Downregulation of Forkhead box F1 gene expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.
OBJECTIVE
High mortality and morbidity in infants born with congenital diaphragmatic hernia (CDH) are attributed to pulmonary hypoplasia and pulmonary hypertension (PH). Forkhead box (Fox) transcription factors are known to be crucial for cell proliferation and homeostasis. FoxF1 is essential for lung morphogenesis, vascular development, and endothelial proliferation. Mutations in FoxF1 and also the Fox family member FoxC2 have been identified in neonates with PH. In human and experimental models of arterial PH, the Fox protein FoxO1 was found to be downregulated. We hypothesized that Fox expression is altered in the lungs of the nitrofen-induced CDH rat model and investigated the expression of FoxF1, FoxC2, and FoxO1.


METHODS
Following ethical approval (Rec 913b), time-pregnant Sprague-Dawley rats received nitrofen or vehicle on gestational day (D9). Fetuses were sacrificed on D21, inspected for CDH and divided into CDH (n = 11) and control group (n = 11). Gene expression of FoxF1, FoxC2, and FoxO1 was evaluated with qRT-PCR. Detected alterations of mRNA levels were subsequently assessed on the protein level by performing western blot analysis and laser scanning confocal microscopy.


RESULTS
The relative mRNA level of FoxF1 was significantly downregulated in CDH lungs compared to controls (FoxF1 CDH 1.047 ± 0.108, FoxF1 Ctrl 1.419 ± 0.01, p = 0.014). Relative mRNA levels of FoxC2 and FoxO1 were not found to be altered between the experimental groups (FoxC2 CDH 30.74 ± 8.925, FoxC2 Ctrl 27.408 ± 7.487, p = 0.776; FoxO1 CDH 0.011 ± 0.002, FoxO1 Ctrl 0.011 ± 0.001, p = 0.809). On the protein level, western blotting demonstrated a reduced pulmonary protein expression of FoxF1 in CDH lungs. Confocal microscopy showed a markedly diminished expression of FoxF1 in the pulmonary vasculature of CDH lungs compared to controls.


CONCLUSIONS
Our study demonstrates a strikingly reduced expression of FoxF1 in the pulmonary vasculature of nitrofen-induced CDH. Altered FoxF1 gene expression during embryogenesis may participate in vascular maldevelopment resulting in PH in this animal model.
National Children's Research Centre, Our Lady's Children's Hospital, Gate 5, Crumlin, Dublin, Ireland.
J
J
Zimmer
National Children's Research Centre, Our Lady's Children's Hospital, Gate 5, Crumlin, Dublin, Ireland.
T
T
Takahashi
National Children's Research Centre, Our Lady's Children's Hospital, Gate 5, Crumlin, Dublin, Ireland.|Department of Pediatric Surgery, Hannover Medical School, Hannover, Germany.
A D
AD
Hofmann
National Children's Research Centre, Our Lady's Children's Hospital, Gate 5, Crumlin, Dublin, Ireland. prem.puri@ncrc.ie.|School of Medicine and Medical Science and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland. prem.puri@ncrc.ie.
Prem
P
Puri
D000818:Animals; D015153:Blotting, Western; D004195:Disease Models, Animal; D015536:Down-Regulation / Q000235:genetics; D005260:Female; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D065630:Hernias, Diaphragmatic, Congenital / Q000235:genetics*; D008168:Lung / Q000098:blood supply*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D010647:Phenyl Ethers; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D000071161:Forkhead Box Protein O1
D009419:Nerve Tissue Proteins; D010647:Phenyl Ethers; C074639:Foxo1 protein, rat; D000071161:Forkhead Box Protein O1; C007350:nitrofen
Congenital diaphragmatic hernia; Fox transcription factors; FoxF1; Nitrofen; Pulmonary hypertension
Am J Med Genet A. 2010 May;152A(5):1257-62
20425831
Physiol Rev. 2007 Jan;87(1):219-44
17237346
Development. 2001 Jan;128(2):155-66
11124112
Circ Res. 2014 Sep 26;115(8):709-20
25091710
Nat Rev Genet. 2009 Apr;10(4):233-40
19274050
Hum Genet. 2016 May;135(5):569-86
27071622
Birth Defects Res C Embryo Today. 2014 Dec;102(4):343-58
25424472
J Pediatr Surg. 2010 Oct;45(10 ):1989-94
20920717
Cardiol Young. 2013 Oct;23(5):697-704
23331759
J Pediatr Surg. 2004 Jun;39(6):825-8; discussion 825-8
15185205
Pediatr Surg Int. 2016 Feb;32(2):147-54
26543024
PLoS One. 2012;7(4):e35364
22539968
Nat Med. 2014 Nov;20(11):1289-300
25344740
Pediatr Surg Int. 2016 Oct;32(10 ):967-73
27480986
J Clin Invest. 2012 Dec;122(12):4306-13
23202738
J Pediatr Surg. 2010 Jul;45(7):1445-54
20638522
Dev Biol. 2001 Jul 15;235(2):489-506
11437453
Am J Physiol Lung Cell Mol Physiol. 2004 Mar;286(3):L521-30
14607778
J Biol Chem. 2009 Feb 27;284(9):5936-44
19049965
Development. 2001 Jun;128(12):2397-406
11493558
Development. 2007 Oct;134(20):3753-61
17881493
Early Hum Dev. 2014 Dec;90(12):941-6
25448786
Hum Genomics. 2010 Jun;4(5):345-52
20650821
Braz J Med Biol Res. 2014 Feb;47(2):171-8
24519134
Am J Pathol. 2003 Feb;162(2):547-55
12547712
Front Surg. 2014 Nov 14;1:44
25593968
Am J Physiol Lung Cell Mol Physiol. 2002 Jul;283(1):L60-6
12060561
Pediatr Surg Int. 2013 Aug;29(8):823-34
23832098
Am J Hum Genet. 2009 Jun;84(6):780-91
19500772
Pediatr Surg Int. 2015 Nov;31(11):1005-13
26403792
J Pediatr Surg. 2016 Jun;51(6):896-9
26996590
Arch Dis Child Fetal Neonatal Ed. 2015 Mar;100(2):F137-44
25411443