Surfactant Turnover Research Articles

Structural and Functional Diversity of the Peroxiredoxin 6 Enzyme Family.

Significance: Peroxiredoxins (Prdxs) with a single peroxidative cysteine (CP) in a conserved motif PXXX(T/S)XXCP within its thioredoxin fold, have been classified as the peroxiredoxin 6 (Prdx6 ) family. All Prdxs can reduce H2O2 and short chain hydroperoxides while Prdx6 in addition, can reduce phospholipid hydroperoxides (PLOOH) due to its ability to interact with peroxidized phospholipid substrate. The single CP of Prdx6 uses various external electron donors including glutathione thioredoxin, and ascorbic acid for resolution of its peroxidized state and, therefore, its peroxidase activity. Prdx6 proteins also exhibit Ca2+-independent phospholipase A2 (PLA2), lysophosphatidylcholine acyltransferase (LPCAT), and chaperone activities that depend on cellular localization and the oxidation and oligomerisation states of the protein. Thus, Prdx6 is a "moonlighting" enzyme. Recent Advance: Physiologically, Prdx6s have been reported to play an important role in protection against oxidative stress, repair of peroxidized cell membranes, mammalian lung surfactant turnover, activation of some NADPH oxidases, the regulation of seed germination in plants, as an indicator of cellular levels of reactive O2 species through Nrf-Klf9 activation, and possibly in male fertility, regulation of cell death through ferroptosis, cancer metastasis, and oxidative stress-related signalling pathways. Critical Issues: This review outlines Prdx6 enzyme unique structural features and explores its wide range of physiological functions. Yet, existing structural data falls short of fully revealing all of human Prdx6 multifunctional roles. Further endeavour is required to bridge this gap in its understanding. Although there are wide variations in both the structure and function of Prdx6 family members in various organisms, all Prdx6 proteins show the unique a long C-terminal extension that is also seen in Prdx1, but not in other Prdxs. Future Directions: As research data continues to accumulate, the potential for detailed insights into the role of C-terminal of Prdx6 in its oligomerisation and activities. There is a need for thorough exploration of structural characteristics of the various biological functions. Additionally, uncovering the interacting partners of Prdx6 and understanding its involvement in signalling pathways will significantly contribute to a more profound comprehension of its role.

The impact of bacterial exposure in early life on lung surfactant gene expression, function and respiratory rate in germ-free mice

Early-life changes to lung and gut microbiota have been linked to alterations in immune responses that may lead to pulmonary diseases later in life. Associations between early-life microbiota, germ-free status, lung gene expression, lung development and function are not well described. In this study, we compare early-life lung gene transcription under germ-free and different perinatal microbial exposures, and analyze with a predetermined focus on lung capacity and lung surfactant. We also analyze the later-in-life physiological measures of breathing patterns and lung surfactant function between the germ-free, gnotophoric and gnotobiotic offspring. To achieve this, we kept pregnant BALB/c germ-free mice in separate germ-free isolators until exposure to either A: no exposure (GF), B: Bifidobacterium animalis ssp. Lactis (BI04) or C: full cecum content harvested from other female SPF mice (Cecum). Subsequently, perinatally exposed offspring were used for the analyses. Lung tissue transcriptomics analysis was done at postnatal day 10 (PNday10) at the first phase of lung alveolar development. Head-out plethysmography for breathing pattern analysis was performed on the siblings at PNday23 followed by lung surfactant collection. The function of the collected lung surfactant was then analyzed ex vivo using the constrained drop surfactometer. Our results show that lung transcriptomics had differentially expressed genes related to surfactant turnover between groups and sex at PNday10. They also show that the GF and BI04 animals had lower respiratory rate than Cecum mice, or compared to age-matched specific pathogen-free (SPF) reference animals. We also see changes in lung surfactant function ex vivo. The overall conclusions are that 10-day-old GF mice do not have a markedly different lung gene transcription compared to gnotophoric or gnotobiotic mice, but genes related to surfactant metabolism are among the few differentially expressed genes. We show here for the first time that early-life microbiome status correlates with early-life surfactant-gene transcription and to later-in-life lung surfactant function and associated respiratory-rate changes in mice.

