Hypoxia-induced retinal angiogenesis in zebrafish as a model to study retinopathy

Abstract

Mechanistic understanding and defining novel therapeutic targets of diabetic retinopathy and age-related macular degeneration (AMD) have been hampered by a lack of appropriate adult animal models. Here we describe a simple and highly reproducible adult fli-EGFP transgenic zebrafish model to study retinal angiogenesis. The retinal vasculature in the adult zebrafish is highly organized and hypoxia-induced neovascularization occurs in a predictable area of capillary plexuses. New retinal vessels and vascular sprouts can be accurately measured and quantified. Orally active anti-VEGF agents including sunitinib and ZM323881 effectively block hypoxia-induced retinal neovascularization. Intriguingly, blockage of the Notch signaling pathway by the inhibitor DAPT under hypoxia, results in a high density of arterial sprouting in all optical arteries. The Notch suppression-induced arterial sprouting is dependent on tissue hypoxia. However, in the presence of DAPT substantial endothelial tip cell formation was detected only in optic capillary plexuses under normoxia. These findings suggest that hypoxia shifts the vascular targets of Notch inhibitors. Our findings for the first time show a clinically relevant retinal angiogenesis model in adult zebrafish, which might serve as a platform for studying mechanisms of retinal angiogenesis, for defining novel therapeutic targets, and for screening of novel antiangiogenic drugs.

Figure 1. Hypoxia-induced retinal angiogenesis in adult fli-EGFP-Tg zebrafish.

Adult fli-EGFP-Tg zebrafish were placed in a hypoxic aquaria and air saturation in the water was controlled at 10% (820 ppb) by an automated device (J). After 12-days exposure to this hypoxic environment, retinal angiogenesis in the capillary plexuses was detected (B, E, H, and K). Corresponding areas of the retinal vasculture exposed to normoxia were used as controls (A, D and G). Numbers of new vascular branches and sprouts, intercapillary distances, and total vascularization areas were accurately quantified (C, F, I, and L). Yellow arrowheads point to vascular sprouts. Yellow arrows point to endothelial tips. Data represents mean determinants of 11–16 randomized samples. ***p<0.001. Bar in panels A and B = 100 µm; in panels D and E = 50 µm; and in G and H = 20 µm.

Figure 2. Time-course of hypoxia-induced retinal neovascularization.

Hypoxia-induced retinal neovascularization in adult fli-EGFP-Tg zebrafish was kinetically monitored (A–H). Angiogenic sprouts were readily formed at day 2 and became overwhelmingly pronounced at day 3 (B) after exposure to hypoxia. The hypoxia-induced retinal angiogenic vessels continued to grow between days 3–12 (B–D). A maximal angiogenic response was detected at day 12. Quantification of new vessel branches (E), sprouts (F) intercapillary distances (G), and total vascularization areas (H) showed significant differences at all time points. Yellow arrowheads point to vascular sprouts. Data represents mean determinants of 11–16 randomized samples. ***p<0.001. Bar = 50 µm.

Figure 3. Dose-dependent hypoxia-induced retinal neovascularization.

Adult fli-EGFP-Tg zebrafish were exposed to 20% or 10% air-saturated water for 6 days. Retinal neovascularization was analyzed using whole-mount confocal analysis and quantified as branching points, numbers of sprouts, intercapillary distances, and total vascularization areas (A–J). Yellow arrowheads point to vascular sprouts. Data represents mean determinants of 11–25 randomized samples. *p<0.05. ***p<0.001. Bar = 50 µm

Figure 4. Inhibition of retinal neovascularization by orally active anti-VEGF drugs.

Adult fli-EGFP-Tg zebrafish were exposed to 10 % hypoxia in the absence (A) or presence of sunitinib (B and E) or ZN323881 (C and F) anti-VEGF small molecules for 14 days. Retinal neovascularization was analyzed using whole-mount confocal analysis and quantified as branching points (G), numbers of sprouts (H), intercapillary distances (I), and total vascularization area (J). Yellow arrowheads point to vascular sprouts. Data represents mean determinants of 11–29 randomized samples. *p<0.05. ***p<0.001. Bar = 50 µm.

Figure 5. Inhibition of Notch under hypoxia and normoxia.

Adult fli-EGFP-Tg zebrafish were exposed to 10 µM DAPT under hypoxia for 5 days (A, E, I, C, G, and K) or under normoxia for 6 days (B, D, F, H, and L). Control optic arteries without exposure to hypoxia or DAPT are shown in (J). E–H are amplified images of the inserts of A–D. Retinal neovascularization was analyzed using whole-mount confocal analysis and quantified as numbers of arterial sprouts (M), capillary tips (N) or branch formations (O).Yellow arrowheads point to vascular sprouts. Yellow arrows point to endothelial tips. The dashed red lines encircle the central optic artery. Data represent mean determinants of 11–13 randomized samples. *p<0.05. ***p<0.001. Bars in A–D = 100 µm; Bars in A–D = 50 µm; and Bars in A–D = 20 µm.