RESUMO
Pulmonary surfactant (PS) is a lipid-protein complex that forms films reducing surface tension at the alveolar air-liquid interface. Surfactant protein C (SP-C) plays a key role in rearranging the lipids at the PS surface layers during breathing. The N-terminal segment of SP-C, a lipopeptide of 35 amino acids, contains two palmitoylated cysteines, which affect the stability and structure of the molecule. The C-terminal region comprises a transmembrane α-helix that contains a ALLMG motif, supposedly analogous to a well-studied dimerization motif in glycophorin A. Previous studies have demonstrated the potential interaction between SP-C molecules using approaches such as Bimolecular Complementation assays or computational simulations. In this work, the oligomerization state of SP-C in membrane systems has been studied using fluorescence spectroscopy techniques. We have performed self-quenching and FRET assays to analyze dimerization of native palmitoylated SP-C and a non-palmitoylated recombinant version of SP-C (rSP-C) using fluorescently labeled versions of either protein reconstituted in different lipid systems mimicking pulmonary surfactant environments. Our results reveal that doubly palmitoylated native SP-C remains primarily monomeric. In contrast, non-palmitoylated recombinant SP-C exhibits dimerization, potentiated at high concentrations, especially in membranes with lipid phase separation. Therefore, palmitoylation could play a crucial role in stabilizing the monomeric α-helical conformation of SP-C. Depalmitoylation, high protein densities as a consequence of membrane compartmentalization, and other factors may all lead to the formation of protein dimers and higher-order oligomers, which could have functional implications under certain pathological conditions and contribute to membrane transformations associated with surfactant metabolism and alveolar homeostasis.
Assuntos
Proteína C Associada a Surfactante Pulmonar , Surfactantes Pulmonares , Proteína C Associada a Surfactante Pulmonar/química , Proteína C Associada a Surfactante Pulmonar/metabolismo , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismo , Transferência Ressonante de Energia de Fluorescência , Lipídeos/química , TensoativosRESUMO
Mycobacterium tuberculosis (Mtb) cultured axenically without detergent forms biofilm-like cords, a clinical identifier of virulence. In lung-on-chip (LoC) and mouse models, cords in alveolar cells contribute to suppression of innate immune signaling via nuclear compression. Thereafter, extracellular cords cause contact-dependent phagocyte death but grow intercellularly between epithelial cells. The absence of these mechanopathological mechanisms explains the greater proportion of alveolar lesions with increased immune infiltration and dissemination defects in cording-deficient Mtb infections. Compression of Mtb lipid monolayers induces a phase transition that enables mechanical energy storage. Agent-based simulations demonstrate that the increased energy storage capacity is sufficient for the formation of cords that maintain structural integrity despite mechanical perturbation. Bacteria in cords remain translationally active despite antibiotic exposure and regrow rapidly upon cessation of treatment. This study provides a conceptual framework for the biophysics and function in tuberculosis infection and therapy of cord architectures independent of mechanisms ascribed to single bacteria.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Animais , Camundongos , Biofilmes , Pulmão/microbiologia , Pulmão/patologia , Mycobacterium tuberculosis/fisiologia , Tuberculose/microbiologia , Tuberculose/patologia , Virulência , Fenômenos BiomecânicosRESUMO
Respiratory disorders caused by allergy have been associated to bronchiolar inflammation leading to life-threatening airway narrowing. However, whether airway allergy causes alveolar dysfunction contributing to the pathology of allergic asthma remains unaddressed. To explore whether airway allergy causes alveolar dysfunction that might contribute to the pathology of allergic asthma, alveolar structural and functional alterations were analyzed during house dust mite (HDM)-induced airway allergy in mice, by flow cytometry, light and electron microscopy, monocyte transfer experiments, assessment of intra-alveolarly-located cells, analysis of alveolar macrophage regeneration in Cx3cr1 cre:R26-yfp chimeras, analysis of surfactant-associated proteins, and study of lung surfactant biophysical properties by captive bubble surfactometry. Our results demonstrate that HDM-induced airway allergic reactions caused severe alveolar dysfunction, leading to alveolar macrophage death, pneumocyte hypertrophy and surfactant dysfunction. SP-B/C proteins were reduced in allergic lung surfactant, that displayed a reduced efficiency to form surface-active films, increasing the risk of atelectasis. Original alveolar macrophages were replaced by monocyte-derived alveolar macrophages, that persisted at least two months after the resolution of allergy. Monocyte to alveolar macrophage transition occurred through an intermediate stage of pre-alveolar macrophage and was paralleled with translocation into the alveolar space, Siglec-F upregulation, and downregulation of CX3CR1. These data support that the severe respiratory disorders caused by asthmatic reactions not only result from bronchiolar inflammation, but additionally from alveolar dysfunction compromising an efficient gas exchange.
