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Doktor F, Figueira RL, Khalaj K, Ijaz A, Lacher M, Blundell M, Antounians L, Zani A. Characterization of the congenital diaphragmatic hernia model in C57BL/6J fetal mice: a step toward lineage tracing experiments. Pediatr Surg Int 2023; 39:296. [PMID: 37981587 DOI: 10.1007/s00383-023-05583-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 10/25/2023] [Indexed: 11/21/2023]
Abstract
PURPOSE Lineage tracing is key to study the fate of individual cells and their progeny especially in developmental biology. To conduct these studies, we aimed to establish a reproducible model of CDH in the most commonly used genetic background strain that is C57BL/6J mice. METHODS CDH was induced in C57BL/6J dams by maternal administration of nitrofen + bisdiamine at E8.5. Fetuses from olive oil-gavaged mothers served as controls. Lungs from CDH and control fetuses were compared for (1) growth via radial airspace count (RAC), mean linear intercept (MLI) and gene expression for Fgf10, Nrp1, and Ctnnb1; (2) maturation (Pdpn, Spc, Ager, Abca3, Eln, Acta2, Pdgfra) via gene and protein expression; (3) vascularization via gene and protein expression (CD31, Vegfa, Vegfr1/2, Epas1, Enos). STATISTICS unpaired t-test or Mann-Whitney test. RESULTS Nitrofen + bisdiamine administration resulted in 36% left-sided CDH (31% mortality). CDH fetuses had hypoplastic lungs and impaired growth (lower RAC, higher MLI, lower Fgf10, Nrp1, Ctnnb1), maturation (decreased Pdpn, Ager, Eln gene expression), and vascularization (decreased Cd31, Vegfr1/2; Epas1 and Enos). Lower protein expression was confirmed for PDPN, ELN and CD31. CONCLUSION Modeling CDH in C57BL/6J mouse fetuses is effective in reproducing the classical CDH hallmarks. This model will be critical for lineage tracing experiments.
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Affiliation(s)
- Fabian Doktor
- Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, M5G 0A4, Canada
- Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, M5G 1X8, Canada
- Department of Pediatric Surgery, University of Leipzig, 04109, Leipzig, Germany
| | - Rebeca Lopes Figueira
- Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, M5G 0A4, Canada
- Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, M5G 1X8, Canada
| | - Kasra Khalaj
- Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, M5G 0A4, Canada
- Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, M5G 1X8, Canada
| | - Aizah Ijaz
- Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, M5G 0A4, Canada
- Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, M5G 1X8, Canada
| | - Martin Lacher
- Department of Pediatric Surgery, University of Leipzig, 04109, Leipzig, Germany
| | - Matisse Blundell
- Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, M5G 0A4, Canada
- Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, M5G 1X8, Canada
| | - Lina Antounians
- Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, M5G 0A4, Canada
- Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, M5G 1X8, Canada
| | - Augusto Zani
- Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, M5G 0A4, Canada.
- Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, M5G 1X8, Canada.
- Department of Surgery, University of Toronto, Toronto, M5T 1P5, Canada.
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Burgos CM, Gupta VS, Conner P, Frenckner B, Lally KP, Ebanks AH, Harting MT. Syndromic congenital diaphragmatic hernia: Current incidence and outcome. Analysis from the congenital diaphragmatic hernia study group registry. Prenat Diagn 2023; 43:1265-1273. [PMID: 37418285 DOI: 10.1002/pd.6407] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2023] [Revised: 06/25/2023] [Accepted: 07/04/2023] [Indexed: 07/08/2023]
Abstract
BACKGROUND The aim of this study was to describe the incidence of Congenital Diaphragmatic Hernia, CDH, associated with known or clinically suspected syndromes, and the postnatal outcomes from a large database for CDH. METHODS Data from the multicenter, multinational database on infants with CDH (Congenital Diaphragmatic Hernia Study Group Registry) born from 1996 to 2020 were analyzed. Patients with known or suspected syndromes were grouped and outcome data were analyzed and compared to those without syndromic features. RESULTS A total of 12,553 patients were entered in the registry during the study period, and 421 had reported known syndromes, representing 3.4% of all CDH cases in the registry. A total of 50 different associated syndromes were reported. In addition to those with clinically suspected genetic conditions, a total rate of genetic syndromes with CDH was 8.2%. The overall survival to discharge for syndromic CDH was 34% and for non-syndromic CDH was 76.7%. The most common were syndromes Fryns syndrome (19.7% of all syndromes, 17% survival), trisomy 18 or Edward syndrome (17.5%, 9% survival), trisomy 21 or Down syndrome (9%, 47% survival), trisomy 13 or Patau syndrome (6.7%, 14% survival), Cornelia de Lange syndrome (6.4% of all syndromes, 22% survival) and Pallister-Killian syndrome (5.5% of all syndromes, 39.1% survival). In addition, 379 cases had reported chromosomal anomalies and 233 cases had clinically suspected syndromes, based on two more dysmorphic features or malformations in addition to CDH, but without molecular diagnosis. The syndromic CDH group had lower birth weight and gestational age at birth and increased incidence of bilateral CDH (2.9%) and rates of non-repair (53%). The length of hospital stay was longer, and larger number of patients needed O2 at 30 days. Extracorporeal life support was used only in 15% of the cases. Those who underwent surgical repair had survival to discharge rates of 73%. CONCLUSION Syndromic CDH is rare and only 3.4% of the reported cases of CDH have a known syndrome or association, but, if including patients with two dysmorphic features malformations, in addition to CDH, altogether as many as 8.2% have a diagnosed or suspected genetic condition. These children have with lower survival rates. Given higher rates of non-repair and decreased extracorporeal life support use, along with a high early mortality, decision-making regarding goals of care clearly influences outcomes. Survival varies depending on the genetic cause. Early genetic diagnosis is important and may influence the decision-making.
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Affiliation(s)
- Carmen Mesas Burgos
- Department of Pediatric Surgery, Karolinska University Hospital, Stockholm, Sweden
- Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden
| | - Vikas S Gupta
- Department of Pediatric Surgery, McGovern Medical School at UT Health and Children's Memorial Hermann Hospital, Houston, Texas, USA
| | - Peter Conner
- Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden
- Center for Fetal Medicine, Karolinska University Hospital, Stockholm, Sweden
| | - Björn Frenckner
- Department of Pediatric Surgery, Karolinska University Hospital, Stockholm, Sweden
- Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden
| | - Kevin P Lally
- Department of Pediatric Surgery, McGovern Medical School at UT Health and Children's Memorial Hermann Hospital, Houston, Texas, USA
| | - Ashley H Ebanks
- Department of Pediatric Surgery, McGovern Medical School at UT Health and Children's Memorial Hermann Hospital, Houston, Texas, USA
| | - Matthew T Harting
- Department of Pediatric Surgery, McGovern Medical School at UT Health and Children's Memorial Hermann Hospital, Houston, Texas, USA
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Doktor F, Antounians L, Miller J, Harb M, Meats T, Bercovitch R, Ireland D, Zani A. Seasonal Variation of Congenital Diaphragmatic Hernia: A Review of the Literature and Database Report from the United States and Canada. Eur J Pediatr Surg 2023; 33:11-16. [PMID: 35858641 DOI: 10.1055/a-1905-4808] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
INTRODUCTION The etiology of congenital diaphragmatic hernia (CDH) remains unknown and only 10 to 30% of patients have a genetic cause. Seasonal variation is known to contribute to the development of some congenital anomalies. Our aim was to investigate whether CDH births have seasonal variation. MATERIALS AND METHODS A literature review was conducted for CDH and seasonality. Moreover, data from the CDH International Patient Registry Database were collected for infants with due dates between 2008 and 2014. Due dates were used to determine seasonal distribution of births. Birth rates per month in the United States and Canada were extracted from publicly available databases. Data were analyzed using analysis of variance and contingency tables. RESULTS First, the literature review revealed 11 articles, of which 3 were eligible for inclusion. These studies reported conflicting results on seasonality of CDH. Second, we extracted due dates from the CDH International Patient Registry Database (1,259 patients) and found that there were fewer due dates in winter months (12.1 ± 4 patients/month) than in summer (16.7 ± 6 patients/month; p = 0.011) and fall months (16.3 ± 5 patients/month; p = 0.022). Although this trend was similar to that of all births in the United States and Canada, a lower incidence was observed in winter for CDH infants (20.2%) than for the general population (24.1%, p = 0.0012). CDH survival rate did not vary by season. CONCLUSION This study provides evidence for a seasonal variation of CDH births. No causative link was established between CDH development and seasonality. Population-based studies with a focus on exposome data are needed to explain seasonal variation in CDH.
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Affiliation(s)
- Fabian Doktor
- Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, Ontario, Canada.,Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, Ontario, Canada
| | - Lina Antounians
- Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, Ontario, Canada.,Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, Ontario, Canada
| | - Jason Miller
- CDH Patient Registry, CDH International, Wake Forest, North Carolina, United States
| | - Maria Harb
- CDH Patient Registry, CDH International, Wake Forest, North Carolina, United States
| | - Tracy Meats
- CDH Patient Registry, CDH International, Wake Forest, North Carolina, United States
| | - Rachel Bercovitch
- Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, Ontario, Canada.,Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, Ontario, Canada
| | - Dawn Ireland
- CDH Patient Registry, CDH International, Wake Forest, North Carolina, United States
| | - Augusto Zani
- Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, Ontario, Canada.,Division of General and Thoracic Surgery, The Hospital for Sick Children, Toronto, Ontario, Canada.,Department of Surgery, University of Toronto, Toronto, Ontario, Canada
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Sbragia L, Oria M, Scorletti F, Romero Lopez MDM, Schmidt AF, Levy B, Peiro JL. A novel surgical toxicological-free model of diaphragmatic hernia in fetal rats. Pediatr Res 2022; 92:118-124. [PMID: 34465875 DOI: 10.1038/s41390-021-01702-4] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/13/2021] [Revised: 07/22/2021] [Accepted: 08/04/2021] [Indexed: 11/10/2022]
Abstract
BACKGROUND Teratogen-induced congenital diaphragmatic hernia (CDH) rat models are commonly used to study the pathophysiology. We have created a new and reliable surgically induced diaphragmatic hernia (DH) model to obtain a purely mechanical DH rat model, and avoid the confounding teratogen-induced effects on the lung development. METHODS Fetal DH was surgically created on fetuses at E18.5 and harvested at E21.5 in rats. Four groups were evaluated (n = 16): control (CONT), control exposed to Nitrofen (CONT NIT), DH surgically created (DH SURG), and CDH Nitrofen (CDH NIT). Body weight, total lung weights, and their ratio (BW, TLW, and TLBR) were compared. Air space (AS), parenchyma (PA), total protein, and DNA contents were measured to verify lung hypoplasia. Medial wall thickness (MWT) of pulmonary arterioles was also analyzed. RESULTS DH SURG showed significant hypoplasia (decreased in total protein and DNA) vs CONT (p < 0.05); DH SURG vs CDH NIT were similar in TLW and TLBR. DH SURG has less AS than CONT (p < 0.05) and similar PA compared to CONT NIT and CDH NIT, MWT were similarly increased in CONT NIT, DH SURG, and CDH NIT. CONCLUSIONS This novel surgical model generates fetal lung hypoplasia contributing to the study of the mechanical compression effect on fetal lung development in DH. IMPACT There is a critical need to develop a surgical model in rat to complement the findings of the well-known Nitrofen-induced CDH model. This experimental study is pioneer and can help to understand better the CDH pathophysiological changes caused by herniated abdominal viscera compression against the lung during the final stage of gestation in CDH fetuses, and also to develop more efficient treatments in near future.
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Affiliation(s)
- Lourenço Sbragia
- Center for Fetal and Placental Research. Division of Pediatric General and Thoracic Surgery. Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, OH, USA.,Laboratory of Experimental Fetal Surgery, Division of Pediatric Surgery, Department of Surgery and Anatomy, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil
| | - Marc Oria
- Center for Fetal and Placental Research. Division of Pediatric General and Thoracic Surgery. Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, OH, USA.,University of Cincinnati College of Medicine, Cincinnati, OH, USA
| | - Federico Scorletti
- Center for Fetal and Placental Research. Division of Pediatric General and Thoracic Surgery. Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, OH, USA
| | - Maria Del Mar Romero Lopez
- Center for Fetal and Placental Research. Division of Pediatric General and Thoracic Surgery. Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, OH, USA.,Perinatal Institute, Division of Neonatology /Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
| | - Augusto F Schmidt
- Division of Neonatology, University of Miami Miller School of Medicine, Miami, FL, USA
| | - Brittany Levy
- Center for Fetal and Placental Research. Division of Pediatric General and Thoracic Surgery. Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, OH, USA
| | - Jose L Peiro
- Center for Fetal and Placental Research. Division of Pediatric General and Thoracic Surgery. Cincinnati Children's Hospital Medical Center (CCHMC), Cincinnati, OH, USA. .,University of Cincinnati College of Medicine, Cincinnati, OH, USA.
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Friedmacher F, Rolle U, Puri P. Genetically Modified Mouse Models of Congenital Diaphragmatic Hernia: Opportunities and Limitations for Studying Altered Lung Development. Front Pediatr 2022; 10:867307. [PMID: 35633948 PMCID: PMC9136148 DOI: 10.3389/fped.2022.867307] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Accepted: 03/18/2022] [Indexed: 11/21/2022] Open
Abstract
Congenital diaphragmatic hernia (CDH) is a relatively common and life-threatening birth defect, characterized by an abnormal opening in the primordial diaphragm that interferes with normal lung development. As a result, CDH is accompanied by immature and hypoplastic lungs, being the leading cause of morbidity and mortality in patients with this condition. In recent decades, various animal models have contributed novel insights into the pathogenic mechanisms underlying CDH and associated pulmonary hypoplasia. In particular, the generation of genetically modified mouse models, which show both diaphragm and lung abnormalities, has resulted in the discovery of multiple genes and signaling pathways involved in the pathogenesis of CDH. This article aims to offer an up-to-date overview on CDH-implicated transcription factors, molecules regulating cell migration and signal transduction as well as components contributing to the formation of extracellular matrix, whilst also discussing the significance of these genetic models for studying altered lung development with regard to the human situation.
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Affiliation(s)
- Florian Friedmacher
- Department of Pediatric Surgery, University Hospital Frankfurt, Goethe University Frankfurt, Frankfurt, Germany
| | - Udo Rolle
- Department of Pediatric Surgery, University Hospital Frankfurt, Goethe University Frankfurt, Frankfurt, Germany
| | - Prem Puri
- Beacon Hospital, University College Dublin, Dublin, Ireland
- Conway Institute of Biomolecular and Biomedical Research, School of Medicine, University College Dublin, Dublin, Ireland
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Jaslove JM, Goodwin K, Sundarakrishnan A, Spurlin JW, Mao S, Košmrlj A, Nelson CM. Transmural pressure signals through retinoic acid to regulate lung branching. Development 2022; 149:274047. [PMID: 35051272 PMCID: PMC8917413 DOI: 10.1242/dev.199726] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2021] [Accepted: 12/10/2021] [Indexed: 01/22/2023]
Abstract
During development, the mammalian lung undergoes several rounds of branching, the rate of which is tuned by the relative pressure of the fluid within the lumen of the lung. We carried out bioinformatics analysis of RNA-sequencing of embryonic mouse lungs cultured under physiologic or sub-physiologic transmural pressure and identified transcription factor-binding motifs near genes whose expression changes in response to pressure. Surprisingly, we found retinoic acid (RA) receptor binding sites significantly overrepresented in the promoters and enhancers of pressure-responsive genes. Consistently, increasing transmural pressure activates RA signaling, and pharmacologically inhibiting RA signaling decreases airway epithelial branching and smooth muscle wrapping. We found that pressure activates RA signaling through the mechanosensor Yap. A computational model predicts that mechanical signaling through Yap and RA affects lung branching by altering the balance between epithelial proliferation and smooth muscle wrapping, which we test experimentally. Our results reveal that transmural pressure signals through RA to balance the relative rates of epithelial growth and smooth muscle differentiation in the developing mouse lung and identify RA as a previously unreported component in the mechanotransduction machinery of embryonic tissues.
