Surfactant Protein C Expression Research Articles

IRF4-mediated Treg phenotype switching can aggravate hyperoxia-induced alveolar epithelial cell injury

Bronchopulmonary dysplasia (BPD) is characterized by alveolar dysplasia, and evidence indicates that interferon regulatory factor 4 (IRF4) is involved in the pathogenesis of various inflammatory lung diseases. Nonetheless, the significance and mechanism of IRF4 in BPD remain unelucidated. Consequently, we established a mouse model of BPD through hyperoxia exposure, and ELISA was employed to measure interleukin-17 A (IL-17 A) and interleukin-6 (IL-6) expression levels in lung tissues. Western blotting was adopted to determine the expression of IRF4, surfactant protein C (SP-C), and podoplanin (T1α) in lung tissues. Flow cytometry was utilized for analyzing the percentages of FOXP3+ regulatory T cells (Tregs) and FOXP3+RORγt+ Tregs in CD4+ T cells in lung tissues to clarify the underlying mechanism. Our findings revealed that BPD mice exhibited disordered lung tissue structure, elevated IRF4 expression, decreased SP-C and T1α expression, increased IL-17 A and IL-6 levels, reduced proportion of FOXP3+ Tregs, and increased proportion of FOXP3+RORγt+ Tregs. For the purpose of further elucidating the effect of IRF4 on Treg phenotype switching induced by hyperoxia in lung tissues, we exposed neonatal mice with IRF4 knockout to hyperoxia. These mice exhibited regular lung tissue structure, increased proportion of FOXP3+ Tregs, reduced proportion of FOXP3+RORγt+ Tregs, elevated SP-C and T1α expression, and decreased IL-17 A and IL-6 levels. In conclusion, our findings demonstrate that IRF4-mediated Treg phenotype switching in lung tissues exacerbates alveolar epithelial cell injury under hyperoxia exposure.

Abnormal low expression of SFTPC promotes the proliferation of lung adenocarcinoma by enhancing PI3K/AKT/mTOR signaling transduction.

The abnormality of surfactant protein C (SFTPC) has been linked to the development of a number of interstitial lung diseases, according to mounting evidence. Nonetheless, the function and mechanism of SFTPC in the biological progression of lung adenocarcinoma (LUAD) remain unclear. Analysis of public datasets and testing of clinical samples suggested that SFTPC expression was abnormally low in LUAD, which was associated with the onset and poor prognosis of LUAD. The SFTPC-related risk score was derived using least absolute shrinkage and selection operator Cox regression as well as multivariate Cox regression. The risk score was highly correlated with tumor purity and tumor mutation burden, and it could serve as an independent prognostic indicator for LUAD. Low-risk LUAD patients may benefit more from CTLA-4 or/and PD-1 inhibitors. Overall, the risk score is useful for LUAD patient prognostication and treatment guidance. Moreover, in vitro and in vivo experiments demonstrated that SFTPC inhibits the proliferation of LUAD by inhibiting PI3K/AKT/mTOR signaling transduction. These results reveal the molecular mechanism by which SFTPC inhibits the proliferation of LUAD and suggest that SFTPC could be a new therapeutic target for LUAD.

A promoterless AAV6.2FF-based lung gene editing platform for the correction of surfactant protein B deficiency

Surfactant protein B (SP-B) deficiency is a rare genetic disease that causes fatal respiratory failure within the first year of life. Currently, the only corrective treatment is lung transplantation. Here, we co-transduced the murine lung with adeno-associated virus 6.2FF (AAV6.2FF) vectors encoding a SaCas9-guide RNA nuclease or donor template to mediate insertion of promoterless reporter genes or the (murine) Sftpb gene in frame with the endogenous surfactant protein C (SP-C) gene, without disrupting SP-C expression. Intranasal administration of 3×1011 vg donor template and 1×1011 vg nuclease consistently edited approximately 6% of lung epithelial cells. Frequency of gene insertion increased in a dose-dependent manner, reaching 20%-25% editing efficiency with the highest donor template and nuclease doses tested. We next evaluated whether this promoterless gene editing platform could extend survival in the conditional SP-B knockout mouse model. Administration of 1×1012 vg SP-B-donor template and 5×1011 vg nuclease significantly extended median survival (p= 0.0034) from 5days in the untreated off doxycycline group to 16days in the donor AAV and nuclease group, with one gene-edited mouse living 243days off doxycycline. This AAV6.2FF-based gene editing platform has the potential to correct SP-B deficiency, as well as other disorders of alveolar type II cells.

