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RESPIRATORY CELL PHYSIOLOGY

FWF project: FWF P17501

1. Zusammenfassung für die Öffentlichkeitsarbeit

Lungenzellen reagieren auf die Nähe und Beschaffenheit von Wasser-Luft-Phasengrenzen mit einer biomechanischen Abschirmung. Diese Zellen, die am Ende der Bronchialaufzweigungen sitzen, sind bemerkenswert: Sie leben in unmittelbarer Nähe zur Luft, und produzieren ein Substanzgemisch das die Oberflächenspannung von Wasser reduziert. Denn diese wäre so hoch dass Alveolen kollabieren, ein durchaus beobachteter Vorfall bei verschiedenen Lungenerkrankungen. Ursache dafür liegt in der Mikrostruktur der luftexponierten Oberflächen: Sie sind stark gekrümmt und von einem dünnen Wasserfilm überzogen. Die von den Zellen produzierte Substanz gehört zur Gruppe der surface active agents, kurz Surfactants. Im Gegensatz zu technischen Pendants (Tenside z.B.) erzeugen sie jedoch einen unlöslichen Festkörper an der Wasseroberfläche, der sich wie eine komprimierte molekulare Feder verhält und der Oberflächenspannung entgegenwirkt. Wir beobachteten, dass die Zellen diese Phasengrenze wahrnehmen: Je näher sie rückt, desto höher steigt der zelluläre Ca^2+--Gehalt. Ca²⁺ Ionen dienen allgemein als Botenstoffe, oder molekulare Schalter. Im konkreten Fall wird dadurch Surfactant freigesetzt, und die Oberflächenspannung sinkt. Damit entfällt dieses Signal und die Zellen werden wieder inaktiv. Wir haben also einen Feedback entdeckt, der aus einer Kraft besteht, die einen biologischen Mechanismus in Gange setzt, der wiederum die physikalisch Umgebung formt. Diese Oberflächenkräfte können u.U. so stark sein, dass sie die Zellen auch zerstören. Wir vermuten, dass Surfactant primär eine Schutzfunktion besaß, dem sekundär andere Funktionen in der Lungenmechanik folgten.

Im vorliegenden Projekt befaßten wir uns hauptsächlich mit einem Teilprozeß, aber einem wichtigen: Wie wird Surfactant aktiviert, denn innerhalb der Zelle ist er noch inaktiv. Um dies zu klären mußten neue Methoden entwickelt werden: Eine so genannte "inverted interface" sowie mehrere Assays, darunter ein "Adsorption Assay" der für industrielle und medizinische Anwendungen von großem Interesse sein könnte. Ein EU-Patent wurde diesbezüglich eingereicht, und führte zu einer engen Zusammenarbeit mit einem Spanischen Biophysika-lischen Institut. Dieser Assay arbeitet nach dem Prinzip, dass Surfactant durch physikalische Prozesse aktiviert wird, ein Phänomen dass wir kurz zuvor durch Mikroskopie entdeckt haben. Und diese Methode erlaubt auch die Untersuchung von Phänomenen die sich unterhalb eines Oberflächenfilms abspielen und bislang kaum untersucht werden konnten: Surfactant bildet nämlich komplexe oberflächenassoziierte Reservoirs mit expandierten und kondensierten Lipiden, die für eine ausreichend rasche Filmadsorption ausschlaggebend sind. Diese Eigenschaft untersuchen zu können ist überaus wichtig, stellt sie doch z.B. einen zusätzlichen Parameter für die Entwicklung und Optimierung neuer Surfactantpräparate dar, als auch einen Parameter auf der Suche nach klinisch relevanten Surfactantinhibitoren.

