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Pleural and tracheal injuries remain significant problems, and an easy to use, effective pleural or tracheal sealant would be a significant advance. The major challenges are requirements for adherence, high strength and elasticity, dynamic durability, appropriate biodegradability, and lack of cell or systemic toxicity. We designed and evaluated two sealant materials comprised respectively of alginate methacrylate and of gelatin methacryloyl, each functionalized by conjugation with dopamine HCl. Both compounds are cross-linked into easily applied as pre-formed hydrogel patches or as in situ hydrogels formed at the wound site utilizing FDA-approved photo-initiators and oxidants. Material testing demonstrates appropriate adhesiveness, tensile strength, burst pressure, and elasticity with no significant cell toxicity in vitro assessments. Air-leak was absent after sealant application to experimentally-induced injuries in ex-vivo rat lung and tracheal models and in ex vivo pig lungs. Sustained repair of experimentally-induced pleural injury was observed for up to one month in vivo rat models and for up to 2 weeks in vivo rat tracheal injury models without obvious air leak or obvious toxicities. The alginate-based sealant worked best in a pre-formed hydrogel patch whereas the gelatin-based sealant worked best in an in situ formed hydrogel at the wound site thus providing two potential approaches. These studies provide a platform for further pre-clinical and potential clinical investigations. Statement of significance Pneumothorax and pleural effusions resulting from trauma and a range of lung diseases and critical illnesses can result in lung collapse that can be immediately life-threatening or result in chronic leaking (bronchopleural fistula) that is currently difficult to manage. This leads to significantly increased morbidity, mortality, hospital stays, health care costs, and other complications. We have developed sealants originating from alginate and gelatin biomaterials, each functionalized by methacryloylation and by dopamine conjugation to have desired mechanical characteristics for use in pleural and tracheal injuries. The sealants are easily applied, non-cytotoxic, and perform well in vitro and in vivo model systems of lung and tracheal injuries. These initial proof of concept investigations provide a platform for further studies.

Summary: Heart failure (HF) and subarachnoid hemorrhage (SAH) chronically reduce cerebral perfusion, which negatively affects clinical outcome. This work demonstrates a strong relationship between cerebral artery cystic fibrosis transmembrane conductance regulator (CFTR) expression and altered cerebrovascular reactivity in HF and SAH. In HF and SAH, CFTR corrector compounds (C18 or lumacaftor) normalize pathological alterations in cerebral artery CFTR expression, vascular reactivity, and cerebral perfusion, without affecting systemic hemodynamic parameters. This normalization correlates with reduced neuronal injury. Therefore, CFTR therapeutics have emerged as valuable clinical tools to manage cerebrovascular dysfunction, impaired cerebral perfusion, and neuronal injury. Key Words: cognitive impairment, corrector compounds, cystic fibrosis transmembrane conductance regulator (CFTR), myogenic vasoconstriction, sphingosine-1-phosphate, tumor necrosis factor, vascular smooth muscle cells

The University of Vermont Larner College of Medicine, in collaboration with the National Heart, Lung, and Blood Institute (NHLBI), the Alpha-1 Foundation, the American Thoracic Society, the Cystic Fibrosis Foundation, the European Respiratory Society, the International Society for Cell & Gene Therapy, and the Pulmonary Fibrosis Foundation, convened a workshop titled “Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases” from July 24 through 27, 2017, at the University of Vermont, Burlington, Vermont. The conference objectives were to review and discuss current understanding of the following topics: 1) stem and progenitor cell biology and the role that they play in endogenous repair or as cell therapies after lung injury, 2) the emerging role of extracellular vesicles as potential therapies, 3) ex vivo bioengineering of lung and airway tissue, and 4) progress in induced pluripotent stem cell protocols for deriving lung cell types and applications in disease modeling. All of these topics are research areas in which significant and exciting progress has been made over the past few years. In addition, issues surrounding the ethics and regulation of cell therapies worldwide were discussed, with a special emphasis on combating the growing problem of unproven cell interventions being administered to patients with lung diseases. Finally, future research directions were discussed, and opportunities for both basic and translational research were identified.

Chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease, interstitial lung disease, and lung cancer, are among the leading causes of morbidity globally and impose major health and financial burdens on patients and society. Effective treatments are scarce, and relevant human model systems to effectively study CLD pathomechanisms and thus discover and validate potential new targets and therapies are needed. Precision-cut lung slices (PCLS) from healthy and diseased human tissue represent one promising tool that can closely recapitulate the complexity of the lung's native environment, and recently, improved methodologies and accessibility to human tissue have led to an increased use of PCLS in CLD research. Here, we discuss approaches that use human PCLS to advance our understanding of CLD development, as well as drug discovery and validation for CLDs. PCLS enable investigators to study complex interactions among different cell types and the extracellular matrix in the native three-dimensional architecture of the lung. PCLS further allow for high-resolution (live) imaging of cellular functions in several dimensions. Importantly, PCLS can be derived from diseased lung tissue upon lung surgery or transplantation, thus allowing the study of CLDs in living human tissue. Moreover, CLDs can be modeled in PCLS derived from normal lung tissue to mimic the onset and progression of CLDs, complementing studies in end-stage diseased tissue. Altogether, PCLS are emerging as a remarkable tool to further bridge the gap between target identification and translation into clinical studies, and thus open novel avenues for future precision medicine approaches.

Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death in the US and is primarily triggered in susceptible individuals by chronic cigarette smoke (CS) exposure. Protease/antiprotease imbalances in COPD leads to destruction of lung matrix. Therapies targeting this imbalance have not yet provided a cure. Mass spectrometry (MS) has been used to analyze the proteome of healthy and COPD lungs, but extracellular matrix (ECM) and matrix bound proteins (i.e. matrisome (MAT) are difficult to detect and thus underrepresented. Decellularized lungs (DEC) have enriched MATs. We hypothesized that the proteomic profile of DEC COPD lungs is distinct from healthy and may provide new insight into COPD pathogenesis. DEC lungs from two murine COPD models (elastase, CS) were analyzed by MS using quantitative TMT-labeling and compared to healthy controls. DEC lungs from 12 normal and 4 COPD human lungs were analyzed using 3 samples from distinct regions per lung. Principal component analysis showed that the proteome of elastase model derived DEC lungs was markedly different compared to normal. Although clustering was not observed for DEC lungs from murine CS or human COPD vs controls, the MAT had detectable differences. Notably, human lung samples had high intra-lung similarity, independent of disease status. Proteome changes in DEC lungs from CS mice positively correlated with those seen in human COPD lungs (Pearson correlation r=0.17, p

Chronic obstructive pulmonary disease (COPD) is characterised by a progressive loss of lung tissue. Inducing repair processes within the adult diseased lung is of major interest and Wnt/β-catenin signalling represents a promising target for lung repair. However, the translation of novel therapeutic targets from model systems into clinical use remains a major challenge.We generated murine and patient-derived three-dimensional (3D) ex vivo lung tissue cultures (LTCs), which closely mimic the 3D lung microenvironment in vivo. Using two well-known glycogen synthase kinase-3β inhibitors, lithium chloride (LiCl) and CHIR 99021 (CT), we determined Wnt/β-catenin-driven lung repair processes in high spatiotemporal resolution using quantitative PCR, Western blotting, ELISA, (immuno)histological assessment, and four-dimensional confocal live tissue imaging.Viable 3D-LTCs exhibited preserved lung structure and function for up to 5 days. We demonstrate successful Wnt/β-catenin signal activation in murine and patient-derived 3D-LTCs from COPD patients. Wnt/β-catenin signalling led to increased alveolar epithelial cell marker expression, decreased matrix metalloproteinase-12 expression, as well as altered macrophage activity and elastin remodelling. Importantly, induction of surfactant protein C significantly correlated with disease stage (per cent predicted forced expiratory volume in 1 s) in patient-derived 3D-LTCs.Patient-derived 3D-LTCs represent a valuable tool to analyse potential targets and drugs for lung repair. Enhanced Wnt/β-catenin signalling attenuated pathological features of patient-derived COPD 3D-LTCs.

