Immature Surfactant Protein B Increases in the Serum of Patients with Calcific Severe Aortic Stenosis.

Valvular disease is a complex pathological condition that impacts countless individuals around the globe. Due to limited treatments, it is crucial to understand its mechanisms to identify new targets. Valve disease may result in pulmonary venous hypertension, which is linked to compromised functioning of the alveolar and capillary membranes and hindered gas exchange. Nonetheless, the correlation between surfactant proteins (SPs) and valve disease remains unexplored. A total of 44 patients were enrolled in this study, with 36 undergoing aortic valve replacement and 8 needing a second aortic valve substitution due to bioprosthetic valve degeneration. Ten healthy subjects were also included. The results showed that patients who underwent both the first valve replacement and the second surgery had significantly higher levels of immature SP-B (proSP-B) compared to control subjects. The levels of the extra-lung collectin SP-D were higher in patients who needed a second surgery due to bioprosthetic valve degeneration, while SP-A levels remained unchanged. The research also showed that there was no reciprocal relationship between inflammation and SP-D as the levels of inflammatory mediators did not differ between groups. The present study demonstrates that circulating proSP-B serves as a reliable marker of alveolar-capillary membrane damage in patients with valvular heart disease.

SFTPB in serum extracellular vesicles as a biomarker of progressive pulmonary fibrosis.

Progressive pulmonary fibrosis (PPF), defined as the worsening of various interstitial lung diseases (ILDs), currently lacks useful biomarkers. To identify novel biomarkers for early detection of patients at risk of PPF, we performed a proteomic analysis of serum extracellular vesicles (EVs). Notably, the identified candidate biomarkers were enriched for lung-derived proteins participating in fibrosis-related pathways. Among them, pulmonary surfactant-associated protein B (SFTPB) in serum EVs could predict ILD progression better than the known biomarkers, serum KL-6 and SP-D, and it was identified as an independent prognostic factor from ILD-gender-age-physiology index. Subsequently, the utility of SFTPB for predicting ILD progression was evaluated further in 2 cohorts using serum EVs and serum, respectively, suggesting that SFTPB in serum EVs but not in serum was helpful. Among SFTPB forms, pro-SFTPB levels were increased in both serum EVs and lungs of patients with PPF compared with those of the control. Consistently, in a mouse model, the levels of pro-SFTPB, primarily originating from alveolar epithelial type 2 cells, were increased similarly in serum EVs and lungs, reflecting pro-fibrotic changes in the lungs, as supported by single-cell RNA sequencing. SFTPB, especially its pro-form, in serum EVs could serve as a biomarker for predicting ILD progression.

The Synthetic Surfactant CHF5633 Restores Lung Function and Lung Architecture in Severe Acute Respiratory Distress Syndrome in Adult Rabbits.

PURPOSE: Acute respiratory distress syndrome (ARDS) is a major cause of hypoxemic respiratory failure in adults. In ARDS extensive inflammation and leakage of fluid into the alveoli lead to dysregulation of pulmonary surfactant metabolism and function. Altered surfactant synthesis, secretion, and breakdown contribute to the clinical features of decreased lung compliance and alveolar collapse. Lung function in ARDS could potentially be restored with surfactant replacement therapy, and synthetic surfactants with modified peptide analogues may better withstand inactivation in ARDS alveoli than natural surfactants. METHODS: This study aimed to investigate the activity in vitro and the bolus effect (200 mg phospholipids/kg) of synthetic surfactant CHF5633 with analogues of SP-B and SP-C, or natural surfactant Poractant alfa (Curosurf®, both preparations Chiesi Farmaceutici S.p.A.) in a severe ARDS model (the ratio of partial pressure arterial oxygen and fraction of inspired oxygen, P/F ratio ≤ 13.3 kPa) induced by hydrochloric acid instillation followed by injurious ventilation in adult New Zealand rabbits. The animals were ventilated for 4 h after surfactant treatment and the respiratory parameters, histological appearance of lung parenchyma and levels of inflammation, oxidative stress, surfactant dysfunction, and endothelial damage were evaluated. RESULTS: Both surfactant preparations yielded comparable improvements in lung function parameters, reductions in lung injury score, pro-inflammatory cytokines levels, and lung edema formation compared to untreated controls. CONCLUSIONS: This study indicates that surfactant replacement therapy with CHF5633 improves lung function and lung architecture, and attenuates inflammation in severe ARDS in adult rabbits similarly to Poractant alfa. Clinical trials have so far not yielded conclusive results, but exogenous surfactant may be a valid supportive treatment for patients with ARDS given its anti-inflammatory and lung-protective effects.

