Rat Surfactant Research Articles

Expression of the pulmonary surfactant in rat with paraquat-induced acute lung injury

To explore the expression of pulmonary surfactant in rats with paraquat-induced acute lung injury. A total of 36 SD rats were randomly divided into the control group (n=6) and paraquat group at 6, 12, 24, 48, 72 h five time points, each time points 6 rats. All of the rats were injected with paraquat (80 mg/kg) for once. The specimens were collected at 0 h (instant) after paraquat intoxication in the control group, and at the corresponding point in time in the paraquat each time point groups. The pathology changes of lung tissue were observed by HE staining, thiobarbituric acid method was used to detect malondialdehyde (MDA) level, and Western blot method was used to analyze the relative expression of surfactant protein (SP)-A and SP-D in bronchoalveolar lavage fluid (BALF) with time. As compared to control, the levels of MDA in BALF were significantly higher at 6, 24, 48 h after paraquat intoxication ((4.19 ± 0.12), (3.33 ± 0.08), (3.52 ± 0.08) vs (2.82 ± 0.15) nmol/mg, all P<0.01), no significantly different at 12, 72 h ((2.76 ± 0.13), (2.79 ± 0.10) nmol/mg, both P>0.05); the relative expression of SP-A in BALF were significantly higher at 6 h after paraquat intoxication (1.32 ± 0.19 vs 1.00 ± 0.19, P<0.01), lower at 24, 48 h (0.43 ± 0.07, 0.67 ± 0.08, both P<0.01), and no significantly different at 12, 72 h (0.98 ± 0.15, 0.79 ± 0.18, both P>0.05); the relative expression of SP-D in BALF were significantly higher at 6, 12 h after paraquat intoxication (1.54 ± 0.33, 1.64 ± 0.37 vs 1.00 ± 0.23, both P<0.01), no significantly different at 24, 48 h (1.07 ± 0.19, 0.97 ± 0.15, both P>0.05), and reached the peak at 72 h (2.15 ± 0.26, P<0.01); the expression of MDA, SP-A and SP-D were characterized by the state of fluctuation with increased first, reduced after, and finally increased. The expression of SP-A, SP-D increase at the early stage of the paraquat-induced acute lung injury in rat, which can reflect the degree of lung injury.

PS-280 Surface Film Fromationin VitroBy Infant And Therapeutic Surfactants: Role Of Surfactant Protein B

Background Essential surfactant properties include transfer to gas-liquid interface, reduction of surface tension and film replenishment during respiratory cycles. Objective To compare component-specific film formation properties of infant and therapeutic surfactants. Design/methods Using a multiwell fluorescence assay, we compared maximal fluorescence (Max), time to reach Max (tMax) and phospholipid concentration for ½ maximal signal (½Max) for calfactant (CAL), poractant (POR), beractant (BER), colfosceryl palmitate (COL), with surfactant from immature infants with RDS. Dose-response studies were performed for addition of SP-B, albumin and budesonide. Results Max and ½Max values for CAL were higher/similar to those of rat surfactant. There were significant differences between CAL and other therapeutic surfactants for Max (CAL >COL >POR >BER) whereas ½Max were similar except for COL. In surfactant from 39 infant tracheal aspirates, ½Max was inversely correlated with SP-B content (p = 0.001). Addition of SP-B to samples with low endogenous content ( Conclusions This assay reveals differences in film formation efficiency for therapeutic surfactants reflecting differences in SP-B content and lipid composition. Film formation by infant surfactant is strongly influenced by SP-B content. The findings support the key physiological role of SP-B and the safety of surfactant as anti-inflammatory drug vehicle.