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Affiliation(s)
- Jacob M. Jaslove
- Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA,Graduate School of Biomedical Sciences, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
| | - Katharine Goodwin
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA
| | - Aswin Sundarakrishnan
- Department of Chemical & Biological Engineering, Princeton University, Princeton, NJ 08544, USA
| | - James W. Spurlin
- Department of Chemical & Biological Engineering, Princeton University, Princeton, NJ 08544, USA,Department of Biosciences, Rice University, Houston, TX 77005, USA
| | - Sheng Mao
- Department of Mechanics and Engineering Science, BIC-ESAT, College of Engineering, Peking University, Beijing 100871, People's Republic of China,Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544, USA
| | - Andrej Košmrlj
- Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544, USA,Princeton Institute for the Science & Technology of Materials, Princeton, NJ 08544, USA
| | - Celeste M. Nelson
- Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA,Department of Chemical & Biological Engineering, Princeton University, Princeton, NJ 08544, USA,Author for correspondence ()
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7
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Rocke AW, Clarke TG, Dalmer TRA, McCluskey SA, Rivas JFG, Clugston RD. Low maternal vitamin A intake increases the incidence of teratogen induced congenital diaphragmatic hernia in mice. Pediatr Res 2022; 91:83-91. [PMID: 33654278 PMCID: PMC8770141 DOI: 10.1038/s41390-021-01409-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Revised: 01/04/2021] [Accepted: 01/27/2021] [Indexed: 01/31/2023]
Abstract
BACKGROUND Congenital diaphragmatic hernia (CDH) is a severe birth defect associated with high perinatal mortality and long-term morbidity. The etiology of CDH is poorly understood although abnormal retinoid signaling has been proposed to contribute to abnormal diaphragm development. Existing epidemiological data suggest that inadequate dietary vitamin A intake is a risk factor for developing CDH. METHODS Using a mouse model of teratogen-induced CDH, the objective of this study was to test the hypothesis that low maternal vitamin A intake contributes to abnormal diaphragm development. To test this hypothesis, we optimized a model of altered maternal dietary vitamin A intake and a teratogenic model of CDH in mice that recapitulates the hallmark features of posterolateral diaphragmatic hernia in humans. RESULTS Our data uniquely show that low maternal dietary vitamin A intake and marginal vitamin A status increases the incidence of teratogen-induced CDH in mice. CONCLUSION Low dietary vitamin A intake and marginal vitamin A status lead to an increased incidence of teratogen-induced CDH in mice, highlighting the importance of adequate dietary vitamin A intake and CDH risk. IMPACT This study describes and validates a mouse model of altered maternal and fetal vitamin A status. This study links existing epidemiological data with a mouse model of teratogen-induced congenital diaphragmatic hernia, highlighting the importance of low maternal vitamin A intake as a risk factor for the development of congenital diaphragmatic hernia. This study supports the Retinoid Hypothesis, which posits that the etiology of congenital diaphragmatic hernia is linked to abnormal retinoid signaling in the developing diaphragm.
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Affiliation(s)
- Ayanna W Rocke
- Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
- Women and Children's Health Research Institute, University of Alberta, Edmonton, AB, Canada
| | - Tianna G Clarke
- Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
- Women and Children's Health Research Institute, University of Alberta, Edmonton, AB, Canada
| | - Timothy R A Dalmer
- Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
- Women and Children's Health Research Institute, University of Alberta, Edmonton, AB, Canada
| | - Sydney A McCluskey
- Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
| | - Juan F Garcia Rivas
- Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
| | - Robin D Clugston
- Department of Physiology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada.
- Women and Children's Health Research Institute, University of Alberta, Edmonton, AB, Canada.
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Bendixen C, Brosens E, Chung WK. Genetic Diagnostic Strategies and Counseling for Families Affected by Congenital Diaphragmatic Hernia. Eur J Pediatr Surg 2021; 31:472-481. [PMID: 34911129 DOI: 10.1055/s-0041-1740337] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
Congenital diaphragmatic hernia (CDH) is a relatively common and severe birth defect with variable clinical outcome and associated malformations in up to 60% of patients. Mortality and morbidity remain high despite advances in pre-, intra-, and postnatal management. We review the current literature and give an overview about the genetics of CDH to provide guidelines for clinicians with respect to genetic diagnostics and counseling for families. Until recently, the common practice was (molecular) karyotyping or chromosome microarray if the CDH diagnosis is made prenatally with a 10% diagnostic yield. Undiagnosed patients can be reflexed to trio exome/genome sequencing with an additional diagnostic yield of 10 to 20%. Even with a genetic diagnosis, there can be a range of clinical outcomes. All families with a child with CDH with or without additional malformations should be offered genetic counseling and testing in a family-based trio approach.
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Affiliation(s)
- Charlotte Bendixen
- Department of General, Visceral, Vascular and Thoracic Surgery, Unit of Pediatric Surgery, Universitätsklinikum Bonn, Bonn, Germany
| | - Erwin Brosens
- Department of Pediatric Surgery, Erasmus MC Sophia Children's Hospital, Rotterdam, the Netherlands
| | - Wendy Kay Chung
- Department of Medicine, Columbia University Irving Medical Center, New York, United States.,Department of Pediatrics, Columbia University Irving Medical Center, New York, United States
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9
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Cannata G, Caporilli C, Grassi F, Perrone S, Esposito S. Management of Congenital Diaphragmatic Hernia (CDH): Role of Molecular Genetics. Int J Mol Sci 2021; 22:ijms22126353. [PMID: 34198563 PMCID: PMC8231903 DOI: 10.3390/ijms22126353] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2021] [Revised: 06/04/2021] [Accepted: 06/11/2021] [Indexed: 12/11/2022] Open
Abstract
Congenital diaphragmatic hernia (CDH) is a relatively common major life-threatening birth defect that results in significant mortality and morbidity depending primarily on lung hypoplasia, persistent pulmonary hypertension, and cardiac dysfunction. Despite its clinical relevance, CDH multifactorial etiology is still not completely understood. We reviewed current knowledge on normal diaphragm development and summarized genetic mutations and related pathways as well as cellular mechanisms involved in CDH. Our literature analysis showed that the discovery of harmful de novo variants in the fetus could constitute an important tool for the medical team during pregnancy, counselling, and childbirth. A better insight into the mechanisms regulating diaphragm development and genetic causes leading to CDH appeared essential to the development of new therapeutic strategies and evidence-based genetic counselling to parents. Integrated sequencing, development, and bioinformatics strategies could direct future functional studies on CDH; could be applied to cohorts and consortia for CDH and other birth defects; and could pave the way for potential therapies by providing molecular targets for drug discovery.
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Affiliation(s)
- Giulia Cannata
- Pediatric Clinic, Pietro Barilla Children’s Hospital, University of Parma, Via Gramsci 14, 43126 Parma, Italy; (G.C.); (C.C.); (F.G.)
| | - Chiara Caporilli
- Pediatric Clinic, Pietro Barilla Children’s Hospital, University of Parma, Via Gramsci 14, 43126 Parma, Italy; (G.C.); (C.C.); (F.G.)
| | - Federica Grassi
- Pediatric Clinic, Pietro Barilla Children’s Hospital, University of Parma, Via Gramsci 14, 43126 Parma, Italy; (G.C.); (C.C.); (F.G.)
| | - Serafina Perrone
- Neonatology Unit, Pietro Barilla Children’s Hospital, Department of Medicine and Surgery, University of Parma, 43126 Parma, Italy;
| | - Susanna Esposito
- Pediatric Clinic, Pietro Barilla Children’s Hospital, University of Parma, Via Gramsci 14, 43126 Parma, Italy; (G.C.); (C.C.); (F.G.)
- Correspondence: ; Tel.: +39-0521-7047
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10
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Abstract
PURPOSE This paper aims to build upon previous work to definitively establish in vitro models of murine pseudoglandular stage lung development. These can be easily translated to human fetal lung samples to allow the investigation of lung development in physiologic and pathologic conditions. METHODS Lungs were harvested from mouse embryos at E12.5 and cultured in three different settings, i.e., whole lung culture, mesenchyme-free epithelium culture, and organoid culture. For the whole lung culture, extracted lungs were embedded in Matrigel and incubated on permeable filters. Separately, distal epithelial tips were isolated by firstly removing mesothelial and mesenchymal cells, and then severing the tips from the airway tubes. These were then cultured either in branch-promoting or self-renewing conditions. RESULTS Cultured whole lungs underwent branching morphogenesis similarly to native lungs. Real-time qPCR analysis demonstrated expression of key genes essential for lung bud formation. The culture condition for epithelial tips was optimized by testing different concentrations of FGF10 and CHIR99021 and evaluating branching formation. The epithelial rudiments in self-renewing conditions formed spherical 3D structures with homogeneous Sox9 expression. CONCLUSION We report efficient protocols for ex vivo culture systems of pseudoglandular stage mouse embryonic lungs. These models can be applied to human samples and could be useful to paediatric surgeons to investigate normal lung development, understand the pathogenesis of congenital lung diseases, and explore novel therapeutic strategies.
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11
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Mous DS, Buscop-van Kempen MJ, Wijnen RMH, Tibboel D, Morty RE, Rottier RJ. Opposing Effects of TGFβ and BMP in the Pulmonary Vasculature in Congenital Diaphragmatic Hernia. Front Med (Lausanne) 2021; 8:642577. [PMID: 33777983 PMCID: PMC7991367 DOI: 10.3389/fmed.2021.642577] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2020] [Accepted: 02/22/2021] [Indexed: 11/24/2022] Open
Abstract
Background: Pulmonary hypertension is the major cause of morbidity and mortality in congenital diaphragmatic hernia (CDH). Mutations in several genes that encode signaling molecules of the transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) pathways have previously been associated with CDH. Since studies on the activation of these pathways in CDH are scarce, and have yielded inconsistent conclusions, the downstream activity of both pathways was assessed in the nitrofen-CDH rat model. Methods and Results: Pregnant Sprague-Dawley rats were treated with nitrofen at embryonic day (E) 9.5 to induce CDH in offspring. At E21, lungs were screened for the expression of key factors of both signaling pathways, at both the mRNA transcript and protein levels. Subsequently, paying particular attention to the pulmonary vasculature, increased phosphorylation of SMAD2, and decreased phosphorylation of Smad5 was noted in the muscular walls of small pulmonary vessels, by immunohistochemistry. This was accompanied by increased proliferation of constituent cells of the smooth muscle layer of these vessels. Conclusions: Increased activation of the TGFβ pathway and decreased activation of the BMP pathway in the pulmonary vasculature of rats with experimentally-induced CDH, suggesting that the deregulated of these important signaling pathways may underlie the development of pulmonary hypertension in CDH.
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Affiliation(s)
- Daphne S Mous
- Department of Pediatric Surgery, Erasmus Medical Center - Sophia Children's Hospital, Rotterdam, Netherlands
| | - Marjon J Buscop-van Kempen
- Department of Pediatric Surgery, Erasmus Medical Center - Sophia Children's Hospital, Rotterdam, Netherlands.,Department of Cell Biology, Erasmus Medical Center, Rotterdam, Netherlands
| | - Rene M H Wijnen
- Department of Pediatric Surgery, Erasmus Medical Center - Sophia Children's Hospital, Rotterdam, Netherlands
| | - Dick Tibboel
- Department of Pediatric Surgery, Erasmus Medical Center - Sophia Children's Hospital, Rotterdam, Netherlands
| | - Rory E Morty
- Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.,Department of Internal Medicine (Pulmonology), University of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany
| | - Robbert J Rottier
- Department of Pediatric Surgery, Erasmus Medical Center - Sophia Children's Hospital, Rotterdam, Netherlands.,Department of Cell Biology, Erasmus Medical Center, Rotterdam, Netherlands
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12
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Brosens E, Peters NCJ, van Weelden KS, Bendixen C, Brouwer RWW, Sleutels F, Bruggenwirth HT, van Ijcken WFJ, Veenma DCM, Otter SCMCD, Wijnen RMH, Eggink AJ, van Dooren MF, Reutter HM, Rottier RJ, Schnater JM, Tibboel D, de Klein A. Unraveling the Genetics of Congenital Diaphragmatic Hernia: An Ongoing Challenge. Front Pediatr 2021; 9:800915. [PMID: 35186825 PMCID: PMC8852845 DOI: 10.3389/fped.2021.800915] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/24/2021] [Accepted: 12/28/2021] [Indexed: 12/16/2022] Open
Abstract
Congenital diaphragmatic hernia (CDH) is a congenital structural anomaly in which the diaphragm has not developed properly. It may occur either as an isolated anomaly or with additional anomalies. It is thought to be a multifactorial disease in which genetic factors could either substantially contribute to or directly result in the developmental defect. Patients with aneuploidies, pathogenic variants or de novo Copy Number Variations (CNVs) impacting specific genes and loci develop CDH typically in the form of a monogenetic syndrome. These patients often have other associated anatomical malformations. In patients without a known monogenetic syndrome, an increased genetic burden of de novo coding variants contributes to disease development. In early years, genetic evaluation was based on karyotyping and SNP-array. Today, genomes are commonly analyzed with next generation sequencing (NGS) based approaches. While more potential pathogenic variants are being detected, analysis of the data presents a bottleneck-largely due to the lack of full appreciation of the functional consequence and/or relevance of the detected variant. The exact heritability of CDH is still unknown. Damaging de novo alterations are associated with the more severe and complex phenotypes and worse clinical outcome. Phenotypic, genetic-and likely mechanistic-variability hampers individual patient diagnosis, short and long-term morbidity prediction and subsequent care strategies. Detailed phenotyping, clinical follow-up at regular intervals and detailed registries are needed to find associations between long-term morbidity, genetic alterations, and clinical parameters. Since CDH is a relatively rare disorder with only a few recurrent changes large cohorts of patients are needed to identify genetic associations. Retrospective whole genome sequencing of historical patient cohorts using will yield valuable data from which today's patients and parents will profit Trio whole genome sequencing has an excellent potential for future re-analysis and data-sharing increasing the chance to provide a genetic diagnosis and predict clinical prognosis. In this review, we explore the pitfalls and challenges in the analysis and interpretation of genetic information, present what is currently known and what still needs further study, and propose strategies to reap the benefits of genetic screening.
