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Concept: Idiopathic pulmonary fibrosis

178

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease.

Concepts: Basement membrane, Molecular biology, Idiopathic pulmonary fibrosis, DNA, Gene expression, Gene, Lung, Epithelium

173

Matrix stiffening and myofibroblast resistance to apoptosis are cardinal features of chronic fibrotic diseases involving diverse organ systems. The interactions between altered tissue biomechanics and cellular signaling that sustain progressive fibrosis are not well defined. In this study, we used ex vivo and in vivo approaches to define a mechanotransduction pathway involving Rho/Rho kinase (Rho/ROCK), actin cytoskeletal remodeling, and a mechanosensitive transcription factor, megakaryoblastic leukemia 1 (MKL1), that coordinately regulate myofibroblast differentiation and survival. Both in an experimental mouse model of lung fibrosis and in human subjects with idiopathic pulmonary fibrosis (IPF), we observed activation of the Rho/ROCK pathway, enhanced actin cytoskeletal polymerization, and MKL1 cytoplasmic-nuclear shuttling. Pharmacologic disruption of this mechanotransduction pathway with the ROCK inhibitor fasudil induced myofibroblast apoptosis through a mechanism involving downregulation of BCL-2 and activation of the intrinsic mitochondrial apoptotic pathway. Treatment with fasudil during the postinflammatory fibrotic phase of lung injury or genetic ablation of Mkl1 protected mice from experimental lung fibrosis. These studies indicate that targeting mechanosensitive signaling in myofibroblasts to trigger the intrinsic apoptosis pathway may be an effective approach for treatment of fibrotic disorders.

Concepts: In vivo, Cancer, Adenosine triphosphate, Signal transduction, Pulmonary fibrosis, Apoptosis, Idiopathic pulmonary fibrosis, Fibrosis

171

Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro.

Concepts: Cell, Fibrosis, Chaperone, Lung, Protein, Heat shock protein, Collagen, Idiopathic pulmonary fibrosis

170

Extracellular matrix deposition and tissue scarring characterize the process of fibrosis. Transforming growth factor beta (TGFβ) and Insulin-like growth factor binding protein-3 (IGFBP-3) have been implicated in the pathogenesis of fibrosis in various tissues by inducing mesenchymal cell proliferation and extracellular matrix deposition. We identified Syndecan-2 (SDC2) as a gene induced by TGFβ in an IGFBP-3-dependent manner. TGFβ induction of SDC2 mRNA and protein required IGFBP-3. IGFBP-3 independently induced production of SDC2 in primary fibroblasts. Using an ex-vivo model of human skin in organ culture expressing IGFBP-3, we demonstrate that IGFBP-3 induces SDC2 ex vivo in human tissue. We also identified Mitogen-activated protein kinase-interacting kinase (Mknk2) as a gene induced by IGFBP-3. IGFBP-3 triggered Mknk2 phosphorylation resulting in its activation. Mknk2 independently induced SDC2 in human skin. Since IGFBP-3 is over-expressed in fibrotic tissues, we examined SDC2 levels in skin and lung tissues of patients with systemic sclerosis (SSc) and lung tissues of patients with idiopathic pulmonary fibrosis (IPF). SDC2 levels were increased in fibrotic dermal and lung tissues of patients with SSc and in lung tissues of patients with IPF. This is the first report describing elevated levels of SDC2 in fibrosis. Increased SDC2 expression is due, at least in part, to the activity of two pro-fibrotic factors, TGFβ and IGFBP-3.

Concepts: Pulmonary fibrosis, Scleroderma, Tissue, Idiopathic pulmonary fibrosis, Extracellular matrix, Fibrosis, Signal transduction, Skin

169

Pirfenidone (PFD) is a novel antifibrotic agent approved for patients with pulmonary fibrosis. However, there are concerns regarding toxicity of the drug. In this meta-analysis, we analyzed the adverse events (AEs) of PFD for the treatment of pulmonary fibrosis.

Concepts: Pharmaceutical industry, Pharmacology, Epidemiology, Pulmonary fibrosis, Randomized controlled trial, Fibrosis, Idiopathic pulmonary fibrosis

168

BACKGROUND: Stress of the endoplasmic reticulum (ER) leading to activation of the unfolded protein response (UPR) and alveolar epithelial cell (AEC) apoptosis may play a role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our objectives were to determine whether circulating caspase-cleaved cytokeratin-18 (cCK-18) is a marker of AEC apoptosis in IPF, define the relationship of cCK-18 with activation of the UPR, and assess its utility as a diagnostic biomarker. METHODS: IPF and normal lung tissues were stained with the antibody (M30) that specifically binds cCK-18. The relationship between markers of the UPR and cCK-18 was determined in AECs exposed in vitro to thapsigargin to induce ER stress. cCK-18 was measured in serum from subjects with IPF, hypersensitivity pneumonitis (HP), nonspecific interstitial pneumonia (NSIP), and control subjects. RESULTS: cCK-18 immunoreactivity was present in AECs of IPF lung, but not in control subjects. Markers of the UPR (phosphorylated IRE-1alpha and spliced XBP-1) were more highly expressed in IPF type II AECs than in normal type II AECs. Phosphorylated IRE-1alpha and cCK-18 increased following thapsigargin-induced ER stress. Serum cCK-18 level distinguished IPF from diseased and control subjects. Serum cCK-18 was not associated with disease severity or outcome. CONCLUSIONS: cCK-18 may be a marker of AEC apoptosis and UPR activation in patients with IPF. Circulating levels of cCK-18 are increased in patients with IPF and cCK-18 may be a useful diagnostic biomarker.