Silica nanoparticle exposure inhibits surfactant protein A and B in A549 cells through ROS-mediated JNK/c-Jun signaling pathway.

Exposure to silica nanoparticles (SiNPs) is related to the dysregulation of pulmonary surfactant that maintains lung stability and function. Nevertheless, there are limited studies concerning the interaction and influence between SiNPs and pulmonary surfactant, and the damage and mechanism are still unclear. Herein, we used A549 cells to develop an in vitro model, with which we investigated the effect of SiNPs exposure on the expression of pulmonary surfactant and the potential regulatory mechanism. The results showed that SiNPs were of cytotoxicity in regarding of reduced cell viability and promoted the production of excessive reactive oxygen species (ROS). Additionally, the JNK/c-Jun signaling pathway was activated, and the expression of surfactant protein A (SP-A) and surfactant protein B (SP-B) was decreased. After the cells being treated with N-acetyl-L-cysteine (NAC), we found that the ROS content was effectively downregulated, and the expression of proteins related to JNK and c-Jun signaling pathways was suppressed. In contrast, the expression of SP-A and SP-B was enhanced. Furthermore, we treated the cells with JNK inhibitor and c-Jun-siRNA and found that the expression of protein related to JNK and c-Jun signaling pathways, as well as SP-A and SP-B, changed in line with that of NAC treatment. These findings suggest that SiNPs exposure can upregulate ROS and activate the JNK/c-Jun signaling pathway in A549 cells, thereby inhibiting the expression of SP-A and SP-B proteins.

Impacts of lipopolysaccharide on fetal lung developmental maturity and surfactant protein B and surfactant protein C protein expression in gestational diabetes mellitus rats.

The rise of bioinformatics based on computer medicine provides a new method to reveal the complex biological data. This experiment is to explore the impacts of lipopolysaccharide on fetal lung developmental maturity and expressions of lung surfactant protein B (SP-B) and lung surfactant protein C (SP-C) in rats with gestational diabetes mellitus (GDM), thereby discussing the mechanism of developmental disorders in rats. Forty-eight conceived female rats were experimental subjects. Twenty-eight rats were randomly selected to construct the GDM models. All conceived rats underwent section on the 21st day of pregnancy. The ultrastructure of alveolar type II epithelial cells and the morphology of lung tissue were observed under a microscope. The protein localization and expression of SP-B and SP-C were determined by immunohistochemistry; the protein levels of SP-B and SP-C were determined by Western blot. Blood glucose and body weight of the GDM group were higher than those of the control group; the number of alveoli and alveolar area in the GDM group was lower than those in the control group; the alveolar interval in the GDM group was significantly higher than that in the control group (P < 0.05). The average absorbance of SP-B and SP-C in fetal lung tissue was significantly lower in the GDM group than that in the control group (P < 0.01). Changes in fetal lung tissue structure of rats were related to SP-B and SP-C, which was one of the main factors that affected the maturation of fetal lung tissue.

Treatment of Respiratory Distress Syndrome with Single Recombinant Polypeptides that Combine Features of SP-B and SP-C.

Treatment of respiratory distress syndrome (RDS) with surfactant replacement therapy in prematurely born infants was introduced more than 30 years ago; however, the surfactant preparations currently in clinical use are extracts from animal lungs. A synthetic surfactant that matches the currently used nature-derived surfactant preparations and can be produced in a cost-efficient manner would enable worldwide treatment of neonatal RDS and could also be tested against lung diseases in adults. The major challenge in developing fully functional synthetic surfactant preparations is to recapitulate the properties of the hydrophobic lung surfactant proteins B (SP-B) and SP-C. Here, we have designed single polypeptides that combine properties of SP-B and SP-C and produced them recombinantly using a novel solubility tag based on spider silk production. These Combo peptides mixed with phospholipids are as efficient as nature-derived surfactant preparations against neonatal RDS in premature rabbit fetuses.

