Proliferation of primary bovine retinal endothelial cells

Summary: A physiologically relevant cellular model to assess angiogenesis.

Model Description

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. 

Species Primary bovine retinal endothelium cells (BRECs) 
Induction rhVEGF165 
Positive control Aflibercept (Eylea®) at 0.01 to 30 nM 
Indicator and Read-outs 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 2h 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). 

Outcomes and Read-Outs 

Imaging

Representative examples of assays on BRECs 

Fig. 1. Typical endothelial cell morphology of BRECs at DIV6 (left) and DIV7 (right).
Fig. 1. 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). 
Fig. 2. 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.
Fig. 2. 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 efficiently than rhVEGF from Life Technologies. Therefore, the higher IC50 value is a result from enhanced BREC proliferation. 

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