Deciphering the impacts of modulating the Wnt-planar cell polarity (PCP) pathway on alveolar repair.

Many adult lung diseases involve dysregulated lung repair. Deciphering the molecular and cellular mechanisms that govern intrinsic lung repair is essential to develop new treatments to repair/regenerate the lungs. Aberrant Wnt signalling is associated with lung diseases including emphysema, idiopathic pulmonary fibrosis and pulmonary arterial hypertension but how Wnt signalling contributes to these diseases is still unclear. There are several alternative pathways that can be stimulated upon Wnt ligand binding, one of these is the Planar Cell Polarity (PCP) pathway which induces actin cytoskeleton remodelling. Wnt5a is known to stimulate the PCP pathway and this ligand is of particular interest in regenerative lung biology because of its association with lung diseases and its role in the alveolar stem cell niche. To decipher the cellular mechanisms through which Wnt5a and the PCP pathway affect alveolar repair we utilised a 3-D ex-vivo model of lung injury and repair, the AIR model. Our results show that Wnt5a specifically enhances the alveolar epithelial progenitor cell population following injury and surprisingly, this function is attenuated but not abolished in Looptail (Lp) mouse lungs in which the PCP pathway is dysfunctional. However, Lp tracheal epithelial cells show reduced stiffness and Lp alveolar epithelial cells are less migratory than wildtype (WT), indicating that Lp lung epithelial cells have a reduced capacity for repair. These findings provide important mechanistic insight into how Wnt5a and the PCP pathway contribute to lung repair and indicate that these components of Wnt signalling may be viable targets for the development of pro-repair treatments.

Links of Cytoskeletal Integrity with Disease and Aging.

Aging is a complex feature and involves loss of multiple functions and nonreversible phenotypes. However, several studies suggest it is possible to protect against aging and promote rejuvenation. Aging is associated with many factors, such as telomere shortening, DNA damage, mitochondrial dysfunction, and loss of homeostasis. The integrity of the cytoskeleton is associated with several cellular functions, such as migration, proliferation, degeneration, and mitochondrial bioenergy production, and chronic disorders, including neuronal degeneration and premature aging. Cytoskeletal integrity is closely related with several functional activities of cells, such as aging, proliferation, degeneration, and mitochondrial bioenergy production. Therefore, regulation of cytoskeletal integrity may be useful to elicit antiaging effects and to treat degenerative diseases, such as dementia. The actin cytoskeleton is dynamic because its assembly and disassembly change depending on the cellular status. Aged cells exhibit loss of cytoskeletal stability and decline in functional activities linked to longevity. Several studies reported that improvement of cytoskeletal stability can recover functional activities. In particular, microtubule stabilizers can be used to treat dementia. Furthermore, studies of the quality of aged oocytes and embryos revealed a relationship between cytoskeletal integrity and mitochondrial activity. This review summarizes the links of cytoskeletal properties with aging and degenerative diseases and how cytoskeletal integrity can be modulated to elicit antiaging and therapeutic effects.

Made by cells for cells - extracellular vesicles as next-generation mainstream medicines.

Current medicine has only taken us so far in reducing disease and tissue damage. Extracellular vesicles (EVs), which are membranous nanostructures produced naturally by cells, have been hailed as a next-generation medicine. EVs deliver various biomolecules, including proteins, lipids and nucleic acids, which can influence the behaviour of specific target cells. Since EVs not only mirror composition of their parent cells but also modify the recipient cells, they can be used in three key areas of medicine: regenerative medicine, disease detection and drug delivery. In this Review, we discuss the transformational and translational progress witnessed in EV-based medicine to date, focusing on two key elements: the mechanisms by which EVs aid tissue repair (for example, skin and bone tissue regeneration) and the potential of EVs to detect diseases at an early stage with high sensitivity and specificity (for example, detection of glioblastoma). Furthermore, we describe the progress and results of clinical trials of EVs and demonstrate the benefits of EVs when compared with traditional medicine, including cell therapy in regenerative medicine and solid biopsy in disease detection. Finally, we present the challenges, opportunities and regulatory framework confronting the clinical application of EV-based products.

