Natalya Subbotina

Targeted Injury of Type II Alveolar Epithelial Cells Induces Pulmonary Fibrosis

RATIONALE Ineffective repair of a damaged alveolar epithelium has been postulated to cause pulmonary fibrosis. In support of this theory, epithelial cell abnormalities, including hyperplasia, apoptosis, and persistent denudation of the alveolar basement membrane, are found in the lungs of humans with idiopathic pulmonary fibrosis and in animal models of fibrotic lung disease. Furthermore, mutations in genes that affect regenerative capacity or that cause injury/apoptosis of type II alveolar epithelial cells have been identified in familial forms of pulmonary fibrosis. Although these findings are compelling, there are no studies that demonstrate a direct role for the alveolar epithelium or, more specifically, type II cells in the scarring process. OBJECTIVES To determine if a targeted injury to type II cells would result in pulmonary fibrosis. METHODS A transgenic mouse was generated to express the human diphtheria toxin receptor on type II alveolar epithelial cells. Diphtheria toxin was administered to these animals to specifically target the type II epithelium for injury. Lung fibrosis was assessed by histology and hydroxyproline measurement. MEASUREMENTS AND MAIN RESULTS Transgenic mice treated with diphtheria toxin developed an approximately twofold increase in their lung hydroxyproline content on Days 21 and 28 after diphtheria toxin treatment. The fibrosis developed in conjunction with type II cell injury. Histological evaluation revealed diffuse collagen deposition with patchy areas of more confluent scarring and associated alveolar contraction. CONCLUSIONS The development of lung fibrosis in the setting of type II cell injury in our model provides evidence for a causal link between the epithelial defects seen in idiopathic pulmonary fibrosis and the corresponding areas of scarring.

Implicating Exudate Macrophages and Ly-6Chigh Monocytes in CCR2-Dependent Lung Fibrosis following Gene-Targeted Alveolar Injury

The alveolar epithelium is characteristically abnormal in fibrotic lung disease, and we recently established a direct link between injury to the type II alveolar epithelial cell (AEC) and the accumulation of interstitial collagen. The mechanisms by which damage to the epithelium induces lung scarring remain poorly understood. It is particularly controversial whether an insult to the type II AEC initiates an inflammatory response that is required for the development of fibrosis. To explore whether local inflammation occurs following a targeted epithelial insult and contributes to lung fibrosis, we administered diphtheria toxin to transgenic mice with type II AEC–restricted expression of the diphtheria toxin receptor. We used immunophenotyping techniques and diphtheria toxin receptor–expressing, chemokine receptor-2–deficient (CCR2−/−) mice to determine the participation of lung leukocyte subsets in pulmonary fibrogenesis. Our results demonstrate that targeted type II AEC injury induces an inflammatory response that is enriched for CD11b+ nonresident exudate macrophages (ExM) and their precursors, Ly-6Chigh monocytes. CCR2 deficiency abrogates the accumulation of both cell populations and protects mice from fibrosis, weight loss, and death. Further analyses revealed that the ExM are alternatively activated and that ExM and Ly-6Chigh monocytes express mRNA for IL-13, TGF-β, and the collagen genes, COL1A1 and COLIIIA1. Furthermore, the accumulated ExM and Ly-6Chigh monocytes contain intracellular collagen, as detected by immunostaining. Together, these results implicate CCR2 and the accumulation of ExM and Ly-6Chigh monocytes as critical determinants of pulmonary fibrosis induced by selective type II AEC injury.

Inhibition of Myocardin-Related Transcription Factor/Serum Response Factor Signaling Decreases Lung Fibrosis and Promotes Mesenchymal Cell Apoptosis

Myofibroblasts are crucial to the pathogenesis of tissue fibrosis. Their formation of stress fibers results in the release of myocardin-related transcription factor (MRTF), a transcriptional coactivator of serum response factor (SRF). MRTF-A (Mkl1)-deficient mice are protected from lung fibrosis. We hypothesized that the SRF/MRTF pathway inhibitor CCG-203971 would modulate myofibroblast function in vitro and limit lung fibrosis in vivo. Normal and idiopathic pulmonary fibrosis lung fibroblasts were treated with/without CCG-203971 (N-[4-chlorophenyl]-1-[3-(2-furanyl)benzoyl]-3-piperidine carboxamide) and/or Fas-activating antibody in the presence/absence of transforming growth factor (TGF)-β1, and apoptosis was assessed. In vivo studies examined the effect of therapeutically administered CCG-203971 on lung fibrosis in two distinct murine models of fibrosis induced by bleomycin or targeted type II alveolar epithelial injury. In vitro, CCG-203971 prevented nuclear localization of MRTF-A; increased the apoptotic susceptibility of normal and idiopathic pulmonary fibrosis fibroblasts; blocked TGF-β1-induced myofibroblast differentiation; and inhibited TGF-β1-induced expression of fibronectin, X-linked inhibitor of apoptosis, and plasminogen activator inhibitor-1. TGF-β1 did not protect fibroblasts or myofibroblasts from apoptosis in the presence of CCG-203971. In vivo, CCG-203971 significantly reduced lung collagen content in both murine models while decreasing alveolar plasminogen activator inhibitor-1 and promoting myofibroblast apoptosis. These data support a central role of the SRF/MRTF pathway in the pathobiology of lung fibrosis and suggest that its inhibition can help resolve lung fibrosis by promoting fibroblast apoptosis.

