Surfactant-associated Protein C Research Articles

Fusu mixture alleviates inflammatory reactions in lipopolysaccharide-induced acute respiratory distress syndrome mice via regulation of surfactant-associated protein C, aquaporin 5, and Notch1.

Acute respiratory distress syndrome (ARDS) is a common life-threatening critical illness with high mortality. Fusu mixture (FSM) can improve the mechanical ventilation in ARDS patients. However, the detailed pharmacological mechanisms and active substances of FSM are still unclear. This study aimed to explore the potential pharmacological mechanisms of FSM for treating ARDS and its chemical compositions. A lipopolysaccharide (LPS)-induced ARDS mouse model was established, and the mice subsequently received FSM (50 mg/kg) orally for 5 days. Then, the blood samples and lung tissues were collected. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in serum, and histopathology examinations were applied to analyze the inflammatory response of lung tissues in ARDS mice. In addition, protein expressions of aquaporin 5 (AQP-5), surfactant-associated protein C (SP-C), and Notch1 were detected by western blot assays and immunohistochemical (IHC) examination. In addition, the chemical compositions of FSM were analyzed by high performance liquid chromatography (HPLC), using standard reference agents. After LPS induction, the serum levels of IL-6 and TNF-α in ARDS mice were significantly increased (P<0.01, vs. Control), and FSM significantly reduced these 2 pro-inflammatory cytokines (IL-6 and TNF-α) compared to the model mice (P<0.01). Histopathology examinations showed FSM significantly attenuated the inflammatory responses in lung tissues. Furthermore, after FSM treatment, the SP-C and AQP-5 were significantly increased, compared to the Model mice (P<0.01), and FSM also up-regulated the Notch1 expressions in lung tissues of ARDS mice (P<0.001, vs. Model). Collectively, it is suggested that FSM alleviates inflammatory reactions and promotes the proliferation of alveolar epithelial cells in LPS-induced ARDS mice via regulation of SP-C, AQP-5, and Notch1 in lung tissues.

Neutrophil extracellular traps promote bronchopulmonary dysplasia-like injury in neonatal mice via the WNT/β-catenin pathway.

Bronchopulmonary dysplasia (BPD) is one of the most common and severe chronic diseases in preterm infants. Premature infants are susceptible to BPD due to immature lungs and adverse perinatal episodes of infection, hyperoxia, and mechanical ventilation. Neutrophils are the first line of host defence, and the release of neutrophil extracellular traps (NETs) is an important strategy to immobilize and kill invading microorganisms. This study examined whether NETs were associated with BPD in preterm infants and contributed to hyperoxia-induced lung injury in neonatal mice via the WNT/β-catenin pathway. In this study, we found that preterm infants with BPD had higher levels of NETs in their tracheal aspirates than those without BPD. Neonatal mice treated with NETs after birth exhibited BPD-like changes in their lungs. Furthermore, the levels of Aquaporin 5 (AQP5) and surfactant-associated protein C (SPC), which represent alveolar differentiation and development, were significantly lower than those in the controls. The WNT/β-catenin pathway is one of the most well-known signalling pathways involved in lung growth. We found that the expression of the target genes c-MYC, cyclin D, and vascular endothelial growth factor (VEGF) and the important proteins WNT3a and β-catenin significantly decreased. Moreover, heparin, which is a NET inhibitor, attenuated changes in gene and protein expression, thereby attenuating BPD-like changes. This finding indicates that NETs are associated with BPD and can induce BPD-like changes in neonatal mice via the WNT/β-catenin pathway.