Structural and functional stability of the sulfur-free surfactant protein B peptide mimic B-YL in synthetic surfactant lipids.

BACKGROUND: Optimal functionality of synthetic lung surfactant for treatment of respiratory distress syndrome in preterm infants largely depends on the quality and quantity of the surfactant protein B (SP-B) peptide mimic and the lipid mixture. B-YL peptide is a 41-residue sulfur-free SP-B mimic with its cysteine and methionine residues replaced by tyrosine and leucine, respectively, to enhance its oxidation resistance. AIM: Testing the structural and functional stability of the B-YL peptide in synthetic surfactant lipids after long-term storage. METHODS: The structural and functional properties of B-YL peptide in surfactant lipids were studied using three production runs of B-YL peptides in synthetic surfactant lipids. Each run was held at 5 °C ambient temperature for three years and analyzed with structural and computational techniques, i.e., MALDI-TOF mass spectrometry, ATR-Fourier Transform Infrared Spectroscopy (ATR-FTIR), secondary homology modeling of a preliminary B-YL structure, and tertiary Molecular Dynamic simulations of B-YL in surfactant lipids, and with functional methods, i.e., captive bubble surfactometry (CBS) and retesting in vivo surface activity in surfactant-deficient young adult rabbits. RESULTS: MALDI-TOF mass spectrometry showed no degradation of the B-YL peptide as a function of stored time. ATR-FTIR studies demonstrated that the B-YL peptide still assumed stable alpha-helical conformations in synthetic surfactant lipids. These structural findings correlated with excellent in vitro surface activity during both quasi-static and dynamic cycling on CBS after three years of cold storage and in vivo surface activity of the aged formulations with improvements in oxygenation and dynamic lung compliance approaching those of the positive control surfactant Curosurf®. CONCLUSIONS: The structure of the B-YL peptide and the in vitro and in vivo functions of the B-YL surfactant were each maintained after three years of refrigeration storage.

Impact of synthetic surfactant CHF5633 with SP-B and SP-C analogues on lung function and inflammation in rabbit model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is associated with diffuse inflammation, alveolar epithelial damage, and leakage of plasma proteins into the alveolar space, which together contribute to inactivation of pulmonary surfactant and respiratory failure. Exogenous surfactant delivery is therefore considered to hold potential for ARDS treatment, but clinical trials with natural derived surfactant or synthetic surfactant containing a surfactant protein C (SP-C) analogue have been negative. Synthetic surfactant CHF5633, containing analogues of SP-B and SP-C, may be effective against ARDS. The aim here was to compare treatment effects of CHF5633 and animal-derived surfactant poractant alfa in animal model of ARDS. ARDS was induced in adult New Zealand rabbits by mild lung lavages followed by injurious ventilation until respiratory failure (P/F ratio <26.7 kPa). The animals were then treated with intratracheal bolus of 200 mg/kg CHF5633 or poractant alfa (Curosurf® ), or air as control. The animals were subsequently ventilated for an additional 4 hr and respiratory parameters were recorded regularly. Postmortem, histological analysis, degree of lung edema, and levels of the cytokines TNFα, IL-6, and IL-8 in lung homogenates were evaluated. Both surfactant preparations improved lung function, reduced the levels of pro-inflammatory cytokines, and degree of lung edema to very similar degrees versus the controls. No significant differences in any of the analyzed parameters were observed between the CHF5633- and poractant alfa-treated groups. This study indicates that single dose of CHF5633 improves lung function and attenuates inflammation as effectively as poractant alfa in experimental ARDS caused by injurious ventilation.

Towards the Molecular Mechanism of Pulmonary Surfactant Protein SP-B: At the Crossroad of Membrane Permeability and Interfacial Lipid Transfer.