“Milking” the way to diagnosis

A 43-year-old non-smoker man presented to the emergency department with exertional dyspnoea and left pleuritic pain for 3 days. He denied fever or cough. He had history of pulmonary embolism, deep venous thrombosis and multiple opportunistic infections. At presentation, his oxygen saturation was 94% with venturi mask at 31% and lung auscultation showed basal crackles. He had no signs of peripheral oedema or deep venous thrombosis. Inflammatory parameters were within normal range. High-resolution chest computed tomography showed diffuse ground-glass opacities with interlobular and intralobular septal thickening — a pattern described as “crazy paving” (Panel A). Bronchoscopy yielded milky appearing lavage fluid (Panel B) and pathological testing revealed proteinaceous material positive on periodic acid–Schiff staining. These findings confirmed a diagnosis of pulmonary alveolar proteinosis (PAP). He underwent volume lavage of both lungs sequentially. At 6-month follow-up, he still maintained substantial improvement, with oxygen saturation of 94% in ambient air. PAP is a rare pulmonary disease caused by impaired surfactant turnover [1]. Although the crazy paving pattern is often associated with PAP [1], it’s not specific and can be seen in other conditions, including acute respiratory distress syndrome, pulmonary haemorrhage, organizing pneumonia, lipoid pneumonia and infections like Pneumocystis jirovecii [2] and SARS-CoV-2 pneumonia.

Chronic pharmacological antagonism of the GM-CSF receptor in mice does not replicate the pulmonary alveolar proteinosis phenotype but does alter lung surfactant turnover.

Granulocyte macrophage colony stimulating factor (GM-CSF) is a key participant in, and a clinical target for, the treatment of inflammatory diseases including rheumatoid arthritis (RA). Therapeutic inhibition of GM-CSF signalling using monoclonal antibodies to the α-subunit of the GM-CSF receptor (GMCSFRα) has shown clear benefit in patients with RA, giant cell arteritis (GCAs) and some efficacy in severe SARS-CoV-2 infection. However, GM-CSF autoantibodies are associated with the development of pulmonary alveolar proteinosis (PAP), a rare lung disease characterised by alveolar macrophage (AM) dysfunction and the accumulation of surfactant lipids. We assessed how the anti-GMCSFRα approach might impact surfactant turnover in the airway. Female C57BL/6J mice received a mouse-GMCSFRα blocking antibody (CAM-3003) twice per week for up to 24 weeks. A parallel, comparator cohort of the mouse PAP model, GM-CSF receptor β subunit (GMCSFRβ) knock-out (KO), was maintained up to 16 weeks. We assessed lung tissue histopathology alongside lung phosphatidylcholine (PC) metabolism using stable isotope lipidomics. GMCSFRβ KO mice reproduced the histopathological and biochemical features of PAP, accumulating surfactant PC in both broncho-alveolar lavage fluid (BALF) and lavaged lung tissue. The incorporation pattern of methyl-D9-choline showed impaired catabolism and not enhanced synthesis. In contrast, chronic supra-pharmacological CAM-3003 exposure (100 mg/kg) over 24 weeks did not elicit a histopathological PAP phenotype despite some changes in lung PC catabolism. Lack of significant impairment of AM catabolic function supports clinical observations that therapeutic antibodies to this pathway have not been associated with PAP in clinical trials.

Co-aerosolized Pulmonary Surfactant and Ambroxol for COVID-19 ARDS Intervention: What Are We Waiting for?