Assuntos
Asma , Hipersensibilidade , Surfactantes Pulmonares , Camundongos , Animais , Macrófagos Alveolares/metabolismo , Hipersensibilidade/complicações , Asma/metabolismo , Inflamação/complicações , TensoativosRESUMO
Pulmonary surfactant (PS) has been proposed as an efficient drug delivery vehicle for inhaled therapies. Its ability to adsorb and spread interfacially and transport different drugs associated with it has been studied mainly by different surface balance designs, typically interconnecting various compartments by interfacial paper bridges, mimicking in vitro the respiratory air-liquid interface. It has been demonstrated that only a monomolecular surface layer of PS/drug is able to cross this bridge. However, surfactant films are typically organized as multi-layered structures associated with the interface. The aim of this work was to explore the contribution of surface-associated structures to the spreading of PS and the transport of drugs. We have designed a novel vehiculization balance in which donor and recipient compartments are connected by a whole three-dimensional layer of liquid and not only by an interfacial bridge. By combining different surfactant formulations and liposomes with a fluorescent lipid dye and a model hydrophobic drug, budesonide (BUD), we observed that the use of the bridge significantly reduced the transfer of lipids and drug through the air-liquid interface in comparison to what can be spread through a fully open interfacial liquid layer. We conclude that three-dimensional structures connected to the surfactant interfacial film can provide an important additional contribution to interfacial delivery, as they are able to transport significant amounts of lipids and drugs during surfactant spreading.
RESUMO
Pulmonary delivery of small interfering RNA (siRNA) using nanoparticle-based delivery systems is promising for local treatment of respiratory diseases. We designed dry powder inhaler formulations of siRNA-loaded lipid-polymer hybrid nanoparticles (LPNs) with aerosolization properties optimized for inhalation therapy. Interactions between LPNs and pulmonary surfactant (PS) determine the fate of inhaled LPNs, but interaction mechanisms are unknown. Here we used surface-sensitive techniques to study how physicochemical properties and pathological microenvironments influence interactions between siRNA-loaded LPNs and supported PS layers. PS was deposited on SiO2 surfaces as single bilayer or multilayers and characterized using quartz crystal microbalance with dissipation monitoring and Fourier-transform infrared spectroscopy with attenuated total reflection. Immobilization of PS as multilayers, resembling the structural PS organization in the alveolar subphase, effectively reduced the relative importance of interactions between PS and the underlying surface. However, the binding affinity between PS and LPNs was identical in the two models. The physicochemical LPN properties influenced the translocation pathways and retention time of LPNs. Membrane fluidity and electrostatic interactions were decisive for the interaction strength between LPNs and PS. Experimental conditions reflecting pathological microenvironments promoted LPN deposition. Hence, these results shed new light on design criteria for LPN transport through the air-blood barrier.
