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Xinyuan Zhang, Wei Liu, Ching Song, Ningli Wang; Function of a soluble VEGF receptor 2 protein in vivo. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):61.
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© ARVO (1962-2015); The Authors (2016-present)
To test the function of a truncated, soluble vascular endothelial growth factor receptor 2 (sVEGFR2) possessing the combined-functional domains 1-3 and 5 in vivo.
The inhibitory function of the sVEGFR2 was test in vivo. A rat Vegfr2 gene restriction fragment encoding a truncated sVEGFR2 protein, which contains the immunoglobulin-like domains 1-3 and 5, was cloned into the eukaryotic expression vector pCMV6 via recombinant DNA technology. The resulting pCMV6-sVegfr2 plasmid was transfected in human embryonic kidney (HEK) 293 cells via liposomal transient transfection. ELISA was used to quantify VEGFA, VEGFR2 and sVEGFR2 protein in the conditioned medium. . Full-length rat VEGFR2 encoded by the pCMV6-rVegfr2 plasmid was used as a control.
Soluble VEGFR2 produced by pCMV6-truncated-rVegfr2 inhibited full-length VEGFR2 protein expression in the cell membrane. The amount of full-length VEGFR2 protein in the pCMV6-truncated-rVegfr2 transfected cells was 20% lower than that in the negative control (non-transfected HEK 293 cells). Expression of VEGFA was significantly decreased in the pCMV6-truncated-rVegfr2 trasfected cells conpared with the negtive control cells ( 25.5± 2.1% vs. 52.2± 1.8 P=0.000). Either pCMV6-truncated-rat-Vegfr2 (pCMV6-truncated-rVegfr2) or pCMV6-rVegfr2 inhibited the expression of intracellular green fluorescent protein. The sVEGFR2 protein content was found to increase by approximately 26% in the transfected cells compared to that in the negative control cells (68.2% ± 1.7% vs. 41.9% ± 2.9%, P = 0.000) and by 18% compared to the negative control cells (68.2% ± 1.7% vs. 50.0% ± 0.5%, P = 0.003). Propidium iodide and Hoechst staining indicated no significant change in the number of HEK293 cells undergoing apoptosis 6 days after pCMV6-trucated-Vegfr2 transfection, compared to the negative control.
Recombinant sVEGFR2 inhibited the expression of endogenous full-length VEGFR2 and VEGFA but was not cytotoxic.This study employed a eukaryotic system to express sVEGFR2 while maintaining its natural configuration and normal function by preserving the glycosylation and disulfide bonds in the extracellular VEGFR2 fragment.
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