Developmental Basis and Consequences of a Key Innovation in Lungless Salamanders

A longstanding goal of evolutionary developmental biology is to document the precise mechanisms that underlie formation of, and differences among, basic body plans, organ systems and physiological functions. Another, less‐studied approach is to reveal how those mechanisms are perturbed to enable, or even mediate, the origin of novel adaptations that are critical for the evolutionary success of individual clades. I will report results of our recent studies of evolutionary lung loss in plethodontid salamanders and of the correlated evolution of cutaneous respiration, which is the principal mode of respiratory gas exchange in all 478 species in this, the largest family of extant urodeles. Surprisingly, plethodontids are not entirely lungless: a lung rudiment forms initially in embryos but is resorbed before hatching. Some embryonic cells express lung‐associated molecular markers, such as the gene Nkx2.1, although at lower levels than in lunged species. Moreover, elevated levels of TGF‐ß signaling, which is known to mediate Nkx2.1 expression during normal lung development, may be part of the genetic mechanism responsible for lung loss. In contrast, we have discovered in salamanders a novel paralog of the gene surfactant‐associated protein C (SFTPC), which is a critical component of pulmonary surfactant expressed exclusively in the lung in other vertebrates. In plethodontids, however, and only in plethodontids, this gene is expressed in novel sites of respiration outside the lungs and at different sites in larvae vs. adults that correlate with gas exchange. These and other changes to the ancestral urodele ontogeny may have contributed to the remarkable evolutionary radiation of the lungless salamanders.

Down expression of lnc-BMP1-1 decreases that of Caveolin-1 is associated with the lung cancer susceptibility and cigarette smoking history.

Lnc-BMP1-1 is a lncRNA transcribed from SFTPC (surfactant associated protein C), a lung tissue specific gene encoding pulmonary-associated surfactant protein C (SPC) that is solely secreted by alveolar typeⅡ epithelial cells, among which the ones with SFTPC+ might be transformed into lung adenocarcinoma cells. Caveolin-1 (Cav-1) is a candidate tumor suppressor gene and is vital for coping with oxidative stress induced by cigarette smoke. When comparing lung cancer tissues with their adjacent normal tissues, the expression of lnc-BMP1-1 were decreased, especially in patients with cigarette smoking history (P=0.027), and positively associated with the expression of Cav-1 (P<0.001). When comparing to A549 cells transfected with empty vector (A549-NC cells), the expression level of Cav-1 in A549 cells with over-expressed lnc-BMP1-1 (A549-BMP cells) was increased along with the decreased level of HDAC2 protein. The drug sensitivity of A549-BMP cells to Doxorubicin hydrochloride (DOX) was increased; the growth and migration capability of A549-BMP cells were inhibited along with the decreased protein level of Bcl-2 and DNMT3a; the growth of tumor in nude mice injected with A549-BMP cells were inhibited, too. Furthermore, the lnc-BMP1-1 and Cav-1 expression was also down-regulated in the human bronchial epithelial (16HBE) cells treated with cigarette smoke extract (CSE).

Bone Marrow Mesenchymal Stem Cell with Up-Regulation of MicroRNA-206 in the Treatment of Bronchopulmonary Dysplasia in Newborn Mice

Background:: Bronchopulmonary dysplasia (BPD) is a chronic lung disease of preterm neonates with a few therapeutic options. Our previous studies suggested that bone marrow-derived mesenchymal stem cell (BMSC) therapy is effective in the treatment of BPD in mouse models. Furthermore, microRNA-206 (miR-206) suggested having a correlation with BPD by regulating the expression of fibronectin 1 (FN1). Objectives:: The aim of this study was to characterize the role of BMSCs with the up-regulation of miR-206 in the treatment of BPD in newborn mice. Methods: The BPD mouse model was induced by 60% oxygen. BMSCs transfected with miR-206 or negative control (NC) plasmid were administered on postnatal day 7 (P7) and every following week through an intraperitoneal injection (105 cells/animal) until P35. The mice were weighed and their lungs were weighed and prepared for histology. The expression levels of miR-206, FN1, and pulmonary surfactant-associated protein-C (SP-C) were determined through the quantitative polymerase chain reaction and under the immunofluorescence. The protein expression levels of transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6) were determined by ELISA. Results: BMSCs with up-regulation of miR-206 reduced the degrees of pulmonary edema, improved pulmonary architecture, and attenuated inflammation in BPD mice compared to the NC plasmid group. Furthermore, the up-regulation of miR-206 in BMSCs down-regulated the expression levels of TGF-β and IL-6 in plasma samples and of SP-C and FN1 in the lung tissue of BPD mice. Conclusions: BMSCs with up-regulation of miR-206 are effective in the treatment of BPD in newborn mice, and the study introduced a novel idea for the treatment of BPD.

