Co-expression of vascular endothelial growth factor and neuropilin-1 in ovine feto-placental artery endothelial cells.
Vascular endothelial growth factor (VEGF) is a key regulator for placental angiogenesis and vascular functions via activating two high affinity tyrosine-kinase receptors, VEGF receptor-1 (VEGFR-1) and -2 (VEGFR-2). Recently, a specific VEGF165 receptor, neuropilin-1 (NP-1), was also identified in endothelial cells and upon VEGF binding, NP-1, synergistically with VEGFR-2, enhances VEGF-induced cell proliferation and migration. To evaluate the role of VEGF and NP-1 in regulating fetoplacental angiogenesis and endothelial function, an ovine fetal placental artery endothelial (OFPAE) cell line was established. In this study, an OFPAE cell cDNA library was constructed. Two positive clones for VEGF and one for NP-1 were isolated from the OFPAE cell cDNA library, and their partial 3' sequences were identified. The sequence of VEGF cDNA insert had 98% homology to the reported ovine VEGF (GenBank accesssion # X89506). The partial NP-1 cDNA sequence included a portion of the protein coding region and a complete 3' untranslated region (UTR), and had 90% homology to human NP-1 (GenBank accession # AF016050). The predicted amino acid sequence of ovine NP-1 was 97-98% identical to human (GenBank accession # AAC12921.1), mouse (GenBank accession # NP_032763), and rat (GenBank accession # AAC53345.1) NP-1. Two CU-rich stabilizing and two consensus destabilizing elements 5'-AUUUA-3' were identified in the 3' UTR of ovine NP-1 cDNA sequence. These elements are the potential binding sites for mRNA-binding proteins which may regulate the stability of NP-1 mRNA. Expression of VEGF and NP-1 in OFPAE cells and fetal placentas was confirmed by Northern and Western blot analyses. Using PCR analysis, we also identified partial sequences of multiple VEGF isoforms (VEGF188, 183, 164, and 120) as well as VEGFR-1, VEGFR-2, and neuropilin-2 (NP-2) from the OFPAE cell cDNA library. These results indicate that multiple isoforms of VEGF are expressed in OFPAE cells. Moreover, we also identified, for the first time, a complete 3' UTR of NP-1 cDNA in any species. Together with expression of VEGF and VEGF receptors in OFPAE cells, we propose that there is an autocrine mechanism by which VEGF regulates fetal placental angiogenesis and other functions of endothelial cells.
Tsoi
Stephen C M
SC
Department of Obstetrics and Gynecology, University of Wisconsin-Madison, 7E Meriter Hospital, 202 S Park St, Madison, WI 53715, USA.
Wen
YunXia
Y
Chung
Jin Young
JY
Chen
DongBao
D
Magness
Ronald R
RR
Zheng
Jing
J
D000818:Animals; D001158:Arteries / Q000166:cytology; D019898:Autocrine Communication; D001483:Base Sequence; D016228:Endothelial Growth Factors / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology*; D005091:Exons; D005260:Female; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008222:Lymphokines / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D039942:Neuropilin-1 / Q000235:genetics / Q000378:metabolism*; D010920:Placenta / Q000098:blood supply; D021041:Placental Circulation; D011247:Pregnancy; D020033:Protein Isoforms; D016415:Sequence Alignment; D017421:Sequence Analysis; D012756:Sheep; D020506:Untranslated Regions; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D016228:Endothelial Growth Factors; D036341:Intercellular Signaling Peptides and Proteins; D008222:Lymphokines; D020033:Protein Isoforms; D020506:Untranslated Regions; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D039942:Neuropilin-1