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. 2014 Sep 2;124(10):4564–4576. doi: 10.1172/JCI74527

Figure 6. AKB-9778 suppresses ischemia-induced retinal NV in the presence of high ANG2 levels.

Figure 6

Tet-opsin-Ang2 mice were placed in 75% oxygen at P7, returned to room air at P12, and divided into 4 groups: Dox+/vehicle (n = 17) received daily s.c. Dox (50 mg/kg) and an intraocular injection of vehicle; Dox+/AKB-9778 (n = 21) received daily s.c. Dox and an intraocular injection of 5 μg AKB-9778; Dox–/vehicle (n = 10) received an intraocular injection of vehicle; Dox–/AKB-9778 (n = 10) received an intraocular injection of 5 μg AKB-9778. At P17, the Dox+/vehicle mice had severe NV (A) that was markedly reduced in the Dox+/AKB-9778 mice (B). The large vessels are hyaloid vessels, but all other green staining is NV. Compared with Dox–/vehicle mice (C), Dox–/AKB-9778 mice appeared to show less NV (D). Scale bar: 500 μm. The area of NV was significantly larger in Dox+/vehicle mice versus that in Dox–/vehicle mice (E) (*P < 0.001 by 1-way ANOVA with Bonferroni’s correction), indicating that ANG2 stimulated NV. The area of NV was significantly smaller in Dox+/AKB-9778 versus Dox+/vehicle mice (**P < 0.001 by 1-way ANOVA with Bonferroni’s correction), indicating that AKB-9778 suppressed NV in the presence of high levels of ANG2 and possibly less so in the absence of high ANG2 levels. In hypoxic HUVECs (incubated in 5% oxygen for 4 or 16 hours), there were marked reductions of ANG1-induced (500 ng/ml) TIE2 phosphorylation (F) and downstream signaling (G); however, AKB-9778 (5 μM) restored TIE2 phosphorylation (F) and downstream signaling (G), even in the presence of exogenous ANG2 (500 ng/ml). Antibodies recognizing the proteins regardless of phosphorylation status were used as loading controls. Similar results were obtained in 3 independent experiments.