Upregulated EFNB2 and EPHB4 promotes lung development in a nitrofen-induced congenital diaphragmatic hernia rat model

Liu,Hao; Li,Xue; Yu,Wen  Qian; Liu,Cai  Xia

doi:10.3892/ijmm.2018.3824

Upregulated EFNB2 and EPHB4 promotes lung development in a nitrofen-induced congenital diaphragmatic hernia rat model

Authors:
- Hao Liu
- Xue Li
- Wen Qian Yu
- Cai Xia Liu
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Affiliations: Department of Gynecology and Obstetrics, Shengjing Hospital Affiliated to China Medical University, Shenyang, Liaoning 110004, P.R. China
Published online on: August 14, 2018 https://doi.org/10.3892/ijmm.2018.3824
Pages: 2373-2382
Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Congenital diaphragmatic hernia (CDH) is a common congenital malformation associated with high mortality rates, mainly due to pulmonary hypoplasia and persistent pulmonary hypertension following birth. The present study aimed to investigate abnormal lung development in a rat CDH model, and examine temporal and spatial changes in the expression of ephrin type‑B receptor 4 (EPHB4) and ephrin‑B2 (EFNB2) during fetal lung development, to elucidate the role of these factors during lung morphogenesis. Pregnant rats received nitrofen on embryonic day (E) 8.5 to induce CDH, and fetal lungs were collected on E13.5, E15.5, E17.5, E19.5, and E21.5. The mean linear intercept (MLI) and mean alveolar number (MAN) were observed in fetal lung tissue at E21.5 following hematoxylin and eosin staining. E13.5 fetal lungs were cultured for 96 h in serum‑free medium and branch development was observed under a microscope. The gene and protein expression levels of EPHB4 and EFNB2 were assessed by reverse transcription‑quantitative polymerase chain reaction analysis, and immunoblotting and immunohistochemistry, respectively. The fetal rat lungs were treated with EFNB2 and the activity of key signaling pathways was assessed. The lung index (lung weight/body weight) at E21.5 was significantly lower in the CDH rats, compared with that in the control fetal rats. The MLI and MAN were also lower in the CDH group. The number of lung terminal buds at E13.5 (embryonic stage), and the lung‑explant perimeter and surface were all smaller in the CDH group rats than in the control group at the same age. Pulmonary hypoplasia was observed following 96 h of in vitro culture. No significant differences were found in the expression levels of EFNB2 and EPHB4 between the CDH and control groups at E13.5 (embryonic stage) or E15.5 (pseudoglandular stage), however, EFNB2 and EPHB4 were significantly upregulated at E17.5 (canalicular stage), and at E19.5 and E21.5 (saccular/alveolar stages). EFNB2 stimulated pulmonary branching and EFNB2 supplementation decreased the activity of p38, c‑Jun NH2‑terminal kinase, extracellular signal‑regulated kinase, and signal transducer and activator of transcription. The CDH fetal rats developed pulmonary dysplasia at an early stage of fetal pulmonary development. Upregulated expression of EFNB2 and EPHB4 was observed in the rat lung of nitrofen‑induced CDH, and the increased expression of EFNB2 promoted rat lung development in the nitrofen‑induced CDH model.

