Effect of hypoxia on lung gene expression and proteomic profile: insights into the pulmonary surfactant response

Exposure of lung to hypoxia has been previously reported to be associated with significant alterations in the protein content of bronchoalveolar lavage (BAL) and lung tissue. In the present work we have used a proteomic approach to describe the changes in protein complement induced by moderate long-term hypoxia (rats exposed to 10% O2 for 72h) in BAL and lung tissue, with a special focus on the proteins associated with pulmonary surfactant, which could indicate adaptation of this system to limited oxygen availability. The analysis of the general proteomic profile indicates a hypoxia-induced increase in proteins associated with inflammation both in lavage and lung tissue. Analysis at mRNA and protein levels revealed no significant changes induced by hypoxia on the content in surfactant proteins or their apparent oligomeric state. In contrast, we detected a hypoxia-induced significant increase in the expression and accumulation of hemoglobin in lung tissue, at both mRNA and protein levels, as well as an accumulation of hemoglobin both in BAL and associated with surface-active membranes of the pulmonary surfactant complex. Evaluation of pulmonary surfactant surface activity from hypoxic rats showed no alterations in its spreading ability, ruling out inhibition by increased levels of serum or inflammatory proteins.Ministerio de Ciencia BIO2012-30733Ministerio de Ciencia CSD2007-00010Gobierno de la Comunidad de Madrid S2009MAT-1507National Institutes of Health NIH HL3478

Lipid–protein and protein–protein interactions in the pulmonary surfactant system and their role in lung homeostasis

Pulmonary surfactant is a lipid/protein complex synthesized by the alveolar epithelium and secreted into the airspaces, where it coats and protects the large respiratory air–liquid interface. Surfactant, assembled as a complex network of membranous structures, integrates elements in charge of reducing surface tension to a minimum along the breathing cycle, thus maintaining a large surface open to gas exchange and also protecting the lung and the body from the entrance of a myriad of potentially pathogenic entities. Different molecules in the surfactant establish a multivalent crosstalk with the epithelium, the immune system and the lung microbiota, constituting a crucial platform to sustain homeostasis, under health and disease. This review summarizes some of the most important molecules and interactions within lung surfactant and how multiple lipid–protein and protein–protein interactions contribute to the proper maintenance of an operative respiratory surface

A small key unlocks a heavy door : the essential function of the small hydrophobic proteins SP-B and SP-C to trigger adsorption of pulmonary surfactant lamellar bodies

The molecular basis involving adsorption of pulmonary surfactant at the respiratory air–liquid interface and the specific roles of the surfactant proteins SP-B and SP-C in this process have not been completely resolved. The reasons might be found in the largely unknown structural assembly in which surfactant lipids and proteins are released from alveolar type II cells, and the difficulties to sample, manipulate and visualize the adsorption of these micron-sized particles at an air–liquid interface under appropriate physiological conditions. Here, we introduce several approaches to overcome these problems. First, by immunofluorescence we could demonstrate the presence of SP-B and SP-C on the surface of exocytosed surfactant particles. Second, by sampling the released particles and probing their adsorptive capacity we could demonstrate a remarkably high rate of interfacial adsorption, whose rate and extent was dramatically affected by treatment with antibodies against SP-B and SP-C. The effect of both antibodies was additive and specific. Third, direct microscopy of an inverted air–liquid interface revealed that the blocking effect is due to a stabilization of the released particles when contacting the air–liquid interface, precluding their transformation and the formation of surface films. We conclude that SP-B and SP-C are acting as essential, preformed molecular keys in the initial stages of surfactant unpacking and surface film formation. We further propose that surfactant activation might be transduced by a conformational change of the surfactant proteins upon contact with surface forces acting on the air–liquid interface

Relaciones estructura-función del sistema surfactante pulmonar: detección de complejos multiproteicos nativos y participación del surfactante en la difusión interfacial de oxígeno

Tesis de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Bioquímica y Biología Molecular I, leída el 7-07-2011El surfactante pulmonar es una mezcla de lípidos y proteínas cuya función principal es disminuir la tensión superficial en la interfase aire-líquido de los alveolos, evitando así su colapso al final de la espiración. En la presente Tesis se han abordado dos objetivos. En primer lugar se ha estudiado la estructura y función de complejos proteicos nativos de surfactante obtenidos a partir de su solubilización con detergentes, prestando especial atención a aquéllos constituidos por la proteína hidrofóbica SP-B. Además se ha generado un modelo teórico de la estructura de dicha proteína basado en la estructura tridimensional conocida de la saposina B. Los resultados obtenidos, así como el modelo propuesto, sugieren una organización supramolecular de la SP-B más compleja que la supuesta hasta el momento. En segundo lugar se ha estudiado el posible papel del surfactante pulmonar en el transporte de oxígeno a través del alveolo, mediante dos aproximaciones distintas. Por un lado se han obtenido cinéticas de difusión de oxígeno a través del surfactante pulmonar y sus fracciones, que sugieren un papel facilitador de la difusión de oxígeno por parte del surfactante pulmonar. Por otro lado se ha estudiado el efecto de la hipoxia en pulmones y surfactante pulmonar de rata. Los resultados han mostrado una composición y función del surfactante pulmonar normal en animales sometidos al 10% de oxígeno durante 72 horas, a pesar del estado inflamatorio del pulmón revelado por experimentos de proteómica. Además, se encontró un aumento en la expresión de hemoglobina en el pulmón y de su presencia asociada al surfactante pulmonar de ratas sometidas a hipoxia.Sección Deptal. de Bioquímica y Biología Molecular (Biológicas)Fac. de Ciencias BiológicasTRUEpu

