Thorium Alters Lung Surfactant Protein Expression in Alveolar Epithelial Cells: In Vitro and In Vivo Investigation.

Thorium-232 (Th), the most abundant naturally occurring nuclear fuel, has been identified as a sustainable source of energy. In view of its large-scale utilization and human evidence of lung disorders and carcinogenicity, it is imperative to understand the effect of Th exposure on lung cells. The present study investigated the effect of Th-dioxide (1-100 µg/mL, 24-48 h) on expression of surfactant proteins (SPs) (SP-A, SP-B, SP-C, and SP-D, which are essential to maintain lung's surface tension and host-defense) in human lung cells (WI26 and A549), representative of alveolar cell type-I and type-II, respectively. Results demonstrated the inhibitory effect of Th on transcriptional expression of SP-A, SP-B, and SP-C. However, Th promoted the mRNA expression of SP-D in A549 and reduced its expression in WI26. To a significant extent, the effect of Th on SPs was found to be in accordance with their protein levels. Moreover, Th exposure altered the extracellular release of SP-D/A from A549, which remained unaltered in WI26. Our results suggested the differential role of oxidative stress and ATM and HSP90 signaling in Th-induced alterations of SPs. These effects of Th were found to be consistent in lung tissues of mice exposed to Th aerosols, suggesting a potential role of SPs in Th-associated lung disorders.

Proteomics of Galápagos Marine Iguanas Links Function of Femoral Gland Proteins to the Immune System.

Communication between individuals via molecules, termed chemosignaling, is widespread among animal and plant species. However, we lack knowledge on the specific functions of the substances involved for most systems. The femoral gland is an organ that secretes a waxy substance involved in chemical communication in lizards. Although the lipids and volatile substances secreted by the femoral glands have been investigated in several biochemical studies, the protein composition and functions of secretions remain completely unknown. Applying a proteomic approach, we provide the first attempt to comprehensively characterize the protein composition of femoral gland secretions from the Galápagos marine iguana. Using samples from several organs, the marine iguana proteome was assembled by next-generation sequencing and MS, resulting in 7513 proteins. Of these, 4305 proteins were present in the femoral gland, including keratins, small serum proteins, and fatty acid-binding proteins. Surprisingly, no proteins with discernible roles in partner recognition or inter-species communication could be identified. However, we did find several proteins with direct associations to the innate immune system, including lysozyme C, antileukoproteinase (ALP), pulmonary surfactant protein (SFTPD), and galectin (LGALS1) suggesting that the femoral glands function as an important barrier to infection. Furthermore, we report several novel anti-microbial peptides from the femoral glands that show similar action against Escherichia coli and Bacillus subtilis such as oncocin, a peptide known for its effectiveness against Gram-negative pathogens. This proteomics data set is a valuable resource for future functional protein analysis and demonstrates that femoral gland secretions also perform functions of the innate immune system.

Heterogeneity in Human Induced Pluripotent Stem Cell-derived Alveolar Epithelial Type II Cells Revealed with ABCA3/SFTPC Reporters.

Alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, are typically identified through the use of the canonical markers, SFTPC and ABCA3. Self-renewing AEC2-like cells have been generated from human induced pluripotent stem cells (iPSCs) through the use of knock-in SFTPC fluorochrome reporters. However, developmentally, SFTPC expression onset begins in the fetal distal lung bud tip and thus is not specific to mature AEC2s. Furthermore, SFTPC reporters appear to identify only those iPSC-derived AEC2s (iAEC2s) expressing the highest SFTPC levels. Here, we generate an ABCA3 knock-in GFP fusion reporter (ABCA3:GFP) that enables the purification of iAEC2s while allowing visualization of lamellar bodies, organelles associated with AEC2 maturation. Using an SFTPCtdTomato and ABCA3:GFP bifluorescent line for in vitro distal lung-directed differentiation, we observe later onset of ABCA3:GFP expression and broader identification of the subsequently emerging iAEC2 population based on ABCA3:GFP expression compared with SFTPCtdTomato expression. Comparing ABCA3:GFP/SFTPCtdTomato double-positive with ABCA3:GFP single-positive (SP) cells by RNA sequencing and functional studies reveals iAEC2 cellular heterogeneity with both populations functionally processing surfactant proteins but the SP cells exhibiting faster growth kinetics, increased clonogenicity, increased expression of progenitor markers, lower levels of SFTPC expression, and lower levels of AEC2 maturation markers. Over time, we observe that each population (double-positive and SP) gives rise to the other and each can serve as the parents of indefinitely self-renewing iAEC2 progeny. Our results indicate that iAEC2s are a heterogeneous population of cells with differing proliferation versus maturation properties, the majority of which can be tracked and purified using the ABCA3:GFP reporter or surrogate cell surface proteins, such as SLC34A2 and CPM.

