sign in sign up
<<
Home , Pulmonary surfactant, Surfactant

Arch Dis Child Fetal Neonatal Ed: first published as 10.1136/archdischild-2018-315413 on 14 December 2018. Downloaded from Review Understanding the principle biophysics concepts of pulmonary in health and disease Chiara Autilio,1,2 Jesús Pérez-Gil1,2

1Department of Biochemistry Abstract stable film at the air- interface, displacing and Molecular Biology, Faculty (PS) is a - complex water molecules from exposure to air and therefore of Biology, Complutense reduces (figure 1A). At the same University, Madrid, Spain essential to stabilise the delicate structure of mammalian 2Research Institute "Hospital alveoli along with successive compression-expansion time, different components of PS constitute a first 12 de Octubre", Complutense respiratory cycles. To do so, surfactant reduces barrier to the access of pathogens to the rest of the University, Madrid, Spain dramatically surface tension at the air-liquid interface, organism via the large respiratory surface. an activity that depends critically on a proper lipid The major components of PS are (~92%), Correspondence to composition and the presence of some specific surfactant mostly saturated (DPPC) Professor Jesús Pérez-Gil, Department of Biochemistry, . Lack or dysfunction of this system is associated (~40%), which is essential for PS surface active Faculty of Biology, Complutense with severe respiratory pathologies, which are in some properties. The saturated nature of acyl chains in University, José Antonio Novais cases treated by supplementation with exogenous DPPC allows for maximal packing on reduction of 12, Madrid 28040, Spain ; surfactant materials. The biophysical function and surface area, and therefore, maximal exclusion of jperezgil@​ ​bio.ucm.​ ​es performance of PS, in health and disease, are directly water molecules and maximal reduction in surface Received 8 October 2018 influenced by its composition, structure and mechanical tension. Unsaturated phosphatidylcholine (PC), Revised 15 November 2018 properties. This review summarises the main biophysics phosphatidylglycerol (PG) and account Accepted 16 November 2018 concepts behind the mechanisms that define surfactant for ~25%, ~10% and ~5–8% of PS total mass, function in a healthy and in pathological situations. respectively, whereas the other lipid species (neutral It also revises some of the most useful biophysical lipids and other (PL)) correspond to techniques that provide information about surfactant- the residual 8–10% (figure 1B).1 The remaining related processes. Finally, translational biophysics will part of PS (~8%) is constituted by four specific

be invoked to illustrate how biophysical studies may proteins, which can be classified into two families: by copyright. contribute to understand the role of surfactant in health SP-B and SP-C (hydrophobic) and SP-A and SP-D and disease and to design better surfactant-based (hydrophilic). SP-A, SP-B and SP-C are associated therapeutic approaches. to PS membranes, mainly binding negative-charged PLs (SP-B, SP-C) and DPPC (SP-A), and are essen- tial to facilitate formation of the PS interfacial film, organising and stabilising the structure, and Pulmonary surfactant in health and ensuring correct recycling (figure 1B). Moreover, disease SP-A and SP-D belong to the protein family Pulmonary surfactant (PS) is a membrane-based and are involved in innate immune defence against system composed of a mixture of lipids and proteins. lung pathogens and allergens.2 3 It is synthetised by alveolar type II pneumocytes The composition, structure and mechanical prop- and secreted into the thin layer of fluid that covers erties of PS layers are directly responsible for the the alveolar epithelium. The main function of PS biophysical function and performance of the system is related to the stabilisation of the delicate struc- in a healthy lung. The lack, deficiency or inactiva- ture of mammalian alveoli by producing a dramatic tion of PS leads to severe respiratory disorders both http://fn.bmj.com/ reduction of the surface tension at the respiratory in neonates and adults, such as neonatal respira- air-liquid interface. At this interface, water mole- tory distress syndrome (RDS) and acute respiratory cules in contact with air cohere more strongly, distress syndrome (ARDS). because the interactions with the water bulk phase RDS is mostly due to lung immaturity and lack of are not compensated with interactions on the air side PS in preterm neonates. The resulting high surface

where water molecules in vapour phase are scarce. tension impedes keeping the alveoli open since on 17 December 2018 by guest. Protected This generates a net intermolecular cohesive , the first breath, causing high neonatal mortality which is defined as the surface tension. Opening (figure 1C left). In the early 1990s, surfactant new surface exposed to air, as required during each replacement therapy (SRT) with an exogenous © Author(s) (or their single inspiration, demands overcoming this cohe- surfactant preparation contributed to decreasing employer(s)) 2018. No sive superficial force, supplying the energy neces- mortality rates,4 as it facilitates respiratory commercial re-use. See rights sary to break the molecular interactions that the mechanics, which stimulates the self-production and permissions. Published 5 6 by BMJ. new molecules need to break through the gas-liquid of PS in newborns. However, SRT is not effec- interface. At high surface tension, therefore, the tive when RDS is due to severe genetic disorders To cite: Autilio C, Pérez-Gil J. work of , that is, to open the 15–20 (for instance, SP-B deficiency due to mutations in Arch Dis Child Fetal Neonatal Ed Epub ahead of print: times/min, would consume a fair proportion of the SFTPB gene or alterations in the ABCA3 gene) [please include Day Month metabolic energy, and would unavoidably end in and particularly challenged in pathologies in which Year]. doi:10.1136/ alveolar collapse. To prevent collapse and reduce both endogenous and therapeutic surfactant result archdischild-2018-315413 the , PS spontaneously forms a inactivated by a direct or indirect damage of their

