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.

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Netrins are secreted molecules involved in axon guidance and angiogenesis. We previously showed that Netrin-4 acts as an anti-angiogenic factor by inhibiting endothelial cell (EC) functions. In this study, we investigated the effects of Netrin-4 on vascular smooth muscle cell (VSMC) activity in vitro and in vivo. We show that exogenous Netrin-4 stimulated VSMC adhesion and migration, and increased their coverage on EC tubes (grown on a Matrigel substrate). siRNA knock-down of endogenous Netrin-4 expression in VSMC decreased their recruitment to EC tubes. VSMC expressed Netrin-4 and three of the six Netrin-1 cognate receptors: DCC, Neogenin, and Unc5B. Silencing of these receptors reduced Netrin-4 adhesion to VSMC, strongly suggesting that these receptors were involved in the recruitment process. We previously showed that Netrin-4 overexpression in PC3 cancer cells delayed tumor growth in a model of subcutaneous xenograft by reducing tumor vessel density. Here, we show that Netrin-4 overexpression improved tumor blood vessel structure and increased VSMC coverage. Thus, Netrin-4 induced mural cell recruitment may play a role in the inhibition of tumor growth. Our data suggest that Netrin-4 is important for blood vessel normalization through the regulation of both endothelial and perivascular cells.

In vivo, bone marrow-derived multipotent mesenchymal stromal cells (MSC) have been identified at sites of tumors, suggesting that specific signals mobilize and activate MSC to migrate to areas surrounding tumors. The signals and migratory mechanisms that guide MSC are not well understood. Here, we investigated the migration of human MSC induced by conditioned medium of Huh-7 hepatoma cells (Huh-7 CM). Using a transwell migration system, we showed that human MSC migration was increased in the presence of Huh-7 CM. Using a human cytokine antibody array, we detected increased levels of MIP-1d and MIP-3a in Huh-7 CM. Recombinant chemokines MIP-1d and MIP-3a induced MSC migration. Anti-MIP-1d and anti-MIP-3a antibodies added to Huh-7 CM decreased MSC migration, further suggesting that MIP-1d and MIP-3a were implicated in the Huh-7 CM-induced MSC migration. By real-time polymerase chain reaction, we observed an absence of chemokine receptors CCR2 and CXCR2 and low expression of CCR1, CCR5, and CCR6 in MSC. Expression of these chemokine receptors was not regulated by Huh-7 CM. Furthermore, matrix metalloproteinase 1 (MMP-1) expression was strongly increased in MSC after incubation with Huh-7 CM, suggesting that MSC migration depends on MMP-1 activity. The signaling pathway MAPK/ERK was activated by Huh-7 CM but its inhibition by PD98059 did not impair Huh-7 CM-induced MSC migration. Further, long-term incubation of MSC with MIP-1d increased a-smooth muscle actin expression, suggesting its implication in the Huh-7 CM-induced evolvement of MSC into myofibroblasts. In conclusion, we report that two inflammatory cytokines, MIP-1d and MIP-3a, are able to increase MSC migration in vitro. These cytokines might be responsible for migration and evolvement of MSC into myofibroblasts around tumors.

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