Pulmonary Surfactant-associated Protein Research Articles

Genetic risk factors associated with respiratory distress syndrome.

Respiratory distress syndrome (RDS) among preterm infants is typically due to a quantitative deficiency of pulmonary surfactant. Aside from the degree of prematurity, diverse environmental and genetic factors can affect the development of RDS. The variance of the risk of RDS in various races/ethnicities or monozygotic/dizygotic twins has suggested genetic influences on this disorder. So far, several specific mutations in genes encoding surfactant-associated molecules have confirmed this. Specific genetic variants contributing to the regulation of pulmonary development, its structure and function, or the inflammatory response could be candidate risk factors for the development of RDS. This review summarizes the background that suggests the genetic predisposition of RDS, the identified mutations, and candidate genetic polymorphisms of pulmonary surfactant proteins associated with RDS.

Regulation of transplanted mesenchymal stem cells by the lung progenitor niche in rats with chronic obstructive pulmonary disease

BackgroundStem cell transplantation is a promising method for the treatment of chronic obstructive pulmonary disease (COPD), and mesenchymal stem cells (MSCs) have clinical potential for lung repair/regeneration. However, the rates of engraftment and differentiation are generally low following MSC therapy for lung injury. In previous studies, we constructed a pulmonary surfactant-associated protein A (SPA) suicide gene system, rAAV-SPA-TK, which induced apoptosis in alveolar epithelial type II (AT II) cells and vacated the AT II cell niche. We hypothesized that this system would increase the rates of MSC engraftment and repair in COPD rats.MethodsThe MSC engraftment rate and morphometric changes in lung tissue in vivo were investigated by in situ hybridization, hematoxylin and eosin staining, Masson’s trichrome staining, immunohistochemistry, and real-time PCR. The expression of hypoxia inducible factor (HIF-1α) and stromal cell-derived factor-1 (SDF-1), and relationship between HIF-1α and SDF-1 in a hypoxic cell model were analyzed by real-time PCR, western blotting, and enzyme-linked immunosorbent assay.ResultsrAAV-SPA-TK transfection increased the recruitment of MSCs but induced pulmonary fibrosis in COPD rats. HIF-1α and SDF-1 expression were enhanced after rAAV-SPA-TK transfection. Hypoxia increased the expression of HIF-1α and SDF-1 in the hypoxic cell model, and SDF-1 expression was augmented by HIF-1α under hypoxic conditions.ConclusionsVacant AT II cell niches increase the homing and recruitment of MSCs to the lung in COPD rats. MSCs play an important role in lung repair and promote collagen fiber deposition after induction of secondary damage in AT II cells by rAAV-SPA-TK, which involves HIF-1α and SDF-1 signaling.

Effects of dexamethasone and oxidative stress on surfactant protein A, B, C and D expressions of H441 cells

Objective To explore the effects of dexamethasone on the expressions of surfactant protein(SP) A,B,C and D in H441 cells injured by oxidative stress.Methods H441 cells were challenged with hydrogen peroxide as oxidative stress damage cell model.Then the cells were divided into control group,groups on different dexamethasone levels (1,10 and 100 umol/L),H2O2 (100 μmol/L) injury group and dexamethasone (100 μmol/L)+ H2 O2 (100 μmol/1) group.All cells were cultured for 24 hours,then the expressions of SP mRNA were analyzed by quantitative real timepolymerase chain reaction,and proteins were detected by Western blot.One-way variant analysis and Dunneu's T3 test were applied as statistical methods.Results (1) The mRNA level of SP-A in H2 O2 injury group was higher than that in the control group (4.0016±0.3618 vs 0.9914±0.4170),while mRNA of SP C and SP-D were lower than those in the control group (SP C:0.2840±0.0586 vs 0.9719±0.1022;SP-D:0.6199±0.0074 vs0.9518±0.0614) (all P＜0.05).Compared with H2O2injury group,the mRNA level of SP-A in dexamethasone + H2O2 group was down-regulated (2.5191±0.3463),but SP-D mRNA was up-regulated (0.9076±0.0167)(all P＜0.05).(2) The SP-A protein level in H2O2 injury group was higher than that in the control group (2.1795±0.0645 vs 0.9728±0.009),while SP-B、SP-C and SP-D protein levels were lower than those in the control group (SP-B:0.2593±0.0086 vs 0.7253±0.0134;SP-C:0.5592±0.0111 vs 0.9472±0.0052;SP-D:1.1550±0.0237 vs 1.2991±0.0298).Compared with H2O2 injury group,the protein level of SP-A in dexamethasone+ H2 O2 group was down-regulated(1.4974 ± 0.0185),while SP-B,SP-C and SP-D levels were up-regulated (0.7389±0.0182,0.9244±0.0282 and 1.3339±0.0136).The differences were statistically significant respectively (all P＜0.05).Conclusions Dexamethasone may protect H441 cells from being damaged by H2O2 through regulating SP mRNA and protein expression. Key words: Oxidative stress; Cell line, tumor; Pulmonary surfactant-associated proteins; Dexamethasone