Autilio C, Pérez-Gil J. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F9. doi:10.1136/archdischild-2018-315413 F1 Arch Dis Child Fetal Neonatal Ed: first published as 10.1136/archdischild-2018-315413 on 14 December 2018. Downloaded from Review

Figure 1 Pulmonary surfactant (PS) in healthy and diseased human lung tissue. (A) Illustrative scheme of PL-mediated reduction of surface by copyright. tension. When a layer of PLs is formed at the air-liquid interface, the PL amphipathic molecules displace water molecules and reduce the air-liquid proximity. (B) A schematic representation of a normal alveolus, with the organisation of PS membranes at the air-liquid interface and the lipid/protein composition of lung surfactant. (C) Collapsed alveolus in RDS due to ATII cell immaturity and injured alveoli both in ARDS and MAS. The resulting inhibition of PS membranes caused by protein exudate, sPLA2, reactive species (ROS) production, cholesterol and acids is shown. (D) A schematic representation of (1) IPF alveolus with a reduction in levels of both SP-C and PLs and (2) PAP alveolus obstruction due to the excess of already used and undegraded PS. aAMφ, activated macrophages; AMφ, alveolar macrophages; aPMN, activated polymorphonucleated cells; ARDS, acute respiratory distress syndrome; ATI, alveolar type I pneumocytes; ATII, alveolar type II pneumocytes; BA, ; CHOL, cholesterol; DPPC, saturated dipalmitoylphosphatidylcholine; FFA, free fatty acids; imATII, immature alveolar type II pneumocytes; IPF, idiopathic pulmonary fibrosis; MAS, meconium aspiration syndrome; MFB, myofibroblasts; MO, monocytes; n-ATI, necrotic alveolar type I pneumocytes; NL, neutral lipid; ox-PLs, oxidised phospholipids; ox-SP-B, oxidised SP-B; ox-SP-C, oxidised SP-C; PAP, pulmonary alveolar proteinosis; PG, phosphatidylglycerol; PL, ; RBC, red blood cell; RDS, respiratory distress syndrome; sPLA2, secretory phospholipase A2; unPC, unsaturated phosphatidylcholine. surface properties.7 Still, SRT seems to exhibit partial efficiency by means of the same compounds described above, which may be in some cases of ARDS. The identification of which patients with found in the meconium mixture.6 11 Besides, a simultaneous action http://fn.bmj.com/ ARDS can benefit the most is an important challenge in clinical of cholesterol and bile acids highly increases surfactant fluidity and research. contributes further to its inactivation (Figure 1C right).12 More- ARDS is an acute respiratory failure triggered by various over, bile acids act as a cofactor of sPLA2, increasing the conditions, characterised by extensive lung inflammation and activity and contributing to lung injury.13 diffuse alveolar injury. The resulting impairment of PS is mainly Although less relevant in a paediatric context, some reports

due to an increase of infiltrating inflammatory cells, necrotic suggest that PS dysfunction could be also behind idiopathic on 17 December 2018 by guest. Protected cell fractions and exosomes. However, higher levels of secretory pulmonary fibrosis (IPF), a chronic and progressive epithelial phospholipase A2 (sPLA2) may also contribute to hydrolyse PLs injury which still nowadays remains unsolved. Specifically, a and nourish the inflammatory cascade.8 9 The release of lyso- reduction in SP-C was described as one of the triggering and phospholipids (LPL) and free fatty acids (FFA) from hydrolysed contributing events to the fibrotic process of this lung disease.7 membranes as well as the presence of reactive oxygen species Moreover, the plasticity of alveolar macrophages seems to play (ROS), cytokines and other oedema proteins contribute to a critical role in the pathogenesis of IPF14 which may lead, directly injure surfactant and/or provide inflammation-medi- in turn, to a resulting unbalance between surfactant degrada- ated damages (Figure 1C center). Moreover, the levels of SP-A tion and recycling (Figure 1D left). The insufficient uptake and SP-B have been found to be lower in patients suffering from and catabolism of PS could also be due to autoantibodies or ARDS, increasing the overall inactivation of the PS.6 mutations in the genes of macrophage colony-stimulating Among the various possible types of neonatal ARDS, meconium factor receptors (CSF2A, CSF2RB). This is the cause of congen- aspiration syndrome (MAS) is caused by of meconium ital pulmonary alveolar proteinosis (PAP), which is associated to and may have a relevant mortality.10 MAS affects surfactant activity abnormal accumulation of old and less functional surfactant in

F2 Autilio C, Pérez-Gil J. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F9. doi:10.1136/archdischild-2018-315413 Arch Dis Child Fetal Neonatal Ed: first published as 10.1136/archdischild-2018-315413 on 14 December 2018. Downloaded from Review

Figure 2 Essential properties of lung surfactant in healthy breathing. On expiration: Pulmonary surfactant (PS) organises in multilayer structures characterised by the presence of DPPC at the air-liquid interface with the consequent drastic reduction in surface tension (<2 mN/m); during inspiration: re-extension of phospholipid (PL) membranes along with the initial of PS aggregates at the air-liquid interface, reaching the equilibrium surface tension of 20 mN/m. This process is mediated by SP-A and SP-B and facilitated by PG, cholesterol and unsaturated PLs. CHOL,

