RESUMO
The extracellular matrix (ECM) of the pulmonary parenchyma must maintain the structural relationships among resident cells during the constant distortion imposed by respiration. This dictates that both the ECM and cells adapt to changes in shape, while retaining their attachment. Membrane-associated integrins and discoidin domain receptors (DDR) bind collagen and transmit signals to the cellular cytoskeleton. Although the contributions of DDR2 to collagen deposition and remodeling during osseous development are evident, it is unclear how DDR2 contributes to lung development. Using mice (smallie, Slie/Slie, DDR2Δ) bearing a spontaneous inactivating deletion within the DDR2 coding region, we observed a decrease in gas-exchange surface area and enlargement of alveolar ducts. Compared with fibroblasts isolated from littermate controls, DDR2Δ fibroblasts, spread more slowly, developed fewer lamellipodia, and were less responsive to the rigidity of neighboring collagen fibers. Activated ß1-integrin (CD29) was reduced in focal adhesions (FA) of DDR2Δ fibroblasts, less phospho-zyxin localized to and fewer FA developed over ventral actin stress fibers, and the adhesions had a lower aspect ratio compared with controls. However, DDR2 deletion did not reduce cellular displacement of the ECM. Our findings indicate that DDR2, in concert with collagen-binding ß1-integrins, regulates the timing and location of focal adhesion formation and how lung fibroblasts respond to ECM rigidity. Reduced rigidity sensing and mechano-responsiveness may contribute to the distortion of alveolar ducts, where the fiber cable-network is enriched and tensile forces are concentrated. Strategies targeting DDR2 could help guide fibroblasts to locations where tensile forces organize parenchymal repair.
Assuntos
Receptor com Domínio Discoidina 2 , Camundongos , Animais , Receptor com Domínio Discoidina 2/metabolismo , Integrinas/metabolismo , Adesões Focais/metabolismo , Receptores com Domínio Discoidina , Colágeno/metabolismo , Pulmão/metabolismoRESUMO
Platelet-derived growth factor receptor-α (PDGFRα) is absolutely required for the development of secondary pulmonary alveolar septa. Our earlier observations indicated that PDGFRα resides intracellularly as well as on the plasma membrane of murine lung fibroblasts (LF). We have examined how neuropilin-1 (Nrp1), a surface receptor without kinase activity, regulates the intracellular trafficking of PDGFRα in LF obtained from mice, some bearing a targeted deletion of Nrp1 in myofibroblasts. Using the proximity ligation assay, we observed that PDGFRα and Nrp1 colocalized in both early antigen-1 (EEA1) containing sorting endosomes and with adaptor protein containing a pleckstrin homology domain and a phosphotyrosine-binding domain-1 (APPL1) in very early endosomes (VEE). These findings were confirmed using live-cell imaging, which demonstrated that recently internalized PDGFRα was observed in Rab5-containing vesicles residing within 100 nm of the plasma membrane. Nrp1 deletion reduced the phosphorylation of Akt (protein kinase B), the major downstream target of PDGFRα, and limited accumulation of inositol-3 phosphates in APPL1-containing endosomes after exposure to PDGFA. PDGFRα co-immunoprecipitated with APPL1, indicating that PDGFRα enters VEE. Targeted deletion of Nrp1 or APPL1-depletion in control LF reduced the activity of an Akt1 biosensor following stimulation with PDGFA. Our findings demonstrate that Nrp1 enhances the entry of PDGFRα into APPL1 containing VEE and that APPL1 enhances PDGFRα signaling. Therefore, Nrp1 promotes endosomal signaling by PDGFRα offering a potential mechanism to explain our prior observation that Nrp1 supports the formation of alveolar ducts and alveoli during secondary septation in mice.
