ABCA3 is critical for lamellar body biogenesis in vivo

Mutations in ATP-binding cassette transporter A3 (human ABCA3) protein are associated with fatal respiratory distress syndrome in newborns. We therefore characterized mice with targeted disruption of the ABCA3 gene. Homozygous Abca3-/- knock-out mice died soon after birth, whereas most of the wild type, Abca3+/+, and heterozygous, Abca3+/-, neonates survived. The lungs from E18.5 and E19.5 Abca3-/- mice were less mature than wild type. Alveolar type 2 cells from Abca3-/- embryos contained no lamellar bodies, and expression of mature SP-B protein was disrupted when compared with the normal lung surfactant system of wild type embryos. Small structural and functional differences in the surfactant system were seen in adult Abca3+/- compared with Abca3+/+ mice. The heterozygotes had fewer lamellar bodies, and the incorporation of radiolabeled substrates into newly synthesized disaturated phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine in both lamellar bodies and surfactant was lower than in Abca3+/+ mouse lungs. In addition, since the fraction of near term Abca3-/- embryos was significantly lower than expected from Mendelian inheritance ABCA3 probably plays roles in development unrelated to surfactant. Collectively, these findings strongly suggest that ABCA3 is necessary for lamellar body biogenesis, surfactant protein-B processing, and lung development late in gestation.     

Fgf10 dosage is critical for the amplification of epithelial cell progenitors and for the formation of multiple mesenchymal lineages during lung development

The key role played by Fgf10 during early lung development is clearly illustrated in Fgf10 knockout mice, which exhibit lung agenesis. However, Fgf10 is continuously expressed throughout lung development suggesting extended as well as additional roles for FGF10 at later stages of lung organogenesis. We previously reported that the enhancer trap Mlcv1v-nLacZ-24 transgenic mouse strain functions as a reporter for Fgf10 expression and displays decreased endogenous Fgf10 expression. In this paper, we have generated an allelic series to determine the impact of Fgf10 dosage on lung development. We report that 80% of the newborn Fgf10 hypomorphic mice die within 24 h of birth due to respiratory failure. These mutant mouse lungs display severe hypoplasia, dilation of the distal airways and large hemorrhagic areas. Epithelial differentiation and proliferation studies indicate a specific decrease in TTF1 and SP-B expressing cells correlating with reduced epithelial cell proliferation and associated with a decrease in activation of the canonical Wnt signaling in the epithelium. Analysis of vascular development shows a reduction in PECAM expression at E14.5, which is associated with a simplification of the vascular tree at E18.5. We also show a decrease in α-SMA expression in the respiratory airway suggesting defective smooth muscle cell formation. At the molecular level, these defects are associated with decrease in Vegfa and Pdgfa expression likely resulting from the decrease of the epithelial/mesenchymal ratio in the Fgf10 hypomorphic lungs. Thus, our results indicate that FGF10 plays a pivotal role in maintaining epithelial progenitor cell proliferation as well as coordinating alveolar smooth muscle cell formation and vascular development.     

ABCA3 inactivation in mice causes respiratory failure, loss of pulmonary surfactant, and depletion of lung phosphatidylglycerol

The highly branched mammalian lung relies on surfactant, a mixture of phospholipids, cholesterol, and hydrophobic proteins, to reduce intraalveolar surface tension and prevent lung collapse. Human mutations in the ABCA3 transporter have been associated with childhood respiratory disease of variable severity and onset. Here, we report the generation of Abca3 null mice, which became lethargic and cyanotic and died within 1 h of birth. Tissue blots found ABCA3 expression was highest in lung but was also detectable in other tissues, including the kidney. Gross development of kidney and lung was normal in neonatal Abca3(-/-) pups, but the mice failed to inflate their lungs, leading to death from atelectatic respiratory failure. Ultrastructural analysis of the Abca3(-/-) lungs revealed an absence of surfactant from the alveolar space and a profound loss of mature lamellar bodies, the intracellular storage organelle for surfactant. Mass spectrometry measurement of >300 phospholipids in lung tissue taken from Abca3(-/-) mice showed a dramatic reduction of phosphatidylglycerol (PG) levels as well as selective reductions in phosphatidylcholine species containing short acyl chains. These results establish a requirement of ABCA3 for lamellar body formation and pulmonary surfactant secretion and suggest a unique and critical role for the transporter in the metabolism of pulmonary PG. They also demonstrate the utility of the Abca3 null mouse as a model for a devastating human disease.     