Hydroxypropyl Cyclodextrin Improves Amiodarone-induced Aberrant Lipid Homeostasis of Alveolar Cells.

Alveolar epithelial type II (AT2) cells secrete pulmonary surfactant via lamellar bodies (LBs). Abnormalities in LBs are associated with pulmonary disorders, including fibrosis. However, high-content screening (HCS) for LB abnormalities is limited by the lack of understanding of AT2 cell functions. In the present study, we have developed LB cells harboring LB-like organelles that secrete surfactant proteins. These cells were more similar to AT2 cells than to parental A549 cells. LB cells recapitulated amiodarone (AMD)-induced LB enlargement, similar to AT2 cells of patients exposed to AMD. To reverse AMD-induced LB abnormalities, we performed HCS of approved drugs and identified 2-hydroxypropyl-ß-cyclodextrin (HPßCD), a cyclic oligosaccharide, as a potential therapeutic agent. A transcriptome analysis revealed that HPßCD modulates lipid homeostasis. In addition, HPßCD inhibited AMD-induced LB abnormalities in human induced pluripotent stem cell-derived AT2 cells. Our results demonstrate that LB cells are useful for HCS and suggest that HPßCD is a candidate therapeutic agent for AMD-induced interstitial pneumonia.

A primate-specific RNA-binding protein (RBMXL3) is involved in glucocorticoid regulation of human pulmonary surfactant protein B (SP-B) mRNA stability.

The ability of pulmonary surfactant to reduce alveolar surface tension requires adequate levels of surfactant protein B (SP-B). Dexamethasone (DEX) increases human SP-B expression, in part, through increased SP-B mRNA stability. A 30-nt-long hairpin element (RBE) in the 3'-untranslated region of human SP-B mRNA mediates both DEX-induced and intrinsic mRNA stabilities, but the mechanism is unknown. Proteomic analysis of RBE-interacting proteins identified a primate-specific protein, RNA-binding motif X-linked-like-3 (RBMXL3). siRNA directed against RBMXL3 reduces DEX-induced SP-B mRNA expression in human bronchoalveolar cells. Human SP-B mRNA stability, measured by our dual cistronic plasmid assay, is unaffected by DEX in mouse lung epithelial cells lacking RBMXL3, but DEX increases human SP-B mRNA stability when RBMXL3 is expressed and requires the RBE. In the absence of DEX, RBE interacts with cellular proteins, reducing intrinsic SP-B mRNA stability in human and mouse lung epithelial cells. RBMXL3 specifically binds the RBE in vitro, whereas RNA immunoprecipitation and affinity chromatography analyses indicate that binding is enhanced in the presence of DEX. These results describe a model where intrinsic stability of human SP-B mRNA is reduced through binding of cellular mRNA decay factors to RBE, which is then relieved through DEX-enhanced binding of primate-specific RBMXL3.

Myeloid cell dynamics in bleomycin-induced pulmonary injury in mice; effects of anti-TNFα antibody.