Assuntos
Endossomos/metabolismo , Fibroblastos/metabolismo , Pulmão/metabolismo , Neuropilina-1/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Endossomos/genética , Fibroblastos/citologia , Pulmão/citologia , Camundongos , Camundongos Knockout , Neuropilina-1/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genéticaRESUMO
Glucocorticoids have been widely used and exert pleiotropic effects on alveolar structure and function, but do not improve the long-term clinical outcomes for patients with bronchopulmonary dysplasia, emphysema, or interstitial lung diseases. Treatments that foster alveolar regeneration could substantially improve the long-term outcomes for such patients. One approach to alveolar regeneration is to stimulate and guide intrinsic alveolar progenitors along developmental pathways used during secondary septation. Other investigators and we have identified platelet-derived growth factor receptor-α-expressing fibroblast subpopulations that are alternatively skewed toward myofibroblast or lipofibroblast phenotypes. In this study, we administered either the glucocorticoid receptor agonist dexamethasone (Dex) or the antagonist mifepristone to mice during the first postnatal week and evaluated their effects on cellular proliferation and adoption of α-smooth muscle actin and lipid droplets (markers of the myofibroblast and lipofibroblast phenotypes, respectively). We observed that Dex increased the relative abundance of fibroblasts with progenitor characteristics, i.e., containing both α-smooth muscle actin and lipid droplets, uncoupling protein-1 (a marker of brown and beige adipocytes), delta-like ligand-1, and stem cell antigen-1. Dex enhanced signaling through the Smad1/5 pathway, which increased uncoupling protein-1 in a lung fibroblast progenitor cell line. We conclude that glucocorticoid receptor manipulation can sustain fibroblast plasticity, and posit that targeting downstream glucocorticoid responsive pathways could steer fibroblast progenitors along more desirable regenerative pathways.
Assuntos
Fibroblastos/citologia , Glucocorticoides/farmacologia , Alvéolos Pulmonares/crescimento & desenvolvimento , Células-Tronco/citologia , Adipócitos Marrons/citologia , Adipócitos Marrons/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Dexametasona/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Mifepristona/farmacologia , Fosforilação/efeitos dos fármacos , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Proteína Desacopladora 1/metabolismoRESUMO
Alveolar development in humans primarily occurs postnatally and requires a carefully orchestrated expansion of distal epithelial and mesenchymal progenitor populations and coordinated differentiation, to create a highly segmented gas-exchange surface. The regulation of alveolarization normally assimilates cues from paracrine cell-cell, cell-extracellular matrix, and mechanical interactions which are superimposed on cells and the extracellular matrix through phasic respiratory movement. In bronchopulmonary dysplasia, the entire process is precociously initiated when cellular and extracellular components are adapted to the saccular stage where movement and circulation are much more limited. This review focuses on mesenchymal cells (fibroblasts, endothelial cells, and pericytes), and epithelial cells are primarily discussed as sources of growth factor ligands or recipients of ligands produced by mesenchymal cells. Some interstitial fibroblasts differentiate to contractile myofibroblasts, containing a smooth muscle-actin rich cytoskeleton, which connects with tensile and elastic elements in the extracellular matrix, and together comprise a load-bearing network that diffuses mechanical forces during respiration. Other interstitial fibroblasts assimilate neutral lipid droplets, which regulate the differentiation of distal epithelial progenitors and surfactant production by alveolar type 2 cells. Pericytes organize and reinforce the capillary network as it expands to match the coverage of type 1 epithelial cells. Hyperoxia and the mechanical load imposed by positive pressure mechanical ventilation disrupt these paracrine interactions, leaving thickened alveolar walls, airways and arterioles, thereby diminishing gas-exchange surface area. Better understanding of these mechanisms of alveolar septation will lead to more effective treatments to preserve and perhaps augment the surface usual sequence of events that drive alveolarization.
