Secretion Of Surfactant Protein Research Articles

Biophysical parameters affecting lung surfactant function, surface tension and the transition from aerosol to droplet exhalation (in relation to COVID-19 infection)

A considerable number of biophysical and biochemical studies has increased our understanding of the surface activity of surfactant proteins B and C (SP-B, SP-C) in the (mammalian) lung and their importance for a healthy, proper breathing system. For instance, it is well-known that these surfactant proteins are released from the lamellar bodies of type II cells of the lung alveoli (type II pneumocytes), and that at compression, the lipid-protein monolayer of the surfactant is squeezed into three-dimensional (3D) stacks, acting as a surfactant reservoir. Moreover, studies have demonstrated the influence of hydrophobic nanoparticles on lung surfactant model systems, well before the outbreak of the COVID-19 pandemic. The potentially devastating effect of SARS-CoV-2 infection on vital lung function, including pneumonia and Acute respiratory distress syndrome (ARDS), meanwhile has been confirmed worldwide by thousands of fatal cases, although the mechanism of its onset is not completely understood. In theory, any virus carrying palmitoylated spike proteins (like beta-coronaviruses in general) might interfere with the alveolar surface activity, when infecting the deep respiratory system. However, it is clearly established that SARS-CoV-2 uses the Angiotensin converting enzyme 2 receptor (ACE2) for entry in the host, which receptor is expressed on the same type II pneumocytes, and, as a result, the virus may directly interfere with the secretion of surfactant proteins. This study aims at elucidating the main targets for containment of the spread of the SARS-CoV-2 virus via the respiratory system, a better understanding of the role of virus-surfactant interactions, the formation of aerosols and role of the inflammatory components of the immune system, as well as the role of positive pressure ventilation systems on particle exhalation, as being used at ICUs. Finally, suggestions are made for important goals for future biophysics research in infectious disease prevention and containment.

MicroRNA‑431 inhibits the expression of surfactant proteins through the BMP4/activin/TGF‑β signaling pathway by targeting SMAD4.

The synthesis and secretion of surfactant proteins (SPs) is an important sign of lung maturation. Furthermore, the morbidity of lung developmental diseases, including respiratory distress syndrome and bronchopulmonary dysplasia which are mainly caused by immature lung development and lack of SPs, is increasing. As is well known, multiple microRNAs (miRs/miRNAs) are able to influence lung development via numerous different signaling pathways. However, few studies examine the association between the miRNAs and lung developmental diseases. A previous study has demonstrated that miR‑431 was significantly (F=33.49; P<0.001) downregulated in the lung tissues of Sprague‑Dawley rats at 3time points, embryonic day19, embryonic day 21 and postnatal day3. The present study reported that the regulation of miR‑431 may influence the expression of SPs. Thus, the further potential mechanisms of miR‑431 in negatively regulating lung development were examined in the present study. Stable A549 cell lines overexpressing or knocking down SMAD family member 4 (SMAD4) transfected with miR‑431 overexpressed or knocked down, and their control groups were established. Subsequently, the expression of bone morphogenetic protein4 (BMP4), SMAD4 and SPs (SP‑A, SP‑B and SP‑C) at the RNA and protein levels were validated respectively by reverse transcription quantitative PCR and western blotting. miR‑431 exhibited a decreased expression, while BMP4 and SPs exhibited increased expression at the mRNA and protein levels in the SMAD4 knockdown group. Meanwhile, the expression of SPs were reduced in the SMAD4‑knockdown group via overexpressing miR‑431 and increased in the SMAD4‑overexpression group via inhibiting miR‑431. The present results indicate that SMAD4 negatively regulates the expression of SPs, and that miR‑431 negatively regulates the expression of SPs through inhibiting the BMP4/activin/transforming growth factor‑β signaling pathway by targeting SMAD4.

Isolation of Alveolar Type II Cells from Adult Bovine Lung.

Alveolar type II (ATII) cells play a key role as part of the distal lung epithelium, including in the innate immune response and as self-renewing progenitors to replace alveolar type I (ATI) cells during epithelial regeneration. Their secretion of surfactant protein helps maintain homeostasis and exerts protective, antimicrobial properties. ATII cells remain difficult to study, partly due to inefficient and expensive isolation methods, a propensity to differentiate into ATI cells, and susceptibility to fibroblast contamination. Published methods of isolation often require specialized technology, negatively impacting the development of in vitro models of disease, including bovine tuberculosis. Presented here is a simple and cost-effective method for generation of bovine primary ATII cells. These cells exhibit an ATII phenotype in 2D and 3D culture and are conducive to further study of the role of ATII cells in bovine respiratory diseases. © 2019 by John Wiley & Sons, Inc.

