Total Lung Collagen Research Articles

A novel BRD4 degrader, ARV-825, attenuates lung fibrosis through senolysis and antifibrotic effect

BackgroundRecent studies suggest that cellular senescence is related to the pathogenesis of idiopathic pulmonary fibrosis. However, cellular senescence has yet to be targeted therapeutically in clinical practice. ARV825, a recently developed BRD4 degrader, has been reported as a novel senolytic drug. Conversely, it has also been reported that BRD4 regulates the pro-fibrotic gene expression of fibroblasts. Therefore, this study focuses on the senolytic and anti-fibrotic effects of ARV825 and evaluated these effects on lung fibrosis. MethodsLung fibroblasts were induced to senescence through serial passage. The expression of senescence markers and pro-fibrotic markers were determined through quantitative PCR or immunoblot analysis. Lung fibrosis was induced in mice through intratracheal administration of bleomycin. Mice treated with ARV825 underwent histological analysis of lung fibrosis using the Ashcroft score. Total lung collagen was quantified through a hydroxyproline assay. Respiratory mechanics analysis was performed using the flexiVent system. ResultsFor senescent cells, ARV825 induced the expression of an apoptosis marker while reducing the expression of BRD4 and senescence markers. On the other hand, for early passage pre-senescent cells, ARV825 reduced the expression of collagen type 1 and α-smooth muscle actin. In an experimental mouse model of lung fibrosis, ARV825 attenuated lung fibrosis and improved lung function. Immunohistochemical staining revealed a significant decrease in the number of senescent alveolar type 2 cells in lung tissue due to ARV825 treatment. ConclusionsThese results suggest that ARV825 may impact the progressive and irreversible course of fibrotic lung diseases.

449 Progression of silica-induced pulmonary fibrosis is arrested after selective ablation of Col1a1+ fibroblasts

OBJECTIVES/GOALS: Silicosis is a highly fatal progressive fibrotic disease of the lungs characterized by accumulation and persistence of fibroblasts that excessively deposit Collagen1a1. We sought to eliminate Collagen1a1-expressing fibroblasts through a targeted genetic ablation strategy and hypothesized that this would arrest the progression of Silicosis. METHODS/STUDY POPULATION: Silicosis was induced with a single intratracheal (i.t.) instillation of silica particles ( RESULTS/ANTICIPATED RESULTS: Targeted ablation of Col1a1+ fibroblast in established Silicosis resulted in a decrease in: 1) Col1a1+ fibroblasts by flow cytometry and within fibrotic nodules by immunofluorescent staining, 2) total lung collagen content by histology and hydroxyproline assay, 3) tissue-associated disease by microCT and an increase in arterial oxygen saturation by pulse oximetry. Cessation of targeted Col1a1+ fibroblast ablation resulted in a rebound effect in Silicosis disease progression. Following ablation, Col1a1+ fibroblasts expanded by proliferation (Ki67+) and total lung collagen levels returned to pre-ablation levels. DISCUSSION/SIGNIFICANCE: Silicosis is a often fatal disease with no FDA approved therapies. These results suggest that targeted loss of Col1a1+ fibroblasts in Silicosis is sufficient to arrest disease progression. Thus, it is essential to understand how targeted loss of pro-fibrotic fibroblasts can alter disease progression as a tool to develop novel therapeutic strategies.

Therapeutic Efficacy of Weissella cibaria CMU and CMS1 on Allergic Inflammation Exacerbated by Diesel Exhaust Particulate Matter in a Murine Asthma Model.

Background and Objectives: Diesel exhaust particulate matter (DEPM) is an air pollutant that is associated with asthma. In this study, the therapeutic efficacy of Weissella cibaria strains CMU (Chonnam Medical University) and CMS (Chonnam Medical School) 1, together with the drug Synatura, an anti-tussive expectorant, was investigated in a murine asthma model exacerbated by DEPM. Materials and Methods: BALB/c mice were sensitized with ovalbumin (OVA) before intranasal challenge with OVA and DEPM. W. cibaria CMU, CMS1, and Synatura were administered orally for 21 days. Results: Neither Synatura nor W. cibaria strains affected spleen, liver, or lung weights. W. cibaria strains CMU and CMS1 significantly reduced the levels of interleukin (IL)-4, OVA-specific immunoglobulin E (IgE), and total lung collagen in bronchoalveolar lavage fluid (BALF), similar to those with Synatura, regardless of the oral dose concentration (p < 0.05). In addition, the W. cibaria CMU strain significantly alleviated IL-1β, IL-6, IL-12, monocyte chemotactic protein-1, and tumor necrosis factor-α in BALF, whereas the CMS1 strain significantly alleviated IL-10 and IL-12 in BALF (p < 0.05); however, Synatura did not show any statistical efficacy against them (p > 0.05). All concentrations of W. cibaria CMU and low concentrations of W. cibaria CMS1 significantly reduced lung bronchiolar changes and inflammatory cell infiltration. Conclusions: In conclusion, W. cibaria CMU in asthmatic mice showed better efficacy than W. cibaria CMS1 in improving asthma exacerbated by DEPM exposure, as well as better results than pharmaceuticals.