Phycocyanin attenuates pulmonary fibrosis via the TLR2-MyD88-NF-\u03baB signaling pathway

Our aim was to investigate the effects of phycocyanin (PC) on bleomycin (BLM)-induced pulmonary fibrosis (PF). In this study, C57 BL/6 wild-type (WT) mice and toll-like receptor (TLR) 2 deficient mice were treated with PC for 28 days following BLM exposure. Serum and lung tissues were collected on days 3, 7 and 28. Data shows PC significantly decreased the levels of hydroxyproline (HYP), vimentin, surfactant-associated protein C (SP-C), fibroblast specific protein-1 (S100A4) and α-smooth muscle actin (α-SMA) but dramatically increased E-cadherin and podoplanin (PDPN) expression on day 28. Moreover, PC greatly decreased the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and myeloperoxidase (MPO) at the earlier time. Reduced expression of key genes in the TLR2 pathway was also detected. Compared with WT mice, TLR2-deficient mice exhibited less injury, and the protective effect of PC was partly diminished in this background. These data indicate the anti-fibrotic effects of PC may be mediated by reducing W/D ratio, MPO, IL-6, TNF-α, protecting type I alveolar epithelial cells, inhibiting fibroblast proliferation, attenuating epithelial-mesenchymal transitions (EMT) and reducing oxidative stress. The TLR2-MyD88-NF-κB pathway plays an important role in PC-mediated reduction in pulmonary fibrosis.

Alveolar Type II Cells Possess the Capability of Initiating Lung Tumor Development

Identifying cells of tumor origin is a fundamental question in tumor biology. Answers to this central question will not only advance our understanding of tumor initiation and progression but also have important therapeutic implications. In this study, we aimed to uncover the cells of origin of lung adenocarcinoma, a major subtype of non-small cell lung cancer. To this end, we developed new mouse models of lung adenocarcinoma that enabled selective manipulation of gene activity in surfactant associated protein C (SPC)-expressing cells, including alveolar type II cells and bronchioalveolar stem cells (BASCs) that reside at the bronchioalveolar duct junction (BADJ). Our findings showed that activation of oncogenic Kras alone or in combination with the removal of the tumor suppressor p53 in SPC+ cells resulted in development of alveolar tumors. Similarly, sustained EGF signaling in SPC+ cells led to alveolar tumors. By contrast, BASCs failed to proliferate or produce tumors under these conditions. Importantly, in a mouse strain in which Kras/p53 activity was selectively altered in type II cells but not BASCs, alveolar tumors developed while BADJs retained normal architecture. These results confirm and extend previous findings and support a model in which lung adenocarcinoma can initiate in alveolar type II cells. Our results establish the foundation for elucidating the molecular mechanisms by which lung cancer initiates and progresses in a specific lung cell type.

Construction of eukaryote expression vector carrying human pulmonary surfactant-associated protein C and its expression in vitro

To clone human pulmonary surfactant-associated protein C (SP-C) and construct an eukaryote expression vector pcDNA3.1(+)/SP-C, and then to examine the SP-C expression in vitro, which may provide a reliable way of massive production of SP-C. The total RNA of normal lung tissue neighboring lung cancer from patients undergoing operation was extracted, and SP-C cDNA was then obtained by reverse transcription-polymerase chain reaction (RT-PCR). Both SP-C cDNA sequence and plasmid pcDNA3.1(+) were digested with NotI and XhoI, then connected by T4 DNA ligase after recycling agarose. After identification by restriction analysis and DNA sequencing, the recombinant was transfected into human breast cancer MCF-7 cells by using lipofectin reagent, and then the expression of SP-C was examined by RT-PCR and Western blotting. Human SP-C cDNA could be correctly cloned into the plasmid pcDNA3.1(+), and SP-C protein may be expressed in MCF-7 cells after transfection. By in vitro recombination, the eukaryote expression vector pcDNA3.1(+)/SP-C was successfully constructed and expressed SP-C in vitro, which rendered preparation for the construction of specific expression vector of human SP-C, and it laid the foundation of massive production of SP-C through mammary gland bioreactor.