cholesterol; DPPC, saturated dipalmitoylphosphatidylcholine; PG, phosphatidylglycerol; unPC, unsaturated phosphatidylcholine. by copyright. the alveoli with a decrease in the pulmonary capacity15 has been proposed that a highly curved structural intermediate (Figure 1D right). is generated between PS vesicles and the interfacial film. SP-B has the major role by facilitating lipid exchange and stabilising S urfaCTAnt biophysical properties this transient structure, especially in the presence of anionic PLs As mentioned above, several pathologies are associated to (mainly PG). As a matter of fact, it has been determined that damage in PS function and, to date, for most of them there is SP-B forms supramolecular structures at the surface of freshly no effective therapy on the horizon. The main reason is the secreted surfactant complexes, ready to open connections that lack of evidence on key pathological mechanisms and how they facilitate rapid flows of PLs on making contact with the inter- impair PS activity. To overcome this situation, it is necessary to face.17 It also seems that unsaturated PLs and cholesterol influ- understand the main concepts of surfactant biophysics, which ence the insertion of surface-active species into the interfacial should facilitate the development of new clinical applications. film.1 Overall, PS adsorption at the air-liquid interface reduces As summarised in figure 2, the surface activity of PS is character- the surface tension from 70 mN/m (at 37°C) to values around ised by three essential properties, which take place on breathing: 20 mN/m (equilibrium surface tension). Then, on expiration, (1) quick movement towards the air-liquid interface (adsorp- the surface tension is further decreased, to reach values of less http://fn.bmj.com/ tion) and transfer of new lipid species to cover the alveolar area than 2mN/m (figure 2), as demonstrated by both in vitro and in during inspiration; (2) proper packing of the interfacial film with vivo studies.18 As mentioned above, this reduction prevents the a compression-driven reorganisation into surface-associated alveolar collapse through the selective reorganisation of PS. The multilayered membrane structures (compressibility) on expira- lipid-protein film reorganises into a three-dimensional multilay- tion, producing a drastic reduction of surface tension; and 3) ered structure characterised by highly packed domains in contact lateral redistribution of packed lipids during periods of expan- with air to avoid fluid-air proximity. A so-called ‘squeeze-out’ on 17 December 2018 by guest. Protected sion of the air-liquid interface (respreading capability) at the model suggests that this reorganisation to fold part of the inter- subsequent inspiration and alveolar re-extension.16 These cycles facial film towards the subphase would allow most of the DPPC of PS compression and re-extension constitute a partially revers- molecules to remain at the surface. These molecules act as a key ible process in which the interaction of the lipids and proteins component of high-ordered domains with maximal packing seems to play a key role. and maximal surface tension reduction. Film reorganisation To adsorb rapidly, first, PS needs to generate large bilay- selectively excludes unsaturated PLs and cholesterol below the er-based aggregates, with the potential to cooperatively trans- interface as part of the more fluid and deformable film regions.1 port and transfer a large mass of surfactant PLs to the interface. However, several studies have demonstrated that some / These surfactant membrane complexes, in many studies known marsupial species can maintain proper respiratory mechanics as large surfactant aggregates or large aggregates, diffuse towards with a surfactant containing relatively low amounts of DPPC and the air-liquid interface, in a process facilitated by both calcium higher proportions of unsaturated PC.19 20 and protein SP-A.1 Once the interface is reached, the PS aggre- During compression (at expiration), SP-B and SP-C are also gates then need to fuse within the surface. For this to happen, it removed from the surface, since they are associated with the less

Autilio C, Pérez-Gil J. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F9. doi:10.1136/archdischild-2018-315413 F3 Arch Dis Child Fetal Neonatal Ed: first published as 10.1136/archdischild-2018-315413 on 14 December 2018. Downloaded from Review compressible PLs by specific lipid-protein interactions. However, (albumin, fibrinogen, C-reactive protein) compete with PS at when the interfacial membranes are overcompressed, SP-B still the air-liquid interface, creating a steric barrier that impedes has an important role in promoting film stability against mechan- the proper adsorption of material.6 Moreover, the ROS gener- 21 ical perturbations, possibly by providing a high intermembrane ated by the inflammatory response modifies the chemical cohesivity to the multilamellar surfactant reservoir associated structure of both lipids and surfactant proteins. The positive 22 with the interfacial film. In this line, SP-B, SP-C and cholesterol charges of SP-B are reduced, affecting its binding to anionic are essential to create the continuous multilayer structure with a PLs, whereas the loss in palmitoyl groups of SP-C leads to stable association with the interface. The two proteins also play a destabilisation of protein conformation and aggregation with crucial role on inspiration, facilitating the respreading of PS and amyloid formation.6 23 The oxidation of unsaturated lipid guiding the concerted reinsertion of excluded and new PLs into 1 species and sterols also results in surfactant impairment and the interfacial surface (figure 2). promotes a concurrent proinflammatory ambient.24 Similarly, The biophysical properties of PS are related to the complex the activity of some sPLA2 isotypes degrades PS membranes, nature of the system, in which several components are involved liberating LPLs, FFA (including arachidonic acid) that increase in different and interconnected roles. In normal alveoli, this the overall inflammation.6 delicate balance is based on a close cooperation between surfac- As regards lipid composition, a decrease in PC and PG levels tant proteins and lipids as well as their simultaneous catabolism was reported in ARDS, including a significant reduction in the and recycling. This ensures that proteins and lipids that are 25 inactivated on exposure to the highly oxidant environment of amount of DPPC. This event along with a lower SP-A and SP-B alveoli are replaced by fully active newly assembled protein/ also affects the surface tension-lowering proper- 6 lipid surfactant complexes. In ARDS and MAS, the inflamma- ties of PS. As for MAS, a further process of inactivation occurs tory injury drastically modifies the alveolar milieu, impairing by the direct insertion of exogenous cholesterol and bile acids the balance with resulting damages to respiratory mechanics into surfactant layers. The two molecules lead to a fluidising (figure 1C). Protein exudates, ROS, sPLA2 as well as meco- effect, which inhibits monolayer-bilayer conversions and reduces nium elements affect either directly or indirectly PS compo- the extent of packing achieved by DPPC-enriched membrane nents, making this complex unstable and impairing adsorption, and film domains. Hence, the formation of stable multilayer re-extension and the generation of multilayered structures at membranes is compromised as well as the reduction of surface high compression rates. For example, several serum proteins tension on expiration.6 by copyright. http://fn.bmj.com/ on 17 December 2018 by guest. Protected

