ABSTRACT
BACKGROUND: 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. METHODS: 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. RESULTS: 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 α6ß1, expressed in HUVEC. However, the angiogenic response of Netrin-1 seems not to be mediated through the RhoA/ROCK pathway. CONCLUSIONS: 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.
Subject(s)
Chorioallantoic Membrane/drug effects , Human Umbilical Vein Endothelial Cells/drug effects , Mesenchymal Stem Cells/metabolism , Neovascularization, Physiologic/drug effects , Nerve Growth Factors/pharmacology , Tumor Suppressor Proteins/pharmacology , Wharton Jelly/metabolism , Animals , Biological Assay , Cell Movement , Chick Embryo , Chorioallantoic Membrane/blood supply , Chorioallantoic Membrane/cytology , Culture Media, Conditioned/chemistry , Culture Media, Conditioned/pharmacology , Dose-Response Relationship, Drug , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Integrin alpha6beta1/genetics , Integrin alpha6beta1/metabolism , Mesenchymal Stem Cells/cytology , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Netrin Receptors , Netrin-1 , Primary Cell Culture , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Signal Transduction , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Wharton Jelly/cytologyABSTRACT
BACKGROUND: Several evidences indicate that hormones and neuropeptides function as immunomodulators. Among these, growth hormone (GH) is known to act on the thymic microenvironment, supporting its role in thymocyte differentiation. The aim of this study was to evaluate the effect of GH on human thymocytes and thymic epithelial cells (TEC) in the presence of laminin. RESULTS: GH increased thymocyte adhesion on BSA-coated and further on laminin-coated surfaces. The number of migrating cells in laminin-coated membrane was higher in GH-treated thymocyte group. In both results, VLA-6 expression on thymocytes was constant. Also, treatment with GH enhanced laminin production by TEC after 24 h in culture. However, VLA-6 integrin expression on TEC remained unchanged. Finally, TEC/thymocyte co-culture model demonstrated that GH elevated absolute number of double-negative (CD4(-)CD8(-)) and single-positive CD4(+) and CD8(+) thymocytes. A decrease in cell number was noted in double-positive (CD4(+)CD8(+)) thymocytes. CONCLUSIONS: The results of this study demonstrate that GH is capable of enhancing the migratory capacity of human thymocytes in the presence of laminin and promotes modulation of thymocyte subsets after co-culture with TEC.
Subject(s)
Epithelial Cells/drug effects , Growth Hormone/pharmacology , Laminin/biosynthesis , Thymocytes/drug effects , Thymus Gland/cytology , Analysis of Variance , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Cell Adhesion/drug effects , Cell Differentiation/drug effects , Cell Movement/drug effects , Cells, Cultured , Child , Child, Preschool , Coculture Techniques , Flow Cytometry/methods , Humans , Immunohistochemistry , Infant , Infant, Newborn , Integrin alpha6beta1/analysis , Integrin alpha6beta1/metabolism , Laminin/drug effects , Reference Values , Thymus Gland/metabolism , Time FactorsABSTRACT
BACKGROUND: Several evidences indicate that hormones and neuropeptides function as immunomodulators. Among these, growth hormone (GH) is known to act on the thymic microenvironment, supporting its role in thymocyte differentiation. The aim of this study was to evaluate the effect of GH on human thymocytes and thymic epithelial cells (TEC) in the presence of laminin. RESULTS: GH increased thymocyte adhesion on BSA-coated and further on laminin-coated surfaces. The number of migrating cells in laminin-coated membrane was higher in GH-treated thymocyte group. In both results, VLA-6 expression on thymocytes was constant. Also, treatment with GH enhanced laminin production by TEC after 24 h in culture. However, VLA-6 integrin expression on TEC remained unchanged. Finally, TEC/thymocyte co-culture model demonstrated that GH elevated absolute number of double-negative (CD4-CD8-) and single-positive CD4+ and CD8+ thymocytes. A decrease in cell number was noted in double-positive (CD4+CD8+) thymocytes. CONCLUSIONS: The results of this study demonstrate that GH is capable of enhancing the migratory capacity of human thymocytes in the presence of laminin and promotes modulation of thymocyte subsets after co-culture with TEC.