Comparative electron microscopic visualization of the lung alveolar epithelial glycocalyx with different staining and labeling methods.

The alveolar surface of the lung is lined by an epithelium consisting of type I (AECI) and type II alveolar epithelial cells (AECII). This epithelium is covered by a liquid alveolar lining layer (ALL). Besides intra-alveolar surfactant, ALL also contains the alveolar epithelial glycocalyx on the apical side of AECI and AECII. To better understand the alveolar epithelial glycocalyx, its ultrastructural visualization by transmission electron microscopy is required. The aim of this study was to systematically re-evaluate routine cytochemical methods for visualization of the alveolar epithelial glycocalyx and specifically its glycan components. For this purpose, we used chemical fixation by vascular perfusion with aldehydes as a common routine approach in mice. After fixation, staining is needed for glycocalyx visualization. Cytochemical staining agents such as alcian blue, ruthenium red, and lanthanum nitrate were compared. In addition, SNL (Sambucus nigra lectin) and UEA1 (Ulex europaeus agglutinin I) were used for sialic acid and fucose-specific labeling. Alcian blue showed the strongest staining, with cloud-like structures, whereas ruthenium red appeared as thread-like structures. On the other hand, lanthanum nitrate did not stain the alveolar epithelial glycocalyx. For specific sialic acid and fucose labeling, both lectins presented a specific signal. In conclusion, these methods can be used routinely for assessing ultrastructural changes of the alveolar epithelial glycocalyx in experimental in vivo models under different physiological and pathological conditions. In addition, cytochemical staining by tissue massage and post-embedding lectin labeling after vascular perfusion support 3R (reduction, refinement, replacement) principles of animal welfare.

Enzymatic Modulation of the Pulmonary Glycocalyx Enhances Susceptibility to Streptococcus pneumoniae.

The pulmonary epithelial glycocalyx is rich in glycosaminoglycans such as hyaluronan and heparan sulfate. Despite their presence, the importance of these glycosaminoglycans in bacterial lung infections remains elusive. To address this, we intranasally inoculated mice with Streptococcus pneumoniae in the presence or absence of enzymes targeting pulmonary hyaluronan and heparan sulfate, followed by characterization of subsequent disease pathology, pulmonary inflammation, and lung barrier dysfunction. Enzymatic degradation of hyaluronan and heparan sulfate exacerbated pneumonia in mice, as evidenced by increased disease scores and alveolar neutrophil recruitment. However, targeting epithelial hyaluronan in combination with Streptococcus pneumoniae infection further exacerbated systemic disease, indicated by elevated splenic bacterial load and plasma levels of pro-inflammatory cytokines. In contrast, enzymatic cleavage of heparan sulfate resulted in increased bronchoalveolar bacterial burden, lung damage and pulmonary inflammation in mice infected with Streptococcus pneumoniae. Accordingly, heparinase-treated mice also exhibited disrupted lung barrier integrity as evidenced by higher alveolar edema scores and vascular protein leakage into the airways. This finding was corroborated in a human alveolus-on-a-chip platform, confirming that heparinase treatment also disrupts the human lung barrier during Streptococcus pneumoniae infection. Notably, enzymatic pre-treatment with either hyaluronidase or heparinase also rendered human epithelial cells more sensitive to pneumococcal-induced barrier disruption, as determined by transepithelial electrical resistance measurements, consistent with our findings in murine pneumonia. Taken together, these findings demonstrate the importance of intact hyaluronan and heparan sulfate in limiting pneumococci-induced damage, pulmonary inflammation, and epithelial barrier function and integrity. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Multivalent, calcium-independent binding of surfactant protein A and D to sulfated glycosaminoglycans of the alveolar epithelial glycocalyx.