Beneficial effects of N-acetylcysteine on protease-antiprotease balance in attenuating bleomycin-induced pulmonary fibrosis in rats.

Objective(s):The role of N-acetylcysteine (NAC) as an anti-oxidant in attenuating bleomycin-induced pulmonary fibrosis has been reported. However, its effect on parenchymal remodeling via regulating the protease-antiprotease balance is not fully defined. Therefore, the present study was designed to explore the possible role of matrix metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP) and transforming growth factor-β1 (TGF-β1) pathway and their modulation by NAC in attenuating bleomycin-induced pulmonary fibrosis in rats.Materials and Methods:Bleomycin sulphate (7 units/kg) was instilled inside the trachea to induce pulmonary fibrosis. The time course of TGF-β1, MMP-9, TIMP-1,3 mRNA and protein expression, TGF-β1 and hydroxyproline levels were evaluated on days 7, 14, and 28. NAC (0.3 mmol/kg and 3 mmol/kg) was administered in bleomycin-instilled animals.Results:NAC treatment significantly attenuated bleomycin-induced histopathological changes by decreasing interstitial inflammation and reducing the deposition of extracellular matrix proteins such as collagen. Moreover, it increased the mRNA and protein expression of MMP-9 and decreased the expression of TIMP-1,3 in alveolar epithelial cells (AECs), interstitial macrophages and inflammatory cells. Indeed, there was decrease in the MMP-9/TIMP ratio in bleomycin-instilled rats, which increased with NAC treatment. Moreover, NAC attenuated bleomycin-induced increased expression of TGF-β1 and total lung collagen levels.Conclusion:NAC attenuates bleomycin-induced pulmonary fibrosis by normalizing the protease-antiprotease balance and favoring the degradation of collegen to reduce fibrosis.

Metformin protects against bleomycin-induced pulmonary fibrosis in mice

Objective: To explore the effect of metformin on murine model of bleomycin (BLM)-induced lung injury and fibrosis. Methods: A total of 30 mice were divided into 3 groups: control, BLM, and BLM with metformin, in accordance with the random number table and each group had 10 mice. To induce the pulmonary fibrosis model, a concentration of 2 mg/ml bleomycin was intratracheally administered in the BLM group and BLM with metformin group with a volume of 1.75 μl/g, while the control group accepted saline with the same volume. Metformin (200 mg/kg) was given to the mice orally once a day from the day before intratracheal instillation of bleomycin to day 14. The daily survival condition of mice was recorded during 14 days. At day 14, HE-staining was used to assess the severity of fibrosis according to the method proposed by Ashcroft. Total lung collagen content was determined by hydroxyproline assay and Masson's trichrome staining. To examine the expression of fibronectin we used the method of immunohistochemistry staining. The changes of Transforming Growth Factor beta 1 (TGF-β(1)) in plasm, bronchoalveolar lavage fluid (BALF) and lung were measured by ELISA. Results: The survival rates of control group, BLM group and BLM with metformin group at day 14 were 10/10, 4/10 and 7/10 respectively. According to the method proposed by Ashcroft the score of metformin treated mice was significantly lower than that of the bleomycin model mice[(3.82±0.58) vs (7.79±0.06), (P<0.05)]. The hydroxyproline level in lung tissue were markedly attenuated in metformin treated mice compared with bleomycin model mice [(0.40±0.05) vs (0.73±0.10) μg/mg, (P<0.05)]. The level of TGF-β(1) in plasma, BALF and lung tissue were also decreased in mice treated with metformin compared with bleomycin model mice [(2.32±0.68) vs (4.59±0.45) ng/ml, (0.81±0.09) vs (1.40±0.06) ng/ml, (17.12±0.83) vs (21.25±0.69) ng/mg, all P<0.05]. Conclusion: Metformin can reduce the severity of pulmonary fibrosis in mice induced by bleomycin.

Men are from Mars, Women are from Venus. Gender specific changes in pulmonary function in 1 year old rats after neonatal hyperoxia exposure

