April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
siRNA-based Suppression Of VEGFC Expression In a Human RPE Cell Line Does Not Elicit Protection From Exposure To a 2- µM Laser
Author Affiliations & Notes
  • Larry Estlack
    Conceptual Mindworks Inc, Brooks-City Base, Texas
  • Kurt J. Schuster
    TASC, Inc, Brooks-City Base, Texas
  • Katharine E. Sheldon
    711HPW/RHDO, AFRL, Brooks-City Base, Texas
  • Brent J. Lavey
    711HPW/RHDO, AFRL, Brooks-City Base, Texas
  • Robert J. Thomas
    711HPW/RHDO, AFRL, Brooks-City Base, Texas
  • Footnotes
    Commercial Relationships  Larry Estlack, None; Kurt J. Schuster, None; Katharine E. Sheldon, None; Brent J. Lavey, None; Robert J. Thomas, None
  • Footnotes
    Support  AFRL - Human Effectiveness Directorate
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 1390. doi:
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      Larry Estlack, Kurt J. Schuster, Katharine E. Sheldon, Brent J. Lavey, Robert J. Thomas; siRNA-based Suppression Of VEGFC Expression In a Human RPE Cell Line Does Not Elicit Protection From Exposure To a 2- µM Laser. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1390.

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

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Purpose: : To study the effect of RNA interference (RNAi) on the expression of VEGFC in RPE cells in association with exposure to a 2-µm laser.

Methods: : Introduction of synthetic short interfering RNA (siRNA) can induce RNA interference in mammalian cells. A human retinal pigment epithelial cell line (hTERT-RPE1) was transfected with RNAi target selection plasmids for VEGFC. The cell line was characterized to determine the relative abundance of VEGFC protein and mRNA levels. A preconditioning heating regime was used to evaluate VEGFC induction due to thermal stress. The RPE VEGFC knockout cells were exposed to 2-µm laser radiation at different irradiances to determine the threshold damage irradiance (ED50). The ED50 values for the RPE knockout cells were then compared to ED50 values of non-transfected control cells.

Results: : siRNAs are 21-25 bp dsRNA with 3’ overhangs that are processed from longer dsRNA by DICER in the RNA interference pathway. Vector-based siRNA approach was used to produce stable gene silencing in RPE cells. The VEGFC knockout cell lines showed a greater than 90% reduction in protein and mRNA expression. However, there was no significant increase in threshold damage irradiance (ED50) in the VEGFC knockout cells (22 W/cm2) versus control (25 W/cm2).

Conclusions: : Using the mechanism of RNAi to silence the expression of the target gene for VEGFC in RPE cells does not elicit a protection pathway for cell survival from exposure to a 2-µm laser, or similar thermal stimuli. The results would indicate that VEGFC is not the sole factor responsible for cellular response to thermal stress, but may still be involved in the signaling pathway.

Keywords: cell survival • stress response • laser 

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