Lung Surfactant Phospholipids Research Articles

Evolution of interfacial mechanics of lung surfactant mimics progression of acute respiratory distress syndrome.

How acute respiratory distress syndrome progresses from underlying disease or trauma is poorly understood, and there are no generally accepted treatments resulting in a 40% mortality rate. However, during the inflammation that accompanies this disease, the phospholipase A2 concentration increases in the alveolar fluids leading to the hydrolysis of bacterial, viral, and lung surfactant phospholipids into soluble lysolipids. We show that if the lysolipid concentration in the subphase reaches or exceeds its critical micelle concentration, the surface tension, γ, of dipalmitoyl phosphatidylcholine (DPPC) or Curosurf monolayers increases and the dilatational modulus, [Formula: see text], decreases to that of a pure lysolipid interface. This is consistent with DPPC being solubilized in lysolipid micelles and being replaced by lysolipid at the interface. These changes lead to [Formula: see text] which is the criterion for the Laplace instability that can lead to mechanical instabilities during lung inflation, potentially causing alveolar collapse. These findings provide a mechanism behind the alveolar collapse and uneven lung inflation during ARDS.

Interactions between inhalable aged microplastics and lung surfactant: Potential pulmonary health risks

The relationship between microplastics (MPs) and human respiratory health has garnered significant attention since inhalation constitutes the primary pathway for atmospheric MP exposure. While recent studies have revealed respiratory risks associated with MPs, virgin MPs used as plastic surrogates in these experiments did not represent the MPs that occur naturally and that undergo aging effects. Thus, the effects of aged MPs on respiratory health remain unknown. We herein analyzed the interaction between inhalable aged MPs with lung surfactant (LS) extracted from porcine lungs vis-à-vis interfacial chemistry employing in-vitro experiments, and explored oxidative damage induced by aged MPs in simulated lung fluid (SLF) and the underlying mechanisms of action. Our results showed that aged MPs significantly increased the surface tension of the LS, accompanied by a diminution in its foaming ability. The stronger adsorptive capacity of the aged MPs toward the phospholipids of LS appeared to produce increased surface tension, while the change in foaming ability might have resulted from a variation in the protein secondary structure and the adsorption of proteins onto MPs. The adsorption of phospholipid and protein components then led to the aggregation of MPs in SLF, where the aged MPs exhibited smaller hydrodynamic diameters in comparison with the unaged MPs, likely interacting with biomolecules in bodily fluids to exacerbate health hazards. Persistent free radicals were also formed on aged MPs, inducing the formation of reactive oxygen species such as superoxide radicals (O2•–), hydrogen peroxide (HOOH), and hydroxyl radicals (•OH); this would lead to LS lipid peroxidation and protein damage and increase the risk of respiratory disease. Our investigation was the first-ever to reveal a potential toxic effect of aged MPs and their actions on the human respiratory system, of great significance in understanding the risk of inhaled MPs on lung health.

Comparison of survival of preterm newborn rabbits at 25-28 days of gestation with perinatal therapies at birth transition.

