Characterization of Commercially Available Human Primary Alveolar Epithelial Cells.

In vitro lung research requires appropriate cell culture models that adequately mimic in vivo structure and function. Previously, researchers extensively used commercially available and easily expandable A549 and NCI-H441 cells, which replicate some but not all features of alveolar epithelial cells. Specifically, these cells are often restricted by terminally altered expression while lacking important alveolar epithelial characteristics. Of late, human primary alveolar epithelial cells (hPAEpCs) have become commercially available but are so far poorly specified. Here, we applied a comprehensive set of technologies to characterize their morphology, surface marker expression, transcriptomic profile, and functional properties. At optimized seeding numbers of 7,500 cells per square centimeter and growth at a gas-liquid interface, hPAEpCs formed regular monolayers with tight junctions and amiloride-sensitive transepithelial ion transport. Electron microscopy revealed lamellar body and microvilli formation characteristic for alveolar type II cells. Protein and single-cell transcriptomic analyses revealed expression of alveolar type I and type II cell markers; yet, transcriptomic data failed to detect NKX2-1, an important transcriptional regulator of alveolar cell differentiation. With increasing passage number, hPAEpCs transdifferentiated toward alveolar-basal intermediates characterized as SFTPC-, KRT8high, and KRT5- cells. In spite of marked changes in the transcriptome as a function of passaging, Uniform Manifold Approximation and Projection plots did not reveal major shifts in cell clusters, and epithelial permeability was unaffected. The present work delineates optimized culture conditions, cellular characteristics, and functional properties of commercially available hPAEpCs. hPAEpCs may provide a useful model system for studies on drug delivery, barrier function, and transepithelial ion transport in vitro.

Role of TLR4-p38 MAPK-Hsp27 signal pathway in LPS-induced pulmonary epithelial hyperpermeability.

BACKGROUND: The breakdown of alveolar barrier dysfunction contributes to Lipopolysaccharide stimulated pulmonary edema and acute lung injury. Actin cytoskeleton has been implicated to be critical in regulation of epithelial barrier. Here, we performed in vivo and in vitro study to investigate role of TLR4-p38 MAPK-Hsp27 signal pathway in LPS-induced ALI. METHODS: For in vivo studies, 6-8-week-old C57 mice were used, Bronchoalveolar lavage Fluid /Blood fluorescent ratio, wet-to-dry lung weight ratio, as well as protein concentrations and neutrophil cell counts in BALF were detected as either directly or indirectly indicators of pulmonary alveolar barrier dysfunction. And hematoxylin and eosin staining was performed to estimate pulmonary injury. The in vitro explorations of transepithelial permeability were achieved through transepithelial electrical resistance measurement and testing of FITC-Dextran transepithelial flux in A549. In addition, cytoskeletal rearrangement was tested through F-actin immunostaining. And SB203580 was used to inhibit p38 MAPK activation, while siRNA was administered to genetically knockdown specific protein. RESULTS: We showed that LPS triggered activation of p38 MAPK, rearrangement of cytoskeleton which resulted in severe epithelial hyperpermeability and lung edema. A549 pretreated with TLR4 siRNA、p38 MAPK siRNA and its inhibitor SB203580 displayed a lower permeability and fewer stress fibers formation after LPS stimulation, accompanied with lower phosphorylation level of p38 MAPK and Hsp27, which verified the involvement of TLR4-p38 MAPK-Hsp27 in LPS-evoked alveolar epithelial injury. Inhibition of p38 MAPK activity with SB203580 in vivo attenuated pulmonary edema formation and hyperpermeability in response to LPS. CONCLUSIONS: Our study demonstrated that LPS increased alveolar epithelial permeability both in vitro and in vivo and that TLR4- p38 MAPK- Hsp27 signal pathway dependent actin remolding was involved in this process.

Protective effects of chebulic acid on alveolar epithelial damage induced by urban particulate matter.

BACKGROUND: Chebulic acid (CA) isolated from T. chebula, which has been reported for treating asthma, as a potent anti-oxidant resources. Exposure to ambient urban particulate matter (UPM) considered as a risk for cardiopulmonary vascular dysfunction. To investigate the protective effect of CA against UPM-mediated collapse of the pulmonary alveolar epithelial (PAE) cell (NCI-H441), barrier integrity parameters, and their elements were evaluated in PAE. METHODS: CA was acquired from the laboratory previous reports. UPM was obtained from the National Institutes of Standards and Technology, and these were collected in St. Louis, MO, over a 24-month period and used as a standard reference. To confirm the protection of PAE barrier integrity, paracellular permeability and the junctional molecules were estimated with determination of transepithelial electrical resistance, Western Blotting, RT-PCR, and fluorescent staining. RESULTS: UPM aggravated the generation of reactive oxygen species (ROS) in PAE and also decreased mRNA and protein levels of junction molecules and barrier integrity in NCI-H441. However, CA repressed the ROS in PAE, also improved barrier integrity by protecting the junctional parameters in NCI-H411. CONCLUSIONS: These data showed that CA resulted in decreased UPM-induced ROS formation, and the protected the integrity of the tight junctions against UPM exposure to PAE barrier.

miR-186-5p improves alveolar epithelial barrier function by targeting the wnt5a/ß-catenin signaling pathway in sepsis-acute lung injury.

