The highly packed and dehydrated structure of preformed unexposed human pulmonary surfactant isolated from amniotic fluid.

By coating the alveolar air-liquid interface, lung surfactant overwhelms surface tension forces that, otherwise, would hinder the lifetime effort of breathing. Years of research have provided a picture of how highly hydrophobic and specialized proteins in surfactant promote rapid and efficient formation of phospholipid-based complex three-dimensional films at the respiratory surface, highly stable under the demanding breathing mechanics. However, recent evidence suggests that the structure and performance of surfactant typically isolated from bronchoalveolar lung lavages may be far from that of nascent, still unused, surfactant as freshly secreted by type II pneumocytes into the alveolar airspaces. In the present work, we report the isolation of lung surfactant from human amniotic fluid (amniotic fluid surfactant, AFS) and a detailed description of its composition, structure, and surface activity in comparison to a natural surfactant (NS) purified from porcine bronchoalveolar lavages. We observe that the lipid/protein complexes in AFS exhibit a substantially higher lipid packing and dehydration than in NS. AFS shows melting transitions at higher temperatures than NS and a conspicuous presence of nonlamellar phases. The surface activity of AFS is not only comparable with that of NS under physiologically meaningful conditions but displays significantly higher resistance to inhibition by serum or meconium, agents that inactivate surfactant in the context of severe respiratory pathologies. We propose that AFS may be the optimal model to study the molecular mechanisms sustaining pulmonary surfactant performance in health and disease, and the reference material to develop improved therapeutic surfactant preparations to treat yet unresolved respiratory pathologies.

Air Space Distension Precedes Spontaneous Fibrotic Remodeling and Impaired Cholesterol Metabolism in the Absence of Surfactant Protein C.

Surfactant protein (SP)-C deficiency is found in samples from patients with idiopathic pulmonary fibrosis, especially in familial forms of this disease. We hypothesized that SP-C may contribute to fibrotic remodeling in aging mice and alveolar lipid homeostasis. For this purpose, we analyzed lung function, alveolar dynamics, lung structure, collagen content, and expression of genes related to lipid and cholesterol metabolism of aging SP-C knockout mice. In addition, in vitro experiments with an alveolar macrophage cell line exposed to lipid vesicles with or without cholesterol and/or SP-C were performed. Alveolar dynamics showed progressive alveolar derecruitment with age and impaired oxygen saturation. Lung structure revealed that decreasing volume density of alveolar spaces was accompanied by increasing of the ductal counterparts. Simultaneously, septal wall thickness steadily increased, and fibrotic wounds appeared in lungs from the age of 50 weeks. This remarkable phenotype is unique to the 129Sv strain, which has an increased absorption of cholesterol, linking the accumulation of cholesterol and the absence of SP-C to a fibrotic remodeling process. The findings of this study suggest that overall loss of SP-C results in an age-dependent, complex, heterogeneous phenotype characterized by a combination of overdistended air spaces and fibrotic wounds that resembles combined emphysema and pulmonary fibrosis in patients with idiopathic pulmonary fibrosis. Addition of SP-C to cholesterol-laden lipid vesicles enhanced the expression of cholesterol metabolism and transport genes in an alveolar macrophage cell line, identifying a potential new lipid-protein axis involved in lung remodeling.

Protein and lipid fingerprinting of native-like membrane complexes by combining TLC and protein electrophoresis.

TLC has traditionally been used to analyze lipids isolated from membrane complexes. Here, we describe a method based on the combination of TLC and SDS-PAGE to qualitatively analyze the protein/lipid profile of membrane complexes such as those of lung surfactant. For this purpose, native lung surfactant was applied onto a silica TLC plate in the form of an aqueous suspension, preserving not only hydrophilic proteins associated with lipids but also native protein-lipid interactions. Using native membrane complexes in TLC allows the differential migration of lipids and their separation from the protein components. As a result, (partly) delipidated protein-enriched bands can be visualized and analyzed by SDS-PAGE to identify proteins originally associated with lipids. Interestingly, the hydrophobic surfactant protein C, which interacts tightly with lipids in native membrane complexes, migrates through the TLC plate, configuring specific bands that differ from those corresponding to lipids or proteins. This method therefore allows the detection and analysis of strong native-like protein-lipid interactions.

Divide & Conquer: Surfactant Protein SP-C and Cholesterol Modulate Phase Segregation in Lung Surfactant.