Eligibility of ventilated preterm rabbit model to investigate extreme pulmonary immaturity at birth transition is unknown. By extending this model to early saccular stage of fetal lung development, we evaluated efficacy in survival, lung maturation, and underlying mechanisms of contemporary perinatal therapies. Pregnant New Zealand White rabbit does were given dexamethasone (DEX), or sham injection as control (NDEX), 48 and 24 h before delivery at gestational age (GA) of 25-28 days. At birth, newborn rabbits were anesthetized and randomly allocated to four groups receiving either surfactant or nonsurfactant for both DEX and NDEX, and mechanically ventilated within low tidal volumes. Ranges of time to maintain survival rate ≥ 50% in GA 25-28 days were 59-136, 138-259, 173-288, and 437 to ≥600 min, respectively, each across the four groups. The benefits of DEX and/or surfactant for survival were more obvious in GA 25-26 days, as judged by improved lung mechanics, lower lung injury scores, higher lung surfactant phospholipid pools, and surfactant protein mRNA expression, with DEX-surfactant combination being the most optimal for the outcome. In contrast, those of GA 27-28 days had variable but meaningful responses to the treatment. Cox regression analysis revealed GA, DEX, and surfactant being independently protective factors whereas pneumothorax was a risk factor. The extremely preterm rabbits at GA 25-26 days markedly responded to the perinatal therapies for longer survival, lung maturation and injury alleviation, and were relevant for study of preterm birth transition-associated morbidities and underlying mechanisms.NEW & NOTEWORTHY An extremely preterm rabbit model with gestational age of 25-26 (term 31) days was established by mechanical ventilation with individually adjusted tidal volume at lower ranges. The administration of antenatal glucocorticoids and/or postnatal surfactant achieved significantly longer duration to maintain 50% survival and facilitated lung maturation and protection at early saccular stage. The usefulness of this model should be validated in future investigation of perinatal and neonatal morbidity and mortality at extremely preterm birth transition.

Encapsulation of a CpG oligonucleotide in cationic liposomes enhances its local antitumor activity following pulmonary delivery in a murine model of metastatic lung cancer

Immunotherapy brings new hope to the fight against lung cancer. General immunostimulatory agents represent an immunotherapy strategy that has demonstrated efficacy with limited toxicity when delivered intratumorally. The goal of this study was to enhance the antitumor efficacy of unmethylated oligodeoxynucleotides containing CpG motifs (CpG) and polyinosinic-polycytidylic acid (poly I:C) double-stranded RNA following their local delivery in lung cancer by encapsulating them in liposomes. Liposomes encapsulation of nucleic acids could increase their uptake by lung phagocytes and thereby the activation of toll-like receptors within endosomes. Liposomes were prepared using a cationic lipid, dioleoyltrimethylammoniumpropane (DOTAP), and dipalmitoylphosphatidylcholine (DPPC), the main phospholipid in lung surfactant. The liposomes permanently entrapped CpG but could not efficiently withhold poly I:C. Both poly I:C and CpG delayed tumor growth in the murine B16F10 model of metastatic lung cancer. However, only CpG increased IFN-γ levels in the lungs. Pulmonary administration of CpG was superior to its intraperitoneal injection to slow the growth of lung metastases and to induce the production of granzyme B, a pro-apoptotic protein, and IFNγ, MIG and RANTES, T helper type 1 cytokines and chemokines, in the lungs. These antitumor activities of CpG were strongly enhanced by CpG encapsulation in DOTAP/DPPC liposomes. Delivery of low CpG doses to the lungs induced increased inflammation markers in the airspaces but the inflammation did not reach the systemic compartment in a significant manner. These data support the use of a delivery carrier to strengthen CpG antitumor activity following its pulmonary delivery in lung cancer.

Molecular dynamics exploring of atmosphere components interacting with lung surfactant phospholipid bilayers

Sulfur dioxide (SO2), nitrogen oxide (NO2) and ozone (O3) in the atmosphere are significantly correlated with various respiratory and cardiovascular diseases. High doses of each of these gases or a mixture can change the physical and chemical properties of the lung membrane, thus leading to an increased pulmonary vascular permeability and structural failure of the alveolar cell membrane. In the present study, detailed molecular dynamic (MD) modeling was applied to investigate the effects of SO2, NO2, O3 and mixtures of these gases on the dipalmitoyl phosphatidylcholine (DPPC) phospholipid bilayer. The results showed that several key physical properties, including the mass density, lipid ordering parameter, lipid diffusion, and electrostatic potential of the cell membrane, have been changed by the binding of different compounds. This resulted in significant variations and more disorder in the DPPC bilayer. The multiple analyses of membrane properties proved the toxicity of NO2, O3, and SO2 to the DPPC bilayer, providing a theoretical basis for the experimental phenomenon that SO2, NO2 and O3 can cause lung cell apoptosis. For the single systems, the damage to DPPC bilayer caused by O3 was more serious than NO2 and SO2. More importantly, the MD simulations using the mixtures of SO2, NO2, and O3 showed a much greater decline of membrane fluidity and the aggravation of membrane damage than the single systems, indicating a synergistic effect when NO2, SO2, and O3 coexisted in the atmosphere, which could lead to much more severe damage and greater toxicities to the lung.

