June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Characterization and function of a soluble VEGF receptor 2 protein
Author Affiliations & Notes
  • Xinyuan Zhang
    Beijing TongRen Hospital, Beijing, China
  • Footnotes
    Commercial Relationships   Xinyuan Zhang, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2968. doi:
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      Xinyuan Zhang; Characterization and function of a soluble VEGF receptor 2 protein. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2968.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : To clone and express a truncated, soluble vascular endothelial growth factor receptor 2 (sVEGFR2) possessing the combined-functional domains 1–3 and 5 in eukaryotic cells and to test the inhibitory effects of full length VEGFR2 and further to evaluate in vivo.

Methods : HEK293 cells were purchased from the Cancer Institute of the Chinese Academy of Medical Sciences Cell Bank. The E. coli DH5α strain was from our laboratory. The plasmid pCMV6-rVegfr2 was constructed according to the nucleotide sequence published in GenBank (GenBank accession number NM_013062.1). pCMV6-rVegfr2 was truncated with the restriction endonuclease EcoRVT. Transient transfection and expression were performed according to the standard protocol. ELISA was applied as previously described.

Results : pCMV6-trunctated-rVegfr2 (6100 bp) was successfully cloned. The transfection experiments showed that eitherpCMV6-truncated-rat-Vegfr2 (pCMV6-truncated-rVegfr2) or pCMV6-rVegfr2 inhibited the expression of intracellular green fluorescent protein. An analysis of the transfected cells revealed that 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). The differences in test results between the transfected and negative control groups were greatest from 24–30 h after transfection. 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. Soluble VEGFR2 produced by pCMV6-truncated-rVegfr2 inhibited full-length VEGFR2 protein expression in the cell membrane.

Conclusions : The use of transient transfection technology greatly improved transfect efficiency. Recombinant sVEGFR2 inhibited the effect of endogenous full-length VEGFR2 but was not cytotoxic.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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