Notch1 Induces Defective Epithelial Surfactant Processing and Pulmonary Fibrosis.

Rationale: Although type II alveolar epithelial cells (AEC2s) are chronically injured in idiopathic pulmonary fibrosis (IPF), they contribute to epithelial regeneration in IPF. Objectives: We hypothesized that Notch signaling may contribute to AEC2 proliferation, dedifferentiation characterized by loss of surfactant processing machinery, and lung fibrosis in IPF. Methods: We applied microarray analysis, kinome profiling, flow cytometry, immunofluorescence analysis, western blotting, quantitative PCR, and proliferation and surface activity analysis to study epithelial differentiation, proliferation, and matrix deposition in vitro (AEC2 lines, primary murine/human AEC2s), ex vivo (human IPF-derived precision-cut lung slices), and in vivo (bleomycin and pepstatin application, Notch1 [Notch receptor 1] intracellular domain overexpression). Measurements and Main Results: We document here extensive SP-B and -C (surfactant protein-B and -C) processing defects in IPF AEC2s, due to loss of Napsin A, resulting in increased intra-alveolar surface tension and alveolar collapse and induction of endoplasmic reticulum stress in AEC2s. In vivo pharmacological inhibition of Napsin A results in the development of AEC2 injury and overt lung fibrosis. We also demonstrate that Notch1 signaling is already activated early in IPF and determines AEC2 fate by inhibiting differentiation (reduced lamellar body compartment, reduced capacity to process hydrophobic SP) and by causing increased epithelial proliferation and development of lung fibrosis, putatively via altered JAK (Janus kinase)/Stat (signal transducer and activator of transcription) signaling in AEC2s. Conversely, inhibition of Notch signaling in IPF-derived precision-cut lung slices improved the surfactant processing capacity of AEC2s and reversed fibrosis. Conclusions: Notch1 is a central regulator of AEC2 fate in IPF. It induces alveolar epithelial proliferation and loss of Napsin A and of surfactant proprotein processing, and it contributes to fibroproliferation.

Clinical characteristics of patients with familial idiopathic pulmonary fibrosis (f-IPF).

BACKGROUND: The aim of this study was to analyze the relative frequency, clinical characteristics, disease onset and progression in f-IPF vs. sporadic IPF (s-IPF). METHODS: Familial IPF index patients and their family members were recruited into the European IPF registry/biobank (eurIPFreg) at the Universities of Giessen and Marburg (UGMLC). Initially, we employed wide range criteria of f-IPF (e.g. relatives who presumably died of some kind of parenchymal lung disease). After narrowing down the search to occurrence of idiopathic interstitial pneumonia (IIP) in at least one first grade relative, 28 index patients were finally identified, prospectively interviewed and examined. Their family members were phenotyped with establishment of pedigree charts. RESULTS: Within the 28 IPF families, overall 79 patients with f-IPF were identified. In the same observation period, 286 f-IIP and s-IIP patients were recruited into the eurIPFreg at our UGMLC sites, corresponding to a familial versus s-IPF of 9.8%. The both groups showed no difference in demographics (61 vs. 79% males), smoking history, and exposure to any environmental triggers known to cause lung fibrosis. The f-IPF group differed by an earlier age at the onset of the disease (55.4 vs. 63.2 years; p < 0.001). On average, the f-IPF patients presented a significantly milder extent of functional impairment at the time point of inclusion vs. the s-IPF group (FVC 75% pred. vs. FVC 62% pred., p = 0.011). In contrast, the decline in FVC was found to be faster in the f-IPF vs. the s-IPF group (4.94% decline in 6 months in f-IPF vs. 2.48% in s-IPF, p = 0.12). The average age of death in f-IPF group was 67 years vs. 71.8 years in s-IPF group (p = 0.059). The f-IIP group displayed diverse inheritance patterns, mostly autosomal-dominant with variable penetrance. In the f-IPF, the younger generations showed a tendency for earlier manifestation of IPF vs. the older generation (58 vs. 66 years, p = 0.013). CONCLUSIONS: The 28 f-IPF index patients presented an earlier onset and more aggressive natural course of the disease. The disease seems to affect consecutive generations at a younger age. TRIAL REGISTRATION: Nr. NCT02951416 http://www.www.clinicaltrials.gov.