Time course of housekeeping gene expression changes in diffuse alveolar damage induced by hyperoxia exposure in mice

We have found diffuse alveolar damage (DAD) has taken place in some patients under mechanical ventilation with high-inspired oxygen concentrations. To clarify the molecular pathophysiology of this, the time course of gene expression changes induced by hyperoxia exposure in mouse lungs was examined using real-time quantitative polymerase chain reaction (real-time qPCR). Our raw data and those normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) showed that: (1) there is a decrease in levels of mRNAs for surfactant-associated protein C (SFTPC), cytochrome P450, 2F2 (CYP2F2), Claudin 1 (CLDN1), membrane-associated zonula occludens protein-1 (ZO-1), lysozyme (LYZS), and this suggests alveolar dysfunction and a disruption of the immune system, (2) we confirmed apoptotic conditions, such as significant up-regulations of mRNA levels in Myc and Galectin-3, and (3) hyperoxic conditions probably yielded reactive oxygen species (ROS), which resulted in a malignant cycle of ROS production by Myc overexpression [Shimada I, Matsui K, Brinkmann B, Hohoff C, Hiraga K, Tabuchi Y, et al. Novel transcript profiling of diffuse alveolar damage induced by hyperoxia exposure in mice: normalization by glyceraldehyde 3-phosphate dehydrogenase. Int J Legal Med 2008;122:373–83]. In this experiment, GAPDH was up-regulated when hyperoxia exposure was continued. Therefore, we reexamined our data and found that: (1) mRNA levels of other housekeeping genes, including β 2-microglobulin (β2M), ribosomal protein: large P2 (RPLP2), and importin 8 (IPO8) altered to a lesser extent, (2) mRNA levels of β2M and IPO8 were down-regulated when hyperoxia exposure was continued, and (3) our previous work was validated by normalization with these three housekeeping genes.

Novel transcript profiling of diffuse alveolar damage induced by hyperoxia exposure in mice: normalization by glyceraldehyde 3-phosphate dehydrogenase

Under mechanical ventilation with high-inspired oxygen concentration, diffuse alveolar damage was found to take place in some patients. To clarify the molecular pathophysiology of this condition, we investigated the time course of gene expression changes induced by hyperoxia exposure in mouse lung using real-time quantitative polymerase chain reaction (qPCR). Our results normalized by glyceraldehyde 3-phosphate dehydrogenase showed that mRNA levels of cysteine rich protein 61 (CYR61) and connective tissue growth factor (CTGF) were significantly upregulated, while those of surfactant-associated protein C (SFTPC), cytochrome P450, 2F2 (CYP2F2), Claudin 1, (CLDN1), membrane-associated zonula occludens protein-1 (ZO-1), lysozyme (LYZS), and P lysozyme structural (LZP-S) were significantly downregulated. Increasing level of mRNAs, each encoding CYR61 and CTGF, suggests a serious risk of fibrosing alveolitis. Decrease in levels of mRNAs for SFTPC, CYP2F2, CLDN1, ZO-1, LYZS, and LZP-S suggests alveolar dysfunction and disruption of the immune system. Moreover, we confirmed apoptotic conditions, such as significant upregulations of mRNA levels in Myc and Galectin-3. Hyperoxic condition probably yielded reactive oxygen species (ROS), which resulted in a malignant cycle of ROS production by Myc overexpression.

Indole-3-carbinol Inhibits 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Plus Benzo(a)pyrene–Induced Lung Tumorigenesis in A/J Mice and Modulates Carcinogen-Induced Alterations in Protein Levels

We tested the chemopreventive efficacy of indole-3-carbinol (I3C), a constituent of Brassica vegetables, and its major condensation product, 3,3'-diindolylmethane (DIM), against lung tumorigenesis induced by a mixture of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP) in A/J mice. The mixture of NNK plus BaP (2 micromol each) was administered by gavage as eight weekly doses, whereas I3C (112 micromol/g diet) and DIM (2 and 30 micromol/g diet in experiments 1 and 2, respectively) were given in the diet for 23 weeks beginning at 50% of carcinogen treatment. I3C reduced NNK plus BaP-induced tumor multiplicity by 78% in experiment 1 and 86% in experiment 2; the respective reductions in tumor multiplicity by DIM were 5% and 66%. Using a quantitative proteomics method, isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry, we identified and quantified at least 250 proteins in lung tissues. Of these proteins, nine showed differences in relative abundance in lung tissues of carcinogen-treated versus untreated mice: fatty acid synthase, transketolase, pulmonary surfactant-associated protein C (SP-C), L-plastin, annexin A1, and haptoglobin increased, whereas transferrin, alpha-1-antitrypsin, and apolipoprotein A-1 decreased. Supplementation of the diet of carcinogen-treated mice with I3C reduced the level of SP-C, L-plastin, annexin A1, and haptoglobin to that of untreated controls. These results were verified using immunoblotting. We show here that tumor-associated signature proteins are increased during NNK plus BaP-induced lung carcinogenesis, and I3C inhibits this effect, suggesting that the lung tumor chemopreventive activity of I3C might be related to modulation of carcinogen-induced alterations in protein levels.

