Abstract
Purpose: :
VEGF receptor 1(VEGFR1) is known to be one of the main receptors for VEGF-A. VEGFR1 gene produces two kinds of isoform, soluble and membrane bound VEGFR1. By stimulation of VEGF-A, membrane bound VEGFR1 leads angiogenesis. On the other hand, soluble VEGFR1 sequestered VEGF-A activity by binding to VEGF-A, so results in anti-angiogenetic effect. These isoforms are generated by alternative splicing. Previously, we succeeded to convert membrane VEGFR1 to soluble VEGFR1 by blocking splicing event with morpholino antisense oligomer(sVEGFR1_MO, Flt morpholino) using culture cells. In this study, we examined Flt morpholino can inhibit angiogenesis in vivo murine corneal suture model
Methods: :
We used a murine corneal suture model to investigate the effect of morpholino promoting sFlt in suppressing neovascularization and lymphangiogenesis. In an inhibition experiment, we placed 3 corneal sutures on day 0, and then injected Flt morpholino, nonspecific(STD) morpholino, and PBS into corneal stroma of each group on day 1 and 4. On day 7, we harvested the cornea . In a regression experiment, we placed 3 corneal suture on day 0 and we injected Flt morpholino, STD morpholino, and PBS into corneal stroma of each group on day 7 and 10 and then we harvested corneas on day 14. After staining of vessels(CD31) and lymphatic vessels(LYVE1) of cornea, we checked with fluorescent microscope and digitally quantified neovascularization.
Results: :
In analysis of angiogenesis(in regression experiment), we can find significant difference between groups(mean area of angiogenesis/triangular corneal area around suture is 0.262±0.182 in Flt morpholino group(N=22), 0.535±0.256 in STD morpholino group(N=29), and 0.364±0.184 in PBS group(N=28)). Flt morpholino group showed less neovascularization than STD morpholino group(p=0.000). In analysis of lymphangiogenesis(in both inhibition and regression experiment), we can find significant difference between groups(in inhibition, mean area of lymphangiogenesis/triangular corneal area around suture is 0.272±0.181 in Flt group(N=29), 0.470±0.206 in STD group(N=20), and 0.347±0.194 in PBS group(N=30)). Flt morpholino group showed less lymphangiogensis than STD morpholino group(p=0.002 in inhibition, p=0.000 in regression)
Conclusions: :
Morpholinos promoting sFlt reduced angiogenesis and lymphangiogenesis compared with nonspecific morpholino and had a tendency of reducing angiogenesis and lymphangiogenesis compared with control PBS
Keywords: neovascularization • cornea: basic science • vascular endothelial growth factor