After more than 225 days of the first reports of the novel coronavirus from China, COVID-19 pandemic is still on surge. The search for an effective and efficient therapeutic and pharmaceutical intervention is as important and urgent now as it was on Day 1. Majority of the efforts in this direction are toward finding small molecule interventions via repurposing or redirecting the therapeutic approaches. This hypothesis proposes a physical intervention approach directed toward rescuing the complex lung pathology observed in COVID-19 related acute respiratory distress syndrome (CARDS). The loss of content as well as the synthesis and turnover of the surfactant in ARDS has been termed as a “collateral damage.” A synergistic, early stage, cost-effective, pharmaceutically viable, safe, and immediately available solution is hence required. The effectiveness of exogenous surfactant treatment in ARDS has been marred with several limitations as pointed out in various clinical trials and require revised protocols related to surfactant dose and mode of delivery. This hypothesis proposes aerosolized surfactant delivery taking the optimal dosing and coating costs into account along with co-delivery of ambroxol to provide synergistic benefits. Ambroxol is reported to have anti-inflammatory, -oxidant, -viral, and -bacterial activities and has a direct impact on the production and secretion of the surfactant from the alveolar Type 2 cells. If aerosolized, atomized, or nebulized in the form of ambroxol-loaded phospholipid nanovesicles at the early stages of ARDS, depleted surfactant levels may be reinstated and surfactant turnover can be initiated and maintained. The ability to deliver both the components in aerosolized-nebulized form may have a huge impact on alleviating the healthcare burden in low resource settings where the availability of ventilators is limited. In conclusion, the surfactant-ambroxol co-aerosolized intervention approach hypothesized here has implications reaching to clinical and pharmaceutical translation worldwide.

Alveolar proteinosis – disease with unpredictable evolution (series of clinical cases)

Abstract Pulmonary alveolar proteinosis (PAP) is a rare disease, certainly underdiagnosed, characterised by the intra-alveolar accumulation of a milky fluid rich in phospholipids and lipoproteins derived from alveolar surfactant, positive in periodic acid-Schiff staining. The alveolar macrophage plays a major role in the pathogenesis of PAP, and its role in the turn-over of alveolar surfactant is being altered by various mechanisms. More than 90% of cases of PAP are primary autoimmune, characterised by the presence in serum of circulating autoantibodies against granulocyte-macrophages colony-stimulating factor. Other causes of PAP are genetic, secondary to other diseases or to exposure to different agents. The evolution of the disease is unpredictable, from spontaneous remission to progression despite treatment towards pulmonary fibrosis and chronic severe respiratory failure. The gold standard of therapy is the whole lung lavage, other treatments are being still in evaluation. The article presents a few cases that illustrate different patterns in the evolution of PAP.

Hepatic Steatosis Accompanies Pulmonary Alveolar Proteinosis.

Maintenance of tissue-specific organ lipid compositions characterizes mammalian lipid homeostasis. The lungs and liver synthesize mixed phosphatidylcholine (PC) molecular species that are subsequently tailored for function. The lungs progressively enrich disaturated PC directed to lamellar body surfactant stores before secretion. The liver accumulates polyunsaturated PC directed to very-low-density lipoprotein assembly and secretion, or to triglyceride stores. In each tissue, selective PC species enrichment mechanisms lie at the heart of effective homeostasis. We tested for potential coordination between these spatially separated but possibly complementary phenomena under a major derangement of lung PC metabolism, pulmonary alveolar proteinosis (PAP), which overwhelms homeostasis and leads to excessive surfactant accumulation. Using static and dynamic lipidomics techniques, we compared (1) tissue PC compositions and contents, and (2) in lungs, the absolute rates of synthesis in both control mice and the granulocyte-macrophage colony-stimulating factor knockout model of PAP. Significant disaturated PC accumulation in bronchoalveolar lavage fluid, alveolar macrophage, and lavaged lung tissue occurred alongside increased PC synthesis, consistent with reported defects in alveolar macrophage surfactant turnover. However, microscopy using oil red O staining, coherent anti-Stokes Raman scattering, second harmonic generation, and transmission electron microscopy also revealed neutral-lipid droplet accumulations in alveolar lipofibroblasts of granular macrophage colony-stimulating factor knockout animals, suggesting that lipid homeostasis deficits extend beyond alveolar macrophages. PAP plasma PC composition was significantly polyunsaturated fatty acid enriched, but the content was unchanged and hepatic polyunsaturated fatty acid-enriched PC content increased by 50% with an accompanying micro/macrovesicular steatosis and a fibrotic damage pattern consistent with nonalcoholic fatty liver disease. These data suggest a hepatopulmonary axis of PC metabolism coordination, with wider implications for understanding and managing lipid pathologies in which compromise of one organ has unexpected consequences for another.

