Second-generation lung-on-a-chip with an array of stretchable alveoli made with a biological membrane.

The air-blood barrier with its complex architecture and dynamic environment is difficult to mimic in vitro. Lung-on-a-chips enable mimicking the breathing movements using a thin, stretchable PDMS membrane. However, they fail to reproduce the characteristic alveoli network as well as the biochemical and physical properties of the alveolar basal membrane. Here, we present a lung-on-a-chip, based on a biological, stretchable and biodegradable membrane made of collagen and elastin, that emulates an array of tiny alveoli with in vivo-like dimensions. This membrane outperforms PDMS in many ways: it does not absorb rhodamine-B, is biodegradable, is created by a simple method, and can easily be tuned to modify its thickness, composition and stiffness. The air-blood barrier is reconstituted using primary lung alveolar epithelial cells from patients and primary lung endothelial cells. Typical alveolar epithelial cell markers are expressed, while the barrier properties are preserved for up to 3 weeks.

Epithelial dysfunction in chronic respiratory diseases, a shared endotype?

PURPOSE OF REVIEW: Epithelial barrier defects are being appreciated in various inflammatory disorders; however, causal underlying mechanisms are lacking. In this review, we describe the disruption of the airway epithelium with regard to upper and lower airway diseases, the role of epigenetic alterations underlying this process, and potential novel ways of interfering with dysfunctional epithelial barriers as a novel therapeutic approach. RECENT FINDINGS: A defective epithelial barrier, impaired innate defence mechanisms or hampered epithelial cell renewal are found in upper and lower airway diseases. Barrier dysfunction might facilitate the entrance of foreign substances, initiating and facilitating the onset of disease. Latest data provided novel insights for possible involvement of epigenetic alterations induced by inflammation or other unknown mechanisms as a potential mechanism responsible for epithelial defects. Additionally, these mechanisms might precede disease development, and represent a novel therapeutic approach for restoring epithelial defects. SUMMARY: A better understanding of the role of epigenetics in driving and maintaining epithelial defects in various inflammatory diseases, using state-of-the-art biology tools will be crucial in designing novel therapies to protect or reconstitute a defective airway epithelial barrier.

Passing the Vascular Barrier: Endothelial Signaling Processes Controlling Extravasation.

A central function of the vascular endothelium is to serve as a barrier between the blood and the surrounding tissue of the body. At the same time, solutes and cells have to pass the endothelium to leave or to enter the bloodstream to maintain homeostasis. Under pathological conditions, for example, inflammation, permeability for fluid and cells is largely increased in the affected area, thereby facilitating host defense. To appropriately function as a regulated permeability filter, the endothelium uses various mechanisms to allow solutes and cells to pass the endothelial layer. These include transcellular and paracellular pathways of which the latter requires remodeling of intercellular junctions for its regulation. This review provides an overview on endothelial barrier regulation and focuses on the endothelial signaling mechanisms controlling the opening and closing of paracellular pathways for solutes and cells such as leukocytes and metastasizing tumor cells.

Comparative View of Lung Vascular Endothelium of Cattle, Horses, and Water Buffalo.

Endothelium plays an important role in maintaining the vascular barrier and physiological homeostasis. Endothelium also is fundamental to the initiation and regulation of inflammation. Endothelium demonstrates phenotypic and functional heterogeneity not only among various organs but also within an organ. One of the striking examples would be the pulmonary endothelium that participates in creating blood-air barrier. Endothelium in large pulmonary blood vessels is distinct in structure and function from that lining of the pulmonary capillaries. This chapter focuses on the comparative aspects of pulmonary endothelium and highlight unique differences such as the presence of pulmonary intravascular macrophages among select species.

The Pulmonary Vascular Barrier: Insights into Structure, Function, and Regulatory Mechanisms.

Pulmonary blood vessels act as a well-regulated barrier to the flux of fluid and solutes between the lumen and the air space. Perturbation of the barrier function results in excessive fluid leak into the interstitium and alveoli, and impairs gas exchange. Recent studies provide deeper insight into the precise control mechanisms involved in the regulation of the barrier. This chapter will highlight these mechanisms and discuss the current understanding on the fluid and solute transport pathways across the vascular endothelial layer. In addition, the chapter summarizes the contributions of extra-endothelial structures such as pericytes and glycocalyx in regulating fluid flux across pulmonary vessels. The chapter concludes with an analysis on the impact of pulmonary endothelial heterogeneity and experimental models on current interpretations of barrier function and regulatory mechanisms.

