RESUMO
Immune cells have been implicated in idiopathic pulmonary fibrosis (IPF), but the phenotypes and effector mechanisms of these cells remain incompletely characterized. We performed mass cytometry to quantify immune cell subsets in lungs of 12 patients with IPF and 15 organ donors without chronic lung disease and used existing single-cell RNA-sequencing data to investigate transcriptional profiles of immune cells overrepresented in IPF. Among myeloid cells, we found increased numbers of alveolar macrophages (AMØs) and dendritic cells (DCs) in IPF, as well as a subset of monocyte-derived DCs. In contrast, monocyte-like cells and interstitial macrophages were reduced in IPF. Transcriptomic profiling identified an enrichment for IFN-γ response pathways in AMØs and DCs from IPF, as well as antigen processing in DCs and phagocytosis in AMØs. Among T cells, we identified three subsets of memory T cells that were increased in IPF, including CD4+ and CD8+ resident memory T cells (TRM) and CD8+ effector memory cells. The response to the IFN-γ pathway was enriched in CD4 TRM and CD8 TRM cells in IPF, together with T cell activation and immune response-regulating signaling pathways. Increased AMØs, DCs, and memory T cells were present in IPF lungs compared with control subjects. In IPF, these cells possess an activation profile indicating increased IFN-γ signaling and upregulation of adaptive immunity in the lungs. Together, these studies highlight critical features of the immunopathogenesis of IPF.
Assuntos
Fibrose Pulmonar Idiopática , Análise de Célula Única , Perfilação da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologia , Macrófagos Alveolares/metabolismoRESUMO
Adaptation of prions to new species is thought to reflect the capacity of the host-encoded cellular form of the prion protein (PrPC) to selectively propagate optimized prion conformations from larger ensembles generated in the species of origin. Here we describe an alternate replicative process, termed nonadaptive prion amplification (NAPA), in which dominant conformers bypass this requirement during particular interspecies transmissions. To model susceptibility of horses to prions, we produced transgenic (Tg) mice expressing cognate PrPC Although disease transmission to only a subset of infected TgEq indicated a significant barrier to EqPrPC conversion, the resulting horse prions unexpectedly failed to cause disease upon further passage to TgEq. TgD expressing deer PrPC was similarly refractory to deer prions from diseased TgD infected with mink prions. In both cases, the resulting prions transmitted to mice expressing PrPC from the species of prion origin, demonstrating that transmission barrier eradication of the originating prions was ephemeral and adaptation superficial in TgEq and TgD. Horse prions produced in vitro by protein misfolding cyclic amplification of mouse prions using horse PrPC also failed to infect TgEq but retained tropism for wild-type mice. Concordant patterns of neuropathology and prion deposition in susceptible mice infected with NAPA prions and the corresponding prion of origin confirmed preservation of strain properties. The comparable responses of both prion types to guanidine hydrochloride denaturation indicated this occurs because NAPA precludes selection of novel prion conformations. Our findings provide insights into mechanisms regulating interspecies prion transmission and a framework to reconcile puzzling epidemiological features of certain prion disorders.
Assuntos
Especificidade de Hospedeiro/fisiologia , Proteínas PrPC/fisiologia , Doenças Priônicas/transmissão , Doenças Priônicas/veterinária , Príons/fisiologia , Animais , Cervos , Guanidina/farmacologia , Cavalos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas PrPC/química , Proteínas PrPC/genética , Príons/química , Conformação Proteica , Desnaturação Proteica , Coelhos , Ovinos , Especificidade da Espécie , Relação Estrutura-AtividadeRESUMO
Alveolar type II (AT2) epithelial cells are uniquely specialized to produce surfactant in the lung and act as progenitor cells in the process of repair after lung injury. AT2 cell injury has been implicated in several lung diseases, including idiopathic pulmonary fibrosis and bronchopulmonary dysplasia. The inability to maintain primary AT2 cells in culture has been a significant barrier in the investigation of pulmonary biology. We have addressed this knowledge gap by developing a three-dimensional (3D) organotypic coculture using primary human fetal AT2 cells and pulmonary fibroblasts. Grown on top of matrix-embedded fibroblasts, the primary human AT2 cells establish a monolayer and have direct contact with the underlying pulmonary fibroblasts. Unlike conventional two-dimensional (2D) culture, the structural and functional phenotype of the AT2 cells in our 3D organotypic culture was preserved over 7 days of culture, as evidenced by the presence of lamellar bodies and by production of surfactant proteins B and C. Importantly, the AT2 cells in 3D cocultures maintained the ability to replicate, with approximately 60% of AT2 cells staining positive for the proliferation marker Ki67, whereas no such proliferation is evident in 2D cultures of the same primary AT2 cells. This organotypic culture system enables interrogation of AT2 epithelial biology by providing a reductionist in vitro model in which to investigate the response of AT2 epithelial cells and AT2 cell-fibroblast interactions during lung injury and repair.