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Affiliation(s)
- Erwin Brosens
- Department of Clinical Genetics, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Nina C J Peters
- Division of Obstetrics and Fetal Medicine, Department of Obstetrics and Gynecology, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Kim S van Weelden
- Department of Clinical Genetics, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands.,Division of Obstetrics and Fetal Medicine, Department of Obstetrics and Gynecology, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands.,Department of Pediatric Surgery and Intensive Care, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Charlotte Bendixen
- Unit of Pediatric Surgery, Department of General, Visceral, Vascular and Thoracic Surgery, University Hospital Bonn, Bonn, Germany
| | - Rutger W W Brouwer
- Center for Biomics, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands.,Department of Cell Biology, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Frank Sleutels
- Department of Clinical Genetics, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Hennie T Bruggenwirth
- Department of Clinical Genetics, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Wilfred F J van Ijcken
- Center for Biomics, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands.,Department of Cell Biology, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Danielle C M Veenma
- Department of Clinical Genetics, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands.,Department of Pediatrics, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Suzan C M Cochius-Den Otter
- Department of Pediatric Surgery and Intensive Care, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Rene M H Wijnen
- Department of Pediatric Surgery and Intensive Care, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Alex J Eggink
- Division of Obstetrics and Fetal Medicine, Department of Obstetrics and Gynecology, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Marieke F van Dooren
- Department of Clinical Genetics, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Heiko Martin Reutter
- Institute of Human Genetics, University Hospital of Bonn, Bonn, Germany.,Neonatology and Pediatric Intensive Care, Department of Pediatrics and Adolescent Medicine, University Hospital Erlangen, Erlangen, Germany
| | - Robbert J Rottier
- Department of Pediatric Surgery and Intensive Care, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands.,Department of Cell Biology, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - J Marco Schnater
- Department of Pediatric Surgery and Intensive Care, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Dick Tibboel
- Department of Pediatric Surgery and Intensive Care, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
| | - Annelies de Klein
- Department of Clinical Genetics, Erasmus MC Sophia Children's Hospital, Rotterdam, Netherlands
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13
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Fernandes-Silva H, Araújo-Silva H, Correia-Pinto J, Moura RS. Retinoic Acid: A Key Regulator of Lung Development. Biomolecules 2020; 10:biom10010152. [PMID: 31963453 PMCID: PMC7022928 DOI: 10.3390/biom10010152] [Citation(s) in RCA: 43] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2019] [Revised: 01/14/2020] [Accepted: 01/15/2020] [Indexed: 12/14/2022] Open
Abstract
Retinoic acid (RA) is a key molecular player in embryogenesis and adult tissue homeostasis. In embryo development, RA plays a crucial role in the formation of different organ systems, namely, the respiratory system. During lung development, there is a spatiotemporal regulation of RA levels that assures the formation of a fully functional organ. RA signaling influences lung specification, branching morphogenesis, and alveolarization by regulating the expression of particular target genes. Moreover, cooperation with other developmental pathways is essential to shape lung organogenesis. This review focuses on the events regulated by retinoic acid during lung developmental phases and pulmonary vascular development; also, it aims to provide a snapshot of RA interplay with other well-known regulators of lung development.
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Affiliation(s)
- Hugo Fernandes-Silva
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal; (H.F.-S.); (H.A.-S.); (J.C.-P.)
- ICVS/3B’s-PT Government Associate Laboratory, 4710-057 Braga/Guimarães, Portugal
- PhDOC PhD Program, ICVS/3B’s, School of Medicine, University of Minho, 4710-057 Braga, Portugal
| | - Henrique Araújo-Silva
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal; (H.F.-S.); (H.A.-S.); (J.C.-P.)
- ICVS/3B’s-PT Government Associate Laboratory, 4710-057 Braga/Guimarães, Portugal
| | - Jorge Correia-Pinto
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal; (H.F.-S.); (H.A.-S.); (J.C.-P.)
- ICVS/3B’s-PT Government Associate Laboratory, 4710-057 Braga/Guimarães, Portugal
- Department of Pediatric Surgery, Hospital of Braga, 4710-243 Braga, Portugal
| | - Rute S Moura
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal; (H.F.-S.); (H.A.-S.); (J.C.-P.)
- ICVS/3B’s-PT Government Associate Laboratory, 4710-057 Braga/Guimarães, Portugal
- Correspondence: ; Tel.: +35-12-5360-4911
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14
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Li X, Liu H, Yu W, Liu X, Liu C. Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia. Biomed Pharmacother 2019; 121:109621. [PMID: 31734580 DOI: 10.1016/j.biopha.2019.109621] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2019] [Revised: 11/01/2019] [Accepted: 11/01/2019] [Indexed: 02/04/2023] Open
Abstract
OBJECTIVE Congenital diaphragmatic hernia (CDH) is a common and often lethal birth defect characterized by congenital lung malformation, which severely affects neonate prognosis and mortality. This study aimed to investigate differences in protein expression in order to elucidate the mechanism of CDH-associated pulmonary hypoplasia during the early stage of lung development using tandem mass tag (TMT) quantitative proteomics. METHODS Nitrofen was administered orally to establish a rat CDH model, and pathological changes were evaluated through hematoxylin-eosin (H&E), PCNA, and Ki67 staining at the pseudoglandular stage. Fetal lungs were then collected, pooled before TMT labeling, and subjected to mass spectrometry. Immunohistochemistry (IHC), Western blotting, and Q-PCR were used to further validate the candidate proteins. RESULTS A total of 79 differentially expressed proteins (DEPs) were identified when CDH and control lungs were compared, and further bioinformatics analysis showed that these proteins play important roles in tight-junctions, phospholipase D signaling, and the HIF-1 signaling pathway. Three differentially expressed proteins, Cldn3, Magi1, and Myh9 are involved in the tight-junction pathway (P < 0.05), and their differential expressions were confirmed by IHC, Western blotting, and Q-PCR. CONCLUSION These findings indicate that alterations of tight-junction protein expression may play an important role in the pathogenesis of abnormal lung development in CDH. Further studies are warranted to verify the mechanism by which these tight-junction proteins influence the pathogenesis of CDH-associated pulmonary hypoplasia.
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Affiliation(s)
- Xue Li
- Department of Gynecology and Obstetrics, Shengjing Hospital of China Medical University, Shenyang, China; Key Laboratory of Maternal-Fetal Medicine of Liaoning Province, Benxi, China.
| | - Hao Liu
- Department of Gynecology and Obstetrics, Shengjing Hospital of China Medical University, Shenyang, China; Key Laboratory of Maternal-Fetal Medicine of Liaoning Province, Benxi, China.
| | - Wenqian Yu
- Department of Gynecology and Obstetrics, Shengjing Hospital of China Medical University, Shenyang, China; Key Laboratory of Maternal-Fetal Medicine of Liaoning Province, Benxi, China.
| | - Xiaomei Liu
- Department of Gynecology and Obstetrics, Shengjing Hospital of China Medical University, Shenyang, China; Key Laboratory of Maternal-Fetal Medicine of Liaoning Province, Benxi, China.
| | - Caixia Liu
- Department of Gynecology and Obstetrics, Shengjing Hospital of China Medical University, Shenyang, China; Key Laboratory of Maternal-Fetal Medicine of Liaoning Province, Benxi, China.
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15
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Figueira RL, Costa KMD, Marsico AL, Milani TMDS, Gonçalves WA, Borges MDC, Silva OCE, Sbragia L. Vascular and ventilatory mechanical responses in three different stages of pulmonary development in the rabbit model of congenital diaphragmatic hernia 1. Acta Cir Bras 2019; 33:879-888. [PMID: 30484497 DOI: 10.1590/s0102-865020180100000002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2018] [Accepted: 09/08/2018] [Indexed: 11/22/2022] Open
Abstract
PURPOSE To evaluate the vascular ventilatory response in different stages of lung development and to compare them to the neonates with congenital diaphragmatic hernia (CDH) in a rabbit model. METHODS New Zealand rabbits were divided into 8 groups (n=5): E25, E27, E30, and CDH. All groups were ventilated on a FlexiVent (Scireq, Montreal, QC, Canada), compounding the other 4 groups. The CDH surgery was performed at E25 and the harvest at E30. Dynamic compliance (CRS), dynamic elastance (ERS) and dynamic resistance (RRS) were measured every 4 min/24 min. Median wall thickness (MWT) and airspace were measured. ANOVA Bonferroni tests were used to perform statistical analysis. Significance was considered when p<0.05. RESULTS CRS was higher in E30 compared to all other groups (p<0.05). CRS and RRS of CDH and E27 were similar and were higher in E25 (p<0.05). MWT was decreased according to the gestational age, was increased in E27V and E30V (p<0.05) and decreased in CDHV (p<0.05), airspace was decreased in E25 and increased in all ventilated groups (p<0.05). CONCLUSIONS The ventilation response of congenital diaphragmatic hernia is like the pseudoglandular stage of the lung development. These findings add information about the physiology of pulmonary ventilation in CDH.
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Affiliation(s)
- Rebeca Lopes Figueira
- Post Doctoral degree, Postgraduate Laboratory of Experimental Fetal Surgery, Division of Pediatric Surgery, Department of Surgery and Anatomy, Ribeirao Preto Medical School, Universidade de São Paulo (USP), Ribeirao Preto-SP, Brazil. Scientific and intellectual content of the study; acquisition, analysis and interpretation of data; technical procedures; histopathological examinations; statistical analysis; manuscript preparation and writing
| | - Karina Miura da Costa
- Fellow PhD degree, Postgraduate Laboratory of Experimental Fetal Surgery, Division of Pediatric Surgery, Department of Surgery and Anatomy, Ribeirao Preto Medical School, USP, Ribeirao Preto-SP, Brazil. Technical procedures, manuscript writing, critical revision
| | - Ana Laura Marsico
- Fellow PhD degree, Postgraduate Laboratory of Experimental Fetal Surgery, Division of Pediatric Surgery, Department of Surgery and Anatomy, Ribeirao Preto Medical School, USP, Ribeirao Preto-SP, Brazil. Technical procedures, histopathological examinations
| | - Thamires Melchiades da Silva Milani
- Fellow Master degree, Postgraduate Laboratory of Experimental Pulmonary Physiopathology, Department of Internal Medicine, Ribeirao Preto Medical School, USP, Ribeirao Preto-SP, Brazil. Technical procedures, collect and organization of the ventilatory paramethers
| | - Walusa Assad Gonçalves
- Full Professor, Department of Pediatrics, Ribeirao Preto Medical School, USP, Ribeirao Preto-SP, Brazil. Scientific, intellectual and design of the study; technical procedures
| | - Marcos de Carvalho Borges
- Full Professor, Laboratory of Experimental Pulmonary Physiopathology, Department of Internal Medicine, Ribeirao Preto Medical School, USP, Ribeirao Preto-SP, Brazil. Scientific and intellectual content of the study, critical revision, final approval
| | - Orlando Castro E Silva
- Full Professor, Laboratory of Liver Transplantation, Department of Surgery and Anatomy, Ribeirao Preto Medical School, USP, Ribeirao Preto-SP, Brazil. Scientific and intellectual content of the study
| | - Lourenço Sbragia
- Associate Professor, Head, Department of Surgery and Anatomy, Laboratory of Experimental Fetal Surgery, Department of Surgery and Anatomy, Ribeirao Preto Medical School, USP, Ribeirao Preto-SP, Brazil. Scientific and intellectual content of the study, manuscript preparation, critical revision, final approval
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16
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Qi H, Yu L, Zhou X, Wynn J, Zhao H, Guo Y, Zhu N, Kitaygorodsky A, Hernan R, Aspelund G, Lim FY, Crombleholme T, Cusick R, Azarow K, Danko ME, Chung D, Warner BW, Mychaliska GB, Potoka D, Wagner AJ, ElFiky M, Wilson JM, Nickerson D, Bamshad M, High FA, Longoni M, Donahoe PK, Chung WK, Shen Y. De novo variants in congenital diaphragmatic hernia identify MYRF as a new syndrome and reveal genetic overlaps with other developmental disorders. PLoS Genet 2018; 14:e1007822. [PMID: 30532227 PMCID: PMC6301721 DOI: 10.1371/journal.pgen.1007822] [Citation(s) in RCA: 73] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2018] [Revised: 12/20/2018] [Accepted: 11/08/2018] [Indexed: 12/24/2022] Open
Abstract
Congenital diaphragmatic hernia (CDH) is a severe birth defect that is often accompanied by other congenital anomalies. Previous exome sequencing studies for CDH have supported a role of de novo damaging variants but did not identify any recurrently mutated genes. To investigate further the genetics of CDH, we analyzed de novo coding variants in 362 proband-parent trios including 271 new trios reported in this study. We identified four unrelated individuals with damaging de novo variants in MYRF (P = 5.3x10(-8)), including one likely gene-disrupting (LGD) and three deleterious missense (D-mis) variants. Eight additional individuals with de novo LGD or missense variants were identified from our other genetic studies or from the literature. Common phenotypes of MYRF de novo variant carriers include CDH, congenital heart disease and genitourinary abnormalities, suggesting that it represents a novel syndrome. MYRF is a membrane associated transcriptional factor highly expressed in developing diaphragm and is depleted of LGD variants in the general population. All de novo missense variants aggregated in two functional protein domains. Analyzing the transcriptome of patient-derived diaphragm fibroblast cells suggest that disease associated variants abolish the transcription factor activity. Furthermore, we showed that the remaining genes with damaging variants in CDH significantly overlap with genes implicated in other developmental disorders. Gene expression patterns and patient phenotypes support pleiotropic effects of damaging variants in these genes on CDH and other developmental disorders. Finally, functional enrichment analysis implicates the disruption of regulation of gene expression, kinase activities, intra-cellular signaling, and cytoskeleton organization as pathogenic mechanisms in CDH.