Figure 3 Main experimental techniques to assay pulmonary surfactant (PS) activity. Methodologies that test the variation in surface tension of surfactant samples include (A) the Wilhelmy trough (WT), (C) the pulsating bubble surfactometer (PBS), (D) the captive bubble surfactometer (CBS), and (E) the constrained sessile drop (CSD) surfactometer. (B) The fluorescence surfactant adsorption test (SAT) analyses the accumulation of PS overtime at the air-liquid interface. BB, Brilliant Black.

F4 Autilio C, Pérez-Gil J. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F9. doi:10.1136/archdischild-2018-315413 Arch Dis Child Fetal Neonatal Ed: first published as 10.1136/archdischild-2018-315413 on 14 December 2018. Downloaded from Review

Figure 4 Methods to study pulmonary surfactant (PS) film and membrane structure, composition and reorganisation. (A) An illustrative diagram of the Langmuir-Blodgett trough subjected to lateral compression and the concurrent transference of a DPPC-reach interfacial film (previously by copyright. doped with the fluorescence probe BODIPY-PC). Several pictures of the microstructures and nanostructures formed by DPPC at the interface are shown. At high surface , DPPC clusters excluding the bulky fluorescent probe are visible by epifluorescence microscopy as black starry- shape domains against a green background (which represents the mixture of unpacked DPPC and probe). Alternatively, AFM allows for detailed analysis of the domains distribution at a submicroscopic scale with the simultaneous topography of the sample surface. (B) Heat transfer inside a differential scanning calorimetry (DSC) and the typical thermograms of DPPC membranes with and without cholesterol. Under normal conditions with increasing , DPPC undergoes a pretransition (33.5°C) in which phospholipids (PL) transit from a highly ordered solid-like ‘-phase’ to an intermediate ‘ripple phase’ before reaching a disordered ‘fluid phase’ at a Tm of around 41°C. Conversely, in the presence of high amounts of cholesterol, a unique fluid phase appears and the enthalpy associated (area under peak) is strongly reduced. AFM, atomic force microscopy; CHOL, cholesterol; Cp, differential heat capacity; DPPC, saturated dipalmitoylphosphatidylcholine; Tm, melting temperature.

Testing PS activity the bottom of which is immersed in the aqueous subphase and The biophysical properties of PS and related molecular mech- in contact with the air-liquid interface (figure 3A). Thus, when anisms have been studied by using both quantitative and qual- surfactant accumulates at the aqueous surface, a force trans- itative in vitro methods, which test synthetic lipid-protein ducer detects the increase in lateral molecular , which is http://fn.bmj.com/ mixtures, therapeutic as well as human and animal PS related to the reduction in surface tension. samples. Some of the most employed biophysical techniques are summarised in the sections below.1 26 Moreover, the implemen- Surfactant adsorption test tation of these techniques to study PS inactivation and possible Surfactant adsorption test (SAT) is a rapid, sensitive and effective therapies are introduced in the last part of this review. high-throughput fluorescent method to test both adsorp-

tion and accumulation of surfactant at the air-liquid interface on 17 December 2018 by guest. Protected Wilhelmy trough (figure 3B).28 The active surface material is labelled with a The Wilhelmy trough is a simple model typically used to study fluorescent PC (BODIPY-PC) at a final molar ratio of 1% (dye/ the adsorption and spreading properties of surface-active mate- surfactant). Then, a few micrograms of this mixture are injected rials. It was first applied by the pioneer work of Clements27 in at the bottom of a microtitre plate, the wells of which contain a the mid-20th century, when the existence of PS as a surface-active quenching , namely Brilliant Black. The reading of fluo- material in the lungs was proven. Figure 3A describes the basic rescence is quickly initiated and continues for 2 hours when the working principles. To test surfactant adsorption, a controlled plate is subjected to shaking cycles. Since the dye is masked until amount of material is injected at the bottom of a small hydro- surfactant adsorbs at the air-liquid interface, the method allows phobic trough (typically made of Teflon) that is filled with an for the detection of both sample adsorption and accumulation aqueous solution. To evaluate the spreading properties, surfactant over time by monitoring the increase in fluorescence. SAT was is distributed directly at the air-liquid interface. The subsequent developed by Ravasio et al [28] in 2008. However, changes in reduction in surface tension is usually detected by a Wilhelmy this protocol were proposed later to improve the assay sensitivity plate, typically a small piece of filter paper or a platinum plate, (lower volume and higher molar dye/surfactant ratio) and to test

Autilio C, Pérez-Gil J. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F9. doi:10.1136/archdischild-2018-315413 F5 Arch Dis Child Fetal Neonatal Ed: first published as 10.1136/archdischild-2018-315413 on 14 December 2018. Downloaded from Review by copyright.

