Adenovirus-mediated decorin gene transfer prevents TGF-β-induced inhibition of lung morphogenesis

1999 ◽  
Vol 277 (2) ◽  
pp. L412-L422 ◽  
Author(s):  
Jingsong Zhao ◽  
Patricia J. Sime ◽  
Pablo Bringas ◽  
Jack Gauldie ◽  
David Warburton
Keyword(s):  
Transforming Growth Factor ◽  
Recombinant Adenovirus ◽  
Ex Vivo ◽  
Surfactant Protein ◽  
Branching Morphogenesis ◽  
Lung Growth ◽  
Lung Morphogenesis ◽  
Ex Vivo Culture ◽  
Lung Branching ◽  
Embryonic Lungs

Excessive transforming growth factor (TGF)-β signaling has been implicated in pulmonary hypoplasia associated with bronchopulmonary dysplasia, a chronic lung disease of human prematurity featuring pulmonary fibrosis. This implies that inhibitors of TGF-β could be useful therapeutic agents. Because exogenous TGF-β ligands are known to inhibit lung branching morphogenesis and cytodifferentiation in mouse embryonic lungs in ex vivo culture, we examined the capacity of a naturally occurring inhibitor of TGF-β activity, the proteoglycan decorin, to overcome the inhibitory effects of exogenous TGF-β. Intratracheal microinjection of a recombinant adenovirus containing decorin cDNA resulted in overexpression of the exogenous decorin gene in airway epithelium. Although exogenous TGF-β efficiently decreased epithelial lung branching morphogenesis in control cultures, TGF-β-induced inhibition of lung growth was abolished after epithelial transfer of the decorin gene. Additionally, exogenous TGF-β-induced antiproliferative effects as well as the downregulation of surfactant protein C were abrogated by decorin in cultured embryonic lungs. Moreover, lung branching inhibition by TGF-β could be restored by the addition of decorin antisense oligodeoxynucleotides in culture, indicating that decorin is both specifically and directly involved in suppressing TGF-β-mediated negative regulation of lung morphogenesis. Our findings suggest that decorin can antagonize bioactive TGF-β during lung growth and differentiation, establishing the rationale for decorin as a candidate therapeutic approach to ameliorate excessive levels of TGF-β signaling in the developing lung.

scholarly journals Abrogation of Transforming Growth Factor-β Type II Receptor Stimulates Embryonic Mouse Lung Branching Morphogenesis in Culture

1996 ◽  
Vol 180 (1) ◽  
pp. 242-257 ◽  
Author(s):  
Jingsong Zhao ◽  
Ding Bu ◽  
Matt Lee ◽  
Harold C. Slavkin ◽  
Frederick L. Hall ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Branching Morphogenesis ◽  
Mouse Lung ◽  
Type Ii ◽  
Embryonic Mouse ◽  
Lung Branching

scholarly journals Chondroitin sulfate proteoglycans are required for lung growth and morphogenesis in vitro

2003 ◽  
Vol 285 (6) ◽  
pp. L1323-L1336 ◽  
Author(s):  
John M. Shannon ◽  
Kathleen McCormick-Shannon ◽  
Michael S. Burhans ◽  
Xiaofei Shangguan ◽  
Kalpana Srivastava ◽  
...  
Keyword(s):  
Chondroitin Sulfate ◽  
Lung Development ◽  
Sodium Chlorate ◽  
Surfactant Protein ◽  
Branching Morphogenesis ◽  
Clara Cell ◽  
Normal Lung ◽  
Lung Growth ◽  
Isolated Lung