RationalePreterm birth affects about 11% of pregnancies worldwide. The Institute of Medicine estimated the cost associated with premature birth to be $36 billion in 2016. Lungs in preterm infants are in the critical saccular stage of lung development and susceptible to the oxygen‐induced chronic lung disease known as bronchopulmonary dysplasia (BPD). BPD is characterized by functional abnormalities including decreased saccular/alveolar septation, decreased microvascular cross‐sectional area with or without pulmonary hypertension, airway injury with or without increased airway reactivity, and abnormalities of control of breathing in various combinations. While the initial inflammation improves, chronic inflammation ensues for a long time. We tested how neonatal hyperoxia exposure affects lung function one year after birth. We postulated an increase in lung collagen levels reflected by reduced pulmonary function.Methods6 newborn male and female rats in each group were exposed to Hyperoxia (Hx; 85% O2) or Normoxia (Nx; 21% O2) for 14 days, then aged to 1 year in Nx. At one year, lung function was measured using a FlexiVent (Scireq, Montreal, Canada). Using a single frequency forced oscillation manoeuvre and a low‐frequency forced oscillation technique the lung was tested for compliance and resistance. Total lung collagen was determined by a colorimetric assay using Ehrlich's reagent.ResultsAnalysis of one year old rats showed no difference in weight and body length between the Nx and the Hx group within each gender. The inspiratory capacity (IC) was significantly higher in the Hx exposed male rats compared to the Nx exposed male rats (12.6±0.6 ml vs 14.0±0.9 ml; p=0.02). The difference in IC in female rats was significantly lower in the Hx group compared to the Nx group (11.3±0.4 vs 10.1±0.6 ml; p=0.002). Dynamic compliance was significantly increased in the male Hx group compared to the male Nx group (1.43±0.05 vs 1.61±0.11 ml/cm H2O; p=0.04). In contrast, the female Hx group showed significantly reduced compliance compared to the female Nx group (1.32±0.05 vs 1.15±0.17 ml/cm H2O). In addition, the PV loop area was higher in the male Hx group compared to the male Nx group and no difference was found between the two female groups. Total collagen in lung tissue was not different between the male Hx and Nx groups but was higher in the female Hx compared to the Nx group.ConclusionOne year after neonatal Hx exposure, the male lung shows increased compliance and inspiratory capacity compared to the Nx exposed rats suggesting a trend to emphysema. In contrast, female rats show signs of lung fibrosis reflected by reduced compliance and increased total collagen content. This data suggests that neonatal oxygen exposure initiates alterations in inflammatory and repair pathways that are different between male and female rats.Support or Funding InformationNHLBI: R01 HL115061‐01 (Eldridge)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intranasal administration of mesenchymoangioblast‐derived mesenchymal stem cells abrogates airway fibrosis and airway hyperresponsiveness associated with chronic allergic airways disease

Structural changes known as airway remodeling (AWR) characterize chronic/severe asthma and contribute to lung dysfunction. Thus, we assessed the in vivo efficacy of induced pluripotent stem cell and mesenchymoangioblast-derived mesenchymal stem cells (MCA-MSCs) on AWR in a murine model of chronic allergic airways disease (AAD)/asthma. Female Balb/c mice were subjected to a 9-wk model of ovalbumin (Ova)-induced chronic AAD and treated intravenously or intranasally with MCA-MSCs from weeks 9 to 11. Changes in airway inflammation (AI), AWR, and airway hyperresponsiveness (AHR) were assessed. Ova-injured mice presented with AI, goblet cell metaplasia, epithelial thickening, increased airway TGF-β1 levels, subepithelial myofibroblast and collagen accumulation, total lung collagen concentration, and AHR (all P < 0.001 vs. uninjured control group). Apart from epithelial thickness, all other parameters measured were significantly, although not totally, decreased by intravenous delivery of MCA-MSCs to Ova-injured mice. In comparison, intranasal delivery of MCA-MSCs to Ova-injured mice significantly decreased all parameters measured (all P < 0.05 vs. Ova group) and, most notably, normalized aberrant airway TGF-β1 levels, airway/lung fibrosis, and AHR to values measured in uninjured animals. MCA-MSCs also increased collagen-degrading gelatinase levels. Hence, direct delivery of MCA-MSCs offers great therapeutic benefit for the AWR and AHR associated with chronic AAD.-Royce, S. G., Rele, S., Broughton, B. R. S., Kelly, K., Samuel, C. S. Intranasal administration of mesenchymoangioblast-derived mesenchymal stem cells abrogates airway fibrosis and airway hyperresponsiveness associated with chronic allergic airways disease.

Abstract 346: Fatty acid synthase is a risk factor for bleomycin-induced lung fibrosis in mice

Abstract Pulmonary fibrosis is a significant complication in cancer patients treated with radiation or various chemotherapeutic agents. One such reagent, bleomycin, is used to treat a variety of cancers including, but not limited to, lymphoma, ovarian cancer, and melanoma. Its efficacy, however, is impacted by the incidence of significant pulmonary fibrosis, which often becomes dose limiting. In studies investigating the role(s) of metabolism in the profibrotic actions of transforming growth factor beta, we previously determined that increased fatty acid synthase (FASN) expression was observed in the fibrotic areas of lungs from idiopathic pulmonary fibrosis (IPF) patients compared to healthy controls. In that FASN levels are significantly increased in numerous human tumors including breast, prostate, colorectal, and ovarian, we wished to integrate these findings by examining whether inhibiting FASN activity could reduce pulmonary fibrosis. To that end, the cerulenin-derived FASN inhibitor C75 was tested in a murine treatment model of bleomycin-induced lung fibrosis. Following addition of C75, there was significant diminution in a number of profibrotic targets including collagen 1α1, fibronectin, connective tissue growth factor, and total lung collagen as assessed by hydroxyproline content. Not only was there a C75 dose-dependent decrease in the aforementioned targets, but lung function as assessed by peripheral blood oxygenation (on room air) was also stabilized/improved. Thus, FASN inhibition might reflect a possible target to maintain normal lung physiology in the context of cancer chemotherapy. This work was supported by Public Health Service grants GM-055816 and GM-054200 from the National Institutes of General Medical Sciences. Citation Format: Mi-Yeon Jung. Fatty acid synthase is a risk factor for bleomycin-induced lung fibrosis in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 346. doi:10.1158/1538-7445.AM2017-346

