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Establishment of a Functional Ovine Fetoplacental Artery Endothelial Cell Line with a Prolonged Life Span¹

Department of Obstetrics and Gynecology, Perinatal Research Laboratories, University of Wisconsin, Madison, Wisconsin 53715

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 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.

cell proliferation, growth factors, kinases, mitogen-activated protein kinase 3/1, nitric oxide, placenta, placental endothelial cells, signal transduction, simian virus 40, v-akt murine thymoma viral oncogen homolog 1

¹Supported in part by the National Institutes of Health grants HL64703 and HD38843S2 to J.Z.

Correspondence: ² Jing Zheng, Department of Obstetrics and Gynecology, Perinatal Research Laboratories, University of Wisconsin, PAB1, Meriter Hospital, 202 South Park St., Madison, WI 53715. FAX: 608 257 1304; e-mail: jzheng{at}wisc.edu

This article has been cited by other articles:

				K. Wang, Y. Song, D.-B. Chen, and J. Zheng Protein Phosphatase 3 Differentially Modulates Vascular Endothelial Growth Factor- and Fibroblast Growth Factor 2-Stimulated Cell Proliferation and Signaling in Ovine Fetoplacental Artery Endothelial Cells Biol Reprod, October 1, 2008; 79(4): 704 - 710. [Abstract] [Full Text] [PDF]