RESUMEN
Progressive pulmonary fibrosis is a devastating consequence of many acute and chronic insults to the lung. Lung injury leads to alveolar epithelial cell (AEC) death, destruction of the basement membrane, and activation of transforming growth factor-ß (TGF-ß). There is subsequent resolution of the injury and a coordinated and concurrent initiation of fibrosis. Both of these processes may involve activation of similar intracellular signaling pathways regulated in part by dynamic changes to the extracellular matrix. Matrix signaling can augment the profibrotic fibroblast response to TGF-ß. However, similar matrix/integrin signaling pathways may also be involved in the inhibition of ongoing TGF-ß-induced AEC apoptosis. Focal adhesion kinase (FAK) is an integrin-associated signaling molecule expressed by many cell types. We used mice with AEC-specific FAK deletion to isolate the epithelial aspect of integrin signaling in the bleomycin model of lung injury and fibrosis. Mice with AEC-specific deletion of FAK did not exhibit spontaneous lung injury but did have significantly greater terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling-positive cells (18.6 vs. 7.1) per ×200 field, greater bronchoalveolar lavage protein (3.2 vs. 1.8 mg/ml), and significantly greater death (77 vs. 19%) after bleomycin injury compared with littermate control mice. Within primary AECs, activated FAK directly associates with caspase-8 and inhibits activation of the caspase cascade resulting in less apoptosis in response to TGF-ß. Our studies support a model in which dynamic changes to the extracellular matrix after injury promote fibroblast activation and inhibition of epithelial cell apoptosis in response to TGF-ß through FAK activation potentially complicating attempts to nonspecifically target this pathway for antifibrotic therapy.
Asunto(s)
Células Epiteliales/enzimología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Lesión Pulmonar/enzimología , Pulmón/patología , Fibrosis Pulmonar/enzimología , Transducción de Señal , Animales , Apoptosis , Bleomicina , Caspasa 8/metabolismo , Activación Enzimática , Células Epiteliales/patología , Eliminación de Gen , Lesión Pulmonar/complicaciones , Lesión Pulmonar/patología , Ratones , Modelos Biológicos , Especificidad de Órganos , Fibrosis Pulmonar/complicaciones , Fibrosis Pulmonar/patología , Proteína C Asociada a Surfactante Pulmonar/metabolismoRESUMEN
Accumulation of apoptosis-resistant fibroblasts is a hallmark of pulmonary fibrosis. We hypothesized that disruption of inhibitor of apoptosis protein (IAP) family proteins would limit lung fibrosis. We first show that transforming growth factor-ß1 and bleomycin increase X-linked IAP (XIAP) and cellular IAP (cIAP)-1 and -2 in murine lungs and mesenchymal cells. Functional blockade of XIAP and the cIAPs with AT-406, an orally bioavailable second mitochondria-derived activator of caspases (Smac) mimetic, abrogated bleomycin-induced lung fibrosis when given both prophylactically and therapeutically. To determine whether the reduction in fibrosis was predominantly due to AT-406-mediated inhibition of XIAP, we compared the fibrotic response of XIAP-deficient mice (XIAP(-/y)) with littermate controls and found no difference. We found no alterations in total inflammatory cells of either wild-type mice treated with AT-406 or XIAP(-/y) mice. AT-406 treatment limited CCL12 and IFN-γ production, whereas XIAP(-/y) mice exhibited increased IL-1ß expression. Surprisingly, XIAP(-/y) mesenchymal cells had increased resistance to Fas-mediated apoptosis. Functional blockade of cIAPs with AT-406 restored sensitivity to Fas-mediated apoptosis in XIAP(-/y) mesenchymal cells in vitro and increased apoptosis of mesenchymal cells in vivo, indicating that the increased apoptosis resistance in XIAP(-/y) mesenchymal cells was the result of increased cIAP expression. Collectively, these results indicate that: (1) IAPs have a role in the pathogenesis of lung fibrosis; (2) a congenital deficiency of XIAP may be overcome by compensatory mechanisms of other IAPs; and (3) broad functional inhibition of IAPs may be an effective strategy for the treatment of lung fibrosis by promoting mesenchymal cell apoptosis.
