Animal Models Of Lung Fibrosis Research Articles

Gene therapy with AAV9-SGPL1 in an animal model of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-β signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-β signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1β as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Epigenetic regulation of IPF fibroblast phenotype by glutaminolysis

ObjectiveExcessive extra-cellular-matrix production and uncontrolled proliferation of the fibroblasts are characteristics of many fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). The fibroblasts have enhanced glutaminolysis with up-regulated glutaminase, GLS1, which converts glutamine to glutamate. Here, we investigated the role of glutaminolysis and glutaminolysis-derived metabolite α-ketoglutarate (α-KG) on IPF fibroblast phenotype and gene expression. MethodsReduced glutamine conditions were carried out either using glutamine-free culture medium or silencing the expression of GLS1 with siRNA, with or without α-KG compensation. Cell phenotype has been characterized under these different conditions, and gene expression profile was examined by RNA-Seq. Specific profibrotic genes (Col3A1 and PLK1) expression were examined by real-time PCR and western blots. The levels of repressive histone H3K27me3, which demethylase activity is affected by glutaminolysis, were examined and H3K27me3 association with promoter region of Col3A1 and PLK1 were checked by ChIP assays. Effects of reduced glutaminolysis on fibrosis markers were checked in an animal model of lung fibrosis. ResultsThe lack of glutamine in the culture medium alters the profibrotic phenotype of activated fibroblasts. The addition of exogenous and glutaminolysis-derived metabolite α-KG to glutamine-free media barely restores the pro-fibrotic phenotype of activated fibroblasts. Many genes are down-regulated in glutamine-free medium, α-KG supplementation only rescues a limited number of genes. As α-KG is a cofactor for histone demethylases of H3K27me3, the reduced glutaminolysis alters H3K27me3 levels, and enriches H3K27me3 association with Col3A1 and PLK1 promoter region. Adding α-KG in glutamine-free medium depleted H3K27me3 association with Col3A1 promoter region but not that of PLK1. In a murine model of lung fibrosis, mice with reduced glutaminolysis showed markedly reduced fibrotic markers. ConclusionsThis study indicates that glutamine is critical for supporting pro-fibrotic fibroblast phenotype in lung fibrosis, partially through α-KG-dependent and –independent mechanisms, and supports targeting fibroblast metabolism as a therapeutic method for fibrotic diseases.

Randomized double-blind clinical study in patients with COVID-19 to evaluate the safety and efficacy of a phytomedicine (P2Et).

BackgroundIt has been proposed that polyphenols can be used in the development of new therapies against COVID-19, given their ability to interfere with the adsorption and entrance processes of the virus, thus disrupting viral replication. Seeds from Caesalpinia spinosa, have been traditionally used for the treatment of inflammatory pathologies and respiratory diseases. Our team has obtained an extract called P2Et, rich in polyphenols derived from gallic acid with significant antioxidant activity, and the ability to induce complete autophagy in tumor cells and reduce the systemic inflammatory response in animal models.MethodsIn this work, a phase II multicenter randomized double-blind clinical trial on COVID-19 patients was designed to evaluate the impact of the P2Et treatment on the clinical outcome and the immunological parameters related to the evolution of the disease. The Trial was registered with the number No. NCT04410510*. A complementary study in an animal model of lung fibrosis was carried out to evaluate in situ lung changes after P2Et in vivo administration. The ability of P2Et to inhibit the viral load of murine and human coronaviruses in cellular models was also evaluated.ResultsPatients treated with P2Et were discharged on average after 7.4 days of admission vs. 9.6 days in the placebo group. Although a decrease in proinflammatory cytokines such as G-CSF, IL-15, IL-12, IL-6, IP10, MCP-1, MCP-2 and IL-18 was observed in both groups, P2Et decreased to a greater extent G-CSF, IL-6 and IL-18 among others, which are related to lower recovery of patients in the long term. The frequency of T lymphocytes (LT) CD3+, LT double negative (CD3+CD4-CD8-), NK cells increased in the P2Et group where the population of eosinophils was also significantly reduced. In the murine bleomycin model, P2Et also reduced lung inflammation and fibrosis. P2Et was able to reduce the viral replication of murine and human coronaviruses in vitro, showing its dual antiviral and anti-inflammatory role, key in disease control.ConclusionsTaken together these results suggest that P2Et could be consider as a good co-adjuvant in the treatment of COVID-19.Clinical trail registrationhttps://clinicaltrials.gov/ct2/show/NCT04410510, identifier: NCT04410510.

