Establishment of a functional ovine fetoplacental artery endothelial cell line with a prolonged life span.
To study mechanisms governing fetoplacental vascular function, we have established a primary ovine fetoplacental artery endothelial (OFPAE) cell line. These OFPAE cells produce nitric oxide (NO), proliferate, and migrate in response to fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF). To overcome the senescence crisis that this primary OFPAE cell line will eventually enter, we attempted to establish a functional OFPAE cell line with a prolonged life span by transfecting cells with plasmids containing a neomycin resistance gene and a simian virus 40 gene (SV40) expressing large T (T) and small t (t) antigens. The OFPAE cells at passage 8 were transfected. After neomycin selection, the surviving OFPAE (designated SV40 OFPAE) cells were expanded up to passage 80. Up to passage 30, these SV40 OFPAE cells maintained a morphology similar to untransfected OFPAE cells. Expression of T and t antigens in SV40 OFPAE cells was confirmed by immunocytochemistry. These SV40 OFPAE cells exhibited positive uptake of acetylated low-density lipoprotein (Ac-LDL) and positive staining for NO synthase 3 (NOS3) and formed capillary-like tube structures on Matrigel. Up to passages 20-23, these SV40 OFPAE cells proliferated (P < 0.05) and produced (P < 0.05) NO in response to both FGF2 and VEGF. Moreover, this cell proliferation stimulated by FGF2 and VEGF was dose-dependently inhibited (P < 0.05) by PD98059 (a selective mitogen-activated protein kinase 1 and 2 [MAP2K1/2, also termed MEK1/2] inhibitor) or by LY294002 (a selective phosphoinositide 3-kinase [PI3K] inhibitor). These data indicate that SV40 OFPAE cells, at least at passage 23, retain endothelial phenotypes and functions similar to their parental, untransfected OFPAE cells. Thus, a functional OFPAE cell line with an extended life span has been successfully established, potentially providing a valuable cell model for studying fetoplacental endothelial function.
Song
Yang
Y
Department of Obstetrics and Gynecology, Perinatal Research Laboratories, University of Wisconsin, Madison, Wisconsin 53715, USA.
Zheng
Jing
J
D000818:Animals; D001158:Arteries / Q000166:cytology* / Q000201:enzymology; D002461:Cell Line, Transformed / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010920:Placenta / Q000098:blood supply*; D010957:Plasmids / Q000235:genetics; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D034561:Sheep, Domestic; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
D016222:Fibroblast Growth Factor 2; D020929:Mitogen-Activated Protein Kinase Kinases; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D042461:Vascular Endothelial Growth Factor A