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Category: Angiogenesis

The inhibition of vascular endothelial cell growth factor A (VEGF) pathway in in vitro proliferation assay is an integral part of the potency assessment of VEGF inhibitor candidates for intraocular vascular diseases. The primary bovine retinal endothelium cells (BRECs) are physiologically relevant cells to assess the angiogenesis.

SUMMARY

Cells

Primary bovine retinal endothelium cells (BRECs)

Induction

rhVEGF165

Positive control

Aflibercept (Eylea®) at 0.01 to 30 nM

Indicator and detection

Indicator: resazurin (7-Hydroxy-3H-phenoxazin-3-one-10-oxide) dye is irreversibly reduced to highly fluorescent metabolite resorufin (7-Hydroxy-3H-phenoxazin-3-one); (Hakkarainen et al., 2016).

 

Detection: After 2 h exposure to resazurin, the amount of resorufin is measured at 560 nm (ex), 590 nm (em) by using Cytation 3 multi-mode reader (BioTek Instruments, Winooski, VT, USA); (Hakkarainen et al., 2016).

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IMAGES

Representative examples of assays on BRECs

Typical endothelial cell morphology of BRECs at DIV6 (left) and DIV7 (right). Scale bar 100 µm. BREC proliferation assay validation was performed as previously described (Yang et al., 2014). The half maximal inhibitory concentration (IC50) of VEGF inhibitor (aflibercept, Eylea®) was assessed using two commercially available rhVEGF165 (R&D Systems and Life Technologies).

A dose-dependent inhibition of rhVEGF-induced (R&D Systems and Life Technologies) BREC proliferation by aflibercept (Eylea®) at concentrations 0.01 to 30 nM. The figure on the left shows proliferative activity of rhVEGF on BREC from R&D Systems and Life Technologies. rhVEGF from R&D Systems seems to enhance the BRECs proliferation more efficienthly than rh VEGF from Life Technologies. Therefore, the higher IC50 value is a result from enhanced BREC proliferation.

SELECTED PUBLICATIONS

  1. Yang J, Wang X, Fuh G, Yu L, Wakshull E, Khosraviani M, Day ES, Demeule B, Liu J, Shire SJ, Ferrara N, Yadav S. Comparison of binding characteristics and in vitro activities of three inhibitors of vascular endothelial growth factor A. Mol Pharm 2014,11(10):3421-3430. doi: 10.1021/mp500160v.

REFERENCES

  1. Hakkarainen JJ, Reinisalo M, Ragauskas S, Seppänen A, Kaja S, Kalesnykas G. Acute cytotoxic effects of marketed ophthalmic formulations on human corneal epithelial cells. Int J Pharm. 2016;511(1):73-78. doi: 10.1016/j.ijpharm.2016.06.135

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