Cationic Nanoliposomes Are Efficiently Taken up by Alveolar Macrophages but Have Little Access to Dendritic Cells and Interstitial Macrophages in the Normal and CpG-Stimulated Lungs.

The purpose of this study was to assess whether cationic nanoliposomes could address tumor vaccines to dendritic cells in the lungs in vivo. Nanoliposomes were prepared using a cationic lipid, dimethylaminoethanecarbamoyl-cholesterol (DC-cholesterol) or dioleoyltrimethylammoniumpropane (DOTAP), and dipalmitoylphosphatidylcholine (DPPC), the most abundant phospholipid in lung surfactant. The liposomes presented a size below 175 nm and they effectively entrapped tumor antigens, an oligodeoxynucletotide containing CpG motifs (CpG) and the fluorescent dye calcein used as a tracer. Although the liposomes could permanently entrap a large fraction of the actives, they could not sustain their release in vitro. Liposomes made of DOTAP were safe to respiratory cells in vitro, while liposomes composed of DC-cholesterol were cytotoxic. DOTAP nanoliposomes were mainly taken up by alveolar macrophages following delivery to the lungs in mice. Few dendritic cells took up the liposomes, and interstitial macrophages did not take up liposomal calcein more than they took up soluble calcein. Stimulation of the innate immune system using liposomal CpG strongly enhanced uptake of calcein liposomes by all phagocytes in the lungs. Although a small percentage of dendritic cells took up the nanoliposomes, alveolar macrophages represented a major barrier to dendritic cell access in the lungs.

The phospholipase A2 activity of peroxiredoxin 6 [S

Peroxiredoxin 6 (Prdx6) is a Ca2+-independent intracellular phospholipase A2 (called aiPLA2) that is localized to cytosol, lysosomes, and lysosomal-related organelles. Activity is minimal at cytosolic pH but is increased significantly with enzyme phosphorylation, at acidic pH, and in the presence of oxidized phospholipid substrate; maximal activity with phosphorylated aiPLA2 is ∼2 µmol/min/mg protein. Prdx6 is a "moonlighting" protein that also expresses glutathione peroxidase and lysophosphatidylcholine acyl transferase activities. The catalytic site for aiPLA2 activity is an S32-H26-D140 triad; S32-H26 is also the phospholipid binding site. Activity is inhibited by a serine "protease" inhibitor (diethyl p-nitrophenyl phosphate), an analog of the PLA2 transition state [1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol (MJ33)], and by two naturally occurring proteins (surfactant protein A and p67phox), but not by bromoenol lactone. aiPLA2 activity has important physiological roles in the turnover (synthesis and degradation) of lung surfactant phospholipids, in the repair of peroxidized cell membranes, and in the activation of NADPH oxidase type 2 (NOX2). The enzyme has been implicated in acute lung injury, carcinogenesis, neurodegenerative diseases, diabetes, male infertility, and sundry other conditions, although its specific roles have not been well defined. Protein mutations and animal models are now available to further investigate the roles of Prdx6-aiPLA2 activity in normal and pathological physiology.

Protective effect of baicalin on fetal lung development in a rabbit model of congenital diaphragmatic hernia.