BACKGROUND: Alveolar epithelial barrier dysfunction is one of the pathological features of sepsis-acute lung injury(ALI). However, the molecular mechanisms that regulate the function of alveolar epithelial barrier remain unclear. This study aimed to determine the regulatory role of miR-186-5p in alveolar epithelial barrier function in sepsis-ALI and its underlying molecular mechanism. METHODS: We established sepsis-ALI models in vivo and in vitro, detected the miR-186-5p and wnt5a/ß-catenin expressions, and observed the functional changes of the alveolar epithelial barrier by miR-186-5p overexpression. We used rescue experiments to clarify whether miR-186-5p works through wnt5a/ß-catenin. RESULTS: miR-186-5p expression was decreased, wnt5a expression was increased, and the wnt5a/ß-catenin signaling pathway was activated in mouse lung tissues and A549 cells after inflammatory stimulation. miR-186-5p overexpression resulted in wnt5a/ß-catenin signaling pathway inhibition, decreased apoptosis in A549 cells, improved alveolar epithelial barrier function, reduced lung tissue injury in ALI mice, decreased IL-6 and TNF-α levels, and increased claudin4 and ZO-1 expression. Using miRNA-related database prediction and dual-luciferase reporter gene analysis, the targeting relationship between miR-186-5p and wnt5a was determined. The protective effect produced by miR-186-5p overexpression on the alveolar barrier was reversed after the application of the wnt5a/ß-catenin activator Licl. CONCLUSION: Our experimental data suggest miR-186-5p targets the wnt5a/ß-catenin pathway, thereby regulating alveolar epithelial barrier function. Furthermore, both miR-186-5p and wnt5a/ß-catenin are potential therapeutic targets that could impact sepsis-ALI.

3D Inkjet-Bioprinted Lung-on-a-Chip.

There is an urgent need for physiologically relevant and customizable biochip models of human lung tissue to provide a niche for lung disease modeling and drug efficacy. Although various lung-on-a-chips have been developed, the conventional fabrication method has been limited in reconstituting a very thin and multilayered architecture and spatial arrangements of multiple cell types in a microfluidic device. To overcome these limitations, we developed a physiologically relevant human alveolar lung-on-a-chip model, effectively integrated with an inkjet-printed, micron-thick, and three-layered tissue. After bioprinting lung tissues inside four culture inserts layer-by-layer, the inserts are implanted into a biochip that supplies a flow of culture medium. This modular implantation procedure enables the formation of a lung-on-a-chip to facilitate the culture of 3D-structured inkjet-bioprinted lung models under perfusion at the air-liquid interface. The bioprinted models cultured on the chip maintained their structure with three layers of tens of micrometers and achieved a tight junction in the epithelial layer, the critical properties of an alveolar barrier. The upregulation of genes involved in the essential functions of alveoli was also confirmed in our model. Our culture insert-mountable organ-on-a-chip is a versatile platform that can be applied to various organ models by implanting and replacing culture inserts. It is amenable to mass production and the development of customized models through the convergence with bioprinting technology.

3D pulmonary fibrosis model for anti-fibrotic drug discovery by inkjet-bioprinting.

Pulmonary fibrosis (PF) is known as a chronic and irreversible disease characterized by excessive extracellular matrix accumulation and lung architecture changes. Large efforts have been made to develop prospective treatments and study the etiology of pulmonary fibrotic diseases utilizing animal models and spherical organoids. As part of these efforts, we created an all-inkjet-printed three-dimensional (3D) alveolar barrier model that can be used for anti-fibrotic drug discovery. Then, we developed a PF model by treating the 3D alveolar barrier with pro-fibrotic cytokine and confirmed that it is suitable for the fibrosis model by observing changes in structural deposition, pulmonary function, epithelial-mesenchymal transition, and fibrosis markers. The model was tested with two approved anti-fibrotic drugs, and we could observe that the symptoms in the disease model were alleviated. Consequently, structural abnormalities and changes in mRNA expression were found in the induced fibrosis model, which were shown to be recovered in all drug treatment groups. The all-inkjet-printed alveolar barrier model was reproducible for disease onset and therapeutic effects in the human body. This finding emphasized that thein vitroartificial tissue with faithfully implemented 3D microstructures using bioprinting technology may be employed as a novel testing platform and disease model to evaluate potential drug efficacy.

