Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton's jelly mesenchymal stem cells (WJ-MSC).

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.

Growth hormone in the presence of laminin modulates interaction of human thymic epithelial cells and thymocytes in vitro.

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.

Growth hormone in the presence of laminin modulates interaction of human thymic epithelial cells and thymocytes in vitro

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.

Differential integrin expression by T lymphocytes: potential role in DMD muscle damage.

The expression and function of integrin-type extracellular matrix receptors, VLA-4 and VLA-5, and laminin receptor VLA-6 on the surface of CD3(+)CD4(+) and CD3(+)CD8(+) defined T cell populations was evaluated in the blood of Duchenne muscular dystrophy (DMD) patients and healthy individuals. Both the number of CD4(+) and CD8(+) T cell subsets expressing VLA-4 or VLA-5 and the fibronectin-driven T cell migration was significantly higher in DMD patients. These data indicate that interactions of VLA-4 and/or VLA-5 with fibronectin may drive T lymphocytes to specific niches within muscle, contributing to tissue damage and fibrosis in DMD patients.

Laminin/VLA-6 interactions and T cell function.

A growing number of investigators consider extracellular matrix (ECM) proteins to be determinant factors in lymphocyte positioning and activation. One major ECM component is laminin, which is constitutively expressed in the thymus as well as in thymus-dependent areas of peripheral lymphoid organs. In the thymus, laminin is produced by epithelial and dendritic cells, and appears to mediate interactions with thymocytes through specific laminin receptors, in particular the integrin VLA-6. This receptor is also expressed by peripheral T cells, and is apparently involved in effector T cell migration and activation. We showed that CD4+ T lymphocytes from chronic chagasic mice exhibited an increase in the absolute and relative number of cells with high VLA-6 expression. Additionally, it is likely that VLA-6/laminin interactions are required for the development of the CD4+T cell-dependent anti-myocardial autoreactive process that occurs in these animals. Lastly, laminin can bind to some cytokines, a fact that may represent an additional mechanism by which this extracellular matrix component modulates the behavior of T lymphocytes. Taken together, the present data strongly indicate that interactions involving laminin and VLA-6 are functionally linked to relevant events in T cell physiology, comprising entrance of pro-thymocytes into the thymus, intrathymic T cell migration and differentiation, as well as the functioning of mature T lymphocytes, including effector cells.

Extracellular matrix components of the mouse thymus microenvironment. V. Interferon-gamma modulates thymic epithelial cell/thymocyte interactions via extracellular matrix ligands and receptors.

Extracellular matrix (ECM) proteins influence cell migration and differentiation in a variety of cell systems. Within the thymus, the ECM distribution pattern is conserved among various mammalian species, but its physiological role is not completely understood. Interferon-gamma (IFN-gamma), a cytokine produced by thymocytes, is able to in vitro modulate ECM production by thymic epithelial cells (TEC) in a dose-dependent biphasic pattern. In the same model, we determined herein that the expression of VLA-5 and VLA-6 (fibronectin and laminin receptors, respectively) were upregulated by low doses of IFN-gamma, whereas high doses of the cytokine induced an opposite effect. In a second in vitro system, we evidenced that thymocyte adhesion to a TEC line was also modulated by IFN-gamma. Importantly, such effects were due to the biphasic modulation of ECM ligands and receptors since they could be specifically prevented by preincubating the TEC cultures with anti-ECM or anti-ECM receptor antibodies. Additionally, spontaneous thymocyte release from thymic nurse cells was similarly biphasically modulated by IFN-gamma. Our data provide support for the notion that thymocyte-derived products can play a role in the dialogue that exists in the thymus, involving differentiating thymocytes and microenvironmental cells. Moreover, we bring arguments indicating that one of the implicated mechanisms involved is the modulation of ECM ligands and receptors by the thymic epithelium.

alpha6beta1-Integrin, a major cell surface carrier of beta1-6-branched oligosaccharides, mediates migration of EJ-ras-transformed fibroblasts on laminin-1 independently of its glycosylation state.

EJ-ras oncogene-induced malignant transformation is characterized by a series of changes in cell surface carbohydrates and cell-cell and cell-matrix interactions. Here, we show that EJ-ras-transformed NIH-3T3 fibroblasts acquired a migratory phenotype on laminin-1 surfaces. Such a phenotype was accompanied by overexpression of: (a) functional alpha6beta1, but not other laminin binding beta1-integrins; and (b) glycoconjugates on the cell surface bearing large oligosaccharides recognized by leukoagglutinin from Phaseolus vulgaris (L-PHA). The internal pool of pre-beta1-integrins was differently regulated in EJ-ras-transformed cells compared with nontransfected fibroblasts. Conversion of pre-beta1- into mature beta1-integrins was faster in EJ-ras-transformed cells, a process associated with the overexpression of the alpha6-chain. Overexpression of L-PHA-reactive oligosaccharides is dependent on the activity of N-acetylglucosaminyltransferase V, which is increased in transformed cells [J. W. Dennis et al., Science (Washington DC), 236: 582-585, 1987]. We show that beta1-integrins were the major carriers of L-PHA-reactive oligosaccharides on the cell surface. This glycosylation pattern, however, was not necessary for either the cell surface expression of beta1-integrins or their functional activity in the migratory response to laminin-1. Moreover, EJ-ras-transformed fibroblasts aggregated spontaneously. These effects were not observed in c-jun-transfected fibroblasts, which were unable to migrate on laminin, did not overexpress either beta1-integrins or L-PHA-reactive oligosaccharides, and did not self-aggregate.

Adhesion to laminin is down-regulated upon retinoic acid-induced F9 cell differentiation: a role for alpha 6/beta 1 integrin.

F9 mouse teratocarcinoma cells have a high capacity to adhere to laminin and we identified alpha 6/beta 1 integrin as the principal laminin-binding protein present in these cells. F9 cells differentiated into parietal endoderm when monolayer cultures were treated with retinoic acid and dibutyryl cyclic AMP. In this process a decreased adherence to laminin was observed due to a lower expression of alpha 6/beta 1 integrin on the cell surface.