Assuntos
Nanopartículas , Surfactantes Pulmonares , Polímeros/química , Dióxido de Silício , RNA Interferente Pequeno/química , Nanopartículas/química , Lipídeos/químicaRESUMO
This work evaluates interaction of pulmonary surfactant (PS) and antimicrobial peptides (AMPs) in order to investigate (i) if PS can be used to transport AMPs, and (ii) to what extent PS interferes with AMP function and vice versa. This, in turn, is motivated by a need to find new strategies to treat bacterial infections in the airways. Low respiratory tract infections (LRTIs) are a leading cause of illness and death worldwide that, together with the problem of multidrug-resistant (MDR) bacteria, bring to light the necessity of developing effective therapies that ensure high bioavailability of the drug at the site of infection and display a potent antimicrobial effect. Here, we propose the combination of AMPs with PS to improve their delivery, exemplified for the hydrophobically end-tagged AMP, GRR10W4 (GRRPRPRPRPWWWW-NH2), with previously demonstrated potent antimicrobial activity against a broad spectrum of bacteria under various conditions. Experiments using model systems emulating the respiratory interface and an operating alveolus, based on surface balances and bubble surfactometry, served to demonstrate that a fluorescently labelled version of GRR10W4 (GRR10W4-F), was able to interact and insert into PS membranes without affecting its biophysical function. Therefore, vehiculization of the peptide along air-liquid interfaces was enabled, even for interfaces previously occupied by surfactants layers. Furthermore, breathing-like compression-expansion dynamics promoted the interfacial release of GRR10W4-F after its delivery, which could further allow the peptide to perform its antimicrobial function. PS/GRR10W4-F formulations displayed greater antimicrobial effects and reduced toxicity on cultured airway epithelial cells compared to that of the peptide alone. Taken together, these results open the door to the development of novel delivery strategies for AMPs in order to increase the bioavailability of these molecules at the infection site via inhaled therapies.
Assuntos
Anti-Infecciosos , Surfactantes Pulmonares , Surfactantes Pulmonares/química , Triptofano , Peptídeos Antimicrobianos , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Antibacterianos/farmacologia , Antibacterianos/química , Monofosfato de Adenosina , Testes de Sensibilidade MicrobianaRESUMO
The disulfide bonds formed in the SAPA domain of a recombinant version of the NH2-terminal propeptide (SP-BN) from the precursor of human pulmonary surfactant protein B (SP-B) were identified through sequential digestion of SP-BN with GluC/trypsin or thermolysin/GluC, followed by mass spectrometry (MS) analysis. MS spectra allowed identification of disulfide bonds between Cys32-Cys49 and Cys40-Cys55, and we propose a disulfide connectivity pattern of 1-3 and 2-4 within the SAPA domain, with the Cys residues numbered according to their position from the N-terminus of the propeptide sequence. The peaks with m/z â¼ 2136 and â¼ 1780 in the MS spectrum of the GluC/trypsin digest were assigned to peptides 24AWTTSSLACAQGPE37 and 45QALQCR50 linked by Cys32-Cys49 and 38FWCQSLE44 and 51ALGHCLQE58 linked by Cys40-Cys55 respectively. Tandem mass spectrometry (MS/MS) analysis verified the position of the bonds. The results of the series ions, immonium ions and internal fragment ions were all compatible with the proposed 1-3/2-4 position of the disulfide bonds in the SAPA domain. This X-pattern differs from the kringle-type found in the SAPB domain of the SAPLIP proteins, where the first Cys in the sequence links to the last, the second to the penultimate and the third to the fourth one. Regarding the SAPB domain of the SP-BN propeptide, the MS analysis of both digests identified the bond Cys100-Cys112, numbered 7-8, which is coincident with the bond position in the kringle motif. SIGNIFICANCE: The SAPLIP (saposin-like proteins) family encompasses several proteins with homology to saposins (sphingolipids activator proteins). These are proteins with mainly alpha-helical folds, compact packing including well conserved disulfide bonds and ability to interact with phospholipids and membranes. There are two types of saposin-like domains termed as Saposin A (SAPA) and Saposin B (SAPB) domains. While disulfide connectivity has been well established in several SAPB domains, the position of disulfide bonds in SAPA domains is still unknown. The present study approaches a detailed proteomic study to determine disulfide connectivity in the SAPA domain of the precursor of human pulmonary surfactant-associated protein SP-B. This task has been a challenge requiring the combination of different sequential proteolytic treatments followed by MS analysis including MALDI-TOF and tandem mass MS/MS spectrometry. The determination for first time of the position of disulfide bonds in SAPA domains is an important step to understand the structural determinants defining its biological functions.