Microvascular fluid flow in ex vivo and engineered lungs.

In recent years, it has become common to experiment with ex vivo perfused lungs for organ transplantation and to attempt regenerative pulmonary engineering using decellularized lung matrices. However, our understanding of the physiology of ex vivo organ perfusion is imperfect; it is not currently well understood how decreasing microvascular barrier affects the perfusion of pulmonary parenchyma. In addition, protocols for lung perfusion and organ culture fluid-handling are far from standardized, with widespread variation on both basic methods and on ideally controlled parameters. To address both of these deficits, a robust, noninvasive, and mechanistic model is needed which is able to predict microvascular resistance and permeability in perfused lungs while providing insight into capillary recruitment. Although validated mathematical models exist for fluid flow in native pulmonary tissue, previous models generally assume minimal intravascular leak from artery to vein and do not assess capillary bed recruitment. Such models are difficult to apply to both ex vivo lung perfusions, in which edema can develop over time and microvessels can become blocked, and to decellularized ex vivo organomimetic cultures, in which microvascular recruitment is variable and arterially perfused fluid enters into the alveolar space. Here, we develop a mathematical model of pulmonary microvascular fluid flow which is applicable in both instances, and we apply our model to data from native, decellularized, and regenerating lungs under ex vivo perfusion. The results provide substantial insight into microvascular pressure-flow mechanics, while producing previously unknown output values for tissue-specific capillary-alveolar hydraulic conductivity, microvascular recruitment, and total organ barrier resistance.NEW & NOTEWORTHY We present a validated model of pulmonary microvascular fluid mechanics and apply this model to study the effects of increased capillary permeability in decellularized and regenerating lungs. We find that decellularization alters microvascular steady-state mechanics and that re-endothelialization partially rescues key biologic parameters. The described model provides powerful insight into intraorgan microvascular dynamics and may be used to guide regenerative engineering experiments. We include all data and derivations necessary to replicate this work.

Alveolar epithelial glycocalyx shedding aggravates the epithelial barrier and disrupts epithelial tight junctions in acute respiratory distress syndrome.

The main pathophysiological mechanism of acute respiratory distress syndrome (ARDS) invovles the increase in alveolar barrier permeability that is primarily caused by epithelial glycocalyx and tight junction (TJ) protein destruction. This study was performed to explore the effects of the alveolar epithelial glycocalyx on the epithelial barrier, specifically on TJ proteins, in ARDS. We used C57BL/6 mice and human lung epithelial cell models of lipopolysaccharide (LPS)-induced ARDS. Changes in alveolar permeability were evaluated via pulmonary histopathology analysis and by measuring the wet/dry weight ratio of the lungs. Degradation of heparan sulfate (HS), an important component of the epithelial glycocalyx, and alterations in levels of the epithelial TJ proteins (occludin, zonula occludens 1, and claudin 4) were assessed via ELISA, immunofluorescence analysis, and western blotting analysis. Real-time quantitative polymerase chain reaction was used to detect the mRNA of the TJ protein. Changes in glycocalyx and TJ ultrastructures in alveolar epithelial cells were evaluated through electron microscopy. In vivo and in vitro, LPS increased the alveolar permeability and led to HS degradation and TJ damage. After LPS stimulation, the expression of the HS-degrading enzyme heparanase (HPA) in the alveolar epithelial cells was increased. The HPA inhibitor N-desulfated/re-N-acetylated heparin alleviated LPS-induced HS degradation and reduced TJ damage. In vitro, recombinant HPA reduced the expression of the TJ protein zonula occludens-1 (ZO-1) and inhibited its mRNA expression in the alveolar epithelial cells. Taken together, our results demonstrate that shedding of the alveolar epithelial glycocalyx aggravates the epithelial barrier and damages epithelial TJ proteins in ARDS, with the underlying mechanism involving the effect of HPA on ZO-1.

Imaging Plasmodium immunobiology in the liver, brain, and lung.

Plasmodium falciparum malaria is responsible for the deaths of over half a million African children annually. Until a decade ago, dynamic analysis of the malaria parasite was limited to in vitro systems with the typical limitations associated with 2D monocultures or entirely artificial surfaces. Due to extremely low parasite densities, the liver was considered a black box in terms of Plasmodium sporozoite invasion, liver stage development, and merozoite release into the blood. Further, nothing was known about the behavior of blood stage parasites in organs such as the brain where clinical signs manifest and the ensuing immune response of the host that may ultimately result in a fatal outcome. The advent of fluorescent parasites, advances in imaging technology, and availability of an ever-increasing number of cellular and molecular probes have helped illuminate many steps along the pathogenetic cascade of this deadly tropical parasite.