Co-culture of human alveolar epithelial (hAELVi) and macrophage (THP-1) cell lines.

The air-blood barrier is mainly composed of alveolar epithelial cells and macrophages. Whereas the epithelium acts as a diffusional barrier, macrophages represent an immunological barrier, in particular for larger molecules and nanoparticles. This paper describes a new co-culture of human cell lines representing both cell types. Acquiring, culturing and maintaining primary alveolar epithelial cells presents significant logistical and technical difficulties. The recently established human alveolar epithelial lentivirus immortalized cell line, hAELVi, when grown on permeable filters, form monolayers with high functional and morphological resemblance to alveolar type I cells. To model alveolar macrophages, the human cell line THP-1 was seeded on pre-formed hAELVi monolayers. The co-culture was characterized regarding cellular morphology, viability and barrier function. Macrophages were homogenously distributed on the epithelium and could be kept in co-culture for up to 7 days. Transmission electron microscopy showed loose contact between THP-1 and hAELVi cells. When grown at air liquid interface, both cells were covered with extracellular matrix-like structure, which was absent in THP-1 mono culture. In co-culture with macrophages, hAELVi cells displayed similar, sometimes even higher, trans-epithelial electrical resistance than in mono-cultures. When exposed to silver and starch NPs, hAELVi mono-cultures were more tolerant to the particles than THP-1 mono-cultures. The viability in the co-culture was similar to that of hAELVi monocultures. Transport studies with sodium fluorescein in presence/absence of EDTA proved that the co culture acts as functional diffusion barrier. These data demonstrate that hAELVi-/THP-1 co-cultures represent a promising model for safety and permeability studies of inhaled chemicals, drugs and nanoparticles.

Using a Novel Microfabricated Model of the Alveolar-Capillary Barrier to Investigate the Effect of Matrix Structure on Atelectrauma.

The alveolar-capillary barrier is composed of epithelial and endothelial cells interacting across a fibrous extracelluar matrix (ECM). Although remodeling of the ECM occurs during several lung disorders, it is not known how fiber structure and mechanics influences cell injury during cyclic airway reopening as occurs during mechanical ventilation (atelectrauma). We have developed a novel in vitro platform that mimics the micro/nano-scale architecture of the alveolar microenvironment and have used this system to investigate how ECM microstructural properties influence epithelial cell injury during airway reopening. In addition to epithelial-endothelial interactions, our platform accounts for the fibrous topography of the basal membrane and allows for easy modulation of fiber size/diameter, density and stiffness. Results indicate that fiber stiffness and topography significantly influence epithelial/endothelial barrier function where increased fiber stiffness/density resulted in altered cytoskeletal structure, increased tight junction (TJ) formation and reduced barrier permeability. However, cells on rigid/dense fibers were also more susceptible to injury during airway reopening. These results indicate that changes in the mechanics and architecture of the lung microenvironment can significantly alter cell function and injury and demonstrate the importance of implementing in vitro models that more closely resemble the natural conditions of the lung microenvironment.

Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier.

This paper describes a new human alveolar epithelial cell line (hAELVi - human Alveolar Epithelial Lentivirus immortalized) with type I-like characteristics and functional tight junctions, suitable to model the air-blood barrier of the peripheral lung. Primary human alveolar epithelial cells were immortalized by a novel regimen, grown as monolayers on permeable filter supports and characterized morphologically, biochemically and biophysically. hAELVi cells maintain the capacity to form tight intercellular junctions, with high trans-epithelial electrical resistance (> 1000 Ω*cm²). The cells could be kept in culture over several days, up to passage 75, under liquid-liquid as well as air-liquid conditions. Ultrastructural analysis and real time PCR revealed type I-like cell properties, such as the presence of caveolae, expression of caveolin-1, and absence of surfactant protein C. Accounting for the barrier properties, inter-digitations sealed with tight junctions and desmosomes were also observed. Low permeability of the hydrophilic marker sodium fluorescein confirmed the suitability of hAELVi cells for in vitro transport studies across the alveolar epithelium. These results suggest that hAELVi cells reflect the essential features of the air-blood barrier, as needed for an alternative to animal testing to study absorption and toxicity of inhaled drugs, chemicals and nanomaterials.

