Lung Surfactant Protein Research Articles

Elucidation of Lipid Binding Sites on Lung Surfactant Protein A Using X-ray Crystallography, Mutagenesis, and Molecular Dynamics Simulations.

Surfactant protein A (SP-A) is a collagenous C-type lectin (collectin) that is critical for pulmonary defense against inhaled microorganisms. Bifunctional avidity of SP-A for pathogen-associated molecular patterns (PAMPs) such as lipid A and for dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant membranes lining the air-liquid interface of the lung, ensures that the protein is poised for first-line interactions with inhaled pathogens. To improve our understanding of the motifs that are required for interactions with microbes and surfactant structures, we explored the role of the tyrosine-rich binding surface on the carbohydrate recognition domain of SP-A in the interaction with DPPC and lipid A using crystallography, site-directed mutagenesis, and molecular dynamics simulations. Critical binding features for DPPC binding include a three-walled tyrosine cage that binds the choline headgroup through cation-π interactions and a positively charged cluster that binds the phosphoryl group. This basic cluster is also critical for binding of lipid A, a bacterial PAMP and target for SP-A. Molecular dynamics simulations further predict that SP-A binds lipid A more tightly than DPPC. These results suggest that the differential binding properties of SP-A favor transfer of the protein from surfactant DPPC to pathogen membranes containing appropriate lipid PAMPs to effect key host defense functions.

Insight into the Structure of Lung Surfactant Protein B (SP-B)

Lung surfactant proteins play crucial role in breathing and also help to defend the lungs from pathogens. Surfactant associated-protein B (SP-B) is the most vital component of lung surfactant. SP-B is essential to life in that it helps reduce the surface tension at the air-water interface of the alveolus to near zero value and consequently prevent the lung from collapse. However, SP-B's overall three-dimensional structure is not yet known, and therefore its mechanism of action remains a mystery. The extreme hydrophobic nature of SP-B makes it difficult to study. Nonetheless, recent advances in our laboratory have made it possible to recombinantly express surfactant protein B (rSP-B) in bacteria. Circular dichroism (CD) confirmed that rSP-B can be reproducibly and stably folded and can maintain its secondary structure over a range of temperatures. Dynamic Light scattering (DLS) indicates two size distributions by intensity, one at the right size range to study by solution nuclear magnetic resonance (NMR) and the other at a bigger size range. rSP-B is also showing promising result in vitro in terms of its functionality when tested with a Langmuir-Blodgett trough. Lipid films spread on water and compressed in the presence of an appropriate amount rSP-B are able to induce monolayer to multilayer transitions at the expected surface pressure of ∼40 to 50 mN/m and also show superior compressibility and faster adsorption rates compared to films compressed without rSP-B. Preliminary one-dimensional proton NMR spectra show signals in the amide peptide backbone region that are similar to those observed in earlier studies of SP-B fragments. Our findings indicate that recombinant SP-B can be stably folded and functional in vitro, and that it may be possible to find conditions to make it possible to determine SP-B's structure using solution NMR spectroscopy.

Combined effect of synthetic protein, Mini-B, and cholesterol on a model lung surfactant mixture at the air–water interface

The overall goal of this work is to study the combined effects of Mini-B, a 34 residue synthetic analog of the lung surfactant protein SP-B, and cholesterol, a neutral lipid, on a model binary lipid mixture containing dipalmitolphosphatidylcholine (DPPC) and palmitoyl-oleoyl-phosphatidylglycerol (POPG), that is often used to mimic the primary phospholipid composition of lung surfactants. Using surface pressure vs. mean molecular area isotherms, fluorescence imaging and analysis of lipid domain size distributions; we report on changes in the structure, function and stability of the model lipid-protein films in the presence and absence of varying composition of cholesterol. Our results indicate that at low cholesterol concentrations, Mini-B can prevent cholesterol's tendency to lower the line tension between lipid domain boundaries, while maintaining Mini-B's ability to cause reversible collapse resulting in the formation of surface associated reservoirs. Our results also show that lowering the line tension between domains can adversely impact monolayer folding mechanisms. We propose that small amounts of cholesterol and synthetic protein Mini-B can together achieve the seemingly opposing requirements of efficient LS: fluid enough to flow at the air–water interface, while being rigid enough to oppose irreversible collapse at ultra-low surface tensions.