The acid injury and repair (AIR) model: A novel ex-vivo tool to understand lung repair.

Research into mechanisms underlying lung injury and subsequent repair responses is currently of paramount importance. There is a paucity of models that bridge the gap between in vitro and in vivo research. Such intermediate models are critical for researchers to decipher the mechanisms that drive repair and to test potential new treatments for lung repair and regeneration. Here we report the establishment of a new tool, the Acid Injury and Repair (AIR) model, that will facilitate studies of lung tissue repair. In this model, injury is applied to a restricted area of a precision-cut lung slice using hydrochloric acid, a clinically relevant driver. The surrounding area remains uninjured, thus mimicking the heterogeneous pattern of injury frequently observed in lung diseases. We show that in response to injury, the percentage of progenitor cells (pro surfactant protein C, proSP-C and TM4SF1 positive) significantly increases in the injured region. Whereas in the uninjured area, the percentage of proSP-C/TM4SF1 cells remains unchanged but proliferating cells (Ki67 positive) increase. These effects are modified in the presence of inhibitors of proliferation (Cytochalasin D) and Wnt secretion (C59) demonstrating that the AIR model is an important new tool for research into lung disease pathogenesis and potential regenerative medicine strategies.

The Planar Polarity Component VANGL2 Is a Key Regulator of Mechanosignaling.

VANGL2 is a component of the planar cell polarity (PCP) pathway, which regulates tissue polarity and patterning. The Vangl2 Lp mutation causes lung branching defects due to dysfunctional actomyosin-driven morphogenesis. Since the actomyosin network regulates cell mechanics, we speculated that mechanosignaling could be impaired when VANGL2 is disrupted. Here, we used live-imaging of precision-cut lung slices (PCLS) from Vangl2 Lp/+ mice to determine that alveologenesis is attenuated as a result of impaired epithelial cell migration. Vangl2 Lp/+ tracheal epithelial cells (TECs) and alveolar epithelial cells (AECs) exhibited highly disrupted actomyosin networks and focal adhesions (FAs). Functional assessment of cellular forces confirmed impaired traction force generation in Vangl2 Lp/+ TECs. YAP signaling in Vangl2 Lp airway epithelium was reduced, consistent with a role for VANGL2 in mechanotransduction. Furthermore, activation of RhoA signaling restored actomyosin organization in Vangl2 Lp/+ , confirming RhoA as an effector of VANGL2. This study identifies a pivotal role for VANGL2 in mechanosignaling, which underlies the key role of the PCP pathway in tissue morphogenesis.

An Ex Vivo Acid Injury and Repair (AIR) Model Using Precision-Cut Lung Slices to Understand Lung Injury and Repair.

Recent advances in cell culture models like air-liquid interface culture and ex vivo models such as organoids have advanced studies of lung biology; however, gaps exist between these models and tools that represent the complexity of the three-dimensional environment of the lung. Precision-cut lung slices (PCLS) mimic the in vivo environment and bridge the gap between in vitro and in vivo models. We have established the acid injury and repair (AIR) model where a spatially restricted area of tissue is injured using drops of HCl combined with Pluronic gel. Injury and repair are assessed by immunofluorescence using robust markers, including Ki67 for cell proliferation and prosurfactant protein C for alveolar type 2/progenitor cells. Importantly, the AIR model enables the study of injury and repair in mouse lung tissue without the need for an initial in vivo injury, and the results are highly reproducible. Here, we present detailed protocols for the generation of PCLS and the AIR model. We also describe methods to analyze and quantify injury in AIR-PCLS by immunostaining with established early repair markers and fluorescence imaging. This novel ex vivo model is a versatile tool for studying lung cell biology in acute lung injury and for semi-high-throughput screening of potential therapeutics. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of precision-cut lung slices Basic Protocol 2: The acid injury and repair model Basic Protocol 3: Analysis of AIR-PCLS: Immunostaining and imaging.