Isolation and characterisation of alveolar type II pneumocytes from adult bovine lung

Alveolar type II (ATII) cells play a key role as part of the distal lung epithelium, including roles in the innate immune response and as self-renewing progenitors to replace alveolar type I (ATI) cells during regeneration of the alveolar epithelium. Their secretion of surfactant protein helps to maintain homeostasis in the distal lung and exert protective, antimicrobial properties. Despite the cell’s crucial roles, they remain difficult to study, in part due to inefficient and expensive isolation methods, a propensity to differentiate into alveolar type I cells in culture and susceptibility to fibroblast overgrowth from primary isolations. Published methods of isolation often require specialist technology, negatively impacting the development of in vitro models of disease, including bovine tuberculosis (BTB), a serious re-emerging disease in both animals and humans worldwide. We present here a simple and cost-effective method that may be utilised in the generation of bovine primary ATII cells. These exhibit an ATII phenotype in 2D and 3D culture in our studies and are conducive to further study of the role of ATII cells in bovine respiratory diseases.

The Expression of Surfactant Proteins A and D in the Intestines and Pancreas of Murine Fetuses

Purpose: Surfactant proteins exist in the digestive tract and may play an important role in the host defense. However, the expression of surfactant proteins in the premature digestive system remains unclear. The aim of this study was to investigate the expression of surfactant proteins in the intes-tines and pancreas of murine fetuses. Methods: Immunostaining for SP-A and SP-D was assessed in the small intestine and pancreas of ICR murine fetuses on days 15, 16, 17 and 18 of gestation (normal duration of pregnancy: 19 - 21 days). RT-PCR was performed to detect the expression of spa and spd mRNA in the small intestine and pancreas on day 16, 17 and 18 of gestation. Results: Immunoreactivity for SP-A and SP-D in the acinar cells of pancreas and intestinal mucosal surface were positive on day 16 of gestation onward. RT-PCR revealed that the expression of spa and spd mRNA was significant in the pancreas but weak in the small intestine. Conclusions: Our data revealed that surfactant proteins are present in the fetal intestines and pancreas and that a significant expression of spa and spd mRNA is detected in the fetal pancreas. Pancreas may be a possible organ involved in the synthesis and secretion of surfactant proteins into the intestinal lumen.

Surfactant proteins A and D in pulmonary diseases of preterm infants

Immaturity of the pulmonary and immune systems represents an important risk factor for increased morbidity and mortality in neonates. Surfactant protein (SP)-A and SP-D, linking molecules between these two systems, are critical for lung homeostasis as they regulate surfactant metabolism and host immunodefense activities in innate and adaptive immunity. Preterm neonates with respiratory distress syndrome showed lower concentrations of SP-A and SP-D, and the administration of exogenous surfactant was found to strengthen the secretion of SPs. Low levels of SP-A and SP-D also correlated with a higher risk of infection and development of bronchopulmonary dysplasia. Moreover, SP-A- and SP-D-enriched surfactant formulations were more resistant to the inhibitory action of the plasmatic proteins in animal models. Based on these assumptions, new-generation surfactants, enriched with SP-A and/or SP-D, may enhance the function of immune system and lungs in neonates, potentially improving the clinical outcome.

Secreted surfactant protein A from fetal membranes induces stress fibers in cultured human myometrial cells

In the present study, we investigated the ability of human fetal membranes (amnion and choriodecidua) to regulate human maternal uterine cell functions through the secretion of surfactant protein (SP)-A and SP-D at the end of pregnancy. We detected the expression of both SP-A (SP-A1 and SP-A2) and SP-D by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry revealed that human fetal membranes expressed both SP-A and SP-D. By Western blot analysis, we demonstrated that SP-A protein expression was predominant in choriodecidua, whereas the amnion predominantly expressed SP-D. Only the secretion of SP-A was evidenced in the culture supernatants of amnion and choriodecidua explants by immunodot blot and confirmed by Western blot. Exogenous human purified SP-A induced stress fiber formation in cultured human myometrial cells via a pathway involving Rho-kinase. Conditioned medium from choriodecidua and amnion explants mimicked the SP-A effect. Treatment of myometrial cells with SP-A-depleted conditioned medium from choriodecidua or amnion explants failed to change the actin dynamic. These data indicate that SP-A released by human fetal membranes is able to exert a paracrine regulation of F-actin filament organization in myometrial cells.