Pulmonary surfactant is a lipid-protein complex that coats the alveolar air-liquid interface, enabling the proper functioning of lung mechanics. The hydrophobic surfactant protein SP-B, in particular, plays an indispensable role in promoting the rapid adsorption of phospholipids into the interface. For this, formation of SP-B ring-shaped assemblies seems to be important, as oligomerization could be required for the ability of the protein to generate membrane contacts and to mediate lipid transfer among surfactant structures. SP-B, together with the other hydrophobic surfactant protein SP-C, also promotes permeability of surfactant membranes to polar molecules although the molecular mechanisms underlying this property, as well as its relevance for the surface activity of the protein, remain undefined. In this work, the contribution of SP-B and SP-C to surfactant membrane permeability has been further investigated, by evaluation of the ability of differently-sized fluorescent polar probes to permeate through giant vesicles with different lipid/protein composition. Our results are consistent with the generation by SP-B of pores with defined size in surfactant membranes. Furthermore, incubation of surfactant with an anti-SP-B antibody not only blocked membrane permeability but also affected lipid transfer into the air-water interface, as observed in a captive bubble surfactometer device. Our findings include the identification of SP-C and anionic phospholipids as modulators required for maintaining native-like permeability features in pulmonary surfactant membranes. Proper permeability through membrane assemblies could be crucial to complement the overall role of surfactant in maintaining alveolar equilibrium, beyond its biophysical function in stabilizing the respiratory air-liquid interface.

Mechanism of Lamellar Body Formation by Lung Surfactant Protein B.

Breathing depends on pulmonary surfactant, a mixture of phospholipids and proteins, secreted by alveolar type II cells. Surfactant requires lamellar bodies (LBs), organelles containing densely packed concentric membrane layers, for storage and secretion. LB biogenesis remains mysterious but requires surfactant protein B (SP-B), which is synthesized as a precursor (pre-proSP-B) that is cleaved during trafficking into three related proteins. Here, we elucidate the functions and cooperation of these proteins in LB formation. We show that the N-terminal domain of proSP-B is a phospholipid-binding and -transfer protein whose activities are required for proSP-B export from the endoplasmic reticulum (ER) and sorting to LBs, the conversion of proSP-B into lipoprotein particles, and neonatal viability in mice. The C-terminal domain facilitates ER export of proSP-B. The mature middle domain, generated after proteolytic cleavage of proSP-B, generates the striking membrane layers characteristic of LBs. Together, our results lead to a mechanistic model of LB biogenesis.

A lung tropic AAV vector improves survival in a mouse model of surfactant B deficiency.

Surfactant protein B (SP-B) deficiency is an autosomal recessive disorder that impairs surfactant homeostasis and manifests as lethal respiratory distress. A compelling argument exists for gene therapy to treat this disease, as de novo protein synthesis of SP-B in alveolar type 2 epithelial cells is required for proper surfactant production. Here we report a rationally designed adeno-associated virus (AAV) 6 capsid that demonstrates efficiency in lung epithelial cell transduction based on imaging and flow cytometry analysis. Intratracheal administration of this vector delivering murine or human proSFTPB cDNA into SP-B deficient mice restores surfactant homeostasis, prevents lung injury, and improves lung physiology. Untreated SP-B deficient mice develop fatal respiratory distress within two days. Gene therapy results in an improvement in median survival to greater than 200 days. This vector also transduces human lung tissue, demonstrating its potential for clinical translation against this lethal disease.

Pulmonary Surfactant Lipid Reorganization Induced by the Adsorption of the Oligomeric Surfactant Protein B Complex.

Surfactant protein B (SP-B) is essential in transferring surface-active phospholipids from membrane-based surfactant complexes into the alveolar air-liquid interface. This allows maintaining the mechanical stability of the surfactant film under high pressure at the end of expiration; therefore, SP-B is crucial in lung function. Despite its necessity, the structure and the mechanism of lipid transfer by SP-B have remained poorly characterized. Earlier, we proposed higher-order oligomerization of SP-B into ring-like supramolecular assemblies. In the present work, we used coarse-grained molecular dynamics simulations to elucidate how the ring-like oligomeric structure of SP-B determines its membrane binding and lipid transfer. In particular, we explored how SP-B interacts with specific surfactant lipids, and how consequently SP-B reorganizes its lipid environment to modulate the pulmonary surfactant structure and function. Based on these studies, there are specific lipid-protein interactions leading to perturbation and reorganization of pulmonary surfactant layers. Especially, we found compelling evidence that anionic phospholipids and cholesterol are needed or even crucial in the membrane binding and lipid transfer function of SP-B. Also, on the basis of the simulations, larger oligomers of SP-B catalyze lipid transfer between adjacent surfactant layers. Better understanding of the molecular mechanism of SP-B will help in the design of therapeutic SP-B-based preparations and novel treatments for fatal respiratory complications, such as the acute respiratory distress syndrome.

