto do Miocárdio/terapia , Adapaleno/análise , Animais , Feminino , Humanos , Imunofenotipagem , Camundongos , Camundongos KnockoutRESUMO
The glucocorticoid receptor (NR3C1, also known as GR) binds to specific DNA sequences and directly induces transcription of anti-inflammatory genes that contribute to cytokine repression, frequently in cooperation with NF-kB. Whether inflammatory repression also occurs through local interactions between GR and inflammatory gene regulatory elements has been controversial. Here, using global run-on sequencing (GRO-seq) in human airway epithelial cells, we show that glucocorticoid signaling represses transcription within 10 min. Many repressed regulatory regions reside within "hyper-ChIPable" genomic regions that are subject to dynamic, yet nonspecific, interactions with some antibodies. When this artifact was accounted for, we determined that transcriptional repression does not require local GR occupancy. Instead, widespread transcriptional induction through canonical GR binding sites is associated with reciprocal repression of distal TNF-regulated enhancers through a chromatin-dependent process, as evidenced by chromatin accessibility and motif displacement analysis. Simultaneously, transcriptional induction of key anti-inflammatory effectors is decoupled from primary repression through cooperation between GR and NF-kB at a subset of regulatory regions. Thus, glucocorticoids exert bimodal restraints on inflammation characterized by rapid primary transcriptional repression without local GR occupancy and secondary anti-inflammatory effects resulting from transcriptional cooperation between GR and NF-kB.
Assuntos
Dexametasona/farmacologia , Inflamação/metabolismo , RNA Mensageiro/genética , Receptores de Glucocorticoides/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Cromatina/metabolismo , Dexametasona/metabolismo , Elementos Facilitadores Genéticos , Células HEK293 , Humanos , NF-kappa B/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Bone marrow (BM)-derived stem cells with their various functions and characteristics have become a well-recognized source for the cell-based therapies. However, knowledge on their therapeutic potential and the shortage for a cross-link between distinct BM-derived stem cells, primed after the onset of myocardial infarction (MI), seems to be still rudimentary. Therefore, the post-examination of the therapeutic characteristics of such primed hematopoietic CD133+ and mesenchymal CD271+ stem cells was the object of the present study. METHODS AND RESULTS: The effects of respective CD133+ and CD271+ mononuclear cells alone as well as in the co-culture model have been explored with focus on their angiogenic potential. The phenotypic analysis revealed a small percentage of isolated cells expressing both surface markers. Moreover, target stem cells isolated with our standardized immunomagnetic isolation procedure did not show any negative alterations following BM storage in regard to cell numbers and/or quality. In vitro network formation relied predominantly on CD271+ stem cells when compared with single CD133+ culture. Interestingly, CD133+ cells contributed in the tube formation, only if they were cultivated in combination with CD271+ cells. Additional to the in vitro examination, therapeutic effects of the primed stem cells were investigated 48 h post MI in a murine model. Hence, we have found a lower expression of transforming growth factor ßeta 3 (TGFß3) as well as an increase of the proangiogenic factors after CD133+ cell treatment in contrast to CD271+ cell treatment. On the other hand, the CD271+ cell therapy led to a lower expression of the inflammatory cytokines. CONCLUSION: The interactions between CD271+ and CD133+ subpopulations the extent to which the combination may enhance cardiac regeneration has still not been investigated so far. We expect that the multiple characteristics and various regenerative effects of CD271+ cells alone as well as in combination with CD133+ will result in an improved therapeutic impact on ischemic heart disease.