Lung CT Densitometry in Idiopathic Pulmonary Fibrosis for the Prediction of Natural Course, Severity, and Mortality.

BACKGROUND: In this study, we retrospectively assessed the relationships among physiologic measurements, survival, and quantitative high-resolution CT (HRCT) scanning indexes in patients with idiopathic pulmonary fibrosis (IPF). METHODS: Seventy patients (48 male; mean [SD] age, 66.4 [9.0] years) with IPF were enrolled in the study. After segmentation of the lungs in thin-section CT scanning with the patient in the supine position at full inspiration, we assessed following parameters: mean lung attenuation (MLA), skewness, kurtosis, peak attenuation, total lung area, inflexion point with slope, and area right of the inflexion point (AROIP). Additionally, FVC, FEV1, total lung capacity, diffusing capacity or transfer factor of the lung for carbon monoxide (Dlco), and 6-min walk distance were analyzed. Univariate and multivariate analysis were used for the prediction of physiologic outcomes by HRCT scanning indexes and then were correlated to survival in a proportional hazards analysis. RESULTS: The strongest correlation was observed between MLA and FEV1, with an r of -0.63. MLA, peak attenuation, slope, attenuation, and AROIP correlated negatively with all physiologic measurements. AROIP was the best predictor of Dlco. Analysis for prediction of mortality showed that AROIP, kurtosis, and FVC were related significantly to survival. Multivariate regression revealed a significant impact of only AROIP (among age, sex, MLA, skewness, kurtosis, FVC, and Dlco) on survival. CONCLUSIONS: These data indicate that HRCT scanning indexes are correlated to physiologic measurements. The newly defined parameter, AROIP, is of additive value for prediction of outcome. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT02951416; URL: www.clinicaltrials.gov.

Aberrant expression and activity of histone deacetylases in sporadic idiopathic pulmonary fibrosis.

BACKGROUND: Activation and differentiation of fibroblasts into contractile protein-expressing myofibroblasts and their acquired apoptosis-resistant phenotype are critical factors towards the development of idiopathic pulmonary fibrosis (IPF), a fatal disease characterised by distorted pulmonary structure and excessive extracellular matrix (ECM) deposition. The molecular mechanisms underlying these processes in IPF remain incompletely understood. We investigated the possible implication of aberrant overexpression and activity of histone deacetylases (HDACs) in IPF. METHODS: We analysed lung tissues from patients with sporadic IPF (n=26) and non-diseased control lungs (n=16) for expression of class I and II HDACs. Primary IPF fibroblasts were treated with HDAC inhibitors (HDACi) LBH589 or valproic acid (VPA). RESULTS: Compared to control lungs, protein levels of class I (HDAC1, HDAC2, HDAC3, HDAC8) and class II HDACs (HDAC4, HDAC 5, HDAC 7, HDAC 9) were significantly elevated in IPF lungs. Using immunohistochemistry, strong induction of nearly all HDAC enzymes was observed in myofibroblasts of fibroblast foci and in abnormal bronchiolar basal cells at sites of aberrant re-epithelialisation in IPF lungs, but not in controls. Treatment of primary IPF fibroblasts with the pan-HDACi LBH589 resulted in significantly reduced expression of genes associated with ECM synthesis, proliferation and cell survival, as well as in suppression of HDAC7, and was paralleled by induction of endoplasmic reticulum stress and apoptosis. The profibrotic and apoptosis-resistant phenotype of IPF fibroblasts was also partly attenuated by the class I HDACi VPA. CONCLUSIONS: Aberrant overexpression of HDACs in basal cells of IPF lungs may contribute to the bronchiolisation process in this disease. Similarly, generation and apoptosis resistance of IPF fibroblasts are mediated by enhanced activity of HDAC enzymes. Therefore, pan-HDAC inhibition by LBH589 may present a novel therapeutic option for patients with IPF.

