Abstract
Purpose :
Vascular endothelial growth factor (VEGF) 121 isoform has potent retinal neuroprotective effects without promoting retinal inflammation. We designed and generated two novel VEGF121 variants (eVEGF) that can promote cell autologous signaling via VEGFR2 and tested their neuroprotective function in vitro using primary mouse retinal ganglion cells (RGC).
Methods :
We engineered two novel VEGF variants, eVEGF-38 and eVEGF-53, that localize to the cell membrane and with myc-tags. We also generated VEGF189 with a myc-tag and packaged all the constructs into adeno-associated virus serotype 2 (AAV2) for efficient transduction and expression in human microvascular endothelial cells (hREC) and RGC. Transgene expression was measured by western blot, qPCR and immunostaining. In hREC, VEGFR2, VCAM-1, E-selectin and tissue factor expression were measured by immunostaining or qPCR. VEGFR2 signaling, axon length, and synapse formation in RGC were measured by immunostaining. RGC survival was measured by TUNEL staining and intracellular [Ca2+] was measured using fura2/AM calcium imaging.
Results :
In hREC, eVEGF-38 and eVEGF-53 expression was detected in cell lysate but not in conditioned media. Increased VEGFR2 phosphorylation and expression was detected at the cell membrane. Pro-inflammatory markers were increased by VEGF189/AAV2 and VEGF165 protein (p<0.05), but not by eVEGF-38/AAV2 or eVEGF-53/AAV2 compared with GFP/AAV2 control. In RGC, high expression levels of all transgenes as well as phosphor-ERK and phospho-AKT were detected, and average lengths of RGC axons were increased by 500% (p<0.001) compared to control, while VEGFR2 inhibition completely blocked axon development in the RGC. Both eVEGFs/AAV2 and VEGF189/AAV2 promoted synapse formation in RGC. eVEGF-38/AAV2, eVEGF-53/AAV2, VEGF189/AAV2, and exogenous VEGF121 protein promoted RGC survival compared to control (45%, 48%, 50%, 62% vs. 22% survival, P<0.001). Expression of eVEGF-38, eVEGF-53, and VEGF189 significantly enhanced RGC survival upon H2O2 treatment (10%, 15%, 18% increased survival compared to control, P<0.05), which was abolished by LY-294002 treatment. Lastly, eVEGF-38 and eVEGF-53 expression decreased intracellular [Ca2+] upon carbachol and NMDA stimulation compared to control.
Conclusions :
The novel engineered VEGF variants significantly promote RGC axon formation, survival as well as synapse formation, and represent potential novel therapeutics for retinal neuroprotection for glaucoma.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.