Activity and biophysical inhibition resistance of a novel synthetic lung surfactant containing Super-Mini-B DATK peptide.

Background/objectives. This study examines the surface activity, resistance to biophysical inhibition, and pulmonary efficacy of a synthetic lung surfactant containing glycerophospholipids combined with Super Mini-B (S-MB) DATK, a novel and stable molecular mimic of lung surfactant protein (SP)-B. The objective of the work is to test whether S-MB DATK synthetic surfactant has favorable biophysical and physiological activity for future use in treating surfactant deficiency or dysfunction in lung disease or injury.Methods. The structure of S-MB DATK peptide was analyzed by homology modeling and by FTIR spectroscopy. The in vitro surface activity and inhibition resistance of synthetic S-MB DATK surfactant was assessed in the presence and absence of albumin, lysophosphatidylcholine (lyso-PC), and free fatty acids (palmitoleic and oleic acid). Adsorption and dynamic surface tension lowering were measured with a stirred subphase dish apparatus and a pulsating bubble surfactometer (20 cycles/min, 50% area compression, 37 °C). In vivo pulmonary activity of S-MB DATK surfactant was measured in ventilated rabbits with surfactant deficiency/dysfunction induced by repeated lung lavages that resulted in arterial PO2 values <100 mmHg.Results. S-MB DATK surfactant had very high surface activity in all assessments. The preparation adsorbed rapidly to surface pressures of 46–48 mN/m at 37 °C (low equilibrium surface tensions of 22–24 mN/m), and reduced surface tension to <1 mN/m under dynamic compression on the pulsating bubble surfactometer. S-MB DATK surfactant showed a significant ability to resist inhibition by serum albumin, C16:0 lyso-PC, and free fatty acids, but surfactant inhibition was mitigated by increasing surfactant concentration. S-MB DATK synthetic surfactant quickly improved arterial oxygenation and lung compliance after intratracheal instillation to ventilated rabbits with severe surfactant deficiency.Conclusions. S-MB DATK is an active mimic of native SP-B. Synthetic surfactants containing S-MB DATK (or related peptides) combined with lipids appear to have significant future potential for treating clinical states of surfactant deficiency or dysfunction, such as neonatal and acute respiratory distress syndromes.

The clinical effect of airway pressure release ventilation for acute lung injury/acute respiratory distress syndrome

