Proteome Analysis of Bevacizumab Intervention in Experimental Central Retinal Vein Occlusion

Abstract

Bevacizumab is a frequently used inhibitor of vascular endothelial growth factor (VEGF) in the management of macular edema in central retinal vein occlusion (CRVO). Studying retinal protein changes in bevacizumab intervention may provide insights into mechanisms of action. In nine Danish Landrace pigs, experimental CRVO was induced in both eyes with argon laser. The right eyes received an intravitreal injection of 0.05 mL bevacizumab (n = 9), while the left control eyes received 0.05 mL saline water (NaCl). Retinal samples were collected 15 days after induced CRVO. Label-free quantification nano-liquid chromatography-tandem mass spectrometry identified 59 proteins that were regulated following bevacizumab treatment. Following bevacizumab intervention, altered levels of bevacizumab components, including the Ig gamma-1 chain C region and the Ig kappa chain C region, were observed. Changes in other significantly regulated proteins ranged between 0.58-1.73, including for the NADH-ubiquinone oxidoreductase chain (fold change = 1.73), protein-transport protein Sec24B (fold change = 1.71), glycerol kinase (fold change = 1.61), guanine-nucleotide-binding protein G(T) subunit-gamma-T1 (fold change = 0.67), and prefoldin subunit 6 (fold change = 0.58). A high retinal concentration of bevacizumab was achieved within 15 days. Changes in the additional proteins were limited, suggesting a narrow mechanism of action.

Keywords: bevacizumab; biomarker; mass spectrometry; proteome; proteomics; retina; retinal vein occlusion; vascular endothelial growth factor.

Figure 1

Fundus images obtained within 30 min of induced CRVO. (A) Fundus image of experimental CRVO with venous dilation and retinal hemorrhages upstream of the occlusion. White arrow: Retinal hemorrhage. Black arrow: Dilated retinal vein. Dark blue arrow: Site where laser is applied to displace thrombotic material toward the lamina cribrosa. (B) Peripheral view of retinal hemorrhages. White arrow: Retinal hemorrhage.

Figure 2

(A) Leakage of fluorescein observed following CRVO. Light blue arrow: Leakage of fluorescein. (B) Leakage of fluorescein upstream of the occlusion site. Light blue arrow: Leakage of fluorescein. Dark blue arrow: Site of occlusion.

Figure 3

Volcano plot. The log2 LFQ ratio (bevacizumab/NaCl) is plotted on the x-axis. On the y-axis, the -log p-value refers to the log10-transformed p-value from the t-test used to test if a protein was significantly regulated. The horizontal line denotes a significance level of 0.05. Statistically significantly regulated proteins are located above the horizontal line. Components of bevacizumab are not included in the volcano plot.