Proteoglycans (PGs) have been shown to play a key role in the development of many tissues. We have investigated the role of sulfated PGs in early rat lung development by treating cultured tissues with 30 mM sodium chlorate, a global inhibitor of PG sulfation. Chlorate treatment disrupted growth and branching of embryonic day 13 lung explants. Isolated lung epithelium (LgE) migrated toward and invaded lung mesenchyme (LgM), and chlorate irreversibly suppressed this response. Chlorate also inhibited migration of LgE toward beads soaked in FGF10. Chlorate severely decreased branching morphogenesis in tissue recombinants consisting of LgM plus either LgE or tracheal epithelium (TrE) and decreased expression of surfactant protein C gene ( SP-C). Chlorate also reduced bone morphogenetic protein-4 expression in cultured tips and recombinants but had no effect on the expression of clara cell 10-kDa protein ( CC10), sonic hedgehog ( Shh), FGF10, and FGF receptor 2IIIb. Chlorate reduced the growth of LgE in mesenchyme-free culture but did not affect SP-C expression. In contrast, chlorate inhibited both rudiment growth and the induction of SP-C in mesenchyme-free cultured TrE. Treatment of lung tips and tissue recombinants with chondroitinase ABC abolished branching morphogenesis. Chondroitinase also suppressed growth of TrE in mesenchyme-free culture. Chondroitinase treatment, however, had no effect on the induction of SP-C expression in any of these cultures. These results demonstrate the overall importance of sulfated PGs to normal lung development and demonstrate a dynamic role for chondroitin sulfate PGs in embryonic lung growth and morphogenesis.

scholarly journals Saracatinib, a Selective Src Kinase Inhibitor, Blocks Fibrotic Responses in In Vitro, In Vivo and Ex Vivo Models of Pulmonary Fibrosis

2022 ◽  
Author(s):  
Farida Ahangari ◽  
Christine Becker ◽  
Daniel G Foster ◽  
Maurizio Chioccioli ◽  
Meghan Nelson ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Recombinant Adenovirus ◽  
Ex Vivo ◽  
Src Kinase ◽  
Lung Fibroblasts

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and often fatal disorder. Two FDA approved anti-fibrotic drugs, nintedanib and pirfenidone, slow the rate of decline in lung function, but responses are variable and side effects are common. Using an in-silico data-driven approach, we identified a robust connection between the transcriptomic perturbations in IPF disease and those induced by saracatinib, a selective Src kinase inhibitor, originally developed for oncological indications. Based on these observations, we hypothesized that saracatinib would be effective at attenuating pulmonary fibrosis. We investigated the anti-fibrotic efficacy of saracatinib relative to nintedanib and pirfenidone in three preclinical models: (i) in vitro in normal human lung fibroblasts (NHLFs); (ii) in vivo in bleomycin and recombinant adenovirus transforming growth factor-beta (Ad-TGF-β) murine models of pulmonary fibrosis; and (iii) ex vivo in precision cut lung slices from these mouse models. In each model, the effectiveness of saracatinib in blocking fibrogenic responses was equal or superior to nintedanib and pirfenidone.

scholarly journals ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Ana N. Gonçalves ◽  
Jorge Correia-Pinto ◽  
Cristina Nogueira-Silva
Keyword(s):  
Western Blot ◽  
Congenital Diaphragmatic Hernia ◽  
Progenitor Cells ◽  
Expression Profile ◽  
Diaphragmatic Hernia ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Fetal Lung ◽  
Branching Morphogenesis ◽  
Lung Growth