Lung surfactant collectins, surfactant protein A (SP-A) and D (SP-D), are oligomeric C-type lectins involved in lung immunity. Through their carbohydrate recognition domain, they recognize carbohydrates at pathogen surfaces and initiate lung innate immune response. Here, we propose that they may also be able to bind to other carbohydrates present in typical cell surfaces, such as the alveolar epithelial glycocalyx. To test this hypothesis, we analyzed and quantified the binding affinity of SP-A and SP-D to different sugars and glycosaminoglycans (GAGs) by microscale thermophoresis (MST). In addition, by changing the calcium concentration, we aimed to characterize any consequences on the binding behavior. Our results show that both oligomeric proteins bind with high affinity (in nanomolar range) to GAGs, such as hyaluronan (HA), heparan sulfate (HS) and chondroitin sulfate (CS). Binding to HS and CS was calcium-independent, as it was not affected by changing calcium concentration in the buffer. Quantification of GAGs in bronchoalveolar lavage (BAL) fluid from animals deficient in either SP-A or SP-D showed changes in GAG composition, and electron micrographs showed differences in alveolar glycocalyx ultrastructure in vivo. Taken together, SP-A and SP-D bind to model sulfated glycosaminoglycans of the alveolar epithelial glycocalyx in a multivalent and calcium-independent way. These findings provide a potential mechanism for SP-A and SP-D as an integral part of the alveolar epithelial glycocalyx binding and interconnecting free GAGs, proteoglycans, and other glycans in glycoproteins, which may influence glycocalyx composition and structure.NEW & NOTEWORTHY SP-A and SP-D function has been related to innate immunity of the lung based on their binding to sugar residues at pathogen surfaces. However, their function in the healthy alveolus was considered as limited to interaction with surfactant lipids. Here, we demonstrated that these proteins bind to glycosaminoglycans present at typical cell surfaces like the alveolar epithelial glycocalyx. We propose a model where these proteins play an important role in interconnecting alveolar epithelial glycocalyx components.

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.

The unremarkable alveolar epithelial glycocalyx: a thorium dioxide-based electron microscopic comparison after heparinase or pneumolysin treatment.

Recent investigations analyzed in depth the biochemical and biophysical properties of the endothelial glycocalyx. In comparison, this complex cell-covering structure is largely understudied in alveolar epithelial cells. To better characterize the alveolar glycocalyx ultrastructure, unaffected versus injured human lung tissue explants and mouse lungs were analyzed by transmission electron microscopy. Lung tissue was treated with either heparinase (HEP), known to shed glycocalyx components, or pneumolysin (PLY), the exotoxin of Streptococcus pneumoniae not investigated for structural glycocalyx effects so far. Cationic colloidal thorium dioxide (cThO2) particles were used for glycocalyx glycosaminoglycan visualization. The level of cThO2 particles orthogonal to apical cell membranes (≙ stained glycosaminoglycan height) of alveolar epithelial type I (AEI) and type II (AEII) cells was stereologically measured. In addition, cThO2 particle density was studied by dual-axis electron tomography (≙ stained glycosaminoglycan density in three dimensions). For untreated samples, the average cThO2 particle level was ≈ 18 nm for human AEI, ≈ 17 nm for mouse AEI, ≈ 44 nm for human AEII and ≈ 35 nm for mouse AEII. Both treatments, HEP and PLY, resulted in a significant reduction of cThO2 particle levels on human and mouse AEI and AEII. Moreover, a HEP- and PLY-associated reduction in cThO2 particle density was observed. The present study provides quantitative data on the differential glycocalyx distribution on AEI and AEII based on cThO2 and demonstrates alveolar glycocalyx shedding in response to HEP or PLY resulting in a structural reduction in both glycosaminoglycan height and density. Future studies should elucidate the underlying alveolar epithelial cell type-specific distribution of glycocalyx subcomponents for better functional understanding.

Ten simple rules for implementing open and reproducible research practices after attending a training course.