Mutations of rat surfactant protein A have distinct effects on its glycosylation, secretion, aggregation and degradation

AimsSurfactant protein A (SP-A) plays critical roles in the innate immune system and surfactant homeostasis of the lung. Mutations in SP-A2 of the carbohydrate recognition domain (CRD) impair its glycosylation and are associated with pulmonary fibrosis in humans. We aim to examine how mutations in SP-A that impair its glycosylation affect its biological properties and lead to disease. Main methodsWe generated rat SP-A constructs with two types of mutations that impair its glycosylation: N-glycosylation site mutations (N21T, N207S and N21T/N207S) and disease-associated CRD mutations (G231V, F198S). We transfected these constructs into Chinese hamster ovary (CHO)-K1 cells and assessed biochemical differences in cellular and secreted wild-type and mutant SP-As by western blot, immunofluorescence, and sensitivity to enzymatic digestion. Key findingsMutations of the CRD completely impaired SP-A secretion, whereas mutations of N-glycosylation sites had little effect. Both types of mutations formed nonidet p-40 (NP-40) insoluble aggregates, but the aggregates only from CRD mutations could be partially rescued by a chemical chaperone, 4-phenylbutyrate acid (4-PBA). The majority of CRD mutant SP-A was retained in the endoplasmic reticulum. Moreover, both types of mutations reduced SP-A stability, with CRD mutant SP-A being more sensitive to chymotrypsin digestion. Both types of soluble mutant SP-A could be degraded by the proteasome pathway, while insoluble aggregates could be additionally degraded by the lysosomal pathway. SignificanceOur data provide evidence that the differential glycosylation of SP-A may play distinct roles in SP-A secretion, aggregation and degradation which may contribute to familial pulmonary fibrosis caused by SP-A2 mutations.

Effect of bile Acid on fetal lung in rat model of intrahepatic cholestasis of pregnancy.

Objective. To determine the correlation between maternal bile acid (BA) level and fetal pulmonary surfactant in rats and study the effects of BA on fetal lung in rat model of intrahepatic cholestasis of pregnancy. Methods. Forty pregnant rats were treated with (A) 5.5 mg/kg BA, (B) 1.4 mg/kg BA, and (C) 1 ml physiological saline. Levels of total bile acid (TBA), ALT, AST, TBIL, DBIL, and SP-A were determined and the lungs of fetal rats were analyzed for pathological changes. Results. Groups A and B intervened with BA showed significant higher level of TBA in both maternal and fetal serum, more mortality rate of fetal rats, more concentration of SP-A in fetal serum, and wider alveolus mesenchyme of fetal rats than the control Group C. Higher level of BA associated with increased fetal risk and lower numerical density of mitochondria in type II alveolar epithelial cells. The levels of TBA in maternal serum were found to have significant positive correlation with those in fetal serum and SP-A level but negatively with the area of alveolus and the numerical density of lamellar body. Conclusions. The TBA level in maternal serum showed significant association with lung pathological changes in fetal rats.

Effects of recombinant human keratinocyte growth factor on surfactant, plasma, and liver phospholipid homeostasis in hyperoxic neonatal rats

Respiratory distress and bronchopulmonary dysplasia (BPD) are major problems in preterm infants that are often addressed by glucocorticoid treatment and increased oxygen supply, causing catabolic and injurious side effects. Recombinant human keratinocyte growth factor (rhKGF) is noncatabolic and antiapoptotic and increases surfactant pools in immature lungs. Despite its usefulness in injured neonatal lungs, the mechanisms of improved surfactant homeostasis in vivo and systemic effects on lipid homeostasis are unknown. We therefore exposed newborn rats to 85% vs. 21% oxygen and treated them systemically with rhKGF for 48 h before death at 7 days. We determined type II pneumocyte (PN-II) proliferation, surfactant protein (SP) mRNA expression, and the pulmonary metabolism of individual phosphatidylcholine (PC) species using [D(9)-methyl]choline and tandem mass spectrometry. In addition, we assessed liver and plasma lipid metabolism, addressing PC synthesis de novo, the liver-specific phosphatidylethanolamine methyl transferase (PEMT) pathway, and triglyceride concentrations. rhKGF was found to maintain PN-II proliferation and increased SP-B/C expression and surfactant PC in both normoxic and hyperoxic lungs. We found increased total PC together with decreased [D(9)-methyl]choline enrichment, suggesting decreased turnover rather than increased secretion and synthesis as the underlying mechanism. In the liver, rhKGF increased PC synthesis, both de novo and via PEMT, underlining the organotypic differences of rhKGF actions on lipid metabolism. rhKGF increased the hepatic secretion of newly synthesized polyunsaturated PC, indicating improved systemic supply with choline and essential fatty acids. We suggest that rhKGF has potential as a therapeutic agent in neonates by improving pulmonary and systemic PC homeostasis.