To evaluate the effect of airway pressure release ventilation (APRV) in patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS), to evaluate the extent of ventilator-induced lung injury (VILI), and to explore its possible mechanism. A prospective study was conducted in the Department of Critical Care Medicine of the First Hospital of Hebei Medical University from December 2010 to February 2012. The patients with ALI/ARDS were enrolled. They were randomly divided into two groups. The patients in APRV group were given APRV pattern, while those in control group were given lung protection ventilation, synchronized intermittent mandatory ventilation with positive end-expiratory pressure (SIMV+PEEP). All patients were treated with AVEA ventilator. The parameters such as airway peak pressure (Ppeak), mean airway pressure (Pmean), pulse oxygen saturation (SpO2), mean arterial pressure (MAP), heart rate (HR), central venous pressure (CVP), arterial blood gas, urine output (UO), the usage of sedation and muscle relaxation drugs were recorded. AVEA ventilator "turning point (Pflex) operation" was used to describe the quasi-static pressure volume curve (P-V curve). High and low inflection point (UIP, LIP) and triangular Pflex volume (Vdelta) were automatically measured and calculated. The ventilation parameters were set, and the 24-hour P-V curve was recorded again in order to be compared with subsequent results. Venous blood was collected before treatment, 24 hours and 48 hours after ventilation to measure lung surfactant protein D (SP-D) and large molecular mucus in saliva (KL-6) by enzyme linked immunosorbent assay (ELISA), and the correlation between the above two parameters and prognosis on 28 days was analyzed by multinomial logistic regression. Twenty-six patients with ALI/ARDS were enrolled, and 22 of them completed the test with 10 in APRV group and 12 in control group. The basic parameters and P-V curves between two groups were similar before the test. After 24 hours and 48 hours, mechanical ventilation was given in both groups. The patients' oxygenation was improved significantly, though there were no significant changes in hemodynamic parameters. The Pmean (cmH2O, 1 cmH2O = 0.098 kPa) in APRV group was significantly higher than that in control group (24 hours: 24.20±4.59 vs. 17.50±3.48, P < 0.01; 48 hours: 18.10±4.30 vs. 15.00±2.59, P < 0.05). After ventilation for 24 hours, the ratio of patients with increased Vdelta in APRV group was higher than that in control group (90% vs. 75%), but without statistical difference (P > 0.05). The SP-D level (μg/L) in serum in APRV group showed a tendency of increase (increased from 19.70±7.34 to 27.61±10.21, P < 0.05), in contrast there was a tendency of decrease in control group (decreased from 21.83±7.31 to 16.58±2.90, P > 0.05), the difference between the two groups was statistically significant (P < 0.05). After 48-hour ventilation, SP-D in APRV group was decreased, but no change was found in control group, and no significant difference was found as compared with that of the control group (16.45±8.17 vs. 17.20±4.59, P > 0.05). There was no significant difference in serum KL-6 between the two groups before and after ventilation. The SP-D and KL-6 levels in serum were unrelated with 28-day survival rate of the patients. The odds ratio (OR) of SP-D were 0.900 [95% confidence interval (95%CI) = 0.719-1.125], 1.054 (95%CI = 0.878-1.266), 1.143 (95%CI = 0.957-1.365), and the OR of KL-6 were 1.356 (95%CI = 0.668-2.754), 0.658 (95%CI = 0.161-2.685), 0.915 (95%CI = 0.350-2.394) before the test, 24 hours and 48 hours after ventilation (all P > 0.05). APRV was similar to lung protective ventilation strategy in oxygenation and improvements in the lung mechanics parameters. APRV with a higher Pmean can recruit alveolar more effectively, and it had no impact on hemo-dynamics, but might exacerbate VILI.

Effects of Tocilizumab on Experimental Severe Acute Pancreatitis and Associated Acute Lung Injury.

To examine the therapeutic effects of tocilizumab, an antibody against interleukin-6 receptor, on experimental severe acute pancreatitis and associated acute lung injury. The optimal dose of tocilizumab and the activation of interleukin-6 inflammatory signaling were also investigated. Randomized experiment. Research laboratory at a university hospital. Experimental severe acute pancreatitis in rats. Severe acute pancreatitis was induced by retrograde injection of sodium taurocholate (50 mg/kg) into the biliopancreatic duct. In dose-study, rats were administered with different doses of tocilizumab (1, 2, 4, 8, and 16 mg/kg) through the tail vein after severe acute pancreatitis induction. In safety-study, rats without severe acute pancreatitis induction were treated with high doses of tocilizumab (8, 16, 32, and 64 mg/kg). Serum and tissue samples of rats in time-study were collected for biomolecular and histologic evaluations at different time points (2, 6, 12, 18, and 24 hr). 1) Under the administration of tocilizumab, histopathological scores of pancreas and lung were decreased, and severity parameters related to severe acute pancreatitis and associated lung injury, including serum amylase, C-reactive protein, lung surfactant protein level, and myeloperoxidase activity, were all significant alleviated in rat models. 2) Dose-study demonstrated that 2 mg/kg tocilizumab was the optimal treatment dose. 3) Basing on multi-organ pathologic evaluation, physiological and biochemical data, no adverse effect and toxicity of tocilizumab were observed in safety-study. 4) Pancreatic nuclear factor-κB and signal transducer and activator of transcription 3 were deactivated, and the serum chemokine (C-X-C motif) ligand 1 was down-regulated after tocilizumab administration. Our study demonstrated tocilizumab, as a marketed drug commonly used for immune-mediated diseases, was safe and effective for the treatment of experimental severe acute pancreatitis and associated acute lung injury. Our findings provide experimental evidences for potential clinical application of tocilizumab in severe acute pancreatitis and associated complications.

