Eric S. White

Myofibroblast Differentiation by Transforming Growth Factor-β1 Is Dependent on Cell Adhesion and Integrin Signaling via Focal Adhesion Kinase

Myofibroblast differentiation and activation by transforming growth factor-β1 (TGF-β1) is a critical event in the pathogenesis of human fibrotic diseases, but regulatory mechanisms for this effect are unclear. In this report, we demonstrate that stable expression of the myofibroblast phenotype requires both TGF-β1and adhesion-dependent signals. TGF-β1-induced myofibroblast differentiation of lung fibroblasts is blocked in non-adherent cells despite the preservation of TGF-β receptor(s)-mediated signaling of Smad2 phosphorylation. TGF-β1 induces tyrosine phosphorylation of focal adhesion kinase (FAK) including that of its autophosphorylation site, Tyr-397, an effect that is dependent on cell adhesion and is delayed relative to early Smad signaling. Pharmacologic inhibition of FAK or expression of kinase-deficient FAK, mutated by substituting Tyr-397 with Phe, inhibit TGF-β1-induced α-smooth muscle actin expression, stress fiber formation, and cellular hypertrophy. Basal expression of α-smooth muscle actin is elevated in cells grown on fibronectin-coated dishes but is decreased on laminin and poly-d-lysine, a non-integrin binding polypeptide. TGF-β1 up-regulates expression of integrins and fibronectin, an effect that is associated with autophosphorylation/activation of FAK. Thus, a safer and more effective therapeutic strategy for fibrotic diseases characterized by persistent myofibroblast activation may be to target this integrin/FAK pathway while not interfering with tumor-suppressive functions of TGF-β1/Smad signaling.

Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function

Respiratory disease is the third leading cause of death in the industrialized world. Consequently, the trachea, lungs, and cardiopulmonary vasculature have been the focus of extensive investigations. Recent studies have provided new information about the mechanisms driving lung development and differentiation. However, there is still much to learn about the ability of the adult respiratory system to undergo repair and to replace cells lost in response to injury and disease. This Review highlights the multiple stem/progenitor populations in different regions of the adult lung, the plasticity of their behavior in injury models, and molecular pathways that support homeostasis and repair.

Acellular Normal and Fibrotic Human Lung Matrices as a Culture System for In Vitro Investigation

RATIONALE Extracellular matrix (ECM) is a dynamic tissue that contributes to organ integrity and function, and its regulation of cell phenotype is a major aspect of cell biology. However, standard in vitro culture approaches are of unclear physiologic relevance because they do not mimic the compositional, architectural, or distensible nature of a living organ. In the lung, fibroblasts exist in ECM-rich interstitial spaces and are key effectors of lung fibrogenesis. OBJECTIVES To better address how ECM influences fibroblast phenotype in a disease-specific manner, we developed a culture system using acellular human normal and fibrotic lungs. METHODS Decellularization was achieved using treatment with detergents, salts, and DNase. The resultant matrices can be sectioned as uniform slices within which cells were cultured. MEASUREMENTS AND MAIN RESULTS We report that the decellularization process effectively removes cellular and nuclear material while retaining native dimensionality and stiffness of lung tissue. We demonstrate that lung fibroblasts reseeded into acellular lung matrices can be subsequently assayed using conventional protocols; in this manner we show that fibrotic matrices clearly promote transforming growth factor-β-independent myofibroblast differentiation compared with normal matrices. Furthermore, comprehensive analysis of acellular matrix ECM details significant compositional differences between normal and fibrotic lungs, paving the way for further study of novel hypotheses. CONCLUSIONS This methodology is expected to allow investigation of important ECM-based hypotheses in human tissues and permits future scientific exploration in an organ- and disease-specific manner.