Atomic force microscopy analysis of rat pulmonary surfactant films

Pulmonary surfactant facilitates breathing by forming a surface tension reducing film at the air–liquid interface of the alveoli. The objective was to characterize the structure of surfactant films using endogenous rat surfactant. Solid-support surfactant films, at different surface pressures, were obtained using a Langmuir balance and were analyzed using atomic force microscopy. The results showed a lipid film structure with three distinct phases: liquid expanded, liquid ordered and liquid condensed. The area covered by the liquid condensed domains increased as surface pressure increased. The presence of liquid ordered phase within these structures correlated with the cholesterol content. At a surface pressure of 50 mN/m, stacks of bilayers appeared. Several structural details of these films differ from previous observations made with goat and exogenous surfactants. Overall, the data indicate that surfactant films demonstrate phase separation at low surface pressures and multilayer formation at higher pressure, features likely important for normal surfactant function.

Increased palmitoyl-myristoyl-phosphatidylcholine in neonatal rat surfactant is lung specific and correlates with oral myristic acid supply

Surfactant predominantly comprises phosphatidylcholine (PC) species, together with phosphatidylglycerols, phosphatidylinositols, neutral lipids, and surfactant proteins-A to -D. Together, dipalmitoyl-PC (PC16:0/16:0), palmitoyl-myristoyl-PC (PC16:0/14:0), and palmitoyl-palmitoleoyl-PC (PC16:0/16:1) make up 75-80% of mammalian surfactant PC, the proportions of which vary during development and in chronic lung diseases. PC16:0/14:0, which exerts specific effects on macrophage differentiation in vitro, increases in surfactant during alveolarization (at the expense of PC16:0/16:0), a prenatal event in humans but postnatal in rats. The mechanisms responsible and the significance of this reversible increase are, however, not understood. We hypothesized that, in rats, myristic acid (C14:0) enriched milk is key to lung-specific PC16:0/14:0 increases in surfactant. We found that surfactant PC16:0/14:0 in suckling rats correlates with C14:0 concentration in plasma chylomicrons and lung tissue triglycerides, and that PC16:0/14:0 fractions reflect exogenous C14:0 supply. Significantly, C14:0 was increased neither in plasma PC, nor in liver triglycerides, free fatty acids, or PC. Lauric acid was also abundant in triglycerides, but was not incorporated into surfactant PC. Comparing a C14:0-rich milk diet with a C14:0-poor carbohydrate diet revealed increased C14:0 and decreased C16:0 in plasma and lung triglycerides, respectively. PC16:0/14:0 enrichment at the expense of PC16:0/16:0 did not impair surfactant surface tension function. However, the PC profile of the alveolar macrophages from the milk-fed animals changed from PC16:0/16:0 rich to PC16:0/14:0 rich. This was accompanied by reduced reactive oxygen species production. We propose that nutritional supply with C14:0 and its lung-specific enrichment may contribute to decreased reactive oxygen species production during alveolarization.

Atomic Force Microscopic Examination of Rat Pulmonary Surfactant Films

The ability of pulmonary surfactant to drive surface tension (ST) to low values near 0 mN/m during compression stabilizes the alveoli at end expiration. The manner in which surfactant phospholipids (PL) (50% unsaturation) attain surface tension near zero is still not clear. Atomic force microscopy (AFM) of rat surfactant extract films at a surface pressure (SP) of 20 mN/m reveals microdomains (mD), apparently composed of liquid-ordered (L0) and tilted-condensed (TC) phases within the predominant liquid-expanded (LE) phase. Numerous nanodomains (nD) were also present. As SP increased to 40 mN/m, the area composed of mD increased but the nD decreased. This contrasts with previous studies with bovine surfactant where the mD appeared to form nD. At 50 mN/m, the film exhibited the formation of numerous stacked multilayers (PL bilayers) which could be incorporated into the monolayer during film expansion. The present results are consistent with the reversible squeeze-out of unsaturated PL into multilayers during compression, resulting in a monolayer highly enriched in gel phase saturated PL components. Furthermore, as with other surfactants, the presence of L0 phase appears related to cholesterol. Reducing the cholesterol content by acetone precipitation resulted in the loss of L0 phase, further implicating this sterol in surfactant phase separation.