Assuntos
Diferenciação Celular/fisiologia , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Comunicação Parácrina/fisiologia , Alvéolos Pulmonares/irrigação sanguínea , Alvéolos Pulmonares/crescimento & desenvolvimento , Alvéolos Pulmonares/metabolismo , Capilares/metabolismo , Humanos , Hiperóxia/metabolismo , Células-Tronco Mesenquimais/citologia , Alvéolos Pulmonares/citologiaRESUMO
BACKGROUND: Secondary pulmonary alveolar septal formation requires platelet derived growth factor (PDGF-A) and platelet derived growth factor receptor-alpha (PDGFRα), and their regulation influences alveolar septal areal density and thickness. Insufficient PDGFRα expression in lung fibroblasts (LF) results in failed septation. METHODS: Mice in which the endogenous PDGFRα-gene regulates expression of the green fluorescent protein were used to temporally and spatially track PDGFRα-signaling. Transition from the G1/G0 to the S-phase of the cell cycle was compared in PDGFRα-expressing and non-expressing LF using flow cytometry. Laser scanning confocal microscopy was used to quantify p27(kip1) and forkhead box "other" 3a (FoxO3a) in the nuclei of alveolar cells from mice bearing the PDGFRα-GFP knock-in, and p27(kip1) in mice with a conditional deletion of PDGFRα-gene function. The effects of PDGF-A on the phosphorylation and the intracellular location of FoxO3a were examined using Western immuoblotting and immunocytochemistry. RESULTS: In neonatal mouse lungs, entry of the PDGFRα-expressing LF subpopulation into the S-phase of the cell cycle diminished sooner than in their non-expressing LF counterparts. This preferential diminution was influenced by PDGFRα-mediated signaling, which phosphorylates and promotes cytoplasmic localization of FoxO3a. Comparative observations of LF at different ages during secondary septation and in mice that lack PDGFRα in alveolar LF demonstrated that nuclear localization of the G1 cyclin-dependent kinase inhibitor p27(kip1) correlated with reduced LF entry into S-phase. CONCLUSIONS: Nuclear localization of FoxO3a, an important regulator of p27(kip1) gene-expression, correlates with diminished proliferation of the PDGFRα-expressing LF subpopulation. These mechanisms for diminishing the effects of PDGFRα-mediated signaling likely regulate secondary septal formation and their derangement may contribute to imbalanced fibroblast cell kinetics in parenchymal lung diseases.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Proteína Forkhead Box O3 , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Transgênicos , Pontos de Checagem da Fase S do Ciclo Celular/fisiologia , Transdução de Sinais/fisiologiaRESUMO
In mice, secondary alveolar septal formation primarily occurs during a brief postnatal period and is accompanied by transient expansion of the interstitial lung fibroblast (LF) population. PDGF-A, which solely signals through PDGF-receptor-alpha (PDGF-Rα), is required for expansion, but the receptor's relevant downstream targets remain incompletely defined. We have evaluated the proliferation, apoptosis, and differential response to the selective protein tyrosine kinase inhibitor, imatinib, by pdgfrα-expressing LF (pdgfrα-LF) and compared them with their nonexpressing LF counterparts. Our objective was to determine whether diminished signaling through PDGF-Rα-mediated pathways regulates the decline in myofibroblasts, which accompanies septal thinning and ensures more efficient alveolar gas exchange. Using quantitative stereology and flow cytometry at postnatal d 12 and 14, we observed that imatinib caused a selective suppression of proliferation and an increase in apoptosis. The number of the alpha smooth muscle actin (αSMA) producing pdgfrα-LF was also reduced. Using cultures of neonatal mouse LF, we showed that imatinib did not suppress PDGF-Rα gene expression but reduced PDGF-A-mediated Akt phosphorylation, potentially explaining the increase in apoptosis. Our findings are relevant to bronchopulmonary dysplasia in which positive pressure ventilation interferes with myofibroblast depletion, septal thinning, and capillary maturation.
Assuntos
Miofibroblastos/enzimologia , Alvéolos Pulmonares/enzimologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Actinas/metabolismo , Fatores Etários , Envelhecimento , Análise de Variância , Animais , Animais Recém-Nascidos , Apoptose , Benzamidas , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Mesilato de Imatinib , Camundongos , Camundongos Transgênicos , Miofibroblastos/patologia , NF-kappa B/metabolismo , Fosforilação , Piperazinas/farmacologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Alvéolos Pulmonares/crescimento & desenvolvimento , Alvéolos Pulmonares/patologia , Pirimidinas/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de Sinais , Ativação TranscricionalRESUMO
Airway hyperresponsiveness (AHR) is influenced by structural components of the bronchial wall, including the smooth muscle and connective tissue elements and the neuromuscular function. AHR is also influenced by parenchymally derived tethering forces on the bronchial wall, which maintain airway caliber by producing outward radial traction. Our previous work has shown that vitamin A-deficient (VAD) rats exhibit cholinergic hyperresponsiveness and a decrease in the expression and function of the muscarinic-2 receptors (M2R). We hypothesized that if decreases in radial traction from airway or parenchymal structures contributed to the VAD-related increase in AHR, then the radial traction would normalize more slowly than VAD-related alterations in neurotransmitter signaling. Rats remained vitamin A sufficient (VAS) or were rendered VAD and then maintained on the VAD diet in the presence or absence of supplementation with all-trans retinoic acid (RA). VAD was associated with an approximately twofold increase in respiratory resistance and elastance compared with VAS rats. Exposure to RA for 12 days but not 4 days restored resistance and elastance to control (VAS) levels. In VAD rats, AHR was accompanied by decreases in bronchial M2R gene expression and function, which were restored after 12 days of RA supplementation. Subepithelial bronchial elastic fibers were decreased by approximately 50% in VAD rats and were significantly restored by RA. The increase in AHR that is associated with VAD is accompanied by decreases in M2R expression and function that can be restored by RA and a reduction in airway elastic fibers that can be partially restored by RA.