Open, reproducible, and replicable research practices are a fundamental part of science. Training is often organized on a grassroots level, offered by early career researchers, for early career researchers. Buffet style courses that cover many topics can inspire participants to try new things; however, they can also be overwhelming. Participants who want to implement new practices may not know where to start once they return to their research team. We describe ten simple rules to guide participants of relevant training courses in implementing robust research practices in their own projects, once they return to their research group. This includes (1) prioritizing and planning which practices to implement, which involves obtaining support and convincing others involved in the research project of the added value of implementing new practices; (2) managing problems that arise during implementation; and (3) making reproducible research and open science practices an integral part of a future research career. We also outline strategies that course organizers can use to prepare participants for implementation and support them during this process.

Human alveolar progenitors generate dual lineage bronchioalveolar organoids.

Mechanisms of epithelial renewal in the alveolar compartment remain incompletely understood. To this end, we aimed to characterize alveolar progenitors. Single-cell RNA-sequencing (scRNA-seq) analysis of the HTII-280+/EpCAM+ population from adult human lung revealed subclusters enriched for adult stem cell signature (ASCS) genes. We found that alveolar progenitors in organoid culture in vitro show phenotypic lineage plasticity as they can yield alveolar or bronchial cell-type progeny. The direction of the differentiation is dependent on the presence of the GSK-3ß inhibitor, CHIR99021. By RNA-seq profiling of GSK-3ß knockdown organoids we identified additional candidate target genes of the inhibitor, among others FOXM1 and EGF. This gives evidence of Wnt pathway independent regulatory mechanisms of alveolar specification. Following influenza A virus (IAV) infection organoids showed a similar response as lung tissue explants which confirms their suitability for studies of sequelae of pathogen-host interaction.

The ultrastructural heterogeneity of lung surfactant revealed by serial section electron tomography: insights into the 3-D architecture of human tubular myelin.

Weibel's hypothetical three-dimensional (3-D) model in 1966 provided first ultrastructural details into tubular myelin (TM), a unique, complex surfactant subtype found in the hypophase of the alveolar lining layer. Although initial descriptions by electron microscopy (EM) were already published in the 1950s, a uniform morphological differentiation from other intra-alveolar surfactant subtypes is still missing and potential structure-function relationships remain enigmatic. Technical developments in volume EM methods now allow a more detailed reinvestigation, to address unanswered ultrastructural questions, we analyzed ultrathin sections of humanized SP-A1/SP-A2 coexpressing mouse and human lung samples by conventional transmission EM. We combined these two-dimensional (2-D) information with 3-D analysis of single- and dual-axis electron tomography of serial sections for high z-resolution (in a range of a few nanometers) and extended volumes of up to 1 µm total z-information, this study reveals that TM constitutes a heterogeneous surfactant organization mainly comprised of distorted parallel membrane planes with local intersections, which are distributed all over the TM substructure. These intersecting membrane planes form, among other various polygons, the well-known 2-D "lattice", respectively 3-D quadratic tubules, which in many analyzed spots of human alveoli appear to be less abundant than also observed nonconcentric 3-D lamellae, the additional application of serial section electron tomography to conventional transmission EM demonstrates a high heterogeneity of TM membrane networks, which indicates dynamic transformations between its substructures. Our method provides an ideal basis for further in and ex vivo structural analyses of surfactant under various conditions at nanometer scale.

Resting time after phorbol 12-myristate 13-acetate in THP-1 derived macrophages provides a non-biased model for the study of NLRP3 inflammasome.