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1	Torfs CP, Curry CJ, Bateson TF and Honoré LH: A population-based study of congenital diaphragmatic hernia. Teratology. 46:555–565. 1992. View Article : Google Scholar : PubMed/NCBI
2	Skari H, Bjornland K, Haugen G, Egeland T and Emblem R: Congenital diaphragmatic hernia: A meta-analysis of mortality factors. J Pediatr Surg. 35:1187–1197. 2000. View Article : Google Scholar : PubMed/NCBI
3	Pober BR, Russell MK and Ackerman KG: Congenital Diaphragmatic Hernia Overview.
4	van Loenhout RB, Tibboel D, Post M and Keijzer R: Congenital diaphragmatic hernia: Comparison of animal models and relevance to the human situation. Neonatology. 96:137–149. 2009. View Article : Google Scholar : PubMed/NCBI
5	Pober BR: Overview of epidemiology, genetics, birth defects, and chromosome abnormalities associated with CDH. Am J Med Genet C Semin Med Genet. 145C:158–171. 2007. View Article : Google Scholar : PubMed/NCBI
6	Peetsold MG, Heij HA, Kneepkens CM, Nagelkerke AF, Huisman J and Gemke RJ: The long-term follow-up of patients with a congenital diaphragmatic hernia: A broad spectrum of morbidity. Pediatr Surg Int. 25:1–17. 2009. View Article : Google Scholar
7	Muratore CS, Kharasch V, Lund DP, Sheils C, Friedman S, Brown C, Utter S, Jaksic T and Wilson JM: Pulmonary morbidity in 100 survivors of congenital diaphragmatic hernia monitored in a multidisciplinary clinic. J Pediatr Surg. 36:133–140. 2001. View Article : Google Scholar
8	Badillo A and Gingalewski C: Congenital diaphragmatic hernia: Treatment and outcomes. Semin Perinatol. 38:92–96. 2014. View Article : Google Scholar : PubMed/NCBI
9	Bargy F, Beaudoin S and Barbet P: Fetal lung growth in congenital diaphragmatic hernia. Fetal Diagn Ther. 21:39–44. 2006. View Article : Google Scholar
10	Acker SN, Mandell EW, Sims-Lucas S, Gien J, Abman SH and Galambos C: Histologic identification of prominent intrapul-monary anastomotic vessels in severe congenital diaphragmatic hernia. J Pediatr. 166:178–183. 2015. View Article : Google Scholar
11	Sluiter I, Reiss I, Kraemer U, Krijger Rd, Tibboel D and Rottier RJ: Vascular abnormalities in human newborns with pulmonary hypertension. Expert Rev Respir Med. 5:245–256. 2011. View Article : Google Scholar : PubMed/NCBI
12	Guilbert TW, Gebb SA and Shannon JM: Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development. Am J Physiol Lung Cell Mol Physiol. 279:L1159–L1171. 2000. View Article : Google Scholar : PubMed/NCBI
13	Hirai H, Maru Y, Hagiwara K, Nishida J and Takaku F: A novel putative tyrosine kinase receptor encoded by the eph gene. Science. 238:1717–1720. 1987. View Article : Google Scholar : PubMed/NCBI
14	Pasquale EB: Eph-ephrin bidirectional signaling in physiology and disease. Cell. 133:38–52. 2008. View Article : Google Scholar : PubMed/NCBI
15	Klein R: Eph/ephrin signalling during development. Development. 139:4105–4109. 2012. View Article : Google Scholar : PubMed/NCBI
16	Kania A and Klein R: Mechanisms of ephrin-eph signalling in development, physiology and disease. Nat Rev Mol Cell Biol. 17:240–256. 2016. View Article : Google Scholar : PubMed/NCBI
17	Lisabeth EM, Falivelli G and Pasquale EB: Eph receptor signaling and ephrins. Cold Spring Harb Perspect Biol. 5:a0091592013. View Article : Google Scholar : PubMed/NCBI
18	Cheng N, Brantley DM and Chen J: The ephrins and eph receptors in angiogenesis. Cytokine Growth Factor Rev. 13:75–85. 2002. View Article : Google Scholar
19	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔ^C^T method. Methods. 25:402–408. 2001. View Article : Google Scholar
20	Downard CD, Jaksic T, Garza JJ, Dzakovic A, Nemes L, Jennings RW and Wilson JM: Analysis of an improved survival rate for congenital diaphragmatic hernia. J Pediatr Surg. 38:729–732. 2003. View Article : Google Scholar : PubMed/NCBI