(629) - Altered Surfactant Turn-Over Immediately after Lung Transplantation Is Associated with Impaired Graft Function at Three Months

(629) - Altered Surfactant Turn-Over Immediately after Lung Transplantation Is Associated with Impaired Graft Function at Three Months

The Innate and Adaptive Immune Response during M. tuberculosis Infection

Mycobacterium tuberculosis, the causative agent of tuberculosis is a facultative intracellular pathogen that infects and resides in humans and is a leading infectious cause of death in many parts of the world with a worrying increase in transmission and resistance to drugs. Surfactant proteins A and D (SP-A and -D) play a role in many acute bacterial, viral, and fungal infections and in acute allergic responses. In vitro, human SPs bind Mycobacterium tuberculosis and alter human and rat macrophage-mediated functions. Here we report the roles of SP-A and SP-D in M. tuberculosis infection following aerosol challenge of SP-A-, SP-D-, and SP-A/-D-deficient mice. These studies surprisingly identified no gross defects in uptake or immune control of M. tuberculosis in SP-A-, SP-D-, and SP-A/-D- deficient mice. While both SP-A- and SP-D-deficient mice exhibited evidence of immunopathologic defects, the CD11b high CD11c high dendritic cell populations and the gamma interferon (IFN-γ)-dependent CD4+ T cell response to M. tuberculosis were unaltered in all genotypes tested. Together, these data indicate that SP-A and SP-D are dispensable for immune control of M. tuberculosis in a low-dose, aerosol challenge, murine model of tuberculosis (TB). This pathogen is generally transmitted by inhalation of infectious aerosols into the lung with deposition in the terminal bronchioles and alveoli. Most affected persons stand an effective immune response that might controls this pathology but does not totally eradicate the primary tuberculosis infection and the reactivation of persistent M. tuberculosis later in life occur frequently in active tuberculosis cases . Many studies are still running up in order to better understand the interactions between M. tuberculosis and the immune environment of the lung. In this review, we describe initial interactions between the lung environment and M. tuberculosis and we summarize the normal surfactant turnover by alveolar macrophages and AEC II in to the uptake of M. tuberculosis in alveolar epithelial cells and macrophages during the innate immune response followed by the T cells initiation of the adaptive immunity in the lung.

Disaturated-phosphatidylcholine and Surfactant protein-B turnover in human acute lung injury and in control patients

BackgroundPatients with Adult Respiratory Distress Syndrome (ARDS) and Acute Lung Injury (ALI) have low concentrations of disaturated-phosphatidylcholine and surfactant protein-B in bronchoalveolar lavage fluid. No information is available on their turnover.ObjectivesTo analyze disaturated-phosphatidylcholine and surfactant protein-B turnover in patients with ARDS/ALI and in human adults with normal lungs (controls).Methods2H2O as precursor of disaturated-phosphatidylcholine-palmitate and 113C-Leucine as precursor of surfactant protein-B were administered intravenously to 12 patients with ARDS/ALI and to 8 controls. Disaturated-phosphatidylcholine and surfactant protein-B were isolated from serial tracheal aspirates, and their fractional synthetic rate was derived from the 2H and 13C enrichment curves, obtained by gas chromatography mass spectrometry. Disaturated-phosphatidylcholine, surfactant protein-B, and protein concentrations in tracheal aspirates were also measured.Results1) Surfactant protein-B turned over at faster rate than disaturated-phosphatidylcholine both in ARDS/ALI patients and in controls. 2) In patients with ARDS/ALI the fractional synthesis rate of disaturated-phosphatidylcholine was 3.1 times higher than in controls (p < 0.01), while the fractional synthesis rate of surfactant protein-B was not different. 3) In ARDS/ALI patients the concentrations of disaturated-phosphatidylcholine and surfactant protein-B in tracheal aspirates were markedly and significantly reduced (17% and 40% of the control values respectively).Conclusions1) Disaturated-phosphatidylcholine and surfactant protein-B have a different turnover both in healthy and diseased lungs. 2) In ARDS/ALI the synthesis of these two surfactant components may be differently regulated.