Background: The activation of NLRP3 inflammasome in macrophages has been proven to play a crucial role in the development of cardiovascular diseases. THP-1 monocytes can be differentiated to macrophages by incubation with phorbol-12-myristate 13-acetate (PMA), providing a suitable model for in vitro studies. However, PMA has been shown to have effects on the levels of IL-1ß, the main mediator of NLRP3 inflammasome, while the effects on the other mediators of the inflammasome have not been reported before. Methods: THP-1 monocytes were incubated without (THP-1), with 5ng/ml PMA for 48h (PMA48h) or with 5ng/ml PMA for 48h plus 24h in fresh medium (PMArest). Morphological changes and the expression of macrophage surface markers (CD14, CD11b, CD36 and CD204) were evaluated by flow cytometry. Changes in intracellular levels of inflammasome components (NLRP3, ASC, pro-caspase-1, pro-IL1ß) were analyzed by western blot and release of mature IL-1ß in cell supernatant was analyzed by ELISA. ASC speck formation was determined by immunofluorescence. Results: After 48h incubation with PMA or subsequent rest in fresh medium, cells became adherent, and the differential expression of CD36, CD11b, CD14 and CD204 compared to THP-1 cells confirmed that PMArest resemble macrophages from a molecular point of view. Changes in the levels were detected in PMA48h group for all the NLRP3-related proteins, with increase of NLRP3 and pro-IL-1ß and secretion of mature IL-1ß. In PMArest, no pro-IL-1ß and lower amounts of mature IL-1ß were detected. No ASC speck was found in PMA treated groups, but the addition of a second stimulus to PMArest resulted in ASC speck formation, together with IL-1ß production, confirming the responsiveness of the model. Conclusion: Differentiation of THP-1 with 5ng/ml PMA followed by 24h resting period provides a model that morphologically and molecularly resembles macrophages. However, even at low concentrations, PMA induces production of IL-1ß. The 24h rest period provides for down-regulation of pro-IL-1ß in PMArest group, without affecting its ability to respond to a second stimulus through activation of inflammasome.

Value of non-apical echocardiographic views in the up-grading of patients with aortic stenosis.

Purpose. Echocardiography assessment from apical five-chamber view (A5CV) is the standard technique for aortic stenosis (AS) grading. Data on non-apical views, such as right parasternal (RPV), subcostal (SCV) and suprasternal notch (SSNV), is scarce and constitutes the aim of our study. Methods. We designed an observational study that included patients with AS recruited prospectively in whom the stenosis was graded by echocardiography from A5CV and non-apical view. The value of non-apical views in up-grading the stenosis severity (primary objective), the prognostic relevance of such reclassification and the feasibility and reproducibility of non-apical views assessment (secondary objectives) was evaluated. Results. Feasibility of AS appraisal from RPV, SCV and SSNV was 78%, 81% and 56%, respectively (SCV vs SSNV, p = .009). AS were up-graded from non-apical views according to peak gradient, mean gradient, area and indexed area by 24%, 17%, 24% and 22%, respectively (p < .0001). Non-apical views reclassified from non-severe to severe AS, from low gradient severe to high gradient severe AS and from non-critical to critical AS 19%, 23% and 3% of cases (p < .0001). The 4-years hard cardiac events rate was 41% in patients with non-severe AS, 67% in patients with severe AS from non-apical views, 68% in patients with severe AS from A5CV and 80% in patients with severe AS from A5CV and non-apical views (p < .001). Reproducibility of AS evaluation from non-apical views was fair to excellent (intraclass correlation coefficients: SSNV = 0.44, RPV = 0.61, SCV = 0.92). Conclusion. Assessment of AS from non-apical views is feasible, reproducible and valuable over A5CV; its use is encouraged.

Improved Alveolar Dynamics and Structure After Alveolar Epithelial Type II Cell Transplantation in Bleomycin Induced Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressively and ultimately fatal lung disease. Previously it has been shown that intratracheal administration of alveolar epithelial type II cells (AE2C) in the animal model of bleomycin-induced pulmonary fibrosis is able to reverse fibrosis and restore surfactant protein levels. However, to date, it has not been evaluated whether these changes involve any improvement in alveolar dynamics. Consequently, the aim of the present work was to study lung physiology after AE2C transplantation at different time points during the development of injury and fibrosis. Lung fibrosis was induced by intratracheal instillation of bleomycin (4U/kg) in rat lungs. The animals were transplanted with AE2C (2.5 × 106 cells/animal) 3 or 7 days after bleomycin instillation. Assessments were done at day 7 and 14 after the induction of fibrosis to plot time dependent changes in lung physiology and mechanics. To assess the pressures and rates at which closed alveoli reopens invasive pulmonary tests using a small-animal mechanical ventilator (Flexivent®, Scireq, Canada) including de-recruitability tests and forced oscillation technique as well as quasi-static pressure volume loops were performed. Afterwards lungs were fixed by vascular perfusion and subjected to design-based stereological evaluation at light and electron microscopy level. AE2C delivered during the lung injury phase (3 days) of the disease are only able to slightly recover the volume of AE2C and volume fraction of LB in AE2C. However, it did not show either positive effects regarding ventilated alveolar surface nor any increase of lung compliance. On the other hand, when AE2C are delivered at the beginning of the fibrotic phase (7 days after bleomycin instillation), an increased ventilated alveolar surface to control levels and reduced septal wall thickness can be observed. Moreover, transplanted animals showed better lung performance, with increased inspiratory capacity and compliance. In addition, a detailed analysis of surfactant active forms [mainly tubular myelin, lamellar body (LB)-like structures and multilamellar vesicles (MLV)], showed an effective recovery during the pro-fibrotic phase due to the healthy AE2C transplantation. In conclusion, AE2C transplantation during fibrogenic phases of the disease improves lung performance, structure and surfactant ultrastructure in bleomycin-induced lung fibrosis.