Differential effects of insulin and dexamethasone on pulmonary surfactant-associated genes and proteins in A549 and H441 cells and lung tissue

In this study, the effects of insulin and dexamethasone on the expression and mRNA transcription of 4 pulmonary surfactant-associated proteins [surfactant protein (SFTP)A, SFTPB, SFTPC and SFTPD] were examined. The commercially available cell lines, A549 and H441, were used as acceptable models of lung surfactant-producing cells. Subsequently, the effects of insulin on the expression of surfactant-associated proteins were examined in patients with lung adenocarcinoma during lung resection. Our results demonstrated the inhibitory effects of insulin on the transcription of the SFTPB, SFTPC and SFTPD genes in H441 cells and the SFTPB gene in A549 cells. Treatment with insulin significantly decreased the protein expression of SFTPA1 and SFTPA2 in the H441 cells and that of proSFTPB in the A549 cells. Dexamethasone promoted the transcription of the SFTPB, SFTPC and SFTPD genes in the A549 and H441 cells and reduced the transcription of the SFTPA1 and SFTPA2 genes in the H441 cells (SFTPA mRNA expression was not detected in A549 cells). Furthermore, we demonstrated that the mRNA levels of the selected genes were significantly lower in the cell lines compared to the lung tissue. A549 and H441 cells represent similar cell types. Yet, in our experiments, these cells reacted differently to insulin and/or dexamethasone treatment, and the mRNA levels of their main protein products, surfactant-associated proteins, were significantly lower than those in real tissue. Therefore, the results obtained in this study challenge the suitability of A549 and H441 cells as models of type II pneumocytes and Clara cells, respectively. However, we successfully demonstrate the possibility of studying the effects of insulin on pulmonary surfactant-associated genes and proteins in patients with lung adenocarcinoma.

Expression of Pulmonary Surfactant-Associated Protein B in Neonatal Respiratory Distress Syndrome

Summary Background: The aim of this study was to investigate the role of pulmonary surfactant-associated protein B (SP-B) expression in the pathogenesis of neonatal respiratory distress syndrome (RDS) via detecting the protein and mRNA expression of SP-B. Methods: A total of 60 unrelated neonates who died of RDS were chosen as the RDS group and then subgrouped into ≤32 weeks group, 32∼37 weeks group and ≥37 weeks group (n=20). Sixty neonates who died of other diseases were enrolled as controls and subdivided into 3 matched groups based on the gestational age. Western blot assay and RT-PCR were employed. Results: In the RDS group, SP-B protein expression was reduced or deficient in 8 neonates of which 6 had no SP-B protein expression. In the control group, only 1 had reduced SP-B protein expression. The reduced or deficient SP-B protein expression in 9 neonates of both groups was noted in the ≥37 weeks group. In the RDS group, the SP-B mRNA expression was significantly lower than that in the control group. In the ≤37 weeks group, SP-B mRNA expression was comparable between the RDS group and control group. In the 32∼37 weeks group, the SP-B mRNA expression in the RDS group was significantly reduced when compared with the control group. In the ≥37 weeks group, the SP-B mRNA expression in the RDS group was dramatically lower than that in the control group. Conclusions: Alteration of SP-B expression is present at transcriptional and translational levels. Reduction of SP-B mRNA and protein expression is involved in the pathogenesis of RDS.

