June 2013
Volume 54, Issue 15
ARVO Annual Meeting Abstract  |   June 2013
Angiogenic profile of hypoxic vascular endothelial cells overexpressing RTEF-1
Author Affiliations & Notes
  • Binoy Appukuttan
    Ophthalmology, Casey Eye Institute - OHSU, Portland, OR
  • Andrew Stempel
    Ophthalmology, Casey Eye Institute - OHSU, Portland, OR
  • Beth Wilmot
    OCTRI, OHSU, Portland, OR
  • Tim Stout
    Ophthalmology, Casey Eye Institute - OHSU, Portland, OR
  • Footnotes
    Commercial Relationships Binoy Appukuttan, None; Andrew Stempel, None; Beth Wilmot, None; Tim Stout, Clayton Foundation (P), Oxford Biomedica (C), AGTC (F), Peregrine Pharmaceuticals Inc (C), Stem Cells Inc (C)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 5570. doi:
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      Binoy Appukuttan, Andrew Stempel, Beth Wilmot, Tim Stout; Angiogenic profile of hypoxic vascular endothelial cells overexpressing RTEF-1. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5570.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: To investigate the angiogenic gene expression profile of hypoxic and normoxic vascular endothelial cells after introduction of RTEF-1. Previously we had identified novel RTEF-1 isoforms within hypoxic human retinal derived vascular endothelial cells. These transcription factors can enhance or inhibit VEGF gene expression. In this study we sought to identify other genes that RTEF-1 may influence expression of within vascular endothelial cells.

Methods: Human vascular endothelial cells were stably transfected with the full length RTEF-1 (kind gift of Jian Li). Cells over expressing RTEF-1 or naive control cells were cultured under normoxic or hypoxic conditions. RNA was isolated with the RNAqueous kit (Ambion), and cDNA was synthesized using the RT2 First Strand Kit (Qiagen). Relative expression of 84 transcripts with established roles in angiogenesis as well as 5 house keeping genes, was determined in triplicate using the human RT2 Profiler PCR Array (Qiagen). Data was obtained using the Chromo4 Thermocycler (Bio-Rad) and data analysis was performed with the RT2 Profiler PCR Array Data Analysis software (Qiagen). Expression of RTEF-1 by these cells was determined to confirm over-expression in the stably transfected line.

Results: Under hypoxic or normoxic conditions, cells overexpressing RTEF-1 significantly increased the expression (7 to 29 fold) of collagen type IV alpha 3 (COL4A3), epiregulin (EREG), and hepatocyte growth factor (HGF). In the hypoxic environment alone, cells overexpressing RTEF-1 induced expression (3 to 10 fold greater than control) of angiopoietin 1 (ANGPT1), vascular endothelial growth factor receptor 1 (FLT1), and laminin alpha-5 (LAMA5), while concurrently reducing expression (16 to 248 fold) of cadherin 5 (CDH5), interleukin 8 (IL8), integrin beta-3 (ITGB3), vascular endothelial growth receptor 2 (KDR), matrix metalloproteinase 2 (MMP2), neuropilin 2 (NRP2), stabilin 1 (STAB1) and transforming growth factor alpha (TGFA).

Conclusions: RTEF-1 influences the expression of various angiogenic promoting genes, other than VEGF-A, while reducing expression of other genes. How these genes and their protein products interact during an angiogenic event and perturb the homeostasis of vascular endothelial cells, will form the basis of future work. RTEF-1 may drive vascular endothelial cells towards a pro-angiogenic pathway while repressing apoptotic and inflammatory initiators.

Keywords: 533 gene/expression • 739 transcription factors • 548 hypoxia  

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