Lung surfactant (LS) is an essential system supporting the respiratory function. Cholesterol can be deleterious for LS function, a condition that is reversed by the presence of the lipopeptide SP-C. In this work, the structure of LS-mimicking membranes has been analyzed under the combined effect of SP-C and cholesterol by deuterium NMR and phosphorus NMR and by electron spin resonance. Our results show that SP-C induces phase segregation at 37°C, resulting in an ordered phase with spectral features resembling an interdigitated state enriched in dipalmitoylphosphatidylcholine, a liquid-crystalline bilayer phase, and an extremely mobile phase consistent with small vesicles or micelles. In the presence of cholesterol, POPC and POPG motion seem to be more hindered by SP-C than dipalmitoylphosphatidylcholine. The use of deuterated cholesterol did not show signs of specific interactions that could be attributed to SP-C or to the other hydrophobic surfactant protein SP-B. Palmitoylation of SP-C had an indirect effect on the extent of protein-lipid perturbations by stabilizing SP-C structure, and seemed to be important to maximize differences among the lipids participating in each phase. These results shed some light on how SP-C-induced lipid perturbations can alter membrane structure to sustain LS functionality at the air-liquid interface.

Effect of Lung Surfactant Protein SP-C and SP-C-Promoted Membrane Fragmentation on Cholesterol Dynamics.

To allow breathing and prevent alveolar collapse, lung surfactant (LS) develops a complex membranous system at the respiratory surface. LS is defined by a specific protein and lipid composition, including saturated and unsaturated phospholipid species and cholesterol. Surfactant protein C (SP-C) has been suggested to be an essential element for sustaining the presence of cholesterol in surfactant without functional impairment. In this work, we used a fluorescent sterol-partitioning assay to assess the effect of the surfactant proteins SP-B and SP-C on cholesterol distribution in membranes. Our results suggest that in the LS context, the combined action of SP-B and SP-C appears to facilitate cholesterol dynamics, whereas SP-C does not seem to establish a direct interaction with cholesterol that could increase the partition of free cholesterol into membranes. Interestingly, SP-C exhibits a membrane-fragmentation behavior, leading to the conversion of large unilamellar vesicles into highly curved vesicles ∼25 nm in diameter. Sterol partition was observed to be sensitive to the bending of bilayers, indicating that the effect of SP-C to mobilize cholesterol could be indirectly associated with SP-C-mediated membrane remodeling. Our results suggest a potential role for SP-C in generating small surfactant structures that may participate in cholesterol mobilization and pulmonary surfactant homeostasis at the alveolar interfaces.

Palmitoylation as a key factor to modulate SP-C-lipid interactions in lung surfactant membrane multilayers.

Surfactant protein C (SP-C) has been regarded as the most specific protein linked to development of mammalian lungs, and great efforts have been done to understand its structure-function relationships. Previous evidence has outlined the importance of SP-C palmitoylation to sustain the proper dynamics of lung surfactant, but the mechanism by which this posttranslational modification aids SP-C to stabilize the interfacial surfactant film along the compression-expansion breathing cycles, is still unrevealed. In this work we have compared the structure, orientation and lipid-protein interactions of a native palmitoylated SP-C with those of a non-palmitoylated recombinant SP-C (rSP-C) form in air-exposed multilayer membrane environments, by means of ATR-FTIR spectroscopy. Palmitoylation does not affect the secondary structure of the protein, which exhibits a full α-helical conformation in partly dehydrated phospholipid multilayer films. However, differences between the Amide I band of the IR spectrum of palmitoylated and non-palmitoylated proteins suggest subtle differences affecting the environment of their helical component. These differences are accompanied by differential effects on the IR bands from phospholipid phosphates, indicating that palmitoylation modulates lipid-protein interactions at the headgroup region of phospholipid layers. On the other hand, the relative dichroic absorption of polarized IR has allowed calculating that the palmitoylated protein adopts a more tilted transmembrane orientation than the non-palmitoylated SP-C, likely contributing to more compact, dehydrated and possibly stable multilayer lipid-protein films. As a whole, the behavior of multilayer films containing palmitoylated SP-C may reflect favorable structural properties for surfactant reservoirs at the air-liquid respiratory interface.

Glycomacropeptide administration attenuates airway inflammation and remodeling associated to allergic asthma in rat.

OBJECTIVE: Glycomacropeptide (GMP) is a bioactive peptide derived from milk that has been reported to exhibit a range of anti-inflammatory and immunomodulatory properties. The aim of this study was to analyze the prophylactic effect of GMP administration on airway inflammation and remodeling in an experimental model of asthmatic rat. METHODS: Animals treated orally with or without GMP (500 mg/kg/day) were ovalbumin-sensitized and -nebulized and several indicators of Th2 response, airway structural changes and inflammatory cells recruitment were evaluated. RESULTS: Treatment with GMP prior and during asthma development resulted in reduction of allergen-specific IgE titers in serum and blood eosinophilia. Also, GMP substantially suppressed the recruitment of inflammatory cells to bronchoalveolar compartment. Histological studies demonstrated that GMP markedly inhibits eosinophils infiltration, goblet cells hyperplasia and collagen deposit in lung tissue. The latter effect was related with an inhibition in transforming growth factor-ß expression. In addition, expression of interleukin-5 and -13 were substantially inhibited in lung while that of interleukin-10 was increased. CONCLUSION: Our results suggest that administration of GMP may prevent the development of an excessive Th2 response in asthma and effectively ameliorates the progression of the disease.