Molecular pathology of pulmonary surfactants and cytokines in drowning compared with other asphyxiation and fatal hypothermia

Drowning involves complex fatal factors, including asphyxiation and electrolyte/osmotic disturbances, as well as hypothermia in cold water. The present study investigated the molecular pathology of pulmonary injury due to drowning, using lung specimens from forensic autopsy cases of drowning (n = 21), acute mechanical asphyxia due to neck compression and smothering (n = 24), and hypothermia (cold exposure, n = 11), as well as those of injury (n = 23), intoxication (n = 13), fire fatality (n = 18), and acute cardiac death (n = 9) for comparison. TaqMan real-time reverse transcription polymerase chain reaction was used to quantify messenger RNA (mRNA) expressions of pulmonary surfactant-associated proteins A and D (SP-A and SP-D), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-10. SP-A and SP-D mRNA levels were lower for drowning, mechanical asphyxiation, fire fatality, and acute cardiac deaths than for hypothermia and injury. TNF-α, IL-1β, and IL-10 mRNA levels were higher for drowning or for drowning and injury than for other groups; there was no significant difference between fire fatality, involving airway injury due to inhalation of hot/irritant gases, and other control groups. These observations suggest characteristic molecular biological patterns of pulmonary injury involving suppression of pulmonary surfactants and activation of early-phase mediators of inflammation in drowning, with high mRNA expression levels of pulmonary surfactants in fatal hypothermia; however, there was no significant difference among these markers in immunohistochemical detection, except for SP-A. These mRNA expressions can be used as markers of pulmonary injury to assist in investigations of the pathophysiology of drowning and fatal hypothermia in combination with other biochemical and biological markers.

Lucinactant for the treatment of respiratory distress syndrome in neonates

Respiratory distress syndrome (RDS) is a leading cause of morbidity and mortality in premature neonates. This syndrome is caused by a lack of endogenous surfactant production in the lungs. Surfactant replacement was established as a safe and effective treatment in the 1990s and has become the standard of care for these infants. Surfactant products are either protein-free synthetic phospholipid compounds or animal-derived lung preparations. Currently, about 90,000 infants a year receive treatment with one of the commercially available animal-derived surfactants. Lucinactant (Surfaxin®) is a new synthetic surfactant with a pulmonary surfactant-associated protein B mimic that recently received FDA approval. The clinical trials that have been performed, although underpowered, may indicate that lucinactant is superior to phospholipid synthetic surfactant preparations and at least as effective as animal-derived surfactants in reducing morbidity and mortality from RDS. This review summarizes the current clinical knowledge about lucinactant.

Roles of vascular endothelial growth factor and dexamethasone in expressions of surfactant protein B and transforming growth factor-β1 in type II alveolar epithelial cells

Objective To investigate the effects of vascular endothelial growth factor (VEGF) and dexamethasone on mRNA expressions of surfactant protein B (SP-B) and transforming growth factor-β1 (TGF-β1) of type Ⅱ alveolar epithelial cell (AECⅡ). Methods AECⅡ were isolated and purified from fetal rat lung tissues,then cultured with different dose of VEGF (25,50 and 100 ng/ml) and dexamethasone (25,50,100 and 200 nmol/ml).The mRNA levels of SP-B and TGF-β1 were detected by real-time quantitative polymerase chain reaction (RT-PCR) and expression of TGF-β1 protein was detected by immunocytochemistry.ANOVA or q-test was applied to compare the difference among groups.Results Compared with control group,SP-B mRNA levels in 25 ng/ml VEGF group and 25,50,100 and 200 nmol/ml dexamethasone groups were higher (13.500±3.172,3.547±0.690,5.219±0.782,3.493±0.335,and 3.981 ± 1.133 vs 1.001 ± 0.059,q=-5.286,-4.943,- 7.228,- 9.906 and - 3.525 respectively,P＜0.05) ; TGF-β1 mRNA expression of 25 ng/ml VEGF group,50,100 and 200 nmol/ml dexamethasone group was lower (0.451 ± 0.078,0.579±0.019,0.422 ± 0.020 and 0.769 ± 0.025 vs 1.019±0.226,q=4.110,3.356,4.551 and 1.901 respectively,P＜0.05) ; other groups had no significant differences compared with control group (P＞0.05).Immunocytochemistry showed that the positive rate of TGF-β1 expression in 25 ng/ml VEGF,50,100 and 200 nmol/ml dexamethasone group was 23％,26％,22％ and 29％,respectively,while in the control group,the expression of TGF-β1 was positive in most of the AECⅡ (80％).Conclusions Both VEGF and dexamethasone could increase the expression of SP-B at mRNA level at appropriate concentrations.At the same time,the expression of TGF-β1 is inhibited.It is suggested that both VEGF and dexamethasone might increase the mRNA expression of SP-B by inhibiting the expression of TGF-β1. Key words: Pulmonary alveoli; Pulmonary surfactant-associated protein B; Transforming growth factor beta 1 ; Vascular endothelial growth factor A; Daxamethasone