Effect of water-soluble C(60) fullerenes on pulmonary fibrosis induced by bleomycin in mice

Objective: To evaluate the role of water-soluble C(60) fullerenes in mice model of lung injury and fibrosis that induced by bleomycin. Methods: A total of 20 healthy C57BL/6J mice were randomly divided into normal control group, bleomycin group, high dose C(60) group, low dose C(60) group, each group with 5 mice. Mice were induced pulmonary fibrosis by intratracheal injection of bleomycin except the normal control group, which was induced by saline instead. In low dose C(60) group and high dose C(60) group, 1 mg·kg(-1)·d(-1) and 10 mg·kg(-1)·d(-1) water-soluble C(60) fullerenes was injected into mice intraperitoneally every day, which began from one day before intratracheal instillation of bleomycin until the end of observation. Saline was given to mice in the same way in normal control and bleomycin group. This study investigated the variation of weight and survival rate of mice for 14 d. HE-staining and Masson's trichrome staining were used to assess the severity of fibrosis according to the method proposed by Ashcroft at 14th day. Total lung collagen content was determined by hydroxyproline assay. The changes of transforming growth factor-β(1) (TGF-β(1)) and tumor necrosis factor α (TNF-α) in plasma, bronchial alveolar lavage fluid (BALF) and lung tissue were measured by enzyme-linked immunosorbent assay (ELISA). And, the amount of reactive oxygen species (ROS) was tested by 2, 7-dichlorofuorescin diacetate (DCFH-DA), and determined by the ratio of fluorescence intensity and protein content (OD/μg). Results: C(60) can protect mice that injured by bleomycin from weight loss. According the method proposed by Ashcroft et al.HE and Masson's trichrome staining showed that collagen deposition in lung tissue were markedly attenuated in C(60) (1 mg·kg(-1)·d(-1) and 10 mg·kg(-1)·d(-1)) treated mice compared with bleomycin model mice[(4.08±0.52), (3.00±0.41) vs (6.75±0.75) points, both P<0.01]. In low dose C(60) group and high dose C(60) group, the content of hydroxyproline in lung tissue were significantly lower than that in bleomycin group[(0.36±0.06), (0.35±0.08) vs (0.55±0.16) μg/mg, both P<0.05]. The level of TGF-β(1) in BALF and lung tissue were also decreased in mice treated with C(60) (10 mg·kg(-1)·d(-1)) compared with bleomycin model mice, but the difference had no statistical significance[(9.38±5.32) vs (23.60±8.96) pg/ml, (2.89±0.35) vs (6.44±2.95) pg/mg, both P>0.05]. Also, in high dose C(60) group, the content of TNF-α in plasma, BALF and lung tissue were significantly lower than those in bleomycin group[(4.56±0.73) vs (7.21±2.26) pg/ml, (34.58±23.30) vs (151.00±27.34) pg/ml, (22.99±5.83) vs (122.90±22.04) pg/mg, all P<0.05]. In addition, Compared with bleomycin group, ROS in lung tissue was significantly decreased after treatment with C(60) (10 mg·kg(-1)·d(-1))[(19.68±0.91) vs (22.92±1.71) OD/μg, P<0.05]. Conclusion: Water-soluble C(60) fullerenes reduce the severity of pulmonary fibrosis induced by bleomycin in mice.

Effect of nintedanib on airway inflammation and remodeling in a murine chronic asthma model