Can Stem Cells Beat COVID-19: Advancing Stem Cells and Extracellular Vesicles Toward Mainstream Medicine for Lung Injuries Associated With SARS-CoV-2 Infections.

A number of medicines are currently under investigation for the treatment of COVID-19 disease including anti-viral, anti-malarial, and anti-inflammatory agents. While these treatments can improve patient's recovery and survival, these therapeutic strategies do not lead to unequivocal restoration of the lung damage inflicted by this disease. Stem cell therapies and, more recently, their secreted extracellular vesicles (EVs), are emerging as new promising treatments, which could attenuate inflammation but also regenerate the lung damage caused by COVID-19. Stem cells exert their immunomodulatory, anti-oxidant, and reparative therapeutic effects likely through their EVs, and therefore, could be beneficial, alone or in combination with other therapeutic agents, in people with COVID-19. In this review article, we outline the mechanisms of cytokine storm and lung damage caused by SARS-CoV-2 virus leading to COVID-19 disease and how mesenchymal stem cells (MSCs) and their secreted EVs can be utilized to tackle this damage by harnessing their regenerative properties, which gives them potential enhanced clinical utility compared to other investigated pharmacological treatments. There are currently 17 clinical trials evaluating the therapeutic potential of MSCs for the treatment of COVID-19, the majority of which are administered intravenously with only one clinical trial testing MSC-derived exosomes via inhalation route. While we wait for the outcomes from these trials to be reported, here we emphasize opportunities and risks associated with these therapies, as well as delineate the major roadblocks to progressing these promising curative therapies toward mainstream treatment for COVID-19.

Placenta Stem/Stromal Cell-Derived Extracellular Vesicles for Potential Use in Lung Repair.

Many acute and chronic lung injuries are incurable and rank as the fourth leading cause of death globally. While stem cell treatment for lung injuries is a promising approach, there is growing evidence that the therapeutic efficacy of stem cells originates from secreted extracellular vesicles (EVs). Consequently, EVs are emerging as next-generation therapeutics. While EVs are extensively researched for diagnostic applications, their therapeutic potential to promote tissue repair is not fully elucidated. By housing and delivering tissue-repairing cargo, EVs refine the cellular microenvironment, modulate inflammation, and ultimately repair injury. Here, the potential use of EVs derived from two placental mesenchymal stem/stromal cell (MSC) lines is presented; a chorionic MSC line (CMSC29) and a decidual MSC cell line (DMSC23) for applications in lung diseases. Functional analyses using in vitro models of injury demonstrate that these EVs have a role in ameliorating injuries caused to lung cells. It is also shown that EVs promote repair of lung epithelial cells. This study is fundamental to advancing the field of EVs and to unlock the full potential of EVs in regenerative medicine.

Isolation and Characterization of Extracellular Vesicles from Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) have received immense attention in the past decade for their diverse use in diagnosis and therapeutics. Enhancing our understanding of EVs and increasing the reliability and reproducibility of EV research demands the use of standard isolation procedures and multiple characterization methods. Here we describe the most commonly used EV isolation method involving ultracentrifugation, and various characterization methods that include nanoparticle tracking analysis, atomic force microscopy and electron microscopy, which measure the size, concentration, and morphology of EVs.

High-fidelity probing of the structure and heterogeneity of extracellular vesicles by resonance-enhanced atomic force microscopy infrared spectroscopy.

Extracellular vesicles (EVs) are highly specialized nanoscale assemblies that deliver complex biological cargos to mediate intercellular communication. EVs are heterogeneous, and characterization of this heterogeneity is paramount to understanding EV biogenesis and activity, as well as to associating them with biological responses and pathologies. Traditional approaches to studying EV composition generally lack the resolution and/or sensitivity to characterize individual EVs, and therefore the assessment of EV heterogeneity has remained challenging. We have recently developed an atomic force microscope IR spectroscopy (AFM-IR) approach to probe the structural composition of single EVs with nanoscale resolution. Here, we provide a step-by-step procedure for our approach and show its power to reveal heterogeneity across individual EVs, within the same population of EVs and between different EV populations. Our approach is label free and able to detect lipids, proteins and nucleic acids within individual EVs. After isolation of EVs from cell culture medium, the protocol involves incubation of the EV sample on a suitable substrate, setup of the AFM-IR instrument and collection of nano-IR spectra and nano-IR images. Data acquisition and analyses can be completed within 24 h, and require only a basic knowledge of spectroscopy and chemistry. We anticipate that new understanding of EV composition and structure through AFM-IR will contribute to our biological understanding of EV biology and could find application in disease diagnosis and the development of EV therapies.

