March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Knockdown Of Splicing Factor Fbp21 Alters Pro - To Anti-angiogenic Vegf Protein Ratio In Primary Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • James G. Carter
    Physiology & Pharmacology,
    University of Bristol, Bristol, United Kingdom
  • Amanda J. Churchill
    Academic Unit of Ophthalmology,
    University of Bristol, Bristol, United Kingdom
  • David O. Bates
    Physiology & Pharmacology,
    University of Bristol, Bristol, United Kingdom
  • Footnotes
    Commercial Relationships  James G. Carter, None; Amanda J. Churchill, None; David O. Bates, None
  • Footnotes
    Support  National Eye Research Centre, UK
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 4119. doi:
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      James G. Carter, Amanda J. Churchill, David O. Bates; Knockdown Of Splicing Factor Fbp21 Alters Pro - To Anti-angiogenic Vegf Protein Ratio In Primary Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4119.

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

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Abstract

Purpose: : Development of angiogenic eye disease is mediated by changes in the balance of Vascular Endothelial Growth Factor (VEGF) isoforms between pro-angiogenic VEGFxxx and anti-angiogenic VEGFxxxb families. Anti-angiogenic borrelidin derivatives target the splice factor FBP21, which may be involved in the splicing switch between pro- and anti-angiogenic VEGF isoforms[1]. This study was to determine the effect of FBP21 knockdown on VEGF isoform expression.

Methods: : Primary retinal pigment epithelial (RPE) cells were treated with siRNA targeted to FBP21. mRNA was extracted to confirm knockdown of FBP21 compared with scrambled siRNA by qRT-PCR. Protein was assessed for changes in VEGF isoform expression by ELISA. The same knockdown experiments were replicated in a luciferase reporter assay in HEK293 cells to determine if changes in VEGF expression profiles were pre- or post- transcriptional. The cells were transfected with reporter constructs containing VEGF165 or VEGF165b 3’-UTRs and the effect of FBP21 siRNA knockdown compared with scrambled siRNA was assessed by changes in luciferase/TK renilla expression.

Results: : Knockdown of FBP21 by siRNA was confirmed by qRT-PCR (p<0.01). This siRNA treatment caused a decrease in overall VEGF protein production (685pg/mg vs. 975pg/mg, p<0.01), but critically a significant decrease in the proportion of VEGFxxxb protein being produced in 1° RPE cells (48.8% vs. 64.8%, p<0.01). There were no significant changes in luciferase expression under FBP21 knockdown for either VEGF165 or VEGF165b constructs in the reporter assay. This shows that these changes in VEGF expression ratios were mediated at the point of transcription, rather than a post-transcriptional/translation regulation event.

Conclusions: : Inhibition of FBP21 alters VEGF isoform expression at the splicing level. Identifying mechanisms involved in FBP21-mediated splice site selection and assessing the possible therapeutic role of borrelidins on these splicing complexes may give an alternative to current anti-VEGF treatments in ocular angiogenesis.1. Woolard, J., et al., Borrelidin modulates the alternative splicing of VEGF in favour of anti-angiogenic isoforms. Chemical Science, 2011. 2(2): p. 273-278.

Keywords: vascular endothelial growth factor • retinal pigment epithelium • gene/expression 
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