Bleomycin is a cancer therapeutic known to cause lung injury which progresses to fibrosis. Evidence suggests that macrophages contribute to this pathological response. Tumor necrosis factor (TNF)α is a macrophage-derived pro-inflammatory cytokine implicated in lung injury. Herein, we investigated the role of TNFα in macrophage responses to bleomycin. Treatment of mice with bleomycin (3 U/kg, i.t.) caused histopathological changes in the lung within 3 d which culminated in fibrosis at 21 d. This was accompanied by an early (3-7 d) influx of CD11b+ and iNOS+ macrophages into the lung, and Arg-1+ macrophages at 21 d. At this time, epithelial cell dysfunction, defined by increases in total phospholipids and SP-B was evident. Treatment of mice with anti-TNFα antibody (7.5 mg/kg, i.v.) beginning 15-30 min after bleomycin, and every 5 d thereafter reduced the number and size of fibrotic foci and restored epithelial cell function. Flow cytometric analysis of F4/80+ alveolar macrophages (AM) isolated by bronchoalveolar lavage and interstitial macrophages (IM) by tissue digestion identified resident (CD11b-CD11c+) and immature infiltrating (CD11b+CD11c-) AM, and mature (CD11b+CD11c+) and immature (CD11b+CD11c-) IM subsets in bleomycin treated mice. Greater numbers of mature (CD11c+) infiltrating (CD11b+) AM expressing the anti-inflammatory marker, mannose receptor (CD206) were observed at 21 d when compared to 7 d post bleomycin. Mature proinflammatory (Ly6C+) IM were greater at 7 d relative to 21 d. These cells transitioned into mature anti-inflammatory/pro-fibrotic (CD206+) IM between 7 and 21 d. Anti-TNFα antibody heightened the number of CD11b+ AM in the lung without altering their activation state. Conversely, it reduced the abundance of mature proinflammatory (Ly6C+) IM in the tissue at 7 d and immature pro-fibrotic IM at 21 d. Taken together, these data suggest that TNFα inhibition has beneficial effects in bleomycin induced injury, restoring epithelial function and reducing numbers of profibrotic IM and the extent of pulmonary fibrosis.

Recombinant expression of the precursor of rat lung surfactant protein B in Escherichia coli and its antibacterial mechanism.

While the discovery of antibiotics has made a huge contribution to medicine, bacteria that are resistant to many antibiotics pose new challenges to medicine. Antimicrobial peptides (AMPs), a new kind of antibiotics, have attracted people's attention because they are not prone to drug resistance. In this study, glutathione transferase (GST) was used as a fusion partner to recombinantly expressed rat lung surfactant protein B precursor (proSP-B) in E. coli pLySs. Cck-8 evaluated the cytotoxicity of the fusion protein and calculated its 50% inhibitory concentration (IC50). The purified peptides showed broad-spectrum antibacterial activity using filter paper method and MIC, and propidium iodide (PI) was used to explore the antibacterial mechanism against Staphylococcus aureus. In addition, the pEGFP-N2-proSP-B vector was constructed to explore the localization of proSP-B in CCL-149 cells. We found that proSP-B has obvious antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and fungi, and has broad-spectrum antibacterial activity. Besides, proSP-B fusion protein has low toxicity and can change the permeability of Staphylococcus aureus cell membrane to realize its antibacterial. For these reasons, proSP-B can be used as a potential natural antibacterial drug.

The History and Mystery of Alveolar Epithelial Type II Cells: Focus on Their Physiologic and Pathologic Role in Lung.

Alveolar type II (ATII) cells are a key structure of the distal lung epithelium, where they exert their innate immune response and serve as progenitors of alveolar type I (ATI) cells, contributing to alveolar epithelial repair and regeneration. In the healthy lung, ATII cells coordinate the host defense mechanisms, not only generating a restrictive alveolar epithelial barrier, but also orchestrating host defense mechanisms and secreting surfactant proteins, which are important in lung protection against pathogen exposure. Moreover, surfactant proteins help to maintain homeostasis in the distal lung and reduce surface tension at the pulmonary air-liquid interface, thereby preventing atelectasis and reducing the work of breathing. ATII cells may also contribute to the fibroproliferative reaction by secreting growth factors and proinflammatory molecules after damage. Indeed, various acute and chronic diseases are associated with intensive inflammation. These include oedema, acute respiratory distress syndrome, fibrosis and numerous interstitial lung diseases, and are characterized by hyperplastic ATII cells which are considered an essential part of the epithelialization process and, consequently, wound healing. The aim of this review is that of revising the physiologic and pathologic role ATII cells play in pulmonary diseases, as, despite what has been learnt in the last few decades of research, the origin, phenotypic regulation and crosstalk of these cells still remain, in part, a mystery.

Sumoylation of CCAAT-enhancer-binding protein α inhibits lung differentiation in Bronchopulmonary Dysplasia model rats.