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Affiliation(s)
- Hongjian Qi
- Department of Systems Biology, Columbia University Medical Center, New York, New York, United States of America
- Department of Applied Mathematics and Applied Physics, Columbia University, New York, New York, United States of America
| | - Lan Yu
- Department of Pediatrics Medical Center, Columbia University, New York, New York, United States of America
| | - Xueya Zhou
- Department of Systems Biology, Columbia University Medical Center, New York, New York, United States of America
- Department of Pediatrics Medical Center, Columbia University, New York, New York, United States of America
| | - Julia Wynn
- Department of Pediatrics Medical Center, Columbia University, New York, New York, United States of America
| | - Haoquan Zhao
- Department of Systems Biology, Columbia University Medical Center, New York, New York, United States of America
- Department of Biomedical Informatics, Columbia University Medical Center, New York, New York, United States of America
| | - Yicheng Guo
- Department of Systems Biology, Columbia University Medical Center, New York, New York, United States of America
| | - Na Zhu
- Department of Systems Biology, Columbia University Medical Center, New York, New York, United States of America
- Department of Pediatrics Medical Center, Columbia University, New York, New York, United States of America
| | - Alexander Kitaygorodsky
- Department of Systems Biology, Columbia University Medical Center, New York, New York, United States of America
- Department of Biomedical Informatics, Columbia University Medical Center, New York, New York, United States of America
| | - Rebecca Hernan
- Department of Pediatrics Medical Center, Columbia University, New York, New York, United States of America
| | - Gudrun Aspelund
- Department of Surgery, Columbia University Medical Center, New York, New York, United States of America
| | - Foong-Yen Lim
- Cincinnati Children's Hospital, Cincinnati, Ohio, United States of America
| | | | - Robert Cusick
- Children's Hospital & Medical Center of Omaha, University of Nebraska College of Medicine, Omaha, Nebraska, United States of America
| | - Kenneth Azarow
- Department of Surgery, Oregon Health & Science University, Portland, Oregon, United States of America
| | - Melissa E Danko
- Monroe Carell Jr. Children's Hospital, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
| | - Dai Chung
- Monroe Carell Jr. Children's Hospital, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
| | - Brad W Warner
- Washington University, St. Louis Children's Hospital, St. Louis, Missouri, United States of America
| | - George B Mychaliska
- University of Michigan, CS Mott Children's Hospital, Ann Arbor, Michigan, United States of America
| | - Douglas Potoka
- Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, United States of America
| | - Amy J Wagner
- Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America
| | - Mahmoud ElFiky
- Department of Pediatric Surgery, Faculty of Medicine, Cairo University, Cairo, Egypt
| | - Jay M Wilson
- Department of Surgery, Boston Children's Hospital, Boston, Massachusetts, United States of America
- Department of Surgery, Harvard Medical School, Boston, Massachusetts, United States of America
| | - Debbie Nickerson
- University of Washington, Seattle, Washington, United States of America
| | - Michael Bamshad
- University of Washington, Seattle, Washington, United States of America
| | - Frances A High
- Department of Surgery, Boston Children's Hospital, Boston, Massachusetts, United States of America
- Department of Surgery, Harvard Medical School, Boston, Massachusetts, United States of America
- Pediatric Surgical Research Laboratories, Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, United States of America
| | - Mauro Longoni
- Department of Surgery, Harvard Medical School, Boston, Massachusetts, United States of America
- Pediatric Surgical Research Laboratories, Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, United States of America
| | - Patricia K Donahoe
- Department of Surgery, Harvard Medical School, Boston, Massachusetts, United States of America
- Pediatric Surgical Research Laboratories, Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, United States of America
| | - Wendy K Chung
- Department of Pediatrics Medical Center, Columbia University, New York, New York, United States of America
- Department of Medicine, Columbia University, New York, New York, United States of America
- Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, New York, United States of America
| | - Yufeng Shen
- Department of Systems Biology, Columbia University Medical Center, New York, New York, United States of America
- Department of Biomedical Informatics, Columbia University Medical Center, New York, New York, United States of America
- JP Sulzberger Columbia Genome Center, Columbia University Medical Center, New York, New York, United States of America
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17
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Gosemann JH, Friedmacher F, Hofmann A, Zimmer J, Kuebler JF, Rittinghausen S, Suttkus A, Lacher M, Alvarez L, Corcionivoschi N, Puri P. Prenatal treatment with rosiglitazone attenuates vascular remodeling and pulmonary monocyte influx in experimental congenital diaphragmatic hernia. PLoS One 2018; 13:e0206975. [PMID: 30418988 PMCID: PMC6231640 DOI: 10.1371/journal.pone.0206975] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2018] [Accepted: 10/23/2018] [Indexed: 01/29/2023] Open
Abstract
Introduction Extensive vascular remodeling causing pulmonary hypertension (PH) represents a major cause of mortality in patients with congenital diaphragmatic hernia (CDH). The chemokine monocyte chemoattractant protein-1 (MCP-1) is a biomarker for the severity of PH and its activation is accompanied by pulmonary influx of monocytes and extensive vascular remodeling. MCP-1 activation can be reversed by application of rosiglitazone (thiazolidinedione). We performed this study to evaluate the role of MCP-1 for the pathogenesis of PH in experimental CDH. We hypothesized that vascular remodeling and MCP-1 activation is accompanied by pulmonary influx of fetal monocytes and can be attenuated by prenatal treatment with rosiglitazone. Methods In a first set of experiments pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D21 and divided into CDH and control. Quantitative real-time polymerase chain reaction, Western blot (WB), and immunohistochemistry (IHC) were used to evaluate MCP-1 expression, activation, and localization. Quantification and localization of pulmonary monocytes/macrophages were carried out by IHC. In a second set of experiments nitrofen-exposed dams were randomly assigned to prenatal treatment with rosiglitazone or placebo on D18+D19. Fetal lungs were harvested on D21, divided into control, CDH+rosiglitazone, and CDH+placebo and evaluated by WB as well as IHC. Results Increased thickness of pulmonary arteries of CDH fetuses was accompanied by increased systemic and perivascular MCP-1 protein expression and significantly higher amounts of pulmonary monocytes/macrophages compared to controls (p<0.01). These effects were reversed by prenatal treatment with rosiglitazone (p<0.01 vs. CDH+P; control). Conclusion Prenatal treatment with rosiglitazone has the potential to attenuate activation of pulmonary MCP-1, pulmonary monocyte influx, and vascular remodeling in experimental CDH. These results provide a basis for future research on prenatal immunomodulation as a novel treatment strategy to decrease secondary effects of PH in CDH.
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MESH Headings
- Animals
- Chemokine CCL2/blood
- Chemokine CCL2/genetics
- Chemokine CCL2/metabolism
- Disease Models, Animal
- Female
- Gene Expression
- Hernias, Diaphragmatic, Congenital/drug therapy
- Hernias, Diaphragmatic, Congenital/etiology
- Hernias, Diaphragmatic, Congenital/metabolism
- Hernias, Diaphragmatic, Congenital/pathology
- Immunohistochemistry
- Lung/metabolism
- Lung/pathology
- Macrophages/immunology
- Macrophages/metabolism
- Monocytes/drug effects
- Monocytes/metabolism
- Phenyl Ethers/adverse effects
- Pregnancy
- Prenatal Care
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Rats
- Rosiglitazone/pharmacology
- Vascular Remodeling/drug effects
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Affiliation(s)
- Jan-Hendrik Gosemann
- National Children’s Research Centre, Our Lady’s Children’s Hospital, Dublin, Ireland
- Department of Pediatric Surgery, University of Leipzig, Leipzig, Germany
- * E-mail:
| | - Florian Friedmacher
- National Children’s Research Centre, Our Lady’s Children’s Hospital, Dublin, Ireland
- The Royal London Hospital, London, United Kingdom
| | - Alejandro Hofmann
- National Children’s Research Centre, Our Lady’s Children’s Hospital, Dublin, Ireland
- Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany
| | - Julia Zimmer
- National Children’s Research Centre, Our Lady’s Children’s Hospital, Dublin, Ireland
- Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany
| | - Joachim F. Kuebler
- Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany
| | - Susanne Rittinghausen
- Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover, Germany
| | - Anne Suttkus
- Department of Pediatric Surgery, University of Leipzig, Leipzig, Germany
| | - Martin Lacher
- Department of Pediatric Surgery, University of Leipzig, Leipzig, Germany
| | - Luis Alvarez
- National Children’s Research Centre, Our Lady’s Children’s Hospital, Dublin, Ireland
- Wellcome Centre Human Genetics, University of Oxford, Oxford, United Kingdom
| | - Nicolae Corcionivoschi
- National Children’s Research Centre, Our Lady’s Children’s Hospital, Dublin, Ireland
- Agri-Food and Biosciences Institute, Belfast, Northern Ireland, United Kingdom
| | - Prem Puri
- National Children’s Research Centre, Our Lady’s Children’s Hospital, Dublin, Ireland
- School of Medicine and Medical Science and Conway Institute of Biomedical Research, University College Dublin, Dublin, Ireland
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18
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Fox ZD, Jiang G, Ho KKY, Walker KA, Liu AP, Kunisaki SM. Fetal lung transcriptome patterns in an ex vivo compression model of diaphragmatic hernia. J Surg Res 2018; 231:411-420. [PMID: 30278961 DOI: 10.1016/j.jss.2018.06.064] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2018] [Revised: 04/26/2018] [Accepted: 06/20/2018] [Indexed: 12/27/2022]
Abstract
BACKGROUND The purpose of this study was to employ a novel ex vivo lung model of congenital diaphragmatic hernia (CDH) to determine how a mechanical compression affects early pulmonary development. METHODS Day-15 whole fetal rat lungs (n = 6-12/group) from nitrofen-exposed and normal (vehicle only) dams were explanted and cultured ex vivo in compression microdevices (0.2 or 0.4 kPa) for 16 h to mimic physiologic compression forces that occur in CDH in vivo. Lungs were evaluated with significance set at P < 0.05. RESULTS Nitrofen-exposed lungs were hypoplastic and expressed lower levels of surfactant protein C at baseline. Although compression alone did not alter the α-smooth muscle actin (ACTA2) expression in normal lungs, nitrofen-exposed lungs had significantly increased ACTA2 transcripts (0.2 kPa: 2.04 ± 0.15; 0.4 kPa: 2.22 ± 0.11; both P < 0.001). Nitrofen-exposed lungs also showed further reductions in surfactant protein C expression at 0.2 and 0.4 kPa (0.53 ± 0.04, P < 0.01; 0.69 ± 0.23, P < 0.001; respectively). Whereas normal lungs exposed to 0.2 and 0.4 kPa showed significant increases in periostin (POSTN), a mechanical stress-response molecule (1.79 ± 0.10 and 2.12 ± 0.39, respectively; both P < 0.001), nitrofen-exposed lungs had a significant decrease in POSTN expression (0.4 kPa: 0.67 ± 0.15, P < 0.001), which was confirmed by immunohistochemistry. CONCLUSIONS Collectively, these pilot data in a model of CDH lung hypoplasia suggest a primary aberration in response to mechanical stress within the nitrofen lung, characterized by an upregulation of ACTA2 and a downregulation in SPFTC and POSTN. This ex vivo compression system may serve as a novel research platform to better understand the mechanobiology and complex regulation of matricellular dynamics during CDH fetal lung development.
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Affiliation(s)
- Zachary D Fox
- Department of Surgery, Michigan Medicine, University of Michigan, Ann Arbor, Michigan
| | - Guihua Jiang
- Department of Surgery, Michigan Medicine, University of Michigan, Ann Arbor, Michigan
| | - Kenneth K Y Ho
- Mechanical Engineering, Michigan Medicine, University of Michigan, Ann Arbor, Michigan
| | - Kendal A Walker
- Department of Surgery, Michigan Medicine, University of Michigan, Ann Arbor, Michigan
| | - Allen P Liu
- Mechanical Engineering, Michigan Medicine, University of Michigan, Ann Arbor, Michigan
| | - Shaun M Kunisaki
- Department of Surgery, Michigan Medicine, University of Michigan, Ann Arbor, Michigan.
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19
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Mous DS, Kool HM, Burgisser PE, Buscop-van Kempen MJ, Nagata K, Boerema-de Munck A, van Rosmalen J, Dzyubachyk O, Wijnen RMH, Tibboel D, Rottier RJ. Treatment of rat congenital diaphragmatic hernia with sildenafil and NS-304, selexipag's active compound, at the pseudoglandular stage improves lung vasculature. Am J Physiol Lung Cell Mol Physiol 2018; 315:L276-L285. [PMID: 29745254 DOI: 10.1152/ajplung.00392.2017] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Patients with congenital diaphragmatic hernia (CDH) often suffer from severe pulmonary hypertension, and the choice of current vasodilator therapy is mostly based on trial and error. Because pulmonary vascular abnormalities are already present early during development, we performed a study to modulate these pulmonary vascular changes at an early stage during gestation. Pregnant Sprague-Dawley rats were treated with nitrofen at day 9.5 of gestation (E9.5) to induce CDH in the offspring, and subsequently, the phosphodiesterase-5 inhibitor sildenafil and/or the novel prostaglandin-I receptor agonist selexipag (active compound NS-304) were administered from E17.5 until E20.5. The clinical relevant start of the treatment corresponds to week 20 of gestation in humans, when CDH is usually detected by ultrasound. CDH pups showed increased density of air saccules that was reverted after the use of only sildenafil. The pulmonary vascular wall was thickened, and right ventricular hypertrophy was present in the CDH group and improved both after single treatment with sildenafil or selexipag, whereas the combination therapy with both compounds did not have additive value. In conclusion, antenatal treatment with sildenafil improved airway morphogenesis and pulmonary vascular development, whereas selexipag only acted positively on pulmonary vascular development. The combination of both compounds did not act synergistically, probably because of a decreased efficiency of both compounds caused by cytochrome- P450 3A4 interaction and induction. These new insights create important possibilities for future treatment of pulmonary vascular abnormalities in CDH patients already in the antenatal period of life.
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Affiliation(s)
- Daphne S Mous
- Department of Pediatric Surgery, Erasmus Medical Center-Sophia Children's Hospital , Rotterdam , The Netherlands
| | - Heleen M Kool
- Department of Pediatric Surgery, Erasmus Medical Center-Sophia Children's Hospital , Rotterdam , The Netherlands
| | - Petra E Burgisser
- Department of Pediatric Surgery, Erasmus Medical Center-Sophia Children's Hospital , Rotterdam , The Netherlands
| | - Marjon J Buscop-van Kempen
- Department of Pediatric Surgery, Erasmus Medical Center-Sophia Children's Hospital , Rotterdam , The Netherlands
| | - Koji Nagata
- Department of Pediatric Surgery, Erasmus Medical Center-Sophia Children's Hospital , Rotterdam , The Netherlands.,Department of Pediatric Surgery, Reproductive and Developmental Medicine, Graduate School of Medical Sciences, Kyushu University , Fukuoka , Japan
| | - Anne Boerema-de Munck
- Department of Pediatric Surgery, Erasmus Medical Center-Sophia Children's Hospital , Rotterdam , The Netherlands
| | - Joost van Rosmalen
- Department of Biostatistics, Erasmus Medical Center , Rotterdam , The Netherlands
| | - Oleh Dzyubachyk
- Department of Radiology, Leiden University Medical Center , Leiden , The Netherlands
| | - Rene M H Wijnen
- Department of Pediatric Surgery, Erasmus Medical Center-Sophia Children's Hospital , Rotterdam , The Netherlands
| | - Dick Tibboel
- Department of Pediatric Surgery, Erasmus Medical Center-Sophia Children's Hospital , Rotterdam , The Netherlands
| | - Robbert J Rottier
- Department of Pediatric Surgery, Erasmus Medical Center-Sophia Children's Hospital , Rotterdam , The Netherlands.,Department of Cell Biology, Erasmus Medical Center , Rotterdam , The Netherlands
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20
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Aydin E, Joshi R, Oria M, Varisco BM, Lim FY, Peiro JL. Fetal tracheal occlusion in mice: a novel transuterine method. J Surg Res 2018; 229:311-315. [PMID: 29937007 DOI: 10.1016/j.jss.2018.04.028] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2017] [Revised: 03/29/2018] [Accepted: 04/13/2018] [Indexed: 11/29/2022]
Abstract
BACKGROUND Fetal tracheal occlusion (TO) is an emerging surgical therapy in congenital diaphragmatic hernia that improves the fetal lung growth. Different animal models of congenital diaphragmatic hernia and TO present advantages and disadvantages regarding ethical issues, cost, surgical difficulty, size, survival rates, and available genetic tools. We developed a minimally invasive murine transuterine TO model, which will be useful in defining how TO impacts lung molecular biology, cellular processes, and overall lung physiology. MATERIALS AND METHODS Time-mated C57BL/6 mice underwent laparotomy at embryonic day 16.5 (E16.5) with transuterine TO performed on two fetuses in each uterine horn. At E18.5, dams were sacrificed and fetuses harvested. The lungs of the TO fetuses were compared with the nonmanipulated counterparts by morphometric and histologic analysis. RESULTS Successful TO was confirmed in 16 of 20 TO fetuses. Twelve of them survived to E18.5 (75%). Fetal weights were comparable, but lung weights were significantly greater in TO (28.41 ± 5.87 versus 23.38 ± 3.09, P = 0.043). Lung to body weight ratio was also greater (0.26 ± 0.003 versus 0.22 ± 0.002, P = 0.006). E18.5 TO lungs demonstrated dilated central and distal airspaces with increased cellularity. DNA/protein and DNA/lung weight ratios were elevated while protein/lung weight ratio was lower in TO compared to control. CONCLUSIONS Mice fetal transuterine TO is feasible with comparable outcomes to other current animal models. The increase in the lung weight, lung to body weight ratio and the DNA/protein ratio indicate organized lung growth rather than edema or cell hypertrophy.
