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Yang Kyung Cho, Hironori Uehara, Jason R. Young, Wei Huang, Ling Luo, Xiaohui Zhang, Bonnie Archer, Jacquelyn Simonis, Thomas Olson, Bala Ambati; Effect of Morpholino Targeting Flt-1 in Mice Corneal Keratoplasty Model. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2950.
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Morpholinos are synthetic molecules similar to DNA oligonucleotides which can bind mRNA and pre-mRNA and sterically block the molecular machinery of translation or alternative splicing. We sought to determine whether morpholinos can target the alternative splicing of Flt-1 pre-mRNA to upregulate the production of sFlt, limiting neovascularization and lymphangiogenesis resulting in increased graft survival in a murine corneal penetrating keratoplasty(PK) model.
We used a murine corneal keratoplasty model to investigate the effect of a morpholino that promotes sFlt to suppress neovascularization and lymphangiogenesis to increase graft survival. We performed mice corneal penetrating keratoplasty in one eye of each Balb/C mouse (C57BL6 mice as donors) and injected 40ng/ul of Flt morpholino(n=16), nonspecific (STD) morpholino(n=18), and PBS(n=11) into the subconjucntival space on the day of PK and weekly until postoperative 4 weeks. Corneal opacity was graded to evaluate graft survival weekly through postoperative week 8, at which point the corneas were harvested. After flat mounting the harvested corneas, we performed immunohistochemistry and fluorescent microscopy to digitally quantify neovascularization. We compared the area of neovascularization and lymphangiogenesis to the total corneal area and graft area
Flt morpholino increased graft survival compared to the PBS group (p=0.043).Flt morpholino group showed less neovascularized area in both total cornea (0.2267±0.0139) and graft (0.1005±0.0173) cornea when compared with STD the morpholino group (0.2771±0.0257 in total cornea, 0.2190±0.0328 in graft cornea, respectively) and PBS group (0.2777±0.0220 in total cornea, 0.1883±0.0384 in graft cornea, respectively) (all p’s<0.05). The Flt morpholino group showed less lymphangiogensis in both total cornea (0.0780±0.0095) and graft cornea (0.0719±0.0109) compared with the STD morpholino group (0.1005±0.0114 in total cornea, 0.1258±0.0198 in graft cornea) and PBS group (0.1199±0.0078 in total cornea, 0.1231±0.0165 in graft cornea) (all p’s<0.05).
We found that morpholinos promoting sFlt reduced angiogenesis and lymphangiogenesis compared with STD morpholino and PBS in a murine corneal penetrating keratoplasty model and increased graft survival compared with PBS.
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