Effect of antioxidant on pulmonary surfactant in acute lung injury rats

Objective To explore the changes of neutrophil elastase (NE) and surfactant protein A (SP-A) in acute lung injury(ALI) rats,and the effect of antioxidant. Methods Sixty healthy mature Wister rats were divided into 2 groups, the control group and treatment group. The rats in two groups all received peritoneal injection of E. coli to establish the ALI animal model. 30 minutes after injection of E. coli,the rats in treatment group were injected reduced glutathione from vena caudalis. The levels of NE in blood and expressions of SP-A in lung tissue were detected at 3,6 and 12 hours after injection of E. coli. Results ALI symptom appeared 3 hours after injection of E. coli in the control group, obvious after 6 hours, the rats vomi-ted pink secretion after 12 hours. Lung edema and bleeding were found by pathologic examination. No obvious symptom was found in treatment group after 3 hours, slight tachypnea after 6 hours, slight edema in pulmonary tissue after 12 hours. After administration of reduced glutathione,levels of NE at 3,6 and 12 hours in the treatment group were lower than those in the control group,and indicated statistical significance in 6 and 12 hours(P <0. 05) ;Levels of SP-A in 3,6 and 12 hours in the treatment group were higher than those in the control group, and indicated statistical significance in 3,6 and 12 hours (P < 0. 05). Conclusion Dysfunction of pulmonary surfactant is secondary in ALI, degradation of SP-A is the one of reasons, the application of reduced glutathione as antioxidant, could effectively suppress NE to decompose basosexine elastin of cells and destroy surface active protein, has protective effect on ALI. Key words: Acute lung injury; Neutrophil elastase; Pulmonary surfactant protein A; Anti-oxidant; Rat

Effect of L-arginine on pulmonary surfactant in the rats with LPS-induced acute lung injury

Objectiye To investigate the effect of L-arginine on pulmonary surfactant (PS) in the rats with lipopolysaccharide (LPS)-induced acute lung injury (ALI).Methods Forty-eight adult male SD rats weighing 220-270 g were randomly divided into 4 groups:group Ⅰ control (group C);group Ⅱ LPS;group Ⅲ LPS 3 h + L-arginine(group L1);group Ⅳ LPS6 h + L-arginine(group L2). ALI was induced by intravenous (Ⅳ)LPS 5 mg/kg in LPS,L1 and L2 groups. L-arginine 500 mg/kg was given intraperitoneally at 3 or 6 h after LPS administration in group L1 or group L2. The animals were sacrificed at 3 h after L-arginine administration, eight animals were sacrificed at 6 h and 9 h after LPS in group C and group LPS. The lungs were immediately removed for determination of surfactant-protein (SP-A) mRNA expression and broncho-alveolar lavage fluid (BALF). The total phospholipid (TPL) and total protein (TP) concentrations in the BALF were measured. Results SP-A mRNA in the lung tissue and TPL concentration in BALF were significantly decreased while TP concentration in BALF was significantly increased in group LPS, as compared with control groups( P ＜ 0.01 ). L-arginine given at 3 h after LPS administration significantly attenuated the LPS-induced changes while L-arginine given at 6 h after LPS did not. Conclusion L-arginine can protect the lungs from LPS-induced injury by up-regulating PS-expression. Key words: Arginine; Endotoxemia; Respiratory distress syndrome, adult; Pulmonary surfactantassociated proteins

Immunomodulatory properties of exogenous surfactant in adult rat alveolar macrophages