Targeted Injury of Type II Alveolar Epithelial Cells Induces Pulmonary Fibrosis

RATIONALE Ineffective repair of a damaged alveolar epithelium has been postulated to cause pulmonary fibrosis. In support of this theory, epithelial cell abnormalities, including hyperplasia, apoptosis, and persistent denudation of the alveolar basement membrane, are found in the lungs of humans with idiopathic pulmonary fibrosis and in animal models of fibrotic lung disease. Furthermore, mutations in genes that affect regenerative capacity or that cause injury/apoptosis of type II alveolar epithelial cells have been identified in familial forms of pulmonary fibrosis. Although these findings are compelling, there are no studies that demonstrate a direct role for the alveolar epithelium or, more specifically, type II cells in the scarring process. OBJECTIVES To determine if a targeted injury to type II cells would result in pulmonary fibrosis. METHODS A transgenic mouse was generated to express the human diphtheria toxin receptor on type II alveolar epithelial cells. Diphtheria toxin was administered to these animals to specifically target the type II epithelium for injury. Lung fibrosis was assessed by histology and hydroxyproline measurement. MEASUREMENTS AND MAIN RESULTS Transgenic mice treated with diphtheria toxin developed an approximately twofold increase in their lung hydroxyproline content on Days 21 and 28 after diphtheria toxin treatment. The fibrosis developed in conjunction with type II cell injury. Histological evaluation revealed diffuse collagen deposition with patchy areas of more confluent scarring and associated alveolar contraction. CONCLUSIONS The development of lung fibrosis in the setting of type II cell injury in our model provides evidence for a causal link between the epithelial defects seen in idiopathic pulmonary fibrosis and the corresponding areas of scarring.

New insights into form and function of Fibronectin Splice Variants

The extracellular matrix (ECM) is a highly dynamic structure that not only provides a physical framework for cells within connective tissues, but also imparts instructive signals for development, tissue homeostasis and basic cell functions through its composition and ability to exert mechanical forces. The ECM of tissues is composed of, in addition to proteoglycans and hyaluronic acid, a number of proteins, most of which are generated after alternative splicing of their pre‐mRNA. However, the precise function of these protein isoforms is still obscure in most cases. Fibronectin (FN), one of the main components of the ECM, is also one of the best‐known examples of a family of proteins generated by alternative splicing, having at least 20 different isoforms in humans. Over the last few years, considerable progress on elucidating the functions of the alternatively spliced FN isoforms has been achieved with the essential development of key engineered mouse strains. Here we summarize the phenotypes of the mouse strains having targeted mutations in the FN gene, which may lead to novel insights linking function of alternatively spliced isoforms of fibronectin to human pathologies. Copyright

The myofibroblast matrix: implications for tissue repair and fibrosis

Myofibroblasts, and the extracellular matrix (ECM) in which they reside, are critical components of wound healing and fibrosis. The ECM, traditionally viewed as the structural elements within which cells reside, is actually a functional tissue whose components possess not only scaffolding characteristics, but also growth factor, mitogenic, and other bioactive properties. Although it has been suggested that tissue fibrosis simply reflects an ‘exuberant’ wound‐healing response, examination of the ECM and the roles of myofibroblasts during fibrogenesis instead suggest that the organism may be attempting to recapitulate developmental programmes designed to regenerate functional tissue. Evidence of this is provided by the temporospatial re‐emergence of embryonic ECM proteins by fibroblasts and myofibroblasts that induce cellular programmatic responses intended to produce a functional tissue. In the setting of wound healing (or physiological fibrosis), this occurs in a highly regulated and exquisitely choreographed fashion which results in cessation of haemorrhage, restoration of barrier integrity, and re‐establishment of tissue function. However, pathological tissue fibrosis, which oftentimes causes organ dysfunction and significant morbidity or mortality, likely results from dysregulation of normal wound‐healing processes or abnormalities of the process itself. This review will focus on the myofibroblast ECM and its role in both physiological and pathological fibrosis, and will discuss the potential for therapeutically targeting ECM proteins for treatment of fibrotic disorders.