Effect of pulmonary surfactant on TNF-α-activated endothelial cells and neutrophil adhesion in vitro

Pulmonary surfactant given to infants and adults with respiratory failure is metabolized and recycled to a large extent. A small proportion also enters the circulation in cases of increased permeability of the alveolar-capillary membrane. We therefore investigated whether exogenous surfactants such as a natural bovine (natSF) or a synthetic (synSF) preparation had an impact on inflammatory conditions involving the adhesion of neutrophils to endothelial cells. Human umbilical cord vein endothelial cells (HUVEC) were plated on coverslips until confluence, activated by tumor necrosis factor-alpha and incubated with or without surfactant in the media. Human neutrophils passed the HUVEC layer in a flow chamber and interactions were visualized using a video microscope. To test if surfactant affected the expression of cell adhesion molecules, RT-PCR analyses were performed for E-selectin, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1). Using concentrations between 50 and 300μg/ml of surfactant in the pre-incubation media the number of adherent neutrophils increased by 10–20% at the higher concentration of the natSF (*P<0.05) whereas the synSF had no effect. Increased neutrophil adhesion was associated with a significant up-regulation of mRNA levels for E-selectin and VCAM-1; mRNA levels for ICAM-1, however, were not affected by the presence of surfactant. These observations indicate that natSF but not synSF might have pro-inflammatory effects when higher amounts of the exogenous dose reach the circulation. This might be explained by different fatty acid profiles, e.g. the presence of arachidonic acid in the natSF or higher concentrations of surfactant-associated protein-C in the synSF.

Structural model for an AxxxG‐mediated dimer of surfactant‐associated protein C

The pulmonary surfactant prevents alveolar collapse and is required for normal pulmonary function. One of the important components of the surfactant besides phospholipids is surfactant-associated protein C (SP-C). SP-C shows complex oligomerization behavior and a transition to beta-amyloid-like fibril structures, which are not yet fully understood. Besides this nonspecific oligomerization, MS and chemical cross-linking data combined with CD spectra provide evidence of a specific, mainly alpha-helical, dimer at low to neutral pH. Furthermore, resistance to CNBr cleavage and dual NMR resonances of porcine and human recombinant SP-C with Met32 replaced by isoleucine point to a dimerization site located at the C-terminus of the hydrophobic alpha-helix of SP-C, where a strictly conserved heptapeptide sequence is found. Computational docking of two SP-C helices, described here, reveals a dimer with a helix-helix interface that strikingly resembles that of glycophorin A and is mediated by an AxxxG motif similar to the experimentally determined GxxxG pattern of glycophorin A. It is highly likely that mature SP-C adopts such a dimeric structure in the lamellar bilayer systems found in the surfactant. Dimerization has been shown in previous studies to have a role in sorting and trafficking of SP-C and may also be important to the surfactant function of this protein.

The N-terminal Propeptide of Lung Surfactant Protein C is Necessary for Biosynthesis and Prevents Unfolding of a Metastable α-Helix

The lung surfactant-associated protein C (SP-C) consists mainly of a polyvaline α-helix, which is stable in a lipid membrane. However, in agreement with the predicted β-strand conformation of a polyvaline segment, helical SP-C unfolds and transforms into β-sheet aggregates and amyloid fibrils within a few days in aqueous organic solvents. SP-C fibril formation and aggregation have been associated with lung disease. Here, we show that in a recently isolated biosynthetic precursor of SP-C (SP-Ci), a 12 residue N-terminal propeptide locks the metastable polyvaline part in a helical conformation. The SP-Ci helix does not aggregate or unfold during several weeks of incubation, as judged by hydrogen/deuterium exchange and mass spectrometry. Hydrogen/deuterium exchange experiments further indicate that the propeptide reduces exchange in parts corresponding to mature SP-C. Finally, in an acidic environment, SP-Ci unfolds and aggregates into amyloid fibrils like SP-C. These data suggest a direct role of the N-terminal propeptide in SP-C biosynthesis.

