| Abstract: |
Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1+ ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1+ ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1+ ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1+ ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1+ ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-κB pathways. Therefore, E4ORF1+ ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis. © 2008 by The National Academy of Sciences of the USA. |
| Keywords: |
signal transduction; mitogen activated protein kinase; controlled study; protein phosphorylation; unclassified drug; human cell; cell proliferation; animals; mice; bone marrow cells; cell survival; cells, cultured; serine; enzyme activation; mice, scid; angiogenesis; phosphorylation; endothelium cell; endothelial cells; fibroblast growth factor 2; umbilical veins; proto-oncogene proteins c-akt; vasculotropin a; murinae; neoplasm transplantation; tumor; mice, inbred nod; endothelium; neovascularization, physiologic; embryonal carcinoma; cell expansion; adenoviridae; lentivirinae; virus protein; adenovirus; stem; sprouting; protein e4orf1; virus induction; adenovirus e4 proteins; carcinoma, embryonal; culture media, serum-free; hl-60 cells; receptors, fibroblast growth factor
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