Relaciones estructura-función del sistema surfactante pulmonar: detección de complejos multiproteicos nativos y participación del surfactante en la difusión interfacial de oxígeno

El surfactante pulmonar es una mezcla de lípidos y proteínas cuya función principal es disminuir la tensión superficial en la interfase aire-líquido de los alveolos, evitando así su colapso al final de la espiración. En la presente Tesis se han abordado dos objetivos. En primer lugar se ha estudiado la estructura y función de complejos proteicos nativos de surfactante obtenidos a partir de su solubilización con detergentes, prestando especial atención a aquéllos constituidos por la proteína hidrofóbica SP-B. Además se ha generado un modelo teórico de la estructura de dicha proteína basado en la estructura tridimensional conocida de la saposina B. Los resultados obtenidos, así como el modelo propuesto, sugieren una organización supramolecular de la SP-B más compleja que la supuesta hasta el momento. En segundo lugar se ha estudiado el posible papel del surfactante pulmonar en el transporte de oxígeno a través del alveolo, mediante dos aproximaciones distintas. Por un lado se han obtenido cinéticas de difusión de oxígeno a través del surfactante pulmonar y sus fracciones, que sugieren un papel facilitador de la difusión de oxígeno por parte del surfactante pulmonar. Por otro lado se ha estudiado el efecto de la hipoxia en pulmones y surfactante pulmonar de rata. Los resultados han mostrado una composición y función del surfactante pulmonar normal en animales sometidos al 10% de oxígeno durante 72 horas, a pesar del estado inflamatorio del pulmón revelado por experimentos de proteómica. Además, se encontró un aumento en la expresión de hemoglobina en el pulmón y de su presencia asociada al surfactante pulmonar de ratas sometidas a hipoxia

Pulmonary surfactant metabolism in the alveolar airspace: Biogenesis,extracellular conversions, recycling

Pulmonary surfactant is a lipid–protein complex that lines and stabilizes the respiratory interface in the alveoli, allowing for gas exchange during the breathing cycle. At the same time, surfactant constitutes the first line of lung defense against pathogens. This review presents an updated view on the processes involved in biogenesis and intracellular processing of newly synthesized and recycled surfactant components, as well as on the extracellular surfactant transformations before and after the formation ofthe surface active film atthe air–water interface. Special attention is paid to the crucial regulation of surfactant homeostasis, because its disruption is associated with several lung pathologies

Pulmonary surfactant layers accelerate O2 diffusion through the air-water interface

AbstractDuring respiration, it is accepted that oxygen diffuses passively from the lung alveolar spaces through the respiratory epithelium until reaching the pulmonary capillaries, where blood is oxygenated. It is also widely assumed that pulmonary surfactant, a lipid–protein complex secreted into alveolar spaces, has a main surface active function, essential to stabilize the air–liquid interface, reducing in this way the work of breathing. The results of the present work show that capillary water layers containing enough density of pulmonary surfactant membranes transport oxygen much faster than a pure water phase or a water layer saturated with purely lipidic membranes. Membranes reconstituted from whole pulmonary surfactant organic extract, containing all the lipids plus the hydrophobic surfactant proteins, permit also very rapid oxygen diffusion, substantially faster than achieved by membranes prepared from the surfactant lipid fraction depleted of proteins. A model is proposed suggesting that protein-promoted membrane networks formed by pulmonary surfactant might have important properties to facilitate oxygenation through the thin water layer covering the respiratory surface

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

Pulmonary surfactant is a lipid-protein complex which coats lung alveoli. It displays the essential function of reducing surface tension at the air-liquid interface, avoiding alveolar collapse during expiration. The optimized biophysical properties of surfactant rely on its defined composition, constituted mainly by phospholipids and tiny amounts of lipid-associated specific proteins. Due to the highly hydrophobic nature of surfactant, organic solvents have been traditionally employed to obtain and characterize surfactant lipids and proteins, very likely leading to disruption of native interactions among its components. In the present work we have addressed the search of native protein complexes in pulmonary surfactant, which could have an essential role in the optimal function of the system. By solubilizing native lipid-protein membranes of surfactant with non-denaturing detergents, and with the use of a two-dimensional electrophoresis strategy, we have been able to detect the presence of supramolecular complexes composed of surfactant proteins SP-A, SP-B and SP-C. Furthermore, by co-immunoprecipitation assays, we have confirmed for the first time the existence of a direct interaction between SP-A and SP-B, an important feature which could explain the known functional cooperation of both proteins in several aspects of surfactant biology