Assuntos
Antígeno AC133/metabolismo , Adapaleno/metabolismo , Diferenciação Celular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Infarto do Miocárdio/terapia , Neovascularização Fisiológica , Animais , Biomarcadores , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Imunofenotipagem , Transplante de Células-Tronco Mesenquimais/métodos , Camundongos , Camundongos Transgênicos , Infarto do Miocárdio/etiologia , RegeneraçãoRESUMO
Corticosteroids (CSs) are commonly used to manage wheezing and asthma in pediatric populations. Although corticosteroids are effective in alleviating airway diseases, some children with more moderate-severe asthma phenotypes show CS resistance and exhibit significant airflow obstruction, persistent inflammation, and more frequent exacerbations. Previous studies have demonstrated that Th1 cytokines, such as TNF-α and IFN-γ, promote CS resistance in adult human airway smooth muscle (ASM). In the present study, using a human fetal ASM cell model, we tested the hypothesis that TNF-α/IFN-γ induces CS resistance. In contrast to TNF-α or IFN-γ alone, the combination of TNF-α/IFN-γ blunted the ability of fluticasone propionate (FP) to reduce expression of the chemokines CCL5 and CXCL10 despite expression of key anti-inflammatory glucocorticoid receptor target genes being largely unaffected by TNF-α/IFN-γ. Expression of the NF-κB subunit p65 and phosphorylation of Stat1 were elevated in cells treated with TNF-α/IFN-γ, an effect that remained in the presence of FP. siRNA knockdown studies demonstrated the effects of TNF-α/IFN-γ on increased p65 are mediated by Stat1, a transcription factor activated by IFN-γ. Expression of TNFAIP3, a negative regulator of NF-κB activity, was not altered by TNF-α/IFN-γ. However, the effects of TNF-α/IFN-γ were partially reduced by overexpression of TNFAIP3 but did not influence p65 expression. Together, these data suggest that IFN-γ augments the effects of TNF-α on chemokines by enhancing expression of key inflammatory pathways in the presence of CS. Interactions between TNF-α- and IFN-γ-mediated pathways may promote inflammation in asthmatic children resistant to CSs.
Assuntos
Corticosteroides/farmacologia , Brônquios/imunologia , Resistência a Medicamentos/efeitos dos fármacos , Fluticasona/farmacologia , Interferon gama/imunologia , Miócitos de Músculo Liso/imunologia , Traqueia/imunologia , Fator de Necrose Tumoral alfa/imunologia , Brônquios/crescimento & desenvolvimento , Criança , Pré-Escolar , Resistência a Medicamentos/imunologia , Feminino , Humanos , Masculino , Células Th1/imunologia , Traqueia/crescimento & desenvolvimentoRESUMO
In asthma, the clinical efficacy of inhaled corticosteroids (ICSs) is enhanced by long-acting ß2-adrenoceptor agonists (LABAs). ICSs, or more accurately, glucocorticoids, promote therapeutically relevant changes in gene expression, and, in primary human bronchial epithelial cells (pHBECs) and airway smooth muscle cells, this genomic effect can be enhanced by a LABA. Modeling this interaction in human bronchial airway epithelial BEAS-2B cells transfected with a 2× glucocorticoid response element (2×GRE)-driven luciferase reporter showed glucocorticoid-induced transcription to be enhanced 2- to 3-fold by LABA. This glucocorticoid receptor (GR; NR3C1)-dependent effect occurred rapidly, was insensitive to protein synthesis inhibition, and was maximal when glucocorticoid and LABA were added concurrently. The ability of LABA to enhance GR-mediated transcription was not associated with changes in GR expression, serine (Ser203, Ser211, Ser226) phosphorylation, ligand affinity, or nuclear translocation. Chromatin immunoprecipitation demonstrated that glucocorticoid-induced recruitment of GR to the integrated 2×GRE reporter and multiple gene loci, whose mRNAs were unaffected or enhanced by LABA, was also unchanged by LABA. Transcriptomic analysis revealed glucocorticoid-induced mRNAs were variably enhanced, unaffected, or repressed by LABA. Thus, events leading to GR binding at target genes are not the primary explanation for how LABAs modulate GR-mediated transcription. As many glucocorticoid-induced genes are independently induced by LABA, gene-specific control by GR- and LABA-activated transcription factors may explain these observations. Because LABAs promote similar effects in pHBECs, therapeutic relevance is likely. These data illustrate the need to understand gene function(s), and the mechanisms leading to gene-specific induction, if existing ICS/LABA combination therapies are to be improved.