April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Antisense Morpholine Oligonucleotide Against FLT Splice Junction Inhibits Murine Corneal Hemangiogenesis (HA) and Lymphangiogenesis (LA)
Author Affiliations & Notes
  • Y. Qazi
    Ophthalmology, John Moran Eye Center/University of Utah, Salt Lake City, Utah
    Ophthalmology, Emory Eye Center/Emory University, Atlanta, Georgia
  • H. Uehara
    Ophthalmology, John Moran Eye Center/University of Utah, Salt Lake City, Utah
  • B. K. Ambati
    Ophthalmology, John Moran Eye Center/University of Utah, Salt Lake City, Utah
  • Footnotes
    Commercial Relationships  Y. Qazi, None; H. Uehara, None; B.K. Ambati, None.
  • Footnotes
    Support  NEI 5R01EY017950
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 2038. doi:
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      Y. Qazi, H. Uehara, B. K. Ambati; Antisense Morpholine Oligonucleotide Against FLT Splice Junction Inhibits Murine Corneal Hemangiogenesis (HA) and Lymphangiogenesis (LA). Invest. Ophthalmol. Vis. Sci. 2010;51(13):2038.

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

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Abstract

Purpose: : To design and test a molecular tool to convert the pro-angiogenic microenvironment of vascularised corneas by manipulating post-transcriptional processing of the FLT gene towards its anti-angiogenic isoform, sFLT-1.

Methods: : Antisense morpholino oligonucleotide (morpholino, MO) was designed complementary to a sequence within the FLT splice junction of exon13-intron13 at the 5' splice site. Gene Tools LLC. prepared the morpholino. Fluorescein-tagged standard morpholino (STD MO) was transfected (electroporation) into HUVECs to test and confirm delivery of morpholinos into the nucleus using fluorescence microscopy.A suture-induced model of corneal neovascularization (KNV) was used. BALB/CJ mice had 3 sutures placed intrastromally.1 week after suture placement, the mice were randomly divided into 3 groups, their corneas photographed and each of the groups received one of the following interventions intracorneally: 200ng sFlt-MO; 200ng STD MO; 5ul phosphate-buffered saline (PBS).1week post-injection, the corneas were photographed and the mice sacrificed. The harvested corneas were were used for flatmounts, ELISA and RT-PCR. Immunostaining was performed for vascular endothelial cells (CD31) and lymphatic endothelial cells (LYVE1). Scion Image was used to quantitate HA and LA from flatmounts imaged by fluorescence microscopy.KNV was induced in mice as above and 1 week after suture placement, the mice were randomly divided into 2 groups, each of which received an intracorneal injection of a combination of either 2ug siRNA.sFlt + 200ng sFlt-MO, or, 2ug siRNA negative control + 200ng sFlt-MO. 1 week post-injection, the corneas were harvested and processed as mentioned above.

Results: : Morpholinos designed to increase sFlt-1 (sFlt-MO) significantly inhibited the progression of both blood and lymph vessels.Treated corneas had 6.9% HA (p= 0.017, n=8) and 4.6% LA (p<0.01, n=8) compared to control STD MO with 65.6% HA and 52.9%. Regression of KNV was also seen.sFlt-MO increased its RNA transcript on RT-PCR and reduced free VEGF-A expression on ELISA. pSEC.siRNA.sFlt-1 abolished the effects of sFlt-MO, confirming specificity of mechanism.

Conclusions: : sFlt-MO promotes alternative splicing of the murine FLT gene to sFLT-1 rendering an anti-angiogenic microenvironment in the cornea. The 5' splice site of FLT gene at e13-i13 junction may be a promising target in the development of molecular antiangiogenic strategies.

Keywords: neovascularization • gene/expression • transcription 
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