1. Summary for public relations work

 Pulmonary alevolar cells sense the presence, proximity and the properties of an air-liquid interface and protect the lung from surface forces. These cells, located at the branching endpoints of the conducting airways, are characterized by two major features: They live at an extremely close distance to air, and they produce a substance that dramatically lowers the surface tension of water. This tension is normally so high that it would force the alveoli to collapse, a scenario seen in several lung diseases. The reason for this potential threat originates from the air-exposed surface of each alveolus, which is highly curved and covered by a thin layer of water. The substance now produced by the cells is called surfactant, an acronym of surface active agent. As implicated in it's name, surfactant molecules avidly seek the air-liquid interface to create something like a solid phase on top. This phase behaves like a compressed molecular spring able to counteract the surface forces. In the course of our investigations we found that the alveolar cells sense the presence of the interface. The closer it is, the higher their intracellular amount of free Ca²⁺-ions. Ca²⁺ ions generally serve as cellular messengers, or molecular switches. In the particular case, Ca²⁺ ions trigger the release of surfactant into the extracellular space. There it reduces the surface tension and the signal for the cells to release further amounts of surfactant is abolished. Thus, we observed a feedback loop, consisting of a physical force that initiates a biological process, which, in turn, leads to a change in the physical environment. The surface forces may as be huge as to destroy the cells. Thus, we speculated that surfactant serves as a protective coat, both for the cells and the lung in total, and may even have evolved in this sequence.

In this project, we focused on the processing of surfactant when released from the cells, i.e. how is surfactant turned to be surface active because it is not when still within the cell. These studies necessitated to simulate the alveolar situation, and for this purpose we invented several new approaches: An "inverted interface", a cell assay to measure exocytosis, a surfactant phospholipid assay, and a microplate based adsorption assay. The latter should be highly useful for industrial and medical purposes. A European patent is currently pending, and it brought us to a very close collaboration with leading Spanish biophysicists. The assay works on the principle that surfactant activation is regulated by pure physical forces, a phenomenon which we discovered before by fluorescence microscopy. And the assay also allows to study sub-surface phenomena, an issue which could hardly be investigated so far, but which sheds new light on surfactant functions: Surfactant creates surface-associated reservoirs, complex arrangements of liquid condensed and liquid expanded phases, which feed the surface when it is expanded. This feature is of outstanding interest because it might be a useful additional parameter to optimize the performance of applied surfactant preparations, and a parameter in the search for clinically relevant surfactant inhibitors.

2. Brief Project Report

2.1. Report on the scientific work

2.1.1. Information on the development of the research work

Experimental work was carried out to ~70% by 1 full time employee (PhD position), ~20% by volunteers, and the applicant. ~50% of the initial goals could be achieved, ~20% are ongoing (e.g. protein labeling), ~20% have been started but turned out to be unfeasible (e.g. AFM, EM), and ~10 have not been started (protein identification). On the other hand, new promising directions emerged and were pursued further. Amongst these, one provided the basis of a new grant (FWF P20472).

Scientifically, the overall concept concerned the processing of pulmonary surfactant. The goal was technical in some parts (development of new approaches), and basic science in others (e.g. mechanical aspects of surfactant membranes). In summary, we investigated the properties of surfactant in it's natural form, directly after release from cells, using unprecedented methods that allow to analyze surfactant transformation on air-liquid interfaces at the level of single particles, in real-time, with reporters for lipid expanded and condensed phases, under full control of physiologically important factors like humidity and temperature, and in combination with the analysis of surface tension, surface topography and viscosity (see below).

Between start and end of the project, there were several changes in the direction of the field. Of importance was the development and the rigorous testing of a new fluorescent microplate based adsorption assay allowing a high sample throughput with a minimum of sample volume. The method is quantitative, cheap, robust and particularly suited for screening purposes in pharmacology and medical research and for industrial applications. Another focus, not envisaged during project application, was the interplay between alveolar cells, surfactant and air-liquid interfaces (now supported by FWF P20472). It emerged in the course of the present project and provides, to our opinion, the opening of a new scientific field. As a matter of fact, both goals, though topically related with the originally formulated ones, lead to a change of experimental plans and time tables. However, they did not lead to personal changes, nor did they demand a change in the requested equipment. Other, non-project related topics have also been started, but will only be briefly mentioned and the end of the following chapter.