Assuntos
Surfactantes Pulmonares , Saposinas , Sequência de Aminoácidos , Dissulfetos/análise , Humanos , Peptídeos/química , Fosfolipídeos , Proteômica , Proteína B Associada a Surfactante Pulmonar , Receptores Fc , Esfingolipídeos , Espectrometria de Massas em Tandem , Termolisina , TripsinaRESUMO
Surfactant protein C (SP-C) has several functions in pulmonary surfactant. These include the transfer of lipids between different membrane structures, a role in surfactant recycling and homeostasis, and involvement in modulation of the innate defense system. Despite these important functions, the structures of functional SP-C complexes have remained unclear. SP-C is known to exist as a primarily α-helical structure with an apparently unstructured N-terminal region, yet there is recent evidence that the functions of SP-C could be associated with the formation of SP-C dimers and higher oligomers. In this work, we used molecular dynamics simulations, two-dimensional umbrella sampling, and well-tempered metadynamics to study the details of SP-C dimerization. The results suggest that SP-C dimerizes in pulmonary surfactant membranes, forming dimers of different topologies. The simulations identified a dimerization motif region V21xxxVxxxGxxxM33 that is much larger than the putative A30xxxG34 motif that is commonly assumed to control the dimerization of some α-helical transmembrane domains. The results provide a stronger basis for elucidating how SP-C functions in concert with other surfactant proteins.
Assuntos
Proteína C Associada a Surfactante Pulmonar , Surfactantes Pulmonares , Dimerização , Proteína C Associada a Surfactante Pulmonar/metabolismo , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , TensoativosRESUMO
The lives of thousands premature babies have been saved along the last thirty years thanks to the establishment and consolidation of pulmonary surfactant replacement therapies (SRT). It took some time to close the gap between the identification of the biophysical and molecular causes of the high mortality associated with respiratory distress syndrome in very premature babies and the development of a proper therapy. Closing the gap required the elucidation of some key questions defining the structure-function relationships in surfactant as well as the particular role of the different molecular components assembled into the surfactant system. On the other hand, the application of SRT as part of treatments targeting other devastating respiratory pathologies, in babies and adults, is depending on further extensive research still required before enough amounts of good humanized clinical surfactants will be available. This review summarizes our current concepts on the compositional and structural determinants defining pulmonary surfactant activity, the principles behind the development of efficient natural animal-derived or recombinant or synthetic therapeutic surfactants, as well as a the most promising lines of research that are already opening new perspectives in the application of tailored surfactant therapies to treat important yet unresolved respiratory pathologies.
Assuntos
Surfactantes Pulmonares , Síndrome do Desconforto Respiratório do Recém-Nascido , Síndrome do Desconforto Respiratório , Animais , Humanos , Recém-Nascido , Surfactantes Pulmonares/química , Surfactantes Pulmonares/uso terapêutico , Síndrome do Desconforto Respiratório do Recém-Nascido/tratamento farmacológico , Tensoativos/farmacologia , Tensoativos/uso terapêuticoRESUMO
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.