A New Immortalized Human Alveolar Epithelial Cell Model to Study Lung Injury and Toxicity on a Breathing Lung-On-Chip System.

The evaluation of inhalation toxicity, drug safety and efficacy assessment, as well as the investigation of complex disease pathomechanisms, are increasingly relying on in vitro lung models. This is due to the progressive shift towards human-based systems for more predictive and translational research. While several cellular models are currently available for the upper airways, modelling the distal alveolar region poses several constraints that make the standardization of reliable alveolar in vitro models relatively difficult. In this work, we present a new and reproducible alveolar in vitro model, that combines a human derived immortalized alveolar epithelial cell line (AXiAEC) and organ-on-chip technology mimicking the lung alveolar biophysical environment (AXlung-on-chip). The latter mimics key features of the in vivo alveolar milieu: breathing-like 3D cyclic stretch (10% linear strain, 0.2 Hz frequency) and an ultrathin, porous and elastic membrane. AXiAECs cultured on-chip were characterized for their alveolar epithelial cell markers by gene and protein expression. Cell barrier properties were examined by TER (Transbarrier Electrical Resistance) measurement and tight junction formation. To establish a physiological model for the distal lung, AXiAECs were cultured for long-term at air-liquid interface (ALI) on-chip. To this end, different stages of alveolar damage including inflammation (via exposure to bacterial lipopolysaccharide) and the response to a profibrotic mediator (via exposure to Transforming growth factor ß1) were analyzed. In addition, the expression of relevant host cell factors involved in SARS-CoV-2 infection was investigated to evaluate its potential application for COVID-19 studies. This study shows that AXiAECs cultured on the AXlung-on-chip exhibit an enhanced in vivo-like alveolar character which is reflected into: 1) Alveolar type 1 (AT1) and 2 (AT2) cell specific phenotypes, 2) tight barrier formation (with TER above 1,000 Ω cm2) and 3) reproducible long-term preservation of alveolar characteristics in nearly physiological conditions (co-culture, breathing, ALI). To the best of our knowledge, this is the first time that a primary derived alveolar epithelial cell line on-chip representing both AT1 and AT2 characteristics is reported. This distal lung model thereby represents a valuable in vitro tool to study inhalation toxicity, test safety and efficacy of drug compounds and characterization of xenobiotics.

Human-Based Advanced in vitro Approaches to Investigate Lung Fibrosis and Pulmonary Effects of COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic has caused considerable socio-economic burden, which fueled the development of treatment strategies and vaccines at an unprecedented speed. However, our knowledge on disease recovery is sparse and concerns about long-term pulmonary impairments are increasing. Causing a broad spectrum of symptoms, COVID-19 can manifest as acute respiratory distress syndrome (ARDS) in the most severely affected patients. Notably, pulmonary infection with Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), the causing agent of COVID-19, induces diffuse alveolar damage (DAD) followed by fibrotic remodeling and persistent reduced oxygenation in some patients. It is currently not known whether tissue scaring fully resolves or progresses to interstitial pulmonary fibrosis. The most aggressive form of pulmonary fibrosis is idiopathic pulmonary fibrosis (IPF). IPF is a fatal disease that progressively destroys alveolar architecture by uncontrolled fibroblast proliferation and the deposition of collagen and extracellular matrix (ECM) proteins. It is assumed that micro-injuries to the alveolar epithelium may be induced by inhalation of micro-particles, pathophysiological mechanical stress or viral infections, which can result in abnormal wound healing response. However, the exact underlying causes and molecular mechanisms of lung fibrosis are poorly understood due to the limited availability of clinically relevant models. Recently, the emergence of SARS-CoV-2 with the urgent need to investigate its pathogenesis and address drug options, has led to the broad application of in vivo and in vitro models to study lung diseases. In particular, advanced in vitro models including precision-cut lung slices (PCLS), lung organoids, 3D in vitro tissues and lung-on-chip (LOC) models have been successfully employed for drug screens. In order to gain a deeper understanding of SARS-CoV-2 infection and ultimately alveolar tissue regeneration, it will be crucial to optimize the available models for SARS-CoV-2 infection in multicellular systems that recapitulate tissue regeneration and fibrotic remodeling. Current evidence for SARS-CoV-2 mediated pulmonary fibrosis and a selection of classical and novel lung models will be discussed in this review.

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.

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.].

A three-hybrid approach to scanning the proteome for targets of small molecule kinase inhibitors.

In this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors. Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats. For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography. The Y3H system described here may prove generally useful in the discovery of candidate drug targets.