Assuntos
Comunicação Celular/fisiologia , Células Epiteliais/metabolismo , Lesão Pulmonar/patologia , Pulmão/patologia , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/metabolismo , Humanos , FenótipoRESUMO
The alveolar compartment, the fundamental gas exchange unit in the lung, is critical for tissue oxygenation and viability. We explored hepatocyte growth factor (HGF), a pleiotrophic cytokine that promotes epithelial proliferation, morphogenesis, migration, and resistance to apoptosis, as a candidate mediator of alveolar formation and regeneration. Mice deficient in the expression of the HGF receptor Met in lung epithelial cells demonstrated impaired airspace formation marked by a reduction in alveolar epithelial cell abundance and survival, truncation of the pulmonary vascular bed, and enhanced oxidative stress. Administration of recombinant HGF to tight-skin mice, an established genetic emphysema model, attenuated airspace enlargement and reduced oxidative stress. Repair in the TSK/+ mouse was punctuated by enhanced akt and stat3 activation. HGF treatment of an alveolar epithelial cell line not only induced proliferation and scattering of the cells but also conferred protection against staurosporine-induced apoptosis, properties critical for alveolar septation. HGF promoted cell survival was attenuated by akt inhibition. Primary alveolar epithelial cells treated with HGF showed improved survival and enhanced antioxidant production. In conclusion, using both loss-of-function and gain-of-function maneuvers, we show that HGF signaling is necessary for alveolar homeostasis in the developing lung and that augmentation of HGF signaling can improve airspace morphology in murine emphysema. Our studies converge on prosurvival signaling and antioxidant protection as critical pathways in HGF-mediated airspace maintenance or repair. These findings support the exploration of HGF signaling enhancement for diseases of the airspace.
Assuntos
Fator de Crescimento de Hepatócito , Homeostase , Proteínas Proto-Oncogênicas c-met , Alvéolos Pulmonares , Animais , Movimento Celular/genética , Proliferação de Células , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fator de Crescimento de Hepatócito/administração & dosagem , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Camundongos , Morfogênese/genética , Morfogênese/fisiologia , Proteínas Proto-Oncogênicas c-met/deficiência , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/fisiologia , Enfisema Pulmonar/genética , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/fisiopatologia , Transdução de Sinais , Sobrevivência de Tecidos/genéticaRESUMO
Bronchopulmonary dysplasia (BPD), a common chronic respiratory disease that occurs after premature birth, is believed to be secondary to oxidative damage from hyperoxia and inflammation, which leads to impaired alveolar formation and chronic lung dysfunction. We hypothesized that extracellular superoxide dismutase (SOD)3, an antioxidant uniquely targeted to the extracellular matrix (ECM) and alveolar fluid, might have a different response (down-regulation) to hyperoxic injury and recovery in room air (RA), thereby contributing to the persistent airspace injury and inflammation. We used a murine BPD model using postnatal hyperoxia (O2) (4 or 5 d) followed by short-term recovery (14 d) in RA, which mimics the durable effects after injury during alveolar development. This was associated with significantly increased mRNA expression for antioxidant genes mediated by nuclear factor erythroid 2-related factor (Nrf2) in the O2 (n = 4) versus RA group (n = 5). SOD3, an Nrf2-independent antioxidant, was significantly reduced in the O2-exposed mice compared with RA. Immunohistochemistry revealed decreased and disrupted SOD3 deposition in the alveolar ECM of O2-exposed mice. Furthermore, this distinct hyperoxic antioxidant and injury profile was reproducible in murine lung epithelial 12 cells exposed to O2. Overexpression of SOD3 rescued the injury measures in the O2-exposed cells. We establish that reduced SOD3 expression correlates with alveolar injury measures in the recovered neonatal hyperoxic lung, and SOD3 overexpression attenuates hyperoxic injury in an alveolar epithelial cell line. Such findings suggest a candidate mechanism for the pathogenesis of BPD that may lead to targeted interventions.