Assuntos
Líquido Amniótico/química , Surfactantes Pulmonares/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Animais , Varredura Diferencial de Calorimetria , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lauratos/química , Lipídeos/química , Membranas , SuínosRESUMO
Lung surfactant (LS) stabilizes the respiratory surface by forming a film at the alveolar air-liquid interface that reduces surface tension and minimizes the work of breathing. Typically, this surface-active agent has been isolated from animal lungs both for research and biomedical applications. However, these materials are constituted by complex membranous architectures including surface-active and inactive lipid/protein assemblies. In this work, we describe the composition, structure and surface activity of discrete membranous entities that are part of a LS preparation isolated from bronchoalveolar lavages of porcine lungs. Seven different fractions could be resolved from whole surfactant subjected to sucrose density gradient centrifugation. Detailed compositional characterization revealed differences in protein and cholesterol content but no distinct saturated:unsaturated phosphatidylcholine ratios. Moreover, no significant differences were detected regarding apparent hydration at the headgroup region of membranes, as reported by the probe Laurdan, and lipid chain mobility analysed by electron spin resonance (ESR) in spite of the variety of membranous assemblies observed by transmission electron microscopy. In addition, six of the seven separated LS subfractions formed similar, essentially disordered-like, interfacial films and performed efficient surface activity, under physiologically relevant conditions. Altogether, our work show that a LS isolated from porcine lungs is comprised by a heterogenous population of membranous assemblies lacking freshly secreted unused LS complexes sustaining highly dehydrated and ordered membranous assemblies as previously reported. We propose that surfactant subfractions may illustrate intermediates in sequential structural steps within the structural transformations occurring along the respiratory compression-expansion cycles.
Assuntos
Lipídeos/química , Pulmão/química , Surfactantes Pulmonares/química , Tensoativos/química , Animais , Brônquios/química , Brônquios/metabolismo , Pulmão/metabolismo , Alvéolos Pulmonares/química , Surfactantes Pulmonares/metabolismo , Tensão Superficial , Tensoativos/metabolismo , SuínosRESUMO
Inhaled substances, such as consumer products, chemicals at the workplace, and nanoparticles, can affect the lung function in several ways. In this paper, we explore the adverse outcome pathway (AOP) that starts when inhaled substances that reach the alveoli inhibit the function of the lung surfactant, and leads to decreased lung function. Lung surfactant covers the inner surface of the alveoli, and regulates the surface tension at the air-liquid interface during breathing. The inhibition of the lung surfactant function leads to alveolar collapse because of the resulting high surface tension at the end of expiration. The collapsed alveoli can be re-opened by inspiration, but this re-opening causes shear stress on cells covering the alveoli. This can damage the alveolar-capillary membrane integrity, allowing blood components to enter the alveolar airspace. Blood components, such as albumin, can interact with the lung surfactant and further inhibit its function. The collapse of the alveoli is responsible for a decrease in the surface area available for blood oxygenation, and it reduces the volume of air that can be inhaled and exhaled. These different key events lead to decreased lung function, characterized by clinical signs of respiratory toxicity and reduced blood oxygenation. Here we present the weight of evidence that supports the AOP, and we give an overview of the methods available in vitro and in vivo to measure each key event of the pathway, and how this AOP can potentially be used in screening for inhalation toxicity.
RESUMO
Polyhydroxyalkanoates (PHA) are polyesters produced intracellularly by many bacterial species as energy storage materials, which are used in biomedical applications, including drug delivery systems, due to their biocompatibility and biodegradability. In this study, we evaluated the potential application of this nanomaterial as a basis of inhaled drug delivery systems. To that end, we assessed the possible interaction between PHA nanoparticles (NPs) and pulmonary surfactant using dynamic light scattering, Langmuir balances, and epifluorescence microscopy. Our results demonstrate that NPs deposited onto preformed monolayers of DPPC or DPPC/POPG bind these surfactant lipids. This interaction facilitated the translocation of the nanomaterial towards the aqueous subphase, with the subsequent loss of lipid from the interface. NPs that remained at the interface associated with liquid expanded (LE)/tilted condensed (TC) phase boundaries, decreasing the size of condensed domains and promoting the intermixing of TC and LE phases at submicroscopic scale. This provided the stability necessary for attaining high surface pressures upon compression, countering the destabilization induced by lipid loss. These effects were observed only for high NP loads, suggesting a limit for the use of these NPs in pulmonary drug delivery.