21 Surfactant Dipalmitoyl Phosphatidylcholine Kinetic is Markedly Impaired in Term Newborns with Pneumonia

Background and aims: The efficacy of exogenous surfactant treatment in term newborns with pneumonia is still undetermined. Aim of the study is to assess surfactant dipalmitoyl phosphatidylcholine (DPPC) kinetics in newborns with pneumonia using U13C-DPPC as tracer. Methods: Nineteen full-term newborns requiring mechanical ventilation were enrolled: 9 had severe pneumonia and 10 no lung disease. Gestational age and birth weights were similar (38.3 ± 2.0 wks and 2782 ± 724 g). All newborns received intratracheally 2 mg kg−1 U13C-DPPC mixed with 2 to 5 mg kg−1 of exogenous surfactant. We collected sequential tracheal aspirates from the start of the study (before the tracer administration) and isotopic enrichments of DPPC palmitate moieties were measured from serial tracheal aspirates by gas chromatography mass spectrometry. Data are expressed as median (interquartile range) and comparisons are performed by Mann-Whitney test. p< 0.05 was regarded as statistically significant. Results: Median and interquartile range of DPPC pool size in newborns with pneumonia was 3 mg kg−1 (1.5-14.0) and 26.7 mg kg−1 (18.3-44.0) in control group. The difference was statistically significant (p=0.041). Median DPPC half life was 10.1 h (7.6-13.9) in pneumonia and 22.5 h (18.2-36.1) in controls. The difference was highly significant (p=0.004). Conclusions: Term newborns with pneumonia exhibited a smaller pool size and shorter DPPC half life than controls suggesting a markedly faster surfactant turnover presumably triggered by neonatal pneumonia.

Role of the PI3-kinase signaling pathway in trafficking of the surfactant protein A receptor P63 (CKAP4) on type II pneumocytes

Surfactant protein A (SP-A) plays an important role in the maintenance of lung lipid homeostasis. Previously, an SP-A receptor, P63 (CKAP4), on type II pneumocyte plasma membranes (PM) was identified by chemical cross-linking techniques. An antibody to P63 blocked the specific binding of SP-A to pneumocytes and the ability of SP-A to regulate surfactant secretion. The current report shows that another biological activity of SP-A, the stimulation of surfactant uptake by pneumocytes, is inhibited by P63 antibody. cAMP exposure resulted in enrichment of P63 on the cell surface as shown by stimulation of SP-A binding, enhanced association of labeled P63 antibody with type II cells, and promotion of SP-A-mediated liposome uptake, all of which were inhibited by competing P63 antibody. Incubation of A549 and type II cells with SP-A also increased P63 localization on the PM. The phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway was explored as a mechanism for the transport of this endoplasmic reticulum (ER)-resident protein to the PM. Treatment with LY-294002, an inhibitor of the PI3-kinase pathway, prevented the SP-A-induced PM enrichment of P63. Exposure of pneumocytes to SP-A or cAMP activated Akt (PKB). Blocking either PI3-kinase or Akt altered SP-A-mediated lipid turnover. The data demonstrate an important role for the PI3-kinase-Akt pathway in intracellular transport of P63. The results add to the growing body of evidence that P63 is critical for SP-A receptor-mediated interactions with type II pneumocytes and the resultant regulation of surfactant turnover.