Roles of Nrf2/HO-1 and ICAM-1 in the Protective Effect of Nano-Curcumin against Copper-Induced Lung Injury.

Copper (Cu) is an essential trace element for maintaining normal homeostasis in living organisms. Yet, an elevated level of Cu beyond homeostatic capacity may lead to oxidative damage of cellular components in several organs, including the lungs. This work investigated the effects of curcumin (Curc) and nano-curcumin (nCurc) against Cu-induced lung injury, accenting the roles of oxidative stress, inflammation, and the nuclear factor erythroid 2-related factor/heme oxygenase-1 Nrf2/HO-1 pathway. Rats were challenged with 100 mg/kg of copper sulfate (CuSO4) while being treated with Curc or nCurc for 7 days. Cu-triggered lung oxidative stress detected as dysregulation of oxidative/antioxidant markers, a downregulation of Nrf-2/HO-1 signaling, and an increase in the inflammatory markers interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and intracellular adhesion molecule-1 (ICAM-1). Additionally, it decreased the expression of lung-specific proteins, surfactant protein-C (SP-C), and mucin-1 (MUC-1), induced apoptosis, and caused changes in lung histology. Curc and nCurc alleviated CuSO4-induced lung injury by suppressing oxidative damage and inflammation and activating Nrf-2/HO-1. They also prevented apoptosis and restored the normal expression of SP-C and MUC-1. We concluded that nCurc exhibited superior efficacy compared with Curc in mitigating CuSO4-induced lung injury. This was associated with reduced oxidative stress, inflammation, and apoptotic responses and increased Nrf2/HO-1 signaling and expression of SP-C and MUC-1.

Activating Transcription Factor 6 (ATF6) Modulates The Proteostatic Response To Mutant Surfactant Protein C Conformers In Alveolar Epithelial Cells

Alveolar Type II (AT2) epithelial cell dysfunction is a hallmark of the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF), a progressive interstitial lung disease. Mutations in the surfactant protein C (SP-C) gene (SFTPC) are a high effect size etiological cause of PF in a subset of these patients. Prior in vitro studies from our lab showed that SFTPC mutations located within the BRICHOS domain of the SP-C proprotein result in production of ER retained and aggregation prone conformers. A major aspect of the proteostasis network is the unfolded protein response (UPR), which is initiated when misfolded proteins activate one or more of 3 ER transmembrane sensing proteins (Protein kinase R-like ER kinase (PERK), the Inositol-Requiring kinase 1 (IRE1) and Activating Transcription Factor 6 (ATF6). Consistent with this, we published that clinical IPF associated BRICHOS SFTPC mutations activate all 3 arms of UPR/ER stress signaling in mice in vivo. The goal of this study was to define AT2 proteostatic responses to SFTPC expression and the contribution of ATF6 to the UPR. For this, we generated stable doxycycline-inducible Mouse Lung Epithelial (MLE-12) cell lines expressing either Wild Type (SP-CWT) or an aggregating mutant (SP-CC121G) which were interrogated using cycloheximide chase and / or treatments with activators or inhibitors of ATF6, IRE1, and PERK as well as ERAD (vp97) or autophagy. Readouts included qPCR, luciferase reporter assays, and immunoblotting to assess UPR signaling and apoptosis, coupled with fluorescence microscopy to localize SP-C expression. Using this model system we found that SP-CC121G was ER retained and activated the UPR within 8 hours of Dox induction expression as measured by BIP expression. Commensurate with this both IRE1a (as spliced XBP1 (sXBP1)) and ATF6 were activated by SP-CC121G. The clearance of both mutant SP-CC121G as well as a portion of proSP-CWT was dependent on ERAD as its disruption using the vp97 inhibitor Eeyarestatin I induced formation of higher molecular weight aggregates while bafilomycin failed to induce SP-CC121G aggregates or block their clearance. ATF6 and IRE1 activation during ERAD inhibition accelerated aggregate protein degradation. When SP-CC121G MLE12 cells were treated with either an ATF6 inhibitor or an IRE1 activator, marked increases in levels of C/EBP Homologous Protein (CHOP) occur while either ATF6 agonism or IRE1 inhibition decreased CHOP expression. Collectively, these our data demonstrate a primary role for ERAD in the proteostatic response to SP-C expression while ATF6 agonism appears to provide protection from SFTPC mutant expression in part by accelerating degradation of SP-C aggregates in the face of IRE1 UPR signaling. NIH R01 HL145408 (MFB), VA Merit Review 2I01BX001176 (MFB) and NIH F32HL160011 (LRR) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Phenotypic Analysis of Fibrogenic Monocyte/Macrophage Subsets in Acute Inflammatory Exacerbations of Lung Disease Driven by Mutant Surfactant Protein-C Expression