ABSTRACTIntroduction: Nintedanib is a multi-tyrosine kinase receptor inhibitor recently approved for treatment of idiopathic pulmonary fibrosis. Although angiogenesis is a key process involved in airway structural changes in patients with bronchial asthma, the effect of nintedanib targeting the angiokinase pathway on airway inflammation and remodeling has not been evaluated. Methods: We used a 3-month ovalbumin (OVA) challenge mouse model of airway remodeling. Nintedanib was orally administrated during the challenge period, and the effects were examined based on the percentage of airway inflammatory cells, airway hyper-reactivity (AHR), peribronchial goblet cell hyperplasia, total lung collagen and smooth muscle area. The expression of growth factor receptors was analyzed in mice lung tissues. Results: The OVA challenged group showed a significant increase in airway eosinophilic inflammation, elevated Th2 cytokines, AHR, and airway remodeling compared to those in the control group. The airway remodeling process, as evaluated by goblet cell hyperplasia, total lung collagen level, and airway smooth muscle area, was suppressed by nintedanib compared to that by OVA. Nintedanib effectively suppressed the phosphorylation of vascular endothelial growth factor/ platelet derived growth factor subunit2/fibroblast growth factor3 receptors in the mice lung. Conclusions: Nintedanib effectively ameliorated airway inflammation and remodeling in an OVA-induced chronic asthma model. These results suggest that nintedanib could be a new treatment agent targeting airway remodeling in patients with severe asthma.

Chronic intermittent hypoxia worsens bleomycin-induced lung fibrosis in rats

Obstructive sleep apnea (OSA) has been linked to increased mortality in pulmonary fibrosis. Its key feature, chronic intermittent hypoxia (CIH), can lead to oxidative stress and inflammation, known to lead to fibrotic pathology in other organs. We tested the effects of CIH in an animal model of bleomycin-induced lung fibrosis. Sprague Dawley rats were instilled intratracheally with bleomycin (Blm) or saline (Sal), and exposed to CIH or normal air (Norm) for 9 or 30 days. Pulmonary function was tested and lungs were harvested for histological and molecular analyses.In Blm-treated animals, 30days of CIH compared to Norm increased total lung collagen content (p=0.008) and reduced Quasi-static lung compliance (p=0.04). CIH upregulated lipid peroxidation and increased NF-κB activation, IL-17 mRNA and Col1α1 mRNA expression. Our results indicate that following Blm-induced lung injury, CIH amplifies collagen deposition via oxidative and inflammatory pathways, culminating in stiffer lungs. Thus, OSA may augment fibrosis in patients with interstitial lung disease.

Unique and Redundant Functions of p70 Ribosomal S6 Kinase Isoforms Regulate Mesenchymal Cell Proliferation and Migration in Pulmonary Fibrosis.

The p70 ribosomal S6 kinase (p70S6K) is a downstream substrate that is phosphorylated and activated by the mammalian target of rapamycin complex and regulates multiple cellular processes associated with pulmonary fibrogenesis. Two isoforms of the p70S6K have been identified (S6K1 and S6K2), but their relative contributions in mediating pulmonary fibrosis are unknown. To interrogate the roles of the p70S6K isoforms, we overexpressed transforming growth factor (TGF)-α in mice deficient for the S6K1 or S6K2 genes and measured changes in lung histology, morphometry, total lung collagen, lung function, and proliferation between wild-type and isoform-deficient mice. Deficiency of S6K1, but not S6K2, had a significant effect on reducing proliferation in subpleural fibrotic lesions during TGF-α-induced fibrosis. Migration was significantly decreased in mesenchymal cells isolated from the lungs of S6K1 knockout mice compared with wild-type or S6K2 knockout mice. Conversely, increases in subpleural thickening were significantly decreased in S6K2-deficient mice compared with wild type. Deficiency of S6K2 significantly reduced phosphorylation of the downstream S6 ribosomal protein in lung homogenates and isolated mesenchymal cells after TGF-α expression. However, deficiency of neither isoform alone significantly altered TGF-α-induced collagen accumulation or lung function decline in vivo. Furthermore, deficiency in neither isoform prevented changes in collagen accumulation or lung compliance decline after administration of intradermal bleomycin. Together, these findings demonstrate that the p70S6K isoforms have unique and redundant functions in mediating fibrogenic processes, including proliferation, migration, and S6 phosphorylation, signifying that both isoforms must be targeted to modulate p70S6K-mediated pulmonary fibrosis.

Secretory leukocyte protease inhibitor gene deletion alters bleomycin-induced lung injury, but not development of pulmonary fibrosis