Background and aim: The study investigates the immune response of the lung alveolar macrophages in adult rats with intratracheal instillation of surfactant, isolated from porcine, rabbit and rat lung as well as the synthetic surfactant.Material and methods: Adult male rats were divided into five groups: Controls were instilled with buffer, similarly P-SF, R-SF, r-SF, and S-SF group of animals were instilled with porcine, rabbit, rat and synthetic surfactant, respectively.Results: After one month, IL-1β secretions were found to be elevated in the supernatant of alveolar macrophages. Further, nitric oxide (NO) level was elevated with porcine and synthetic surfactant, while reactive oxygen species (ROS) generation was found to be inhibited significantly in all the treatment groups. Apoptoses were studied by using fluorescence dyes acridine orange/ethidium bromide, where percent of the apoptotic cells were found to be decreased in all the treated groups.Conclusions: Exogenous surfactant elevates secretion of pro-inflammatory cytokine IL-1β and also the NO as signaling molecule. The inhibitory effects of surfactants on ROS generation and on apoptosis may result from the interruption in the cell signaling pathway. In the treatment of respiratory distress syndrome, the anti-inflammatory response of surfactant may thus depend upon the specific preparation of the surfactant.

Comparative Studies On Bovine And Rat Pulmonary Surfactants Using AFM

Recent studies employing a variety of microscopic techniques have revealed that phospholipid (PL) phase separations and transitions may play important roles in determining the biophysical properties of pulmonary surfactant. Most of these microscopic studies used model systems which were composed of simple mixtures of PL with or without hydrophobic surfactant proteins and cholesterol. The present work compared modified natural (lipid extract) bovine and rat surfactants using atomic force microscopy (AFM). AFM revealed PL phase separation upon compression of both surfactant monolayers, and a monolayer-to-multilayer transition at surface pressure 40-50 mN/m. Similar to bovine surfactant, the tilted-condensed (TC) phase in rat surfactant consisted of domains both on micrometer and nanometer scales. Upon film compression, the microdomains were dissociated into nanodomains, thus forming a more homogeneous two-phase mixture. Differences between rat and bovine surfactants were: (1) more TC domains were formed at lower surface pressures in rat than in bovine surfactant; and (2) an interesting domain-in-domain structure was exclusively observed in rat surfactant. These structural differences were attributed to the higher cholesterol content of rat surfactant (∼ 10 vs ∼2.5 wt%). To further investigate the effects of cholesterol on the structure of surfactant films, we have studied cholesterol-depleted bovine surfactant (∼ 0%) prepared by repetitive acetone extraction. Removal of cholesterol from bovine surfactant induced significant variations in film structure. More importantly, the film structure can be effectively restored by recombining cholesterol with the cholesterol-depleted bovine surfactant. Recombinant bovine surfactant with 10% cholesterol showed domain-in-domain structures similar to those found with rat surfactant. These interspecies studies of the micro- and nano- structures of natural pulmonary surfactants add insight into the biophysical interpretation of phospholipid phase transition and separation, in particular the role of cholesterol.

Developmental changes in rat surfactant lipidomics in the context of species variability

Lung surfactant comprises mainly phosphatidylcholine (PC) species together with phosphatidylglycerols and surfactant proteins (SP) SP-A to -D. Changes in the concentrations of its principal components dipalmitoyl-PC, palmitoylmyristoyl-PC, palmitoylpalmitoleoyl-PC relative to developmental, structural and physiological differences are only partially understood. Particularly, their attribution to differences in air-liquid interface curvature, compared with dynamic parameters, such as respiratory rate, are controversial. We postulated that during alveolarization the changes in these principal PC components of surfactant differ from those in other phospholipid parameters, and that across endothermic vertebrates their concentrations are related to lung physiology rather than structure. We therefore investigated in rats from postnatal day (d)1 to d42 the pattern of surfactant phospholipids relative to alveolarization (d4-d14), and we discuss these changes in terms of molecular adaptation to pulmonary structure or physiology. Contrary to mammals with advanced alveolarization and increased respiratory rate (RR) at term, concentrations of dipalmitoyl-PC (49-52%) and palmitoylmyristoyl-PC (7-9%) in lung lavage fluid were identical at d1 and d42. At d7-d14, when in rats RR is increased, palmitoyl-myristoyl-PC transiently increased by 2.5- to 3.9-fold at the expense of dipalmitoyl-PC (-32% to 34%) and palmitoyl-palmitoleoyl-PC (-16%). Other lipidomic changes followed essentially different patterns of increase or decrease. Palmitoyl-myristoyl-PC was increased in large aggregates suggesting that it is an integral component of active surfactant. In the overall context of vertebrates, irrespective of age and lung structure, fractions of palmitoyl-myristoyl-PC, dipalmitoyl-PC and palmitoyl-palmitoleoyl-PC correlate with differences in RR rather than alveolar curvature. In adult mammals, however, only concentrations of palmitoyl-palmitoleoyl-PC correlate with RR.