Abstract Acute inflammatory exacerbations (AIE) represent precipitous deteriorations of a number of chronic lung conditions, including pulmonary fibrosis (PF), COPD, and asthma. AIEs are marked by diffuse and persistent polycellular alveolitis that profoundly accelerate lung function decline and mortality. Excess monocyte mobilization during AIE, and their persistence in the lung are linked to poor disease outcome. Guided by clinical evidence, we have developed an inducible model of pulmonary fibrosis leveraging the PF-linked missense isoleucine to threonine mutation at position 73 [I73T] in the alveolar type-2 cell-restricted Surfactant Protein-C [SP-C] gene [SFTPC]. We have previously shown that ablation of CCR2+ monocytes was protective with respect to lung histopathology, mouse survival, and overall inflammation following SP-C mutant induction. Here we identify and phenotypically characterize resident and recruited monocytes/macrophages populations intervening during the initiation and progression of AIE-PF. Single cell sequencing shows pro-inflammatory and pro-fibrotic signatures originating from immature recruited clusters (itgax-itgam+ ccr2+ cx3cr1+)as well as activated macrophages (cd68+ itgax+ itgam+ arg1+)14 d after SP-C I73Tinduction. Using CellChat Explorer database, these cells were predicted to coordinate intense tgfb1, fn1/fibronectinand spp1/osteopontinsignaling. Together, these results provide a clear picture of the fibrogenic role of specific activated monocyte/macrophage subsets responding to alveolar epithelial stress. NIEHS R01ES032553 (AV); ALSAM Foundation for Research Initiatives Grant (AV)

Decreased expression of surfactant Protein-C and CD74 in alveolar epithelial cells during influenza virus A(H1N1)pdm09 and H3N2 infection

The primary replication site of Influenza A virus (IAV) is type II alveolar epithelial cells (AECII), which are central to normal lung function and present important immune functions. Surfactant components are synthesized primarily by AECII, which play a crucial role in host defense against infection.The aim of this study was to analyze if the impact of influenza infection is differential between A(H1N1)pdm09 and A/Victoria/3/75 (H3N2) on costimulatory molecules and ProSP-C expression in AECII from BALB/c mice infected and A549 cell line infected with both strains.Pandemic A(H1N1)pdm09 and A/Victoria/3/75 (H3N2) were used to infect BALB/c mice and the A549 cell line. We evaluated the surface expression of co-stimulatory molecules (CD45/CD31/CD74/ProSP-C) in AECII and A549 cell lines. Our results showed a significant decrease in ProSP-C+ CD31− CD45– and CD74+ CD31− CD45− expression in AECII and A549 cell line with the virus strain A(H1N1)pdm09 versus A/Victoria/3/75 (H3N2) and controls (non-infection conditions).Our findings indicate that changes in the expression of ProSP-C in AECII and A549 cell lines in infection conditions could result in dysfunction leading to decreased lung compliance, increased work of breathing and increased susceptibility to injury.

A Potent Histone Deacetylase Inhibitor MPT0E028 Mitigates Emphysema Severity via Components of the Hippo Signaling Pathway in an Emphysematous Mouse Model.

BackgroundChronic obstructive pulmonary disease (COPD) is a major cause of chronic mortality. The objective of this study was to investigate the therapeutic potential of a novel potent histone deacetylase (HDAC) inhibitor MPT0E028 on emphysema.Materials and MethodsA mouse model of porcine pancreatic elastase (PPE)-induced emphysema was orally administered 0, 25, or 50 mg/kg body weight (BW) of the MPT0E028 five times/week for 3 weeks. Pulmonary function, mean linear intercept (MLI), chest CT, inflammation, yes-associated protein (YAP), transcriptional coactivator with PDZ-binding motif (TAZ), surfactant protein C (SPC), T1-α, p53, and sirtuin 1 (SIRT1) levels were examined.Results50 mg/kg BW of the MPT0E028 significantly decreased the tidal volume in emphysematous mice (p < 0.05). Emphysema severity was significantly reduced from 26.65% (PPE only) to 13.83% (50 mg/kg BW of the MPT0E028). Total cell counts, neutrophils, lymphocytes, and eosinophils significantly decreased with both 25 and 50 mg/kg BW of the MPT0E028 (p < 0.05). Also, 50 mg/kg BW of the MPT0E028 significantly decreased the levels of KC, TNF-α, and IL-6 in lung tissues and serum (p < 0.05). Expressions of p-TAZ/TAZ in lung tissues significantly decreased with 50 mg/kg BW of the MPT0E028 (p < 0.05). Expressions of p53 significantly decreased in alveolar regions with 50 mg/kg BW of the MPT0E028 (p < 0.05), and the expression of SPC increased in alveolar regions with 50 mg/kg BW of the MPT0E028 (p < 0.05).ConclusionsOur study showed that the potent HDAC inhibitor MPT0E028 reduced the severity and inflammation of emphysema with improvement in lung function, which could be regulated by Hippo signaling pathway. The MPT0E028 may have therapeutic potential for emphysema.