Histoplasma capsulatum surmounts obstacles to intracellular pathogenesis.

The fungal pathogen Histoplasma capsulatum causes respiratory and disseminated disease, even in immunocompetent hosts. In contrast to opportunistic pathogens, which are readily controlled by phagocytic cells, H. capsulatum yeasts are able to infect macrophages, survive antimicrobial defenses, and proliferate as an intracellular pathogen. In this review, we discuss some of the molecular mechanisms that enable H. capsulatum yeasts to overcome obstacles to intracellular pathogenesis. H. capsulatum yeasts gain refuge from extracellular obstacles such as antimicrobial lung surfactant proteins by engaging the β-integrin family of phagocytic receptors to promote entry into macrophages. In addition, H. capsulatum yeasts conceal immunostimulatory β-glucans to avoid triggering signaling receptors such as the β-glucan receptor Dectin-1. H. capsulatum yeasts counteract phagocyte-produced reactive oxygen species by expression of oxidative stress defense enzymes including an extracellular superoxide dismutase and an extracellular catalase. Within the phagosome, H. capsulatum yeasts block phagosome acidification, acquire essential metals such as iron and zinc, and utilize de novo biosynthesis pathways to overcome nutritional limitations. These mechanisms explain how H. capsulatum yeasts avoid and negate macrophage defense strategies and establish a hospitable intracellular niche, making H. capsulatum a successful intracellular pathogen of macrophages.

Expression and Localization of Lung Surfactant Proteins in Human Testis.

BackgroundSurfactant proteins (SPs) have been described in various tissues and fluids including tissues of the nasolacrimal apparatus, airways and digestive tract. Human testis have a glandular function as a part of the reproductive and the endocrine system, but no data are available on SPs in human testis and prostate under healthy and pathologic conditions.ObjectiveThe aim of the study was the detection and characterization of the surfactant proteins A, B, C and D (SP-A, SP-B, SP-C, SP-D) in human testis. Additionally tissue samples affected by testicular cancer were investigated.ResultsSurfactant proteins A, B, C and D were detected using RT-PCR in healthy testis. By means of Western blot analysis, these SPs were detected at the protein level in normal testis, seminoma and seminal fluid, but not in spermatozoa. Expression of SPs was weaker in seminoma compared to normal testicular tissue. SPs were localized in combination with vimentin immunohistochemically in cells of Sertoli and Leydig.ConclusionSurfactant proteins seem to be inherent part of the human testis. By means of physicochemical properties the proteins appear to play a role during immunological and rheological process of the testicular tissue. The presence of SP-B and SP-C in cells of Sertoli correlates with their function of fluid secretion and may support transportation of spermatozoa. In seminoma the expression of all SP's was generally weaker compared to normal germ cells. This could lead to a reduction of immunomodulatory and rheology processes in the germ cell tumor.

Environmental Pollutant Ozone Causes Damage to Lung Surfactant Protein B (SP-B).

Lung surfactant protein B (SP-B) is an essential protein found in the surfactant fluid at the air–water interface of the lung. Exposure to the air pollutant ozone could potentially damage SP-B and lead to respiratory distress. We have studied two peptides, one consisting of the N-terminus of SP-B [SP-B(1–25)] and the other a construct of the N- and C-termini of SP-B [SP-B(1–25,63–78)], called SMB. Exposure to dilute levels of ozone (∼2 ppm) of monolayers of each peptide at the air–water interface leads to a rapid reaction, which is evident from an increase in the surface tension. Fluorescence experiments revealed that this increase in surface tension is accompanied by a loss of fluorescence from the tryptophan residue at the interface. Neutron and X-ray reflectivity experiments show that, in contrast to suggestions in the literature, the peptides are not solubilized upon oxidation but rather remain at the interface with little change in their hydration. Analysis of the product material reveals that no cleavage of the peptides occurs, but a more hydrophobic product is slowly formed together with an increased level of oligomerization. We attributed this to partial unfolding of the peptides. Experiments conducted in the presence of phospholipids reveal that the presence of the lipids does not prevent oxidation of the peptides. Our results strongly suggest that exposure to low levels of ozone gas will damage SP-B, leading to a change in its structure. The implication is that the oxidized protein will be impaired in its ability to interact at the air–water interface with negatively charged phosphoglycerol lipids, thus compromising what is thought to be its main biological function.