Membrane mediated development of the vertebrate blood-gas-barrier.

During embryonic lung development, establishment of the gas-exchanging units is guided by epithelial tubes lined by columnar cells. Ultimately, a thin blood-gas barrier (BGB) is established and forms the interface for efficient gas exchange. This thin BGB is achieved through processes, which entail lowering of tight junctions, stretching, and thinning in mammals. In birds the processes are termed peremerecytosis, if they involve cell squeezing and constriction, or secarecytosis, if they entail cutting cells to size. In peremerecytosis, cells constrict at a point below the protruding apical part, resulting in fusion of the opposing membranes and discharge of the aposome, or the cell may be squeezed by the more endowed cognate neighbors. Secarecytosis may entail formation of double membranes below the aposome, subsequent unzipping and discharge of the aposome, or vesicles form below the aposome, fuse in a bilateral manner, and release the aposome. These processes occur within limited developmental windows, and are mediated through cell membranes that appear to be of intracellular in origin. In addition, basement membranes (BM) play pivotal roles in differentiation of the epithelial and endothelial layers of the BGB. Laminins found in the BM are particularly important in the signaling pathways that result in formation of squamous pneumocytes and pulmonary capillaries, the two major components of the BGB. Some information exists on the contribution by BM to BGB formation, but little is known regarding the molecules that drive peremerecytosis, or even the origins and composition of the double and vesicular membranes involved in secarecytosis.

Autophagy protects against ischemia/reperfusion-induced lung injury through alleviating blood-air barrier damage.

BACKGROUND: Understanding the role and underlying regulation mechanism of autophagy in ischemia/reperfusion (I/R)-induced lung injury may provide potentially new pharmacologic targets for treatment of acute lung injury. The aim of this study was to adjust autophagy with pharmacologic agents to determine its functional significance in I/R-induced lung injury. METHODS: Human pulmonary microvascular endothelial cells (HPMVECs) and mice were pre-conditioned with autophagy inhibitor chloroquine or promoter rapamycin before they were challenged with oxygen-glucose deprivation/oxygen-glucose restoration (OGD) and lung I/R, respectively. Extracellular signal-regulated kinase (ERK)1/2 inhibitor U0126 was pre-injected into I/R-induced mice to test the role of ERK1/2 in regulating autophagy. RESULTS: OGD caused tight conjunction damage and cell death in HPMVECs, which was further aggravated by blocking autophagy, yet ameliorated through promoting autophagy. On a consistent basis, inhibiting autophagy aggravated I/R-induced lung edema and tissue inflammation, which was significantly alleviated by promoting autophagy with rapamycin. In addition, inhibition of ERK1/2 increased expression of active mammalian target-of-rapamycin and thus decreased I/R-induced autophagy. CONCLUSIONS: It appears that autophagy plays a protective role in I/R-induced lung injury and this effect may be enhanced by moderately improving autophagy level. Meanwhile, the ERK1/2 signal pathway has a positively regulating role in lung I/R-induced autophagy.

Mathematical model of a heterogeneous pulmonary acinus structure.

The pulmonary acinus is a gas exchange unit distal to the terminal bronchioles. A model of its structure is important for the computational investigation of mechanical phenomena at the acinus level. We propose a mathematical model of a heterogeneous acinus structure composed of alveoli of irregular sizes, shapes, and locations. The alveoli coalesce into an intricately branched ductal tree, which meets the space-filling requirement of the acinus structure. Our model uses Voronoi tessellation to generate an assemblage of the alveolar or ductal airspace, and Delaunay tessellation and simulated annealing for the ductal tree structure. The modeling condition is based on average acinar and alveolar volume characteristics from published experimental information. By applying this modeling technique to the acinus of healthy mature rats, we demonstrate that the proposed acinus structure model reproduces the available experimental information. In the model, the shape and size of alveoli and the length, generation, tortuosity, and branching angle of the ductal paths are distributed in several ranges. This approach provides a platform for investigating the heterogeneous nature of the acinus structure and its relationship with mechanical phenomena at the acinus level.

Engineering an in vitro air-blood barrier by 3D bioprinting.