Distinct Epithelial Cell Profiles in Normal Versus Induced-Congenital Diaphragmatic Hernia Fetal Lungs.

BackgroundRecent studies identified a great diversity of cell types in precise number and position to create the architectural features of the lung that ventilation and respiration at birth depend on. With damaged respiratory function at birth, congenital diaphragmatic hernia (CDH) is one of the more severe causes of fetal lung hypoplasia with unspecified cellular dynamics.Objectives To characterize the epithelial cell tissue in hypoplastic lungs, a careful analysis regarding pulmonary morphology and epithelial cell profile was conducted from pseudoglandular-to-saccular phases in normal versus nitrofen-induced CDH rat lungs.DesignOur analysis comprises three experimental groups, control, nitrofen (NF) and CDH, in which the relative expression levels (western blot) by group and developmental stage were analyzed in whole lung. Spatiotemporal distribution (immunohistochemistry) was revealed by pulmonary structure during normal and hypoplastic fetal lung development. Surfactant protein-C (SP-C), calcitonin gene-related peptide (CGRP), clara cell secretory protein (CCSP), and forkhead box J1 (FOXJ1) were the used molecular markers for alveolar epithelial cell type 2 (AEC2), pulmonary neuroendocrine, clara, and ciliated cell profiles, respectively.ResultsGenerally, we identified an aberrant expression of SP-C, CGRP, CCSP, and FOXJ1 in nitrofen-exposed lungs. For instance, the overexpression of FOXJ1 and CGRP in primordia of bronchiole defined the pseudoglandular stage in CDH lungs, whereas the increased expression of CGRP in bronchi; FOXJ1 and CGRP in terminal bronchiole; and SP-C in BADJ classified the canalicular and saccular stages in hypoplastic lungs. We also described higher expression levels in NF than CDH or control groups for both FOXJ1 in bronchi, terminal bronchiole and BADJ at canalicular stage, and SP-C in bronchi and terminal bronchiole at canalicular and saccular stages. Finally, we report an unexpected expression of FOXJ1 in BADJ at canalicular and saccular stages, whereas the multi cilia observed in bronchi were notably absent at embryonic day 21.5 in induced-CDH lungs.ConclusionThe recognized alterations in the epithelial cell profile contribute to a better understanding of neonatal respiratory insufficiency in induced-CDH lungs and indicate a problem in the epithelial cell differentiation in hypoplastic lungs.

Effects of Different Ligands in the Notch Signaling Pathway on the Proliferation and Transdifferentiation of Primary Type II Alveolar Epithelial Cells.

Background: Transdifferentiation of type II alveolar epithelial cells (AECII) into type I alveolar epithelial cells (AECI) is involved in neonatal respiratory distress syndrome (NRDS). Different ligands of the Notch pathway could have different effects on AECII transdifferentiation.Objective: To investigate the effects of Dlk1 and Jagged1 on the proliferation and transdifferentiation of AECII.Methods: Fetal AECIIs (19 days of gestation) were divided: control group, Dlk1 group, rhNF-κB group. Proliferation was tested using the MTT assay. Expression of surfactant protein C (SP-C) and aquaporin 5 (AQP5) was examined by immunofluorescence. mRNA and protein levels of SP-C, AQP5, Nortch1, Dlk1, Jagged1, and Hes1 were examined by RT-PCR and western blot.Results: In response to Dlk1, cell number and proliferation were increased (P < 0.05), and mRNA and protein levels of SP-C, Dlk1, Notch1, and Hes1 were up-regulated, while AQP and Jagged1 were decreased. In response to rhNF-κB, the cell number and proliferation were reduced, and mRNA and protein levels of Jagged1 and Notch1 were up-regulated, while Dlk1, and SP-C were downregulated. In the Dlk1 group, SP-C, and AQP5 expression patterns suggested that the cells were still transdifferentiating by 96 h, while in the rhNF-κB group, most cells had transdifferentiated by 72 h and were close to apoptosis by 96 h.Conclusion: These results suggest that Dlk1 promoted proliferation of AECIIs and inhibited cell transdifferentiation, while Jagged1 treatment inhibited proliferation of AECIIs and promoted transdifferentiation to AECIs. These results provide some clue for the eventual management of NDRS.