Molecular determinants for amyloid fibril formation: lessons from lung surfactant protein C.

Amyloid fibrils are polymers composed of proteins in beta-sheet conformation, which are found in at least 20 diseases for which no cure is available. These diseases include Alzheimer's disease and spongiform encephalopathies, in which the amyloid beta-peptide and the prion protein (PrP), respectively, form amyloid. All fibrils are morphologically very similar although the native structures of the corresponding proteins are widely different. Proteins that are not known to form fibrils in vivo can do so under conditions where unfolding intermediates are well populated. This indicates that fibril formation can arise from most, if not all, polypeptide chains under certain conditions, and that nature has found ways to avoid fibrillogenic protein conformations. In support of this, it was recently found that unpaired beta-strands present in native proteins are prevented from forming intermolecular beta-sheets, by strategic placement of prolines and charged residues for example. Structural studies of the lung surfactant-associated protein C (SP-C) have revealed determinants for amyloid fibril formation. The poly-valine alpha-helix of SP-C spontaneously converts to beta-sheet aggregates in vitro and SP-C amyloid fibrils are found in pulmonary alveolar proteinosis. A beta, PrP, and SP-C harbour an alpha-helix which is strongly predicted to form a beta-strand, and in all cases investigated so far such alpha-helix/beta-sheet discordance correlates with the ability to form beta-sheet aggregates and fibrils.

Molecular determinants for amyloid fibril formation: lessons from lung surfactant protein C.

Amyloid fibrils are polymers composed of proteins in beta-sheet conformation, which are found in at least 20 diseases for which no cure is available. These diseases include Alzheimer's disease and spongiform encephalopathies, in which the amyloid beta-peptide and the prion protein (PrP), respectively, form amyloid. All fibrils are morphologically very similar although the native structures of the corresponding proteins are widely different. Proteins that are not known to form fibrils in vivo can do so under conditions where unfolding intermediates are well populated. This indicates that fibril formation can arise from most, if not all, polypeptide chains under certain conditions, and that nature has found ways to avoid fibrillogenic protein conformations. In support of this, it was recently found that unpaired beta-strands present in native proteins are prevented from forming intermolecular beta-sheets, by strategic placement of prolines and charged residues for example. Structural studies of the lung surfactant-associated protein C (SP-C) have revealed determinants for amyloid fibril formation. The poly-valine alpha-helix of SP-C spontaneously converts to beta-sheet aggregates in vitro and SP-C amyloid fibrils are found in pulmonary alveolar proteinosis. A beta, PrP, and SP-C harbour an alpha-helix which is strongly predicted to form a beta-strand, and in all cases investigated so far such alpha-helix/beta-sheet discordance correlates with the ability to form beta-sheet aggregates and fibrils.

Changes in lung-specific molecular expression during differentiation of hamster embryonic M3E3/C3 cell line

To clarify the differentiation mechanisms of bronchiolar epithelial cells, changes at the transcription level of epithelial cell-specific proteins were examined using M3E3/C3, a cell line derived from hamster fetal lung. During a 9-day incubation period with 24 microg/mL of retinol, the cells became attached to each other and formed large bud-like structures which could be detected with periodic acid-Schiff staining. During the incubation period, the mRNA level of surfactant-associated protein-B significantly increased 2.6- and 5.4-fold higher than cells incubated without retinol on days 3 and 9, respectively. The Clara cell-specific secretory protein mRNA level also increased and peaked at 5.1-fold (P < 0.05) on day 5 compared with control cells. In contrast, mRNA for surfactant-associated protein-C, an alveolar type II cell-specific protein, decreased. Moreover, the expression of the gene for hepatocyte nuclear factor 3alpha, a putative transactivating factor for lung-related genes, was up-regulated resulting in consistently higher levels (2.4- to 5.6-fold) compared with controls, while those for transmembrane-type mucin-1 and glyceraldehyde-3-phosphate dehydrogenase were constantly expressed during the incubation. The present study confirms that at the gene transcription level M3E3/C3 cells differentiate into Clara-like cells with mucus granules in the presence of retinol.

Differential effects of halothane and thiopental on surfactant protein C messenger RNA in vivo and in vitro in rats.