Idiopathic pulmonary fibrosis is a progressive, fatal disease with limited treatment options. Protease-mediated transforming growth factor-β (TGF-β) activation has been proposed as a pathogenic mechanism of lung fibrosis. Protease activity in the lung is tightly regulated by protease inhibitors, particularly secretory leukocyte protease inhibitor (SLPI). The bleomycin model of lung fibrosis was used to determine the effect of increased protease activity in the lungs of Slpi−/− mice following injury. Slpi−/−, and wild-type, mice received oropharyngeal administration of bleomycin (30 IU) and the development of pulmonary fibrosis was assessed. Pro and active forms of matrix metalloproteinase (MMP)-2 and MMP-9 were measured. Lung fibrosis was determined by collagen subtype-specific gene expression, hydroxyproline concentration, and histological assessment. Alveolar TGF-β activation was measured using bronchoalveolar lavage cell pSmad2 levels and global TGF-β activity was assessed by pSmad2 immunohistochemistry. The active-MMP-9 to pro-MMP-9 ratio was significantly increased in Slpi−/− animals compared with wild-type animals, demonstrating enhanced metalloproteinase activity. Wild-type animals showed an increase in TGF-β activation following bleomycin, with a progressive and sustained increase in collagen type I, alpha 1 (Col1α1), III, alpha 1(Col3α1), IV, alpha 1(Col4α1) mRNA expression, and a significant increase in total lung collagen 28 days post bleomycin. In contrast Slpi−/− mice showed no significant increase of alveolar TGF-β activity following bleomycin, above their already elevated levels, although global TGF-β activity did increase. Slpi−/− mice had impaired collagen gene expression but animals demonstrated minimal reduction in lung fibrosis compared with wild-type animals. These data suggest that enhanced proteolysis does not further enhance TGF-β activation, and inhibits sustained Col1α1, Col3α1, and Col4α1 gene expression following lung injury. However, these changes do not prevent the development of lung fibrosis. Overall, these data suggest that the absence of Slpi does not markedly modify the development of lung fibrosis following bleomycin-induced lung injury.

Combining an epithelial repair factor and anti-fibrotic with a corticosteroid offers optimal treatment for allergic airways disease.

We evaluated the extent to which individual versus combination treatments that specifically target airway epithelial damage [trefoil factor-2 (TFF2)], airway fibrosis [serelaxin (RLX)] or airway inflammation [dexamethasone (DEX)] reversed the pathogenesis of chronic allergic airways disease (AAD). Following induction of ovalbumin (OVA)-induced chronic AAD in 6–8 week female Balb/c mice, animals were i.p. administered naphthalene (NA) on day 64 to induce epithelial damage, then received daily intranasal administration of RLX (0.8 mg·mL(−1)), TFF2 (0.5 mg·mL(−1)), DEX (0.5 mg·mL(−1)), RLX + TFF2 or RLX + TFF2 + DEX from days 67–74. On day 75, lung function was assessed by invasive plethysmography, before lung tissue was isolated for analyses of various measures. The control group was treated with saline + corn oil (vehicle for NA). OVA + NA-injured mice demonstrated significantly increased airway inflammation, airway remodelling (AWR) (epithelial damage/thickness; subepithelial myofibroblast differentiation, extracellular matrix accumulation and fibronectin deposition; total lung collagen concentration), and significantly reduced airway dynamic compliance (cDyn). RLX + TFF2 markedly reversed several measures of OVA + NA-induced AWR and normalized the reduction in cDyn. The combined effects of RLX + TFF2 + DEX significantly reversed peribronchial inflammation score, airway epithelial damage, subepithelial extracellular matrix accumulation/fibronectin deposition and total lung collagen concentration (by 50–90%) and also normalized the reduction of cDyn. Combining an epithelial repair factor and anti-fibrotic provides an effective means of treating the AWR and dysfunction associated with AAD/asthma and may act as an effective adjunct therapy to anti-inflammatory corticosteroids

Radix puerariae extracts ameliorate paraquat-induced pulmonary fibrosis by attenuating follistatin-like 1 and nuclear factor erythroid 2p45-related factor-2 signalling pathways through downregulation of miRNA-21 expression.

BackgroundPuerarin, extracted from Radix puerariae, was reported to ameliorate airway inflammation, lung injury and lung fibrosis induced by paraquat (PQ) in mice. However, effects of Radix puerariae extracts (RPEs) on lung fibrosis or signalling pathways in PQ-induced lung injury have not been well studied. Therefore, the goals of our study were to investigate whether Radix puerariae extracts are antifibrotic in a paraquat (PQ) induced lung fibrosis model in mice and to propose possible mechanisms of action of the RPE effects.MethodsWe used a long-term exposure model of PQ-induced lung fibrosis in mice to evaluate effects of antioxidant-containing RPE. We examined effects of miR-21 on follistatin-like 1 (Fstl 1) pathways and oxidative stress in the lung. Gene expression levels of miR-21, Fstl 1, transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), collagen-1 and collagen III were measured by real-time PCR. Protein expression levels of Fstl 1(FSTL1), heme oxygenase-1 (HO-1), nuclear factor erythroid 2p45-related factor-2 (Nrf2), Smad2/3, p38MAPK, nuclear factor-κB 65 (NF-κB65), and matrix metalloproteinase-9 were detected by western blotting. FSTL1 andalpha-smooth muscle actin (α-SMA) in lung tissue were detected by immunohistochemistry. Malondialdehyde, superoxide dismutase (SOD), reduced (GSH) and oxidised (GSSH) glutathione and reactive oxygen species levels, hydroxyproline and total lung collagen were also determined.ResultsLong-term challenge with PQ enhanced miRNA-21 (miR-21), Fstl 1 pathways, oxidative stress and development of fibrotic features in the lungs. RPE reduced features of lung fibrosis by blocking Fstl 1 pathways and oxidative stress through decreased miR-21 expression. This was accompanied by suppression of CTGF, TGF-β1, vascular endothelial growth factor, collagen I, and collagen III. In addition, PQ-induced activation of NF-κB, Nrf2 and α-SMA were enhanced by puerarin. We also found that puerarin increased HO-1, SOD and GSH levels.ConclusionsThese findings demonstrated that RPEs blocked PQ-induced Fstl 1 pathways and oxidative stress by inhibiting miR-21 expression, leading to attenuation of PQ-induced lung fibrosis.