Congenital diaphragmatic hernia as a potential target for transamniotic stem cell therapy

PurposeWe sought to determine whether TRASCET could impact congenital diaphragmatic hernia (CDH). MethodsTwelve pregnant dams received Nitrofen on gestational day 9.5 (E9; term = 22 days) to induce fetal CDH. Fetuses were divided into three groups: untreated (n = 31) and two groups receiving volume-matched intraamniotic injections of either saline (n = 37) or a suspension of 2 × 106 cells/mL of amniotic fluid-derived mesenchymal stem cells (afMSCs; n = 65) on E17. Animals were euthanized at term. Expression of fibroblast growth factor-10 (FGF-10), vascular endothelial growth factor-A (VEGF-A), and surfactant protein-C (SPC) was quantified by qRT-PCR. Statistical analysis was by the Mann–Whitney U test with Bonferroni adjusted criterion (p ≤ 0.01). ResultsAmong survivors with CDH (n = 27/133), the TRASCET group showed significant downregulation of FGF-10 and VEGF-A gene expressions compared to the untreated (p < 0.001 for both) and saline groups (p = 0.005 and p = 0.004, respectively). SPC expression was higher in the TRASCET group compared to the untreated group (p = 0.01), but not the saline group (p = 0.043). Lung laterality had minimal impact on these comparisons. ConclusionsTransamniotic stem cell therapy affects select processes of lung development in experimental congenital diaphragmatic hernia. Further scrutiny into this novel therapy as a potential component of the prenatal management of this disease is warranted. Level of evidenceN/A (animal and laboratory study).

Effects of C/EBP\u03b1 overexpression on alveolar epithelial type II cell proliferation, apoptosis and surfactant protein-C expression after exposure to hyperoxia

BackgroundThis study aims to investigate the effects of CCAAT/enhancer binding protein alpha (C/EBPα) overexpression on cell proliferation, apoptosis and surfactant protein-C(SP-C) in alveolar epithelial type II (AEC II) cells after exposure to hyperoxia.MethodspcDNA3.1(+)-C/EBPα plasmid or air-empty vector were transfected into AEC II cells with or without hyperoxia. AEC II cells were divided into air group, air+pcDNA3.1-C/EBPα group, air-empty vector group, hyperoxia group, hyperoxia+pcDNA3.1-C/EBPα group, and hyperoxia-empty vector group. Cell proliferation was analyzed using Cell Counting Kit-8. The mRNA level and protein expression were measured using PCR and Western blot techniques, respectively. The cell cycle and apoptosis were analyzed using flow cytometry.ResultsAfter 48 h of post-transfection, significantly higher protein expression of C/EBPα was observed in the C/EBPα transfection group with or without hyperoxia compared to the others (P < 0.05). Compared to the air group, hyperoxia decreased cell proliferation, increased apoptosis, decreased SP-C expression, decreased percentage of cells in G1 phase, and increased percentage of cells in the S and G2 phases (P < 0.05); however, reversed by C/EBPα transfection (P < 0.05). No significant changes were observed in cell proliferation, SP-C expression, and apoptosis rates in the C/EBPα transfection group as compared to the controls air-empty vector group.ConclusionC/EBPα overexpression significantly upregulates the expression of SP-C, promotes cell proliferation, and inhibits apoptosis in AEC II cells after exposure to hyperoxia. Hence, this data suggests that C/EBPα overexpression may reverse the damage and exert a protective role in hyperoxia-induced lung injury.

MiR-629-3p-induced downregulation of SFTPC promotes cell proliferation and predicts poor survival in lung adenocarcinoma

The long-term prognosis of patients with lung cancer remains poor and thus it is imminent to further elucidate the molecular mechanism for the oncogenesis of lung cancer. In this study, we observed that surfactant protein C (SFTPC) expression was downregulated in human lung adenocarcinoma tissues and cell lines, and low SFTPC expression correlated with poor overall survival of lung adenocarcinoma patients. Moreover, we found that overexpression of SFTPC could inhibit lung cancer cell proliferation in vitro and in vivo, but downregulation of SFTPC showed the opposite results. Besides, it was observed that miR-629-3p expression was upregulated in human lung adenocarcinoma tissues and cell lines. More importantly, we found that miR-629-3p could downregulate SFTPC expression by directly binding to the SFTPC 3'-UTR and inhibit the regulatory effect of SFTPC on lung adenocarcinoma cell proliferation. In conclusion, these data suggested that miR-629-3p-meditated downregulation of SFTPC may promote lung adenocarcinoma progression.