Expression of pulmonary surfactant protein A mRNA in lungs of fetal rats with maternal intrahepatic cholestasis

Objective To investigate the pulmonary morphologic changes and pulmonary surfactant protein A (SP-A) mRNA expression in fetal rats with maternal intrahepatic cholestasis of pregnancy (ICP).Methods Animal models of intrahepatic cholestasis of pregnancy were induced by 17-α-ethinylestradiol and progesterone.Histopathologic examination of the fetal lungs was performed under light and electronic microscopes.The expression of SP-A mRNA in the fetal lungs was measured by reverse transcription-polymerase chain reaction technique.Results (1) Compared with the control group,histopathologic examination showed that fetal pulmonary tissues had dilated and congested interstitial lung capillaries,thickened alveolar septum,mild focal inflammatory exudation and focal hemorrhage in alveolus.Furthermore,reduced microvilli,mitochondrion vacuolization,cytoplasm disintegration and increased lamellar body evacuation were observed in alveolar epithelial cell II in ICP group under light and electronic microscopes.(2) Expression of SP-A mRNA in fetal lungs of ICP group (0.71 + 0.10) was significantly lower than that in control group (1.00±0.27),t=3.093,P＜0.05.Conclusions Hyperbileacidemia in ICP maternal rat might lead to pathological changes in fetal pulmonary tissues.Low expression of SP-A mRNA might contribute to lesions of fetal lung with ICP. Key words: Pregnancy complications; Cholestasis,intrahepatic; Pulmonary surfactantassociated protein A; RNA,messenger; Rats

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

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

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

Effects of different ventilation modes on efficacy of exogenous pulmonary surfactant for treatment of rats with ventilator-induced lung injury

Objectlve To investigate the effects of different ventilation modes on the efficacy of exogenous pulmonary surfactant(PS)for the treatment of rats with ventilator-induced lung injury(VILI).Methods Forty-two male Wistar rats weighing 310-356 g were randomly divided into 6 groups(n=7 each):group CVT6,group SVT6,group CVT10,group SVT10,group CVT14 and group SVT14.The tidal volume(VT)was set at 6,10 and 14 ml/kg respectively and the respiratory rate(RR) was 75,45 and 32 bpm respectively.The animals were anesthetized with intraperitoneal 3% Pentobarbital 50 mg/kg,then tracheostomized and intubated.VILI model was induced by high-pressure ventilation (HPV) with peak inspimtory pressure (PIP) 40 cm H2O and without positive end-expiratory pressure (PEEP).The air was injected into the trachea via the airway at the end ofexpiration before HPV (T0,baseline value) and 15-25 min of HPV,the airway pressure monitored and the lung compliance(C) calculated.When C was decreased to half of the baseline value,PEEP was increased to 7.5 cm H20.After the tracheal edema fluid was removed,the PS 100 mg/kg was immediately injected into the trachea in group SVT6,SVT10 and SVT14.The equal volume of air was injected into the trachea in group CVT6,CVT10 and CVr14 instead of PS.Then the rats in different groups were ventilated with the corresponding ventilation modes.MAP was monitored and blood samples were token from femoral artery for blood gas analysis at T0, 5 min after HPV (T1 ), and 15, 30, 60, 90, 120 min (T2-6) after administration of PS. The tracheal edema fluid was collected at T1 and T6.The rats were killed at T6 and the lung tissues taken for microscopic examination. Results With the same ventilation mode, the VILI was significantly alleviated after administration of PS. With different ventilation modes,the lung injury was significantly reduced in group SVT 10 compared with the other groups. Conclusion The efficacy of PS for the treatment of rats with VILI is good using the ventilation strategy with VT of 10 ml/kg and RR of 45 bpm. Key words: Pulmonary surfactant-associated protein B; Pulmonary surfactant-associated protein C; Respiratory distress syndrome, adult; Respiration, artificial

