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Yubin Qiu, Chad E Bigelow, Luciana Ferrara, Siyuan Shen, Michael P Maker, Elizabeth Fassbender, Yong Kim, Andrea DeErkenez, Barrett Leehy, Stephen H Poor; Recombinant human vascular endothelial growth factor165(VEGF) derived from different expression systems does not affect in vitro, but does impact in vivo activity profiles. Invest. Ophthalmol. Vis. Sci. 2014;55(13):600. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To characterized the in vitro and in vivo profiles of VEGF prepared using different expression systems
VEGF was expressed in both E.coli and in CHO cells. Activity profiles were determined in human retinal microvascular endothelial cell (HRMVEC) proliferation assays. HRMVECs were serum starved and then treated for 48 hours with VEGF (8 or 10 doses in triplicate) in 0.1% FBS containing medium . Cell proliferation was measured by BrdU incorporation. Rabbits were challenged with an intravitreal injection of VEGF at doses from 200 ng/eye to 2000 ng/eye in six studies (92 rabbits; n=2-4 rabbit/group or 4-8 eye/group). Vessel leakage was assessed 48 hours post-challenge. Vessels were labeled with an i.v. injection of 2000kD FITC-dextran and images acquired of both retinas. Subsequently, rabbits were injected i.v. with sodium fluorescein and images acquired at 3 minutes in one eye and at 4.5-6 minutes in the fellow eye post injection. Vascular permeability was assessed by processing the FITC-dextran image in conjunction with the corresponding fluorescein image from the same eye. The normalized, co-registered, masked and randomized images were subtracted to yield an image representing the extravasated dye.
The effects on proliferation were similar in the HRMVEC assay. The EC50 of E.coli VEGF was 0.44 ng/mL; The EC50 of VEGF from CHO cells was 0.29 ng/mL. In vivo, VEG from both sources induced dose dependent retinal vascular leakage. Doses of ≥400 ng of E.coli VEGF achieved a plateaued maximal leakage; ≥ 800 ng of VEGF from CHO cells achieved a comparable level of leakage. The leakage induced by 400 ng of E. coli VEGF is 0.37 arbitrary units (n = 42 eyes); the average leakage induced by 400 ng VEGF from CHO cells is 0.24 arbitrary units (n =22 eyes, p < 0.001).
VEGF generated from different expression systems demonstrated different potency in vivo, even though they exhibited similar activity in vitro, VEGF derived from E.coli demonstrated a greater in vivo potency then VEGF derived from CHO cells in fluorescein leakage from rabbit retinal vessels.
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