2.1.2. Most important results and brief description of their significance

Our studies showed that following secretion, surfactant particles (LBPs) maintain much of their packed structure, but ultimately spread into a surface coat upon contact with an air-liquid interface. The process is rapid, self-contained, and terminates when surface tension decreased <30 mN/m, or when the surface is pre-occupied with already deposited material. Therefore, LBPs are able to transfer the lipids/proteins directly into the interface without prior transformations (tubular myelin; enzymatic conversion), hypotheses which had been postulated in the past. Thus, LPBs are surface active, but dynamic film compression and refinement are important additional parameters to reduce surface tension below its equilibrium value. Interfacial conversion is tension-dependent, indicating that the architecture of LBPs may be particularly suited to respond to surface forces. This leads to the hypothesis that local surface tension is auto-regulated, by an interplay between cohesive forces within LBPs (which might be constant) and the radial tension of the interface (which might change during ventilatory excursions). Ref. 1.

Surface tension of the inverted air-liquid interface could be assessed by analyzing the pattern of backreflected light upon epiillumination. The principle behind this technique is the fact that the radius of curvature of a fluid meniscus is related to surface tension. The technique requires a surface area of 0,07 mm² only and works non-invasively. Thus, it is particularly suited to be combined with time-resolved imaging of interfacial processes of small samples, as it is the case for LBPs. The impact of this new method is described below.

LBPs stably associate with the interface at low surface tensions. This leads to compact surface-associated reservoirs and explains the close apposition of LBPs to the interface found in vivo. This observation also argues against a pure monolayer situation in surfactant structure. Furthermore, LBPs formed multilayered membranes, with complex 3-D topographies. Most remarkably, this was not seen in clinically (e.g. Curosurf) or native surfactants. Surface topographies were highly sensitive with regard to temperature and humidity. Finally, multilayer membrane formation could be detected directly (e.g. by bright field), or indirectly by a progressive "stiffening" and a slowing-down of embedded beads, or by confocal FRAP. Interestingly, this membrane showed some resistance to mechanical agitation, fostering the idea that in small dimensions, surfactant has to be considered as a solid state structure. In our opinion, this supports an old concept (geodesic dome model), proposed in the 50ies but largely neglected in the literature. Ref. 5.

At an air-liquid interface, LBPs undergo a phase separation between ordered and disordered states, and both coexist for long times. Within the cell, phase separation was not evident. Phase separation (or lipid-reorganization) is an important aspect in lipid polymorphism, and particularly relevant in cell biology. In contrast to e.g. the raft theory, phase separation in pulmonary surfactant is thought to be driven by specific proteins, notably SP-C. For pulmonary function, it is essential to know how the basic mechanisms work to get the lipids and proteins organized to create a functional film. In a sense, we observed a self-organization principle that could be highly relevant for other biological disciplines.

Adsorption processes can be analyzed by fluorescence microscopy. However, quantitative studies, e.g. those used for the assessment of new therapeutic products, would greatly benefit from assay systems allowing a routine evaluation of surfactant performance independent onlabor intense, costly and technically demanding procedures. An outcome of this project was a method to measure surfactant adsorption kinetics into interfacial air-liquid interfaces based on a microplate reader. The method has been tested and validated by using surfactants of different origins, surfactant inhibitors, and comparisons with Wilhelmy balance measurements. The method is suited to be implemented in high throughput screening routines for conditions affecting, or improving, surfactant film formation. Furthermore, this method gives a direct read out of the amount of surfactant adsorbing into the interface, including the functionally important amount of material firmly associating with the interfacial film. Ref. 3.

