title: Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton's jelly mesenchymal stem cells (WJ-MSC).
abstract: Angiogenesis, 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.
  
  
  
  Mesenchymal 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.
  
  
  
  The 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.
  
  
  
  Thus, 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.
authors.affiliation: Laboratory of Stem Cells and Developmental Biology, Faculty of Sciences, University of Chile, Santiago de Chile, Chile.
authors.forename: Catalina P
authors.initials: CP
authors.lastname: Prieto
authors.affiliation: Laboratory of Stem Cells and Developmental Biology, Faculty of Sciences, University of Chile, Santiago de Chile, Chile.
authors.forename: María Carolina
authors.initials: MC
authors.lastname: Ortiz
authors.affiliation: Laboratory of Stem Cells and Developmental Biology, Faculty of Sciences, University of Chile, Santiago de Chile, Chile.
authors.forename: Andrea
authors.initials: A
authors.lastname: Villanueva
authors.affiliation: Laboratory of Stem Cells and Developmental Biology, Faculty of Sciences, University of Chile, Santiago de Chile, Chile.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Villarroel
authors.affiliation: Laboratory of Stem Cells and Developmental Biology, Faculty of Sciences, University of Chile, Santiago de Chile, Chile.
authors.forename: Sandra S
authors.initials: SS
authors.lastname: Edwards
authors.affiliation: Laboratory of Stem Cells and Developmental Biology, Faculty of Sciences, University of Chile, Santiago de Chile, Chile.
authors.forename: Matías
authors.initials: M
authors.lastname: Elliott
authors.affiliation: Campus Oriente, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Chile, Santiago de Chile, Chile.
authors.forename: José
authors.initials: J
authors.lastname: Lattus
authors.affiliation: Campus Oriente, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Chile, Santiago de Chile, Chile.
authors.forename: Sócrates
authors.initials: S
authors.lastname: Aedo
authors.affiliation: Laboratory of Stem Cells and Developmental Biology, Faculty of Sciences, University of Chile, Santiago de Chile, Chile.
authors.forename: Daniel
authors.initials: D
authors.lastname: Meza
authors.affiliation: Laboratory of Stem Cells and Developmental Biology, Faculty of Sciences, University of Chile, Santiago de Chile, Chile.
authors.forename: Pablo
authors.initials: P
authors.lastname: Lois
authors.affiliation: Laboratory of Stem Cells and Developmental Biology, Faculty of Sciences, University of Chile, Santiago de Chile, Chile. vpalma@uchile.cl.
authors.forename: Verónica
authors.identifier: 0000-0002-4320-7525
authors.initials: V
authors.lastname: Palma
mesh_terms: D000818:Animals; D001681:Biological Assay; D002465:Cell Movement; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply / Q000166:cytology / Q000187:drug effects*; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D039121:Integrin alpha6beta1 / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D009414:Nerve Growth Factors / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D000075386:Netrin Receptors; D000075388:Netrin-1; D061251:Primary Cell Culture; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D059631:Wharton Jelly / Q000166:cytology / Q000378:metabolism*
chemical_list: D017077:Culture Media, Conditioned; D039121:Integrin alpha6beta1; C000617351:NTN1 protein, human; D009414:Nerve Growth Factors; D000075386:Netrin Receptors; D011956:Receptors, Cell Surface; D025521:Tumor Suppressor Proteins; C532445:UNC5B protein, human; D000075388:Netrin-1
keywords: Angiogenesis; CAM assay; HUVEC; Netrin-1; Tissue repair; WJ-MSC