Mesenchymal stem cells and serelaxin synergistically abrogate established airway fibrosis in an experimental model of chronic allergic airways disease

This study determined if the anti-fibrotic drug, serelaxin (RLN), could augment human bone marrow-derived mesenchymal stem cell (MSC)-mediated reversal of airway remodeling and airway hyperresponsiveness (AHR) associated with chronic allergic airways disease (AAD/asthma). Female Balb/c mice subjected to the 9-week model of ovalbumin (OVA)-induced chronic AAD were either untreated or treated with MSCs alone, RLN alone or both combined from weeks 9–11. Changes in airway inflammation (AI), epithelial thickness, goblet cell metaplasia, transforming growth factor (TGF)-β1 expression, myofibroblast differentiation, subepithelial and total lung collagen deposition, matrix metalloproteinase (MMP) expression, and AHR were then assessed. MSCs alone modestly reversed OVA-induced subepithelial and total collagen deposition, and increased MMP-9 levels above that induced by OVA alone (all p<0.05 vs OVA group). RLN alone more broadly reversed OVA-induced epithelial thickening, TGF-β1 expression, myofibroblast differentiation, airway fibrosis and AHR (all p<0.05 vs OVA group). Combination treatment further reversed OVA-induced AI and airway/lung fibrosis compared to either treatment alone (all p<0.05 vs either treatment alone), and further increased MMP-9 levels. RLN appeared to enhance the therapeutic effects of MSCs in a chronic disease setting; most likely a consequence of the ability of RLN to limit TGF-β1-induced matrix synthesis complemented by the MMP-promoting effects of MSCs.

Effects of Aging and Respiratory Syncytial Virus Infection on the uPA/Plasmin System in Pulmonary Fibrosis

RationaleIdiopathic pulmonary fibrosis (IPF) is a serious disease often resulting in death. Aging, infection, pulmonary type 2 (Th2) immune responses and impaired fibrolytic activity play central roles in IPF. We hypothesize that aging and viral infection reduces lung urokinase (uPA) levels and increases uPA inhibitor PAI‐1 and that this leads to altered immune responses and TGF‐beta signaling, resulting in worsened fibrosis.MethodsWe compared the severity of bleomycin‐induced pulmonary fibrosis (BLM‐IPF) in mice deficient for uPA (uPA‐/‐) and its receptor (uPAR‐/‐) to wild type (WT) young and aged mice in the presence or absence of RSV infection.ResultsWT mice have diminished uPA expression following infection and BLM challenge, uPA activity levels and uPA total antigen levels diminished in the serum and lung tissues of infected BLM challenged WT aged mice compared to young mice. Whereas the PAI‐1 expression was significantly higher in uPA‐/‐ or uPAR‐/‐ aged BLM challenged mice compared to the WT mice. uPA or uPAR deficient viral infected and BLM treated mice showed increased fibrosis compared to WT as shown by hydroxyproline, total lung collagen, lung histology, and increased TGF‐beta expression. Lung T cells from infected uPA‐/‐ or uPAR‐/‐ aged mice produced lower levels of Th1, Th17 and higher levels of Th2 cytokines compared to WT mice.ConclusionsThis novel study provided both in vivo and in vitro data that showed how age‐infection‐related changes in the uPA/plasmin system impact the development of IPF. Supported by a VA Merit Review Grant (MRG.)

The anti-fibrotic effect of TGFβ/ALK5 inhibition in experimental pulmonary fibrosis is attenuated in the presence of concurrent γherpesvirus infection

ABSTRACTTGFβ-ALK5 pro-fibrotic signalling and herpesvirus infections have been implicated in the pathogenesis and exacerbation of pulmonary fibrosis. In this study we addressed the role of TGFβ-ALK5 signalling during the progression of fibrosis in a two-hit mouse model of murine γ-herpesvirus 68 (MHV-68) infection on the background of pre-existing bleomycin-induced pulmonary fibrosis. Assessment of total lung collagen levels in combination with ex vivo micro-computed tomography (µCT) analysis of whole lungs demonstrated that MHV-68 infection did not enhance lung collagen deposition in this two-hit model but led to a persistent and exacerbated inflammatory response. Moreover, µCT reconstruction and analysis of the two-hit model revealed distinguishing features of diffuse ground-glass opacities and consolidation superimposed on pre-existing fibrosis that were reminiscent of those observed in acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF). Virally-infected murine fibrotic lungs further displayed evidence of extensive inflammatory cell infiltration and increased levels of CCL2, TNFα, IL-1β and IL-10. Blockade of TGFβ-ALK5 signalling attenuated lung collagen accumulation in bleomycin-alone injured mice, but this anti-fibrotic effect was reduced in the presence of concomitant viral infection. In contrast, inhibition of TGFβ-ALK5 signalling in virally-infected fibrotic lungs was associated with reduced inflammatory cell aggregates and increased levels of the antiviral cytokine IFNγ. These data reveal newly identified intricacies for the TGFβ-ALK5 signalling axis in experimental lung fibrosis, with different outcomes in response to ALK5 inhibition depending on the presence of viral infection. These findings raise important considerations for the targeting of TGFβ signalling responses in the context of pulmonary fibrosis.