Pulmonary surfactant protein SP-B nanorings induce the multilamellar organization of surfactant complexes.

Surfactant protein SP-B is absolutely required for the generation of functional pulmonary surfactant, a unique network of multilayered membranes, which stabilizes the respiratory air-liquid interface. It has been proposed that SP-B assembles into hydrophobic rings and tubes that facilitate the rapid transfer of phospholipids from membrane stores into the interface and the formation of multilayered films, ensuring the stability of the alveoli against physical forces leading to their collapse. To elucidate the molecular organization of SP-B-promoted multilamellar membrane structures, time-resolved Förster Resonance Energy Transfer (FRET) experiments between BODIPY-PC or BODIPY-derivatized SP-B (BODIPY/SP-B), as donor probes, and octadecylrhodamine B, as acceptor probe, were performed in liposomes containing SP-B or BODIPY/SP-B. Our results show that both SP-B and fluorescently labeled SP-B oligomers mediate the connection of adjacent bilayers. Furthermore, by applying rational models to the FRET data, we have been able to provide quantitative details of the structure of SP-B-induced multilayered membrane arrays at the nanometer scale, defining interactions between SP-B rings as key elements for connecting surfactant membranes. The data sustain the structural model and the mechanism of action of SP-B assemblies to sustain the crucial surfactant function.

A novel in vitro model of primary human pediatric lung epithelial cells.

BACKGROUND: Current in vitro human lung epithelial cell models derived from adult tissues may not accurately represent all attributes that define homeostatic and disease mechanisms relevant to the pediatric lung. METHODS: We report methods for growing and differentiating primary Pediatric Human Lung Epithelial (PHLE) cells from organ donor infant lung tissues. We use immunohistochemistry, flow cytometry, quantitative RT-PCR, and single cell RNA sequencing (scRNAseq) analysis to characterize the cellular and transcriptional heterogeneity of PHLE cells. RESULTS: PHLE cells can be expanded in culture up to passage 6, with a doubling time of ~4 days, and retain attributes of highly enriched epithelial cells. PHLE cells can form resistant monolayers, and undergo differentiation when placed at air-liquid interface. When grown at Air-Liquid Interface (ALI), PHLE cells expressed markers of airway epithelial cell lineages. scRNAseq suggests the cultures contained 4 main sub-phenotypes defined by expression of FOXJ1, KRT5, MUC5B, and SFTPB. These cells are available to the research community through the Developing Lung Molecular Atlas Program Human Tissue Core. CONCLUSION: Our data demonstrate that PHLE cells provide a novel in vitro human cell model that represents the pediatric airway epithelium, which can be used to study perinatal developmental and pediatric disease mechanisms.

Surfactant Protein B Deficiency Induced High Surface Tension: Relationship between Alveolar Micromechanics, Alveolar Fluid Properties and Alveolar Epithelial Cell Injury.

High surface tension at the alveolar air-liquid interface is a typical feature of acute and chronic lung injury. However, the manner in which high surface tension contributes to lung injury is not well understood. This study investigated the relationship between abnormal alveolar micromechanics, alveolar epithelial injury, intra-alveolar fluid properties and remodeling in the conditional surfactant protein B (SP-B) knockout mouse model. Measurements of pulmonary mechanics, broncho-alveolar lavage fluid (BAL), and design-based stereology were performed as a function of time of SP-B deficiency. After one day of SP-B deficiency the volume of alveolar fluid V(alvfluid,par) as well as BAL protein and albumin levels were normal while the surface area of injured alveolar epithelium S(AEinjure,sep) was significantly increased. Alveoli and alveolar surface area could be recruited by increasing the air inflation pressure. Quasi-static pressure-volume loops were characterized by an increased hysteresis while the inspiratory capacity was reduced. After 3 days, an increase in V(alvfluid,par) as well as BAL protein and albumin levels were linked with a failure of both alveolar recruitment and airway pressure-dependent redistribution of alveolar fluid. Over time, V(alvfluid,par) increased exponentially with S(AEinjure,sep). In conclusion, high surface tension induces alveolar epithelial injury prior to edema formation. After passing a threshold, epithelial injury results in vascular leakage and exponential accumulation of alveolar fluid critically hampering alveolar recruitability.