An ambitious goal was devoted to cell-air interactions. We found that AT II contacting an air-liquid interface deform it by several nm followed by rupture of the cell membrane (measured by interferometry). In contrast, cells survived if the interface was conditioned to near physiological situations, i.e. high relative humidity and/or preexisting surfactant films. Nevertheless, cells still responded to the change in interface conditions by a sustained rise in [Ca²⁺]_i. By micromanipulation techniques, we found that this increase in [Ca²⁺]_i was a graded response correlating with the proximity of the interface. Furthermore, we showed that this stimulus resulted in increased surfactant release. When cells were cultured at the interface overnight, they produced an ECM with liquid expanded and condensed phases. Phase coexistence and multilayer domains resulted in highly structured and dynamic topographies, similarly to that seen after adsorption of isolated LBPs. These observations have several implications, and allow to formulate several new hypotheses: 1) AT II cells sense the presence, proximity or property of an air-liquid interface. The nature of the sensory mechanism is still elusive, but may be related with plasma membrane structures and/or mechanosensing ion-channels. 2) Surface forces might be a threat and a stimulus to regulate local surface tensions in the lung, acting on the cells and the released particles. 3) The surfactant system might have evolved to protect cells against the harmful nature of surface forces, a pre-adaptation for it's later role in lung mechanics. This aspect is not mentioned in the literature despite conclusive evidences in comparative physiology. 4) The sensory function might be relevant for other regulatory systems, e.g. regulation of alveolar fluid balance. Refs. 8,9.

VEGF(vascular endothelial growth factor)-, LPS (Lipopolysaccharide)- and PFC (perfluorocarbon)-effects on alveolar cells were parallel studies in collaboration with the Dept. of Pediatrics (Innsbruck) and CNRS (Paris). They emerged during the project, but are not directly linked with it. However, they should be mentioned because techniques developed in this project turned out to be indispensable for these investigation: The study of VEGF- and LPS-effects would have been impracticable without the new assays for exocytosis and phospholipids (see below). For the study of PFC-effects, we used a technique to precisely position and manipulate phase transitions (either liquid/liquid or gas/liquid) at sub-µm distances to living cells. Also this technique was developed within this project. Direct effects of PFC on cells are still in question. The results obtained by our approach already point to the requirement of a careful re-evaluation of the supposed actions of PFCs and their suitability in liquid ventilation. Refs. 4,7,10.

Methods: We invented a non-invasive method to measure surface tension by simple microscopy (Ref. 1). The principle was patented (AT500215), and it's novelty and significance appreciated in Biophotonics Int. (August 2005, 58-59), and in a review on methods in surfactant research (Chem. Phys. Lipids; 141: 105-118; 2006). The principle is also used in optical physics (e.g. Optics Express 14:6342-6352; 2006), and implemented into a humidity, air and temperature controllable (physiological-like) microscopic stage (Ref. 5). Another new method is a fluorescent microplate assay for exocytosis in alveolar type II cells (Ref. 2). Since this method is suited to reduce the amount of animal testing, it is routinely used in our and other labs (Dept. Physiology,Ulm; ISPRA) and recognized by companies (e.g. Across Barriers, Saarbrücken). Furthermore, an alternative to radioactive choline measurements was developed and will be published soon (Ref. 4). A fourth new method is the microplate based adsorption assay (Ref. 3). Currently, we have a EU-wide patent pending with collaborators in Madrid (Profs. Perez-Gil and Crux). This method has the potential of being of considerable interest for pharmaceutical companies and medical/clinical departments: It allows large scale screening of surfactant performance, important in surfactant improvement and quality control, exploration of inhibitors/activators and medical diagnosis. Furthermore, the method may open a new field in surfactant research as it allows dynamic quantitation of surface-associated reservoirs, an issue which could not be easily measured so far.

Relevance for other areas of science: The principle of the inverted interface was of considerable usefulness in pure and applied physical-optical research (e.g. development of optical traps and holographic optical tweezers by Prof. M. Ritsch-Marte, Innsbruck). The microplate based adsorption assay may have implications in surface chemistry, surface biophysics and lipid research.

2.1.3. Information on the running of the project and the use of the available funding

Project duration was 3 years. Beside the costs for personnel, it was funded with ~€ 17.200/p.a. Almost half of it (~40%) was devoted to chemicals, consumables and cell preparation, ~43% haven been used for equipment, and ~9% for travel expenses including congress fees. Consumables constituted the main part, largely due to the costs for the animals, animal transport and housing, cell preparations and cell culture. The funding was sufficient to keep our intended experimental plan of approx. <1 preparation/week. Acquisition of equipment changed: Instead of a CASY cell counter (~€19.500) we purchased a MultiSpec Microimager (~€15.000). The change was necessary because the Microimager was highly needed whereas the cell counter turned out to be of less importance. Other parts were a temperature-controlled microscopic stage and smaller equipments. Travel expenses, which mainly consisted of 2 congress participations (Biophys. Soc.; Salt Lake City and Baltimore) for 2 persons (applicant and the PhD), in addition to an incipient cooperation with Universidad Complutense (Madrid), exceeded the amount that has been estimated in the proposal. On the other hand, the sought collaborations with J. Geibel (New Haven) and H. Oberleithner (Münster) have been postponed due to the inherent difficulty of using the AFM for inspecting inverted air-liquid interfaces. The use of personnel costs was according to the grant proposal, with 1 PhD for 3 years. A small contract for work and labor has been assigned for 1 month (€400).