Assuntos
Antineoplásicos/farmacologia , Hiper-Reatividade Brônquica/tratamento farmacológico , Hiper-Reatividade Brônquica/etiologia , Tretinoína/farmacologia , Deficiência de Vitamina A/complicações , Deficiência de Vitamina A/fisiopatologia , Animais , Broncoconstritores/farmacologia , Elasticidade , Feminino , Expressão Gênica , Cloreto de Metacolina/farmacologia , Agonistas Muscarínicos/farmacologia , Pilocarpina/farmacologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/fisiologia , Ratos , Ratos Endogâmicos Lew , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Organismos Livres de Patógenos EspecíficosRESUMO
Sphingolipids represent a diverse group of bioactive lipid species that are generated intracellularly in response to tumor necrosis factor-alpha (TNF-alpha) and are implicated as potential mediators of acute lung injury. The purpose of these studies was to determine whether there was an extracellular, TNF-alpha-regulated pool of sphingolipids in the alveolus that modulates the surface tension lowering capacity of pulmonary surfactant. Intratracheal instillation of TNF-alpha in adult rats led to a twofold increase in the amount of surfactant-associated ceramide and tended to decrease levels of sphingomyelin without significantly altering sphingosine or sphinganine content. TNF-alpha induction of alveolar ceramide was associated with nearly an 80% increase in acid sphingomyelinase activity recovered in cell-free alveolar lavage. Ceramide administered in a dose-dependent manner potently antagonized the surface tension lowering effects of natural surfactant in vitro. Intratracheal TNF-alpha and ceramide treatment of rats significantly increased lung permeability, as was evidenced by extravasation of Evans blue dye into alveolar lavage and lung tissue. Thus these studies are the first to demonstrate the existence of a cytokine-regulated alveolar pool of sphingomyelin hydrolysis products that impairs the biophysical properties of the alveolar surfactant film. The results also suggest the presence of a secretory alveolar sphingomylinase that is TNF-alpha responsive and mediates effects of the cytokine on alveolar sphingolipid metabolism.
Assuntos
Alvéolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , Esfingolipídeos/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Fenômenos Biofísicos , Biofísica , Permeabilidade Capilar/efeitos dos fármacos , Ceramidas/farmacologia , Corantes/farmacocinética , Azul Evans/farmacocinética , Masculino , Circulação Pulmonar/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Esfingomielina Fosfodiesterase/metabolismo , Tensão SuperficialRESUMO
Tumor necrosis factor (TNF)-alpha is a major cytokine implicated in inducing acute and chronic lung injury, conditions associated with surfactant phosphatidylcholine (PtdCho) deficiency. Acutely, TNF-alpha decreases PtdCho synthesis but stimulates surfactant secretion. To investigate chronic effects of TNF-alpha, we investigated PtdCho metabolism in a murine transgenic model exhibiting lung-specific TNF-alpha overexpression. Compared with controls, TNF-alpha transgenic mice exhibited a discordant pattern of PtdCho metabolism, with a decrease in PtdCho and disaturated PtdCho (DSPtdCho) content in the lung, but increased levels in alveolar lavage. Transgenics had lower activities and increased immunoreactive levels of cytidylyltransferase (CCT), a key PtdCho biosynthetic enzyme. Ceramide, a CCT inhibitor, was elevated, and linoleic acid, a CCT activator, was decreased in transgenics. Radiolabeling studies revealed that alveolar reuptake of DSPtdCho was significantly decreased in transgenic mice. These observations suggest that chronic expression of TNF-alpha results in a complex pattern of PtdCho metabolism where elevated lavage PtdCho may originate from alveolar inflammatory cells, decreased surfactant reuptake, or altered surfactant secretion. Reduced parenchymal PtdCho synthesis appears to be attributed to CCT enzyme that is physiologically inactivated by ceramide or by diminished availability of activating lipids.