All-Atom Molecular Dynamics Simulations of Dimeric Lung Surfactant Protein B in Lipid Multilayers.

Although lung surfactant protein B (SP-B) is an essential protein that plays a crucial role in breathing, the details of its structure and mechanism are not well understood. SP-B forms covalent homodimers, and in this work we use all-atom molecular dynamics simulations to study dimeric SP-B's structure and its behavior in promoting lipid structural transitions. Four initial system configurations were constructed based on current knowledge of SP-B's structure and mechanism, and the protein maintained a helicity consistent with experiment in all systems. Several SP-B-induced lipid reorganization behaviors were observed, and regions of the protein particularly important for these activities included SP-B's "central loop" and "hinge" regions. SP-B dimers with one subunit initially positioned in each of two adjacent bilayers appeared to promote close contact between two bilayers. When both subunits were initially positioned in the same bilayer, SP-B induced the formation of a defect in the bilayer, with water penetrating into the centre of the bilayer. Similarly, dimeric SP-B showed a propensity to interact with preformed interpores in the bilayer. SP-B dimers also promoted bilayer thinning and creasing. This work fleshes out the atomistic details of the dimeric SP-B structures and SP-B/lipid interactions that underlie SP-B's essential functions.

Mass spectrometry imaging as a tool for evaluating the pulmonary distribution of exogenous surfactant in premature lambs.

BACKGROUND: The amount of surfactant deposited in the lungs and its overall pulmonary distribution determine the therapeutic outcome of surfactant replacement therapy. Most of the currently available methods to determine the intrapulmonary distribution of surfactant are time-consuming and require surfactant labelling. Our aim was to assess the potential of Mass Spectrometry Imaging (MSI) as a label-free technique to qualitatively and quantitatively evaluate the distribution of surfactant to the premature lamb. METHODS: Twelve preterm lambs (gestational age 126-127d, term ~150d) were allocated in two experimental groups. Seven lambs were treated with an intratracheal bolus of the synthetic surfactant CHF5633 (200 mg/kg) and 5 lambs were managed with mechanical ventilation for 120 min, as controls. The right lung lobes of all lambs were gradually frozen while inflated to 20 cmH2O pressure for lung cryo-sections for MSI analysis. The intensity signals of SP-C analog and SP-B analog, the two synthetic peptides contained in the CHF5633 surfactant, were used to locate, map and quantify the intrapulmonary exogenous surfactant. RESULTS: Surfactant treatment was associated with a significant improvement of the mean arterial oxygenation and lung compliance (p < 0.05). Nevertheless, the physiological response to surfactant treatment was not uniform across all animals. SP-C analog and SP-B analog were successfully imaged and quantified by means of MSI in the peripheral lungs of all surfactant-treated animals. The intensity of the signal was remarkably low in untreated lambs, corresponding to background noise. The signal intensity of SP-B analog in each surfactant-treated animal, which represents the surfactant distributed to the peripheral right lung, correlated well with the physiologic response as assessed by the area under the curves of the individual arterial partial oxygen pressure and dynamic lung compliance curves of the lambs. CONCLUSIONS: Applying MSI, we were able to detect, locate and quantify the amount of exogenous surfactant distributed to the lower right lung of surfactant-treated lambs. The distribution pattern of SP-B analog correlated well with the pulmonary physiological outcomes of the animals. MSI is a valuable label-free technique which is able to simultaneously evaluate qualitative and quantitative drug distribution in the lung.

Native supramolecular protein complexes in pulmonary surfactant: Evidences for SP-A/SP-B interactions.