Effects of positive end expiratory pressure ventilation upon respiratory function and hydrophobic surfactants proteins in rabbit with seawater respiratory distress syndrome

To investigate the effects of positive end-expiratory pressure (PEEP) ventilation upon respiratory function and lung surfactant protein B (SP-B)/SP-C in seawater respiratory distress syndrome (SW-RDS) rabbit. Twenty-four female New Zealand rabbits were randomly equally divided into three groups:conventional mechanical ventilation (CMV) group, PEEP group, and control group. After anesthesia and tracheal intubation followed by 20 minutes of CMV, SW-RDS model was established by lung perfusion of artificial seawater through tracheal intubation. Then the CMV group ventilated for 6 hours (each parameter unchanged); PEEP group ventilated with PEEP of 8 cm H2O for 2 hours based on prior parameters and subsequently with CMV for 4 hours; control group without ventilation after modeling. Oxygenation indices and lung compliance were continuously monitored during ventilation. After ventilation, the rabbits in study groups were sacrificed while those dying a natural death in the control group selected. Bronchoalveolar lavage fluid of the left lung was collected to determine the alveolar surface tension and the right lung was harvested to measure the mRNA expression of SP-B/SP-C by real-time PCR as well as SP-B protein by Western blotting. After modeling, the data of lung compliance and oxygenation indices became significantly worse in every group but without statistical difference in three groups. All rabbits in control group died within 15 minutes of ventilator withdrawal. After receiving PEEP ventilation for 30 minutes, these indices significantly improved compared with CMV group in which there was no significant change of the indices (P < 0.05). Minimal alveolar surface tension in CMV group (mN/m, 30.8 +/- 6.3) was greater than in PEEP group (21.1 +/- 4.4, P < 0.05) and control group (23.6 +/- 4.6, P < 0.05); SP-B/SP-C mRNA relative expression (0.37 +/- 0.15/0.60 +/- 0.19) and SP-B relative protein abundance (0.38 +/- 0.17) in CMV group were obviously lower than in PEEP group (0.73 +/- 0.15/0.92 +/- 0.40, 0.52 +/- 0.22, P < 0.05). PEEP ventilation can improve the oxygenation indices and lung compliance in SW-RDS animals. And such an effect is correlated with both the mRNA expressions of SP-B/SP-C and mechanical distension.

Glycoprotein analysis of porcine bronchoalveolar lavage fluid reveals potential biomarkers corresponding to resistance toActinobacillus pleuropneumoniaeinfection

Biomarkers facilitating both pathogen-independent diagnosis of respiratory health and breeding selection of pigs with increased resistance to respiratory tract infections would be of considerable interest to the pig industry. Following this concept we performed a comparative glycoproteome analysis of bronchoalveolar lavage fluid (BALF) from healthy pigs and pigs 4 days (acute) and 20 days (chronic) after an experimental infection with Actinobacillus pleuropneumoniae. In order to identify possible differences in BALF glycoprotein patterns we investigated pigs of three different breeding lines (German Landrace, Piétrain, Hampshire). In total, 12 glycosylated proteins (alpha-1-acid glycoprotein, fetuin A, properdin, haptoglobin precursor, haptoglobin, hemoglobin, hyaluronidase, inter-alpha-trypsin inhibitor family heavy chain-related protein, alpha-1-antichymotrypsin 3, pulmonary surfactant-associated protein D (SP-D), transferrin, and alpha-1B-glycoprotein) were identified as being differentially expressed depending on the health status of the animal. Fetuin A levels were consistently low in chronically infected pigs thereby being a potential marker for chronic infection. Hyaluronidase levels were consistently high in all pigs after experimental infection independent on isolation of the pathogen thereby being a potential marker for previous pathogen contact and latent infection. High levels of fetuin A as well as low levels of haptoglobin and pulmonary SP-D correlated with the absence of lung lesions in pigs of the Hampshire breeding line, implying a potential application as selection markers for breeding programmes.