Figure 5 Pulmonary surfactant inactivation and drug delivery. (A) Schematic illustration of the basic mechanisms leading to pulmonary surfactant (PS) membrane inactivation by serum or meconium. (B) In vitro protocols to study the inhibition by serum in the Wilhelmy trough and the captive bubble surfactometer (CBS). (C) Double balance set-up to recreate PS and PS/drug spreading along the air-liquid interface, mimicking in vitro surfactant distribution from upper to lower airways (modified from40 ). the human samples of different origin,29 30 converting SAT in a leakage of material towards the capillary at high compression useful tool to design a bedside point of care method. rates.1

Pulsating bubble surfactometer Captive bubble surfactometer http://fn.bmj.com/ Pulsating bubble surfactometer (PBS) has a simple design and is a The captive bubble surfactometer (CBS) was designed commercially available technique, which evaluates the compres- by Schürch and colleagues to circumvent PBS limitations sion/re-expansion properties of surfactant under quasiphys- (figure 3D).32 This technique is presently considered the most iological conditions. This method, developed by Adams and effective methodology to study dynamic changes in surface Enhörning,31 recreates the geometry of the alveolus, allowing tension in breathing-like conditions (up to 60 cycles/min). The

a breathing simulation within the range of 0.02–80 cycles/min. capability of surfactant to adsorb, re-extend and be compressed on 17 December 2018 by guest. Protected As shown in figure 3C, a small plastic capillary is used to create is evaluated at the air-liquid interface between an air bubble an air bubble (0.8–1.1 mm), which is connected to the atmo- and a buffer solution, contained in a glass chamber. The small sphere face upwards and to surfactant suspension (contained in bubble (2–7 mm) is created and floats against an agarose roof a chamber) face downwards. Hence, the surface active material with no connection to the atmosphere, preventing the leakage adsorbs into the air-liquid interface, distributing all over the of material. At a controlled temperature, surfactant suspension bubble surface. This is periodically expanded and compressed can be dispersed inside the buffer solution or applied locally by a piston pulsator, modifying the pressure inside the capillary. onto the bubble surface by using a small capillary tube (permit- The resulting changes in the bubble radius (from 0.4 to 0.55 mm) ting to increase sample ). Then, the chamber is are continuously monitored by a microscope and transformed sealed and the bubble is compressed and expanded periodically into values of surface tension that define the activity of tested by varying the hydrostatic pressure with a piston. The changes surfactant. However, two main disadvantages are observed: (1) in shape of the bubble are recorded continuously, which allows the incapability to assess surfactant samples with concentration for the calculation of volume, area and surface tension at any above a certain limit (maximum of 1–2 mg/mL) and (2) the time.

F6 Autilio C, Pérez-Gil J. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F9. doi:10.1136/archdischild-2018-315413 Arch Dis Child Fetal Neonatal Ed: first published as 10.1136/archdischild-2018-315413 on 14 December 2018. Downloaded from Review

Constrained sessile drop Thus, sufficient levels of cholesterol promote organisation of a Constrained sessile drop (CSD) is an alternative method to unique, intermediate phase, the so-called ‘liquid ordered’ phase detect variations in surface tension under conditions of simu- (figure 4B). PS composition has evolved for its membranes and lated breathing (figure 3E).33 Briefly, a sessile aqueous drop is films to sustain a simultaneous coexistence of ordered and disor- created on the top of a small stainless steel pedestal, which is dered membrane phases at close to physiological connected to a liquid store. The sample can be applied directly values.35 The existence of these different phases and membrane at the air-liquid interface from either the water reservoir or the organisations and their interconversion as a function of tempera- air above, thus permitting the study of all physiological and ther- ture, the determination of Tm values governing the phase transi- apeutic conditions. A syringe is used to continuously modify tions and the associated energy (enthalpy) can all be characterised the volume of the drop moving liquid to and from the store, by differential scanning calorimetry (DSC). DSC thermograms subjecting the sample to compression-expansion cycles. The from surfactant membranes can be obtained in a microcalorim- resulting changes in the drop shape are recorded and trans- eter, which measures the differential heat (Cp) required to raise formed into surface tension values. the temperature of the sample (surfactant) to the same value reached by a reference (buffer) along a ramp of increasing (or 36 Understanding the molecular mechanisms of decreasing) temperature. As schematised in figure 4B, when PS dysfunction the sample undergoes an endothermic molecular reorganisation, Apart from SAT, which measures PS accumulation at the the Cp increases, revealing both the Tm (peak temperature at air-liquid interface, all methods described above analyse surfac- which the Cp occurs) and the energy needed to complete the tant biophysical activity as changes in surface tension with phase transition (area under peak). different set-ups. However, PS molecular structure, composition and reorganisation at particular environmental conditions also S urfaCTAnt biophysics in clinical research influence surfactant function both in health and disease. Alternative protocols of classic in vitro methods have been proposed to create and assess the occurrence of surfactant inhi- Langmuir-Blodgett trough bition associated with several lung diseases. The design includes The use of Langmuir-Blodgett (LB) troughs allows for the study testing PS from animal sources (ie, porcine or mice surfactants), 1 26 of both structure and dynamic properties of PS (figure 4A). It which can be easily collected, to better understand the process enables the compression and expansion of the adsorbed/spread of surfactant inactivation along with possible reverting strate- surfactant film by moving the horizontal barrier that delimits gies. In this regard, two key inhibition mechanisms must be