Developing PI3K Inhibitors for Respiratory Diseases.

A number of different experimental models using both non-selective and selective PI3K inhibitors have shown that many pathogenic steps of respiratory disorders, such as bronchial asthma, Chronic Obstructive Pulmonary Disease (COPD), Idiopathic Pulmonary Fibrosis (IPF), Acute Respiratory Distress Syndrome (ARDS) and Lung Cancer (LC) are, at least in part, regulated by the PI3K signaling pathway, suggesting that the inhibition of PI3K could represent an ideal therapeutic target for the treatment of respiratory diseases. This chapter summarizes the current state of the therapeutic strategies aimed to exploit the inhibition of PI3K in this context. In animal models of asthma, selective δ and γ inhibitors have shown to be effective, and when administered by inhalation, reasonably safe. Nevertheless, very few clinical trials have been performed so far. The efficacy of current traditional therapies for allergic bronchial asthma has likely diminished the need for new alternative treatments. Surprisingly, in COPD, where instead there is an urgent need for new and more effective therapeutic approaches, the number of clinical studies is still low and not capable yet, with the exception for an acceptable safety profile, to show a significant improvement of clinical outcomes. In IPF, a disease with a disappointing prognosis, PI3K inhibitors have been bound to a FAP ligand with the aim to selectively target myofibroblasts, showing to significantly reduce collagen production and the development of lung fibrosis in an animal model of lung fibrosis. Due to its role in cell activation and cell replication, the PI3K pathway is obviously largely involved in lung cancer. Several studies, currently ongoing, are testing the effect of PI3K inhibitors mainly in NSCLC. Some evidence in the treatment of cancer patients suggests the possibility that PI3K inhibitors may enhance the response to conventional treatment. The involvement of PI3Kδ in the modulation of airway neutrophil recruitment and bronchial epithelial functional alterations also suggest a potential role in the treatment of ARDS, but at the current state the ongoing trials are aimed to the treatment of ARDS in COVID-19 patients. In general, few clinical trials investigating PI3K inhibitors in respiratory disorders have been performed so far. This relatively new approach of treatment is just at its beginning and certainly needs further efforts and additional studies.

IGF-1 Receptor Signaling Regulates Type II Pneumocyte Senescence and Resulting Macrophage Polarization in Lung Fibrosis

Type II pneumocyte (alveolar epithelial cells type II [AECII]) senescence has been implicated in the progression of lung fibrosis. The capacity of senescent cells to modulate pulmonary macrophages to drive fibrosis is unexplored. Insulin-like growth factor-1 receptor (IGF-1R) signaling has been implicated as a regulator of senescence and aging. Mice with an AECII-specific deletion of IGF-1R received thoracic irradiation (n ≥ 5 per condition), and the effect of IGF-1R deficiency on radiation-induced AECII senescence and macrophage polarization to an alternatively activated phenotype (M2) was investigated. IGF-1R signaling, macrophage polarization, and senescence were evaluated in surgically resected human lung (n = 63). IGF-1R deficient mice demonstrated reduced AECII senescence (senescent AECII/field; intact: 7.25% ± 3.5% [mean ± SD], deficient: 2.75% ± 2.8%, P = .0001), reduced accumulation of M2 macrophages (intact: 24.7 ± 2.2 cells/field, deficient: 15.5 ± 1.2 cells/field, P = .0086), and fibrosis (hydroxyproline content; intact: 71.9 ± 21.7 μg/lung, deficient: 31.7 ± 7.9, P = .0485) after irradiation. Senescent AECII enhanced M2 polarization in a paracrine fashion (relative Arg1 mRNA, 0 Gy: 1.0 ± 0.4, 17.5 Gy: 7.34 ± 0.5, P < .0001). Evaluation of surgical samples from patients treated with chemoradiation demonstrated increased expression of IGF-1 (unirradiated: 10.2% ± 4.9% area, irradiated: 15.1% ± 11.5%, P = .0377), p21 (unirradiated: 0.013 ± 0.02 histoscore, irradiated: 0.084 ± 0.09 histoscore, P = .0002), IL-13 (unirradiated: 13.7% ± 2.8% area, irradiated: 21.7% ± 3.8%, P < .0001), and M2 macrophages in fibrotic regions relative to nonfibrotic regions (unirradiated: 11.4 ± 12.2 CD163 + cells/core, irradiated: 43.1 ± 40.9 cells/core, P = .0011), consistent with findings from animal models of lung fibrosis. This study demonstrates that senescent AECII are necessary for the progression of pulmonary fibrosis and serve as a targetable, chronic stimuli for macrophage activation in fibrotic lung.