Bacalin has been reported to improve fetal lung growth by increasing fetal lung surfactant phospholipids. Therefore, the present study evaluated the effect of transplacental treatment of baicalin during lung development in rabbits with congenital diaphragmatic hernia (DH) rabbit. DH was induced in fetal rabbit on gestational day 23 and baicalin injections were administered daily until term period. Fetal survival rate, body weight and lung-to-body weight ratio (LBWR) were subsequently measured, and the expression of surfactant protein B (SPB) and Ki-67, and morphometry analysis was determined in lung tissues. It was observed that in baicalin treated group the fetal survival rate and LBWR were decreased compared with the control without DH group. Lung morphometry results suggested that treatment with baicalin did not significantly ameliorate congenital DH, and adventitial and medial thickness were similar to those in the control groups, and less muscularization of vessels measuring 30–60 µm. Furthermore, baicalin treatment did not markedly affect the expressions of Ki-67 and SPB in fetuses with DH. Baicalin was demonstrated to improve the morphology of lung in rabbits; however, as it did not induce marked airway changes this present study suggests that baicalin is not suitable for the management of congenital DH.

Mutation of Serine 32 to Threonine in Peroxiredoxin 6 Preserves Its Structure and Enzymatic Function but Abolishes Its Trafficking to Lamellar Bodies

Peroxiredoxin 6 (Prdx6), a bifunctional protein with phospholipase A2 (aiPLA2) and GSH peroxidase activities, protects lungs from oxidative stress and participates in lung surfactant phospholipid turnover. Prdx6 has been localized to both cytosol and lamellar bodies (LB) in lung epithelium, and its organellar targeting sequence has been identified. We propose that Prdx6 LB targeting facilitates its role in the metabolism of lung surfactant phosphatidylcholine (PC). Ser-32 has been identified as the active site in Prdx6 for aiPLA2 activity, and this activity was abolished by the mutation of serine 32 to alanine (S32A). However, aiPLA2 activity was unaffected by mutation of serine 32 in Prdx6 to threonine (S32T). Prdx6 protein expression and aiPLA2 activity were normal in the whole lung of a "knock-in" mouse model carrying an S32T mutation in the Prdx6 gene but were absent from isolated LB. Analyses by proximity ligation assay in lung sections demonstrated the inability of S32T Prdx6 to bind to the chaperone protein, 14-3-3ϵ, that is required for LB targeting. The content of total phospholipid, PC, and disaturated PC in lung tissue homogenate, bronchoalveolar lavage fluid, and lung LB was increased significantly in Prdx6-S32T mutant lungs, whereas degradation of internalized [(3)H]dipalmitoyl-PC was significantly decreased. Thus, Thr can substitute for Ser for the enzymatic activities of Prdx6 but not for its targeting to LB. These results confirm an important role for LB Prdx6 in the degradation and remodeling of lung surfactant phosphatidylcholine.

Serine 32 to threonine mutation preserves peroxiredoxin 6 structure and enzymatic function but abolishes its trafficking to lamellar bodies in vivo

Peroxiredoxin 6 (Prdx6), a bifunctional protein with lysosomal‐type phospholipase A2 (aiPLA2) and GSH peroxidase activities, protects lungs from oxidative stress and participates in lung surfactant phospholipid (PL) turnover. Prdx6 has been localized to both cytosol and acidic organelles (lamellar bodies [LB] and lysosomes) in lung epithelium and its organellar targeting sequence has been identified (31DSWGILFSHP40, Am.J.Physiol.297 : L871, 2009). Interestingly, an S32T mutation fully preserved secondary structure of recombinant protein and had no effect on either its peroxidase or aiPLA2 activity. In a “knock‐in” mouse model carrying the S32T mutation in the Prdx6 gene, Prdx6 protein and aiPLA2 activity were present in lung epithelial cells. However,the protein was absent from epithelial cell LB. Lung surfactant dipalmitoylphosphatidylcholine (DPPC) is endocytosed by alveolar epithelial cells and degraded by aiPLA2. The content of total phospholipid, PC, and disaturated phosphatidylcholine (DSPC) in bronchoalveolar lavage fluid, lung lamellar bodies, and lung homogenate was increased in S32T mutant animals and degradation of internalized [3H]DPPC in isolated mouse lungs was significantly decreased. Thus, Thr can substitute for Ser for the enzymatic activities of Prdx6 but not for its targeting to LB. These results confirm an important role for lamellar body Prdx6 in the degradation and remodeling of lung surfactant phosphatidylcholine. [HL‐019737, HL102016 and HL105509]