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/metabolismo , Mucosa Respiratória/metabolismo , Transcrição Gênica/fisiologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Células Cultivadas , Preparações de Ação Retardada , Relação Dose-Resposta a Droga , Humanos , Receptores de Glucocorticoides/genética , Mucosa Respiratória/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Different subtypes of bone marrow-derived stem cells are characterized by varying functionality and activity after transplantation into the infarcted heart. Improvement of stem cell therapeutics requires deep knowledge about the mechanisms that mediate the benefits of stem cell treatment. Here, we demonstrated that co-transplantation of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) led to enhanced synergistic effects on cardiac remodeling. While HSCs were associated with blood vessel formation, MSCs were found to possess transdifferentiation capacity. This cardiomyogenic plasticity of MSCs was strongly promoted by a gap junction-dependent crosstalk between myocytes and stem cells. The inhibition of cell-cell coupling significantly reduced the expression of the cardiac specific transcription factors NKX2.5 and GATA4. Interestingly, we observed that small non-coding RNAs are exchanged between MSCs and cardiomyocytes in a GJ-dependent manner that might contribute to the transdifferentiation process of MSCs within a cardiac environment. Our results suggest that the predominant mechanism of HSCs contribution to cardiac regeneration is based on their ability to regulate angiogenesis. In contrast, transplanted MSCs have the capability for intercellular communication with surrounding cardiomyocytes, which triggers the intrinsic program of cardiogenic lineage specification of MSCs by providing cardiomyocyte-derived cues.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Infarto do Miocárdio/terapia , Transdução de Sinais , Animais , Comunicação Celular , Transdiferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Junções Comunicantes/metabolismo , Humanos , Camundongos SCID , Miócitos Cardíacos/fisiologia , Neovascularização FisiológicaRESUMO
PURPOSE OF REVIEW: The recent developments and clinical applications of natural orifice translumenal endoscopic surgery (NOTES)-procedures and technologies are going to be presented. RECENT FINDINGS: In experimental as well as clinical settings, NOTES-procedures are predominantly performed in hybrid technique. Current experimental studies focus on the implementation of new surgical approaches as well as on the training of procedures. One emphasis in the clinical application is transrectal and transanal interventions. Transanal total mesorectal excision is equivalent to laparoscopic procedures but with the benefit of an even less invasive access. Transvaginal cholecystectomy can achieve results that are comparable to surgeries that are performed with laparoscopic techniques alone. An analysis of the German NOTES-Register concerning appendectomies as well as the national performance of NOTES-interventions in Switzerland is presented. Apart from intraabdominal approaches, several centers proclaim transoral thyroidectomies and transoral mediastinoscopies. SUMMARY: NOTES-procedures are performed in animal experiments as well as in clinical setting although with less frequency. At this time, hybrid techniques using rigid instruments are mainly applied.
Assuntos
Colecistectomia/métodos , Laparoscopia , Cirurgia Endoscópica por Orifício Natural , Tireoidectomia/métodos , Colecistectomia/instrumentação , Colecistectomia/tendências , Humanos , Laparoscopia/tendências , Cirurgia Endoscópica por Orifício Natural/tendências , Duração da Cirurgia , Seleção de Pacientes , Tireoidectomia/instrumentação , Tireoidectomia/tendênciasRESUMO
Glucocorticoids exert important therapeutic effects on airway smooth muscle (ASM), yet few direct targets of glucocorticoid signaling in ASM have been definitively identified. Here, we show that the transcription factor, Krüppel-like factor 15 (KLF15), is directly induced by glucocorticoids in primary human ASM, and that KLF15 represses ASM hypertrophy. We integrated transcriptome data from KLF15 overexpression with genome-wide analysis of RNA polymerase (RNAP) II and glucocorticoid receptor (GR) occupancy to identify phospholipase C delta 1 as both a KLF15-regulated gene and a novel repressor of ASM hypertrophy. Our chromatin immunoprecipitation sequencing data also allowed us to establish numerous direct transcriptional targets of GR in ASM. Genes with inducible GR occupancy and putative antiinflammatory properties included IRS2, APPL2, RAMP1, and MFGE8. Surprisingly, we also observed GR occupancy in the absence of supplemental ligand, including robust GR binding peaks within the IL11 and LIF loci. Detection of antibody-GR complexes at these areas was abrogated by dexamethasone treatment in association with reduced RNA polymerase II occupancy, suggesting that noncanonical pathways contribute to cytokine repression by glucocorticoids in ASM. Through defining GR interactions with chromatin on a genome-wide basis in ASM, our data also provide an important resource for future studies of GR in this therapeutically relevant cell type.