Alterations of the composition and metabolism of pulmonary surfactant phospholipids induced by experimental peritonitis in rats

Pulmonary complications often accompany the development of acute peritonitis. In this study, we analyzed the alterations of alveolar surfactant phospholipids in rats with experimentally induced peritonitis. The results showed a reduction of almost all phospholipid fractions in pulmonary surfactant of experimental animals. The most abundant alveolar phospholipids—phosphatidylcholine and phosphatidylglycerol were reduced significantly in surfactant of rats with experimental peritonitis. In addition, analysis of the fatty acid composition of these two phospholipids revealed marked differences between experimental and control animals. The activity of phospholipase A2, which is localized in the hydrophyllic phase of alveolar surfactant, was higher in rats with experimental peritonitis compared to sham-operated ones. Also, a weak acyl-CoA:lysophospholipid acyltransferase activity was detected in alveolar surfactant of rats with experimental peritonitis, whereas in control animals this activity was not detectable. The lipid-transfer activity was quite similar in pulmonary surfactant of control and experimental rats. The total number of cells and the percentage of neutrophils were strongly increased in broncho-alveolar lavage fluid from rats with peritonitis. Thus, our results showed that the development of peritonitis was accompanied by pulmonary pathophysiological processes that involved alterations of the phospholipid and fatty acid composition of alveolar surfactant. We suggest that the increased populations of inflammatory cells, which basically participate in internalization and secretion of surfactant components, contributed to the observed alterations of alveolar phospholipids. These studies would be useful for clarification of the pathogenic mechanisms underlying the occurrence of pulmonary disorders that accompany acute inflammatory conditions, such as peritonitis and sepsis.

Surfactant as an Airway Smooth Muscle Relaxant

A variety of clinical and experimental evidence indicates that surfactant may be important in the pathogenesis and treatment of asthma. The purpose of this study was to determine the pharmacologic effect of pulmonary surfactant and its major lipid and protein constituents on bronchial smooth muscle. First-generation bronchi from male Sprague-Dawley rats were contracted with methacholine and exposed to two kinds of surfactant: whole rat surfactant and two bovine surfactant extracts in clinical use. The latter lack the hydrophilic surfactant-associated proteins (SP)-A and SP-D. All the surfactants relaxed the rat bronchi in a concentration-dependent manner; however, whole rat surfactant was more potent than the bovine extracts. Both surfactant lipids and SP-A contributed to the bronchial relaxation. The relaxation response produced by the highest concentration (0.5 mg/ml) of whole rat surfactant was equivalent to that caused by substance P (5 microM) and approximately half of that caused by 1 microM isoproterenol. The relaxation response was epithelium-dependent and blocked by indomethacin but not by N-omega-nitro-L-arginine methyl ester. We conclude that surfactant can relax airway smooth muscle directly via a prostanoid-mediated, epithelium-dependent process that does not involve nitric oxide synthase.

Aerosolized surfactant and dextran for experimental acute respiratory distress syndrome caused by acidified milk in rats.