Assuntos
Displasia Broncopulmonar/patologia , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Lesão Pulmonar/metabolismo , Pulmão/patologia , Superóxido Dismutase/metabolismo , Animais , Animais Recém-Nascidos , Antioxidantes/química , Displasia Broncopulmonar/enzimologia , Feminino , Hiperóxia , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo , Oxigênio/química , Mucosa Respiratória/metabolismoRESUMO
Common genetic variants confer substantial risk for chronic lung diseases, including pulmonary fibrosis. Defining the genetic control of gene expression in a cell-type-specific and context-dependent manner is critical for understanding the mechanisms through which genetic variation influences complex traits and disease pathobiology. To this end, we performed single-cell RNA sequencing of lung tissue from 66 individuals with pulmonary fibrosis and 48 unaffected donors. Using a pseudobulk approach, we mapped expression quantitative trait loci (eQTLs) across 38 cell types, observing both shared and cell-type-specific regulatory effects. Furthermore, we identified disease interaction eQTLs and demonstrated that this class of associations is more likely to be cell-type-specific and linked to cellular dysregulation in pulmonary fibrosis. Finally, we connected lung disease risk variants to their regulatory targets in disease-relevant cell types. These results indicate that cellular context determines the impact of genetic variation on gene expression and implicates context-specific eQTLs as key regulators of lung homeostasis and disease.
Assuntos
Fibrose Pulmonar , Locos de Características Quantitativas , Humanos , Locos de Características Quantitativas/genética , Fibrose Pulmonar/genética , Regulação da Expressão Gênica/genética , Pulmão , Herança Multifatorial , Estudo de Associação Genômica Ampla/métodos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Whereas prion replication involves structural rearrangement of cellular prion protein (PrP(C)), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27-30), a protease-resistant counterpart of the pathogenic scrapie form (PrP(Sc)) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126-218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27-30) both renature to a common structure that reconstitutes the globular domain.
Assuntos
Epitopos/genética , Proteínas PrPC/genética , Proteínas PrPSc/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais Murinos/biossíntese , Anticorpos Monoclonais Murinos/isolamento & purificação , Bovinos , Sequência Conservada , Cervos , Evolução Molecular Direcionada , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Humanos , Hibridomas , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Proteínas PrPC/química , Proteínas PrPC/imunologia , Proteínas PrPSc/química , Proteínas PrPSc/imunologia , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Saimiri , Deleção de Sequência , OvinosRESUMO
A hallmark of idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases is dysregulated repair of the alveolar epithelium. The Hippo pathway effector transcription factors YAP and TAZ are implicated as essential for type 1 and type 2 alveolar epithelial cell (AT1 and AT2) differentiation in the developing lung, yet aberrant activation of YAP/TAZ is a prominent feature of the dysregulated alveolar epithelium in IPF. In these studies, we sought to define the functional role of YAP/TAZ activity during alveolar regeneration. We demonstrated that Yap and Taz were normally activated in AT2 cells shortly after injury, and deletion of Yap/Taz in AT2 cells led to pathologic alveolar remodeling, failure of AT2-to-AT1 cell differentiation, increased collagen deposition, exaggerated neutrophilic inflammation, and increased mortality following injury induced by a single dose of bleomycin. Loss of Yap/Taz activity prior to an LPS injury prevented AT1 cell regeneration, led to intraalveolar collagen deposition, and resulted in persistent innate inflammation. These findings establish that AT2 cell Yap/Taz activity is essential for functional alveolar epithelial repair and prevention of fibrotic remodeling.