FGF‐8b induces growth and rich vascularization in an orthotopic PC‐3 model of prostate cancer

Fibroblast growth factor 8 (FGF‐8) is expressed at an increased level in a high proportion of prostate cancers and it is associated with a poor prognosis of the disease. Our aim was to study the effects of FGF‐8b on proliferation of PC‐3 prostate cancer cells and growth of PC‐3 tumors, and to identify FGF‐8b‐associated molecular targets. Expression of ectopic FGF‐8b in PC‐3 cells caused a 1.5‐fold increase in cell proliferation in vitro and a four‐ to fivefold increase in the size of subcutaneous and orthotopic prostate tumors in nude mice. Tumors expressing FGF‐8b showed a characteristic morphology with a very rich network of capillaries. This was associated with increased spread of the cancer cells to the lungs as measured by RT‐qPCR of FGF‐8b mRNA. Microarray analyses revealed significantly altered, up‐ and downregulated, genes in PC‐3 cell cultures (169 genes) and in orthotopic PC‐3 tumors (61 genes). IPA network analysis of the upregulated genes showed the strongest association with development, cell proliferation (CRIP1, SHC1), angiogenesis (CCL2, DDAH2), bone metastasis (SPP1), cell‐to‐cell signaling and energy production, and the downregulated genes associated with differentiation (DKK‐1, VDR) and cell death (CYCS). The changes in gene expression were confirmed by RT‐qPCR. In conclusion, our results demonstrate that FGF‐8b increases the growth and angiogenesis of orthotopic prostate tumors. The associated gene expression signature suggests potential mediators for FGF‐8b actions on prostate cancer progression and metastasis. J. Cell. Biochem. 107: 769–784, 2009. © 2009 Wiley‐Liss, Inc.     

Fibroblast Growth Factor 9 Regulation by MicroRNAs Controls Lung Development and Links DICER1 Loss to the Pathogenesis of Pleuropulmonary Blastoma

Pleuropulmonary Blastoma (PPB) is the primary neoplastic manifestation of a pediatric cancer predisposition syndrome that is associated with several diseases including cystic nephroma, Wilms tumor, neuroblastoma, rhabdomyosarcoma, medulloblastoma, and ovarian Sertoli-Leydig cell tumor. The primary pathology of PPB, epithelial cysts with stromal hyperplasia and risk for progression to a complex primitive sarcoma, is associated with familial heterozygosity and lesion-associated epithelial loss-of-heterozygosity of DICER1. It has been hypothesized that loss of heterozygosity of DICER1 in lung epithelium is a non-cell autonomous etiology of PPB and a critical pathway that regulates lung development; however, there are no known direct targets of epithelial microRNAs (miRNAs) in the lung. Fibroblast Growth Factor 9 (FGF9) is expressed in the mesothelium and epithelium during lung development and primarily functions to regulate lung mesenchyme; however, there are no known mechanisms that regulate FGF9 expression during lung development. Using mouse genetics and molecular phenotyping of human PPB tissue, we show that FGF9 is overexpressed in lung epithelium in the initial multicystic stage of Type I PPB and that in mice lacking epithelial Dicer1, or induced to overexpress epithelial Fgf9, increased Fgf9 expression results in pulmonary mesenchymal hyperplasia and a multicystic architecture that is histologically and molecularly indistinguishable from Type I PPB. We further show that miR-140 is expressed in lung epithelium, regulates epithelial Fgf9 expression, and regulates pseudoglandular stages of lung development. These studies identify an essential miRNA-FGF9 pathway for lung development and a non-cell autonomous signaling mechanism that contributes to the mesenchymal hyperplasia that is characteristic of Type I PPB.     