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.

Influence of ambroxol on paraquat-induced lung tissue injury and change of pulmonary surfactant-associated protein A in the experimental rats

To investigate the influence of ambroxol on paraquat poisoning induced acute lung tissue injury and the change of pulmonary surfactant associated protein A in the experimental rats. One hundred and twenty healthy adult male Sprague-Dawley rats were randomizedly assigned into normal saline (NS) group (n = 24), paraquat poisoning induced lung tissue injury model (PQ) group (n = 48) and ambroxol treatment (AT) group (n = 48). The indexes were observed among the three groups comprising the mortality rate, the change of arterial blood PaCO(2) and PaO(2), the ratio of wet to dry lung tissue (W/D), the change of the lung tissue under light and electric microscope respectively, and the expression of pulmonary surfactant associated protein A. The mortality rate of rats in the PQ group was 50.0% on the seventh day while the mortality rate in the AT group was 25.0%. The level of arterial blood PaCO(2) in the PQ group (6.94 +/- 0.8) kPa was significantly higher than that in the AT group (6.12 +/- 0.5) kPa and the NS group (4.6 +/- 0.4) kPa. The level of arterial blood PaO(2) in the PQ group (6.98 +/- 1.1) kPa was significantly lower than that in the AT group (8.25 +/- 0.7) kPa and the NS group (12.7 +/- 0.8) kPa. There were significant differences among the groups (P < 0.05). The degree of lung tissue injury was severe in PQ group and relieved in AT group. The expression of pulmonary surfactant associated protein A was significantly decreased in PQ group 13.22% +/- 2.21% on the seventh day, compared with that in the AT group (21.82% +/- 3.67%) (P < 0.05). The expression of pulmonary surfactant associated protein A in AT group was significantly higher in the AT group (18.97% +/- 0.91%) than that in the PQ group on the seventh day (P < 0.05). Ambroxol plays a role in facilitating synthesis and secretion of pulmonary surfactant protein A and relieves the lung tissue injury induced by paraquat poisoning.

A Signal from Surfactant

Condon et al. uncovered a new role for lung surfactant as a signal whereby a developing fetus announces that it's ready for birth. Labor that starts too early or too late can endanger the fetus. For example, premature infants are at risk of respiratory distress syndrome, a serious condition that results from a lack of surfactant in the immature lung. Labor is associated with an inflammatory response, in which leukocytes infiltrate the myometrium, but the initiating stimulus and source of these leukocytes--as well as the trigger for labor--have remained unclear. Condon et al. found that secretion of surfactant protein A (SP-A) mRNA in mouse amniotic fluid (AF) increased late in gestation along with production of interleukin-1β (IL-1β, a marker for activation) in AF macrophages and the number of macrophages in the uterine wall. The authors used wild-type mice carrying transgenic embryos to demonstrate that at least some of these uterine macrophages were of fetal origin. Intra-amniotic injection of SP-A promoted migration of fetal macrophages into the uterine wall and caused preterm labor, whereas intra-amniotic injection of antibodies to SP-A delayed labor. SP-A stimulated the expression of IL-1β and nuclear factor κB (NF-κB) in macrophages isolated from AF. NF-κB p50 and p65 proteins redistributed from uterine cytoplasm to nucleus late in gestation, an effect that was mimicked by intra-amniotic injection of SP-A. Intra-amniotic injection of an NF-κB inhibitor delayed the onset of labor and prevented premature labor in response to SP-A. Thus, the authors propose that SP-A from fetal lung acts as a labor-initiating signal to the myometrium of fetal maturity. J. C. Condon, P. Jeyasuria, J. M. Faust, C. R. Mendelsohn, Surfactant protein secreted by the maturing mouse fetal lung acts as a hormone that signals the initiation of parturition. Proc. Natl. Acad. Sci. U.S.A. 101 , 4978-4983 (2004). [Abstract] [Full Text]

Maintenance of surfactant protein A and D secretion by rat alveolar type II cells in vitro