Bronchopulmonary dysplasia (BPD) is a major cause of mortality and morbidity in premature infants, characterized by alveolar simplification, surfactant deficiency, and respiratory distress. In the present study, we have investigated the functional roles of sumoylated CCAAT/enhancer binding protein alpha (C/EBPα) in the BPD rat model. A significant increase in small ubiquitin-like modifier 1 (SUMO1) and sumoylated C/EBPα protein levels were observed in BPD rats, and the levels of the sumoylated C/EBPα were associated with the pulmonary surfactant proteins (SPs). In order to confirm the role of sumoylated C/EBPα in BPD rats, SUMO1 was knocked down by lentiviral transfection of neonatal rat lungs with SUMO1-RNAi-LV. We found that the expression of C/EBPα and surfactant proteins increased following SUMO1 knockdown. Furthermore, the relatively low decrease in the levels of C/EBPα sumoylation was correlated with reduced glycogen consumption. Besides, co-immunoprecipitation assays revealed that sumoylation is involved in the regulation of the interaction between C/EBPα and TGFß2 in the lung. In conclusion, our findings indicate that sumoylation may act as a negative regulator of the C/EBPα-mediated transactivation in BPD rats.

Preterm birth impairs postnatal lung development in the neonatal rabbit model.

BACKGROUND: Bronchopulmonary dysplasia continues to cause important respiratory morbidity throughout life, and new therapies are needed. The common denominator of all BPD cases is preterm birth, however most preclinical research in this area focusses on the effect of hyperoxia or mechanical ventilation. In this study we investigated if and how prematurity affects lung structure and function in neonatal rabbits. METHODS: Pups were delivered on either day 28 or day 31. For each gestational age a group of pups was harvested immediately after birth for lung morphometry and surfactant protein B and C quantification. All other pups were hand raised and harvested on day 4 for the term pups and day 7 for the preterm pups (same corrected age) for lung morphometry, lung function testing and qPCR. A subset of pups underwent microCT and dark field imaging on day 0, 2 and 4 for terms and on day 0, 3, 5 and 7 for preterms. RESULTS: Preterm pups assessed at birth depicted a more rudimentary lung structure (larger alveoli and thicker septations) and a lower expression of surfactant proteins in comparison to term pups. MicroCT and dark field imaging revealed delayed lung aeration in preterm pups, in comparison to term pups. Preterm birth led to smaller pups, with smaller lungs with a lower alveolar surface area on day 7/day 4. Furthermore, preterm birth affected lung function with increased tissue damping, tissue elastance and resistance and decreased dynamic compliance. Expression of vascular endothelial growth factor (VEGFA) was significantly decreased in preterm pups, however in the absence of structural vascular differences. CONCLUSIONS: Preterm birth affects lung structure and function at birth, but also has persistent effects on the developing lung. This supports the use of a preterm animal model, such as the preterm rabbit, for preclinical research on BPD. Future research that focuses on the identification of pathways that are involved in in-utero lung development and disrupted by pre-term birth, could lead to novel therapeutic strategies for BPD.

Quantitative Lipidomics in Pulmonary Alveolar Proteinosis.

Rationale: Pulmonary alveolar proteinosis (PAP) is characterized by filling of the alveolar spaces by lipoprotein-rich material of ill-defined composition, and is caused by molecularly different and often rare diseases that occur from birth to old age.Objectives: To perform a quantitative lipidomic analysis of lipids and the surfactant proteins A, B, and C in lavage fluids from patients with proteinosis of different causes in comparison with healthy control subjects.Methods: During the last two decades, we have collected BAL samples from patients with PAP due to autoantibodies against granulocyte-macrophage colony-stimulating factor; genetic mutations in CSF2RA (colony-stimulating factor 2 receptor α-subunit), MARS (methionyl aminoacyl-tRNA synthetase), FARSB (phenylalanine-tRNA synthetase, ß-subunit), and NPC2 (Niemann-Pick disease type C2); and secondary to myeloid leukemia. Their lipid composition was quantified.Measurements and Main Results: Free cholesterol was largely increased by 60-fold and cholesteryl esters were increased by 24-fold. There was an excessive, more than 130-fold increase in ceramide and other sphingolipids. In particular, the long-chain ceramides d18:1/20:0 and d18:1/24:0 were elevated and likely contributed to the proapoptotic environment observed in PAP. Cellular debris lipids such as phosphatidylethanolamine and phosphatidylserine were only moderately increased, by four- to sevenfold. The surfactant lipid class phosphatidylcholine expanded 17-fold, lysophosphatidylcholine expanded 54-fold, and the surfactant proteins A, B, and C expanded 144-, 4-, and 17-fold, respectively. These changes did not differ among the various diseases that cause PAP.Conclusions: This insight into the alveolar lipidome may provide monitoring tools and lead to new therapeutic strategies for PAP.