Dietary β-carotene regulates interleukin-1β-induced expression of apolipoprotein E in astrocytes isolated from stroke-prone spontaneously hypertensive rats

Stroke-prone spontaneously hypertensive rats (SHRSP) have an abnormality in cholesterol synthesis, but the pathological relevance of this to stroke and related neuronal disorders is not yet clear. The induction of astrocyte-derived cholesterol transportation to neurons by apolipoprotein E (apoE) promotes neuronal repair after brain injuries such as stroke. Such repair is reduced by interleukin-1 beta (IL-1β) and stroke conditions. Furthermore, fibroblast growth factor 1 (FGF1) regulates the production of apoE–cholesterol-rich high density lipoproteins (HDL) and induces gliosis of astrocytes. On the other hand, high levels of plasma carotenoids reduce the risk of ischemic stroke. Thus, we investigated the expression of apoE in primary astrocytes that had been treated with IL-1β or β-carotene. In addition, we compared the expression levels of Apoe genes in astrocytes from SHRSP/Izm and normal control rats, Wistar–Kyoto rats (WKY/Izm) following hypoxia/reoxygenation (H/R). The expression levels of genes and proteins were investigated by RT-PCR, Western blotting (WB), and immunofluorescence analysis. IL-1β decreased the expression levels of the Apoe gene. Conversely, β-carotene significantly enhanced the expression levels of genes related to cholesterol regulation, including Abca1, Abcg1, Hmgcr as well as Apoe. During H/R, the gene expression levels of Apoe were decreased in the SHRSP/Izm rats in comparison with the WKY/Izm rats. These results suggest that IL-1β decreases Apoe expression levels, whereas β-carotene strongly elevates Apoe levels and inhibits FGF1-mediated gliosis of astrocytes. Furthermore, under hypoxic stress, astrocytes isolated from SHRSP/Izm rats displayed altered regulation of Apoe compared with those from WKY/Izm rats.     

Impaired Elastin Deposition in Fstl1?/? Lung Allograft under the Renal Capsule

Lung alveolar development in late gestation is a process important to postnatal survival. Follistatin-like 1 (Fstl1) is a matricellular protein of the Bmp antagonist class, which is involved in the differentiation/maturation of alveolar epithelial cells during saccular stage of lung development. This study investigates the role of Fstl1 on elastin deposition in mesenchyme and subsequent secondary septation in the late gestation stage of terminal saccular formation. To this aim, we modified the renal capsule allograft model for lung organ culture by grafting diced E15.5 distal lung underneath the renal capsule of syngeneic host and cultured up to 7 days. The saccular development of the diced lung allografts, as indicated by the morphology, epithelial and vascular developments, occurred in a manner similar to that in utero. Fstl1 deficiency caused atelectatic phenotype companied by impaired epithelial differentiation in D3 Fstl1^−/− lung allografts, which is similar to that of E18.5 Fstl1^−/− lungs, supporting the role of Fstl1 during saccular stage. Inhibition of Bmp signaling by intraperitoneal injection of dorsomorphin in the host mice rescued the pulmonary atelectasis of D3 Fstl1^−/− allografts. Furthermore, a marked reduction in elastin expression and deposition was observed in walls of air sacs of E18.5 Fstl1^−/− lungs and at the tips of the developing alveolar septae of D7 Fstl1^−/− allografts. Thus, in addition to its role on alveolar epithelium, Fstl1 is crucial for elastin expression and deposition in mesenchyme during lung alveologenesis. Our data demonstrates that the modified renal capsule allograft model for lung organ culture is a robust and efficient technique to increase our understanding of saccular stage of lung development.     