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Affiliation(s)
- Emrah Aydin
- The Center for Fetal, Cellular and Molecular Therapy, Cincinnati Fetal Center, Division of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
| | - Rashika Joshi
- The Division of Critical Care Medicine, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | - Marc Oria
- The Center for Fetal, Cellular and Molecular Therapy, Cincinnati Fetal Center, Division of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | - Brian Michael Varisco
- The Division of Critical Care Medicine, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | - Foong-Yen Lim
- The Center for Fetal, Cellular and Molecular Therapy, Cincinnati Fetal Center, Division of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
| | - Jose Luis Peiro
- The Center for Fetal, Cellular and Molecular Therapy, Cincinnati Fetal Center, Division of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
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21
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Loo CKC, Pearen MA, Pereira TN, Perry-Keene J, Payton D, Ramm GA. Lung and liver growth and retinoic acid status in human fetuses with congenital diaphragmatic hernia. Early Hum Dev 2018; 116:17-23. [PMID: 29096166 DOI: 10.1016/j.earlhumdev.2017.10.005] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/06/2017] [Revised: 10/23/2017] [Accepted: 10/25/2017] [Indexed: 10/18/2022]
Abstract
BACKGROUND Abnormal retinoic acid (RA) signalling is considered a major cause of congenital diaphragmatic hernia (CDH). Pulmonary hypoplasia and pulmonary hypertension are the major causes of morbidity and mortality in infants born with CDH. Experimental studies in animals have found that RA signalling is involved in lung and liver development, but animal models of CDH do not directly correlate with CDH in human fetuses. This study investigated if RA status is also linked to lung and liver growth in human fetuses with CDH. STUDY DESIGN AND PATIENTS Hepatic stellate cells (HSC) in autopsy human fetal liver tissue were identified using cRBP-1 immunohistochemistry and the numbers of HSC manually counted. In mammals, RA is principally stored in HSC complexed to cRBP-1 and therefore cRBP-1+ HSC numbers were used as an indicator of fetal RA status. The number of HSCs was correlated with liver and lung weights, calculated relative to either normal biometric values or fetal body weight. RESULTS The number of cRBP-1+ HSCs correlated with lung weight contralateral to the side of the diaphragmatic hernia (r=0.82, p=0.025) and combined lung weight (r=0.78, p=0.039) but not with ipsilateral lung weight (r=0.43, p=0.33), in fetuses with right and left CDH and a case of giant omphalocoele. Liver growth was influenced by contact with diaphragm but not significantly correlated with cRBP-1 expression (r=0.52, p=0.056). CONCLUSION Fetal RA stores, reflected in the number of cRBP-1+ HSCs, influence lung growth as well as diaphragm development in human fetuses with CDH. Contact with diaphragm influenced liver growth.
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Affiliation(s)
- Christine K C Loo
- Dept of Anatomical Pathology, NSW Health Pathology, Prince of Wales Hospital, Sydney, Australia; Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, Australia
| | - Michael A Pearen
- Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, Australia
| | - Tamara N Pereira
- Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, Australia
| | | | - Diane Payton
- Anatomical Pathology, Pathology Queensland, Australia
| | - Grant A Ramm
- Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, Australia; Faculty of Medicine and Biomedical Sciences, The University of Queensland, Brisbane, Australia.
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22
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Donahoe PK, Longoni M, High FA. Polygenic Causes of Congenital Diaphragmatic Hernia Produce Common Lung Pathologies. THE AMERICAN JOURNAL OF PATHOLOGY 2016; 186:2532-43. [PMID: 27565037 DOI: 10.1016/j.ajpath.2016.07.006] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/31/2016] [Revised: 06/20/2016] [Accepted: 07/13/2016] [Indexed: 12/12/2022]
Abstract
Congenital diaphragmatic hernia (CDH) is one of the most common and lethal congenital anomalies, and significant evidence is available in support of a genetic contribution to its etiology, including single-gene knockout mice associated with diaphragmatic defects, rare monogenetic disorders in humans, familial aggregation, and association of CDH with chromosomal abnormalities. Structural lung defects in the form of lung hypoplasia are almost invariably seen in patients with CDH and frequently in animal models of this condition. Better understanding of the mechanisms of pulmonary defects in CDH has the potential for creating targeted therapies, particularly in postnatal stages, when therapeutics can have maximum clinical impact on the surviving cohorts. Successful treatment of CDH is dependent on the integration of human genomic and genetic data with developmental expression profiling, mouse knockouts, and gene network and pathway modeling, which have generated a large number of candidate genes and pathways for follow-up studies. In particular, defective alveolarization appears to be a common and potentially actionable phenotype in both patients and animal models.
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Affiliation(s)
- Patricia K Donahoe
- Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, Massachusetts; Department of Surgery, Harvard Medical School, Boston, Massachusetts; Broad Institute of the Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts.
| | - Mauro Longoni
- Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, Massachusetts; Department of Surgery, Harvard Medical School, Boston, Massachusetts
| | - Frances A High
- Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, Massachusetts; Department of Surgery, Harvard Medical School, Boston, Massachusetts; Department of Pediatrics, Massachusetts General Hospital, Boston, Massachusetts; Department of Surgery, Boston Children's Hospital, Boston, Massachusetts
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23
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Engels AC, Brady PD, Kammoun M, Finalet Ferreiro J, DeKoninck P, Endo M, Toelen J, Vermeesch JR, Deprest J. Pulmonary transcriptome analysis in the surgically induced rabbit model of diaphragmatic hernia treated with fetal tracheal occlusion. Dis Model Mech 2016; 9:221-8. [PMID: 26744354 PMCID: PMC4770142 DOI: 10.1242/dmm.021626] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2015] [Accepted: 12/29/2015] [Indexed: 01/25/2023] Open
Abstract
Congenital diaphragmatic hernia (CDH) is a malformation leading to pulmonary hypoplasia, which can be treated in utero by fetal tracheal occlusion (TO). However, the changes of gene expression induced by TO remain largely unknown but could be used to further improve the clinically used prenatal treatment of this devastating malformation. Therefore, we aimed to investigate the pulmonary transcriptome changes caused by surgical induction of diaphragmatic hernia (DH) and additional TO in the fetal rabbit model. Induction of DH was associated with 378 upregulated genes compared to controls when allowing a false-discovery rate (FDR) of 0.1 and a fold change (FC) of 2. Those genes were again downregulated by consecutive TO. But DH+TO was associated with an upregulation of 157 genes compared to DH and controls. When being compared to control lungs, 106 genes were downregulated in the DH group and were not changed by TO. Therefore, the overall pattern of gene expression in DH+TO is more similar to the control group than to the DH group. In this study, we further provide a database of gene expression changes induced by surgical creation of DH and consecutive TO in the rabbit model. Future treatment strategies could be developed using this dataset. We also discuss the most relevant genes that are involved in CDH. Summary: Rabbit fetuses with induced diaphragmatic hernia and treated with prenatal tracheal occlusion have a similar pulmonary transcriptome as unaffected controls. This study describes a valuable database of gene expressions in this model.
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Affiliation(s)
- Alexander C Engels
- Department of Development and Regeneration, Organ System Cluster, Faculty of Medicine, KU Leuven, 3000 Leuven, Belgium Clinical Department of Obstetrics and Gynaecology, Division Woman and Child, University Hospitals KU Leuven, 3000 Leuven, Belgium
| | - Paul D Brady
- Department of Human Genetics, Centre for Human Genetics, University Hospitals KU Leuven, 3000 Leuven, Belgium
| | - Molka Kammoun
- Department of Human Genetics, Centre for Human Genetics, University Hospitals KU Leuven, 3000 Leuven, Belgium
| | - Julio Finalet Ferreiro
- Department of Human Genetics, Centre for Human Genetics, University Hospitals KU Leuven, 3000 Leuven, Belgium
| | - Philip DeKoninck
- Department of Development and Regeneration, Organ System Cluster, Faculty of Medicine, KU Leuven, 3000 Leuven, Belgium Clinical Department of Obstetrics and Gynaecology, Division Woman and Child, University Hospitals KU Leuven, 3000 Leuven, Belgium
| | - Masayuki Endo
- Department of Development and Regeneration, Organ System Cluster, Faculty of Medicine, KU Leuven, 3000 Leuven, Belgium
| | - Jaan Toelen
- Department of Development and Regeneration, Organ System Cluster, Faculty of Medicine, KU Leuven, 3000 Leuven, Belgium Clinical Department of Pediatrics, Division Woman and Child, University Hospitals KU Leuven, 3000 Leuven, Belgium
| | - Joris R Vermeesch
- Department of Human Genetics, Centre for Human Genetics, University Hospitals KU Leuven, 3000 Leuven, Belgium
| | - Jan Deprest
- Department of Development and Regeneration, Organ System Cluster, Faculty of Medicine, KU Leuven, 3000 Leuven, Belgium Clinical Department of Obstetrics and Gynaecology, Division Woman and Child, University Hospitals KU Leuven, 3000 Leuven, Belgium
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24
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Paris ND, Coles GL, Ackerman KG. Wt1 and β-catenin cooperatively regulate diaphragm development in the mouse. Dev Biol 2015; 407:40-56. [PMID: 26278035 DOI: 10.1016/j.ydbio.2015.08.009] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2015] [Revised: 08/11/2015] [Accepted: 08/12/2015] [Indexed: 01/19/2023]
Abstract
The developing diaphragm consists of various differentiating cell types, many of which are not well characterized during organogenesis. One important but incompletely understood tissue, the diaphragmatic mesothelium, is distinctively present from early stages of development. Congenital Diaphragmatic Hernia (CDH) occurs in humans when diaphragm tissue is lost during development, resulting in high morbidity and mortality postnatally. We utilized a Wilms Tumor 1 (Wt1) mutant mouse model to investigate the involvement of the mesothelium in normal diaphragm signaling and development. Additionally, we developed and characterized a Wt1(CreERT2)-driven β-catenin loss-of-function model of CDH after finding that canonical Wnt signaling and β-catenin are reduced in Wt1 mutant mesothelium. Mice with β-catenin loss or constitutive activation induced in the Wt1 lineage are only affected when tamoxifen injection occurs between E10.5 and E11.5, revealing a critical time-frame for Wt1/ β-catenin activity. Conditional β-catenin loss phenocopies the Wt1 mutant diaphragm defect, while constitutive activation of β-catenin on the Wt1 mutant background is sufficient to close the diaphragm defect. Proliferation and apoptosis are affected, but primarily these genetic manipulations appear to lead to a change in normal diaphragm differentiation. Our data suggest a fundamental role for mesothelial signaling in proper formation of the diaphragm.
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Affiliation(s)
- Nicole D Paris
- Department of Biomedical Genetics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
| | - Garry L Coles
- Department of Biomedical Genetics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
| | - Kate G Ackerman
- Department of Biomedical Genetics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA; Department of Pediatrics, Center for Pediatric Biomedical Research, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
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25
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Pereira-Terra P, Moura RS, Nogueira-Silva C, Correia-Pinto J. Neuroendocrine factors regulate retinoic acid receptors in normal and hypoplastic lung development. J Physiol 2015; 593:3301-11. [PMID: 26096456 DOI: 10.1113/jp270477] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2015] [Accepted: 05/29/2015] [Indexed: 01/09/2023] Open
Abstract
KEY POINTS Retinoic acid (RA) and ghrelin levels are altered in human hypoplastic lungs when compared to healthy lungs. Although considerable data have been obtained about RA, ghrelin and bombesin in the congenital diaphragmatic hernia (CDH) rat model, neuroendocrine factors have never been associated with the RA signalling pathway in this animal model. In this study, the interaction between neuroendocrine factors and RA was explored in the CDH rat model. The authors found that normal fetal lung explants treated with RA, bombesin and ghrelin showed an increase in lung growth. Hypoplastic lungs presented higher expression levels of the RA receptors α and γ. Moreover bombesin and ghrelin supplementation, in vitro, to normal lungs increased RA receptor α/γ expression whereas administration of bombesin and ghrelin antagonists to normal and hypoplastic lungs decreased it. These data reveal for the first time that there is a link between neuroendocrine factors and RA, and that neuroendocrine factors sensitise the lung to the RA action through RA receptor modulation. ABSTRACT Congenital diaphragmatic hernia (CDH) is characterised by a spectrum of lung hypoplasia and consequent pulmonary hypertension, leading to high morbidity and mortality rates. Moreover, CDH has been associated with an increase in the levels of pulmonary neuroendocrine factors, such as bombesin and ghrelin, and a decrease in the action of retinoic acid (RA). The present study aimed to elucidate the interaction between neuroendocrine factors and RA. In vitro analyses were performed on Sprague-Dawley rat embryos. Normal lung explants were treated with bombesin, ghrelin, a bombesin antagonist, a ghrelin antagonist, dimethylsulfoxide (DMSO), RA dissolved in DMSO, bombesin plus RA and ghrelin plus RA. Hypoplastic lung explants (nitrofen model) were cultured with bombesin, ghrelin, bombesin antagonist or ghrelin antagonist. The lung explants were analysed morphometrically, and retinoic acid receptor (RAR) α, β and γ expression levels were assessed via Western blotting. Immunohistochemistry analysis of RAR was performed in normal and hypoplastic lungs 17.5 days post-conception (dpc). Compared with the controls, hypoplastic lungs exhibited significantly higher RARα/γ expression levels. Furthermore considering hypoplastic lungs, bombesin and ghrelin antagonists decreased RARα/γ expression. Normal lung explants (13.5 dpc) treated with RA, bombesin plus RA, ghrelin plus RA, bombesin or ghrelin exhibited increased lung growth. Moreover, bombesin and ghrelin increased RARα/γ expression levels, whereas the bombesin and ghrelin antagonists decreased RARα/γ expression. This study demonstrates for the first time that neuroendocrine factors function as lung growth regulators, sensitising the lung to the action of RA through up-regulation of RARα and RARγ.