Inhibition of pulmonary surfactant by plasma-derived proteins is an important pathogenetic factor of acute respiratory distress syndrome (ARDS). Inhalation of aerosolized surfactant may be suitable for early treatment of ARDS. However, requirement of a high dose is a drawback. Because dextran reverses surfactant inhibition, we examined whether dextran improves the therapeutic effects of aerosolized surfactant in rats with experimental ARDS. Acidified milk (pH 1.8, 1.5 ml kg(-1)) was injected into the trachea of the rats ventilated with pure oxygen using 2.45 kPa peak inspiratory pressure and 0.74 kPa positive end-expiratory pressure. When PaO2 decreased to <13 kPa, the rats were assigned to four groups: control group (n = 8), receiving no material; D-only group (n = 6), receiving aerosolized dextran for 45 min; S-only group (n = 8), receiving aerosolized modified natural surfactant (MNS) for 30 min; and S-plus-D group (n = 9), receiving aerosolized MNS for 30 min followed by aerosolized dextran for 15 min. In the control group and D-only groups, the mean PaO2 remained at <10 kPa for 180 min. In the S-only and S-plus-D groups, the PaO2 increased to 50 kPa (P < 0.01 vs. untreated). The PaO2 of the surfactant-only group gradually decreased to <17 kPa at 180 min, whereas the PaO2 of the S-plus-D group was maintained at >38 kPa for 180 min (P < 0.01 vs. S-only group). Inhalation of aerosolized dextran potentiates the effects of aerosolized surfactant by prolonging the therapeutic response.

Vitamin E deficiency reduces surfactant lipid biosynthesis in alveolar type II cells.

Reactive oxygen species play an important role in development of lung injury. Neonates exhibit a high risk of developing acute and/or chronic lung disorder, often associated with surfactant deficiency, and in parallel they show low vitamin E concentration. We investigated whether the vitamin E status of adult rats affects the content of phospholipids (PL) in bronchoalveolar lavage and alveolar type II cells. Phosphatidylcholine (PtdCho) is the dominant and functional most important PL in lung surfactant. Therefore, we determined its formation via de novo synthesis and reacylation of lyso-PtdCho in type II cells. Vitamin E depletion caused a decrease of PL content in bronchoalveolar lavage and type II cells and decreased glycerol-3-phosphate O-acyltransferase (G3P-AT) activity, de novo synthesis of PtdCho, and reacylation of lyso-PtdCho in type II cells. Preincubation of type II cell homogenates with dithiothreitol restored the activity of G3P-AT and de novo synthesis but inhibited reacylation. Reacylation was strongly reduced by chelerythrine-mediated inhibition of protein kinase C. We conclude that antioxidant and PKC-modulating properties of vitamin E regulate de novo synthesis of PtdCho and reacylation of lyso-PtdCho in alveolar type II cells. Vitamin E depletion reduced the two pathways of PL synthesis and caused a decrease of PL content in alveolar surfactant of rats.

Biosynthesis of Surfactant Protein C (SP-C): SORTING OF SP-C PROPROTEIN INVOLVES HOMOMERIC ASSOCIATION VIA A SIGNAL ANCHOR DOMAIN