Potent and Selective Human Prostaglandin F (FP) Receptor Antagonist (BAY-6672) for the Treatment of Idiopathic Pulmonary Fibrosis (IPF).

Idiopathic pulmonary fibrosis (IPF) is a rare and devastating chronic lung disease of unknown etiology. Despite the approved treatment options nintedanib and pirfenidone, the medical need for a safe and well-tolerated antifibrotic treatment of IPF remains high. The human prostaglandin F receptor (hFP-R) is widely expressed in the lung tissue and constitutes an attractive target for the treatment of fibrotic lung diseases. Herein, we present our research toward novel quinoline-based hFP-R antagonists, including synthesis and detailed structure-activity relationship (SAR). Starting from a high-throughput screening (HTS) hit of our corporate compound library, multiple parameter improvements-including increase of the relative oral bioavailability Frel from 3 to ≥100%-led to a highly potent and selective hFP-R antagonist with complete oral absorption from suspension. BAY-6672 (46) represents-to the best of our knowledge-the first reported FP-R antagonist to demonstrate in vivo efficacy in a preclinical animal model of lung fibrosis, thus paving the way for a new treatment option in IPF.

Lipid Mediators Regulate Pulmonary Fibrosis: Potential Mechanisms and Signaling Pathways.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease of unknown etiology characterized by distorted distal lung architecture, inflammation, and fibrosis. The molecular mechanisms involved in the pathophysiology of IPF are incompletely defined. Several lung cell types including alveolar epithelial cells, fibroblasts, monocyte-derived macrophages, and endothelial cells have been implicated in the development and progression of fibrosis. Regardless of the cell types involved, changes in gene expression, disrupted glycolysis, and mitochondrial oxidation, dysregulated protein folding, and altered phospholipid and sphingolipid metabolism result in activation of myofibroblast, deposition of extracellular matrix proteins, remodeling of lung architecture and fibrosis. Lipid mediators derived from phospholipids, sphingolipids, and polyunsaturated fatty acids play an important role in the pathogenesis of pulmonary fibrosis and have been described to exhibit pro- and anti-fibrotic effects in IPF and in preclinical animal models of lung fibrosis. This review describes the current understanding of the role and signaling pathways of prostanoids, lysophospholipids, and sphingolipids and their metabolizing enzymes in the development of lung fibrosis. Further, several of the lipid mediators and enzymes involved in their metabolism are therapeutic targets for drug development to treat IPF.