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Affiliation(s)
- Patrícia Pereira-Terra
- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Braga, Portugal.,Life and Health Sciences Research Institute/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal
| | - Rute S Moura
- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Braga, Portugal.,Life and Health Sciences Research Institute/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal
| | - Cristina Nogueira-Silva
- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Braga, Portugal.,Life and Health Sciences Research Institute/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.,Department of Obstetrics and Gynecology, Hospital de Braga, Braga, Portugal
| | - Jorge Correia-Pinto
- Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Braga, Portugal.,Life and Health Sciences Research Institute/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal.,Department of Pediatric Surgery, Hospital de Braga, Braga, Portugal
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26
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Schilders K, Ochieng JK, van de Ven CP, Gontan C, Tibboel D, Rottier RJ. Role of SOX2 in foregut development in relation to congenital abnormalities. World J Med Genet 2014; 4:94-104. [DOI: 10.5496/wjmg.v4.i4.94] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/28/2014] [Revised: 07/14/2014] [Accepted: 09/10/2014] [Indexed: 02/06/2023] Open
Abstract
The uptake of the two essential ingredients for life, oxygen and nutrients, occurs primarily through the oral cavity, but these two lifelines need to be separated with high accuracy once inside the body. The two systems, the gas exchange pulmonary system and the gastro-intestinal feeding system, are derived from the same primitive embryonic structure during development, the foregut, which need to be separated before birth. In certain newborns, this separation occurs not or insufficiently, leading to life threatening conditions, sometimes incompatible with life. The development of the foregut, trachea and lungs is influenced and coordinated by a multitude of signaling cascades and transcription factors. In this review, we will highlight the development of the foregut and pulmonary system and focus on associated congenital abnormalities in light of known genetic alterations with specific attention to the transcription factor SOX2.
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27
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Kool H, Mous D, Tibboel D, de Klein A, Rottier RJ. Pulmonary vascular development goes awry in congenital lung abnormalities. ACTA ACUST UNITED AC 2014; 102:343-58. [PMID: 25424472 DOI: 10.1002/bdrc.21085] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2014] [Accepted: 10/29/2014] [Indexed: 01/04/2023]
Abstract
Pulmonary vascular diseases of the newborn comprise a wide range of pathological conditions with developmental abnormalities in the pulmonary vasculature. Clinically, pulmonary arterial hypertension (PH) is characterized by persistent increased resistance of the vasculature and abnormal vascular response. The classification of PH is primarily based on clinical parameters instead of morphology and distinguishes five groups of PH. Congenital lung anomalies, such as alveolar capillary dysplasia (ACD) and PH associated with congenital diaphragmatic hernia (CDH), but also bronchopulmonary dysplasia (BPD), are classified in group three. Clearly, tight and correct regulation of pulmonary vascular development is crucial for normal lung development. Human and animal model systems have increased our knowledge and make it possible to identify and characterize affected pathways and study pivotal genes. Understanding of the normal development of the pulmonary vasculature will give new insights in the origin of the spectrum of rare diseases, such as CDH, ACD, and BPD, which render a significant clinical problem in neonatal intensive care units around the world. In this review, we describe normal pulmonary vascular development, and focus on four diseases of the newborn in which abnormal pulmonary vascular development play a critical role in morbidity and mortality. In the future perspective, we indicate the lines of research that seem to be very promising for elucidating the molecular pathways involved in the origin of congenital pulmonary vascular disease.
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Affiliation(s)
- Heleen Kool
- Department of Pediatric Surgery of the Erasmus MC, Sophia Children's Hospital, Rotterdam, the Netherlands
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28
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Friedmacher F, Fujiwara N, Hofmann AD, Takahashi H, Gosemann JH, Puri P. Evidence for decreased lipofibroblast expression in hypoplastic rat lungs with congenital diaphragmatic hernia. Pediatr Surg Int 2014; 30:1023-9. [PMID: 25023943 DOI: 10.1007/s00383-014-3549-z] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 07/03/2014] [Indexed: 11/30/2022]
Abstract
PURPOSE Pulmonary hypoplasia (PH) is a serious condition in newborns with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial fibroblasts (LIFs) play an essential role in fetal lung maturation by stimulating alveolarization and lipid homeostasis. In rodents, LIFs are first evident during the canalicular phase of lung development with a significant increase over the last 4 days of gestation. Adipocyte differentiation-related protein (ADRP), a functional lipogenic molecular marker characterizing LIFs, is highly expressed in fetal lungs during this critical time period. We hypothesized that LIF expression in hypoplastic rat lungs is decreased in the nitrofen-induced CDH model, which is accompanied by reduced alveolar ADRP expression and lipid content. METHODS On embryonic day 9.5 (E9.5), time-mated rats received either nitrofen or vehicle. Fetuses were sacrificed on selected time points E18.5 and E21.5, and dissected lungs were divided into controls and CDH-associated PH. Pulmonary gene expression levels of ADRP were determined by quantitative real-time polymerase chain reaction. ADRP immunohistochemistry and oil red O staining were used to assess pulmonary protein expression and lipid content. Immunofluorescence double staining for alpha smooth muscle actin, which is known to be absent in LIFs, and lipid droplets was performed to evaluate the pulmonary expression of this specific subset of fibroblasts. RESULTS Relative mRNA expression of ADRP was significantly reduced in lungs of CDH-associated PH on E18.5 and E21.5 compared to controls. ADRP immunoreactivity and lipid staining were markedly diminished in alveolar mesenchymal cells of CDH-associated PH on E18.5 and E21.5 compared to controls. Confocal laser scanning microscopy demonstrated markedly decreased LIF expression in alveolar interstitium of CDH-associated PH on E18.5 and E21.5 compared to controls. CONCLUSION Decreased pulmonary LIF expression during late gestation suggests impaired LIF functioning in the nitrofen-induced CDH model, which may cause disruption in fetal alveolarization and lipid homeostasis, and thus contribute to the development of PH.
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Affiliation(s)
- Florian Friedmacher
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland,
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Hofmann AD, Friedmacher F, Hunziker M, Takahashi H, Duess JW, Gosemann JH, Puri P. Upregulation of serotonin-receptor-2a and serotonin transporter expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia. J Pediatr Surg 2014; 49:871-4; discussion 874-5. [PMID: 24888825 DOI: 10.1016/j.jpedsurg.2014.01.015] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/12/2014] [Accepted: 01/27/2014] [Indexed: 01/08/2023]
Abstract
PURPOSE Congenital diaphragmatic hernia (CDH) is attributed to severe pulmonary hypoplasia and pulmonary hypertension (PH). PH is characterized by structural changes resulting in vascular remodeling. Serotonin, a potent vasoconstrictor, plays a central role in the development of PH. It exerts its constricting effects on the vessels via Serotonin receptor 2A (5-HT2A) and induces pulmonary smooth muscle cell proliferation via the serotonin transporter (5-HTT). This study was designed to investigate expressions of 5-HT2A and 5-HTT in the pulmonary vasculature of rats with nitrofen-induced CDH. METHODS Rats were exposed to nitrofen or vehicle on D9. Fetuses were sacrificed on D21 and divided into nitrofen and control group (n=32). Pulmonary RNA was extracted and mRNA level of 5HT2A was determined by qRT-PCR. Protein expression of 5HT2A and 5-HTT was investigated by western blotting. Confocal immunofluorescence double-staining for 5-HT2A, 5-HTT, and alpha smooth muscle actin were performed. RESULTS Pulmonary 5-HT2A gene expression levels were significantly increased in nitrofen-induced CDH compared to controls. Western blotting and confocal microscopy confirmed increased pulmonary protein expression in CDH lungs compared to controls. CONCLUSION Increased gene and protein expression of 5HT2A and 5-HTT in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that 5HT2A and 5-HTT are important mediators of PH in nitrofen-induced CDH.
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MESH Headings
- Animals
- Blotting, Western
- Disease Models, Animal
- Female
- Gene Expression Regulation, Developmental
- Hernias, Diaphragmatic, Congenital/embryology
- Hernias, Diaphragmatic, Congenital/genetics
- Hernias, Diaphragmatic, Congenital/metabolism
- Lung/abnormalities
- Lung/drug effects
- Lung/embryology
- Microscopy, Confocal
- Phenyl Ethers/toxicity
- Pregnancy
- Pregnancy, Animal
- RNA, Messenger/genetics
- Rats
- Rats, Sprague-Dawley
- Receptor, Serotonin, 5-HT2A/biosynthesis
- Receptor, Serotonin, 5-HT2A/genetics
- Reverse Transcriptase Polymerase Chain Reaction
- Serotonin Plasma Membrane Transport Proteins/biosynthesis
- Serotonin Plasma Membrane Transport Proteins/genetics
- Up-Regulation
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Affiliation(s)
- Alejandro D Hofmann
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | - Florian Friedmacher
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | - Manuela Hunziker
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | - Hiromizu Takahashi
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | - Johannes W Duess
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | - Jan-Hendrik Gosemann
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland; Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany
| | - Prem Puri
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland; School of Medicine and Medical Science and Conway Institute of Biomedical Research, University College Dublin, Ireland.
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Friedmacher F, Fujiwara N, Hofmann AD, Takahashi H, Alvarez LAJ, Gosemann JH, Puri P. Prenatal retinoic acid increases lipofibroblast expression in hypoplastic rat lungs with experimental congenital diaphragmatic hernia. J Pediatr Surg 2014; 49:876-81; discussion 881. [PMID: 24888826 DOI: 10.1016/j.jpedsurg.2014.01.017] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/12/2014] [Accepted: 01/27/2014] [Indexed: 10/25/2022]
Abstract
BACKGROUND/PURPOSE Prenatal administration of all-trans retinoic acid (ATRA) has been shown to stimulate alveolarization in nitrofen-induced pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial lipofibroblasts (LIFs), characterized by adipocyte differentiation-related protein (ADRP), play a critical role in alveolar development by coordinating lipid homeostasis. Previous studies have demonstrated that ATRA positively affects LIF expression in developing lungs. We hypothesized that pulmonary LIF expression is increased after prenatal ATRA treatment in the nitrofen model of CDH-associated PH. METHODS Timed-pregnant rats were treated with nitrofen or vehicle on E9.5, followed by injection of ATRA or placebo on E18.5, E19.5, and E20.5. Fetal lungs were dissected on E21.5 and divided into Control+Placebo, Control+ATRA, Nitrofen+Placebo, and Nitrofen+ATRA. Pulmonary gene expression levels of ADRP were analyzed by quantitative real-time polymerase chain reaction, and LIF expression was investigated by ADRP immunohistochemistry, oil-red-O-, and immunofluorescence-double-staining. RESULTS Relative mRNA expression of pulmonary ADRP was significantly increased in Nitrofen+ATRA compared to Nitrofen+Placebo (0.31±0.02 vs. 0.08±0.01; P<0.0001). ADRP immunoreactivity and oil-red-O-staining were markedly increased in alveolar interstitium of Nitrofen+ATRA compared to Nitrofen+Placebo. Immunofluorescence-double-staining confirmed markedly increased LIF expression in alveolar walls of Nitrofen+ATRA compared to Nitrofen+Placebo. CONCLUSIONS Increased LIF expression after prenatal treatment with ATRA in nitrofen-induced PH suggests that ATRA may have a therapeutic potential in attenuating CDH-associated PH by stimulating alveolar development.
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Affiliation(s)
- Florian Friedmacher
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland; Department of Pediatric and Adolescent Surgery, Medical University Graz, Graz, Austria
| | - Naho Fujiwara
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | - Alejandro D Hofmann
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | - Hiromizu Takahashi
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | - Luis A J Alvarez
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | - Jan-Hendrik Gosemann
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | - Prem Puri
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland; Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College Dublin, Dublin, Ireland.
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Friedmacher F, Fujiwara N, Hofmann AD, Takahashi H, Gosemann JH, Puri P. Expression of Eya1 and Six1 is decreased in distal airways of rats with experimental pulmonary hypoplasia. J Pediatr Surg 2014; 49:301-4. [PMID: 24528972 DOI: 10.1016/j.jpedsurg.2013.11.043] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/05/2013] [Accepted: 11/10/2013] [Indexed: 10/26/2022]
Abstract
BACKGROUND/PURPOSE Pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH) represents one of the major challenges in neonatal intensive care. Eyes absent 1 (Eya1) and sine oculis homebox 1 (Six1) have been identified as essential components of the gene network that regulates foetal lung development. Eya1 and Six1 are expressed in distal epithelial tips of branching airways as well as in surrounding mesenchymal cells, highlighting their important role during branching morphogenesis. Lungs of Eya1(-/-) and Six1(-/-) knockouts display PH with reduced epithelial branching, appearing to be arrested in the pseudoglandular stage. We hypothesized that Eya1 and Six1 expression is decreased in branching airways of nitrofen-induced PH. METHODS Time-mated rats received either nitrofen or vehicle on E9.5. Foetal lungs were dissected on E15.5 and divided into control and nitrofen groups, whereas lungs harvested on E18.5 were divided into controls, PH without CDH [PH(-)], and PH with CDH [PH(+)]. Pulmonary gene expression levels of Eya1 and Six1 were analyzed by quantitative real-time PCR. Immunofluorescence staining was performed to investigate Eya1 and Six1 protein expression and localization by confocal laser scanning microscopy (CLSM). RESULTS Relative mRNA expression of Eya1 and Six1 was significantly decreased in PH(-) and PH(+) on E18.5 compared to controls. CLSM confirmed markedly diminished immunofluorescence of Eya1 and Six1 in distal airway epithelium as well as in surrounding mesenchymal cells of nitrofen-induced PH on E18.5 compared to controls. CONCLUSIONS Downregulation of Eya1 and Six1 gene expression in nitrofen-induced PH suggests that decreased Eya1 and Six1 expression during the late pseudoglandular stage may interfere with epithelial branching and distal airway maturation, thus resulting in PH.
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Affiliation(s)
- Florian Friedmacher
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | - Naho Fujiwara
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | | | - Hiromizu Takahashi
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | - Jan-Hendrik Gosemann
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
| | - Prem Puri
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland; Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College Dublin, Dublin, Ireland.
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Disruption of THY-1 signaling in alveolar lipofibroblasts in experimentally induced congenital diaphragmatic hernia. Pediatr Surg Int 2014; 30:129-35. [PMID: 24374733 DOI: 10.1007/s00383-013-3444-z] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
PURPOSE Pulmonary hypoplasia (PH), characterized by alveolar immaturity, remains the main cause of neonatal mortality and long-term morbidity in infants with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial fibroblasts (LIFs) are critically important for normal alveolar development. Thymocyte antigen 1 (Thy-1) is a highly expressed cell-surface protein in this specific subset of lung fibroblasts, which plays a key role in fetal alveolarization by coordinating the differentiation and lipid homeostasis of alveolar LIFs. Thy-1 increases the lipid content of LIFs by upregulation of adipocyte differentiation-related protein (ADRP), a lipogenic molecular marker characterizing pulmonary LIFs. Thy-1 (-/-) mice further show impaired alveolar development with reduced proliferation of pulmonary LIFs, resulting in a PH-similar phenotype. We hypothesized that pulmonary Thy-1 signaling is disrupted in experimentally induced CDH, which may has an adverse effect on the lipid content of alveolar LIFs. METHODS Timed-pregnant Sprague-Dawley rats were treated with either 100 mg nitrofen or vehicle on embryonic day 9.5 (E9.5). Fetuses were killed on E21.5, and lungs were divided into controls (n = 14) and CDH-associated PH (n = 14). Pulmonary gene expression levels of Thy-1 and ADRP were assessed by quantitative real-time PCR. ADRP immunohistochemistry and oil-red-O staining were used to localize alveolar LIF expression and lipid droplets. Immunofluorescence double staining for Thy-1 and oil-red-O was performed to evaluate Thy-1 expression and lipid content in alveolar LIFs. RESULTS Radial alveolar count was significantly reduced in CDH-associated PH with significant downregulation of pulmonary Thy-1 and ADRP mRNA expression compared to controls. ADRP immunoreactivity and lipid droplets were markedly diminished in alveolar interstitial cells, which coincided with decreased alveolar LIF expression in CDH-associated PH compared to controls. Confocal laser scanning microscopy confirmed markedly decreased Thy-1 expression and lipid content in alveolar LIFs of CDH-associated PH compared to controls. CONCLUSION Our study provides strong evidence that disruption of pulmonary Thy-1 signaling results in reduced lipid droplets in alveolar LIFs and may thus contribute to PH in the nitrofen-induced CDH model. Treatment modalities aimed at increasing lipid content in alveolar LIFs may therefore have a therapeutic potential in attenuating CDH-associated PH.