Asunto(s)
Bleomicina/toxicidad , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Fibrosis Pulmonar/prevención & control , Animales , Apoptosis , Azocinas/farmacología , Compuestos de Bencidrilo/farmacología , Proteínas Inhibidoras de la Apoptosis/genética , Interferón gamma/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Quimioatrayentes de Monocitos/metabolismo , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta1/administración & dosificaciónRESUMEN
Transforming growth factor-ß (TGF-ß) is a critical driver of acute lung injury and fibrosis. Injury leads to activation of TGF-ß, which regulates changes in the cellular and matrix makeup of the lung during the repair and fibrosis phase. TGF-ß can also initiate alveolar epithelial cell (AEC) apoptosis. Injury leads to destruction of the laminin-rich basement membrane, which is replaced by a provisional matrix composed of arginine-glycine-aspartate (RGD) motif-containing plasma matrix proteins, including vitronectin and fibronectin. To determine the role of specific matrix proteins on TGF-ß-induced apoptosis, we studied primary AECs cultured on different matrix conditions and utilized mice with deletion of vitronectin (Vtn(-/-)) or mice in which the vitronectin RGD motif is mutated to nonintegrin-binding arginine-glycine-glutamate (RGE) (Vtn(RGE/RGE)). We found that AECs cultured on fibronectin and vitronectin or in wild-type mouse serum are resistant to TGF-ß-induced apoptosis. In contrast, AECs cultured on laminin or in serum from Vtn(-/-) or Vtn(RGE/RGE) mice undergo robust TGF-ß-induced apoptosis. Plasminogen activator inhibitor-1 (PAI-1) sensitizes AECs to greater apoptosis by disrupting AEC engagement to vitronectin. Inhibition of integrin-associated signaling proteins augments AEC apoptosis. Mice with transgenic deletion of PAI-1 have less apoptosis after bleomycin, but deletion of vitronectin or disruption of the vitronectin RGD motif reverses this protection, suggesting that the proapoptotic function of PAI-1 is mediated through vitronectin inhibition. Collectively, these data suggest that integrin-matrix signaling is an important regulator of TGF-ß-mediated AEC apoptosis and that PAI-1 functions as a natural regulator of this interaction.
Asunto(s)
Células Epiteliales Alveolares/fisiología , Apoptosis , Factor de Crecimiento Transformador beta/fisiología , Vitronectina/fisiología , Secuencias de Aminoácidos , Animales , Células Cultivadas , Integrinas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Vitronectina/químicaRESUMEN
Pulmonary fibrosis is pathologic remodeling of lung tissue that can result in difficulty breathing, reduced quality of life, and a poor prognosis for patients. Fibrosis occurs as a result of insult to lung tissue, though mechanisms of this response are not well-characterized. The disease is driven in part by dysregulation of fibroblast proliferation and differentiation into myofibroblast cells, as well as pro-fibrotic mediator-driven epithelial cell apoptosis. The most well-characterized pro-fibrotic mediator associated with pulmonary fibrosis is TGF-ß1. Excessive synthesis of, and sensitivity to, pro-fibrotic mediators as well as insufficient production of and sensitivity to anti-fibrotic mediators has been credited with enabling fibroblast accumulation. Available treatments neither halt nor reverse lung damage. In this study we have two aims: to identify molecular and cellular scale mechanisms driving fibroblast proliferation and differentiation as well as epithelial cell survival in the context of fibrosis, and to predict therapeutic targets and strategies. We combine in vitro studies with a multi-scale hybrid agent-based computational model that describes fibroblasts and epithelial cells in co-culture. Within this model TGF-ß1 represents a pro-fibrotic mediator and we include detailed dynamics of TGF-ß1 receptor ligand signaling in fibroblasts. PGE2 represents an anti-fibrotic mediator. Using uncertainty and sensitivity analysis we identify TGF-ß1 synthesis, TGF-ß1 activation, and PGE2 synthesis among the key mechanisms contributing to fibrotic outcomes. We further demonstrate that intervention strategies combining potential therapeutics targeting both fibroblast regulation and epithelial cell survival can promote healthy tissue repair better than individual strategies. Combinations of existing drugs and compounds may provide significant improvements to the current standard of care for pulmonary fibrosis. Thus, a two-hit therapeutic intervention strategy may prove necessary to halt and reverse disease dynamics.
RESUMEN
Thirteen bridged triazolium and imidazolium dicationic salts, which uniquely pair closo-icosahedral perfluoroborane [B(12)F(12)](2-), borane [B(12)H(12)](2-), or carborane [CB(11)H(12)](-) anionic species with unsaturated bridged heterocyclium dications, were synthesized using an aqueous benchtop method. This considerably extends the scope of a reported aqueous synthesis of binary [heterocyclium](2)[B(12)H(12)] and [heterocyclium][CB(11)H(12)] salts. Also, the one-step preparation of five new precursor bridged heterocyclium dicationic dihalide salts using conventional procedures and in one case a microwave-assisted method is described.
RESUMEN
Ten new salts that pair triazolium and imidazolium cations with closo-icosahedral anions [B(12)H(12)](2-) and [CB(11)H(12)](-) were synthesized in water solvent using an open-air, benchtop method. These unreported [Heterocyclium](2)[B(12)H(12)] and [Heterocyclium][CB(11)H(12)] salts extend reports of [Imidazolium][CB(11)H(12)] and [Pyridinium][CB(11)H(12)] salts that were synthesized in anhydrous organic solvents under an inert atmosphere with glovebox or Schlenk techniques. Spectroscopic data, melting points, and densities are reported for each salt. Single-crystal X-ray structures are provided for the five new [B(12)H(12)](2-) salts.