21	Kinane TB: Lung development and implications for hypoplasia found in congenital diaphragmatic hernia. Am J Med Genet C Semin Med Genet. 145C:117–124. 2007. View Article : Google Scholar : PubMed/NCBI
22	Warburton D, Schwarz M, Tefft D, Flores-Delgado G, Anderson KD and Cardoso WV: The molecular basis of lung morphogenesis. Mech Dev. 92:55–81. 2000. View Article : Google Scholar : PubMed/NCBI
23	Cardoso WV: Molecular regulation of lung development. Annu Rev Physiol. 63:471–494. 2001. View Article : Google Scholar : PubMed/NCBI
24	Groenman F, Unger S and Post M: The molecular basis for abnormal Human lung development. Biol Neonate. 87:164–177. 2005. View Article : Google Scholar
25	Cardoso WV and Lü J: Regulation of early lung morphogenesis: Questions, facts and controversies. Development. 133:1611–1124. 2006. View Article : Google Scholar : PubMed/NCBI
26	Ware LB and Matthay MA: Keratinocyte and hepatocyte growth factors in the lung: Roles in lung development inflammation and repair. Am J Physiol Lung Cell Mol Physiol. 282:L924–L940. 2002. View Article : Google Scholar : PubMed/NCBI
27	Wang Y, Nakayama M, Pitulescu ME, Schmidt TS, Bochenek ML, Sakakibara A, Adams S, Davy A, Deutsch U, Lüthi U, et al: Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature. 465:483–486. 2010. View Article : Google Scholar : PubMed/NCBI
28	Vadivel A, van Haaften T, Alphonse RS, Rey-Parra GJ, Ionescu L, Haromy A, Eaton F, Michelakis E and Thébaud B: Critical role of the axonal guidance cue EphrinB2 in lung growth, angiogenesis, and repair. Am J Respir Crit Care Med. 185:564–574. 2012. View Article : Google Scholar
29	Bennett KM, Afanador MD, Lal CV, Xu H, Persad E, Legan SK, Chenaux G, Dellinger M, Savani RC, Dravis C, et al: Ephrin-B2 reverse signaling increases α5β1 integrin mediated fibronectin deposition and reduces distal lung compliance. Am J Respir Cell Mol Biol. 49:680–687. 2013. View Article : Google Scholar : PubMed/NCBI
30	Sikkema AH, den Dunnen WF, Hulleman E, van Vuurden DG, Garcia-Manero G, Yang H, Scherpen FJ, Kampen KR, Hoving EW, Kamps EW, et al: EphB2 activity plays a pivotal role in pediatric medulloblastoma cell adhesion and invasion. Neuro Oncol. 14:1125–1135. 2012. View Article : Google Scholar : PubMed/NCBI
31	Nogueira-Silva C, Piairo P, Carvalho-Dias E, Veiga C, Moura RS and Correia-Pinto J: The role of glycoprotein 130 family of cytokines in fetal rat lung development. PLoS One. 8:e676072013. View Article : Google Scholar : PubMed/NCBI
32	Nogueira-Silva C, Piairo P, Carvalho-Dias E, Peixoto FO, Moura RS and Correia-Pinto J: Leukemia inhibitory factor in rat fetal lung development: Expression and functional studies. PLoS One. 7:e305172012. View Article : Google Scholar : PubMed/NCBI
33	Kling DE, Lorenzo HK, Trbovich AM, Kinane TB, Donahoe PK and Schnitzer JJ: MEK-1/2 inhibition reduces branching morphogenesis and causes mesenchymal cell apoptosis in fetal rat lungs. Am J Physiol Lung Cell Mol Physiol. 282:L370–L378. 2002. View Article : Google Scholar : PubMed/NCBI
34	Piairo P, Moura RS, Nogueira-Silva C and Correia-Pinto J: The apelinergic system in the developing lung: Expression and signaling. Peptides. 32:2474–2483. 2011. View Article : Google Scholar : PubMed/NCBI
35	Boucherat O, Nadeau V, Bérubé-Simard FA, Charron J and Jeannotte L: Crucial requirement of ERK/MAPK signaling in respiratory tract development. Development. 21:3197–3211. 2014. View Article : Google Scholar
36	Boucherat O, Landry-Truchon K, Aoidi R, Houde N, Nadeau V, Charron J and Jeannotte L: Lung development requires an active ERK/MAPK pathway in the lung mesenchyme. Dev Dyn. 246:72–82. 2017. View Article : Google Scholar