The limited available treatment options for patients with chronic lung diseases, such as fibrosis, lead to poor prognosis after diagnosis and short survival rates. An exciting new bioengineering approach utilizes de- and recellularization of lung tissue to potentially overcome donor organ shortage and immune reactions toward the received transplant. The goal of decellularization is to create a scaffold which contains the necessary framework for stability and functionality for regenerating lung tissue while removing immunomodulatory factors by removal of cells. After decellularization, the scaffold could be re-functionalized by repopulation with the patient's own stem/progenitor cells to create a fully functional organ or can be used as ex vivo models of disease. In this chapter the decellularization of lung tissue from multiple species (i.e., rodents, pigs, and humans) as well as disease states such as fibrosis is described. We discuss and describe the various quality control measures which should be used to characterize decellularized scaffolds, methods for protein analysis of the remaining scaffold, and methods for recellularization of scaffolds.

Cigarette smoke is the most relevant risk factor for the development of lung cancer and chronic obstructive pulmonary disease. Many of its more than 4500 chemicals are highly reactive, thereby altering protein structure and function. Here, we used subcellular fractionation coupled to label-free quantitative MS to globally assess alterations in the proteome of different compartments of lung epithelial cells upon exposure to cigarette smoke extract. Proteomic profiling of the human alveolar derived cell line A549 revealed the most pronounced changes within the cellular secretome with preferential downregulation of proteins involved in wound healing and extracellular matrix organization. In particular, secretion of secreted protein acidic and rich in cysteine, a matricellular protein that functions in tissue response to injury, was consistently diminished by cigarette smoke extract in various pulmonary epithelial cell lines and primary cells of human and mouse origin as well as in mouse ex vivo lung tissue cultures. Our study reveals a previously unrecognized acute response of lung epithelial cells to cigarette smoke that includes altered secretion of proteins involved in extracellular matrix organization and wound healing. This may contribute to sustained alterations in tissue remodeling as observed in lung cancer and chronic obstructive pulmonary disease.

Chronic lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) are all characterized by structural changes of the airways and/or lungs that limit airflow and/or gas exchange. Currently, there is no therapy available that adequately targets the structural remodeling of the airways and lungs in these diseases. This underscores the great need for insight into the mechanisms that underpin the development of airway remodeling, fibrosis and emphysema in these diseases, in order to identify suitable drug targets. It is increasingly evident that structural cell cell communication within the lung is central to the development of remodeling, indicating that a more integrative approach should be considered when studying molecular and cellular mechanisms of remodeling. Therefore, there is a great need to study molecular and cellular physiological and pathophysiological mechanisms in as much detail as possible, but with as little as possible loss of the physiological context. Here, we will review the use of models such as cellular co-culture, tissue culture, and lung slice culture, in which cell cell communication and tissue architecture are better preserved or mimicked than in cell culture, and zoom in on the usefulness of molecular and cellular biological tools in these complex model systems to read out or control signaling and gene/protein regulation. (C) 2011 Elsevier Ltd. All rights reserved.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of yet unknown etiology. Recently, (re)activation of developmental pathways, such as Wnt/β-catenin in the alveolar epithelium has been linked to disease development. Regulation of Wnt/β-catenin activation has been shown to be dependent on Heparan Sulfate Proteoglycans (HSPGs). The role of Wnt-HSPG interaction in IPF, however, remains to be elusive. Methods: Murine and human lung epithelial cells were treated with Heparin, Desulfated (DS) Heparins or Heparinase II and Chondroitinase ABC, respectively. A Heparin binding assay was used to assess binding of Wnt3a to Heparin and DS Heparins. Immunofluorescence staining of ex vivo lung tissue for Wnt proteins and HSPGs was conducted. Results: Immunofluorescence staining of ex vivo tissue lung slices revealed upregulated WNT3a and HSPG expression in bleomycin-treated mouse lungs compared to controls. Heparin treatment of murine lung epithelial cells resulted in inhibition of TOP/FOP-flash reporter, indicating decreased canonical WNT signaling. In contrast, Heparinase II and Chondroitinase ABC treatment resulted in upregulated β-catenin-dependent gene transcription. Interestingly, 6-O-DS Heparin treatment of A549 cells did not decrease WNT signaling, while binding of WNT3a to 6-O-DS Heparin was not altered, thereby suggesting less inhibition of WNT signaling upon binding to 6-O-DS Heparin. Conclusion: Taken together, our findings revealed that HS sulfation and degradation modulates WNT/β-catenin signal activity. Altered sulfation states during ECM remodeling in IPF pathology might contribute to (re)activation of developmental pathways like WNT/β-catenin.