Rescue from Pseudomonas aeruginosa Airway Infection via Stem Cell Transplantation.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to impaired ion transport in epithelial cells. Although lung failure due to chronic infection is the major comorbidity in individuals with cystic fibrosis, the role of CFTR in non-epithelial cells has not been definitively resolved. Given the important role of host defense cells, we evaluated the Cftr deficiency in pulmonary immune cells by hematopoietic stem cell transplantation in cystic fibrosis mice. We transplanted healthy bone marrow stem cells and could reveal a stable chimerism of wild-type cells in peripheral blood. The outcome of stem cell transplantation and the impact of healthy immune cells were evaluated in acute Pseudomonas aeruginosa airway infection. In this study, mice transplanted with wild-type cells displayed better survival, lower lung bacterial numbers, and a milder disease course. This improved physiology of infected mice correlated with successful intrapulmonary engraftment of graft-derived alveolar macrophages, as seen by immunofluorescence microscopy and flow cytometry of graft-specific leucocyte surface marker CD45 and macrophage marker CD68. Given the beneficial effect of hematopoietic stem cell transplantation and stable engraftment of monocyte-derived CD68-positive macrophages, we conclude that replacement of mutant Cftr macrophages attenuates airway infection in cystic fibrosis mice.

Mechanical ventilation-induced alterations of intracellular surfactant pool and blood-gas barrier in healthy and pre-injured lungs.

Mechanical ventilation triggers the manifestation of lung injury and pre-injured lungs are more susceptible. Ventilation-induced abnormalities of alveolar surfactant are involved in injury progression. The effects of mechanical ventilation on the surfactant system might be different in healthy compared to pre-injured lungs. In the present study, we investigated the effects of different positive end-expiratory pressure (PEEP) ventilations on the structure of the blood-gas barrier, the ultrastructure of alveolar epithelial type II (AE2) cells and the intracellular surfactant pool (= lamellar bodies, LB). Rats were randomized into bleomycin-pre-injured or healthy control groups. One day later, rats were either not ventilated, or ventilated with PEEP = 1 or 5 cmH2O and a tidal volume of 10 ml/kg bodyweight for 3 h. Left lungs were subjected to design-based stereology, right lungs to measurements of surfactant proteins (SP-) B and C expression. In pre-injured lungs without ventilation, the expression of SP-C was reduced by bleomycin; while, there were fewer and larger LB compared to healthy lungs. PEEP = 1 cmH2O ventilation of bleomycin-injured lungs was linked with the thickest blood-gas barrier due to increased septal interstitial volumes. In healthy lungs, increasing PEEP levels reduced mean AE2 cell size and volume of LB per AE2 cell; while in pre-injured lungs, volumes of AE2 cells and LB per cell remained stable across PEEPs. Instead, in pre-injured lungs, increasing PEEP levels increased the number and decreased the mean size of LB. In conclusion, mechanical ventilation-induced alterations in LB ultrastructure differ between healthy and pre-injured lungs. PEEP = 1 cmH2O but not PEEP = 5 cmH2O ventilation aggravated septal interstitial abnormalities after bleomycin challenge.