Abstract Background Characterized by abnormal lung growth or maturation, congenital diaphragmatic hernia (CDH) affects 1:3000 live births. Cellular studies report proximal (SOX2+) and distal (SOX9+) progenitor cells as key modulators of branching morphogenesis and epithelial differentiation, whereas transcriptome studies demonstrate ROBO/SLIT as potential therapeutic targets for diaphragm defect repair in CDH. In this study, we tested the hypothesis that (a) experimental-CDH could changes the expression profile of ROBO1, ROBO2, SOX2 and SOX9; and (b) ROBO1 or ROBO2 receptors are regulators of branching morphogenesis and SOX2/SOX9 balance. Methods The expression profile for receptors and epithelial progenitor markers were assessed by Western blot and immunohistochemistry in a nitrofen-induced CDH rat model. Immunohistochemistry signals by pulmonary structure were also quantified from embryonic-to-saccular stages in normal and hypoplastic lungs. Ex vivo lung explant cultures were harvested at E13.5, cultures during 4 days and treated with increasing doses of recombinant rat ROBO1 or human ROBO2 Fc Chimera proteins for ROBO1 and ROBO2 inhibition, respectively. The lung explants were analyzed morphometrically and ROBO1, ROBO2, SOX2, SOX9, BMP4, and β-Catenin were quantified by Western blot. Results Experimental-CDH induces distinct expression profiles by pulmonary structure and developmental stage for both receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) that provide evidence of the impairment of proximodistal patterning in experimental-CDH. Ex vivo functional studies showed unchanged branching morphogenesis after ROBO1 inhibition; increased fetal lung growth after ROBO2 inhibition in a mechanism-dependent on SOX2 depletion and overexpression of SOX9, non-phospho β-Catenin, and BMP4. Conclusions These studies provided evidence of receptors and epithelial progenitor cells which are severely affected by CDH-induction from embryonic-to-saccular stages and established the ROBO2 inhibition as promoter of branching morphogenesis through SOX2/SOX9 balance.

Transient induction of TGF-α disrupts lung morphogenesis, causing pulmonary disease in adulthood

2004 ◽  
Vol 287 (4) ◽  
pp. L718-L729 ◽  
Author(s):  
T. D. Le Cras ◽  
W. D. Hardie ◽  
G. H. Deutsch ◽  
K. H. Albertine ◽  
M. Ikegami ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Inflammatory Cells ◽  
Surfactant Protein ◽  
Lung Compliance ◽  
Capillary Endothelium ◽  
Adult Mice ◽  
Vascular Morphogenesis ◽  
Lung Morphogenesis

Clinical studies have associated increased transforming growth factor (TGF)-α and EGF receptor with lung remodeling in diseases including bronchopulmonary dysplasia (BPD). BPD is characterized by disrupted alveolar and vascular morphogenesis, inflammation, and remodeling. To determine whether transient increases in TGF-α are sufficient to disrupt postnatal lung morphogenesis, we utilized neonatal transgenic mice conditionally expressing TGF-α. Expression of TGF-α from postnatal days 3 to 5 disrupted postnatal alveologenesis, causing permanent enlargement of distal air spaces in neonatal and adult mice. Lung volume-to-body weight ratios and lung compliance were increased in adult TGF-α transgenic mice, whereas tissue and airway elastance were reduced. Elastin fibers in the alveolar septae were fragmented and disorganized. Pulmonary vascular morphogenesis was abnormal in TGF-α mice, with attenuated and occasionally tortuous arterial branching. The ratios of right ventricle weight to left ventricle plus septal weight were increased in TGF-α mice, indicating pulmonary hypertension. Electron microscopy showed gaps in the capillary endothelium and extravasation of erythrocytes into the alveolar space of TGF-α mice. Hemorrhage and inflammatory cells were seen in distal air spaces at 1 mo of age. In adult TGF-α mice, alveolar remodeling, nodules, proteinaceous deposits, and inflammatory cells were seen. Immunostaining for pro-surfactant protein C showed that type II cells were abundant in the nodules, as well as neutrophils and macrophages. Trichrome staining showed that pulmonary fibrosis was minimal, apart from areas of nodular remodeling in adult TGF-α mice. Transient induction of TGF-α during early alveologenesis permanently disrupted lung structure and function and caused chronic lung disease.

scholarly journals Lung branching morphogenesis is accompanied by temporal metabolic changes towards a glycolytic preference

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hugo Fernandes-Silva ◽  
Marco G. Alves ◽  
Henrique Araújo-Silva ◽  
Ana M. Silva ◽  
Jorge Correia-Pinto ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Glucose Consumption ◽  
Branching Morphogenesis ◽  
Explant Culture ◽  
Metabolic Changes ◽  
Specific Expression ◽  
Expression Levels ◽  
Early Stages ◽  
Lung Branching

