BackgroundAngiogenesis, the process in which new blood vessels are formed from preexisting ones, is highly dependent on the presence of classical angiogenic factors. Recent evidence suggests that axonal guidance proteins and their receptors can also act as angiogenic regulators. Netrin, a family of laminin-like proteins, specifically Netrin-1 and 4, act via DCC/Neogenin-1 and UNC5 class of receptors to promote or inhibit angiogenesis, depending on the physiological context.MethodsMesenchymal stem cells secrete a broad set of classical angiogenic factors. However, little is known about the expression of non-canonical angiogenic factors such as Netrin-1. The aim was to characterize the possible secretion of Netrin ligands by Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC). We evaluated if Netrin-1 presence in the conditioned media from these cells was capable of inducing angiogenesis both in vitro and in vivo, using human umbilical vein endothelial cells (HUVEC) and chicken chorioallantoic membrane (CAM), respectively. In addition, we investigated if the RhoA/ROCK pathway is responsible for the integration of Netrin signaling to control vessel formation.ResultsThe paracrine angiogenic effect of the WJ-MSC-conditioned media is mediated at least in part by Netrin-1 given that pharmacological blockage of Netrin-1 in WJ-MSC resulted in diminished angiogenesis on HUVEC. When HUVEC were stimulated with exogenous Netrin-1 assayed at physiological concentrations (10–200 ng/mL), endothelial vascular migration occurred in a concentration-dependent manner. In line with our determination of Netrin-1 present in WJ-MSC-conditioned media we were able to obtain endothelial tubule formation even in the pg/mL range. Through CAM assays we validated that WJ-MSC-secreted Netrin-1 promotes an increased angiogenesis in vivo. Netrin-1, secreted by WJ-MSC, might mediate its angiogenic effect through specific cell surface receptors on the endothelium, such as UNC5b and/or integrin α6β1, expressed in HUVEC. However, the angiogenic response of Netrin-1 seems not to be mediated through the RhoA/ROCK pathway.ConclusionsThus, here we show that stromal production of Netrin-1 is a critical component of the vascular regulatory machinery. This signaling event may have deep implications in the modulation of several processes related to a number of diseases where angiogenesis plays a key role in vascular homeostasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0494-5) contains supplementary material, which is available to authorized users.

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Neogenin-1 (NEO1) is a transmembrane receptor involved in axonal guidance, angiogenesis, neuronal cell migration and cell death, during both embryonic development and adult homeostasis. It has been described as a dependence receptor, because it promotes cell death in the absence of its ligands (Netrin and Repulsive Guidance Molecule (RGM) families) and cell survival when they are present. Although NEO1 and its ligands are involved in tumor progression, their precise role in tumor cell survival and migration remain unclear. Public databases contain extensive information regarding the expression of NEO1 and its ligands Netrin-1 (NTN1) and Netrin-4 (NTN4) in primary neuroblastoma (NB) tumors. Analysis of this data revealed that patients with high expression levels of both NEO1 and NTN4 have a poor survival rate. Accordingly, our analyses in NB cell lines with different genetic backgrounds revealed that knocking-down NEO1 reduces cell migration, whereas silencing of endogenous NTN4 induced cell death. Conversely, overexpression of NEO1 resulted in higher cell migration in the presence of NTN4, and increased apoptosis in the absence of ligand. Increased apoptosis was prevented when utilizing physiological concentrations of exogenous Netrin-4. Likewise, cell death induced after NTN4 knock-down was rescued when NEO1 was transiently silenced, thus revealing an important role for NEO1 in NB cell survival. In vivo analysis, using the chicken embryo chorioallantoic membrane (CAM) model, showed that NEO1 and endogenous NTN4 are involved in tumor extravasation and metastasis. Our data collectively demonstrate that endogenous NTN4/NEO1 maintain NB growth via both pro-survival and pro-migratory molecular signaling.

BackgroundWharton’s jelly-derived mesenchymal stem cells (WJ-MSC) show remarkable therapeutic potential to repair tissue upon injury via paracrine signaling by secreting diverse trophic factors that promote angiogenesis. However, the mechanisms and signaling pathways that regulate the induction of these specific factors are still mostly unknown. Emerging evidence suggests that Sonic hedgehog (SHH) plays a central role in angiogenesis and tissue maintenance. However, its contribution to the angiogenic potential of MSC has not been fully addressed. The aim of this work was to characterize the expression of the SHH pathway components in WJ-MSC primary cultures and to evaluate their angiogenic responsiveness to SHH signaling.MethodsPrimary cell cultures obtained from human umbilical cords were treated with pharmacological modulators of the SHH pathway. We evaluated the modulation of diverse trophic factors in cell lysates, conditioned medium, and functional in vitro assays. In addition, we determined the angiogenic potential of the SHH pathway in the chicken chorioallantoic membrane, an in vivo model.ResultsOur results show that WJ-MSC express components of the canonical SHH pathway and are activated by its signaling. In fact, we provide evidence of basal autocrine/paracrine SHH signaling in WJ-MSC. SHH pathway stimulation promotes the secretion of angiogenic factors such as activin A, angiogenin, angiopoietin 1, granulocyte-macrophage colony-stimulating factor, matrix metallometallopeptidase -9, and urokinase-type plasminogen activator, enhancing the pro-angiogenic capabilities of WJ-MSC both in vitro and in vivo.ConclusionWJ-MSC are a cell population responsive to SHH pathway stimulation. Basal SHH signaling is in part responsible for the angiogenic inductive properties of WJ-MSC. Overall, exogenous activation of the SHH pathway enhances the angiogenic properties of WJ-MSC, making this cell population an ideal target for treating tissue injury.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0653-8) contains supplementary material, which is available to authorized users.

Gestational diabetes mellitus (GDM) is a common metabolic disorder, defined by high blood glucose levels during pregnancy, which affects foetal and post-natal development. However, the cellular and molecular mechanisms of this detrimental condition are still poorly understood. A dysregulation in circulating angiogenic trophic factors, due to a dysfunction of the feto-placental unit, has been proposed to underlie GDM. But even the detailed study of canonical pro-angiogenic factors like vascular endothelial growth factor (VEGF) or basic Fibroblast Growth Factor (bFGF) has not been able to fully explain this detrimental condition during pregnancy. Netrins are non-canonical angiogenic ligands produced by the stroma have shown to be important in placental angiogenesis. In order to address the potential role of Netrin signalling in GDM, we tested the effect of Netrin-1, the most investigated member of the family, produced by Wharton’s Jelly Mesenchymal Stem Cells (WJ-MSC), on Human Umbilical Vein Endothelial Cells (HUVEC) angiogenesis. WJ-MSC and HUVEC primary cell cultures from either healthy or GDM pregnancies were exposed to physiological (5 mM) or high (25 mM) d-glucose. Our results reveal that Netrin-1 is secreted by WJ-MSC from healthy and GDM and both expression and secretion of the ligand do not change with distinct experimental glucose conditions. Noteworthy, the expression of its anti-angiogenic receptor UNC5b is reduced in GDM HUVEC compared with its expression in healthy HUVEC, accounting for an increased Netrin-1 signalling in these cells. Consistently, in healthy HUVEC, UNC5b overexpression induces cell retraction of the sprouting phenotype.