Increased lung prolyl hydroxylase and decreased glucocorticoid receptor are related to decreased surfactant protein in the growth-restricted sheep fetus

Experimental placental restriction (PR) by carunclectomy in fetal sheep results in intrauterine growth restriction (IUGR), chronic hypoxemia, increased plasma cortisol, and decreased lung surfactant protein (SP) expression. The mechanisms responsible for decreased SP expression are unknown but may involve decreased glucocorticoid (GC) action or changes in hypoxia signaling. Endometrial caruncles were removed from nonpregnant ewes to induce PR. Lungs were collected from control and PR fetuses at 130-135 (n = 19) and 139-145 (n = 28) days of gestation. qRT-PCR and Western blotting were used to quantify lung mRNA and protein expression, respectively, of molecular regulators and downstream targets of the GC and hypoxia-signaling pathways. We confirmed a decrease in SP-A, -B, and -C, but not SP-D, mRNA expression in PR fetuses at both ages. There was a net downregulation of GC signaling with a reduction in GC receptor (GR)-α and -β protein expression and a decrease in the cofactor, GATA-6. GC-responsive genes including transforming growth factor-β1, IL-1β, and β2-adrenergic receptor were not stimulated. Prolyl hydroxylase domain (PHD)2 mRNA and protein and PHD3 mRNA expression increased with a concomitant increase in hypoxia-inducible factor-1α (HIF-1α) and HIF-1β mRNA expression. There was an increase in mRNA expression of several, but not all, hypoxia-responsive genes. Hence, both GC and hypoxia signaling may contribute to reduced SP expression. Although acute hypoxia normally inactivates PHDs, chronic hypoxemia in the PR fetus increased PHD abundance, which normally prevents HIF signaling. This may represent a mechanism by which chronic hypoxemia contributes to the decrease in SP production in the IUGR fetal lung.

Expression of lung surfactant proteins SP-B and SP-C and their modulating factors in fetal lung of FGR rats.

This study investigated the expression of lung surfactant proteins SP-B and SP-C, and their modulating factors TTF-1 and PLAGL2 in the fetal lung of rats with fetal growth restriction (FGR). The rat FGR model was established by prenatal hypoxia in the first stage of pregnancy, 180 rats for experiment served as hypoxia group, and 197 healthy rats served as normal control group. The FGR incidence in hypoxia was compared with that in normal control group. The histological changes in the fetal lung were observed under the light microscope and electronic microscope in two groups. The SP-B, SP-C, TTF-1 and PLAGL2 proteins were determined in the fetal lung of two groups immunohistochemically. The expression levels of SP-B, SP-C, TTF-1 and PLAGL2 protein and mRNA in the fetal lung of two groups were detected by using Western blotting and RT-PCR respectively. The FGR rat model was successfully established by using hypoxia. Pathologically the fetal lung developed slowly, and the expression levels of SP-B, SP-C, TTF-1 and PLAGL2 protein and mRNA in the fetal lung were significantly reduced in hypoxia group as compared with those in normal control group. It was suggested that maternal hypoxia in the first stage of pregnancy could induce FGR, and reduce the expression of SP-B and SP-C, resulting in the disorder of fetal lung development and maturation.

Complex approach for diagnosing acute respiratory distress syndrome in nosocomial pneumonia

Acute respiratory distress syndrome (ARDS) develops on the basis of nosocomial pneumonia (NP) in 30 to 35% of cases. A complex clinical approach is required for diagnosing it. The aim of this study was to investigate into the role of the PaO2/FiO2 ratio (P/F), extravascular lung water index (EVLWI) and surfactant protein D (SPD) as a complex diagnostic tool for ARDS in NP.