Abstract Background Lung branching morphogenesis is characterized by epithelial-mesenchymal interactions that ultimately define the airway conducting system. Throughout this process, energy and structural macromolecules are necessary to sustain the high proliferative rates. The extensive knowledge of the molecular mechanisms underlying pulmonary development contrasts with the lack of data regarding the embryonic lung metabolic requirements. Here, we studied the metabolic profile associated with the early stages of chicken pulmonary branching. Methods In this study, we used an ex vivo lung explant culture system and analyzed the consumption/production of extracellular metabolic intermediates associated with glucose catabolism (alanine, lactate, and acetate) by 1H-NMR spectroscopy in the culture medium. Then, we characterized the transcript levels of metabolite membrane transporters (glut1, glut3, glut8, mct1, mct3, mct4, and mct8) and glycolytic enzymes (hk1, hk2, pfk1, ldha, ldhb, pdha, and pdhb) by qPCR. ldha and ldhb mRNA spatial localization was determined by in situ hybridization. Proliferation was analyzed by directly assessing DNA synthesis using an EdU-based assay. Additionally, we performed western blot to analyze LDHA and LDHT protein levels. Finally, we used a Clark-Type Electrode to assess the lung explant's respiratory capacity. Results Glucose consumption decreases, whereas alanine, lactate, and acetate production progressively increase as branching morphogenesis proceeds. mRNA analysis revealed variations in the expression levels of key enzymes and transporters from the glycolytic pathway. ldha and ldhb displayed a compartment-specific expression pattern that resembles proximal–distal markers. In addition, high proliferation levels were detected at active branching sites. LDH protein expression levels suggest that LDHB may account for the progressive rise in lactate. Concurrently, there is a stable oxygen consumption rate throughout branching morphogenesis. Conclusions This report describes the temporal metabolic changes that accompany the early stages of chicken lung branching morphogenesis. Overall, the embryonic chicken lung seems to shift to a glycolytic lactate-based metabolism as pulmonary branching occurs. Moreover, this metabolic rewiring might play a crucial role during lung development.

scholarly journals Maintenance of murine long-term repopulating stem cells in ex vivo culture is affected by modulation of transforming growth factor-beta but not macrophage inflammatory protein-1 alpha activities

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4561-4567 ◽  
Author(s):  
T Soma ◽  
JM Yu ◽  
CE Dunbar
Keyword(s):  
Stem Cell ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Tgf Beta ◽  
Inflammatory Protein ◽  
Growth Factor Beta ◽  
Ex Vivo Culture ◽  
Macrophage Inflammatory Protein

Transforming growth factor-beta (TGF-beta) and macrophage inflammatory protein-l alpha (MIP-1 alpha) are both well-described inhibitors of committed and multipotential hematopoietic progenitors. The effect of these cytokines; on true stem cell activity in ex vivo culture systems as assayed by murine long-term repopulating activity (LTRA) has not been examined. We studied the stem cell effects of the addition of these cytokines to ex vivo cultures containing interleukin-3 (IL-3), IL- 6, and stem cell factor (SCF), using the murine competitive repopulation assay. We also tested the impact of adding an anti-TGF- beta neutralizing antibody, to ask whether abrogation of autocrine/paracrine TGF-beta may protect or enhance the survival of LTRA during ex vivo culture. TGF-beta 1 had significant suppressive effects on both short- and long-term repopulating activities, and anti- TGF-beta antibody had enhancing effects compared with control cultures containing IL-3, IL-6, and SCF alone. MIP-1 alpha had no significant effects on either short- or long-term repopulating ability. These data suggest that abrogation of TGF-beta during suspension culture may allow enhanced survival or even expansion of primitive cells ex vivo, with implications for many applications, including gene therapy.

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