Prenatal,but not postnatal,inhibition of fibroblast growth factor receptor signaling causes emphysema

Although fibroblast growth factor (FGF) signaling is required for the formation of the lung in the embryonic period, it is unclear whether FGF receptor activity influences lung morphogenesis later in development. We generated transgenic mice expressing a soluble FGF receptor (FGFR-HFc) under conditional control of the lung-specific surfactant protein C promoter (SP-C-rtTA), to inhibit FGF activity at various times in late gestation and postnatally. Although expression of FGFR-HFc early in development caused severe fetal lung hypoplasia, activation of the transgene in the postnatal period did not alter alveolarization, lung size, or histology. In contrast, expression of the transgene at post-conception day E14.5 decreased lung tubule formation before birth and caused severe emphysema at maturity. FGFR-HFc caused mild focal emphysema when expressed from E16.5 but did not alter alveolarization when expressed after birth. Although FGF signaling was required for branching morphogenesis early in lung development, postnatal alveolarization was not influenced by FGFR-HFc.     

The Effect of Statins on Blood Gene Expression in COPD

BackgroundCOPD is currently the fourth leading cause of death worldwide. Statins are lipid lowering agents with documented cardiovascular benefits. Observational studies have shown that statins may have a beneficial role in COPD. The impact of statins on blood gene expression from COPD patients is largely unknown.ObjectiveIdentify blood gene signature associated with statin use in COPD patients, and the pathways underpinning this signature that could explain any potential benefits in COPD.MethodsWhole blood gene expression was measured on 168 statin users and 451 non-users from the ECLIPSE study using the Affymetrix Human Gene 1.1 ST microarray chips. Factor Analysis for Robust Microarray Summarization (FARMS) was used to process the expression data. Differential gene expression analysis was undertaken using the Linear Models for Microarray data (Limma) package adjusting for propensity score and surrogate variables. Similarity of the expression signal with published gene expression profiles was performed in ProfileChaser.Results25 genes were differentially expressed between statin users and non-users at an FDR of 10%, including LDLR, CXCR2, SC4MOL, FAM108A1, IFI35, FRYL, ABCG1, MYLIP, and DHCR24. The 25 genes were significantly enriched in cholesterol homeostasis and metabolism pathways. The resulting gene signature showed correlation with Huntington’s disease, Parkinson’s disease and acute myeloid leukemia gene signatures.ConclusionThe blood gene signature of statins’ use in COPD patients was enriched in cholesterol homeostasis pathways. Further studies are needed to delineate the role of these pathways in lung biology.     

Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

Background

Heparan sulfate (HS) is present on the surface of virtually all mammalian cells and is a major component of the extracellular matrix (ECM), where it plays a pivotal role in cell-cell and cell-matrix cross-talk through its large interactome. Disruption of HS biosynthesis in mice results in neonatal death as a consequence of malformed lungs, indicating that HS is crucial for airway morphogenesis. Neonatal mortality (~50%) in newborns with congenital diaphragmatic hernia (CDH) is principally associated with lung hypoplasia and pulmonary hypertension. Given the importance of HS for lung morphogenesis, we investigated developmental changes in HS structure in normal and hypoplastic lungs using the nitrofen rat model of CDH and semi-synthetic bacteriophage ('phage) display antibodies, which identify distinct HS structures.

Results

The pulmonary pattern of elaborated HS structures is developmentally regulated. For example, the HS4E4V epitope is highly expressed in sub-epithelial mesenchyme of E15.5 - E17.5 lungs and at a lower level in more distal mesenchyme. However, by E19.5, this epitope is expressed similarly throughout the lung mesenchyme. We also reveal abnormalities in HS fine structure and spatiotemporal distribution of HS epitopes in hypoplastic CDH lungs. These changes involve structures recognised by key growth factors, FGF2 and FGF9. For example, the EV3C3V epitope, which was abnormally distributed in the mesenchyme of hypoplastic lungs, is recognised by FGF2.