Lipid-Protein and Protein-Protein Interactions in the Pulmonary Surfactant System and Their Role in Lung Homeostasis.

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

Short-Term versus Long-Term Culture of A549 Cells for Evaluating the Effects of Lipopolysaccharide on Oxidative Stress, Surfactant Proteins and Cathelicidin LL-37.

Alveolar epithelial type II (ATII) cells and their proper function are essential for maintaining lung integrity and homeostasis. However, they can be damaged by lipopolysaccharide (LPS) during Gram-negative bacterial infection. Thus, this study evaluated and compared the effects of LPS on short and long-term cultures of A549 cells by determining the cell viability, levels of oxidative stress and antimicrobial peptide cathelicidin LL-37 and changes in the expression of surfactant proteins (SPs). Moreover, we compared A549 cell response to LPS in the presence of different serum concentrations. Additionally, the effect of N-acetylcysteine (NAC) on LPS-induced oxidative stress as a possible treatment was determined. Our results indicate that A549 cells are relatively resistant to LPS and able to maintain integrity even at high LPS concentrations. Their response to endotoxin is partially dependent on serum concentration. NAC failed to lower LPS-induced oxidative stress in A549 cells. Finally, LPS modulates SP gene expression in A549 cells in a time dependent manner and differences between short and long-term cultures were present. Our results support the idea that long-term cultivation of A549 cells could promote a more ATII-like phenotype and thus could be a more suitable model for ATII cells, especially for in vitro studies dealing with surfactant production.

miR-20a-5p regulates pulmonary surfactant gene expression in alveolar type II cells.

MicroRNA (miRNA) critically controls gene expression in many biological processes, including lung growth and pulmonary surfactant biosynthesis. The present study was conducted to investigate whether miR-20a-5p had such regulatory functions on alveolar type II (AT-II) cells. To accomplish this, miR-20a-5p-overexpressed and miR-20a-5p-inhibited adenoviral vectors were constructed and transfected into cultured AT-II cells that were isolated from rat foetal lungs of 19 days' gestation. Transfection efficiency was confirmed by observing the fluorescence of green fluorescent protein (GFP) carried by the viral vector, whereas miR-20a-5p levels were verified by real-time PCR. The CCK-8 assay was used to compare the proliferation ability of AT-II cells that had over- or underexpressed miR-20a-5p. The expression of surfactant-associated proteins (SPs) and phosphatase and tensin homolog (PTEN) was measured by real-time PCR and Western blotting. In AT-II cells, transfection resulted in over- or under-regulation of miR-20a-5p. While overexpression of miR-20a-5p promoted pulmonary surfactant gene expression, its underexpression inhibited it. Consistent with its role in negatively regulating the pulmonary surfactant gene, an opposite pattern was observed for miR-20a-5p regulation of PTEN. As a result, when miR-20a-5p was rendered overexpressed, PTEN was down-regulated. By contrast, when miR-20a-5p was underexpressed, PTEN was up-regulated. Neither overexpression nor underexpression of miR-20a-5p altered the cell proliferation. miR-20a-5p plays no role in proliferation of foetal AT-II cells but is a critical regulator of surfactant gene expression. The latter appears to be achieved through a regulatory process that implicates expression of PTEN.

Retention of EsxA in the Capsule-Like Layer of Mycobacterium tuberculosis Is Associated with Cytotoxicity and Is Counteracted by Lung Surfactant.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, primarily infects macrophages but withstands the host cell's bactericidal effects. EsxA, also called virulence factor 6-kDa early secretory antigenic target (ESAT-6), is involved in phagosomal rupture and cell death. We provide confocal and electron microscopy data showing that M. tuberculosis bacteria grown without detergent retain EsxA on their surface. Lung surfactant has detergent-like properties and effectively strips off this surface-associated EsxA, which advocates a novel mechanism of lung surfactant-mediated defense against pathogens. Upon challenge of human macrophages with these M. tuberculosis bacilli, the amount of surface-associated EsxA rapidly declines in a phagocytosis-independent manner. Furthermore, M. tuberculosis bacteria cultivated under exclusion of detergent exert potent cytotoxic activity associated with bacterial growth. Together, this study suggests that the surface retention of EsxA contributes to the cytotoxicity of M. tuberculosis and highlights how cultivation conditions affect the experimental outcome.

