Intrauterine pulmonary hypertension impairs angiogenesis in vitro: role of vascular endothelial growth factor nitric oxide signaling.
BACKGROUND
Mechanisms that impair angiogenesis in neonatal persistent pulmonary hypertension (PPHN) are poorly understood.


OBJECTIVE
To determine if PPHN alters fetal pulmonary artery endothelial cell (PAEC) phenotype and impairs growth and angiogenesis in vitro, and if altered vascular endothelial growth factor-nitric oxide (VEGF-NO) signaling contributes to this abnormal phenotype.


METHODS
Proximal PAECs were harvested from fetal sheep that had undergone partial ligation of the ductus arteriosus in utero (PPHN) and age-matched control animals. Growth and tube formation +/- VEGF and NO stimulation and inhibition were studied in normal and PPHN PAECs. Western blot analysis was performed for VEGF, VEGF receptor-2 (VEGF-R2), and endothelial NO synthase (eNOS) protein content. NO production with VEGF administration was measured in normal and PPHN PAECs.


RESULTS
PPHN PAECs demonstrate decreased growth and tube formation in vitro. VEGF and eNOS protein expression were decreased in PPHN PAECs, whereas VEGF-R2 protein expression was not different. VEGF and NO increased PPHN PAEC growth and tube formation to values achieved in normal PAECs. VEGF inhibition decreased growth and tube formation in normal and PPHN PAECs. NOS inhibition decreased growth in normal and PPHN PAECs, but tube formation was only reduced in normal PAECs. NO reversed the inhibitory effects of VEGF-R2 inhibition on tube formation in normal and PPHN PAECs. VEGF increased NO production in normal and PPHN PAECs.


CONCLUSIONS
PPHN in utero causes sustained impairment of PAEC phenotype in vitro, with reduced PAEC growth and tube formation and down-regulation of VEGF and eNOS protein. VEGF and NO enhanced growth and tube formation of PPHN PAECs.
Gien
Jason
J
Section of Neonatology, Pediatric Heart Lung Center, Department of Pediatrics, University of Colorado School of Medicine, Denver, Colorado, USA. jason.gien@uchsc.edu
Seedorf
Gregory J
GJ
Balasubramaniam
Vivek
V
Markham
Neil
N
Abman
Steven H
SH
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000473:pathology; D004730:Endothelium, Vascular / Q000196:embryology / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047109:Fetal Development; D005315:Fetal Diseases / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D006976:Hypertension, Pulmonary / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D018919:Neovascularization, Physiologic / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism* / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D019335:Nitroarginine / Q000494:pharmacology; D010641:Phenotype; D011651:Pulmonary Artery / Q000196:embryology* / Q000473:pathology; D012756:Sheep; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism* / Q000494:pharmacology