Conclusions

The observed spatiotemporal changes in HS structure during normal lung development will likely reflect altered activities of many HS-binding proteins regulating lung morphogenesis. Abnormalities in HS structure and distribution in hypoplastic lungs can be expected to perturb HS:protein interactions, ECM microenvironments and crucial epithelial-mesenchyme communication, which may contribute to lung dysmorphogenesis. Indeed, a number of epitopes correlate with structures recognised by FGFs, suggesting a functional consequence of the observed changes in HS in these lungs. These results identify a novel, significant molecular defect in hypoplastic lungs and reveals HS as a potential contributor to hypoplastic lung development in CDH. Finally, these results afford the prospect that HS-mimetic therapeutics could repair defective signalling in hypoplastic lungs, improve lung growth, and reduce CDH mortality.     

In vivo potential effects of adenovirus type 5 E1A and E1B on lung carcinogenesis and lymphoproliferative inflammation

Triggering uncontrolled cellular proliferation, chronic inflammation, and/or disruption of p53 activity is critical for tumorigenesis initiated by latent viral oncogenes. The adenovirus type 5 (Ad5) early genes E1A and E1B can maintain lifelong latency in the lungs of patients with chronic pulmonary diseases. To determine the in vivo effects of the latent Ad5 E1A and E1B oncogenes, we have examined the influence of Ad5 E1A and E1B gene products on mouse lung carcinogenesis and inflammation by generation and characterization of lung-specific transgenic mouse models. Here, we show that either the Ad5 E1A 243-amino-acid (aa) protein or the E1B 58-kDa protein was dominantly expressed in the transgenic lung. Preferential expression of Ad5 E1A 243-aa protein alone was not sufficient to induce lung carcinogenesis but resulted in low-grade cellular proliferation and high-grade lymphoproliferative inflammation in the lung. The presence of Ad5 E1B dramatically enhanced the expression of the E1A 243-aa protein, in addition to impairing p53 and apoptosis response, resulting in uncontrolled cellular proliferation, lymphoproliferative inflammation, and metastatic carcinomas in the lung after a period of latency. Our studies may provide clues to understanding the potential in vivo biological effects of Ad5 E1A and E1B latent in the lung and a new scope for assessing in vivo functions of viral genes latent in the infection target tissue.     

Loss of ABCA8B decreases myelination by reducing oligodendrocyte precursor cells in mice

The myelin sheath, which is wrapped around axons, is a lipid-enriched structure produced by mature oligodendrocytes. Disruption of the myelin sheath is observed in several neurological diseases, such as multiple sclerosis. A crucial component of myelin is sphingomyelin, levels of which can be increased by ABCA8, a member of the ATP-binding cassette transporter family. ABCA8 is highly expressed in the cerebellum, specifically in oligodendroglia. However, whether ABCA8 plays a role in myelination and mechanisms that would underlie this role remain unknown. Here, we found that the absence of Abca8b, a mouse ortholog of ABCA8, led to decreased numbers of cerebellar oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes in mice. We show that in oligodendrocytes, ABCA8 interacts with chondroitin sulfate proteoglycan 4 (CSPG4), a molecule essential for OPC proliferation, migration, and myelination. In the absence of Abca8b, localization of CSPG4 to the plasma membrane was decreased, contributing to reduced cerebellar CSPG4 expression. Cerebellar CSPG4+ OPCs were also diminished, leading to decreased mature myelinating oligodendrocyte numbers and cerebellar myelination levels in Abca8b^?/? mice. In addition, electron microscopy analyses showed that the number of nonmyelinated cerebellar axons was increased, whereas cerebellar myelin thickness (g-ratio), myelin sheath periodicity, and axonal diameter were all decreased, indicative of disordered myelin ultrastructure. In line with disrupted cerebellar myelination, Abca8b^?/? mice showed lower cerebellar conduction velocity and disturbed locomotion. In summary, ABCA8 modulates cerebellar myelination, in part through functional regulation of the ABCA8-interacting protein CSPG4. Our findings suggest that ABCA8 disruption may contribute to the pathophysiology of myelin disorders.     