Concepts: Unfolded protein response, Epithelium, Immune system, Endoplasmic reticulum, Pulmonary fibrosis, Pulmonology, Lung, Idiopathic pulmonary fibrosis

167

Macrophage G2A and CD36 lipid receptors are thought to mediate efferocytosis following tissue injury and thereby prevent excessive inflammation which could compromise tissue repair. To test this, we subjected mice lacking G2A or CD36 receptors to bleomycin-induced lung injury and measured efferocytosis, inflammation and fibrosis. Loss of CD36 (but not G2A) delayed clearance of apoptotic alveolar cells (mean 78% increase in apoptotic cells 7 days post-injury), potentiated inflammation (mean 56% increase in lung neutrophils and 75% increase in lung KC levels 7 days post-injury, 51% increase in lung macrophages 14 days post-injury) and reduced lung fibrosis (mean 41% and 29% reduction 14 and 21 days post-injury respectively). Reduced fibrosis in CD36-/- mice was associated with lower levels of pro-fibrotic TH2 cytokines (IL-9, IL-13, IL-4), decreased expression of the M2 macrophage marker Arginase-1 and reduced interstitial myofibroblasts. G2A, on the other hand, was required for optimal clearance of apoptotic neutrophils during zymosan-induced peritoneal inflammation (50.3% increase in apoptotic neutrophils and 30.6% increase in total neutrophils 24 hours following zymosan administration in G2A-/- mice). Thus, CD36 is required for timely removal of apoptotic cells in the context of lung injury and modulates subsequent inflammatory and fibrotic processes relevant to fibrotic lung disease.

Concepts: Idiopathic pulmonary fibrosis, Cell biology, White blood cell, Fibrosis, Monocyte, Immune system, Macrophage, Apoptosis

31

The Phase II TOMORROW trial and two Phase III INPULSIS(®) trials investigated the efficacy and safety of nintedanib versus placebo in patients with idiopathic pulmonary fibrosis (IPF). To obtain an overall estimate of the treatment effect of nintedanib 150 mg twice daily (bid), pooled and meta-analyses of data from these three trials were conducted.

Concepts: Clinical trial, Idiopathic pulmonary fibrosis

31

Idiopathic pulmonary fibrosis is a devastating, age-related lung disease of unknown cause that has few treatment options. This disease was once thought to be a chronic inflammatory process, but current evidence indicates that the fibrotic response is driven by abnormally activated alveolar epithelial cells (AECs). These cells produce mediators that induce the formation of fibroblast and myofibroblast foci through the proliferation of resident mesenchymal cells, attraction of circulating fibrocytes, and stimulation of the epithelial to mesenchymal transition. The fibroblast and myofibroblast foci secrete excessive amounts of extracellular matrix, mainly collagens, resulting in scarring and destruction of the lung architecture. The mechanisms that link idiopathic pulmonary fibrosis with ageing and aberrant epithelial activation are unknown; evidence suggests that the abnormal recapitulation of developmental pathways and epigenetic changes have a role. In this Seminar, we review recent data on the clinical course, therapeutic options, and underlying mechanisms thought to be involved in the pathogenesis of idiopathic pulmonary fibrosis.

Concepts: Asthma, Pulmonary fibrosis, Epithelium, Lung, Idiopathic pulmonary fibrosis, Collagen, Extracellular matrix, Fibrosis

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ABSTRACT BACKGROUND: Idiopathic pulmonary fibrosis is a progressive lung disease with pulmonary vasculopathy. OBJECTIVE: To determine whether sildenafil improves six minute walk distance (6MWD) in subjects with right ventricular dysfunction. MEASUREMENTS: The IPFnet conducted a randomized trial examining the effect of sildenafil on 6MWD in patients with advanced IPF, defined by DLCO <35% predicted. A substudy examined 119/180 randomized subjects where echocardiograms were available for independent review by two cardiologists. Right ventricular (RV) hypertrophy (RVH), right ventricular systolic dysfunction (RVSD) and right ventricular systolic pressure (RVSP) were assessed. Multivariable linear regression models estimated the relationship between RV abnormality, sildenafil treatment and changes in 6MWD, St. George’s Respiratory Score (SGRQ), EuroQol and SF36 from enrollment to 12 weeks. RESULTS: The prevalence of RVH and RVSD were 12.8% and 18.6%, respectively. RVSP was measurable in 71/119 subjects, 60%; mean RVSP was 42.5 mmHg. In the subgroup of subjects with RVSD, sildenafil-treated subjects experienced less decrement in 6MWD (99.3 meters, p=0.01) and greater improvement in SGRQ (13.4 points, p=0.005) and EuroQol visual analog scores (17.9 points, p=0.04) than placebo-treated subjects. In the subgroup with RVH, sildenafil was not associated with change in 6MWD (p=0.13), but was associated with greater relative improvement in SGRQ (14.8 points, p=0.02) versus placebo-treated subjects. Sildenafil treatment in those with RVSD and RVH was not associated with change in SF36. CONCLUSIONS: Sildenafil treatment in IPF with RVSD results in better preservation of exercise capacity as compared to placebo; sildenafil also improves quality of life in subjects with RVH and RVSD.

Concepts: Pulmonology, Improve, Pulmonary fibrosis, Better, Fibrosis, Linear regression, Regression analysis, Idiopathic pulmonary fibrosis