Abstract
Purpose :
Retinal neovascularization is the leading cause of vision impairment in conditions such as diabetic retinopathy, age-related macular degeneration and retinopathy of prematurity. Studies in human neovascular retinas have revealed an imbalance between angiogenesis promotors, vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), and angiogenesis inhibitors, including pigment epithelial-derived factor (PEDF). Therefore, restoring the balance between these factors could constitute a therapeutic approach. To test this hypothesis, we have devised a dual gene therapy approach that simultaneously promotes PEDF overexpression and silences PlGF expression.
Methods :
Human retinal pigment epithelium (RPE) cells were transfected with a self-replicating episomal vector (pEPito) for PEDF overexpression and/or a siRNA targeting PlGF gene and the respective culture media were collected. Protein expression levels in this conditioned media were analyzed by ELISA and gene expression levels in cells by real-time polymerase chain reaction. The conditioned media was used to culture human umbilical vein endothelial cells (HUVEC) and their proliferation rate (CellTrace Violet proliferation kit), migration capacity (scratch assay) and apoptosis (flow cytometry analysis) were analyzed. The angiogenesis inhibitory potential of this approach was assessed by an endothelial cell tube formation assay. N≥ 3, with statistical analysis by Two-way ANOVA followed by a post hoc Bonferroni’s multiple comparison test or Student’s t-test.
Results :
We have previously described the effectiveness of the pEPito vector in the long-term expression in the retina of diabetic mice after a single subretinal injection, ameliorating diabetic retinopathy hallmarks. In this work, pEPito-driven PEDF overexpression in combination with PlGF silencing in RPE cells leads to an enhanced anti-angiogenic effect. We also observed a significant decrease in endothelial cells’ migration (p< 0.05), proliferation (p< 0.05), and an increase in apoptosis induction (p< 0.05). Additionally, a significant inhibitory effect on tube formation was observed (p< 0.05).
Conclusions :
Our findings demonstrate that restoring the secretion capacity of the RPE to correct the imbalance between anti- and pro-angiogenic factors can represent a promising therapeutic approach for retinal neovascular diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.