Reduction in Fibrotic Tissue Formation in Mice Genetically Deficient in Plasminogen Activator Inhibitor-1

Mice with homozygous deletion of the plasminogen activator inhibitor-1 gene (PAI-1(-/-)) are relatively protected from bleomycin-induced pulmonary fibrosis. At least part of the protective effect appears to occur during the latter stages of the pathological process when fibrotic tissue is being deposited. To investigate the effect of PAI-1 deficiency on fibrosis, we studied the accumulation of fibrotic tissue within subcutaneously implanted polyvinyl alcohol sponges. Similar to the effect of PAI-1 deficiency on bleomycin-induced pulmonary fibrosis, the accumulation of fibrotic tissue within implanted sponges occurred more slowly in PAI-1(-/-) compared to wild-type mice. Another striking difference observed in the PAI-1(-/-) mice was the rapid removal of a fibrin-rich matrix that formed within the sponges by 1 day after implantation in both wild-type and PAI-1(-/-) mice. The pattern of connective tissue invasion also differed: cells in wild-type mice infiltrated as individually penetrating cells whereas in PAI-1(-/-) mice they did so as a well-demarcated advancing front. Providing an alternative provisional matrix by impregnating sponges with a low concentration of collagen before implantation corrected the changes induced by PAI-1 deficiency. In conclusion, PAI-1 deficiency appears to affect fibrotic tissue formation in part by altering the provisional matrix that forms soon after tissue injury.

PAI-1 promotes the accumulation of exudate macrophages and worsens pulmonary fibrosis following type II alveolar epithelial cell injury.

Fibrotic disorders of the lung are associated with perturbations in the plasminogen activation system. Specifically, plasminogen activator inhibitor‐1 (PAI‐1) expression is increased relative to the plasminogen activators. A direct role for this imbalance in modulating the severity of lung scarring following injury has been substantiated in the bleomycin model of pulmonary fibrosis. However, it remains unclear whether derangements in the plasminogen activation system contribute more generally to the pathogenesis of lung fibrosis beyond bleomycin injury. To answer this question, we employed an alternative model of lung scarring, in which type II alveolar epithelial cells (AECs) are specifically injured by administering diphtheria toxin (DT) to mice genetically engineered to express the human DT receptor (DTR) off the surfactant protein C promoter. This targeted AEC injury results in the diffuse accumulation of interstitial collagen. In the present study, we found that this targeted type II cell insult also increases PAI‐1 expression in the alveolar compartment. We identified AECs and lung macrophages to be sources of PAI‐1 production. To determine whether this elevated PAI‐1 concentration was directly related to the severity of fibrosis, DTR+ mice were crossed into a PAI‐1‐deficient background (DTR+: PAI‐1−/−). DT administration to DTR+: PAI‐1−/− animals caused significantly less fibrosis than was measured in DTR+ mice with intact PAI‐1 production. PAI‐1 deficiency also abrogated the accumulation of CD11b+ exudate macrophages that were found to express PAI‐1 and type‐1 collagen. These observations substantiate the critical function of PAI‐1 in pulmonary fibrosis pathogenesis and provide new insight into a potential mechanism by which this pro‐fibrotic molecule influences collagen accumulation. Copyright

The vitronectin-binding function of PAI-1 exacerbates lung fibrosis in mice

Plasminogen activator inhibitor-1 (PAI-1) is increased in the lungs of patients with pulmonary fibrosis, and animal studies have shown that experimental manipulations of PAI-1 levels directly influence the extent of scarring that follows lung injury. PAI-1 has 2 known properties that could potentiate fibrosis, namely an antiprotease activity that inhibits the generation of plasmin, and a vitronectin-binding function that interferes with cell adhesion to this extracellular matrix protein. To determine the relative importance of each PAI-1 function in lung fibrogenesis, we administered mutant PAI-1 proteins that possessed either intact antiprotease or vitronectin-binding activity to bleomycin-injured mice genetically deficient in PAI-1. We found that the vitronectin-binding capacity of PAI-1 was the primary determinant required for its ability to exacerbate lung scarring induced by intratracheal bleomycin administration. The critical role of the vitronectin-binding function of PAI-1 in fibrosis was confirmed in the bleomycin model using mice genetically modified to express the mutant PAI-1 proteins. We conclude that the vitronectin-binding function of PAI-1 is necessary and sufficient in its ability to exacerbate fibrotic processes in the lung.

Targeting Inhibitor of Apoptosis Proteins Protects from Bleomycin-Induced Lung Fibrosis.

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-β1 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-1β 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.

Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury

Fibrosis of the lung constitutes a major clinical challenge and novel therapies are required to alleviate the associated morbidity and mortality. Investigating the antifibrotic efficacy of drugs that are already in clinical practice offers an efficient strategy to identify new therapies. The phosphodiesterase 4 (PDE4) inhibitors, approved for the treatment of chronic obstructive pulmonary disease, harbor therapeutic potential for pulmonary fibrosis by augmenting the activity of endogenous antifibrotic mediators that signal through cyclic AMP. In this study, we tested the efficacy of several PDE4 inhibitors including a novel compound (Compound 1) in a murine model of lung fibrosis that results from a targeted type II alveolar epithelial cell injury. We also compared the antifibrotic activity of PDE4 inhibition to the two therapies that are FDA‐approved for idiopathic pulmonary fibrosis (pirfenidone and nintedanib). We found that both preventative (day 0–21) and therapeutic (day 11–21) dosing regimens of the PDE4 inhibitors significantly ameliorated the weight loss and lung collagen accumulation that are the sequelae of targeted epithelial cell damage. In a therapeutic protocol, the reduction in lung fibrosis with PDE4 inhibitor administration was equivalent to pirfenidone and nintedanib. Treatment with this class of drugs also resulted in a decrease in plasma surfactant protein D concentration, a reduction in the plasma levels of several chemokines implicated in lung fibrosis, and an in vitro inhibition of fibroblast profibrotic gene expression. These results motivate further investigation of PDE4 inhibition as a treatment for patients with fibrotic lung disease.