Trimeric coiled-coil domain of human pulmonary surfactant protein D enhances zinc-binding ability and biologic activity of soluble TRAIL

Unlike other TNF ligand family members, the homotrimeric tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) contains a buried zinc atom coordinated by three Cys230 residues from each subunit. The bound zinc ion is essential for maintaining the structure stability and bioactivity of TRAIL. To improve characteristics of TRAIL by modification to enhance its zinc-binding ability, we constructed a new variant of TRAIL in which the extracellular region of the ligand was N-terminally fused with a trimeric coiled-coil domain derived from human pulmonary surfactant-associated protein D (ST), and compared its characteristics with that of native TRAIL. Circular dichroism (CD) studies and metal assays showed that two versions of TRAIL folded as predicted into secondary and tertiary structures, and contained stoichiometric Zn 2+ through optimizing bacterial expression and purification. The addition of the human trimeric coiled-coil domain, however, significantly increased the antitumor activity of soluble TRAIL in vitro and in vivo. The accelerated thermal stability studies demonstrated that human serum album (HSA) promoted the aggregation and degradation of native TRAIL, but not ST, and the addition of ZnSO 4 to the solution of native TRAIL with HSA partially inhibited its aggregation, suggesting ST is more difficult to lose its bound zinc ion than native TRAIL. The issue was further confirmed by dialysis assess. This is the first example of modified TRAIL with enhanced zinc-binding ability.

Expression of surfactant protein-C and Ki67 in pulmonary tissues of premature infants died of hyaline membrane disease

Objective To determine the expression of surfactant protein-c (SP-C) and Ki67 in autopsy lung tissues of premature infants died of hyaline membrane disease (HMD) who were exposed to mechanical ventilation and high oxygen concentrations/pulmonary surfactant (PS). The possible influence of surfactant on the expression of SP-C and Ki67 was also investigated. Methods Thirty preterm infants diagnosed as HMD by clinical data and pathology were selected. Mechanical ventilation and supplemental oxygen (FiO2 0.6-1.0) were given to these infants within 6 hours after birth. According to the duration of ventilation at high oxygen concentrations, all subjects were diveded into four groups: ventilation for 1-3 days, 4-8 days, 9-16 days and >16 days. Five premature infants died within 1 day after delivery for none pulmonary reasons served as controls. The expression of SP-C and Ki67 in lungs were detected by immunobistochemistry. Results The pulmonary pathology in these HMD infants at different phases was consisitent with the main features of different stages during the progress from HMD towards bronchopulmonary dysplasia. The positive staining of SP-C was restricted to type Ⅱ alveolar epithelial cells, and Ki67 positive were preferentially localized in nuclei of alveolar and bronchiolar epithelial cells and fihroblasts. Compared with the control group, the expression of SP-C and Ki67 in HMD infants decreased significantly after 1-3 days of ventialation, but increased after 4 days and reached the peak value after 9-16 days. The expression of SP-C and Ki67 in control group, 1-3 days and 9-16 days of ventialation were 0. 1891±0. 0253, 0. 1576±0. 0327 and 0. 2271±0. 0238 for SP-C and 0. 2297±0. 0380, 0.1929±0. 0403 and 0. 2849±0. 0368 for Ki67, respectively. No significant difference in the expression of SP-C and Ki67 was found between infants treated with PS and those without (P>0.05). Conclusions SP-C and Ki67 may have participated in the pulmonary pathological process in ventilated/oxygen treated preterm infants with HMD, and exogenous surfactant had no effect on the expression of SP-C and Ki67. Key words: Hyaline membrane disease; Pulmonary surfactant-associated protein C; Ki-67 antigen; Infant, premature