Cellular NAD+, fibroblast senescence and pulmonary fibrosis

Aging is a risk factor in fibrotic interstitial lung diseases, including idiopathic pulmonary fibrosis (IPF). Cellular senescence is observed in IPF and animal models of lung fibrosis along with telomere dysfunction, although the potential differential pathogenic significance of their cell type specific localization is unknown. Removal of senescent cells ameliorates fibrosis in animal models and are considered as potential therapeutic approaches. However the pathogenic role of aging and/or cellular senescence in the development of lung fibrosis are not fully understood. Declining cellular nicotinamide adenine dinucleotide (NAD+) levels as seen in aging, may impair the activity of senescence suppressor Sirt1, an NAD+‐dependent deacetylase, hence boosting the levels of NAD+ have been proposed to delay or inhibit aging related illnesses and improved longevity. To elucidate the role of NAD+ in cellular senescence in pathogenesis of pulmonary fibrosis, we investigated the impact of increasing or reducing NAD+ levels on bleomycin (BLM)‐induced mouse pulmonary fibrosis by supplementation with nicotinamide mononucleotide (NMN) by i.p injection, or nicotinamide phosphoribosyltransferase (Nampt) deficiency, respectively. The latter was induced by treating transgenic mice bearing the floxed Nampt and Col1a2‐CreERT transgenes with tamoxifen, resulting in mesenchymal cell specific Nampt deletion. The results showed that pre‐treatment of NMN injection before BLM injury significantly reduced lung myofibroblast differentiation as manifested by decreased Acta2 mRNA, which was accompanied by decreased numbers of senescent type II alveolar epithelial cells (SPC+/p16+), and BM‐derived monocytic myeloid cells. In contrast, deletion of Nampt in mesenchymal cells with consequent reduction of NAD+ levels caused increased myofibroblast differentiation, and more severe lung fibrosis measured by lung hydroxyproline and histology after BLM injury when compared to the response in wild type animals. In contrast to wild type cells, Nampt‐deficient lung fibroblasts exhibited elevated expression of p16, and senescence‐associated secretory phenotype (SASP) cytokines, including TNFα, IL‐1β, and IL‐6. Notably enhanced recruitment of BM‐derived myeloid cells into BLM‐injured lung was also observed. These findings suggest that NAD+ may play a significant role in prevention of cellular senescence and protection from pulmonary injury/fibrosis.Support or Funding InformationThis study is supported by NIH grants HL112880 and HL 138417.

Thymoquinone treatment modulates the Nrf2/HO-1 signaling pathway and abrogates the inflammatory response in an animal model of lung fibrosis

The present study investigates the therapeutic potential of thymoquinone (TQ) in bleomycin-induced lung fibrosis (BMILF) and elucidates the target-signaling pathway for its effect. Lung fibrosis was induced in rats by a single intra-tracheal instillation of bleomycin (BM) (6.5 U/kg) followed by thymoquinone treatment (10 and 20 mg/kg p.o.) for 28 days. Control rats received saline instead of TQ. Changes in body weight, inflammatory cells count, cytokines levels, and biochemical parameters of the broncho-alveolar lavage fluid (BALF) were recorded. In addition, a histopathology examination and western blotting were performed on lung tissues. BM administration resulted in a significant weight loss, which was ameliorated by TQ treatment. BMILF was associated with a reduction in the antioxidant mechanisms and increased lipid peroxidation. Furthermore, elevated levels of inflammatory cytokines, MMP-7 expression, apoptotic markers (caspase 3, Bax, and Bcl-2), and fibrotic changes including TGF-β and hydroxyproline levels in lung tissues were evident. These abnormalities were diminished with TQ treatment. Likewise, altered total and differential cell count in BALF was significantly improved in rats treated with TQ. TQ also produced a dose-dependent reduction in the expressions of Nrf2, Ho-1 and TGF-β. These results propose that the Nrf2/Ho-1 signaling pathway is a principal target for TQ protective effect against BMILF in rats. Furthermore, TQ decreases inflammatory oxidative stress possibly through the modulation of nuclear factor Kappa-B (NF-κB) and thereby minimization of collagen deposition in the lung. Therefore, TQ can be developed as a potential therapeutic modularity in BMILF for human use.

Bleomycin Induces Drug Efflux in Lungs. A Pitfall for Pharmacological Studies of Pulmonary Fibrosis.