one side (or two sides, if two symmetrical barriers are set) of considered: (1) the direct insertion of membrane-perturbing by copyright. the trough. At the same time, the interface can be transferred compounds into surfactant layers (this occurs for cholesterol and onto a solid support (ie, glass or mica) and the microstructures bile acids present into meconium mixture), and (2) the compe- and nanostructures formed by PS at the interface can be eval- tition of surface-active molecules for reaching the interface (this uated at different surface pressures, for example, by epifluo- is the case of serum proteins, especially albumin) (figure 5A). rescence (which can be applied in situ at the trough) or atomic To recreate these conditions, the following experimental strat- 1 force microscopy. This innovation converts the LB trough into egies were proposed12 37 (figure 5B): preincubating samples a highly versatile and effective method, which facilitates the with meconium (1 hour at 37°C, permitting the insertion of its understanding of the molecular mechanisms of surfactant and its components into surfactant membranes) before testing surfac- structural organisation. Nevertheless, this device exhibits several tant activity, or (2) injecting human/animal serum into the bubble drawbacks for testing PS activity, namely the relatively slow speed (CBS) or at the air-liquid interface (Wilhelmy and LB troughs), of dynamic cycles compared with physiological breathing rates, waiting a few minutes to allow for protein adsorption. After this, the large amount of material needed for each replicate and the the sample of interest is applied without touching the interface. possible sample leakage at the trough/barrier junction. Problems This subtlety avoids perturbations to the interfacial barrier of with leakage are reduced considerably by using a balance, which serum proteins. Hence, the surfactant capability to displace ‘this is specifically designed, with a special continuous Teflon ribbon obstacle’ during approximation to the air-liquid interface can be http://fn.bmj.com/ barrier that encloses the whole area, which can be reduced or evaluated in detail. As for CSD, the set-up can also be modified 34 enlarged, and has no junctions. with an additional capillary for introducing inhibiting molecules into the water reservoir.38 Simultaneously, aerosol particles can Differential scanning calorimetry be continuously introduced into the chamber, which increases PLs and lipid-protein membranes, such as those taking part the versatility of the device.39 The aforementioned models can in PS complexes, may exhibit different molecular organisation be easily applied to study sensibility/resistance of therapeutic on 17 December 2018 by guest. Protected or phases as a function of temperature. Typically, membranes surfactants to inactivation by serum or meconium. Similarly, the and interfacial films have a high degree of order at low tempera- prevention/revertion of inhibition by several compounds such ture, with limited rotational and translational molecular diffu- as hyaluronic acid37 may be investigated along with the capa- sion. Conversely, at temperatures above a defined threshold, bility of clinical surfactants (either natural or synthetic) to act the so-called melting temperature (Tm), lipid molecules gain as carriers for drug delivery. With this objective, a novel set-up mobility and become disordered (figure 4B). This reversible based on the use of Wilhelmy troughs was designed, permitting transition and the energy related to it (enthalpy of the system) the recreation in vitro of conditions as close as possible to the depend mainly on changes in Van der Waals which, in turn, diffusion of surfactant with or without carrying drugs, allergens are determined by the length of the acyl chains and the degree or particles, over the respiratory surface (figure 5C).40 Briefly, an of unsaturation as well as by the protein-lipid interactions. Addi- additional trough and a second sensor are added to the device tionally, sterols (such as cholesterol) modulate the system organ- along with a paper bridge that connects the interface of the two isation, intercalating between PLs in the gel phase, making them troughs. Surfactant can be injected into one of the troughs (the gain mobility and packaging them tighter in the liquid phase. donor) that would somehow mimic the upper airways at the