Site-specific and endothelial-mediated dysfunction of the alveolar-capillary barrier in response to lipopolysaccharides.

Infectious agents such as lipopolysaccharides (LPS) challenge the functional properties of the alveolar-capillary barrier (ACB) in the lung. In this study, we analyse the site-specific effects of LPS on the ACB and reveal the effects on the individual cell types and the ACB as a functional unit. Monocultures of H441 epithelial cells and co-cultures of H441 with endothelial cells cultured on Transwells® were treated with LPS from the apical or basolateral compartment. Barrier properties were analysed by the transepithelial electrical resistance (TEER), by transport assays, and immunostaining and assessment of tight junctional molecules at protein level. Furthermore, pro-inflammatory cytokines and immune-modulatory molecules were evaluated by ELISA and semiquantitative real-time PCR. Liquid chromatography-mass spectrometry-based proteomics (LS-MS) was used to identify proteins and effector molecules secreted by endothelial cells in response to LPS. In co-cultures treated with LPS from the basolateral compartment, we noticed a significant reduction of TEER, increased permeability and induction of pro-inflammatory cytokines. Conversely, apical treatment did not affect the barrier. No changes were noticed in H441 monoculture upon LPS treatment. However, LPS resulted in an increased expression of pro-inflammatory cytokines such as IL-6 in OEC and in turn induced the reduction of TEER and an increase in SP-A expression in H441 monoculture, and H441/OEC co-cultures after LPS treatment from basolateral compartment. LS-MS-based proteomics revealed factors associated with LPS-mediated lung injury such as ICAM-1, VCAM-1, Angiopoietin 2, complement factors and cathepsin S, emphasizing the role of epithelial-endothelial crosstalk in the ACB in ALI/ARDS.

Irx1 regulates dental outer enamel epithelial and lung alveolar type II epithelial differentiation.

The Iroquois genes (Irx) appear to regulate fundamental processes that lead to cell proliferation, differentiation, and maturation during development. In this report, the Iroquois homeobox 1 (Irx1) transcription factor was functionally disrupted using a LacZ insert and LacZ expression demonstrated stage-specific expression during embryogenesis. Irx1 is highly expressed in the brain, lung, digits, kidney, testis and developing teeth. Irx1 null mice are neonatal lethal and this lethality it due to pulmonary immaturity. Irx1-/- mice show delayed lung maturation characterized by defective surfactant protein secretion and Irx1 marks a population of SP-C expressing alveolar type II cells. Irx1 is specifically expressed in the outer enamel epithelium (OEE), stellate reticulum (SR) and stratum intermedium (SI) layers of the developing tooth. Irx1 mediates dental epithelial cell differentiation in the lower incisors resulting in delayed growth of the lower incisors. Irx1 is specifically and temporally expressed during developmental stages and we have focused on lung and dental development in this report. Irx1+ cells are unique to the development of the incisor outer enamel epithelium, patterning of Lef-1+ and Sox2+ cells as well as a new marker for lung alveolar type II cells. Mechanistically, Irx1 regulates Foxj1 and Sox9 to control cell differentiation during development.

TGF beta inhibits expression of SP-A, SP-B, SP-C, but not SP-D in human alveolar type II cells.

TGF beta is a multifunctional cytokine that regulates alveolar epithelial cells as well as immune cells and fibroblasts. TGF beta inhibits surfactant protein A, B and C expression in fetal human lung and can inhibit type II cell proliferation induced by FGF7 (KGF). However, little is known about direct effects of TGF beta on adult human type II cells. We cultured alveolar type II cells under air/liquid interface conditions to maintain their state of differentiation with or without TGF beta. TGF beta markedly decreased expression of SP-A, SP-B, SP-C, fatty acid synthase, and the phospholipid transporter ABCA3. However, TGF beta increased protein levels of SP-D with little change in mRNA levels, indicating that it is regulated independently from other components of surfactant. TGF beta is a negative regulator of both the protein and the phospholipid components of surfactant. TGF beta did not induce EMT changes in highly differentiated human type II cells. SP-D is an important host defense molecule and regulated independently from the other surfactant proteins. Taken together these data are the first report of the effect of TGF beta on highly differentiated adult human type II cells. The effects on the surfactant system are likely important in the development of fibrotic lung diseases.

Low-dose betamethasone-acetate for fetal lung maturation in preterm sheep.