The PI 3‐kinase/Akt pathway plays a role in the trafficking of the surfactant protein‐A (SP‐A) receptor P63 (CKAP4) to the cell surface of type II pneumocytes

P63 (CKAP4), a 63 kDa type II transmembrane protein, is an SP‐A receptor on type II pneumocytes. An antibody to P63 previously has been shown to block both the specific binding of SP‐A to pneumocytes and the ability of SP‐A to regulate surfactant secretion. In the current work, the P63 antibody blocked the ability of SP‐A to stimulate surfactant uptake by pneumocytes. P63 is located on both the plasma membrane (PM) and the endoplasmic reticulum (ER). Cyclic AMP exposure resulted in enrichment of P63 on the cell surface as shown by stimulation of SP‐A binding; enhanced association of labeled P63 antibody with type II cells; and promotion of SP‐A‐mediated liposome uptake, all of which were inhibited by competing P63 antibody. Treatment of type II cells with LY294002, an inhibitor of the phosphatidylinositol‐3‐kinase (PI3‐kinase) signaling pathway, prevented the SP‐A‐induced PM enrichment of P63. Exposure of pneumocytes to SP‐A or cAMP activated Akt (protein kinase B). Blocking either PI3‐kinase or Akt with inhibitors prevented SP‐A‐mediated lipid turnover. The data demonstrate an important role for the PI3‐kinase/Akt signaling pathway in the intracellular trafficking of P63. The results add further evidence that P63 is critical for SP‐A receptor‐mediated interactions with type II pneumocytes and the resultant regulation of surfactant turnover. [HL 19737]

Metabolic precursors of surfactant disaturated-phosphatidylcholine in preterms with respiratory distress

Our objective was to study the metabolic precursors of surfactant disaturated-phosphatidylcholine (DSPC) in preterm infants with respiratory distress syndrome (RDS) on mechanical ventilation. We performed 46 DSPC kinetic studies in 23 preterms on fat-free parenteral nutrition and mechanical ventilation (birth weight = 1167 +/- 451 g, gestational age = 28.5 +/- 2.0 weeks). Eight infants received a simultaneous intravenous infusion of U(13)C-glucose and [16,16,16](2)H-palmitate, eight infants received U(13)C-glucose and (2)H(2)O, and seven received U(13)C-palmitate and (2)H(2)O. Surfactant DSPC kinetics were calculated from the isotopic enrichments of DSPC-palmitate from sequential tracheal aspirates and its metabolic precursors in plasma or urine. DSPC fractional synthesis rate (FSR) was 17 +/- 11, 21 +/- 16, and 15 +/- 6%/day from glucose, palmitate, and body water, respectively (P = 0.36). DSPC-FSR from U(13)C-glucose and (2)H(2)O were significantly correlated and yielded similar estimates (difference of -0.1 +/- 3%) (P = 0.91). The difference in the 15 infants receiving palmitate versus (2)H(2)O or palmitate versus glucose was +6.0 +/- 12%/day (P = 0.21). There was a significant correlation between DSPC-FSRs from plasma glucose and plasma FFA. The contribution of glucose versus palmitate to DSPC-FSR was 49 +/- 20% versus 51 +/- 20%, respectively. Plasma glucose and FFA showed similar contributions to DSPC-FSR in infants with RDS and fat-free parenteral nutrition. FSRs from (2)H(2)O or glucose were highly correlated.

Forward transport of the receptor P63 is necessary for secretagogue‐enhanced surfactant protein‐A (SP‐A) binding to type II pneumocytes

SP‐A plays an important role in surfactant lipid turnover in the lung. P63 (CKAP4) located on both the plasma membrane (PM) and ER of type II pneumocytes has been identified as an SP‐A receptor. Interference with specific calcium (Ca++)‐dependent binding of SP‐A to type II cells occurs in competition studies using anti‐P63 pAb and upon treatment with siRNA to reduce P63 protein levels. A 2.5‐fold enhancement of 125I‐SP‐A Ca++‐dependent binding to type II cells plated on microporous membranes was achieved by exposure of the cells to the secretagogue cAMP (0.1mM), without effect on non‐specific (Ca++‐independent) SP‐A cell association. Anti‐P63 Ab blocked the cAMP‐augmented specific binding of SP‐A, evidence for the importance of P63 in this process. Inhibitors of receptor recycling were utilized to evaluate whether the forward transport of the ER‐resident P63 to the PM was necessary for receptor function. Monensin, a carboxylic ionophore, increases the pH of intracellular vesicles and brefeldin A (BFA) blocks formation of transport vesicles. In the presence of monensin (2uM) or BFA (10ug/ml), cAMP‐augmentation of the specific binding of SP‐A was abolished. Thus, secretagogue stimulation of SP‐A binding required the movement of P63 protein from the ER to the PM. Our results provide further evidence for the role of P63 as a SP‐A receptor protein on lung type II cells. [HL 19737]