Infection Tunes Stability of Key Molecules in Lung Diseases

efficiently fulfill a vast range of life processes. In the processes, proteins are dynamic in responding to the environmental changes. If a given protein is needed, de novo synthesis from mRNA will be augmented. Of note, a less emphasized but probably more efficacious way is to enhance the protein stability through preventing from degradation. If redundant, proteins will be promptly dumped out by degradation. Regulation of protein stability via degradation plays an important role in a vast range of life processes. Most proteins destined for turnover are via ubiquitin proteasomal/lysosomal degradation machinery. Protein turnover is not a randomly happened event but highly regulated and is a substrate-specific signal transduction cascade. Firstly, targeted proteins are posttranslational modified for recognition by the ubiquitin proteasomal/lysosomal degradation machinery. A well-studied prototype is phosphorylation that may directly affects ubiquitin proteasome system recognition of the substrate. Other posttranslational modifications, such as acetylation, methylation, or palmitoylation are important modifications involved in ubiquitination process as well. Secondly, the destined protein is ubiquitinated. Ubiquitination covalently adds a ubiquitin or polyubiquitin moieties to a lysine residue within the protein substrate. The process of ubiquitination is an enzymatic cascade involving E1 ubiquitin activation enzyme, E2 ubiquitin conjugation enzyme, and E3 ubiquitin ligase. Among the ubiquitin enzymes, E3 ubiquitin ligase recognizes the substrate that is substrate specific. Strikingly, the ubiquitin can be removed from the ubiquitinated protein by a family of enzymes called de ubiquitin enzymes. Finally, the ubiquitinated protein is then escorted into proteasome or lysosome for degradation. Bacterial infection fine-turns proteins involving in cell surface sensors, signal mediator proteins, and effect or proteins. For example, endotoxin lipopolysaccharide treatment or bacterial infection regulates IL22R, IL33, kinases, and TRAF proteins in lung epithelial cells. Epigenetic enzymes that control essential life process such as DNA replication and gene transcription are also deregulated in these models. Enzymes involved in active lung surfactant or other phospholipids synthesis, metabolism, and energy production are also regulated in this system. Understanding the molecular mechanism(s) of protein turnover in infectious lung disease has shed light on the development of new pharmaceutical therapeutic approaches. Successful results have been achieved to screen small molecules against novel molecular targets including E3 ubiquitin ligases in infectious lung diseases.

Molecular dynamics of dibenz[a,h]anthracene and its metabolite interacting with lung surfactant phospholipid bilayers.

The interaction of dibenz[a,h]anthracene and its ultimate carcinogenic 3,4-diol-1,2-epoxide with lung surfactant phospholipid bilayers was successfully performed using molecular dynamics. The DPPC/DPPG/cholesterol bilayer (64 : 64 : 2) was used as the lung surfactant phospholipid bilayer model and compared with the DPPC bilayer as a reference. Dibenz[a,h]anthracene and its 3,4-diol-1,2-epoxide were inserted in water and lipid phases in order to investigate their interactions with the lung surfactant phospholipid bilayers. The radial distribution function between two P atoms in polar heads shows that the 3,4-diol-1,2-epoxide affects the order between the P atoms in the DPPC/DPPG/cholesterol model more than dibenz[a,h]anthracene, which is a consequence of its preference for the polar heads and dibenz[a,h]anthracene prefers to be located in the hydrocarbon chain of the phospholipid bilayers. Dibenz[a,h]anthracene and its 3,4-diol-1,2-epoxide may form aggregates in water and lipid phases, and in the water-lipid interface. The implications for the possible effect of dibenz[a,h]anthracene and its 3,4-diol-1,2-epoxide in the lung surfactant phospholipid bilayers are discussed.