Pulmonary surfactant is a complex mixture of proteins and phospholipids synthetized by alveolar type II cells. Volatile anesthetics have been shown to reduce surfactant phospholipid biosynthesis by rat alveolar type II cells. Surfactant-associated protein C (SP-C) is critical for the alveolar surfactant functions. Our goal was to evaluate the effects of halothane and thiopental on SP-C messenger RNA (mRNA) expression in vitro in rat alveolar type II cells and in vivo in mechanically ventilated rats. In vitro, freshly isolated alveolar type II cells were exposed to halothane during 4 h (1, 2, 4%) and 8 h (1%), and to thiopental during 4 h (10, 100 micrometer) and 8 h (100 micrometer). In vivo, rats were anesthetized with intraperitoneal thiopental or inhaled 1% halothane and mechanically ventilated for 4 or 8 h. SP-C mRNA expression was evaluated by ribonuclease protection assay. In vitro, 4-h exposure of alveolar type II cells to thiopental 10 and 100 micrometer increased their SP-C mRNA content to 145 and 197%, respectively, of the control values. In alveolar type II cells exposed for 4 h to halothane 1, 2, and 4%, the SP-C mRNA content increased dose-dependently to 160, 235, and 275%, respectively, of the control values. In vivo, in mechanically ventilated rats, 4 h of halothane anesthesia decreased the lung SP-C mRNA content to 53% of the value obtained in control (nonanesthetized, nonventilated) animals; thiopental anesthesia increased to 150% the lung SP-C mRNA content. These findings indicate that halothane and thiopental used at clinically relevant concentrations modulate the pulmonary SP-C mRNA content in rats. In vivo, the additive role of mechanical ventilation is suggested.

Distribution of the Surfactant-Associated Protein C within a Lung Surfactant Model Film Investigated by Near-Field Optical Microscopy

Lung surfactant films at the air/water interface exhibit the particularity that surfactant molecules are expelled from the surface monolayer into a surface associated multilamellar phase during compression. They are able to re-enter the surface film during the following expansion. The underlying mechanism for this behavior is not fully understood yet. However, an important role is ascribed to the surfactant-associated protein C (SP-C). Here, we studied a model lung surfactant, consisting of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and SP-C, by means of scanning near-field optical microscopy (SNOM). Attaching a fluorescent dye to the protein allowed the localization of its lateral distribution at various surface pressures with high resolution. At an early stage of compression, the film appears demixed into a pure lipid phase and a protein-enriched phase. Within the latter phase, protein aggregations are revealed. They show a uniform density, having three times the fluorescence intensity of their surroundings. Across the phase boundary between the lipid phase and the protein-rich phase, there is a protein density gradient rather than an abrupt border. When the film is highly compressed, we observe the formation of multilamellar structures that are fluorescent. They are often surrounded by a slightly fluorescent monolayer. The fluorescence of the multilayer stacks (i.e., the protein content per unit area) is proportional to the height of the stacks.

Changes in lung-specific molecular expression during differentiation of hamster embryonic M3E3/C3 cell line

To clarify the differentiation mechanisms of bronchiolar epithelial cells, changes at the transcription level of epithelial cell-specific proteins were examined using M3E3/C3, a cell line derived from hamster fetal lung. During a 9-day incubation period with 24 microg/mL of retinol, the cells became attached to each other and formed large bud-like structures which could be detected with periodic acid-Schiff staining. During the incubation period, the mRNA level of surfactant-associated protein-B significantly increased 2.6- and 5.4-fold higher than cells incubated without retinol on days 3 and 9, respectively. The Clara cell-specific secretory protein mRNA level also increased and peaked at 5.1-fold (P < 0.05) on day 5 compared with control cells. In contrast, mRNA for surfactant-associated protein-C, an alveolar type II cell-specific protein, decreased. Moreover, the expression of the gene for hepatocyte nuclear factor 3alpha, a putative transactivating factor for lung-related genes, was up-regulated resulting in consistently higher levels (2.4- to 5.6-fold) compared with controls, while those for transmembrane-type mucin-1 and glyceraldehyde-3-phosphate dehydrogenase were constantly expressed during the incubation. The present study confirms that at the gene transcription level M3E3/C3 cells differentiate into Clara-like cells with mucus granules in the presence of retinol.