Greater cellular stiffness in fibroblasts from patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease involving degenerative breathing capacity. Fibrotic disease is driven by dysregulation in mechanical forces at the organ, tissue, and cellular level. While it is known that, in certain pathologies, diseased cells are stiffer than healthy cells, it is not known if fibroblasts derived from patients with IPF are stiffer than their normal counterparts. Using IPF patient-derived cell cultures, we measured the stiffness of individual lung fibroblasts via high-resolution force maps using atomic force microscopy. Fibroblasts from patients with IPF were stiffer and had an augmented cytoskeletal response to transforming growth factor-ß1 compared with fibroblasts from donors without IPF. The results from this novel study indicate that the increased stiffness of lung fibroblasts of IPF patients may contribute to the increased rigidity of fibrotic lung tissue.

None of us is the same as all of us: resolving the heterogeneity of extracellular vesicles using single-vesicle, nanoscale characterization with resonance enhanced atomic force microscope infrared spectroscopy (AFM-IR).

Extracellular vesicles (EVs) are highly specialized, nanoscale messengers that deliver biological signals and in doing so mediate intercellular communication. Increasing evidence shows that within populations of EVs, important properties including morphology, membrane composition, and content vary substantially. This heterogeneity arises in response to the nature, state, and environmental conditions of the cell source. However, currently there are no effective approaches, which unequivocally discriminate differences between individual EVs, which critically hampers progress in this emerging scientific area. Measuring EV heterogeneity is paramount to our understanding of how EVs influence the physiological and pathological functions of their target cells. Moreover, understanding EV heterogeneity is essential for their application as diagnostics and therapeutics. We propose an innovative approach using resonance enhanced atomic force microscope infrared spectroscopy (AFM-IR) to identify the nanoscale structural composition of EVs, as demonstrated and validated using EVs derived from two types of placenta stem cells. The particular strength of this approach is that it is a label-free and ultra-high sensitivity technique that has the power to measure individual EV heterogeneity. New insights gained by this method into EV heterogeneity will have a profound impact not only on our basic understanding of EV biology but also on disease diagnostics and the emerging area of EV-therapies.

Animal models of smoke inhalation injury and related acute and chronic lung diseases.

Smoke inhalation injury leads to various acute and chronic lung diseases and thus is the dominant cause of fire-related fatalities. In a search for an effective treatment and validation of therapies different classes of animal models have been developed, which include both small and large animals. These models have advanced our understanding of the mechanism of smoke inhalation injury, enabling a better understanding of pathogenesis and pathophysiology and development of new therapies. However, none of the animal models fully mirrors human lungs and their pathologies. All animal models have their limitations in replicating complex clinical conditions associated with smoke inhalation injury in humans. Therefore, for a correct interpretation of the results and to avoid bias, a precise understanding of similarities and differences of lungs between different animal species and humans is critical. We have reviewed and presented comprehensive comparison of different animal models and their clinical relevance. We presented an overview of methods utilized to induce smoke inhalation injuries, airway micro-/macrostructure, advantages and disadvantages of the most commonly used small and large animal models.

Atomized Human Amniotic Mesenchymal Stromal Cells for Direct Delivery to the Airway for Treatment of Lung Injury.