Pulmonary surfactant is a lipid-protein complex which coats lung alveoli. It displays the essential function of reducing surface tension at the air-liquid interface, avoiding alveolar collapse during expiration. The optimized biophysical properties of surfactant rely on its defined composition, constituted mainly by phospholipids and tiny amounts of lipid-associated specific proteins. Due to the highly hydrophobic nature of surfactant, organic solvents have been traditionally employed to obtain and characterize surfactant lipids and proteins, very likely leading to disruption of native interactions among its components. In the present work we have addressed the search of native protein complexes in pulmonary surfactant, which could have an essential role in the optimal function of the system. By solubilizing native lipid-protein membranes of surfactant with non-denaturing detergents, and with the use of a two-dimensional electrophoresis strategy, we have been able to detect the presence of supramolecular complexes composed of surfactant proteins SP-A, SP-B and SP-C. Furthermore, by co-immunoprecipitation assays, we have confirmed for the first time the existence of a direct interaction between SP-A and SP-B, an important feature which could explain the known functional cooperation of both proteins in several aspects of surfactant biology. SIGNIFICANCE: This paper deepens for the first time in the existence of complex interaction networks of surfactant proteins in native surfactant membranes. By the use of non-denaturing detergents, two-dimensional electrophoresis and immunoprecipitation, we have been able to make progress in the elucidation of native protein complexes in this essential system, that had been previously hindered by the classical purification protocols employing organic solvents. In this work, we have described the presence of interactions between SP-B and SP-A, two important proteins whose functional cooperation has been broadly reported in the literature. Pioneer determination of such native complexes could have potential implications for understanding the wide variety of roles of pulmonary surfactant system.

MCRIP1 promotes the expression of lung-surfactant proteins in mice by disrupting CtBP-mediated epigenetic gene silencing.

Proper regulation of epigenetic states of chromatin is crucial to achieve tissue-specific gene expression during embryogenesis. The lung-specific gene products, surfactant proteins B (SP-B) and C (SP-C), are synthesized in alveolar epithelial cells and prevent alveolar collapse. Epigenetic regulation of these surfactant proteins, however, remains unknown. Here we report that MCRIP1, a regulator of the CtBP transcriptional co-repressor, promotes the expression of SP-B and SP-C by preventing CtBP-mediated epigenetic gene silencing. Homozygous deficiency of Mcrip1 in mice causes fatal respiratory distress due to abnormal transcriptional repression of these surfactant proteins. We found that MCRIP1 interferes with interactions of CtBP with the lung-enriched transcriptional repressors, Foxp1 and Foxp2, thereby preventing the recruitment of the CtBP co-repressor complex to the SP-B and SP-C promoters and maintaining them in an active chromatin state. Our findings reveal a molecular mechanism by which cells prevent inadvertent gene silencing to ensure tissue-specific gene expression during organogenesis.

Quantitative Lipidomics in Pulmonary Alveolar Proteinosis.

Rationale: Pulmonary alveolar proteinosis (PAP) is characterized by filling of the alveolar spaces by lipoprotein-rich material of ill-defined composition, and is caused by molecularly different and often rare diseases that occur from birth to old age.Objectives: To perform a quantitative lipidomic analysis of lipids and the surfactant proteins A, B, and C in lavage fluids from patients with proteinosis of different causes in comparison with healthy control subjects.Methods: During the last two decades, we have collected BAL samples from patients with PAP due to autoantibodies against granulocyte-macrophage colony-stimulating factor; genetic mutations in CSF2RA (colony-stimulating factor 2 receptor α-subunit), MARS (methionyl aminoacyl-tRNA synthetase), FARSB (phenylalanine-tRNA synthetase, ß-subunit), and NPC2 (Niemann-Pick disease type C2); and secondary to myeloid leukemia. Their lipid composition was quantified.Measurements and Main Results: Free cholesterol was largely increased by 60-fold and cholesteryl esters were increased by 24-fold. There was an excessive, more than 130-fold increase in ceramide and other sphingolipids. In particular, the long-chain ceramides d18:1/20:0 and d18:1/24:0 were elevated and likely contributed to the proapoptotic environment observed in PAP. Cellular debris lipids such as phosphatidylethanolamine and phosphatidylserine were only moderately increased, by four- to sevenfold. The surfactant lipid class phosphatidylcholine expanded 17-fold, lysophosphatidylcholine expanded 54-fold, and the surfactant proteins A, B, and C expanded 144-, 4-, and 17-fold, respectively. These changes did not differ among the various diseases that cause PAP.Conclusions: This insight into the alveolar lipidome may provide monitoring tools and lead to new therapeutic strategies for PAP.