RESUMO
Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disease with significant neurological and skeletal pathologies. Respiratory dysfunction is a secondary pathology contributing to mortality in MPS IIIA patients. Pulmonary surfactant is crucial to optimal lung function and has not been investigated in MPS IIIA. We measured heparan sulphate (HS), lipids and surfactant proteins (SP) in pulmonary tissue and bronchoalveolar lavage fluid (BALF), and surfactant activity in healthy and diseased mice (20 weeks of age). Heparan sulphate, ganglioside GM3 and bis(monoacylglycero)phosphate (BMP) were increased in MPS IIIA lung tissue. There was an increase in HS and a decrease in BMP and cholesteryl esters (CE) in MPS IIIA BALF. Phospholipid composition remained unchanged, but BALF total phospholipids were reduced (49.70%) in MPS IIIA. There was a reduction in SP-A, -C and -D mRNA, SP-D protein in tissue and SP-A, -C and -D protein in BALF of MPS IIIA mice. Captive bubble surfactometry showed an increase in minimum and maximum surface tension and percent surface area compression, as well as a higher compressibility and hysteresis in MPS IIIA surfactant upon dynamic cycling. Collectively these biochemical and biophysical changes in alveolar surfactant are likely to be detrimental to lung function in MPS IIIA.
Assuntos
Heparitina Sulfato/metabolismo , Mucopolissacaridose III/metabolismo , Alvéolos Pulmonares/metabolismo , Surfactantes Pulmonares/metabolismo , Animais , Fenômenos Biofísicos , Líquido da Lavagem Broncoalveolar , Colesterol/metabolismo , Cromatografia Líquida , Gangliosídeo G(M3)/metabolismo , Regulação da Expressão Gênica , Lisofosfolipídeos/metabolismo , Camundongos Endogâmicos C57BL , Monoglicerídeos/metabolismo , Fosfolipídeos/metabolismo , Padrões de Referência , Espectrometria de Massas em TandemRESUMO
RNA therapeutics are poised to revolutionize medicine. To unlock the full potential of RNA drugs, safe and efficient (nano)formulations to deliver them inside target cells are required. Endosomal sequestration of nanocarriers represents a major bottleneck in nucleic acid delivery. Gaining more detailed information on the intracellular behavior of RNA nanocarriers is crucial to rationally develop delivery systems with improved therapeutic efficiency. Surfactant protein B (SP-B) is a key component of pulmonary surfactant (PS), essential for mammalian breathing. In contrast to the general belief that PS should be regarded as a barrier for inhaled nanomedicines, we recently discovered the ability of SP-B to promote gene silencing by siRNA-loaded and lipid-coated nanogels. However, the mechanisms governing this process are poorly understood. The major objective of this work was to obtain mechanistic insights into the SP-B-mediated cellular delivery of siRNA. To this end, we combined siRNA knockdown experiments, confocal microscopy, and focused ion beam scanning electron microscopy imaging in an in vitro non-small-cell lung carcinoma model with lipid mixing assays on vesicles that mimic the composition of (intra)cellular membranes. Our work highlights a strong correlation between SP-B-mediated fusion with anionic endosomal membranes and cytosolic siRNA delivery, a mode of action resembling that of certain viruses and virus-derived cell-penetrating peptides. Building on these gained insights, we optimized the SP-B proteolipid composition, which dramatically improved delivery efficiency. Altogether, our work provides a mechanistic understanding of SP-B-induced perturbation of intracellular membranes, offering opportunities to fuel the rational design of SP-B-inspired RNA nanoformulations for inhalation therapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Proteína B Associada a Surfactante Pulmonar , Animais , Linhagem Celular Tumoral , RNA Interferente Pequeno/genéticaRESUMO
Lung surfactant (LS) is an outstanding example of how a highly regulated and dynamic membrane-based system has evolved to sustain a wealth of structural reorganizations in order to accomplish its biophysical function, as it coats and stabilizes the respiratory air-liquid interface in the mammalian lung. The present review dissects the complexity of the structure-function relationships in LS through an updated description of the lipid-protein interactions and the membrane structures that sustain its synthesis, secretion, interfacial performance and recycling. We also revise the current models and the biophysical techniques employed to study the membranous architecture of LS. It is important to consider that the structure and functional properties of LS are often studied in bulk or under static conditions, in spite that surfactant function is strongly connected with a highly dynamic behaviour, sustained by very polymorphic structures and lipid-lipid, lipid-protein and protein-protein interactions that reorganize in precise spatio-temporal coordinates. We have tried to underline the evidences available of the existence of such structural dynamism in LS. A last important aspect is that the synthesis and assembly of LS is a strongly regulated intracellular process to ensure the establishment of the proper interactions driving LS surface activity, while protecting the integrity of other cell membranes. The use of simplified lipid models or partial natural materials purified from animal tissues could be too simplistic to understand the true molecular mechanisms defining surfactant function in vivo. In this line, we will bring into the attention of the reader the methodological challenges and the questions still open to understand the structure-function relationships of LS at its full biological relevance.