BACKGROUND: Antenatal steroids are standard of care for women who are at risk of preterm delivery; however, antenatal steroid dosing and formulation have not been evaluated adequately. The standard clinical 2-dose treatment with betamethasone-acetate+betamethasone-phosphate is more effective than 2 doses of betamethasone-phosphate for the induction of lung maturation in preterm fetal sheep. We hypothesized that the slowly released betamethasone-acetate component induces similar lung maturation to betamethasone-phosphate+betamethasone-acetate with decreased dose and fetal exposure. OBJECTIVE: The purpose of this study was to investigate pharmacokinetics and fetal lung maturation of antenatal betamethasone-acetate in preterm fetal sheep. STUDY DESIGN: Groups of 10 singleton-pregnant ewes received 1 or 2 intramuscular doses 24 hours apart of 0.25 mg/kg/dose of betamethasone-phosphate+betamethasone-acetate (the standard of care dose) or 1 intramuscular dose of 0.5 mg/kg, 0.25 mg/kg, or 0.125 mg/kg of betamethasone-acetate. Fetuses were delivered 48 hours after the first injection at 122 days of gestation (80% of term) and ventilated for 30 minutes, with ventilator settings, compliance, vital signs, and blood gas measurements recorded every 10 minutes. After ventilation, we measured static lung pressure-volume curves and sampled the lungs for messenger RNA measurements. Other groups of pregnant ewes and fetuses were catheterized and treated with intramuscular injections of betamethasone-phosphate 0.125 mg/kg, betamethasone-acetate 0.125 mg/kg, or betamethasone-acetate 0.5 mg/kg. Maternal and fetal betamethasone concentrations in plasma were measured for 24 hours. RESULTS: All betamethasone-treated groups had increased messenger RNA expression of surfactant proteins A, B, and C, ATP-binding cassette subfamily A member 3, and aquaporin-5 compared with control animals. Treatment with 1 dose of intramuscular betamethasone-acetate 0.125mg/kg improved dynamic and static lung compliance, gas exchange, and ventilation efficiency similarly to the standard treatment of 2 doses of 0.25 m/kg of betamethasone-acetate+betamethasone-phosphate. Betamethasone-acetate 0.125 mg/kg resulted in lower maternal and fetal peak plasma concentrations and decreased fetal exposure to betamethasone compared with betamethasone-phosphate 0.125 mg/kg. CONCLUSION: A single dose of betamethasone-acetate results in similar fetal lung maturation as the 2-dose clinical formulation of betamethasone-phosphate+betamethasone-acetate with decreased fetal exposure to betamethasone. A lower dose of betamethasone-acetate may be an effective alternative to induce fetal lung maturation with less risk to the fetus.

Expression of Surfactant Proteins in the Human Canaliculus: Evidence and Potential Insights Into the Tear Flow Dynamics.

PURPOSE: To investigate the presence and distribution patterns of 6 surfactant proteins (SPs) in the human lacrimal canaliculus. METHODS: The study was performed on fresh frozen cadaveric samples of canaliculi. Immunohistochemical labeling was performed for assessing the presence and distribution of SP: SP-A, SP-B, SP-C, SP-D, SP-G/SFTA2, and SP-H/SFTA3. Immunofluorescence double staining was performed using the respective fluorescein-conjugated antibodies and the results were scored as positive or negative and the distribution pattern within the canalicular system was assessed. Western blot analysis was performed on the protein content which was resolved by reducing 15% sodium dodecyl sulfate-polyacrylamide electrophoresis and bands were studied following staining with primary and secondary antibodies. Human lung tissues were used as controls. RESULTS: Fluorescence double staining with 4,6-diamidino 2-pheynlindole and SPs showed strong immunostaining for SP-A, SP-B, SP-C, SP-D, and SP-H/SFTA3. The positive immunofluorescence was noticed across all the layers of the epithelium but not the subepithelial structures. The expression was noted on the surfaces and superficial cytoplasm of the superficial and deep epithelial cells. There was no expression of SP-G/SFTA2 across the canalicular system. Western blot analysis of the proteins confirmed and concurred with the immunofluorescence findings. CONCLUSIONS: This study provides a proof of principle for the presence of SPs known from lungs in the canalicular system and hypothesizes their possible functions and also their potential role in the tear flow dynamics between the ocular surface and the lacrimal drainage system.