Rat surfactant protein C (SP-C) is synthesized as a 194-amino acid propeptide (SP-C-(1-194)) that is directed to the distal secretory pathway and proteolytically processed as an integral membrane protein to yield its mature form. We had shown previously that trafficking of proSP-C is mediated both by a signal anchor domain contained within the mature SP-C sequence and by a targeting domain in the NH(2)-flanking propeptide. Based on evidence from other integral membrane proteins, we hypothesized that proSP-C targeting is effected by oligomerization of proSP-C monomers. To evaluate this in vitro, cDNA constructs encoding for either wild type proSP-C (pcDNA3/SP-C-(1-194)) or heterologous fusion proteins containing green fluorescent protein (EGFP) linked to SP-C-(1-194) (EGFP/SP-C-(1-194)), to mutant proSP-C lacking the NH(2) targeting domain (EGFP/SP-C-(24-194)), or to mature SP-C alone (EGFP/SP-C-(24-58)) were produced. In transfected A549 cells, fluorescence microscopy revealed that pcDNA3/SP-C-(1-194) and EGFP/SP-C-(1-194) were each expressed in CD63 (+), EEA1 (-) cytoplasmic vesicles. Expression of EGFP/SP-C-(24-194) or EGFP/SP-C-(24-58) resulted in translocation but retention in early compartments. When co-transfected with pcDNA3/SP-C-(1-194), both EGFP/SP-C-(24-194) and EGFP/SP-C-(24-58) were directed to CD63 (+) vesicles that also contained SP-C-(1-194). In contrast, trafficking of a folding mutant that forms juxtanuclear aggregates, EGFP/SP-C(C122/186G), was not corrected by cotransfection with pcDNA3/SP-C-(1-194). Chemical cross-linking studies of transfected cell lysates with bismaleimidohexane produced multimeric forms of both EGFP/SP-C-(1-194) and EGFP/SP-C-(24-58). These results indicate that sorting involves oligomeric association of proSP-C monomers mediated by the mature SP-C domain. Heteromeric assembly allows wild type proSP-C to facilitate trafficking of SP-C mutants with intact transmembrane domains but lacking targeting signals. We speculate that heterotypic oligomerization of wild type with SP-C folding mutants produces a dominant negative thus contributing to the pathology of chronic lung disease associated with patients heterozygous for mutant SP-C alleles.

Members of the C/EBP transcription factor family stimulate expression of the human and rat surfactant protein A (SP-A) genes

Members of the CCAAT enhancer binding protein (C/EBP) transcription factor family were detected in fetal lung of both human and rat. In rat lung, the level of C/EBPs increased with time of gestation, peaking around birth. In adult rat lung, C/EBPs were localized to the alveolar type II cells. The effect of C/EBPs on pulmonary surfactant protein A (SP-A), which is also expressed late in gestation, was investigated. In contrast to control plasmids, C/EBPδ expressing plasmids reversed the action of a transcriptional silencer just upstream of the rat SP-A promoter. In order to test the effect of C/EBPs on endogenous SP-A gene expression, cells that express SP-A were exposed to a phosphorothioate-substituted, double-stranded oligonucleotide matching the consensus C/EBP binding site (decoy oligonucleotide) at concentrations from 0.5 to 10 μM for 72 h. A mutant oligonucleotide with an 8-base pair (bp) substitution served as a control. The decoy oligonucleotide reduced SP-A mRNA as much as 75% compared to a mutant oligonucleotide both in the human lung cell line, NCI-H441, and in primary human fetal alveolar type II cells. The data indicate that C/EBPs facilitate SP-A gene expression, possibly by overcoming transcriptional silencing.

Processing of Type II Procollagen Amino Propeptide by Matrix Metalloproteinases

In many embryonic tissues, type IIA procollagen is synthesized and deposited into the extracellular matrix containing the NH(2)-propeptide, the cysteine-rich domain of which binds to bone morphogenic proteins. To investigate whether matrix metalloproteinases (MMPs) synthesized during development and disease can cleave the NH(2) terminus of type II procollagens, we tested eight types of enzymes. Recombinant trimeric type IIA collagen NH(2)-propeptide encoded by exons 1-8 fused to the lectin domain of rat surfactant protein D was used as a substrate. The latter allowed trimerization of the propeptide domain and permitted isolation by saccharide affinity chromatography. Although MMPs 1, 2, and 8 did not show cleavage, MMPs 3, 7, 9, 13, and 14 cleaved the recombinant protein both at the telopeptide region and at the procollagen N-proteinase cleavage site. MMPs 7 and 13 demonstrated other cleavage sites in the type II collagen-specific region of the N-propeptide; MMP-7 had another cleavage site close to the COOH terminus of the cysteine-rich domain. To prove that an MMP can cleave the native type IIA procollagen in situ, we demonstrated that MMP-7 removes the NH(2)-propeptide from collagen fibrils in the extracellular matrix of fetal cartilage and identified the cleavage products. Because the N-proteinase and telopeptidase cleavage sites are present in both type IIA and type IIB procollagens and the telopeptide cleavage site is retained in the mature collagen fibril, this processing could be important to type IIB procollagen and to mature collagen fibrils as well.