Secretion of surfactant proteins A and D (SP-A and SP-D) has been difficult to study in vitro because a culture system for maintaining surfactant secretion has been difficult to establish. We evaluated several growth factors, corticosteroids, rat serum, and a fibroblast feeder layer for the ability to produce and maintain a polarized epithelium of type II cells that secretes SP-A and SP-D into the apical medium. Type II cells were plated on a filter insert coated with an extracellular matrix and were cultured at an air-liquid interface. Keratinocyte growth factor (KGF) stimulated type II cell proliferation and secretion of SP-A and SP-D more than fibroblast growth factor-10 (FGF-10), hepatocyte growth factor (HGF), or heparin-binding epidermal-like growth factor (HB-EGF). Cells cultured in the presence of KGF and rat serum with or without fibroblasts had high surfactant protein mRNA levels and exhibited a high level of SP-A and SP-D secretion. Dexamethasone inhibited type II cell proliferation but increased expression of SP-B. In the presence of KGF, rat serum, and dexamethasone, the mRNAs for the surfactant proteins were maintained at high levels. Secretion of SP-A and SP-D was found to be independent of phospholipid secretion.

Lung Surfactant Disaturated Phosphatidylcholine and Surfactant Proteins A and B in Dietary-Induced Obesity in Immature Rats † 1625

Dietary-induced obesity in young rats is associated with increases in body weight and the fat pad weight/body weight ratio (a measure of obesity) and decreases in alveolar surface area per alveolar volume (Inselman, et al.JAP 1988;65:2291). A reduction in dynamic lung compliance also occurs. The present study investigated the effect of dietary-induced obesity on disaturated phosphatidyl-choline (DSPC) and surfactant proteins A (SP-A) and B (SP-B) levels. Newborn rats raised in litters of 4 pups/dam were fed a high fat diet post-weaning (obese group, O). A control group (C) consisted of newborn rats raised in litters of 14 pups/dam which were fed a normal rat diet after weaning. Male rats (O=13, C=21) were killed at age 8 weeks, and the lungs were perfused until visibly blood-free. The lungs were then lavaged with normal saline to recover bronchoalveolar lavage fluid, which was separated into large and small aggregates of surfactant by differential centrifugation. The lipid extracts of large and small aggregates of surfactant and lavaged lung tissue were treated with osmium tetroxide, and DSPC was quantified following chromatography on neutral alumina columns. Western blot analyses were performed for SP-A and SP-B on the large aggregates and lung homogenates. Obesity in O animals was confirmed by increases in body weight (24%, P<0.001) and the fat pad weight/body weight ratio (359%, P<0.001) when compared with C animals. When compared with the C group, DSPC in the O rats was elevated by 28% in the lung [O: 177±6; C: 138±4 μg phospholipid phosphorus (PLPi/)/lung] (mean±SE) (P<0.0001), 15% in the large aggregates (O: 17.6±0.8; C: 15.3±0.7 μg PLPi/lung)(P<0.05) and 35% in the small aggregates (O: 8.9±0.8; C: 6.6±0.4 μg PLPi/lung) (P<0.01). SP-A and SP-B levels were increased in large aggregates of surfactant in O rats but not in lung tissue. In addition, surfactant SP-A and SP-B were elevated in O animals when compared with C animals, while lung tissue SP-A and SP-B levels were similar between the two groups. These observations indicate that this model of obesity results in changes in synthesis and secretion of surfactant proteins and phospholipids. These studies suggest that additional factors may be responsible for the alterations in lung function since increased levels of surfactant proteins and phospholipids are insufficient to improve dynamic lung compliance in immature rats rendered obese by dietary changes.

Secretion of surfactant protein A from rat type II pneumocytes

Secretion of surfactant phosphatidylcholine has been extensively studied and there is evidence that it is a regulated process that can be influenced by a variety of physiological factors and pharmacological agents. In contrast, secretion of the major surfactant protein, surfactant protein A (SP-A), has been investigated to much lesser extent. It is not known whether SP-A secretion is constitutive or regulated and, if regulated, whether its regulation is similar to that of phosphatidylcholine. To address those questions we measured SP-A secretion in primary cultures of type II pneumocytes under conditions identical to those used to study phosphatidylcholine secretion. Freshly isolated cells from adult rats were cultured overnight, washed, and then incubated in fresh medium in the presence and absence of surfactant phospholipid secretagogues. As previously reported for phosphatidylcholine, SP-A secretion was linear with time for up to 4 h. However, the rate of SP-A secretion, approximately 6% of total SP-A (cells+medium) released into the medium per hour, was more than sixfold greater than that of the lipid. Although freshly isolated cells contained 70% more SP-A than cells that were cultured overnight, the rate of SP-A secretion was not significantly different. Secretion of SP-A by freshly isolated or cultured type II cells was not increased by a combination of ATP, terbutaline, the adenosine A2 receptor agonist 5'(N-ethylcarboxyamido)adenosine, 12-O-tetradecanoylphorbol-13-acetate, and ionomycin at concentrations that optimally stimulated phosphatidylcholine secretion. We conclude that secretion of the major lipid and protein components of surfactant are independently regulated.