Pulmonary glycogen deficiency as a new potential cause of respiratory distress syndrome.

The glycogenin knockout mouse is a model of Glycogen Storage Disease type XV. These animals show high perinatal mortality (90%) due to respiratory failure. The lungs of glycogenin-deficient embryos and P0 mice have a lower glycogen content than that of wild-type counterparts. Embryonic lungs were found to have decreased levels of mature surfactant proteins SP-B and SP-C, together with incomplete processing of precursors. Furthermore, non-surviving pups showed collapsed sacculi, which may be linked to a significantly reduced amount of surfactant proteins. A similar pattern was observed in glycogen synthase1-deficient mice, which are devoid of glycogen in the lungs and are also affected by high perinatal mortality due to atelectasis. These results indicate that glycogen availability is a key factor for the burst of surfactant production required to ensure correct lung expansion at the establishment of air breathing. Our findings confirm that glycogen deficiency in lungs can cause respiratory distress syndrome and suggest that mutations in glycogenin and glycogen synthase 1 genes may underlie cases of idiopathic neonatal death.

Hidden Microatelectases Increase Vulnerability to Ventilation-Induced Lung Injury.

Mechanical ventilation of lungs suffering from microatelectases may trigger the development of acute lung injury (ALI). Direct lung injury by bleomycin results in surfactant dysfunction and microatelectases at day 1 while tissue elastance and oxygenation remain normal. Computational simulations of alveolar micromechanics 1-day post-bleomycin predict persisting microatelectases throughout the respiratory cycle and increased alveolar strain during low positive end-expiratory pressure (PEEP) ventilation. As such, we hypothesize that mechanical ventilation in presence of microatelectases, which occur at low but not at higher PEEP, aggravates and unmasks ALI in the bleomycin injury model. Rats were randomized and challenged with bleomycin (B) or not (H = healthy). One day after bleomycin instillation the animals were ventilated for 3 h with PEEP 1 (PEEP1) or 5 cmH2O (PEEP5) and a tidal volume of 10 ml/kg bodyweight. Tissue elastance was repetitively measured after a recruitment maneuver to investigate the degree of distal airspace instability. The right lung was subjected to bronchoalveolar lavage (BAL), the left lung was fixed for design-based stereology at light- and electron microscopic level. Prior to mechanical ventilation, lung tissue elastance did not differ. During mechanical ventilation tissue elastance increased in bleomycin-injured lungs ventilated with PEEP = 1 cmH2O but remained stable in all other groups. Measurements at the conclusion of ventilation showed the largest time-dependent increase in tissue elastance after recruitment in B/PEEP1, indicating increased instability of distal airspaces. These lung mechanical findings correlated with BAL measurements including elevated BAL neutrophilic granulocytes as well as BAL protein and albumin in B/PEEP1. Moreover, the increased septal wall thickness and volume of peri-bronchiolar-vascular connective tissue in B/PEEP1 suggested aggravation of interstitial edema by ventilation in presence of microatelectases. At the electron microscopic level, the largest surface area of injured alveolar epithelial was observed in bleomycin-challenged lungs after PEEP = 1 cmH2O ventilation. After bleomycin treatment cellular markers of endoplasmic reticulum stress (p-Perk and p-EIF-2α) were positive within the septal wall and ventilation with PEEP = 1 cmH2O ventilation increased the surface area stained positively for p-EIF-2α. In conclusion, hidden microatelectases are linked with an increased pulmonary vulnerability for mechanical ventilation characterized by an aggravation of epithelial injury.

Corrigendum: Alveolar Dynamics and Beyond - the Importance of Surfactant Protein C and Cholesterol in Lung Homeostasis and Fibrosis.

[This corrects the article DOI: 10.3389/fphys.2020.00386.].

Spermidine supplementation and voluntary activity differentially affect obesity-related structural changes in the mouse lung.