Mutation of serine 32 to threonine in peroxiredoxin 6 preserves enzymatic activity but prevents trafficking to lamellar bodies in vivo (950.1)

Peroxiredoxin 6 (Prdx6), a peroxidase that also expresses phospholipase A2 (aiPLA2) activity, protects lungs from oxidative stress and participates in lung surfactant phospholipid (PL) turnover. Prdx6 has been localized to both cytosolic and acidic (lamellar body [LB] and lysosomal) compartments in lung epithelium. We propose that Prdx6 LB targeting facilitates its role in the degradation and remodeling of lung surfactant phosphatidylcholine. Although, we have previously shown that mutation of serine 32 (S32) to alanine abolishes Prdx6 aiPLA2 activity, we now show that serine to threonine mutation has no effect on either Prdx6 peroxidase or aiPLA2 activity. Since S32 is a necessary component of the amino acid motif that mediates Prdx6 LB targeting, we studied the effect of the S32T mutation on Prdx6 LB localization. In a “knock‐in” mouse model carrying an S32T mutation in the Prdx6 gene, there was phospholipid (PL) accumulation in the lung and a marked decrease in aiPLA2 activity in LB. Prdx6 was absent from LB of mutant mice by both western blot and immunofluorescence analysis despite normal protein expression in lung epithelial cells. Thus, the S32T mutation preserves the PLA2 activity of Prdx6 but does not support its targeting to LB, which results in PL accumulation, despite the normal Prdx6 PLA2 activity in lung tissue.Grant Funding Source: HL102016 and HL105509

Does lung development differ in male and female fetuses?

ABSTRACTPreterm male infants have a higher incidence of morbidity and mortality due to respiratory insufficiency than females of the same gestational age. This male disadvantage could be due to differences in lung architecture; however, few studies have compared lung architecture in male and female fetuses during late gestation. Our principal objectives were to compare the morphology of the fetal lung and the maturity of the surfactant system in preterm male and female fetuses. Lungs from male (n = 9) and female (n = 11) fetal sheep were collected at 0.9 of term (131 days of the 145-day gestation) for morphological and molecular analyses. In separate groups, tracheal liquid was obtained from male (n = 9) and female (n = 9) fetuses at 0.9 of term for determination of surfactant phospholipid composition. We found no sex-related differences in body weight, lung weight, right lung volume, lung tissue and airspace fractions, mean linear intercept, septal crest density, septal thickness, the proportion of proliferating and apoptotic cells, and the percentages of collagen or elastin. The gene expression of surfactant protein -A, -B, -C, and -D and tropoelastin was similar between sexes. There were no differences in the proportion of the major phospholipid classes in the tracheal liquid between sexes; however there was a significantly higher percentage of the phospholipid species phosphatidylinositol 38:5 in males. The greater morbidity and mortality in preterm male lambs do not appear to be related to differences in lung structure or surfactant phospholipid synthesis before birth, but may relate to physiological adaptation to air-breathing at birth.

Advanced spray-dried design, physicochemical characterization, and aerosol dispersion performance of vancomycin and clarithromycin multifunctional controlled release particles for targeted respiratory delivery as dry powder inhalation aerosols