ATP-binding cassette (ABC) transporters are evolutionarily conserved membrane proteins that pump a variety of endogenous substrates across cell membranes. Certain subfamilies are known to interact with pharmaceutical compounds, potentially influencing drug delivery and treatment efficacy. However, the role of drug resistance-associated ABC transporters has not been examined in idiopathic pulmonary fibrosis (IPF) or its animal model: the bleomycin (BLM)-induced murine model. Here, we investigate the expression of two ABC transporters, P-gp (permeability glycoprotein) and BCRP (breast cancer resistance protein), in human IPF lung tissue and two different BLM-induced mouse models of pulmonary fibrosis. We obtained human IPF specimens from patients during lung transplantation and administered BLM to male C57BL/6J mice either by oropharyngeal aspiration (1 U/kg) or subcutaneous osmotic infusion (100 U/kg over 7 d). We report that P-gp and BCRP expression in lungs of patients with IPF was comparable to controls. However, murine lungs expressed increased levels of P-gp and BCRP after oropharyngeal and subcutaneous BLM administration. We localized this upregulation to multiple pulmonary cell types, including alveolar fibroblasts, endothelial cells, and type 2 epithelial cells. Functionally, this effect reduced murine lung exposure to nintedanib, a U.S. Food and Drug Administration-approved IPF therapy known to be a P-gp substrate. The study reveals a discrepancy between IPF pathophysiology and the common animal model of lung fibrosis. BLM-induced drug efflux in the murine lungs may present an uncontrolled confounding variable in the preclinical study of IPF drug candidates, and these findings will facilitate disease model validation and enhance new drug discoveries that will ultimately improve patient outcomes.

Development of an Excipient-Free Peptide Dry Powder Inhalation for the Treatment of Pulmonary Fibrosis.

The caveolin scaffolding domain peptide (CSP) is being developed for the therapeutic intervention of a lethal lung disease, idiopathic pulmonary fibrosis. While direct respiratory delivery of CSP7 (a 7-mer fragment of CSP) is considered an effective route, proper formulation and processing of the peptide are required. First, air-jet milling technology was performed in order to micronize the neat peptide powder. Next, the fine particles were subjected to a stability study with physical and chemical characterizations. In addition, the in vivo efficacy of processed CSP7 powder was evaluated in an animal model of lung fibrosis. The results revealed that, with jet milling, the particle size of CSP7 was reduced to a mass median aerodynamic diameter of 1.58 ± 0.1 μm and 93.3 ± 3.3% fine particle fraction, optimal for deep lung delivery. A statistically significant reduction of collagen was observed in diseased lung tissues of mice that received CSP7 powder for inhalation. The particles remained chemically and physically stable after micronization and during storage. This work demonstrated that jet milling is effective in the manufacturing of a stable, excipient-free CSP7 inhalation powder for the treatment of pulmonary fibrosis.

Low‐dose administration of bleomycin leads to early alterations in lung mechanics

What is the central question of this study? When do alterations in pulmonary mechanics occur following chronic low-dose administration of bleomycin? What is the main finding and its importance? Remarkably, we report changes in lung mechanics as early as day7 that corresponded to parameters determined from single-frequency forced oscillation manoeuvres and pressure-volume loops. These changes preceded substantial histological changes or changes in gene expression levels. These findings are significant to refine drug discovery in idiopathic pulmonary fibrosis, where preclinical studies using lung function parameters would enhance the translational potential of drug candidates where lung function readouts are routinely performed in the clinic. Idiopathic pulmonary fibrosis (IPF) is the most widespread form of interstitial lung disease and, currently, there are only limited treatment options available. In preclinical animal models of lung fibrosis, the effectiveness of experimental therapeutics is often deemed successful via reductions in collagen deposition and expression of profibrotic genes in the lung. However, in clinical studies, improvements in lung function are primarily used to gauge the success of therapeutics directed towards IPF. Therefore, we examined whether changes in respiratory system mechanics in the early stages of an experimental model of lung fibrosis can be used to refine drug discovery approaches for IPF. C57BL/6J mice were administered bleomycin (BLM) or a vehicle control i.p. twice a week for 4weeks. At 7, 14, 21, 28 and 33days into the BLM treatment regimen, indices of respiratory system mechanics and pressure-volume relationships were measured. Concomitant with these measurements, histological and gene analyses relevant to lung fibrosis were performed. Alterations in respiratory system mechanics and pressure-volume relationships were observed as early as 7days after the start of BLM administration. Changes in respiratory system mechanics preceded the appearance of histological and molecular indices of lung fibrosis. Administration of BLM leads to early changes in respiratory system mechanics that coincide with the appearance of representative histological and molecular indices of lung fibrosis. Consequently, these data suggest that dampening the early changes in respiratory system mechanics might be used to assess the effectiveness of experimental therapeutics in preclinical animal models of lung fibrosis.

