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Hironori Uehara, Judd Cahoon, Ling Luo, YangKyung Cho, Jackie Simonis, Bala Ambati; Membrane Bound Vegf Receptor2 Can Be Shifted To Soluble Vegf Receptor2 Using Morpholino Antisense Oligomer, Which Suppresses Angiogenesis And Lymphangiogenesis In Murine Corneal Suture Models. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4868.
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Vascular endothelial growth factor-A (VEGF-A) is a potent initiator of angiogenesis and VEGF receptor-2 (VEGFR2) is its primary angiogenic receptor. In addition to membrane-bound VEGFR2 (mb-VEGFR2), the VEGFR2 gene produces a soluble isoform protein, which contains only partial extracellular domain, because of alternative polyadenylation. Soluble VEGFR2 (sVEGFR2) is known to bind VEGF-C and exert anti-lymphangiogenesis. We tried to shift mRNA expression of mb-VEGFR2 to sVEGFR2 using morpholino-oligomer to manipulate splicing.
We designed morpholino-oligomer targeted to exon13-intron13 junction (VEGFR2e13_MO). Using human umbilical vein endothelial cells, we assessed expression of mb-VEGFR-2 and sVEGFR-2 by real-time RT-PCR, flow cytometry and western blot. We determined a polyadenylation site induced by VEGFR2e13_MO using 3`RACE. Furthermore, we tested potential anti-angiogenic effects of VEGFR2e13_MO in murine corneal suture angiogenesis. CD31 and LYVE-1 stains were used to evaluate neovascularization and lymphangiogenesis.
VEGFR2e13_MO increases sVEGFR2 mRNA 17 times (p<0.001) and decreases mb-VEGFR2 35% (p<0.05) compared with the controls. Flow cytometry showed VEGFR2e13_MO reduced VEGFR2 positive cells 44% compared to the controls. In western blot for sVEGFR2, we detected sVEGFR2 protein from the culture medium of VEGFR2e13_MO transfected cells, but not control cells. By 3`RACE, we confirmed that one of the polyadenylation sites is induced by VEGFR2e13_MO. VEGFR2e13_MO injection reduced neovascularization 50% compared to the controls (p<0.001) one week after corneal suture injury, and reduced lymphangiogenesis 30% compared to the controls (p<0.05) two weeks after corneal suture injury.
A morpholino targeting the exon13-intron13 junction of the VEGFR2 gene preferentially promoted expression of sVEGFR2, while suppressing mb-VEGFR2 and angiogenesis in corneal injury models. By modifying splicing factors, we can activate a latent polyadenylation signal and produce a different isoform of the target protein. This may become a new approach for developing for anti-angiogenic drugs.
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