Hypercapnic acidosis modulates inflammation, lung mechanics, and edema in the isolated perfused lung

Objective Low tidal volume (V T) ventilation strategies may be associated with permissive hypercapnia, which has been shown by ex vivo and in vivo studies to have protective effects. We hypothesized that hypercapnic acidosis may be synergistic with low V T ventilation; therefore, we studied the effects of hypercapnia and V T on unstimulated and lipopolysaccharide-stimulated isolated perfused lungs. Materials and Methods Isolated perfused rat lungs were ventilated for 2 hours with low (7 mL/kg) or moderately high (20 mL/kg) V T and 5% or 20% CO 2, with lipopolysaccharide or saline added to the perfusate. Results Hypercapnia resulted in reduced pulmonary edema, lung stiffness, tumor necrosis factor α (TNF- α) and interleukin 6 (IL-6) in the lavage and perfusate. The moderately high V T did not cause lung injury but increased lavage IL-6 and perfusate IL-6 as well as TNF- α. Pulmonary edema and respiratory mechanics improved, possibly as a result of a stretch-induced increase in surfactant turnover. Lipopolysaccharide did not induce significant lung injury. Conclusions We conclude that hypercapnia exerts a protective effect by modulating inflammation, lung mechanics, and edema. The moderately high V T used in this study stimulated inflammation but paradoxically improved edema and lung mechanics with an associated increase in surfactant release.

Surfactant disaturated-phosphatidylcholine kinetics in acute respiratory distress syndrome by stable isotopes and a two compartment model

BackgroundIn patients with acute respiratory distress syndrome (ARDS), it is well known that only part of the lungs is aerated and surfactant function is impaired, but the extent of lung damage and changes in surfactant turnover remain unclear. The objective of the study was to evaluate surfactant disaturated-phosphatidylcholine turnover in patients with ARDS using stable isotopes.MethodsWe studied 12 patients with ARDS and 7 subjects with normal lungs. After the tracheal instillation of a trace dose of 13C-dipalmitoyl-phosphatidylcholine, we measured the 13C enrichment over time of palmitate residues of disaturated-phosphatidylcholine isolated from tracheal aspirates. Data were interpreted using a model with two compartments, alveoli and lung tissue, and kinetic parameters were derived assuming that, in controls, alveolar macrophages may degrade between 5 and 50% of disaturated-phosphatidylcholine, the rest being lost from tissue. In ARDS we assumed that 5–100% of disaturated-phosphatidylcholine is degraded in the alveolar space, due to release of hydrolytic enzymes. Some of the kinetic parameters were uniquely determined, while others were identified as lower and upper bounds.ResultsIn ARDS, the alveolar pool of disaturated-phosphatidylcholine was significantly lower than in controls (0.16 ± 0.04 vs. 1.31 ± 0.40 mg/kg, p < 0.05). Fluxes between tissue and alveoli and de novo synthesis of disaturated-phosphatidylcholine were also significantly lower, while mean resident time in lung tissue was significantly higher in ARDS than in controls. Recycling was 16.2 ± 3.5 in ARDS and 31.9 ± 7.3 in controls (p = 0.08).ConclusionIn ARDS the alveolar pool of surfactant is reduced and disaturated-phosphatidylcholine turnover is altered.