Expression of mutant Sftpc in murine alveolar epithelia drives spontaneous lung fibrosis.

Epithelial cell dysfunction is postulated as an important component in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mutations in the surfactant protein C (SP-C) gene (SFTPC), an alveolar type II (AT2) cell-restricted protein, have been found in sporadic and familial IPF. To causally link these events, we developed a knockin mouse model capable of regulated expression of an IPF-associated isoleucine-to-threonine substitution at codon 73 (I73T) in Sftpc (SP-CI73T). Tamoxifen-treated SP-CI73T cohorts developed rapid increases in SftpcI73T mRNA and misprocessed proSP-CI73T protein accompanied by increased early mortality (days 7-14). This acute phase was marked by diffuse parenchymal lung injury, tissue infiltration by monocytes, polycellular alveolitis, and elevations in bronchoalveolar lavage and AT2 mRNA content of select inflammatory cytokines. Resolution of alveolitis (2-4 weeks), commensurate with a rise in TGF-β1, was followed by aberrant remodeling marked by collagen deposition, AT2 cell hyperplasia, α-smooth muscle actin-positive (α-SMA-positive) cells, and restrictive lung physiology. The translational relevance of the model was supported by detection of multiple IPF biomarkers previously reported in human cohorts. These data provide proof of principle that mutant SP-C expression in vivo causes spontaneous lung fibrosis, strengthening the role of AT2 cell dysfunction as a key upstream driver of IPF pathogenesis.

Surfactant protein C dampens inflammation by decreasing JAK/STAT activation during lung repair.

Surfactant protein C (SPC), a key component of pulmonary surfactant, also plays a role in regulating inflammation. SPC deficiency in patients and mouse models is associated with increased inflammation and delayed repair, but the key drivers of SPC-regulated inflammation in response to injury are largely unknown. This study focuses on a new mechanism of SPC as an anti-inflammatory molecule using SPC-TK/SPC-KO (surfactant protein C-thymidine kinase/surfactant protein C knockout) mice, which represent a novel sterile injury model that mimics clinical acute respiratory distress syndrome (ARDS). SPC-TK mice express the inducible suicide gene thymidine kinase from by the SPC promoter, which targets alveolar type 2 (AT2) cells for depletion in response to ganciclovir (GCV). We compared GCV-induced injury and repair in SPC-TK mice that have normal endogenous SPC expression with SPC-TK/SPC-KO mice lacking SPC expression. In contrast to SPC-TK mice, SPC-TK/SPC-KO mice treated with GCV exhibited more severe inflammation, resulting in over 90% mortality; there was only 8% mortality of SPC-TK animals. SPC-TK/SPC-KO mice had highly elevated inflammatory cytokines and granulocyte infiltration in the bronchoalveolar lavage (BAL) fluid. Consistent with a proinflammatory phenotype, immunofluorescence revealed increased phosphorylated signal transduction and activation of transcription 3 (pSTAT3), suggesting enhanced Janus kinase (JAK)/STAT activation in inflammatory and AT2 cells of SPC-TK/SPC-KO mice. The level of suppressor of cytokine signaling 3, an anti-inflammatory mediator that decreases pSTAT3 signaling, was significantly decreased in the BAL fluid of SPC-TK/SPC-KO mice. Hyperactivation of pSTAT3 and inflammation were rescued by AZD1480, a JAK1/2 inhibitor. Our findings showing a novel role for SPC in regulating inflammation via JAK/STAT may have clinical applications.

The effect of meconium exposure on the expression and differentiation of amniotic fluid mesenchymal stem cells.