Respirable microparticles/nanoparticles of the antibiotics vancomycin (VCM) and clarithromycin (CLM) were successfully designed and developed by novel organic solution advanced spray drying from methanol solution. Formulation optimization was achieved through statistical experimental design of pump feeding rates of 25% (Low P), 50% (Medium P) and 75% (High P). Systematic and comprehensive physicochemical characterization and imaging were carried out using scanning electron microscopy (SEM), hot-stage microscopy (HSM), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), Karl Fischer titration (KFT), laser size diffraction (LSD), gravimetric vapor sorption (GVS), confocal Raman microscopy (CRM) and spectroscopy for chemical imaging mapping. These novel spray-dried (SD) microparticulate/nanoparticulate dry powders displayed excellent aerosol dispersion performance as dry powder inhalers (DPIs) with high values in emitted dose (ED), respirable fraction (RF), and fine particle fraction (FPF). VCM DPIs displayed better aerosol dispersion performance compared to CLM DPIs which was related to differences in the physicochemical and particle properties of VCM and CLM. In addition, organic solution advanced co-spray drying particle engineering design was employed to successfully produce co-spray-dried (co-SD) multifunctional microparticulate/nanoparticulate aerosol powder formulations of VCM and CLM with the essential lung surfactant phospholipid, dipalmitoylphosphatidylcholine (DPPC), for controlled release pulmonary nanomedicine delivery as inhalable dry powder aerosols. Formulation optimization was achieved through statistical experimental design of molar ratios of co-SD VCM:DPPC and co-SD CLM:DPPC. XRPD and DSC confirmed that the phospholipid bilayer structure in the solid-state was preserved following spray drying. Co-SD VCM:DPPC and co-SD CLM:DPPC dry powder aerosols demonstrated controlled release of antibiotic drug that was fitted to various controlled release mathematical fitting models. The Korsmeyer-Peppas model described the best data fit for all powders suggesting super case-II transport mechanism of controlled release. Excellent aerosol dispersion performance for all co-SD microparticulate/nanoparticulate DPIs was higher than the SD antibiotic drugs suggesting that DPPC acts as an aerosol performance enhancer for these antibiotic aerosol dry powders. Co-SD VCM:DPPC DPIs had higher aerosol dispersion parameters compared to co-SD CLM:DPPC which was related to differences in the physicochemical properties of VCM and CLM.

Serine 32 targeted mutation abolishes Prdx6 trafficking to lamellar bodies in vivo

Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A2 (aiPLA2) activities, participates in lung surfactant phospholipid (PL) turnover and protects lung epithelium from oxidative stress. Prdx6 null mice accumulate PL in lung tissue, compatible with the role of PLA2 activity in lung phospholipid turnover. Prdx6 has been localized to cytosolic and acidic compartments (lamellar bodies (LB) and lysosomes) in lung epithelium. Our previous studies indicated that serine at position 32 (S32) is necessary for proper Prdx6 targeting to LB. We have generated a mouse model with an S32T mutation in the Prdx6 LB targeting sequence. Homozygous mice carrying the S32T mutation exhibited phospholipid (PL) accumulation in LB (PL/protein ratio 14.36) vs. WT (PL/protein ratio 9.51). Western blot analysis demonstrated absence of Prdx6 in LBs isolated from S32T mice despite equal amounts of Prdx6 in lung homogenates. Immunofluorescence analysis of mouse lung tissue sections showed co‐localization of Prdx6 with LAMP1, a marker of lysosome‐related organelles, in wild type but not in S32T mouse alveolar type II cells. These findings indicate that S32 is required for Prdx6 targeting to LB in mouse lung epithelium. [HL102016 and HL19737]

Suitability of polymer materials for production of pulmonary microparticles using a PGSS supercritical fluid technique: Thermodynamic behaviour of fatty acids, PEGs and PEG-fatty acids

