Abstract
Purpose :
Vascular endothelial growth factor A (VEGF) is upregulated in neovascular ocular conditions, including diabetic retinopathy (DR). To study the pathological processes in DR and develop novel treatments, VEGF-induced animal models have been established. Intravitreal injections of recombinant VEGF have been shown to mimic many of the complex DR mechanisms. However, the effects are transient due to the short half-life of injected peptides. The aim of this study was to generate an inducible VEGF expression the human retinal pigment epithelium (ARPE-19) cells.
Methods :
ARPE-19 cells were transduced with lentivirus (LV) expressing a SparQTM all-in-one cumate-inducible plasmid (System Biosciences) expressing VEGF-A165 at a multiplicity of infection (MOI) of 20. To induce VEGF expression, cumate (30 μg/ml) was added 3d post-transduction. After 48h, VEGF levels were determined in the culture medium by ELISA (R&D Systems) and by qPCR from cell lysates. Cell viability was evaluated by MTT assay; cell motility was measured by scratch assays. Effects of VEGF expression on ARPE19 permeability were performed in Transwell® inserts (Corning Inc.) using conditioned media and quantified using fluorescent permeability standard, 6-carboxyfluorescein (6-CF).
Results :
Cumate induction resulted in a 72% increase in VEGF levels as assessed by ELISA (MOI 20: 2005 ± 270 pg/ml vs. Control: 1165 ± 192 pg/ml, p < 0.05). qPCR revealed a 2.5-fold increase of VEGF mRNA levels compared with control (p < 0.01). MTT assay showed significantly increased number of cells at MOI 20 (115 ± 5%) compared with Control (100 ± 1%, p < 0.05), suggesting increased rate of proliferation. Permeability across the RPE monolayer was significantly increased in LV-transduced cells. Treatment of ARPE19 cells with conditioned media resulted in a significant increase in the apparent permeability coefficients (Papp) for 6-CF (0.66 ± 0.08 vs. 1.80 ± 0.41 x 105 cm/s, p < 0.05). Scratch assays revealed increased motility and presence of tube-like structures in transduced cells, suggesting the presence of VEGF.
Conclusions :
Cumate-inducible VEGF expression is a feasible approach for mechanistic studies evaluating the dose-dependent effects of VEGF on ARPE19 in preclinical drug discovery studies. Future work will generate stably expressing cell lines and evaluate the utility of cumate-inducible VEGF-expressing LV in in vivo systems.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.