ATP and adenosine 3',5'-cyclic monophosphate stimulate the synthesis of surfactant protein A in rat lung.

Synthesis and secretion of surfactant protein A (SP-A) were studied in the isolated perfused rat lung using Trans35S-label (approximately 85% methionine, 15% cysteine) in the perfusate with or without 1 mM ATP or 0.1 mM 8-bromoadenosine 3',5',-cyclic monophosphate (8-BrcAMP) for up to 6 h of perfusion. By enzyme-linked immunosorbent assay, the SP-A content was 36 +/- 0.3% of total protein in extracellular surfactant and 10.8 +/- 1.9% of total protein in lamellar bodies of control lungs; these relativr proportions were maintained in the presence of ATP or 8-BrcAMP. Incorporation of [35S]methionine (cysteine) into the surfactant and lamellar body protein fraction could be detected at 4 h of perfusion. At 6 h, specific activity of total protein [disintegrations per minute (dpm)/micrograms)] was significantly increased in both the surfactant (54%) and lamellar body fractions (30%) under the influence of either secretagogue compared with control conditions. In the presence of ATP, there was a significant increase in the SP-A immunoprecipitable counts of 61 and 72% in extra- and intracellular compartments, respectively. However, no significant change was observed in the relative abundance of SP-A mRNA between control and secretagogue-treated lungs. This dissociation of SP-A mRNA abundance and label incorporation into protein indicates that alteration in translational efficiency or posttranslational factors may be involved in the secretagogue-induced stimulation of SP-A synthesis.

Secretion of surfactant protein A and phosphatidylcholine from type II cells of human fetal lung.

Surfactant protein A (SP-A) appears to have a role in lung immune defense as well as generation and metabolism of the alveolar surface-active film. Previous studies indicated that lamellar bodies isolated from lung tissue had a relatively low content of SP-A and that exogenous SP-A was needed for rapid formation of a surface-active film in vitro. We therefore tested the hypothesis that SP-A was secreted from type II cells primarily by a pathway separate from lamellar bodies. Cells were isolated from explants of human fetal lung that had been cultured with hormones to promote differentiation of type II cells, and secretion of surfactant lipid and SP-A were compared. Cultured cells secreted labeled phosphatidylcholine in a nearly linear fashion for 48 h. Basal secretion of SP-A, assayed by enzyme-linked immunosorbent assay, was linear for only 12 h after plating of cells; during this time, there was no accumulation of intracellular SP-A. Addition of secretagogues (phorbol ester, calcium ionophore, and beta-adrenergic agonist) stimulated phosphatidylcholine secretion approximately 4-fold. In contrast, the secretion rate of SP-A was not significantly affected by secretagogues. These findings indicate that a relatively small amount of secreted SP-A (approximately 10%) is released with lamellar bodies. Most SP-A is released by constitutive secretion and may be important for both surfactant- and nonsurfactant-related functions.

Regulation of lung surfactant secretion

Secretion of lung surfactant is the direct step in release of the lipoprotein-like product, synthesized in lung epithelial type II cells, onto the alveolar surface. Release of surfactant phosphatidylcholine (PC) proceeds via formation of surface pores during exocytosis of lamellar bodies. Surfactant secretion is regulated locally in the lung by changes in ventilation rate, possibly mediated by distension and altered intracellular pH. Secretion is also stimulated by various agents, including agonists for beta-adrenergic, purinoceptors, and vasopressin receptors and is associated with increased cytosolic Ca2+, cellular adenosine 3',5'-cyclic monophosphate, and activation of protein kinases. Limited studies suggest that secretion of surfactant protein A may be regulated by both cAMP-dependent and protein kinase C-dependent pathways. The integration of these various mechanisms for the in vivo regulation of surfactant secretion remains largely unexplored. Future research into the mechanisms involved in lamellar body fusion with the plasma membrane, role of protein phosphorylation, transient changes in cAMP and Ca2+, and coordination between the secretion of phospholipid and protein components of surfactant should enhance our understanding of secretion of surfactant "lipoprotein."