The thermodynamic behaviour of selected polymeric components for preparation of controlled release microparticles using supercritical carbon dioxide (scCO(2)) processing was investigated. The polymeric materials selected were egg lecithin (a model for the lung surfactant phospholipid), poly(ethyleneglycol) (PEG) of different molecular weights, fatty acids (C18, C16, and C14), and physical blends of PEGs and fatty acids. In addition a range of PEG-stearates was also assessed. Analysis of thermodynamic behaviour was performed by differential scanning calorimetry (DSC) and by assessment of their interaction with scCO(2) in a high-pressure variable volume view cell. The key criterion was to demonstrate a strong interaction with scCO(2) and to show liquefaction of the polymeric material at acceptable processing temperatures and pressures. Positive results should then indicate the suitability of these materials for processing by the Particle from Gas Saturated Solutions (PGSS) technique using scCO(2) to create microparticles for pulmonary administration. It was found that the materials tested interacted with scCO(2) and showed a sufficient lowering of their melting temperature (T(m)) to make them suitable for use in the PGSS microparticle production rig. Fatty acids of low T(m) were shown to act as a plasticising agent and to lower the T(m) of PEG further during interaction with scCO(2).

Negative allometry of docosahexaenoic acid in the fowl lung and pulmonary surfactant phospholipids

In a recent study (Comp. Biochem. Physiol. B. (2010)155: 301-308) we reported that the fatty acids (FA) of the avian (7 species) total lung phospholipids (PL) (i.e. lung parenchyma and surfactant together) provide allometric properties. To test whether this allometric scaling also occurs in either of the above components, in six gallinaceous species, in a body weight range from 150 g (Japanese quail, Coturnix coturnix japonica) to 19 kg (turkey, Meleagris gallopavo) the PL FA composition (mol%) was determined in the pulmonary surfactant, in native and in thoroughly lavaged lungs (referred to as lung parenchyma). In all three components docosahexaenoic acid (DHA) showed significant and negative allometric scaling (B = -0.056, -0.17 and -0.1, respectively). Surfactant PLs provided further negative allometry for palmitic acid and the opposite was found for palmitoleate and arachidonate. In the lung parenchymal PLs increasing body weight was matched with shorter chain FAs (average FA chain length) and competing n6 and n3 end-product fatty acids (positive allometry for arachidonic acid and negative for DHA). Negative allometric scaling was found for the tissue malondialdehyde concentration in the native and lavaged lungs (B = -0.1582 and -0.1594, respectively). In these tissues strong correlation was found between the MDA concentration and DHA proportion (r = 0.439 and 0.679, respectively), denoting the role of DHA in shaping the allometric properties and influencing the extent of in vivo lipid peroxidation of membrane lipids in fowl lungs.

KL4 Peptide Induces Reversible Collapse Structures on Multiple Length Scales in Model Lung Surfactant

We investigated the effects of KL4, a 21-residue amphipathic peptide approximating the overall ratio of positively charged to hydrophobic amino acids in surfactant protein B (SP-B), on the structure and collapse of dipalmitoylphosphatidylcholine and palmitoyl-oleoyl-phosphatidylglycerol monolayers. As reported in prior work on model lung surfactant phospholipid films containing SP-B and SP-B peptides, our experiments show that KL4 improves surfactant film reversibility during repetitive interfacial cycling in association with the formation of reversible collapse structures on multiple length scales. Emphasis is on exploring a general mechanistic connection between peptide-induced nano- and microscale reversible collapse structures (silos and folds).

MALDI imaging MS of phospholipids in the mouse lung

Lipid mediators are important in lung biochemistry and are derived from the enzymatic oxidation of arachidonic and docosahexaenoic acids, which are PUFAs that are present in phospholipids in cell membranes. In this study, MALDI imaging MS was used to determine the localization of arachidonate- and docosahexaenoate-containing phospholipids in mouse lung. These PUFA-containing phospholipids were determined to be uniquely abundant at the lining of small and large airways, which were unequivocally identified by immunohistochemistry. In addition, it was found that the blood vessels present in the lung were characterized by sphingomyelin molecular species, and lung surfactant phospholipids appeared evenly distributed throughout the lung parenchyma, indicating alveolar localization. This technique revealed unexpected high concentrations of arachidonate- and docosahexaenoate-containing phospholipids lining the airways in pulmonary tissue, which could serve as precursors of lipid mediators affecting airways biology.