Intensive efforts in recent years to develop and commercialize in vitro alternatives in the field of risk assessment have yielded new promising two- and three dimensional (3D) cell culture models. Nevertheless, a realistic 3D in vitro alveolar model is not available yet. Here we report on the biofabrication of the human air-blood tissue barrier analogue composed of an endothelial cell, basement membrane and epithelial cell layer by using a bioprinting technology. In contrary to the manual method, we demonstrate that this technique enables automatized and reproducible creation of thinner and more homogeneous cell layers, which is required for an optimal air-blood tissue barrier. This bioprinting platform will offer an excellent tool to engineer an advanced 3D lung model for high-throughput screening for safety assessment and drug efficacy testing.

Oxygen-induced plasticity in tracheal morphology and discontinuous gas exchange cycles in cockroaches Nauphoeta cinerea.

The function and mechanism underlying discontinuous gas exchange in terrestrial arthropods continues to be debated. Three adaptive hypotheses have been proposed to explain the evolutionary origin or maintenance of discontinuous gas exchange cycles (DGCs), which may have evolved to reduce respiratory water loss, facilitate gas exchange in high CO2 and low O2 micro-environments, or to ameliorate potential damage as a result of oversupply of O2. None of these hypotheses have unequivocal support, and several non-adaptive hypotheses have also been proposed. In the present study, we reared cockroaches Nauphoeta cinerea in selected levels of O2 throughout development, and examined how this affected growth rate, tracheal morphology and patterns of gas exchange. O2 level in the rearing environment caused significant changes in tracheal morphology and the exhibition of DGCs, but the direction of these effects was inconsistent with all three adaptive hypotheses: water loss was not associated with DGC length, cockroaches grew fastest in hyperoxia, and DGCs exhibited by cockroaches reared in normoxia were shorter than those exhibited by cockroaches reared in hypoxia or hyperoxia.

Regulation and repair of the alveolar-capillary barrier in acute lung injury.

Considerable progress has been made in understanding the basic mechanisms that regulate fluid and protein exchange across the endothelial and epithelial barriers of the lung under both normal and pathological conditions. Clinically relevant lung injury occurs most commonly from severe viral and bacterial infections, aspiration syndromes, and severe shock. The mechanisms of lung injury have been identified in both experimental and clinical studies. Recovery from lung injury requires the reestablishment of an intact endothelial barrier and a functional alveolar epithelial barrier capable of secreting surfactant and removing alveolar edema fluid. Repair mechanisms include the participation of endogenous progenitor cells in strategically located niches in the lung. Novel treatment strategies include the possibility of cell-based therapy that may reduce the severity of lung injury and enhance lung repair.

Platelets in lung biology.

Platelets and the lungs have an intimate relationship. Platelets are anucleate mammalian blood cells that continuously circulate through pulmonary vessels and that have major effector activities in hemostasis and inflammation. The lungs are reservoirs for megakaryocytes, the requisite precursor cell in thrombopoiesis, which is the intricate process by which platelets are generated. Platelets contribute to basal barrier integrity of the alveolar capillaries, which selectively restricts the transfer of water, proteins, and red blood cells out of the vessels. Platelets also contribute to pulmonary vascular repair. Although platelets bolster hemostatic and inflammatory defense of the healthy lung, experimental evidence and clinical evidence indicate that these blood cells are effectors of injury in a variety of pulmonary disorders and syndromes. Newly discovered biological capacities of platelets are being explored in the context of lung defense, disease, and remodeling.

The TAT peptide endows PACAP with an enhanced ability to traverse bio-barriers.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potential therapeutic neuropeptide. The 11-amino acid human immunodeficiency virus TAT protein transduction domain is able to deliver protein cargoes across the cell membrane and the blood-brain barrier. A novel fusion protein PACAP-TAT, containing TAT at the C-terminus of PACAP was therefore produced and studied for the ability to cross blood barriers. The gene encoding PACAP-TAT was cloned into the expression vector pKYB, and the target peptide PACAP-TAT was purified using the Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) system. The results of cell assays showed that PACAP-TAT stimulated the cell viability of PAC1-CHO cells with the same potency as PACAP, which indicated that the fusion of TAT did not affect the ability of PACAP-TAT to activate the PACAP-specific receptor PAC1. The transfer efficiencies of PACAP-TAT and PACAP across the blood-brain barrier (BBB), blood-air barrier (BAB) and blood-testis barrier (BTB) were assayed using peptides labeled with fluorescein isothiocyanate (FITC). The results showed that PACAP-TAT traversed blood barriers with an efficiency approximately 2.5-fold greater than PACAP. Fluorescence microscopic examination showed that PACAP-TAT traversed the BBB significantly more efficiently than PACAP. Furthermore, intraperitoneal (i.p.) injection of PACAP-TAT induced a stronger inhibitory effect on food intake than PACAP (p<0.01, PACAP-TAT vs. PACAP), which indicated that TAT helped to increase the localization of PACAP-TAT in the brain. Preparation of PACAP-TAT with the enhanced ability to cross biological barriers will improve its route of administration and expand its scope of application.