Assuntos
Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Surfactantes Pulmonares/química , Surfactantes Pulmonares/metabolismo , HumanosRESUMO
Surfactant protein C (SP-C) is a protein present in the pulmonary surfactant system that is involved in the biophysical properties of this lipoprotein complex, but it also has a role in lung defense and homeostasis. In this article, we propose that the link between both functions could rely on the ability of SP-C to induce fragmentation of phospholipid membranes and generate small vesicles that serve as support to present different ligands to cells in the lungs. Our results using bimolecular fluorescence complementation and tunable resistive pulse sensing setups suggest that SP-C oligomerization could be the triggering event that causes membrane budding and nanovesiculation. As shown by fluorescence microscopy and flow cytometry, these vesicles are differentially assimilated by alveolar macrophages and alveolar type II cells, indicating distinct roles of these alveoli-resident cells in the processing of the SP-C- induced vesicles and their cargo. These results depict a more accurate picture of the mechanisms of this protein, which could be relevant for the comprehension of pulmonary pathologies and the development of new therapeutic approaches.
Assuntos
Proteína C Associada a Surfactante Pulmonar/metabolismo , Lipossomas Unilamelares/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Dimerização , Endocitose , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Domínios Proteicos , Multimerização Proteica , Proteína C Associada a Surfactante Pulmonar/química , Proteína C Associada a Surfactante Pulmonar/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Lipossomas Unilamelares/químicaRESUMO
Pediatric (PARDS) and neonatal (NARDS) acute respiratory distress syndrome have different age-specific characteristics and definitions. Trials on surfactant for ARDS in children and neonates have been performed well before the PARDS and NARDS definitions and yielded conflicting results. This is mainly due to heterogeneity in study design reflecting historic lack of pathobiology knowledge. We reviewed the available clinical and preclinical data to create an expert consensus aiming to inform future research steps and advance the knowledge in this area. Eight trials investigated the use of surfactant for ARDS in children and ten in neonates, respectively. There were improvements in oxygenation (7/8 trials in children, 7/10 in neonates) and mortality (3/8 trials in children, 1/10 in neonates) improved. Trials were heterogeneous for patients' characteristics, surfactant type and administration strategy. Key pathobiological concepts were missed in study design. Consensus with strong agreement was reached on four statements: 1. There are sufficient preclinical and clinical data to support targeted research on surfactant therapies for PARDS and NARDS. Studies should be performed according to the currently available definitions and considering recent pathobiology knowledge. 2. PARDS and NARDS should be considered as syndromes and should be pre-clinically studied according to key characteristics, such as direct or indirect (primary or secondary) nature, clinical severity, infectious or non-infectious origin or patients' age. 3. Explanatory should be preferred over pragmatic design for future trials on PARDS and NARDS. 4. Different clinical outcomes need to be chosen for PARDS and NARDS, according to the trial phase and design, trigger type, severity class and/or surfactant treatment policy. We advocate for further well-designed preclinical and clinical studies to investigate the use of surfactant for PARDS and NARDS following these principles.