A Combined Effect of Proteins SP-B and SP-C and Membrane Curvature on Cholesterol Partition in Lung Surfactant Membranes: Answers from Fluorescence

Cholesterol is the major neutral lipid present in lung surfactant, a lipid-protein complex responsible for the stabilization of the air-liquid interface in mammalian lungs, which is essential for the breathing function. It has been proposed that cholesterol content is tightly regulated in surfactant and that protein SP-C could have a role in this respect. To further analyse the affinity of surfactant membranes for cholesterol in the absence or presence of proteins, we have used cyclodextrin as a cholesterol solubilising agent and a fluorescent analog of cholesterol, cholestatrienol (CTL), whose partition can be followed in membrane vesicles with different lipid/protein compositions and sizes. Our results suggest that there is a direct effect of vesicle size/curvature on the affinity of membranes for CTL. This is translated into lower partition coefficients (Kx) for membranes with higher curvatures, possibly caused by an increased exposure of CTL in more curved bilayers. Surfactant hydrophobic proteins SP-B and SP-C were found to have a curvature-independent effect on CTL mobility, as well as in the mobility of other probes such as transparinaric acid-sphingomyelin (tPA-SM) or diphenylhexatriene-phosphatidylcholine (DPH-PC) in membranes of the same composition. Furthermore, in the absence of SP-B, SP-C-containing liposomes were found to spontaneously split into 30 nm vesicles increasing membrane curvature regardless lipid composition, which correlated with a reduction of Kx compared to pure lipid systems. Taken all together, our results indicate that curvature alters cholesterol accessibility in surfactant lipid bilayers and that lung surfactant proteins disturb membranes in cholesterol containing domains, which may have important physiological consequences for cholesterol mobilization in lung surfactant membranes and films.

Experimental and Computational Studies of Pulmonary Surfactant Protein SP-B Interacting with Lipid Bilayers

Pulmonary lung surfactant protein B (SP-B) is a 79 residue hydrophobic protein, from the Saposin superfamily. Saposin super family proteins share common features including 3 intra-chain disulfide bonds and 4-5 helical regions. SP-B appears to carry out its essential functions in respiration by binding to and modifying the structures of phospholipid bilayers and monolayers at the air-water interface. Due to difficulties arising from SP-B's extreme hydrophobicity, the 3D structure of full SP-B is not yet known. Thus we are using computational methods in combination with solid-state NMR to investigate the structure of SP-B. Our approach is to generate candidate structures via computational methods, predict the 15N spectra for the computed configurations and then compare them to the experimental NMR spectra.For the computational studies, we employ Mini-b, a construct of SP-B with known structure, and add the rest of residues to make a full length SP-B. We use several positions of SP-B in a POPC bilayer as initial structures. Calculations are carried out using GROMACS, with OPLS-aa, an all-atom force field, as well as with PACE, a hybrid model force field. Simulation methods include Molecular Dynamics (MD) and Replica Exchange Molecular Dynamics (REMD). For the experimental part, we use recombinantly expressed 15N labeled SP-B in mechanically oriented POPC bilayers to collect 1D 15N solid-state NMR spectra.

Changes in lung surfactant proteins in rats with lipopolysaccharide-induced fever.

The study was designed to prove the hypothesis that lipopolysaccharide (LPS)-induced fever elicits the changes in surfactant specific proteins, potentially related to thermal tachypnea. In adult rats fever was induced by intraperitoneal administration of LPS at a dose 100 microg/kg of body weight; control group received saline. Respiratory parameters, arterial blood gases and pH and colonic body temperature (BT) were recorded. Five hours later, surfactant proteins (SP) A, B, C and D were evaluated in bronchoalveolar lavage fluid (BALF) and lung tissue (LT). LPS evoked monophasic thermic response (at 300 min 38.7+/-0.2 vs. 36.4+/-0.3 °C, P 0.05) and an increase in minute ventilation due to changes in breathing rate and tidal volume. LPS-instilled animals had higher levels of SP-A and SP-D in LT (P 0.05 and 0.01), and higher SP-D in BALF (P 0.01) than controls. SP-B increased in LT and SP-C in BALF of animals with LPS (both P 0.05 vs. controls). The changes in all surfactant specific proteins are present in LPS-induced fever. Alterations of proteins related to local immune mechanisms (SP-A, SP-D) are probably a part of general inflammatory response to pyrogen. Changes in proteins related to surface activity (SP-B and SP-C) might reflect the effort of the body to stabilize the lungs in thermal challenge.