Assuntos
Lesão Pulmonar Aguda , Fibrose Pulmonar Idiopática , Proteínas de Sinalização YAP , Humanos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Colágeno/metabolismo , Fibrose Pulmonar Idiopática/patologia , Inflamação , Regeneração , Transdução de Sinais , Proteínas de Sinalização YAP/metabolismoRESUMO
Common genetic variants confer substantial risk for chronic lung diseases, including pulmonary fibrosis (PF). Defining the genetic control of gene expression in a cell-type-specific and context-dependent manner is critical for understanding the mechanisms through which genetic variation influences complex traits and disease pathobiology. To this end, we performed single-cell RNA-sequencing of lung tissue from 67 PF and 49 unaffected donors. Employing a pseudo-bulk approach, we mapped expression quantitative trait loci (eQTL) across 38 cell types, observing both shared and cell type-specific regulatory effects. Further, we identified disease-interaction eQTL and demonstrated that this class of associations is more likely to be cell-type specific and linked to cellular dysregulation in PF. Finally, we connected PF risk variants to their regulatory targets in disease-relevant cell types. These results indicate that cellular context determines the impact of genetic variation on gene expression, and implicates context-specific eQTL as key regulators of lung homeostasis and disease.
RESUMO
The human lung is structurally complex, with a diversity of specialized epithelial, stromal and immune cells playing specific functional roles in anatomically distinct locations, and large-scale changes in the structure and cellular makeup of this distal lung is a hallmark of pulmonary fibrosis (PF) and other progressive chronic lung diseases. Single-cell transcriptomic studies have revealed numerous disease-emergent/enriched cell types/states in PF lungs, but the spatial contexts wherein these cells contribute to disease pathogenesis has remained uncertain. Using sub-cellular resolution image-based spatial transcriptomics, we analyzed the gene expression of more than 1 million cells from 19 unique lungs. Through complementary cell-based and innovative cell-agnostic analyses, we characterized the localization of PF-emergent cell-types, established the cellular and molecular basis of classical PF histopathologic disease features, and identified a diversity of distinct molecularly-defined spatial niches in control and PF lungs. Using machine-learning and trajectory analysis methods to segment and rank airspaces on a gradient from normal to most severely remodeled, we identified a sequence of compositional and molecular changes that associate with progressive distal lung pathology, beginning with alveolar epithelial dysregulation and culminating with changes in macrophage polarization. Together, these results provide a unique, spatially-resolved characterization of the cellular and molecular programs of PF and control lungs, provide new insights into the heterogeneous pathobiology of PF, and establish analytical approaches which should be broadly applicable to other imaging-based spatial transcriptomic studies.
RESUMO
Alveolar enlargement, which is characteristic of bronchopulmonary dysplasia, congenital matrix disorders, and cigarette smoke-induced emphysema, is thought to result from enhanced inflammation and ensuing excessive matrix proteolysis. Although there is recent evidence that cell death and oxidative stress punctuate these diseases, the mechanistic link between abnormal lung extracellular matrix and alveolar enlargement is lacking. We hypothesized that the tight-skin (TSK) mouse, which harbors a spontaneous internal duplication in the microfibrillar glycoprotein fibrillin-1, might show whether matrix alterations are sufficient to promote oxidative stress and cell death, injury cascades central to the development of clinical emphysema. We observed no evidence of increased metalloprotease activation by histochemical and zymographic methods. We did find initial oxidative stress followed by increased apoptosis in the postnatal TSK lung. Both blunted antioxidant production and reduced extracellular superoxide dismutase activity were evident in the neonatal lung. High-dose antioxidant treatment with N-acetylcysteine improved airspace caliber and attenuated oxidative stress and apoptosis in neonatal and adult TSK mice. These data establish that an abnormal extracellular matrix without overt elastolysis is sufficient to confer susceptibility to postnatal normoxia, reminiscent of bronchopulmonary dysplasia. The resultant oxidative stress and apoptosis culminate in profound airspace enlargement. The TSK lung exemplifies the critical interplay between extracellular matrix, oxidative stress, and cell-death cascades that may contribute to genetic and acquired airspace enlargement.