Nanoscale dysregulation of collagen structure-function disrupts mechano-homeostasis and mediates pulmonary fibrosis.

Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.

Fibrocytes in Human Pulmonary Fibrosis: Double-Blind Placebo-Controlled Crossover Pilot Study of Sirolimus in Idiopathic Pulmonary Fibrosis

SESSION TITLE: Diffuse Lung Disease I SESSION TYPE: Original Investigation Slide PRESENTED ON: Sunday, October 29, 2017 at 03:15 PM - 04:15 PM PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a progressive disease associated with poor prognosis. Fibrocytes are a novel population of bone marrow-derived circulating cells that traffic to the lungs and contribute to fibrosis in animal models of pulmonary fibrosis via the interaction of the chemokine CXCL12, made in the lung, and the chemokine receptor CXCR4, expressed on fibrocytes. In human interstitial lung disease, the concentration of CXCR4+ circulating fibrocytes correlates with survival. The expression of fibrocyte CXCR4 is dependent on the mTOR pathway, and is reduced by in vitro treatment with the mTOR inhibitor, sirolimus. In animal models of lung fibrosis, sirolimus therapy reduces both the trafficking of fibrocytes to the lungs and the extent of lung fibrosis. The effect of sirolimus in human IPF has not been examined. We hypothesized that therapy with the mTOR inhibitor sirolimus reduces the number of circulating fibrocytes in patients with idiopathic pulmonary fibrosis and is associated with an acceptable side-effect profile. METHODS: We performed a short-term randomized double-blinded placebo-controlled crossover study of 30 subjects diagnosed with IPF by consensus definition. Subjects were randomized to placebo or sirolimus for a 1-3 week run-in period with dose adjustment to achieve therapeutic drug levels, followed by 4 weeks of dose maintenance, and then a 4 week washout period. Subjects were then crossed over to the alternate treatment (placebo or sirolimus) for same length run-in, maintenance and washout periods. The concentration of circulating fibrocytes plasma chemokine ligands, pulmonary function tests, and adverse events were measured at specific time points. Adverse events were classified as serious per the NCI CTCAE criteria and were adjudicated as unrelated or possibly related to the study drug by an independent DSMB. RESULTS: We enrolled 30 subjects between 2011 and 2015; 2 withdrew prior to randomization. The remaining 28 had a median age of 69 (IQR 65-73), 22 were men and 8 were on concurrent therapy with pirfenidone or nintedanib. Sirolimus therapy resulted in a statistically significant reduction in the concentration of circulating fibrocytes (median change -62%, IQR -40% to -29%) whereas placebo did not (median change -32%; IQR -47% to +6%). A total of 49 adverse events occurred during treatment with sirolimus and 29 with placebo, of which 35 (2 serious: elevated liver enzymes and angioedema) were adjudicated as possibly related to therapy during sirolimus and 15 (1 serious: worsened dyspnoea) during placebo treatments. The incidence of total adverse events, serious adverse events, and total and serious adverse events that were potentially related to therapy did not differ significantly during therapy with drug and placebo. CONCLUSIONS: As compared with placebo, short-term treatment with sirolimus resulted in reduction of circulating fibrocyte concentrations in subjects with IPF and was associated with an acceptable safety profile. CLINICAL IMPLICATIONS: Given the benefit of sirolimus in animal models of pulmonary fibrosis and evidence of similar mechanism of action in human IPF in this study, future studies should assess the effect of long-term sirolimus treatment on the natural history of IPF. DISCLOSURE: The following authors have nothing to disclose: Dierdre Axell-House, Victor Yu, Zhimin Zhang, Marie Burdick, Robert Strieter, Borna Mehrad No Product/Research Disclosure Information

