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Hironori Uehara, YangKyung Cho, Judd Cahoon, Bonnie Archer, Bala Ambati; Splicing Shift From Membrane Bound Vegf Receptor 2 To Soluble Vegfr2 Using Morpholino Oligomer Suppresses Suture Induced Corneal Angiogenesis And Lymphangiogenesis, And Suppresses Corneal Graft Rejection. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2406.
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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 (mVEGFR2), the VEGFR2 gene produces a soluble isoform protein, which contains only a partial extracellular domain, because of alternative polyadenylation. Previously, we succeeded in shifting mVEGFR2 to soluble VEGFR2 (sVEGFR2) by manipulating splicing using morpholino oligomers (VEGFR2-MOe13). In this study, we examined whether VEGFR2-MOe13 could suppress suture induced corneal angiogenesis & lymphangiogenesis, and graft rejection in a mouse corneal transplantation model.
For the suture induced corneal model, we harvested the corneas at two timepoints (7 and 14 days) after suture placements. 15μl of standard morpholino (40ng/μl), VEGFR2-MOe13 (40ng/μl) or DPBS was injected subconjunctivally after suture placement. CD31 and LYVE-1 immunostaining were used to evaluate neovascularization and lymphangiogenesis area in cornea, respectively. For the corneal transplantation model, we injected DPBS, standard morpholino or VEGFR2-MOe13 into the conjunctiva 1, 2, 3 and 4 weeks after corneal transplantation (graft: C57Bl/6, recipient: Balb/c). Graft rejection was evaluated by corneal opacity every week up to 8 weeks. At 8 weeks, we evaluated neovascularization & lymphangiogenesis by CD31 and LYVE-1.
VEGFR2-MOe13 suppressed suture-induced neovascularization by 52.2% (7days) and 29.6% (14days) compared to DPBS (p<0.001 and 0.05, respectively). Seven days after suture placement, VEGFR2-MOe13 did not suppress lymphangiogenesis. However, 14days after suture placement, VEGFR2-MOe13 suppressed lymphangiogenesis by 27.8% compared to DPBS (p<0.05). In the corneal transplantation model, VEGFR2-MOe13 increased graft survival compared with DPBS and standard morpholino (log rank test: p=0.0186 and 0.0610, respectively). Concordantly, VEGFR2-MOe13 decreased neovascularization & lymphangiogenesis significantly at 8 weeks.
We demonstrated a splicing shift from mVEGFR2 to sVEGFR2 using VEGFR2-MOe13 suppressed suture induced corneal neovascularization and lymphangiogenesis. We also succeeded in suppressing mouse corneal graft rejection using VEGFR2-MOe13. This may become a new approach for developing anti-angiogenic therapy.
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