Autilio C, Pérez-Gil J. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F9. doi:10.1136/archdischild-2018-315413 F7 Arch Dis Child Fetal Neonatal Ed: first published as 10.1136/archdischild-2018-315413 on 14 December 2018. Downloaded from Review moment of receiving a dose of exogenous surfactant or a surfac- Funding Research in the laboratory of the authors is currently funded by grants tant/drug combination. After several minutes, the spreading of from the Spanish Ministry of Science and Innovation (BIO2015-67930-R) and the Regional Government of Madrid (P2013/MIT-2807). material can be evaluated by an increase in surface pressure also in the second trough (the recipient), reflecting the interfacial trip Competing interests None declared. of surfactant towards distal alveoli. Patient consent for publication Not required. As expected, the possibility to analyse human samples may Provenance and peer review Commissioned; externally peer reviewed. open interesting horizons, yet different drawbacks must be considered.41 Surfactant of human bronchoalveolar lavages (BAL) is usually collected from the upper airways and only References partially reflects the interfacial properties of the whole lungs. 1 Parra E, Pérez-Gil J. Composition, structure and mechanical properties define performance of pulmonary surfactant membranes and films. Chem Phys Lipids The collected material has been already used at the air-liquid 2015;185:153–75. interface with a subsequent lower activity compared with mate- 2 Perez-Gil J, Weaver TE. Pulmonary surfactant pathophysiology: models and rials obtained from animal models, where whole lung lavage is open questions. Physiology 2010;25:132–41. accessible. Additionally, apart from PAP, the amount of recov- 3 Kuroki Y, Akino T. Pulmonary surfactant protein A (SP-A) specifically binds dipalmitoylphosphatidylcholine. J Biol Chem 1991;266:3068–73. ered surfactant is quite low, making it difficult to replicate 4 Singh N, Halliday HL, Stevens TP, et al. Comparison of animal-derived surfactants for experiments, manipulate samples and to apply certain mate- the prevention and treatment of respiratory distress syndrome in preterm infants. rial-demanding techniques. A last caveat is that the composi- Cochrane Database Syst Rev 2015:Cd010249. tion and concentration of the inhibitory environment affecting 5 Cavicchioli P, Zimmermann LJ, Cogo PE, et al. Endogenous surfactant turnover in preterm infants with respiratory distress syndrome studied with stable isotope lipids. surfactant in vivo, including the possible presence of infiltrated Am J Respir Crit Care Med 2001;163:55–60. immune/inflammatory cells and their secretions, can only be 6 Echaide M, Autilio C, Arroyo R, et al. Restoring pulmonary surfactant membranes and recuperated (to be recreated ex vivo) partially. films at the respiratory surface. Biochim Biophys Acta Biomembr 2017;1859:1725–39. In spite of the limitations described above, several confir- 7 Lopez-Rodriguez E, Gay-Jordi G, Mucci A, et al. Lung surfactant : early in life, early in disease and target in cell therapy. Cell Tissue Res 2017;367:721–35. mations have already proven the huge potential of biophysics 8 Touqui L, Arbibe L. A role for phospholipase A2 in ARDS pathogenesis. Mol Med Today to seek effective new strategies useful in clinical practice or 1999;5:244–9. to improve the current ones. Different biological tests have 9 Günther A, Ruppert C, Schmidt R, et al. Surfactant alteration and replacement in acute been proposed to estimate the level of surfactant function in respiratory distress syndrome. Respir Res 2001;2:353–64. 10 De Luca D, van Kaam AH, Tingay DG, et al. The Montreux definition of neonatal ARDS: different lung physiopathological contexts. Determination biological and clinical background behind the description of a new entity. Lancet of the / ratio or of the level of PG, the Respir Med 2017;5:657–66. 11 De Luca D, Minucci A, Tripodi D, et al. Role of distinct phospholipases A2 and their microbubble test or an automated lamellar body count have by copyright. been employed to detect the insurgence of RDS, but these modulators in meconium aspiration syndrome in human neonates. Intensive Care Med 2011;37:1158–65. are not useful to guide the clinical management of patients at 12 Lopez-Rodriguez E, Echaide M, Cruz A, et al. Meconium impairs pulmonary surfactant the delivery room. On the contrary, preliminary results have by a combined action of cholesterol and bile acids. Biophys J 2011;100:646–55. demonstrated the reliability of SAT to predict the need for 13 De Luca D, Minucci A, Zecca E, et al. Bile acids cause secretory phospholipase A2 SRT in preterm babies who failed continuous positive airway activity enhancement, revertible by exogenous surfactant administration. Intensive 30 Care Med 2009;35:321–6. pressure. SAT is a rapid and accurate assay, which provides 14 Desai O, Winkler J, Minasyan M, et al. The role of immune and inflammatory cells in results within the first hours of life. Moreover, it can be easily idiopathic pulmonary fibrosis. Front Med 2018;5:43. employed to test the quality of interfacial material in both 15 Whitsett JA, Wert SE, Weaver TE. Diseases of pulmonary surfactant homeostasis. Annu BALs and amniotic fluids. Although unworkable at the bedside, Rev Pathol 2015;10:371–93. other biophysical techniques have been also applied in clinical 16 Serrano AG, Pérez-Gil J. Protein-lipid interactions and surface activity in the pulmonary surfactant system. Chem Phys Lipids 2006;141:105–18. research by testing human samples. CBS and PBS have been 17 Hobi N, Giolai M, Olmeda B, et al. A small key unlocks a heavy door: The used to evaluate surfactant dysfunction in a variety of condi- essential function of the small hydrophobic proteins SP-B and SP-C to trigger tions such as neonatal42 43 and adult ARDS,44 cystic fibrosis,45 adsorption of pulmonary surfactant . Biochim Biophys Acta PAP46 or asthma.47 CBS was also employed to test BALs of 2016;1863:2124–34. 18 Schürch S, Bachofen H, Possmayer F. Surface activity in situ, in vivo, and in the asphyxiated neonates without lung disease, demonstrating that captive bubble surfactometer. Comp Biochem Physiol A Mol Integr Physiol http://fn.bmj.com/ whole body could be a therapeutic option for 2001;129:195–207. improving surfactant performance.41 19 Lang CJ, Postle AD, Orgeig S, et al. Dipalmitoylphosphatidylcholine is not the major These promising data highlight the need to bridge knowledge surfactant phospholipid species in all . Am J Physiol Regul Integr Comp Physiol 2005;289:R1426–R1439. and expertise gaps between biophysical investigations and clin- 20 Bernhard W, Schmiedl A, Koster G, et al. Developmental changes in rat ical practice. surfactant lipidomics in the context of species variability. Pediatr Pulmonol

2007;42:794–804. on 17 December 2018 by guest. Protected 21 Schürch D, Ospina OL, Cruz A, et al. Combined and independent action of proteins Conclusion SP-B and SP-C in the surface behavior and mechanical stability of pulmonary surfactant films. Biophys J 2010;99:3290–9. Basic research permits to understand normal and disease condi- 22 Bernardino de la Serna J, Vargas R, Picardi V, et al. Segregated ordered lipid tions, but rarely translates this knowledge into effective clinical phases and protein-promoted membrane cohesivity are required for pulmonary applications. For this reason, translational research is essential surfactant films to stabilize and protect the respiratory surface. Faraday Discuss to integrate basic concepts with clinical observations, towards 2013;161:535–48. 23 Rodríguez-Capote K, Manzanares D, Haines T, et al. Reactive oxygen species promoting the conversion from ‘bench to bedside’. In this inactivation of surfactant involves structural and functional alterations to surfactant context, understanding the main biophysical concepts of PS proteins SP-B and SP-C. Biophys J 2006;90:2808–21. both in health and disease is becoming ever more critical to heal 24 Fessler MB, Summer RS. Surfactant lipids at the host-environment interface. metabolic the rift between lung physicians and basic science researchers. sensors, suppressors, and effectors of inflammatory lung disease. Am J Respir Cell Mol A constant feedback should promote and motivate the ongoing Biol 2016;54:624–35. 25 Günther A, Siebert C, Schmidt R, et al. Surfactant alterations in severe pneumonia, investigations for translational science in cross-disciplinary areas acute respiratory distress syndrome, and cardiogenic lung edema. Am J Respir Crit that have been little explored, such as biophysics. Care Med 1996;153:176–84.

