Suppression of retinal neovascularization by lentivirus-mediated netrin-1 small hairpin RNA

Abstract

Objectives: The function of netrin-1 in pathological angiogenesis and its role in retinal neovascularization were investigated in the retinas of oxygen-induced retinopathy (OIR) mice by inhibition of netrin-1.

Methods: Expression of netrin-1 mRNA and protein in the retinas of OIR mice was analyzed by quantitative RT-PCR and immunoblotting. Inhibition of retinal neovascularization was achieved by lentivirus-mediated netrin-1 small hairpin RNA (shRNA) infection. Retinal neovascularization was examined by fluorescein angiography and quantification of preretinal neovascular nuclei in retinal sections.

Results: Both mRNA and protein expression of netrin-1 were significantly upregulated in postnatal day 17 OIR mouse retinas. Treatment of OIR mice with specific lentivirus-mediated netrin-1 shRNA dramatically reduced neovascular outgrowth into the inner limiting membrane. Neovascular tufts and nonperfused areas were also reduced.

Conclusions: High expression of netrin-1 was detected in the retina under ischemic conditions and played a significant role in pathological retinal angiogenesis. Therefore, netrin-1 represents a potential therapeutic target for diabetic retinopathy, retinopathy of prematurity and other ocular neovascular diseases.

Fig. 1

Western blot analysis of netrin-1 expression in murine retinas: netrin-1 mRNA (a), representative Western blot (b), and netrin-1 protein (c). Results are expressed as means ± SEM and analyzed by one-way ANOVA followed by the LSD t test to evaluate significance (n = 6/group). Both mRNA and protein expression of netrin-1 were significantly elevated in the retinas of P17 OIR mice compared with age-matched N17 controls (p < 0.01).

Fig. 2

Western blot analysis of the effect of RNAi on netrin-1 expression in bEnd.3 cells: representative Western blot (a) and relative expression of netrin-1 in bEnd.3 cells (b). Results are expressed as means ± SEM and analyzed by one-way ANOVA followed by the LSD t test to evaluate significance (n = 3/group). Netrin-1 expression was significantly decreased in bEnd.3 cells transfected with netrin-1 shRNA (p < 0.01).

Fig. 3

Western blot analysis of the effect of RNAi on netrin-1 expression in murine retinas: representative Western blot (a) and relative expression of netrin-1 in murine retinas (b). Results are expressed as means ± SEM and analyzed by one-way ANOVA followed by the LSD t test to evaluate significance (n = 6/group). Netrin-1 expression was significantly decreased in the retinas of P17 OIR mice transfected with netrin-1 shRNA compared with scrambled shRNA (p < 0.01).

Fig. 4

Fluorescein angiography demonstrating the effect of the lentivirus-mediated netrin-1 shRNA on retinal neovascularization in OIR mice: retinas injected with scrambled shRNA (a) and retinas transfected with netrin-1 shRNA (b). The numbers of neovascular tufts (marked by white arrows in a) were markedly reduced, and nonperfusion areas were significantly diminished in the retinas transfected with netrin-1 shRNA.

Fig. 5

Inhibition of ischemia-induced retinal neovascularization by netrin-1 shRNA. a A P17 OIR retina injected with scrambled shRNA on P12. b A P17 OIR retina transfected with netrin-1 shRNA. c Quantification of retinal neovascularization (n = 6/group). Netrin-1 shRNA resulted in approximately 50% reduction of preretinal neovascularization. GCL = Ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer. Scale bar = 50 μm.