Epithelial-Derived Inflammation Disrupts Elastin Assembly and Alters Saccular Stage Lung Development.

The highly orchestrated interactions between the epithelium and mesenchyme required for normal lung development can be disrupted by perinatal inflammation in preterm infants, although the mechanisms are incompletely understood. We used transgenic (inhibitory κB kinase ß transactivated) mice that conditionally express an activator of the NF-κB pathway in airway epithelium to investigate the impact of epithelial-derived inflammation during lung development. Epithelial NF-κB activation selectively impaired saccular stage lung development, with a phenotype comprising rapidly progressive distal airspace dilation, impaired gas exchange, and perinatal lethality. Epithelial-derived inflammation resulted in disrupted elastic fiber organization and down-regulation of elastin assembly components, including fibulins 4 and 5, lysyl oxidase like-1, and fibrillin-1. Fibulin-5 expression by saccular stage lung fibroblasts was consistently inhibited by treatment with bronchoalveolar lavage fluid from inhibitory κB kinase ß transactivated mice, Escherichia coli lipopolysaccharide, or tracheal aspirates from preterm infants exposed to chorioamnionitis. Expression of a dominant NF-κB inhibitor in fibroblasts restored fibulin-5 expression after lipopolysaccharide treatment, whereas reconstitution of fibulin-5 rescued extracellular elastin assembly by saccular stage lung fibroblasts. Elastin organization was disrupted in saccular stage lungs of preterm infants exposed to systemic inflammation. Our study reveals a critical window for elastin assembly during the saccular stage that is disrupted by inflammatory signaling and could be amenable to interventions that restore elastic fiber assembly in the developing lung.

Endothelial HIF signaling regulates pulmonary fibrosis-associated pulmonary hypertension.

Pulmonary hypertension (PH) complicating chronic parenchymal lung disease, such as idiopathic pulmonary fibrosis, results in significant morbidity and mortality. Since the hypoxia-inducible factor (HIF) signaling pathway is important for development of pulmonary hypertension in chronic hypoxia, we investigated whether HIF signaling in vascular endothelium regulates development of PH related to pulmonary fibrosis. We generated a transgenic model in which HIF is deleted within vascular endothelial cells and then exposed these mice to chronic intraperitoneal bleomycin to induce PH associated with lung fibrosis. Although no differences in the degree of fibrotic remodeling were observed, we found that endothelial HIF-deficient mice were protected against development of PH, including right ventricle and pulmonary vessel remodeling. Similarly, endothelial HIF-deficient mice were protected from PH after a 4-wk exposure to normobaric hypoxia. In vitro studies of pulmonary vascular endothelial cells isolated from the HIF-targeted mice and controls revealed that endothelial HIF signaling increases endothelial cell expression of connective tissue growth factor, enhances vascular permeability, and promotes pulmonary artery smooth muscle cell proliferation and wound healing ability, all of which have the potential to impact the development of PH in vivo. Taken together, these studies demonstrate that vascular endothelial cell HIF signaling is necessary for development of hypoxia and pulmonary fibrosis associated PH. As such, HIF and HIF-regulated targets represent a therapeutic target in these conditions.

The ZIP8/SIRT1 axis regulates alveolar progenitor cell renewal in aging and idiopathic pulmonary fibrosis.

Type 2 alveolar epithelial cells (AEC2s) function as progenitor cells in the lung. We have shown previously that failure of AEC2 regeneration results in progressive lung fibrosis in mice and is a cardinal feature of idiopathic pulmonary fibrosis (IPF). In this study, we identified deficiency of a specific zinc transporter, SLC39A8 (ZIP8), in AEC2s from both IPF lungs and lungs of old mice. Loss of ZIP8 expression was associated with impaired renewal capacity of AEC2s and enhanced lung fibrosis. ZIP8 regulation of AEC2 progenitor function was dependent on SIRT1. Replenishment with exogenous zinc and SIRT1 activation promoted self-renewal and differentiation of AEC2s from lung tissues of IPF patients and old mice. Deletion of Zip8 in AEC2s in mice resulted in impaired AEC2 renewal, increased susceptibility to bleomycin injury, and development of spontaneous lung fibrosis. Therapeutic strategies to restore zinc metabolism and appropriate SIRT1 signaling could improve AEC2 progenitor function and mitigate ongoing fibrogenesis.

Categorization of lung mesenchymal cells in development and fibrosis.

Pulmonary mesenchymal cells are critical players in both the mouse and human during lung development and disease states. They are increasingly recognized as highly heterogeneous, but there is no consensus on subpopulations or discriminative markers for each subtype. We completed scRNA-seq analysis of mesenchymal cells from the embryonic, postnatal, adult and aged fibrotic lungs of mice and humans. We consistently identified and delineated the transcriptome of lipofibroblasts, myofibroblasts, smooth muscle cells, pericytes, mesothelial cells, and a novel population characterized by Ebf1 expression. Subtype selective transcription factors and putative divergence of the clusters during development were described. Comparative analysis revealed orthologous subpopulations with conserved transcriptomic signatures in murine and human lung mesenchymal cells. All mesenchymal subpopulations contributed to matrix gene expression in fibrosis. This analysis would enhance our understanding of mesenchymal cell heterogeneity in lung development, homeostasis and fibrotic disease conditions.