Obesity is associated with lung function impairment and respiratory diseases; however, the underlying pathophysiological mechanisms are still elusive, and therapeutic options are limited. This study examined the effects of prolonged excess fat intake on lung mechanics and microstructure and tested spermidine supplementation and physical activity as intervention strategies. C57BL/6N mice fed control diet (10% fat) or high-fat diet (HFD; 60% fat) were left untreated or were supplemented with 3 mM spermidine, had access to running wheels for voluntary activity, or a combination of both. After 30 wk, lung mechanics was assessed, and left lungs were analyzed by design-based stereology. HFD exerted minor effects on lung mechanics and resulted in higher body weight and elevated lung, air, and septal volumes. The number of alveoli was higher in HFD-fed animals. This was accompanied by an increase in epithelial, but not endothelial, surface area. Moreover, air-blood barrier and endothelium were significantly thicker. Neither treatment affected HFD-related body weights. Spermidine lowered lung volumes as well as endothelial and air-blood barrier thicknesses toward control levels and substantially increased the endothelial surface area under HFD. Activity resulted in decreased volumes of lung, septa, and septal compartments but did not affect vascular changes in HFD-fed mice. The combination treatment showed no additive effect. In conclusion, excess fat consumption induced alveolar capillary remodeling indicative of impaired perfusion and gas diffusion. Spermidine alleviated obesity-related endothelial alterations, indicating a beneficial effect, whereas physical activity reduced lung volumes apparently by other, possibly systemic effects.

Alveolar Dynamics and Beyond - The Importance of Surfactant Protein C and Cholesterol in Lung Homeostasis and Fibrosis.

Surfactant protein C (SP-C) is an important player in enhancing the interfacial adsorption of lung surfactant lipid films to the alveolar air-liquid interface. Doing so, surface tension drops down enough to stabilize alveoli and the lung, reducing the work of breathing. In addition, it has been shown that SP-C counteracts the deleterious effect of high amounts of cholesterol in the surfactant lipid films. On its side, cholesterol is a well-known modulator of the biophysical properties of biological membranes and it has been proven that it activates the inflammasome pathways in the lung. Even though the molecular mechanism is not known, there are evidences suggesting that these two molecules may interplay with each other in order to keep the proper function of the lung. This review focuses in the role of SP-C and cholesterol in the development of lung fibrosis and the potential pathways in which impairment of both molecules leads to aberrant lung repair, and therefore impaired alveolar dynamics. From molecular to cellular mechanisms to evidences in animal models and human diseases. The evidences revised here highlight a potential SP-C/cholesterol axis as target for the treatment of lung fibrosis.

On Top of the Alveolar Epithelium: Surfactant and the Glycocalyx.

Gas exchange in the lung takes place via the air-blood barrier in the septal walls of alveoli. The tissue elements that oxygen molecules have to cross are the alveolar epithelium, the interstitium and the capillary endothelium. The epithelium that lines the alveolar surface is covered by a thin and continuous liquid lining layer. Pulmonary surfactant acts at this air-liquid interface. By virtue of its biophysical and immunomodulatory functions, surfactant keeps alveoli open, dry and clean. What needs to be added to this picture is the glycocalyx of the alveolar epithelium. Here, we briefly review what is known about this glycocalyx and how it can be visualized using electron microscopy. The application of colloidal thorium dioxide as a staining agent reveals differences in the staining pattern between type I and type II alveolar epithelial cells and shows close associations of the glycocalyx with intraalveolar surfactant subtypes such as tubular myelin. These morphological findings indicate that specific spatial interactions between components of the surfactant system and those of the alveolar epithelial glycocalyx exist which may contribute to the maintenance of alveolar homeostasis, in particular to alveolar micromechanics, to the functional integrity of the air-blood barrier, to the regulation of the thickness and viscosity of the alveolar lining layer, and to the defence against inhaled pathogens. Exploring the alveolar epithelial glycocalyx in conjunction with the surfactant system opens novel physiological perspectives of potential clinical relevance for future research.