Vascular Endothelial Growth Factor Receptor 1 morpholino decreases angiogenesis in a murine corneal suture model

Purpose: This study sought to determine whether a VEGFR1-specific morpholino could induce the alternative splicing of Flt-1 pre-mRNA to down-regulate membrane-bound Flt-1 (mFlt-1) and increase the production of soluble Flt-1 (sFlt-1), thereby limiting angiogenesis and inflammation in a mouse corneal suture injury model. Methods: A murine corneal suture model was used to investigate the effects of a VEGFR1-specific morpholino in vivo. Western blot analysis and RT-PCR were used to compare the impact of the Flt morpholino on mFlt-1 and sFlt-1 levels. For vascular regression modeling, two corneal sutures were placed and injected with Flt morpholino, standard morpholino, and PBS on days 8 and 10. Corneas were harvested on day 14. The grade of neovascularization (graded 0-5; 0 - no neovascularization; 5 - thick tortous new vessel growth over the suture and towards the center of the cornea) was compared on days 8, 10, and 14. Immunohistochemistry, fluorescent microscopy, and confocal microscopy were used to digitally quantify the area and volume of neovascularization and inflammatory infiltration. Results: Western Blot analysis revealed that the Flt morpholino decreased mFlt-1 levels while increasing sFlt-1 levels. An increased sFlt-1/mFlt-1 ratio in the Flt morpholino group was seen with RT-PCR. Based on the neovascularization grading, there was a decrease in neovascularization area in the Flt morpholino group (3.29±0.19 to 2.92±0.13) from day 8 to 14 (p<0.05) compared to the standard morpholino (2.68±0.19 to 3.14±0.22)) and PBS (2.96±0.14 to 3.42±0.19) groups, both of which showed an increase in neovascularization (p<0.05). The Flt morpholino group also showed reduced neovascularization volume compared to the PBS (p=0.001) and STD morpholino groups (p=0.000). Conclusions: Flt morpholinos decrease mFlt-1 and increase sFlt-1 levels, resulting in decreased neovascularization in a murine corneal suture model.