Pregnancy

Establishment of a Functional Ovine Fetoplacental Artery Endothelial Cell Line with a Prolonged Lifespan

Abstract
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 senescence crisis which this primary OFPAE cell line will eventually enter, in this study, we attempted to establish a functional OFPAE cell line with a prolonged lifespan 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. OFPAE cells at passage 8 were transfected. After neomycin selection, survived OFPAE (designated as SV40 OFPAE) cells were expanded up to passage 80 so far. These SV40 OFPAE cells up to passage 30 maintained morphology similar to untransfected OFPAE cells. Expression of T and t antigens in SV40 OFPAE cells was confirmed using 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. These SV40 OFPAE cells at least at passages 20-23 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 as MEK1/2] inhibitor), or 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 lifespan has been successfully established, so potentially providing a valuable cell model for studying fetoplacental endothelial function.

Key words: Growth factors • Kinases • Nitric oxide • Placenta • Signal transduction

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