Assuntos
Apoptose/fisiologia , Matriz Extracelular/patologia , Estresse Oxidativo/fisiologia , Enfisema Pulmonar/patologia , Enfisema Pulmonar/fisiopatologia , Animais , Antioxidantes/metabolismo , Western Blotting , Matriz Extracelular/metabolismo , Fibrilina-1 , Fibrilinas , Perfilação da Expressão Gênica , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Mutantes , Proteínas dos Microfilamentos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Enfisema Pulmonar/genéticaRESUMO
OBJECTIVE(S): Neurologic and neurocognitive dysfunction after cardiopulmonary bypass (CPB) have been shown in both clinical and experimental settings. Although short-term outcome has been evaluated in rats, the assessment of neurocognitive dysfunction with long-term follow-up has not been reported in experimental CPB models. The objective of this study was to evaluate the effects of CPB on long-term neurocognitive function in the rat. DESIGN: Prospective, interventional study. SETTING: A university research laboratory. PARTICIPANTS: Male Sprague-Dawley rats. INTERVENTIONS: Sprague-Dawley rats were randomized to either CPB (n = 19) or sham-operated groups (n = 17). On days 3, 7, and 14 and at 6 weeks after surgery, the rats were submitted to standardized neurologic testing (Neuroscore). In addition, the animals underwent cognitive testing in the Morris water maze (MWM), including basic, probe, and reversal trial protocols during the first 19 postoperative days (short-term cognitive outcome) and then repeated 6 weeks after surgery (long-term cognitive outcome). MEASUREMENTS AND MAIN RESULTS: The CPB group had worse Neuroscores (day 3, 5[2]; day 7, 7[2]; day 14, 5[1]; 6 weeks, 5[1]) compared with the sham group (day 3, 7[2]; day 7, 7[1]; day 14, 7[1]; 6 weeks, 7[1]) at all time points tested (p < 0.05). In the MWM, the CPB group showed both short-term and persistent long-term neurocognitive dysfunction. CONCLUSIONS: Compared with sham-operated controls, rats undergoing CPB showed worse neurologic and neurocognitive outcome early after surgery. Importantly, long-term deficits also persisted at 6 weeks after surgery.
Assuntos
Ponte Cardiopulmonar/efeitos adversos , Transtornos Cognitivos/diagnóstico , Complicações Pós-Operatórias/diagnóstico , Animais , Ponte Cardiopulmonar/psicologia , Transtornos Cognitivos/fisiopatologia , Transtornos Cognitivos/psicologia , Masculino , Aprendizagem em Labirinto/fisiologia , Complicações Pós-Operatórias/fisiopatologia , Complicações Pós-Operatórias/psicologia , Estudos Prospectivos , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Pulmonary fibrosis (PF) is a form of chronic lung disease characterized by pathologic epithelial remodeling and accumulation of extracellular matrix (ECM). To comprehensively define the cell types, mechanisms, and mediators driving fibrotic remodeling in lungs with PF, we performed single-cell RNA sequencing of single-cell suspensions from 10 nonfibrotic control and 20 PF lungs. Analysis of 114,396 cells identified 31 distinct cell subsets/states. We report that a remarkable shift in epithelial cell phenotypes occurs in the peripheral lung in PF and identify several previously unrecognized epithelial cell phenotypes, including a KRT5- /KRT17 + pathologic, ECM-producing epithelial cell population that was highly enriched in PF lungs. Multiple fibroblast subtypes were observed to contribute to ECM expansion in a spatially discrete manner. Together, these data provide high-resolution insights into the complexity and plasticity of the distal lung epithelium in human disease and indicate a diversity of epithelial and mesenchymal cells contribute to pathologic lung fibrosis.
Assuntos
Fibrose Pulmonar , Matriz Extracelular/metabolismo , Fibrose , Humanos , Pulmão/metabolismo , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Análise de Sequência de RNARESUMO
ER stress in type II alveolar epithelial cells (AECs) is common in idiopathic pulmonary fibrosis (IPF), but the contribution of ER stress to lung fibrosis is poorly understood. We found that mice deficient in C/EBP homologous protein (CHOP), an ER stress-regulated transcription factor, were protected from lung fibrosis and AEC apoptosis in 3 separate models where substantial ER stress was identified. In mice treated with repetitive intratracheal bleomycin, we identified localized hypoxia in type II AECs as a potential mechanism explaining ER stress. To test the role of hypoxia in lung fibrosis, we treated mice with bleomycin, followed by exposure to 14% O2, which exacerbated ER stress and lung fibrosis. Under these experimental conditions, CHOP-/- mice, but not mice with epithelial HIF (HIF1/HIF2) deletion, were protected from AEC apoptosis and fibrosis. In vitro studies revealed that CHOP regulates hypoxia-induced apoptosis in AECs via the inositol-requiring enzyme 1α (IRE1α) and the PKR-like ER kinase (PERK) pathways. In human IPF lungs, CHOP and hypoxia markers were both upregulated in type II AECs, supporting a conclusion that localized hypoxia results in ER stress-induced CHOP expression, thereby augmenting type II AEC apoptosis and potentiating lung fibrosis.