The forkhead box transcription factor-2 (Foxa2) and lung disease

Foxa2 [The forkhead box transcription factor-2; also known as HNF3B(hepatocyte nuclear factors-3), or TCF3B(transcription factor-3b) ] [Costa, 1989 #64], is a member of the Forkhead family of nuclear transcription factors that is highly expressed in respiratory epithelial cells lining conducting airways and in alveolar type Ⅱ cells in the development and mature lung. Foxa2 plays critical roles in lung development, which is required for normal lung maturation, surfactant protein and lipid synthesis, epithelial differentiation, and cross talking between epithelium and innate immune. Foxa2 can determine gene expression and cell fate in the lung, and is required for the suppression of Th2 immunity, mucus production, and goblet cell metaplasia in the developing lung in a process determined in part by its regulation of the Cysteinyl leukotriene (CysLT) pathway. Recent studies have implicated that Foxa2 is a suppressor of lung cancer. This review will summarize the role of Foxa2 in lung development, pulmonary diseases, and also the possible signaling pathways regulated by Foxa2.

Synoviolin inhibitor LS-102 reduces endoplasmic reticulum stress-induced collagen secretion in an in vitro model of stress-related interstitial pneumonia.

The deletion mutation of exon 4 in surfactant protein C (SP-C), a lung surfactant protein, has been identified in parent-child cases of familial interstitial pneumonia. It has been shown that this mutation induces endoplasmic reticulum (ER) stress. Synoviolin is an E3 ubiquitin ligase that is localized to the ER and is an important factor in the degradation of ER-related proteins. It has been demonstrated that synoviolin is involved in liver fibrosis. In the present study, we investigated the involvement of synoviolin in the pathogenesis of interstitial pneumonia caused by the exon 4 deletion in the SP-C gene. We transfected wild-type and exon 4-deleted SP-C genes into A549 human lung adenocarcinoma cells and measured the secretion of collagen, which is a representative extracellular matrix protein involved in fibrosis. Secreted collagen levels were increased in the culture medium in SP-C mutants compared to the wild-type cells. Furthermore, the transcription of mRNAs coding for factors associated with fibrosis was increased. Subsequently, to assess the involvement of synoviolin, we constructed plasmids with a luciferase gene under the control of the synoviolin promoter. The A549 cells were transfected with the construct along with the exon 4-deleted SP-C plasmid for use in the luciferase assay. We found a 1.6-fold increase in luciferase activity in the cells carrying exon 4 deleted SP-C, as well as an increase in intrinsic synoviolin expression at the mRNA and protein levels. Collagen secretion was decreased by the addition of LS-102, a synoviolin inhibitor, to the A549 culture medium following transfection with wild-type and exon 4-deleted SP-C. These results demonstrate that synoviolin is involved in the onset of interstitial pneumonia induced by exon 4-deleted SP-C, which suggests that synoviolin inhibitors may be used in the treatment of the disease.

Intravenous insulin therapy during lung resection does not affect lung function or surfactant proteins

BackgroundThe surgical resection of lung disrupts glucose homeostasis and causes hyperglycemia, as in any other major surgery or critical illness. We performed a prospective study where we carefully lowered hyperglycemia by insulin administration during the surgery, and for the first time we monitored immediate insulin effects on lung physiology and gene transcription.MethodsThe levels of blood gases (pH, pCO2, pO2, HCO3-, HCO3- std, base excess, FiO2, and pO2/FiO2) were measured at the beginning of surgery, at the end of surgery, and two hours after. Samples of healthy lung tissue surrounding the tumour were obtained during the surgery, anonymized and sent for subsequent blinded qPCR analysis (mRNA levels of surfactant proteins A1, A2, B, C and D were measured). This study was done on a cohort of 64 patients who underwent lung resection. Patients were randomly divided, and half of them received insulin treatment during the surgery.ResultsWe demonstrated for the first time that insulin administered intravenously during lung resection does not affect levels of blood gases. Furthermore, it does not induce immediate changes in the expression of surfactant proteins.ConclusionAccording to our observations, short insulin treatment applied intravenously during resection does not affect the quality of breathing.