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Hofmann AD, Friedmacher F, Takahashi H, Hunziker M, Gosemann JH, Puri P. Decreased apelin and apelin-receptor expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia. Pediatr Surg Int 2014; 30:197-203. [PMID: 24363088 DOI: 10.1007/s00383-013-3450-1] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
BACKGROUND The high morbidity and mortality in congenital diaphragmatic hernia (CDH) are attributed to severe pulmonary hypoplasia and persistent pulmonary hypertension (PH). PH is characterized by structural changes in pulmonary arteries, resulting in adventitial and medial thickness. These effects are triggered by abnormal apoptosis and proliferation of pulmonary vascular endothelial and smooth muscle cells (SMCs). Apelin (APLN), a target gene of bone morphogenic protein receptor 2 (BMPR2), is known to play an important and manifold role in regulating pulmonary homeostasis promoting endothelial cell (EC) survival, proliferation and migration. In addition to these autocrine effects of apelin, it displays a paracrine function attenuating the response of pulmonary SMCs to growth factors and promoting apoptosis. Apelin exerts its effect via its G-protein-coupled receptor (APLNR) and is solely expressed by pulmonary vascular EC, whereas APLNR is co-localized in pulmonary ECs and SMCs. Dysfunction of BMPR2 and downstream signalling have been shown to disturb the crucial balance of proliferation of SMCs contributing to the pathogenesis of human and experimentally induced PH. We designed this study to investigate the hypothesis that apelin and APLNR signalling are disrupted in the pulmonary vasculature of rats in nitrofen-induced CDH. METHODS Pregnant rats were exposed to nitrofen or vehicle on D9 of gestation. Foetuses were sacrificed on D21 and divided into nitrofen and control group (n = 32). Pulmonary RNA was extracted and mRNA levels of APLN and APLNR were determined by quantitative real-time PCR. Protein expression of apelin and APLNR was investigated by western blotting. Confocal immunofluorescence double staining for apelin, APLNR and SMCs were performed. RESULTS Relative mRNA level of APLN and APLNR were significantly decreased in the CDH group compared to control lungs. Western blotting and confocal microscopy confirmed the qRT-PCR results showing decreased pulmonary protein expression of apelin and APLNR in lungs of nitrofen-exposed foetuses compared to controls. CONCLUSION This study provides striking evidence of markedly decreased gene and protein expression of apelin and its receptor APLNR in the pulmonary vasculature of nitrofen-induced CDH. The disruption of the apelin-APLNR signalling axis in the pulmonary vasculature may lead to extensive vascular remodelling and contribute to PPH in the nitrofen-induced CDH model.
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MESH Headings
- Animals
- Apelin
- Apelin Receptors
- Blotting, Western/methods
- Cell Survival/genetics
- Disease Models, Animal
- Female
- Gene Expression/genetics
- Hernia, Diaphragmatic/chemically induced
- Hernia, Diaphragmatic/genetics
- Hernia, Diaphragmatic/metabolism
- Hernias, Diaphragmatic, Congenital
- Intercellular Signaling Peptides and Proteins/genetics
- Intercellular Signaling Peptides and Proteins/metabolism
- Lung/blood supply
- Lung/metabolism
- Microscopy, Confocal/methods
- Phenyl Ethers
- Pregnancy
- Pulmonary Artery/metabolism
- Pulmonary Veins/metabolism
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- Rats
- Rats, Sprague-Dawley
- Real-Time Polymerase Chain Reaction/methods
- Receptors, G-Protein-Coupled/genetics
- Receptors, G-Protein-Coupled/metabolism
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Affiliation(s)
- Alejandro D Hofmann
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland
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Madurga A, Mizíková I, Ruiz-Camp J, Morty RE. Recent advances in late lung development and the pathogenesis of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 2013; 305:L893-905. [PMID: 24213917 DOI: 10.1152/ajplung.00267.2013] [Citation(s) in RCA: 113] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
In contrast to early lung development, a process exemplified by the branching of the developing airways, the later development of the immature lung remains very poorly understood. A key event in late lung development is secondary septation, in which secondary septa arise from primary septa, creating a greater number of alveoli of a smaller size, which dramatically expands the surface area over which gas exchange can take place. Secondary septation, together with architectural changes to the vascular structure of the lung that minimize the distance between the inspired air and the blood, are the objectives of late lung development. The process of late lung development is disturbed in bronchopulmonary dysplasia (BPD), a disease of prematurely born infants in which the structural development of the alveoli is blunted as a consequence of inflammation, volutrauma, and oxygen toxicity. This review aims to highlight notable recent developments in our understanding of late lung development and the pathogenesis of BPD.
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Affiliation(s)
- Alicia Madurga
- Dept. of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Parkstrasse 1, D-61231 Bad Nauheim, Germany.
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Friedmacher F, Gosemann JH, Fujiwara N, Takahashi H, Hofmann A, Puri P. Expression of Sproutys and SPREDs is decreased during lung branching morphogenesis in nitrofen-induced pulmonary hypoplasia. Pediatr Surg Int 2013; 29:1193-8. [PMID: 23982388 DOI: 10.1007/s00383-013-3385-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
PURPOSE Pulmonary hypoplasia (PH) is a life-threatening condition associated with congenital diaphragmatic hernia (CDH), characterized by defective lung development. Sproutys and Sprouty-related proteins (SPREDs) play a key role in lung branching morphogenesis through modification of epithelial-mesenchymal interactions. During the pseudoglandular stage, Sproutys are highly expressed in distal airway epithelium, while SPREDs within the surrounding mesenchyme. Sprouty2/4 knockouts show severe defects in branching morphogenesis with reduced number of distal airways. SPRED-1 and SPRED-2 are strongly expressed in regions of new airway formation, highlighting their important function in branching pattern. We hypothesized that expression of Sprouty2, Sprouty4, SPRED-1 and SPRED-2 is decreased during lung branching morphogenesis in nitrofen-induced PH. METHODS Timed-pregnant rats received either nitrofen or vehicle on E9.5. On E15.5 (n = 16), fetal lungs were micro-dissected and divided into controls and PH, while on E18.5 (n = 24) groups were: control, PH without CDH [CDH(-)], and PH with CDH [CDH(+)]. Pulmonary gene expression levels of Sprouty2, Sprouty4, SPRED-1 and SPRED-2 were analyzed by qRT-PCR. Immunohistochemistry was performed to evaluate protein expression/distribution. RESULTS On E18.5, relative mRNA expression levels of Sprouty2, Sprouty4, SPRED-1 and SPRED-2 were significantly decreased in CDH(-) and CDH(+) groups compared to controls (P < 0.05). Immunoreactivity of Sprouty2, Sprouty4, SPRED-1 and SPRED-2 was markedly diminished on E18.5 in nitrofen-induced PH. CONCLUSION Decreased expression of Sproutys and SPREDs during the terminal pseudoglandular stage may disrupt lung branching morphogenesis by interfering with epithelial-mesenchymal interactions contributing to PH.
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Affiliation(s)
- Florian Friedmacher
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland
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Friedmacher F, Gosemann JH, Fujiwara N, Alvarez LAJ, Corcionivoschi N, Puri P. Spatiotemporal alterations in Sprouty-2 expression and tyrosine phosphorylation in nitrofen-induced pulmonary hypoplasia. J Pediatr Surg 2013; 48:2219-25. [PMID: 24210189 DOI: 10.1016/j.jpedsurg.2013.07.003] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/09/2013] [Revised: 06/26/2013] [Accepted: 07/01/2013] [Indexed: 11/26/2022]
Abstract
BACKGROUND/PURPOSE Pulmonary hypoplasia (PH) is a life-threatening condition of newborns presenting with congenital diaphragmatic hernia (CDH). Sprouty-2 functions as a key regulator of fibroblast growth factor receptor (FGFR) signalling in developing foetal lungs. It has been reported that FGFR-mediated alveolarization is disrupted in nitrofen-induced PH. Sprouty-2 knockouts show severe defects in lung morphogenesis similar to nitrofen-induced PH. Upon FGFR stimulation, Sprouty-2 is tyrosine-phosphorylated, which is essential for its physiological function during foetal lung development. We hypothesized that Sprouty-2 expression and tyrosine phosphorylation are altered in nitrofen-induced PH. METHODS Time-pregnant rats received either nitrofen or vehicle on gestation day 9 (D9). Foetal lungs were dissected on D18 and D21. Pulmonary Sprouty-2 gene and protein expression levels were analyzed by qRT-PCR, Western blotting and immunohistochemical staining. RESULTS Relative mRNA expression of Sprouty-2 was significantly decreased in hypoplastic lungs without CDH (0.1050±0.01 vs. 0.3125±0.01; P<.0001) and with CDH (0.1671±0.01 vs. 0.3125±0.01; P<.0001) compared to controls on D18. Protein levels of Sprouty-2 were markedly decreased in hypoplastic lungs on D18 with decreased tyrosine phosphorylation levels on D18 and D21 detected at the molecular weight of Sprouty-2 consistent with Sprouty-2 tyrosine phosphorylation. Sprouty-2 immunoreactivity was markedly decreased in hypoplastic lungs on D18 and D21. CONCLUSION Spatiotemporal alterations in pulmonary Sprouty-2 expression and tyrosine phosphorylation during the late stages of foetal lung development may interfere with FGFR-mediated alveolarization in nitrofen-induced PH.
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Affiliation(s)
- Florian Friedmacher
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland
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Arrington CB, Bleyl SB, Brunelli L, Bowles NE. Family-based studies to identify genetic variants that cause congenital heart defects. Future Cardiol 2013; 9:507-18. [PMID: 23834692 DOI: 10.2217/fca.13.40] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Congenital heart defects (CHDs) are the most common congenital abnormalities. Analysis of large multigenerational families has led to the identification of a number of genes for CHDs. However, identifiable variations in these genes are the cause of a small proportion of cases of CHDs, suggesting significant genetic heterogeneity. In addition, large families with CHDs are rare, making the identification of additional genes difficult. Next-generation sequencing technologies will provide an opportunity to identify more genes in the future. However, the significant genetic variation between individuals will present a challenge to distinguish between 'pathogenic' and 'benign' variants. We have demonstrated that the analysis of multiple individuals in small families using combinations of algorithms can reduce the number of candidate variants to a small, manageable number. Thus, the analysis of small nuclear families or even distantly related 'sporadic' cases may begin to uncover the 'dark matter' of CHD genetics.
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Affiliation(s)
- Cammon B Arrington
- Department of Pediatrics (Cardiology) University of Utah School of Medicine, Eccles Institute of Human Genetics, 15 North 2030 East, Room 7110B, Salt Lake City, UT 84112, USA
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Gosemann JH, Friedmacher F, Fujiwara N, Alvarez LAJ, Corcionivoschi N, Puri P. Disruption of the bone morphogenetic protein receptor 2 pathway in nitrofen-induced congenital diaphragmatic hernia. ACTA ACUST UNITED AC 2013; 98:304-9. [PMID: 23780850 DOI: 10.1002/bdrb.21065] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2013] [Accepted: 04/19/2013] [Indexed: 11/07/2022]
Abstract
BACKGROUND/PURPOSE Congenital diaphragmatic hernia (CDH) remains a major therapeutic challenge despite advances in neonatal resuscitation and intensive care. The high mortality and morbidity in CDH has been attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PH). Bone morphogenetic protein receptor 2 (BMPR2) plays a key role in pulmonary vasculogenesis during the late stages of fetal lung development. BMPR2 is essential for control of endothelial and smooth muscle cell proliferation. Dysfunction of BMPR2 and downstream signaling have been shown to disturb the crucial balance of proliferation of smooth muscle cells contributing to the pathogenesis of human and experimental PH. We designed this study to investigate the hypothesis that BMPR2 signaling is disrupted in nitrofen-induced CDH. METHODS Pregnant rats were treated with nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into CDH and control. Quantitative real-time polymerase chain reaction, Western blotting, and confocal-immunofluorescence were performed to determine pulmonary gene expression levels and protein expression of BMPR2 and related proteins. RESULTS Pulmonary Bmpr2 gene expression levels were significantly decreased in nitrofen-induced CDH compared to controls. Western blotting and confocal microscopy revealed decreased pulmonary BMPR2 protein expression and increased activation of p38(MAPK) in CDH compared to controls. CONCLUSION The observed disruption of the BMPR2 signaling pathway may lead to extensive vascular remodeling and contribute to PH in the nitrofen-induced CDH model. BMPR2 may therefore represent a potential target for the treatment of PH in CDH.
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Affiliation(s)
- Jan-Hendrik Gosemann
- National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland; Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany
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Coles GL, Ackerman KG. Kif7 is required for the patterning and differentiation of the diaphragm in a model of syndromic congenital diaphragmatic hernia. Proc Natl Acad Sci U S A 2013; 110:E1898-905. [PMID: 23650387 PMCID: PMC3666741 DOI: 10.1073/pnas.1222797110] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Congenital diaphragmatic hernia (CDH) is a common birth defect that results in a high degree of neonatal morbidity and mortality, but its pathological mechanisms are largely unknown. Therefore, we performed a forward genetic screen in mice to identify unique genes, models, and mechanisms of abnormal diaphragm development. We identified a mutant allele of kinesin family member 7 (Kif7), the disorganized diaphragm (dd). Embryos homozygous for the dd allele possess communicating diaphragmatic hernias, central tendon patterning defects, and increased cell proliferation with diaphragmatic tissue hyperplasia. Because the patterning of the central tendon is undescribed, we analyzed the expression of genes regulating tendonogenesis in dd/dd mutant embryos, and we determined that retinoic acid (RA) signaling was misregulautted. To further investigate the role of Kif7 and RA signaling in the development of the embryonic diaphragm, we established primary mesenchymal cultures of WT embryonic day 13.5 diaphragmatic cells. We determined that RA signaling is necessary for the expression of tendon markers as well as the expression of other CDH-associated genes. Knockdown of Kif7, and retinoic acid receptors alpha (Rara), beta (Rarb), and gamma (Rarg) indicated that RA signaling is dependent on these genes to promote tendonogenesis within the embryonic diaphragm. Taken together, our results provide evidence for a model in which inhibition of RA receptor signaling promotes CDH pathogenesis through a complex gene network.
