Netrin-4 inhibits angiogenesis via binding to neogenin and recruitment of Unc5B

Abstract

Netrins are secreted molecules with roles in axon guidance and angiogenesis. We identified Netrin-4 as a gene specifically overexpressed in VEGF-stimulated endothelial cells (EC) in vitro as well as in vivo. Knockdown of Netrin-4 expression in EC increased their ability to form tubular structures on Matrigel. To identify which receptor is involved, we showed by quantitative RT-PCR that EC express three of the six Netrin-1 cognate receptors: neogenin, Unc5B, and Unc5C. In contrast to Netrin-1, Netrin-4 bound only to neogenin but not to Unc5B or Unc5C receptors. Neutralization of Netrin-4 binding to neogenin by blocking antibodies abolished the chemotactic effect of Netrin-4. Furthermore, the silencing of either neogenin or Unc5B abolished Netrin-4 inhibitory effect on EC migration, suggesting that both receptors are essential for its function in vitro. Coimmunoprecipitation experiments demonstrated that Netrin-4 increased the association between Unc5B and neogenin on VEGF- or FGF-2-stimulated EC. Finally, we showed that Netrin-4 significantly reduced pathological angiogenesis in Matrigel and laser-induced choroidal neovascularization models. Interestingly, Netrin-4, neogenin, and Unc5B receptor expression was up-regulated in choroidal neovessel EC after laser injury. Moreover, Netrin-4 overexpression delayed tumor angiogenesis in a model of s.c. xenograft. We propose that Netrin-4 acts as an antiangiogenic factor through binding to neogenin and recruitment of Unc5B.

Fig. 1.

Overexpression of N4 in angiogenic EC. Shown are Northern blots of RNA extracted from bovine aortic EC (Fig. S1) or from retinal pigment epithelium (RPE) that have been exposed to VEGF (V) or not (0). Blots were probed for thrombospondin-1 (TSP-1), NPC-2, and N4. Band intensity ratios were normalized to 1 for non-VEGF-exposed cells.

Fig. 2.

N4 inhibits EC tube formation and migration induced by VEGF. (A) HUAEC were transfected with control siRNA or N4 siRNA before being incubated on top of Matrigel. Silencing the N4 gene in HUAEC increased both the branching and their ability to form tubular structures on Matrigel compared with nontransfected cells (NT) or with cells transfected with the control siRNA. RT-PCR analysis indicates that the N4 gene has been selectively silenced in N4 siRNA-treated HUAEC. (B) N4 does not affect the basal migration of growth-arrested HUAEC (C) but inhibits the migration induced by VEGF (V) as well as N1 (N1). (C) N1 and N4 inhibit the VEGF-induced FAK phosphorylation in HUAEC (Upper). (Lower) The total FAK immunoprecipitated. The ratio of phosphorylated FAK to total FAK is normalized to unstimulated HUAEC. **, P < 0.01; *, P < 0.05.

Fig. 3.

N4 is a neogenin ligand. (A) HUAEC and HUVEC express Unc5B (UB), Unc5C (UC), and neogenin (NE). The results are expressed relative to their abundance in human fetal brain and normalized to the β-actin mRNA content. (B) Immunoprecipitation of N1 and N4 by chimera of human Fc IgG fused to neogenin (NE), Unc5B (UB), or Unc5C (UC). VEGFR2 (VR2) is used as a negative control. (C) VEGF-driven HUAEC migration is inhibited by N4 and N1. The activity of N4 is almost totally abolished by addition of the chimera NE but not by chimera UB or UC. In contrast, N1 activity is inhibited by the three chimeras.

Fig. 4.

Neogenin and Unc5B are required for N1- and N4-induced inhibition of HUAEC migration. (A) Anti-neogenin (red) antibody staining shows that NE is localized in filipodia of HUAEC. Upon incubation with N4, NE staining is relocalized in the perinuclear area. Nuclei are labeled in blue with DAPI. (Scale bars: 10 μm.) (B) Silencing either Unc5B or NE is sufficient to abrogate the chemotactic effect of N1 or N4. (C) The inhibition of N4 binding to NE abolishes its function whereas the inhibition of its binding to Unc5B or Unc5C has no effect. (D) Neogenin is equally detected in N4-unstimulated (0) and Netrin-4-stimulated HUAEC (0N4) or in VEGF-stimulated (V) or FGF-2-stimulated (F) HUAEC, whereas N4 increases the interaction between UNC5B and neogenin in VEGF-stimulated (VN4) or FGF-2-stimulated (FN4) HUAEC. Detection of neogenin in CHO-transfected cells served as a control (far right lane, NEO).

Fig. 5.

N4 has an antiangiogenic activity in vivo. (A–C) Antiangiogenic effect of Netrin-4 on choroidal neovascularization. (A and B) Laser-induced choroidal neovascularization is visualized in vehicle-treated PBS (n = 10) (A) or N4-treated (n = 10) (B) mice 7 and 10 days after the onset of angiogenesis. At day 14 the neovascularization was visualized by systemic injection of fluorescent dextran on retinas. (Scale bars: 50 μm.) (C) Quantification of the size of the CNV complex area, scored by morphometric analysis, shows that the choroidal neovascularization is significantly reduced in N4-treated eyes. (D–G) Netrin-4 inhibits PC3 angiogenesis in xenograft tumors. (D) Time course of tumor growth of N4-transfected PC3 (N4 PC3 clones 1 and 5) and empty vector-transfected PC3 cells (EV PC3). N4 PC3 Cl 1 and Cl 5 cells give rise to smaller tumors (n = 8 per group) in nude mice than EV PC3. (E–G) CD31 staining was performed on four distinct tumors of each group [EV PC3 (E) and N4 PC3 (F)]. (G) Five fields were examined by immunofluorescence microscopy, the data were analyzed by Image J software, and results are expressed as mean pixels by square micrometers. Mean vessel density (20 fields per condition) is represented (P < 0.05).