BACKGROUND:The goal of this study was to determine if exposure to meconium would alter the phenotype of amniotic fluid mesenchymal stem cells (AF-MSCs) and the ability of these cells to be differentiated into distal airway type cells.METHODS:Meconium was collected, lyophilized and resuspended in PBS at 3 different concentrations (high, medium, and low). AF-MSCs were cultured in the presence of this meconium suspension for 8 hours and then analyzed for changes in gene expression. Additionally, AF-MSCs exposed to meconium were differentiated for 14 days using modified small airway growth medium (mSAGM) and gene expression was determined. As a spontaneous differentiation control, meconium exposed AF-MSCs were cultured in amniotic fluid stem cell medium (AF medium).RESULTS:After 8 hours of exposure in culture, AF-MSCs had increased expression of distal airway genes aquaporin 5 (AQP5) and surfactant protein c (SPC) when cultured in AF medium containing meconium. These gene expression levels were similar to that of AF-MSCs that were differentiated in mSAGM for 14 days. Furthermore, there was an up regulation of pluripotency genes NANOG and OCT4 in response to low meconium concentration for 8 hours. Following 14 days of culture in mSAGM, there was an upregulation of TTF1, SPC and AQP5 expression in the control, as well as in the low and medium meconium exposed groups indicating that these cells were still able to be differentiated. High meconium concentration did, however, appear to influence the level of distal airway gene expression after 14 days in mSAGM. After 14 days in AF medium, there was significant downregulation in pluripotency and mesenchymal markers as well as distal airway gene expression in all groups.CONCLUSION:The phenotype of AF-MSCs is modulated by meconium exposure; however, the cells were still able to differentiate into distal airway gene and protein expression. This result supports the hypothesis that progenitor cells exist in the amniotic fluid and the presence of meconium may affect their initial phenotype. However, these cells were still able to be differentiated to a distal lung phenotype.

Resveratrol efficiently improves pulmonary function via stabilizing mast cells in a rat intestinal injury model

BackgroundIntestinal ischemia/reperfusion (IIR) leads to acute lung injury (ALI) distally by aggravating pulmonary oxidative stress. Resveratrol is effective in attenuating ALI through its antioxidant capacity. This study aimed to determine the effects of resveratrol on IIR-induced ALI and to explore the role of mast cells (MCs) activation in a rat model of IIR. MethodsAdult Sprague-Dawley rats were subjected to IIR by occluding the superior mesenteric artery for 60min followed by 4-hour reperfusion. Resveratrol was intraperitoneally injected at a dose of 15mg/kg for 5days before IIR. MCs stabilizer/inhibitor cromolyn sodium and degranulator compound 48/80 were used to explore the interaction between resveratrol and MCs. Lung tissues were collected for pathological detection and MCs staining. Pulmonary protein expression of surfactant protein-C (SP-C), tryptase, p47phox and gp91phox (two NADPH oxidase subunits), ICAM-1(intercellular adhesion molecule-1) and P-selectin were detected. The levels of oxidative stress markers (SOD, MDA, H2O2 and MPO) and β-hexosaminidase were also measured. ResultsAt the end of IIR, lung injury was significantly increased and was associated with decreased expression of SP-C and increased lung oxidative stress. Increased inflammation as well as activation of MCs was also observed in the lungs after IIR. All these changes were prevented or reversed by resveratrol pretreatment or MCs inhibition with cromolyn sodium. However, these protective effects of resveratrol or cromolyn sodium were reduced by MCs degranulator compound 48/80. ConclusionsThese findings reveal that resveratrol attenuates IIR-induced ALI by reducing NADPH oxidase protein expression and inflammation through stabilizing MCs.

Regulation of p53-mediated changes in the uPA-fibrinolytic system and in lung injury by loss of surfactant protein C expression in alveolar epithelial cells.

Pulmonary surfactant protein C (SP-C) expression by type II alveolar epithelial cells (AECs) is markedly reduced in diverse types of lung injuries and is often associated with AEC apoptosis. It is unclear whether loss of SP-C contributes to the increased p53 and urokinase-type plasminogen activator (uPA) system cross-talk and apoptosis of AECs. Therefore, we inhibited SP-C expression in human and murine AECs using lentivirus vector expressing shRNA and tested p53 and downstream changes in the uPA-fibrinolytic system. Inhibition of SP-C expression in AECs induced p53 and activated caspase-3, indicating AEC apoptosis. We also found that bleomycin or cigarette smoke exposure failed to inhibit SP-C expression or apoptosis in AECs in p53- and plasminogen activator inhibitor-1 (PAI-1)-deficient mice. Depletion of SP-C expression by lentiviral SP-C shRNA in PAI-1-deficient mice failed to induce p53 or apoptosis in AECs, whereas it increased both AEC p53 and apoptosis in wild-type and uPA-deficient mice. SP-C inhibition in AECs also increased in CXCL1 and CXCL2 and their receptor CXCR2 as well as ICAM-1 expression, which is indicative of a proinflammatory response. Overexpression of p53-binding 3'-UTR sequences in AECs inhibited PAI-1 induction while maintaining uPA and uPAR protein and mRNA expression. Furthermore, caveolin-1 expression and phosphorylation were increased in AECs, indicating an intricate link between caveolin-1 and Src kinase-mediated cell signaling and AEC apoptosis due to loss of SP-C expression through p53 and uPA system-mediated cross-talk. The role of uPA, PAI-1, and p53 in the regulation of AEC apoptosis after injury was also determined in knockout mice.