Assuntos
Estresse do Retículo Endoplasmático , Fibrose Pulmonar Idiopática/patologia , Alvéolos Pulmonares/patologia , Fator de Transcrição CHOP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Bleomicina/toxicidade , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Endorribonucleases/metabolismo , Feminino , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Masculino , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/metabolismo , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Fator de Transcrição CHOP/genética , eIF-2 Quinase/metabolismoRESUMO
Reactive oxygen species play a role in the response of brain to ischemia. The effects of metalloporphyrin catalytic antioxidants (AEOL 10113 and AEOL 10150) were examined after murine middle cerebral artery occlusion (MCAO). Ninety minutes after reperfusion from 90 min MCAO in the rat, AEOL 10113, AEOL 10150, or vehicle were given intracerebroventricularly. AEOL 10113 and AEOL 10150 similarly reduced infarct size (35%) and neurologic deficit. AEOL 10113 caused behavioral side effects at twice the neuroprotective dose while AEOL 10150 required a 15-fold increase from the neuroprotective dose to cause behavioral changes. AEOL 10150, given 6 h after 90 min MCAO, reduced total infarct size by 43% without temperature effects. Brain AEOL 10150 elimination t(1/2) was 10 h. In the mouse, intravenous AEOL 10150 infusion post-MCAO reduced both infarct size (25%) and neurologic deficit. Brain AEOL 10150 uptake, greater in the ischemic hemisphere, was dose- and time-dependent. AEOL 10150 had direct effects on proteomic events and ameliorated changes caused by ischemia. In primary mixed neuronal/glial cultures exposed to 2 h of O(2)/glucose deprivation, AEOL 10150 reduced lactate dehydrogenase release dose-dependently and selectively preserved aconitase activity in concentrations consistent with neuroprotection in vivo. AEOL 10150 is an effective neuroprotective compound offering a wide therapeutic window with a large margin of safety against adverse behavioral side effects.
Assuntos
Antioxidantes/farmacologia , Isquemia Encefálica/tratamento farmacológico , Encéfalo/patologia , Hemodinâmica/efeitos dos fármacos , Metaloporfirinas/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Isquemia Encefálica/fisiopatologia , Isquemia Encefálica/prevenção & controle , Artéria Carótida Interna , Infarto Cerebral/patologia , Infarto Cerebral/prevenção & controle , Modelos Animais de Doenças , Injeções Intraventriculares , Manganês/farmacologia , Metaloporfirinas/administração & dosagem , RatosRESUMO
Chronic obstructive pulmonary disease (COPD) is a prevalent smoking-related disease for which no disease-altering therapies currently exist. As dysregulated TGF-ß signaling associates with lung pathology in patients with COPD and in animal models of lung injury induced by chronic exposure to cigarette smoke (CS), we postulated that inhibiting TGF-ß signaling would protect against CS-induced lung injury. We first confirmed that TGF-ß signaling was induced in the lungs of mice chronically exposed to CS as well as in COPD patient samples. Importantly, key pathological features of smoking-associated lung disease in patients, e.g., alveolar injury with overt emphysema and airway epithelial hyperplasia with fibrosis, accompanied CS-induced alveolar cell apoptosis caused by enhanced TGF-ß signaling in CS-exposed mice. Systemic administration of a TGF-ß-specific neutralizing antibody normalized TGF-ß signaling and alveolar cell death, conferring improved lung architecture and lung mechanics in CS-exposed mice. Use of losartan, an angiotensin receptor type 1 blocker used widely in the clinic and known to antagonize TGF-ß signaling, also improved oxidative stress, inflammation, metalloprotease activation and elastin remodeling. These data support our hypothesis that inhibition of TGF-ß signaling through angiotensin receptor blockade can attenuate CS-induced lung injury in an established murine model. More importantly, our findings provide a preclinical platform for the development of other TGF-ß-targeted therapies for patients with COPD.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Doença Pulmonar Obstrutiva Crônica/prevenção & controle , Fumar/efeitos adversos , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Losartan/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos AKR , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Receptor Tipo 1 de Angiotensina/metabolismo , Mecânica Respiratória/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Respiratory dysfunction is a major contributor to morbidity and mortality in aged populations. The susceptibility to pulmonary insults is attributed to "low pulmonary reserve", ostensibly reflecting a combination of age-related musculoskeletal, immunologic and intrinsic pulmonary dysfunction. METHODS/PRINCIPAL FINDINGS: Using a