Comparative proteome analysis of lung tissue from patients with idiopathic pulmonary fibrosis (IPF), non-specific interstitial pneumonia (NSIP) and organ donors.

Among the idiopathic interstitial pneumonias (IIP), the two entities IPF and NSIP seem to be clinically related, but NSIP has a better outcome. The proteomic signatures which distinguish NSIP from IPF remain still elusive. We therefore performed comparative proteomic analysis of peripheral lung tissue from patients with sporadic IPF (n=14) and fibrotic NSIP (fNSIP, n=8) and organ donors (Controls, n=10), by using the 2-dimensional DIGE technique and MALDI-TOF-MS. The study revealed that the proteomic profiles of IPF and fNSIP were quite similar. Among the upregulated proteins in IPF and fNSIP were stress-induced genes involved in the ER stress-pathway, whereas downregulated proteins in IPF and fNSIP included antiapoptotic factors and antifibrotic molecules. The comparison fNSIP versus IPF indicated upregulation of subunits of the proteasome activator complex and antioxidant enzymes of the peroxiredoxin family. We conclude, that only few protein expression changes exist between IPF and fNSIP, and that epithelial ER- and oxidative stress play a major role in the pathogenesis of both diseases. In contrast to IPF, intracellular clearance of ROS and misfolded protein carbonyls seem to be enhanced in fNSIP due to enhanced expression of antioxidant acting proteins, and may explain the better outcome and survival in patients with fNSIP. BIOLOGICAL SIGNIFICANCE: IPF and fibrotic NSIP (fNSIP) belong to the idiopathic interstitial pneumonias and are usually fatal, but fNSIP has a better outcome. In order to identify molecular mechanisms and differences between IPF and fNSIP, we herein present results of a comparative proteome analysis of IPF, fNSIP and control lung tissue. Our data including validation experiments suggest that ER stress and a general stress-response as well as the decline of antioxidant capacity in alveolar epithelium is key in the pathogenesis of IPF and fNSIP. In addition, we could observe a signature of an increased alveolar epithelial protection against oxidative and ER-stress in fNSIP as compared to IPF, which could help to explain the better outcome of fNSIP patients.

Unravelling the progressive pathophysiology of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a life-threatening condition, with a median survival of <3 yrs. The pathophysiology is not fully understood, but chronic injury of alveolar epithelial type II cells (AECII) is considered key. In IPF, disturbed folding and processing of surfactant proteins and impaired DNA repair may represent underlying reasons for maladaptive endoplasmic reticulum stress responses, increased reactive oxygen species production and/or DNA damage. Excessive AECII apoptosis occurs, leading to permanently perturbed epithelial homeostasis. The role of secondary hits also becomes evident. These may aggravate the disease and result in increased epithelial turnover, exhausting the regenerative capacity of progenitors and disturbing epithelial-mesenchymal interactions. Fibroblast proliferation, transdifferentiation and matrix deposition may be mediated through various mechanisms including epithelial-mesenchymal transition, fibrocyte invasion or expansion of a local fibroblast population. Treatment modalities aiming to attenuate epithelial injury are currently in early pre-clinical development and may reach the clinical arena in only a few years. Meanwhile, novel drugs acting on highly activated fibroblasts such as pirfenidone, an anti-fibrotic drug authorised for IPF in the European Union, or BIBF 1120, a novel triple-kinase inhibitor (blocking vascular endothelial growth factor, platelet-derived growth factor and fibroblast growth factor) currently under clinical investigation, seem to attenuate the progression of IPF.