Recent advances into understanding some aspects of the structure and function of mammalian and avian lungs.

Recent findings are reported about certain aspects of the structure and function of the mammalian and avian lungs that include (a) the architecture of the air capillaries (ACs) and the blood capillaries (BCs); (b) the pulmonary blood capillary circulatory dynamics; (c) the adaptive molecular, cellular, biochemical, compositional, and developmental characteristics of the surfactant system; (d) the mechanisms of the translocation of fine and ultrafine particles across the airway epithelial barrier; and (e) the particle-cell interactions in the pulmonary airways. In the lung of the Muscovy duck Cairina moschata, at least, the ACs are rotund structures that are interconnected by narrow cylindrical sections, while the BCs comprise segments that are almost as long as they are wide. In contrast to the mammalian pulmonary BCs, which are highly compliant, those of birds practically behave like rigid tubes. Diving pressure has been a very powerful directional selection force that has influenced phenotypic changes in surfactant composition and function in lungs of marine mammals. After nanosized particulates are deposited on the respiratory tract of healthy human subjects, some reach organs such as the brain with potentially serious health implications. Finally, in the mammalian lung, dendritic cells of the pulmonary airways are powerful agents in engulfing deposited particles, and in birds, macrophages and erythrocytes are ardent phagocytizing cellular agents. The morphology of the lung that allows it to perform different functions-including gas exchange, ventilation of the lung by being compliant, defense, and secretion of important pharmacological factors-is reflected in its "compromise design."

Emodin enhances alveolar epithelial barrier function in rats with experimental acute pancreatitis.

AIM: To investigate the effect of emodin on expression of claudin-4, claudin-5 and occludin, as well as the alveolar epithelial barrier in rats with pancreatitis induced by sodium taurocholate. METHODS: Experimental pancreatitis was induced by retrograde injection of 5% sodium taurocholate into the biliopancreatic duct. Emodin was injected via the external jugular vein 3 h after induction of acute pancreatitis. Rats from sham operation group and acute pancreatitis group were injected with normal saline (an equivalent volume as emodin) at the same time point. Samples of lung and serum were obtained 6 h after drug administration. Pulmonary morphology was examined with HE staining. Pulmonary edema was estimated by measuring water content in lung tissue samples. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) level were measured by enzyme-linked immunospecific assay. Serum amylase and pulmonary myeloperoxidase (MPO) activity were detected by spectrophotometry. Alveolar epithelial barrier was assessed by pulmonary dye extravasation. Expression of claudin-4, claudin-5 and occludin in lung tissue samples was examined by immunohistology, quantitative real-time reverse transcription polymerase chain reaction and Western blotting analysis, respectively. RESULTS: Pancreatitis-associated lung injury was characterized by pulmonary edema, leukocyte infiltration, alveolar collapse, and elevated serum amylase level. The pulmonary damage, pulmonary pathological scores, serum amylase and MPO activity, TNF-alpha and IL-6 levels, and wet/dry ratio were decreased in rats after treatment with emodin. Immunostaining of claudin-4, claudin-5 and occludin was detected in lung tissue samples from rats in sham operation group, which was distributed in alveolar epithelium, vascular endothelium, and bronchial epithelium, respectively. The mRNA and protein expression levels of claudin-4, claudin-5 and occludin in lung tissue samples were markedly decreased, the expression level of claudin-4, claudin-5 and occluding was increased, and the pulmonary dye extravasation was reduced in lung tissue samples from rats with acute pancreatitis after treatment with emodin. CONCLUSION: Emodin attenuates pulmonary edema and inflammation, enhances alveolar epithelial barrier function, and promotes expression of claudin-4, claudin-5 and occludin in lung tissue samples from rats with acute pancreatitis.