Postmortem serum levels of pulmonary surfactant-associated proteins A and D with regard to the cause of death in medicolegal autopsy

Pulmonary surfactant-associated proteins A and D (SP-A and -D) are tissue-specific components. Previous studies showed an increase in the postmortem serum SP-A level due to acute pulmonary alveolar damage and acute respiratory distress. The present study comparatively investigated serum SP-A and SP-D levels with regard to the cause of death in serial medicolegal autopsy cases ( n = 679, within 48 h postmortem). SP-A and SP-D levels were usually higher in left cardiac blood than at other sites, independent of postmortem interval. The left-to-right difference was significantly larger for mechanical asphyxiation, drowning, intoxication and spontaneous cerebral hemorrhage. Both SP-A and -D levels in bilateral cardiac blood were significantly higher for drowning and secondary pulmonary damage involving ARDS after traumas, but were lower for hypothermia (cold exposure). SP-A was predominantly elevated in fire fatality and delayed deaths from injury and fires, while pneumonia showed a predominant elevation of SP-D. These findings suggest that comparative analysis of serum SP-D and SP-A is useful for investigating primary or secondary pulmonary alveolar damage in the death process.

Oxidative Stress and Asthma: Proteome Analysis of Chitinase-like Proteins and FIZZ1 in Lung Tissue and Bronchoalveolar Lavage Fluid

Oxidative stress plays an important role in the development of airway inflammation and hyperreactivity in asthma. The identification of oxidative stress markers in bronchoalveolar lavage fluid (BALF) and lung tissue from ovalbumin (OVA) sensitized mice could provide new insight into disease pathogenesis and possible use of antioxidants to alleviate disease severity. We used two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the impact of the thiol antioxidant, N-acetylcysteine (NAC), on protein expression in a murine OVA model. At least six proteins or protein families were found to be significantly increased in BALF from OVA-challenged mice compared to a control group: Chitinase 3-like protein 3 (Yml), Chitinase 3-like protein 4 (Ym2), acidic mammalian Chitinase (AMCase), pulmonary surfactant-associated protein D (SP-D), resistin-like molecule alpha (RELMalpha) or "found in inflammatory 1" (FIZZ1), and haptoglobin alpha-subunit. A total of nine proteins were significantly increased in lung tissue from the murine asthma model, including Yml, Ym2, FIZZ1, and other lung remodeling-related proteins. Western blotting confirmed increased Yml/Ym2, SP-D, and FIZZ1 expression measured from BAL fluid and lung tissue from OVA-challenged mice. Intraperitoneal NAC administration prior to the final OVA challenge inhibited Yml/Ym2, SP-D, and FIZZ1 expression in BALF and lung tissue. The oxidative stress proteins, Ym1/Ym2, FIZZ1, and SP-D, could play an important role in the pathogenesis of asthma and may be useful oxidative stress markers.

Aberrant Processing Forms of Lung Surfactant Proteins SP–B and SP–C Revealed by High-Resolution Mass Spectrometry

The mutation (g.1286T>C) of the pulmonary surfactant-associated protein C gene (SFTPC) leads to the I73T substitution in the precursor protein (pro-SP-C) and results in interstitial lung disease with the histological pattern of non-specific interstitial pneumonia and pulmonary alveolar proteinosis. Central for the disease is the abnormal processing of the SP-C pro-protein to mature SP-C; however little is known about the nature of intermediates and processing products. We report here the application of high resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry to the characterization of processing intermediates of hydrophobic pulmonary surfactant proteins SP-B and SP-C in intra- alveolar surfactant material of a patient with I73T mutation. SP-C and SP-B processing forms were separated from broncho-alveolar lavage fluid using chloroform/methanol extraction and sodium dodecyl sulfate poly acrylamide gel electrophoreis, detected by Western blot and identified by electrospray- and matrix-assisted laser desorption/ionization-FT-ICR mass spectrometry. The mass spectrometric and immuno-analytical results show the intra-alveolar accumulation of an aberrant C-terminal SP-C processing products in which the mature SP-C protein part is missing and aberrant processing intermediates of SP-B.

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

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