F8 Autilio C, Pérez-Gil J. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F9. doi:10.1136/archdischild-2018-315413 Arch Dis Child Fetal Neonatal Ed: first published as 10.1136/archdischild-2018-315413 on 14 December 2018. Downloaded from Review

26 Zuo YY, Veldhuizen RA, Neumann AW, et al. Current perspectives in pulmonary 38 Saad SM, Policova Z, Dang A, et al. A double injection ADSA-CSD methodology surfactant--inhibition, enhancement and evaluation. Biochim Biophys Acta for lung surfactant inhibition and reversal studies. Colloids Surf B Biointerfaces 2008;1778:1947–77. 2009;73:365–75. 27 Clements JA. Surface tension of lung extracts. Proc Soc Exp Biol Med 1957;95:170–2. 39 Sørli JB, Da Silva E, Bäckman P, et al. A proposed in vitro method to assess 28 Ravasio A, Cruz A, Pérez-Gil J, et al. High-throughput evaluation of pulmonary effects of inhaled particles on lung surfactant Function. Am J Respir Cell Mol Biol surfactant adsorption and surface film formation. J Lipid Res 2008;49:2479–88. 2016;54:306–11. 29 De Luca D, Vázquez-Sánchez S, Minucci A, et al. Effect of whole body hypothermia 40 Hidalgo A, Salomone F, Fresno N, et al. Efficient interfacially driven vehiculization of on inflammation and surfactant function in asphyxiated neonates. Eur Respir J corticosteroids by pulmonary surfactant. Langmuir 2017;33:7929–39. 2014;44:1708–10. 41 Autilio C, Echaide M, De Luca D, et al. Controlled hypothermia may improve 30 Autilio C, Echaide M, Benachi A, et al. A noninvasive surfactant adsorption test surfactant function in asphyxiated neonates with or without meconium aspiration predicting the need for surfactant therapy in preterm infants treated with continuous syndrome. PLoS One 2018;13:e0192295. positive airway pressure. J Pediatr 2017;182:66–73. 42 Autilio C, Echaide M, Dell’Orto V, et al. Effect of whole body hypothermia on 31 Adams FH, Enhörning G. Surface properties of lung extracts: I. A dynamic alveolar surfactant function when amniotic fluid is meconium stained. Ther Hypothermia Temp model. Acta Physiol Scand 1966;68:23–7. Manag 2017. 32 Schürch S, Bachofen H, Goerke J, et al. A captive bubble method reproduces the in 43 De Luca D, Lopez-Rodriguez E, Minucci A, et al. Clinical and biological role of situ behavior of lung surfactant monolayers. J Appl Physiol 1989;67:2389–96. secretory phospholipase A2 in acute respiratory distress syndrome infants. Crit Care 33 Yu LM, Lu JJ, Chan YW, et al. Constrained sessile drop as a new configuration 2013;17:R163. to measure low surface tension in lung surfactant systems. J Appl Physiol 44 Seeds MC, Grier BL, Suckling BN, et al. Secretory phospholipase A2-mediated 2004;97:704–15. depletion of phosphatidylglycerol in early acute respiratory distress syndrome. Am J 34 Cruz A, Pérez-Gil J. Langmuir films to determine lateral surface pressure on lipid Med Sci 2012;343:446–51. segregation. Methods Mol Biol 2007;400:439–57. 45 Gunasekara L, Al-Saiedy M, Green F, et al. Pulmonary surfactant dysfunction in 35 Pérez-Gil J. Structure of pulmonary surfactant membranes and films: the role of pediatric cystic fibrosis: Mechanisms and reversal with a lipid-sequestering drug. J proteins and lipid-protein interactions. Biochim Biophys Acta 2008;1778:1676–95. Cyst Fibros 2017;16:565–72. 36 Chiu MH, Prenner EJ. Differential scanning calorimetry: An invaluable tool for a 46 Hamvas A, Nogee LM, White FV, et al. Progressive lung disease and surfactant detailed thermodynamic characterization of macromolecules and their interactions. J dysfunction with a deletion in gene. Am J Respir Cell Mol Biol Pharm Bioallied Sci 2011;3:39. 2004;30:771–6. 37 López-Rodríguez E, Ospina OL, Echaide M, et al. Exposure to polymers reverses 47 Hohlfeld JM, Ahlf K, Enhorning G, et al. Dysfunction of pulmonary surfactant inhibition of pulmonary surfactant by serum, meconium, or cholesterol in the captive in asthmatics after segmental allergen challenge. Am J Respir Crit Care Med bubble surfactometer. Biophys J 2012;103:1451–9. 1999;159:1803–9. by copyright. http://fn.bmj.com/ on 17 December 2018 by guest. Protected

Autilio C, Pérez-Gil J. Arch Dis Child Fetal Neonatal Ed 2018;0:F1–F9. doi:10.1136/archdischild-2018-315413 F9