Residue specific partitioning of KL4 into phospholipid bilayers

KL4, which has demonstrated success in the treatment of respiratory distress, is a synthetic helical, amphipathic peptide mimetic of lung surfactant protein B. The unusual periodicity of charged residues within KL4 and its relatively high hydrophobicity distinguish it from canonical amphipathic helical peptides. Here we utilized site specific spin labeling of both lipids and the peptide coupled with EPR spectroscopy to discern the effects of KL4 on lipid dynamics, the residue specific dynamics of hydrophobic regions within KL4, and the partitioning depths of specific KL4 residues into the DPPC/POPG and POPC/POPG lipid bilayers under physiologically relevant conditions. KL4 induces alterations in acyl chain dynamics in a lipid-dependent manner, with the peptide partitioning more deeply into DPPC-rich bilayers. Combined with an earlier NMR study of changes in lipid dynamics on addition of KL4 (V.C. Antharam et al., 2009), we are able to distinguish how KL4 affects both collective bilayer motions and intramolecular acyl chain dynamics in a lipid-dependent manner. EPR power saturation results for spin labeled lipids demonstrate that KL4 also alters the accessibility profiles of paramagnetic colliders in a lipid-dependent manner. Measurements of dynamics and depth parameters for individual spin-labeled residues within KL4 are consistent with a model where the peptide partitions deeply into the lipid bilayers but lies parallel to the bilayer interface in both lipid environments; the depth of partitioning is dependent on the degree of lipid acyl chain saturation within the bilayer.

In vivo effects of TGF-β1 in lung surfactant regulation, lung meachanics amd structure

Transforming growth factor beta 1 (TGF-β1) is a signalling protein with a wide range of biological activities. TGF-β1 is thought to have a pivotal role in fibrogenesis, where TGF-β1 induces myofibroblast migration and increases extracellular matrix synthesis, including collagen. Moreover, it has been described that TGF-β1 is a negative modulator of the regulation of surfactant associated proteins A (SP-A), B (SP-B) and C (SP-C) in vitro. Pulmonary surfactant is a lipid-protein complex that lowers surface tension at the respiratory air-liquid interface, stabilizing the lungs against physical forces tending to collapse alveoli. SP-B and SP-C deficiency has been found in patients suffering from lung diseases and related to potential mechanical stress of the lung epithelium. We have characterized lung surfactant protein composition 1 and 2 weeks after adenoviral mediated gene transfer of active TGF-β1 into lungs. Gene expression of surfactant proteins is down-regulated pointing at deficient transcriptional regulation that might include a deficient activity of TTF-1, during TGF-β1 overexpression. Deficiency on SP-B and SP-C at early stages correlates with high surface tension under dynamic cycling of isolated surfactant in Captive Bubble Surfactometry (CBS). In addition, high surface tension correlates with decreased quasistatic lung compliance and increased collapsibility of distal airspaces. Stereological data demonstrate a correlation between septal wall thickness and quasistatic compliance 2 weeks after gen-transfer. At the ultrastructural level thickening of septal walls, could be attributed to an increase in interstitial cells, formation of dense alveolar oedema and increase in profiles of epithelial type II (AEII) cells. The latter could be also attributed to Epithelial to Mesenchymal Transition (EMT), indicated by down-regulation of epithelial markers associated to up-regulation of mesenchymal molecular markers. We can conclude that in vivo TGF-β1 is a strong negative regulator of surfactant metabolism, originating a mechanical stress that may contribute to EMT at following stages.