Characterization of a novel model incorporating airway epithelial damage and related fibrosis to the pathogenesis of asthma

Asthma develops from injury to the airways/lungs, stemming from airway inflammation (AI) and airway remodeling (AWR), both contributing to airway hyperresponsiveness (AHR). Airway epithelial damage has been identified as a new etiology of asthma but is not targeted by current treatments. Furthermore, it is poorly studied in currently used animal models of AI and AWR. Therefore, this study aimed to incorporate epithelial damage/repair with the well-established ovalbumin (OVA)-induced model of chronic allergic airway disease (AAD), which presents with AI, AWR, and AHR, mimicking several features of human asthma. A 3-day naphthalene (NA)-induced model of epithelial damage/repair was superimposed onto the 9-week OVA-induced model of chronic AAD, before 6 weeks of OVA nebulization (NA+OVA group), during the second last OVA nebulization period (OVA/NA group) or 1 day after the 6-week OVA nebulization period (OVA+NA group), using 6–8-week-old female Balb/c mice (n=6–12/group). Mice subjected to the 9-week OVA model, 3-day NA model or respective vehicle treatments (saline and corn oil) were used as appropriate controls. OVA alone significantly increased epithelial thickness and apoptosis, goblet cell metaplasia, TGF-β1, subepithelial collagen (assessed by morphometric analyses of various histological stains), total lung collagen (hydroxyproline analysis), and AHR (invasive plethysmography) compared with that in saline-treated mice (all P<0.05 vs saline treatment). NA alone caused a significant increase in epithelial denudation and apoptosis, TGF-β1, subepithelial, and total lung collagen compared with respective measurements from corn oil-treated controls (all P<0.01 vs corn oil treatment). All three combined models underwent varying degrees of epithelial damage and AWR, with the OVA+NA model demonstrating the greatest increase in subepithelial/total lung collagen and AHR (all P<0.05 vs OVA alone or NA alone). These combined models of airway epithelial damage/AAD demonstrated that epithelial damage is a key contributor to AWR, fibrosis and related AHR, and augments the effects of AI on these parameters.

Intranasally administered serelaxin abrogates airway remodelling and attenuates airway hyperresponsiveness in allergic airways disease.

The peptide hormone relaxin plays a key role in the systemic hemodynamic and renovascular adaptive changes that occur during pregnancy, which is linked to its antiremodelling effects. Serelaxin (a recombinant form of human gene-2 relaxin) has been shown to inhibit lung fibrosis in various disease models and reverse airway remodelling and airway hyperresponsiveness (AHR) in allergic airways disease (AAD). Although continuous systemic delivery of exogenous serelaxin alleviates allergic fibrosis and AHR, more direct routes for administration into the lung have not been investigated. Thus, intranasal administration of serelaxin was evaluated for its ability to reverse airway remodelling and AHR associated with AAD. Female Balb/c mice were subjected to a 9-week model of chronic AAD. Subgroups of animals (n=12/group) were then treated intranasally with serelaxin (0.8mg/mL) or vehicle once daily for 14days (from weeks 9-11). Saline-sensitized/challenged mice treated with intranasal saline served as additional controls. Differential bronchoalveolar lavage (BAL) cell counts, ovalbumin (OVA)-specific IgE levels, tissue inflammation, parameters of airway remodelling and AHR were then assessed. Chronic AAD was associated with significant increases in differential BAL cell counts, OVA-specific IgE levels, inflammation, epithelial thickening, goblet cell metaplasia, TGF-β1 expression, epithelial Smad2 phosphorylation (pSmad2), subepithelial collagen thickness, total lung collagen concentration and AHR (all P<0.05 vs. respective measurements from saline-treated mice). Daily intranasal delivery of serelaxin significantly diminished AAD-induced epithelial thickening, epithelial pSmad2, subepithelial and total lung collagen content (fibrosis) and AHR (all P<0.05 vs. vehicle-treated AAD mice). Intranasal delivery of serelaxin can effectively reduce airway remodelling and AHR, when administered once daily. Respirable preparations of serelaxin may have therapeutic potential for the prevention and/or reversal of established airway remodelling and AHR in asthma.