murine model of the aging lung, senescent DBA/2 mice, we correlated a longitudinal survey of airspace size and injury measures with a transcriptome from the aging lung at 2, 4, 8, 12, 16 and 20 months of age. Morphometric analysis demonstrated a nonlinear pattern of airspace caliber enlargement with a critical transition occurring between 8 and 12 months of age marked by an initial increase in oxidative stress, cell death and elastase activation which is soon followed by inflammatory cell infiltration, immune complex deposition and the onset of airspace enlargement. The temporally correlative transcriptome showed exuberant induction of immunoglobulin genes coincident with airspace enlargement. Immunohistochemistry, ELISA analysis and flow cytometry demonstrated increased immunoglobulin deposition in the lung associated with a contemporaneous increase in activated B-cells expressing high levels of TLR4 (toll receptor 4) and CD86 and macrophages during midlife. These midlife changes culminate in progressive airspace enlargement during late life stages. CONCLUSION/SIGNIFICANCE: Our findings establish that a tissue-specific aging program is evident during a presenescent interval which involves early oxidative stress, cell death and elastase activation, followed by B lymphocyte and macrophage expansion/activation. This sequence heralds the progression to overt airspace enlargement in the aged lung. These signature events, during middle age, indicate that early stages of the aging immune system may have important correlates in the maintenance of tissue morphology. We further show that time-course analyses of aging models, when informed by structural surveys, can reveal nonintuitive signatures of organ-specific aging pathology.
Assuntos
Envelhecimento/patologia , Homeostase , Pulmão/patologia , Pulmão/fisiopatologia , Animais , Linfócitos B/imunologia , Morte Celular , Imunoglobulinas/metabolismo , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Monócitos/imunologia , Estresse Oxidativo , FenótipoRESUMO
Despite the importance of airspace integrity in vertebrate gas exchange, the molecular pathways that instruct distal lung formation are poorly understood. Recently, we found that fibrillin-1 deficiency in mice impairs alveolar formation and recapitulates the pulmonary features of human Marfan syndrome. To further elucidate effectors involved in distal lung formation, we performed expression profiling analysis comparing the fibrillin-1-deficient and wild-type developing lung. NeuroD, a basic helix-loop-helix transcription factor, fulfilled the expression criteria for a candidate mediator of distal lung development. We investigated its role in murine lung development using genetically targeted NeuroD-deficient mice. We found that NeuroD deficiency results in both impaired alveolar septation and altered morphology of the pulmonary neuroendocrine cells. NeuroD-deficient mice had enlarged alveoli associated with reduced epithelial proliferation in the airway and airspace compartments during development. Additionally, the neuroendocrine compartment in these mice manifested an increased number of neuroepithelial bodies but a reduced number of solitary pulmonary neuroendocrine cells in the neonatal lung. Overexpression of NeuroD in a murine lung epithelial cell line conferred a neuroendocrine phenotype characterized by the induction of neuroendocrine markers as well as increased proliferation. These results support an unanticipated role for NeuroD in the regulation of pulmonary neuroendocrine and alveolar morphogenesis and suggest an intimate connection between the neuroendocrine compartment and distal lung development.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica , Pulmão/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Células Epiteliais/citologia , Fibrilina-1 , Fibrilinas , Perfilação da Expressão Gênica , Heterozigoto , Humanos , Camundongos , Proteínas dos Microfilamentos/metabolismo , Modelos Biológicos , Fenótipo , TransfecçãoRESUMO
Aortic aneurysm and dissection are manifestations of Marfan syndrome (MFS), a disorder caused by mutations in the gene that encodes fibrillin-1. Selected manifestations of MFS reflect excessive signaling by the transforming growth factor-beta (TGF-beta) family of cytokines. We show that aortic aneurysm in a mouse model of MFS is associated with increased TGF-beta signaling and can be prevented by TGF-beta antagonists such as TGF-beta-neutralizing antibody or the angiotensin II type 1 receptor (AT1) blocker, losartan. AT1 antagonism also partially reversed noncardiovascular manifestations of MFS, including impaired alveolar septation. These data suggest that losartan, a drug already in clinical use for hypertension, merits investigation as a therapeutic strategy for patients with MFS and has the potential to prevent the major life-threatening manifestation of this disorder.