BACKGROUND: Current treatment regimens for inhalation injury are mainly supportive and rely on self-regeneration processes for recovery. Cell therapy with mesenchymal stromal cells (MSCs) is increasingly being investigated for the treatment of inhalation injury. Human amniotic MSCs (hAMSCs) were used in this study due to their potential use in inflammatory and fibrotic conditions of the lung. This study aimed at demonstrating that hAMSCs can be atomized with high viability, for the purpose of achieving a more uniform distribution of cells throughout the lung. Another aim of this study was to set ground for future application to healthy and diseased lungs by demonstrating that hAMSCs were able to survive after being sprayed onto substrates with different stiffness. METHODS: Two methods of atomization were evaluated, and the LMA MAD780 device was selected for atomizing hAMSCs for optimized delivery. To mimic the stiffness of healthy and diseased lungs, gelatin gel (10% w/v) and tissue culture plastic were used as preliminary models. Poly-l-lysine (PLL) and collagen I coatings were used as substrates on which the hAMSCs were cultured after being sprayed. RESULTS: The feasibility of atomizing hAMSCs was demonstrated with high cell viability (81 ± 3.1% and 79 ± 11.6% for cells sprayed onto plastic and gelatin, respectively, compared with 85 ± 4.8% for control/nonsprayed cells) that was unaffected by the different stiffness of substrates. The presence of the collagen I coating on which the sprayed cells were cultured yielded higher cell proliferation compared with both PLL and no coating. The morphology of sprayed cells was minimally compromised in the presence of the collagen I coating. CONCLUSIONS: This study demonstrated that hAMSCs are able to survive after being sprayed onto substrates with different stiffness, especially in the presence of collagen I. Further studies may advance the effectiveness of cell therapy for lung regeneration.

Formulation of Biologically-Inspired Silk-Based Drug Carriers for Pulmonary Delivery Targeted for Lung Cancer.

The benefits of using silk fibroin, a major protein in silk, are widely established in many biomedical applications including tissue regeneration, bioactive coating and in vitro tissue models. The properties of silk such as biocompatibility and controlled degradation are utilized in this study to formulate for the first time as carriers for pulmonary drug delivery. Silk fibroin particles are spray dried or spray-freeze-dried to enable the delivery to the airways via dry powder inhalers. The addition of excipients such as mannitol is optimized for both the stabilization of protein during the spray-freezing process as well as for efficient dispersion using an in vitro aerosolisation impactor. Cisplatin is incorporated into the silk-based formulations with or without cross-linking, which show different release profiles. The particles show high aerosolisation performance through the measurement of in vitro lung deposition, which is at the level of commercially available dry powder inhalers. The silk-based particles are shown to be cytocompatible with A549 human lung epithelial cell line. The cytotoxicity of cisplatin is demonstrated to be enhanced when delivered using the cross-linked silk-based particles. These novel inhalable silk-based drug carriers have the potential to be used as anti-cancer drug delivery systems targeted for the lungs.

The future perspectives of natural materials for pulmonary drug delivery and lung tissue engineering.

INTRODUCTION: Search for new, functional biomaterials that can be used to synergistically deliver a drug, enhance its adsorption and stimulate the post-injury recovery of tissue function, is one of the priorities in biomedicine. Currently used materials for drug delivery fail to satisfy one or more of these functionalities, thus they have limited potential and new classes of materials are urgently needed. AREAS COVERED: Natural materials, due to their origin, physical and chemical structure can potentially fulfill these requirements and there is already strong evidence of their usefulness in drug delivery. They are increasingly utilized in various therapeutic applications due to the obvious advantages over synthetic materials. Particularly in pulmonary drug delivery, there have been limitations in the use of synthetic materials such as polymers and lipids, leading to an increase in the use of natural and protein-based materials such as silk, keratin, elastin and collagen. Literature search in each specialized field, namely, silk, keratin and collagen was conducted, and the benefits of each material for future application in pulmonary drug delivery are highlighted. EXPERT OPINION: The natural materials discussed in this review have been well established in their use for other applications, yet further studies are required in the application of pulmonary drug delivery. The properties exhibited by these natural materials seem positive for their application in lung tissue engineering, which may allow for more extensive testing for validation of pulmonary drug delivery systems.