M10, a caspase cleavage product of the hepatocyte growth factor receptor, interacts with Smad2 and demonstrates antifibrotic properties in vitro and in vivo

Hepatocyte growth factor receptor, also known as cellular mesenchymal-epithelial transition factor (c-MET, MET), is an important antifibrotic molecule that protects various tissues, including lung, from injury and fibrosis. The intracellular cytoplasmic tail of MET contains a caspase-3 recognition motif "DEVD-T" that on cleavage by caspase-3 generates a 10-amino acid peptide, TRPASFWETS, designated as "M10". M10 contains at its N-terminus the uncharged amino acid proline (P) directly after a cationic amino acid arginine (R) which favors the transport of the peptide through membranes. M10, when added to cell culture medium, remains in the cytoplasm and nuclei of cells for up to 24 hours. M10 effectively decreases collagen in both scleroderma and TGFβ-stimulated normal lung and skin fibroblasts. M10 interacts with the Mad Homology 2 domain of Smad2 and inhibits TGFβ-induced Smad2 phosphorylation, suggesting that the antifibrotic effects of M10 are mediated in part by counteracting Smad-dependent fibrogenic pathways. In the bleomycin murine model of pulmonary fibrosis, M10 noticeably reduced lung inflammation and fibrosis. Ashcroft fibrosis scores and lung collagen content were significantly lower in bleomycin-treated mice receiving M10 as compared with bleomycin-treated mice receiving scrambled peptide. We conclude that M10 peptide interacts with Smad2 and demonstrates strong antifibrotic effects in vitro and in vivo in an animal model of lung fibrosis and should be considered as a potential therapeutic agent for systemic sclerosis and other fibrosing diseases.

Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease characterised by fibrosis of the lung parenchyma and loss of lung function. Although the pathogenic pathways involved in IPF have not been fully elucidated, IPF is believed to be caused by repetitive alveolar epithelial cell injury and dysregulated repair, in which there is uncontrolled proliferation of lung fibroblasts and differentiation of fibroblasts into myofibroblasts, which excessively deposit extracellular matrix (ECM) proteins in the interstitial space. A number of profibrotic mediators including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and transforming growth factor-β are believed to play important roles in the pathogenesis of IPF. Nintedanib is a potent small molecule inhibitor of the receptor tyrosine kinases PDGF receptor, FGF receptor and vascular endothelial growth factor receptor. Data from in vitro studies have shown that nintedanib interferes with processes active in fibrosis such as fibroblast proliferation, migration and differentiation, and the secretion of ECM. In addition, nintedanib has shown consistent anti-fibrotic and anti-inflammatory activity in animal models of lung fibrosis. These data provide a strong rationale for the clinical efficacy of nintedanib in patients with IPF, which has recently been demonstrated in phase III clinical trials.

Noninvasive imaging of experimental lung fibrosis.

Small animal models of lung fibrosis are essential for unraveling the molecular mechanisms underlying human fibrotic lung diseases; additionally, they are useful for preclinical testing of candidate antifibrotic agents. The current end-point measures of experimental lung fibrosis involve labor-intensive histological and biochemical analyses. These measures fail to account for dynamic changes in the disease process in individual animals and are limited by the need for large numbers of animals for longitudinal studies. The emergence of noninvasive imaging technologies provides exciting opportunities to image lung fibrosis in live animals as often as needed and to longitudinally track the efficacy of novel antifibrotic compounds. Data obtained by noninvasive imaging provide complementary information to histological and biochemical measurements. In addition, the use of noninvasive imaging in animal studies reduces animal usage, thus satisfying animal welfare concerns. In this article, we review these new imaging modalities with the potential for evaluation of lung fibrosis in small animal models. Such techniques include micro-computed tomography (micro-CT), magnetic resonance imaging, positron emission tomography (PET), single photon emission computed tomography (SPECT), and multimodal imaging systems including PET/CT and SPECT/CT. It is anticipated that noninvasive imaging will be increasingly used in animal models of fibrosis to gain insights into disease pathogenesis and as preclinical tools to assess drug efficacy.