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Affiliation(s)
| | - Kate G. Ackerman
- Department of Biomedical Genetics and
- Department of Pediatrics, Center for Pediatric Biomedical Research, University of Rochester Medical Center, Rochester, NY 14642
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Friedmacher F, Gosemann JH, Takahashi H, Corcionivoschi N, Puri P. Decreased pulmonary c-Cbl expression and tyrosine phosphorylation in the nitrofen-induced rat model of congenital diaphragmatic hernia. Pediatr Surg Int 2013; 29:19-24. [PMID: 23143077 DOI: 10.1007/s00383-012-3191-6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
PURPOSE The high morbidity of newborn infants with congenital diaphragmatic hernia (CDH) is attributed to pulmonary hypoplasia (PH), which is characterized by a failure of alveolar development. The nitrofen-induced CDH model has been widely used to investigate the pathogenesis of PH in CDH. It has previously been shown that the fibroblast growth factor receptor (FGFR) pathway, which is essential for a proper lung development, is disrupted during late gestation of nitrofen-induced CDH. Casitas B-lineage lymphoma (c-Cbl) proteins are known regulators of signal transduction through FGFRs, indicating their important role during alveolarization in developing lungs. Furthermore, it has been demonstrated that tyrosine phosphorylation of c-Cbl proteins has a pivotal role for their physiological function and activity during fetal lung development. We designed this study to test the hypothesis that pulmonary c-Cbl expression and tyrosine phosphorylation status are decreased in the nitrofen-induced CDH model. METHODS Timed-pregnant rats received either 100 mg nitrofen or vehicle on gestation day 9 (D9). Fetuses were harvested on D18 and D21, and lungs were divided into two groups: control and hypoplastic lungs with CDH (CDH(+)) (n = 10 at each time-point, respectively). Pulmonary gene expression levels of c-Cbl were analyzed by quantitative real-time polymerase chain reaction. Western blotting combined with densitometry analysis was used for semi-quantification of protein levels of pulmonary c-Cbl and tyrosine phosphorylation status. Confocal-immunofluorescence staining was performed to evaluate c-Cbl protein expression and distribution. RESULTS Relative mRNA expression levels of pulmonary c-Cbl were significantly decreased in CDH(+) on D18 and D21 compared to controls. Western blotting showed markedly decreased protein levels of pulmonary c-Cbl and tyrosine phosphorylation status in CDH(+) on D18 and D21. Confocal-immunofluorescence analysis confirmed decreased c-Cbl expression in CDH(+) on D18 and D21 mainly in the distal alveolar epithelium compared to controls. CONCLUSION Decreased pulmonary c-Cbl gene and protein expression accompanied by a decreased tyrosine phosphorylation status during the late stages of fetal lung development may result in reduced c-Cbl activity, and thus interfere with the FGFR-mediated alveolarization in the nitrofen-induced CDH model.
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Affiliation(s)
- Florian Friedmacher
- National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland
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Arrington CB, Bleyl SB, Matsunami N, Bowles NE, Leppert TI, Demarest BL, Osborne K, Yoder BA, Byrne JL, Schiffman JD, Null DM, DiGeronimo R, Rollins M, Faix R, Comstock J, Camp NJ, Leppert MF, Yost HJ, Brunelli L. A family-based paradigm to identify candidate chromosomal regions for isolated congenital diaphragmatic hernia. Am J Med Genet A 2012; 158A:3137-47. [PMID: 23165927 DOI: 10.1002/ajmg.a.35664] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2011] [Accepted: 08/21/2012] [Indexed: 11/09/2022]
Abstract
Congenital diaphragmatic hernia (CDH) is a developmental defect of the diaphragm that causes high newborn mortality. Isolated or non-syndromic CDH is considered a multifactorial disease, with strong evidence implicating genetic factors. As low heritability has been reported in isolated CDH, family-based genetic methods have yet to identify the genetic factors associated with the defect. Using the Utah Population Database, we identified distantly related patients from several extended families with a high incidence of isolated CDH. Using high-density genotyping, seven patients were analyzed by homozygosity exclusion rare allele mapping (HERAM) and phased haplotype sharing (HapShare), two methods we developed to map shared chromosome regions. Our patient cohort shared three regions not previously associated with CDH, that is, 2q11.2-q12.1, 4p13 and 7q11.2, and two regions previously involved in CDH, that is, 8p23.1 and 15q26.2. The latter regions contain GATA4 and NR2F2, two genes implicated in diaphragm formation in mice. Interestingly, three patients shared the 8p23.1 locus and one of them also harbored the 15q26.2 segment. No coding variants were identified in GATA4 or NR2F2, but a rare shared variant was found in intron 1 of GATA4. This work shows the role of heritability in isolated CDH. Our family-based strategy uncovers new chromosomal regions possibly associated with disease, and suggests that non-coding variants of GATA4 and NR2F2 may contribute to the development of isolated CDH. This approach could speed up the discovery of the genes and regulatory elements causing multifactorial diseases, such as isolated CDH.
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Affiliation(s)
- Cammon B Arrington
- Department of Pediatrics (Cardiology), University of Utah School of Medicine, Salt Lake City, Utah 84112, USA
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Sluiter I, van der Horst I, van der Voorn P, Boerema-de Munck A, Buscop-van Kempen M, de Krijger R, Tibboel D, Reiss I, Rottier RJ. Premature differentiation of vascular smooth muscle cells in human congenital diaphragmatic hernia. Exp Mol Pathol 2012; 94:195-202. [PMID: 23018129 DOI: 10.1016/j.yexmp.2012.09.010] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2012] [Accepted: 09/15/2012] [Indexed: 10/27/2022]
Abstract
BACKGROUND Congenital diaphragmatic hernia (CDH) is a rare congenital anomaly characterized by the herniation of abdominal organs into the chest cavity. The high mortality and morbidity of CDH patients are primarily caused by the associated pulmonary hypertension (PH), characterized by the thickening of the vascular media and adventitia. The media consist of heterogeneous populations of vascular smooth muscle cells (VSMC), ranging from synthetic to the characteristic contractile cells. VSMCs are influenced by developmental and environmental cues and may play a role in the development of the structural changes observed in CDH patients. Therefore, we hypothesized that the distribution of the VSMC populations may already be different at the origin of CDH development. METHODOLOGY We analyzed the protein expression of specific markers associated with synthetic and contractile VSMC phenotypes in human lungs at different developmental stages. Next, we compared lungs of premature and term CDH patients, as well as patients with lung hypoplasia due to renal agenesis or PROM, with age-matched controls. RESULTS Synthetic and contractile VSMCs are distributed in a temporal and spatial specific pattern along the proximodistal axis of the lung. CDH patients have more abundant contractile VSMCs which are also more distally distributed. This different distribution pattern is already observed from 19 weeks of gestation onwards. CONCLUSION Our data suggest that the more extensive distribution of contractile VSMCs is associated with an early maturation of the pulmonary vasculature, contrasting the concept that CDH might be the result of delayed maturation of the epithelium.
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Affiliation(s)
- Ilona Sluiter
- Department of Pediatric Surgery, Erasmus MC Rotterdam, The Netherlands.
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van Loenhout RB, Tseu I, Fox EK, Huang Z, Tibboel D, Post M, Keijzer R. The Pulmonary Mesenchymal Tissue Layer Is Defective in an in Vitro Recombinant Model of Nitrofen-Induced Lung Hypoplasia. THE AMERICAN JOURNAL OF PATHOLOGY 2012; 180:48-60. [DOI: 10.1016/j.ajpath.2011.09.032] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/06/2011] [Revised: 08/17/2011] [Accepted: 09/06/2011] [Indexed: 11/25/2022]
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Abstract
During the development of the pulmonary vasculature in the fetus, many structural and functional changes occur to prepare the lung for the transition to air breathing. The development of the pulmonary circulation is genetically controlled by an array of mitogenic factors in a temporo-spatial order. With advancing gestation, pulmonary vessels acquire increased vasoreactivity. The fetal pulmonary vasculature is exposed to a low oxygen tension environment that promotes high intrinsic myogenic tone and high vasocontractility. At birth, a dramatic reduction in pulmonary arterial pressure and resistance occurs with an increase in oxygen tension and blood flow. The striking hemodynamic differences in the pulmonary circulation of the fetus and newborn are regulated by various factors and vasoactive agents. Among them, nitric oxide, endothelin-1, and prostaglandin I2 are mainly derived from endothelial cells and exert their effects via cGMP, cAMP, and Rho kinase signaling pathways. Alterations in these signaling pathways may lead to vascular remodeling, high vasocontractility, and persistent pulmonary hypertension of the newborn.
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Affiliation(s)
- Yuansheng Gao
- Department of Physiology and Pathophysiology, Peking University, Health Science Center, Beijing, China; and Department of Pediatrics, University of Illinois, College of Medicine at Chicago, Chicago, Illinois
| | - J. Usha Raj
- Department of Physiology and Pathophysiology, Peking University, Health Science Center, Beijing, China; and Department of Pediatrics, University of Illinois, College of Medicine at Chicago, Chicago, Illinois
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Beurskens LWJE, Tibboel D, Steegers-Theunissen RÃPM. Role of nutrition, lifestyle factors, and genes in the pathogenesis of congenital diaphragmatic hernia: human and animal studies. Nutr Rev 2009; 67:719-30. [DOI: 10.1111/j.1753-4887.2009.00247.x] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
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Abstract
AIM To review provide an overview of the etiology and current strategies in the management of congenital diaphragmatic hernia (CDH). METHODS We did a comprehensive review of research trends, evidence based studies and epidemiologic studies. RESULTS CDH is a life-threatening pathology in infants, and a major cause of death due to the pulmonary hypoplasia and pulmonary hypertension. There is much research related to elucidating the etiology of CDH and developing management strategies to improve the outcomes in these infants. CONCLUSION An early diagnosis with increased understanding of this disease is a crucial factor for a timely approach to managing the critically ill infant, and to offer the potential for improved outcomes and substantial reductions in morbidity.
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Affiliation(s)
- Alejandra Gaxiola
- Universidad Autonoma de Baja California, Tijuana, Baja California, Mexico
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Abstract
PURPOSE OF REVIEW To evaluate the impact of recent research on the management of congenital diaphragmatic hernia in the light of new theories on embryological development, earlier antenatal diagnosis, fetal and postnatal interventions together with advances in perinatal intensive care. RECENT FINDINGS The year 2007 provided in excess of 200 publications that address various aspects of congenital diaphragmatic hernia. The genetic basis and the causes of pulmonary hypoplasia at the molecular level are slowly being unravelled. Fetal MRI of lung volume, lung-head ratio, liver position and size of diaphragmatic defect have all been evaluated as early predictors of outcome and with a view to prenatal counselling. The impact of fetal interventions such as fetal endoluminal tracheal occlusion, the mode of delivery, the surgical techniques and agents for treating pulmonary hypertension were evaluated. The influence of associated anomalies and therapeutic interventions on the outcome and quality of life of survivors continue to be appraised. SUMMARY Deferred surgery after stabilization with gentle ventilation and reversal of pulmonary hypertension remain the cornerstones of management. Optimal presurgery and postsurgery ventilatory settings remain unproven. Continued improvement in neonatal intensive care raises the bar against which any intervention such as fetal endoluminal tracheal occlusion and extracorporeal membrane oxygenation will be judged.
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Abstract
Congenital diaphragmatic hernia (CDH) is a common major malformation affecting 1/3000-1/4000 births, which continues to be associated with significant perinatal mortality. Much current research is focused on elucidating the genetics and pathophysiology contributing to CDH to develop more effective therapies. The latest data suggest that many cases of CDH are genetically determined and also indicate that CDH is etiologically heterogeneous. The present review will provide a brief summary of diaphragm development and model organism work most relevant to human CDH and will primarily describe important human phenotypes associated with CDH and also provide recommendations for diagnostic evaluation of a fetus or infant with CDH.
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Affiliation(s)
- B R Pober
- Center for Human Genetics, Massachusetts General Hospital, Boston, MA 02114, USA.
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Clugston RD, Zhang W, Greer JJ. Gene expression in the developing diaphragm: significance for congenital diaphragmatic hernia. Am J Physiol Lung Cell Mol Physiol 2008; 294:L665-75. [PMID: 18263670 DOI: 10.1152/ajplung.00027.2008] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023] Open
Abstract
Congenital diaphragmatic hernia (CDH) is a frequently occurring birth defect and a source of potentially fatal neonatal respiratory distress. Recently, through the application of detailed karyotyping methods, several CDH-critical regions within the human genome have been identified. These regions typically contain several genes. Here we focused on genes from 15q26, the best-characterized CDH-critical region, as well as FOG2 and GATA4, genes singled out from CDH-critical regions at 8q22-8q23 and 8p23.1, respectively. We tested the hypothesis that these putative CDH-related genes are expressed within the developing diaphragm at the time of the hypothesized initial defect. Our results show that 15q26 contains a cluster of genes that are expressed in the developing rodent diaphragm, consistent with an association between deletions in this region and CDH. We then examined the protein expression pattern of positively identified genes within the developing diaphragm. Two major themes emerged. First, those factors strongly associated with CDH are expressed only in the nonmuscular, mesenchymal component of the diaphragm, supporting the hypothesis that CDH has its origins in a mesenchymal defect. Second, these factors are all coexpressed in the same cells. This suggests that cases of CDH with unique genetic etiology may lead to a common defect in these cells and supports the hypothesis that these factors may be members of a common pathway. This study is the first to provide a detailed examination of how genes associated with CDH are expressed in the developing diaphragm and provides an important foundation for understanding how the deletion of specific genes may contribute to abnormal diaphragm formation.
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Affiliation(s)
- Robin D Clugston
- University of Alberta, Department of Physiology, Edmonton, Alberta, Canada
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Muehlethaler V, Kunig AM, Seedorf G, Balasubramaniam V, Abman SH. Impaired VEGF and nitric oxide signaling after nitrofen exposure in rat fetal lung explants. Am J Physiol Lung Cell Mol Physiol 2008; 294:L110-20. [DOI: 10.1152/ajplung.00407.2007] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
We hypothesized that abnormal fetal lung growth in experimental congenital diaphragmatic hernia after maternal nitrofen exposure alters lung structure due to impaired VEGF signaling, which can be reversed with VEGF or nitric oxide (NO) treatment. Timed-pregnant Sprague-Dawley rats were treated with nitrofen on embryonic day 9 (E9), and fetal lungs were harvested for explant culture on E15. Explants were maintained in 3% O2for 3 days and were treated with NO gas or recombinant human VEGF protein for 3 days. To determine the effects of VEGF inhibition on lung structure, normal fetal lung explants were treated with SU-5416, a VEGF receptor inhibitor, with or without exogenous NO or VEGF. We found that nitrofen treatment impaired lung structure, as evidenced by decreased branching at day 0, but lung structure was not different from controls after 3 days in culture. Nitrofen reduced lung VEGF but not endothelial NO synthase protein level. Treatment with NO enhanced lung growth in control and nitrofen-exposed lungs; however, the response to NO in the nitrofen-treated lungs was reduced when compared with controls. VEGF treatment did not cause a further increase in lung complexity after nitrofen exposure. SU-5416 treatment altered lung structure, which improved with NO but not VEGF treatment. Both nitrofen and SU-5416 treatment increased apoptosis in the mesenchyme of fetal lung explants. We conclude that nitrofen exposure increased apoptosis, decreased lung growth and reduced VEGF expression, and that exogenous NO but not VEGF treatment enhances lung growth. Disruption of lung architecture after VEGF receptor blockade was similar to nitrofen-induced changes but was more responsive to NO.
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