Expression of fibroblast growth factor 10 and cognate receptors in the developing bovine ovary

In the mammalian ovary, FGF10 is expressed in oocytes and theca cells and is a candidate for paracrine signaling to the developing granulosa cells. To gain insight into the participation of FGF10 in the regulation of fetal folliculogenesis, we assessed mRNA expression patterns of FGF10 and its receptors, FGFR1B and FGFR2B, in relation to fetal follicle dynamics and localized FGF10 protein in bovine fetal ovaries at different ages. Primordial, primary, secondary, and antral follicles were first observed on Days 75, 90, 150, and 210 of gestation, respectively. The levels of GDF9 and BMP15 mRNA, markers for primordial and primary follicles, respectively, increased during fetal ovary development in a consistent manner with fetal follicle dynamics. CYP17A1 mRNA abundance increased from Day 60 to Day 75 and then from Day 120 to Day 150, coinciding with the appearance of secondary follicles. FGF10 mRNA abundance increased from Day 90, and this increase was temporally associated with increases in FGFR1B mRNA abundance and in the population of primary follicles. In contrast, FGFR2B mRNA expression was highest on Day 60 and decreased thereafter. FGF10 protein was localized to oogonia and oocytes and surrounding granulosa cells at all fetal ages. The present data suggest a role for FGF10 in the control of fetal folliculogenesis in cattle.     

Correlation of Versican Expression,Accumulation, and Degradation during Embryonic Development by Quantitative Immunohistochemistry

Versican, a chondroitin sulfate proteoglycan, is important in embryonic development, and disruption of the versican gene is embryonically lethal in the mouse. Although several studies show that versican is increased in various organs during development, a focused quantitative study on versican expression and distribution during lung and central nervous system development in the mouse has not previously been performed. We tracked changes in versican (Vcan) gene expression and in the accumulation and degradation of versican. Vcan expression and quantitative immunohistochemistry performed from embryonic day (E) 11.5 to E15.5 showed peak Vcan expression at E13.5 in the lungs and brain. Quantitative mRNA analysis and versican immunohistochemistry showed differences in the expression of the versican isoforms in the embryonic lung and head. The expression of Vcan mRNA and accumulation of versican in tissues was complementary. Immunohistochemistry demonstrated co-localization of versican accumulation and degradation, suggesting distinct roles of versican deposition and degradation in embryogenesis. Very little versican mRNA or protein was found in the lungs of 12- to 16-week-old mice but versican accumulation was significantly increased in mice with Pseudomonas aeruginosa lung infection. These data suggest that versican plays an important role in fundamental, overlapping cellular processes in lung development and infection.     

Identification of Fibroblast Growth Factor-18 as a Molecule to Protect Adult Articular Cartilage by Gene Expression Profiling

To identify genes that maintain the homeostasis of adult articular cartilage and regenerate its lesions, we initially compared four types of chondrocytes: articular (AA) versus growth plate (AG) cartilage chondrocytes in adult rats, and superficial layer (IS) versus deep layer (ID) chondrocytes of epiphyseal cartilage in infant rats. Microarray analyses revealed that 40 and 186 genes had ≥10-fold higher expression ratios of AA/AG and IS/ID, respectively, and 16 genes showed ≥10-fold of both AA/AG and IS/ID ratios. The results were validated by real-time RT-PCR analysis. Among them, Hoxd1, Fgf18, and Esm1 were expressed more strongly in AA than in IS. Fgf18 was the extracellular and secreted factor that decreased glycosaminoglycan release and depletion from the cartilage, and enhanced proliferation of articular chondrocytes. Fgf18 was strongly expressed in the articular cartilage chondrocytes of adult rats. In a surgical rat osteoarthritis model, a once-weekly injection of recombinant human FGF18 (rhFGF18) given 3 weeks after surgery prevented cartilage degeneration in a dose-dependent manner at 6 and 9 weeks after surgery, with significant effect at 10 μg/week of rhFGF18. As the underlying mechanism, rhFGF18 strongly up-regulated Timp1 expression in the cell and organ cultures, and inhibition of aggrecan release by rhFGF18 was restored by addition of an antibody to Timp1. In conclusion, we have identified Fgf18 as a molecule that protects articular cartilage by gene expression profiling, and the anticatabolic effects may at least partially be mediated by the Timp1 expression.