Role of Bcl-xL in hepatocyte growth factor-elicited epithelial protection in idiopathic pulmonary fibrosis

Hepatocyte growth factor (HGF) is a cytokine with pleiotropic functions during wound healing and repair. Its anti-fibrotic effects were shown in animal models of lung fibrosis and linked to improved cellular survival and proliferation and reduced myofibroblast accumulation. HGF-elicited, pro-survival pathways have yet not been investigated in detail in lung epithelial cells. Based on literature, our study is focused on Bcl-xL, pro-survival protein involved in mitochondrial control of apoptosis.

Mesenchymal Stem Cells and Idiopathic Pulmonary Fibrosis. Potential for Clinical Testing

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating, and fatal lung disease characterized by interstitial fibrosis with decreasing lung volumes and hypoxemic respiratory failure. The prognosis for patients with IPF is poor and the quest to find effective therapies has been unsuccessful. Despite several clinical trials over the past decade, there are no U.S. Food and Drug Administration-approved treatments for patients with IPF and thus no standard of care. In terms of pathogenesis, IPF is characterized by alveolar epithelial cell injury and activation with interstitial inflammation, fibroblast proliferation with extracellular matrix collagen deposition, and loss of lung function. Because mesenchymal stem cells (MSCs) home to sites of injury, inhibit inflammation, and contribute to epithelial tissue repair, their use has been suggested as a therapy for the treatment of IPF. MSCs have potential as a novel therapeutic agent in multiple diseases and they have been safely administered in a number of clinical trials. Some, but not all, preclinical studies in animal models of lung fibrosis suggest that MSCs might be effective in the treatment of IPF. Given the safety and ease of MSC administration in other patient populations, the results in preclinical animal models of IPF, and the major need for novel therapeutic options in this devastating disease, we propose that carefully designed clinical trials of MSCs for the treatment of patients with IPF are appropriate. Establishing safety in the setting of IPF is the first priority in early clinical trials followed by clinical and biological measures of efficacy.

Therapeutic efficacy of Sunitinib and other broad spectrum receptor tyrosine kinase inhibitors (RTKI) in bleomycin-induced pulmonary fibrosis

Platelet-derived growth factor (PDGF) has ample evidence to support its importance in pulmonary fibrosis. Expression of PDGF in the lungs of IPF patients as well as in BAL samples from animal models of lung fibrosis support its involvement during disease development. Vascular endothelial growth factor (VEGF) being a key regulator of angiogenesis and its receptors have been shown to be involved in the pathogenesis of pulmonary sarcoidosis and fibrosis. The hypothesis under investigation is that receptor tyrosine kinase inhibitors (e.g. Sutent) will slow both the fibrotic processes associated with PDGF signaling and the angiogenic processes regulated by VEGF. We tested two RTKi compounds from Pfizer’s internal portfolio (SU-11248 (Sutent), SU-14813) in the bleomycin-induced pulmonary fibrosis mouse model. Prophylactic as well as therapeutic dosing protocols were conducted. Endpoints included histology, body weights, wet lung weights, lung HO-Pro levels and tissue and serum samples for biomarker and lung exposure analyses. Statistically significant reductions in histological changes were demonstrated in lungs treated with both RTK inhibitors in both percentage of total collagen and inflammatory collagen present. RTK inhibitors demonstrated a statistically significant reduction in both wet lung weight and lung HO-Pro content when dosed prophylactically and therapeutically compared to controls. We have demonstrated that broad-spectrum RTK inhibition is efficacious in inhibiting established pulmonary fibrosis in the bleomycin-induced mouse model. Sutent and other broad spectrum RTKis may provide therapeutic benefit to IPF patients by slowing both the fibrotic and angiogenic processes regulated by tyrosine kinases.