Fig 1.
rAAV2-sVEGFRv-1 transgene expression.
(a) Schematic of rAAV2-sVEGFRv-1, which was able to infect HUVECs to express a transgene product that was identified via RT-PCR (b) and ELISA (c) as sVEGFRv-1, with protein levels increasing with time. Data represented as mean ± SEM (n = 3). * p < 0.001.
Fig 2.
rAAV2-sVEGFRv-1 treatment does not result in cytotoxicity.
Potential cytotoxic effects were evaluated via LHD activity assays on both shorter (a) and longer in vitro timescales (b), in addition to a cell viability assay (c), which helped demonstrate the safety of the therapeutic virus vector. Data represented as mean ± SEM (n = 3).
Fig 3.
rAAV2-sVEGFRv-1 ligand binding.
The ability of sVEGFRv-1 to interact with the natural binding partners of VEGFR1, including VEGF-A (a), VEGF-B (b), and PlGF (c) was determined via ELISA. Data represented as mean ± SEM (n = 3). * p < 0.05.
Fig 4.
Effects of rAAV2-sVEGFRv-1 on HUVECs under VEGF-A stimulation.
(a) The therapeutic virus vector reduced endothelial cell proliferation, a major contributor to the pathology of angiogenic ocular conditions induced by VEGF-A and via VEGFR2 activity, which may be due to its ability to block the phosphorylation (b) and activation of protein kinase D (PKD). (c) Quantification of the relative phosphorylation of PKD. Data represented as mean ± SEM (n = 3). * p < 0.05.
Fig 5.
Tight junction protein 1 expression visualized in vitro.
(a) Immunocytochemistry using HUVECs demonstrated that VEGF-A stimulation led to a relative absence of ZO-1 and affected its localization. These effects were mainly protected against by rAAV2-sVEGFRv-1 treatment, with the intensity of the fluorescence being quantified (b). Western blot (c) and the quantification thereof (d) was then used to confirm ZO-1 expression levels. Scale bar = 10 μm. Data represented as mean ± SEM (n = 3). * p < 0.05.
Fig 6.
Effects of rAAV2-sVEGFRv-1 on interleukin production.
HMC3 microglial cells were used to show that the therapeutic virus may be anti-inflammatory, as rAAV2-sVEGFRv-1 was able to protect against increases seen in IL-1β (a, b) and IL-6 production (c, d) induced by PlGF in cells treated with rAAV2-GFP, as compared to unstimulated control cells. (e) Western blot of the respective cytokine expression levels and the quantifications (f, g) thereof. Scale bars = 10 μm. Data represented as mean ± SEM (n = 3). * p < 0.05.