Fibrotic extracellular matrix activates a profibrotic positive feedback loop

Pathological remodeling of the extracellular matrix (ECM) by fibroblasts leads to organ failure. Development of idiopathic pulmonary fibrosis (IPF) is characterized by a progressive fibrotic scarring in the lung that ultimately leads to asphyxiation; however, the cascade of events that promote IPF are not well defined. Here, we examined how the interplay between the ECM and fibroblasts affects both the transcriptome and translatome by culturing primary fibroblasts generated from IPF patient lung tissue or nonfibrotic lung tissue on decellularized lung ECM from either IPF or control patients. Surprisingly, the origin of the ECM had a greater impact on gene expression than did cell origin, and differences in translational control were more prominent than alterations in transcriptional regulation. Strikingly, genes that were translationally activated by IPF-derived ECM were enriched for those encoding ECM proteins detected in IPF tissue. We determined that genes encoding IPF-associated ECM proteins are targets for miR-29, which was downregulated in fibroblasts grown on IPF-derived ECM, and baseline expression of ECM targets could be restored by overexpression of miR-29. Our data support a model in which fibroblasts are activated to pathologically remodel the ECM in IPF via a positive feedback loop between fibroblasts and aberrant ECM. Interrupting this loop may be a strategy for IPF treatment.

In vitro generation of human pluripotent stem cell derived lung organoids

Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here, we report the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids, which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung, organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing, we show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles, suggesting that HLOs are an excellent model to study human lung development, maturation and disease. DOI: http://dx.doi.org/10.7554/eLife.05098.001

Pathogenetic mechanisms in usual interstitial pneumonia/idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive, usually fatal, form of interstitial lung disease characterized by failure of alveolar re‐epithelialization, persistence of fibroblasts/myofibroblasts, deposition of extracellular matrix, and distortion of lung architecture which ultimately results in respiratory failure. Clinical IPF is associated with a histopathological pattern of usual interstitial pneumonia (UIP) on surgical lung biopsy. Therapy for this disease with glucocorticoids and other immunomodulatory agents is largely ineffective and recent trials of newer anti‐fibrotic agents have been disappointing. While the inciting event(s) leading to the initiation of scar formation in UIP remain unknown, recent advances in our understanding of the mechanisms underlying both normal and aberrant wound healing have shed some light on pathogenetic mechanisms that may play significant roles in this disease. Unlike other fibrotic diseases of the lung, such as those associated with collagen vascular disease, occupational exposure, or chemotherapeutic agents, UIP is not associated with a significant inflammatory response; rather, dysregulated epithelial–mesenchymal interactions predominate. Identification of pathways crucial to fibrogenesis might offer potentially novel therapeutic targets to slow or halt the progression of IPF. This review focuses on evolving concepts of cellular and molecular mechanisms in the pathogenesis of UIP/IPF. Copyright

Human iPS cell–derived alveolar epithelium repopulates lung extracellular matrix

The use of induced pluripotent stem cells (iPSCs) has been postulated to be the most effective strategy for developing patient-specific respiratory epithelial cells, which may be valuable for lung-related cell therapy and lung tissue engineering. We generated a relatively homogeneous population of alveolar epithelial type II (AETII) and type I (AETI) cells from human iPSCs that had phenotypic properties similar to those of mature human AETII and AETI cells. We used these cells to explore whether lung tissue can be regenerated in vitro. Consistent with an AETII phenotype, we found that up to 97% of cells were positive for surfactant protein C, 95% for mucin-1, 93% for surfactant protein B, and 89% for the epithelial marker CD54. Additionally, exposing induced AETII to a Wnt/β-catenin inhibitor (IWR-1) changed the iPSC-AETII-like phenotype to a predominantly AETI-like phenotype. We found that of induced AET1 cells, more than 90% were positive for type I markers, T1α, and caveolin-1. Acellular lung matrices were prepared from whole rat or human adult lungs treated with decellularization reagents, followed by seeding these matrices with alveolar cells derived from human iPSCs. Under appropriate culture conditions, these progenitor cells adhered to and proliferated within the 3D lung tissue scaffold and displayed markers of differentiated pulmonary epithelium.