Study of the variation in expression and the significance of markers for alveolar epithelial cells in bronchopulmonary dysplasia newborn rats

Objective To investigate the variation in expression and the significance of markers indicating typeⅠalveolar epithelial cells(AECⅠ) and type Ⅱ alveolar epithelial cells(AECⅡ) in hyperoxia induced bronchopulmonary dysplasia(BPD) model. Methods A total of 80 term normal Wistar rats were randomly devided into model group (85% oxygen) or control group (room air) within 12 h after birth, with 40 rats in each group.On day 7, day 14, day 21 after exposure, the pathological characteristics of lung tissues were observed using HE staining, the expression and location of AECⅠ marker aquaporin 5(AQP5) and AECⅡ marker surfactant protein-C (SP-C) were examined using immunofluorescence double staining.Western blot analysis was employed to examine the expressions levels of AQP5 and SP-C proteins, while real-time PCR was used to evaluate the mRNA expression of AQP5 and SP-C. Results Alveolar developmental disorder was observed in lung tissues of the model group, including fewer, larger, simplified alveoli, thicker alveolar walls, and fewer alveolar secondary septa.Immunofluorescence double staining showed increased and disorganized AQP5 and SP-C expression, with significantly higher ratio of double-stained cells/SP-C positive cells in the model group (P<0.001). Comparing to the control group, the expression of AQP5 and SP-C protein increased from 7 d after hyperoxia exposure, which continued to 21 d. The mRNA expression levels of these two markers both significantly increased in the model group compared with the control group, with AQP5 starting from 7 d while SP-C starting from 14 d after hyperoxia exposure (P<0.05), and the difference between two groups became more significant with the exposure time extending. Conclusion The expression of AECⅠ marker AQP5 and AECⅡ marker SP-C both increase in the lung tissues of hyperoxia induced BPD newborn rats, with more AECⅡ transdifferentiated into AECⅠ.These changes of the markers indicate that there is excessive transdifferentiation of AECⅡ in the recovery process after BPD lung injury. Key words: Transdifferentiation; Alveolar epithelial cell; Bronchopulmonary dysplasia; Hyperoxia; Rats

Follistatin-like 1 expression is decreased in the alveolar epithelium of hypoplastic rat lungs with nitrofen-induced congenital diaphragmatic hernia

Background/purposePulmonary hypoplasia (PH), characterized by incomplete alveolar development, remains a major therapeutic challenge associated with congenital diaphragmatic hernia (CDH). Follistatin-like 1 (Fstl1) is a crucial regulator of alveolar formation and maturation, which is strongly expressed in distal airway epithelium. Fstl1-deficient mice exhibit reduced airspaces, impaired alveolar epithelial cell differentiation, and insufficient production of surfactant proteins similar to PH in human CDH. We hypothesized that pulmonary Fstl1 expression is decreased during alveolarization in the nitrofen-induced CDH model. MethodsTimed-pregnant rats received nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D18 and D21 and divided into control−/nitrofen-exposed specimens. Alveolarization was assessed using morphometric analysis techniques. Pulmonary gene expression of Fstl1 was determined by qRT-PCR. Immunofluorescence-double-staining for Fstl1 and alveolar epithelial marker surfactant protein C (SP-C) was performed to evaluate protein expression/localization. ResultsRadial alveolar count was significantly reduced in hypoplastic lungs of nitrofen-exposed fetuses with significant down regulation of Fstl1 mRNA expression on D18 and D21 compared to controls. Confocal-laser-scanning-microscopy revealed strikingly diminished Fstl1 immunofluorescence and SP-C expression in distal alveolar epithelium of nitrofen-exposed fetuses with CDH-associated PH on D18 and D21 compared to controls. ConclusionsDecreased expression of Fstl1 in alveolar epithelium may disrupt alveolarization and pulmonary surfactant production, thus contributing to the development of PH in the nitrofen-induced CDH model. Level of evidence2b (Centre for Evidence-Based Medicine, Oxford).