Epithelial Injury and Dysfunction in the Pathogenesis of Idiopathic PulmonaryFibrosis.

Idiopathic pulmonary fibrosis is a disease of older adults leading to progressive dyspnea and reduced exercise capacity, typically resulting in death within 3-5years of diagnosis. Underlying genetic susceptibility combined with environmental insults is proposed to trigger a chronic wound repair response, leading to activation of the fibrotic cascade. Perturbations in several molecular pathways mediate vulnerability of the alveolar epithelium to injurious agents, including the unfolded protein response, autophagy, mitophagy, and cellular senescence. These cellular responses are intricately intertwined and link genetic susceptibility to the progressive fibrotic phenotype. Ongoing studies investigating these pathways in type II alveolar epithelial cells show promise for identifying new targeted interventions that could prevent or halt the progression of IPF.

Endoplasmic reticulum stress in pulmonary fibrosis.

Endoplasmic reticulum (ER) stress is associated with development and progression of fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). ER stress was first implicated in the pathogenesis of IPF >15 years ago with the discovery of disease-causing mutations in surfactant protein C, which result in a misfolded gene product in type II alveolar epithelial cells (AECs). ER stress and the unfolded protein response (UPR) have been linked to lung fibrosis through regulation of AEC apoptosis, epithelial-mesenchymal transition, myofibroblast differentiation, and M2 macrophage polarization. Although progress has been made in understanding the causes and consequences of ER stress in IPF and a number of chronic fibrotic disorders, further studies are needed to identify key factors that induce ER stress in important cell types and define critical down-stream processes and effector molecules that mediate ER stress-related phenotypes. This review discusses potential causes of ER stress induction in the lungs and current evidence linking ER stress to fibrosis in the context of individual cell types: AECs, fibroblasts, and macrophages. As our understanding of the relationship between ER stress and lung fibrosis continues to evolve, future studies will examine new strategies to modulate UPR pathways for therapeutic benefit.

Localized hypoxia links ER stress to lung fibrosis through induction of C/EBP homologous protein.

ER stress in type II alveolar epithelial cells (AECs) is common in idiopathic pulmonary fibrosis (IPF), but the contribution of ER stress to lung fibrosis is poorly understood. We found that mice deficient in C/EBP homologous protein (CHOP), an ER stress-regulated transcription factor, were protected from lung fibrosis and AEC apoptosis in 3 separate models where substantial ER stress was identified. In mice treated with repetitive intratracheal bleomycin, we identified localized hypoxia in type II AECs as a potential mechanism explaining ER stress. To test the role of hypoxia in lung fibrosis, we treated mice with bleomycin, followed by exposure to 14% O2, which exacerbated ER stress and lung fibrosis. Under these experimental conditions, CHOP-/- mice, but not mice with epithelial HIF (HIF1/HIF2) deletion, were protected from AEC apoptosis and fibrosis. In vitro studies revealed that CHOP regulates hypoxia-induced apoptosis in AECs via the inositol-requiring enzyme 1α (IRE1α) and the PKR-like ER kinase (PERK) pathways. In human IPF lungs, CHOP and hypoxia markers were both upregulated in type II AECs, supporting a conclusion that localized hypoxia results in ER stress-induced CHOP expression, thereby augmenting type II AEC apoptosis and potentiating lung fibrosis.

Expression of mutant bone morphogenetic protein receptor II worsens pulmonary hypertension secondary to pulmonary fibrosis.

Pulmonary fibrosis is often complicated by pulmonary hypertension (PH), and previous studies have shown a potential link between bone morphogenetic protein receptor II (BMPR2) and PH secondary to pulmonary fibrosis. We exposed transgenic mice expressing mutant BMPR2 and control mice to repetitive intraperitoneal injections of bleomycin for 4 weeks. The duration of transgene activation was too short for mutant BMPR2 mice to develop spontaneous PH. Mutant BMPR2 mice had increased right ventricular systolic pressure compared to control mice, without differences in pulmonary fibrosis. We found increased hypoxia-inducible factor (HIF)1-α stabilization in lungs of mutant-BMPR2-expressing mice compared to controls following bleomycin treatment. In addition, expression of the hypoxia response element protein connective tissue growth factor was increased in transgenic mice as well as in a human pulmonary microvascular endothelial cell line expressing mutant BMPR2. In mouse pulmonary vascular endothelial cells, mutant BMPR2 expression resulted in increased HIF1-α and reactive oxygen species production following exposure to hypoxia, both of which were attenuated with the antioxidant TEMPOL. These data suggest that expression of mutant BMPR2 worsens secondary PH through increased HIF activity in vascular endothelium. This pathway could be therapeutically targeted in patients with PH secondary to pulmonary fibrosis.