2.2. Personnel development – importance of the project for the scientific careers of those involved

A. Ravasio held a 3-years full-time PhD position and was responsible for most of the experimental work. He was trained to fully cope with scientific tasks, including formulation of own and meaningful scientific concepts, design, conduction, and interpretation of experimental work, and communication of results by invited lectures, poster presentations, or as corresponding author of manuscripts. Mr. Ravasio still needs to complete the experimental work to apply for a PhD degree this year. Beside his doctoral thesis, the project will be of great importance for his career development because he got in personal contact with national/international scientists who already expressed their interest to offer him a PostDoc position (unfortunately, Mag. Ravasio is not able to be paid by the FWF further; he will have a contract for an additional 9 month in FWF P20472, where he is the co-applicant).

The project also provided the opportunity for volunteers to start with, or to complete their education (P. Schullian, MD; C. Bertocchi, PhD), and for visiting scientists to get familiar with our methods (Mrs. N. Ivanova, Ulm; Mrs. A. Gonzalez-Serrano, B. Olmeda, E. Jimenez and L. Zarapusch, Madrid; Mrs. M. Mshvildadze, Tbilisi).

The project established and intensified international collaborations and transdisciplinary research. Of great importance is our collaboration with Profs. J. Perez-Gil and A. Crux from Universidad Complutense, Madrid. At present, we have a joint publication (Ref. 3), and another is considered for a high ranked journal this year. Together, our groups share an EU patent concerning the above mentioned adsorption assay. Another close collaboration emerged with I. Garcia-Verdugo and Prof. Kanellopoulos from CNRS Paris. A joint publication (Ref. 4) is on the way. The collaboration with Prof. P. Dietl, Ulm, could be further intensified, and a collaboration related with translational research (PFC-effects) started with Prof. M. Rüdiger und Dr. A. Wemhöner from Dept. of Pediatrics, Innsbruck (now Dept. Pediatrics, Leipzig).

For the applicant, the project was of considerable importance because it's outcome constituted the basis for a further FWF granted project (P20472), and it provided the basis for the foundation, consolidation, and now the continuation of our research group 'respiratory cell physiology' within the Dept. of Physiology, which would have been hardly possible without it.

2.3. Effects of the project outside the scientific field

Supplementation of the lungs of premature babies with an exogenous surfactant preparation has decreased the mortality and morbidity associated with the development of Infant Respiratory Distress Syndrome considerably. Most clinical surfactants currently in use, however, consist of relatively crude extracts from animal sources and several attempts have been made towards the development of new, entirely synthetic human-like therapeutic preparations. On the other hand, clinical surfactants available at present have proven not to be effective enough to ameliorate patients with Acute Respiratory Distress Syndrome, the major cause of mortality in critical care units. Both the detailed evaluation of pathological factors affecting pulmonary surfactant function and the development of new improved clinical surfactants are critically dependent on the availability of reliable methods to qualitatively and quantitatively estimate surfactant adsorption and surface film formation. At present, these experiments are labor intense, costly and technically demanding procedures. Therefore, our pending patent on a microplate based adsorption assay, allowing high throughput evaluation of surfactant adsorption and surface film formation, should be of considerable interest even outside the scientific field, e.g. for pharmaceutical companies.

The project also had some (minor) implications for teaching. The applicant, and the project collaborator A. Ravasio, frequently addressed some issues from the project in the frame of seminars held in our Dept. as well as in main and specialized physiology lectures.