Positional specificity of lysosomal phospholipase A2

Lysosomal phospholipase A(2) (Lpla2) is highly expressed in alveolar macrophages and may mediate the phospholipid metabolism of surfactant. Studies on the properties of this phospholipase are consistent with the presence of both phospholipase A(1) and phospholipase A(2) activities. These activities were studied through the production of O-acyl compounds, produced by the transacylase activity of Lpla2. Liposomes containing POPC and N-acetylsphingosine (NAS) were incubated with the soluble fraction obtained from MDCK cells stably transfected with the mouse Lpla2 gene. Two 1-O-acyl-NASs, 1-O-palmitoyl-NAS and 1-O-oleoyl-NAS, were produced by Lpla2. The formation rate of 1-O-oleoyl-NAS was 2.5-fold that of 1-O-palmitoyl-NAS. When 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) was used, the formation rate of 1-O-oleoyl-NAS was 5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acyl groups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acyl group from the sn-2 position to NAS was preferred to that of the palmitoyl group from the sn-1 position. An exception was observed for 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), for which the formation rate of 1-O-palmitoyl-NAS from PAPC was 4-fold greater than that of 1-O-arachidonoyl-NAS. Thus, Lpla2 has broad positional specificity for the sn-1 and sn-2 acyl groups in phosphatidylcholine and phosphatidylethanolamine.

Lung phospholipid metabolism in transgenic mice overexpressing peroxiredoxin 6

Previous studies with peroxiredoxin 6 (Prdx6) null mice demonstrated that the phospholipase A 2 activity of this enzyme plays a major role in lung phospholipid metabolism. This study evaluated lung phospholipid metabolism in transgenic mice that over-express Prdx6. Lung lysosomal type PLA 2 activity in transgenic mice was 222% of wild type in lung homogenate and 280% in isolated lamellar bodies. Total phospholipid, phosphatidylcholine (PC) and disaturated PC were decreased approximately 20–35% in bronchoalveolar lung fluid, lung homogenate, and lung lamellar bodies in transgenic mice although lung compliance and type 2 cell ultrastructure were unaltered. To study metabolism, unilamellar liposomes ( 3H-DPPC: PC: cholesterol: PG, 10: 5: 3: 2 mol fraction) were instilled endotracheally in anesthetized mice and lungs were removed for perfusion. Compared to wild type, transgenic mice showed similar net uptake of liposomes in 2 h, but significantly increased 3H-DPPC degradation (38.9 ± 1.1 vs. 29.0 ± 1.3% of recovered dpm). The PLA 2 competitive inhibitor MJ33 decreased degradation to 15% of recovered dpm in both transgenic and wild type lungs. Incorporation of [ 14C] palmitate into DSPC at 24 h after its intravenous injection was markedly increased in both the lung surfactant (+100%) and lamellar bodies (+188%) while incorporation of [ 3H] choline was increased by only 10–20%. These results indicate increased DPPC degradation and synthesis by the reacylation pathway with Prdx6 overexpression and provide additional evidence that the PLA 2 activity of Prdx6 has an important role in lung surfactant turnover.

Relationship between pressure-volume curve and markers for collagen turn-over in early acute respiratory distress syndrome

In acute respiratory distress syndrome, the relationships between changes in the elastic behavior of the respiratory system and biological markers of extra-cellular matrix or surfactant turn-over could give some insights into its pathophysiological determinants. In 17 patients with acute respiratory distress syndrome, we assessed the relationship between chord compliance measured on pressure-volume curves obtained at two levels of positive end-expiratory pressure (0 and 10[Symbol: see text]cm[Symbol: see text]H(2)O) and biological markers of collagen turn-over or surfactant degradation in bronchoalveolar lavage fluid obtained simultaneously in the early phase of the disease (first 4 days). The compliance of the respiratory system obtained from the pressure-volume curves was significantly correlated with markers for collagen turn-over (type III procollagen peptide and matrix metalloproteinase 2) and with markers of surfactant degradation (type-IIA secretory phospholipase A2). The correlations were stronger when the curve was traced from positive end-expiratory pressure, suggesting that this condition may improve the assessment of tissue mechanics. A logarithmic relationship best described the correlation between compliance and type III procollagen peptide, in agreement with a collagen-dependent model of maximal distension. The marker for surfactant degradation was associated with ongoing alveolar inflammation (cellularity of bronchoalveolar lavage fluid and tumor necrosis factor-alpha concentration). Interleukin-10, an anti-inflammatory mediator, showed no correlation with compliance. These preliminary data suggest that a severe reduction in compliance in the early phase of acute respiratory distress syndrome is associated with both collagen deposition and surfactant degradation.