MAB1952Z Sigma-AldrichAnti-Integrin α3 Antibody, clone P1B5, azide free

The synaptic protein Neuroligin 1 (NLGN1), a cell adhesion molecule, is critical for the formation and consolidation of synaptic connectivity and is involved in vascular development. The mechanism through which NLGN1 acts, especially in vascular cells, is unknown. Here, we aimed at deepening our knowledge on the cellular activities and molecular pathways exploited by endothelial NLGN1 both in vitro and in vivo. We analyzed the phenotypic consequences of NLGN1 expression modulation in endothelial cells through in vitro angiogenesis assays and the mouse postnatal retinal angiogenesis model. We demonstrate that NLGN1, whereas not affecting endothelial cell proliferation or migration, modulates cell adhesion to the vessel stabilizing protein laminin through cooperation with the α6 integrin, a specific laminin receptor. Finally, we show that in vivo, NLGN1 and α6 integrin preferentially colocalize in the mature retinal vessels, whereas NLGN1 deletion causes an aberrant VE-cadherin, laminin and α6 integrin distribution in vessels, along with significant structural defects in the vascular tree.

Growth cones integrate a remarkably complex concert of chemical cues to guide axons to their appropriate destinations. Recent work suggests that integrins contribute to axon guidance by interacting with a wide range of extracellular molecules including axon guidance molecules, by mechanisms that are not fully understood. Here, we describe an interaction between integrins and netrin-1 in growth cones that contributes to growth cone collapse. Our data show that netrin-1 causes growth cone collapse in a substratum-specific manner and is integrin-dependent. Netrin-1 causes collapse of cultured chick dorsal root ganglion (DRG) growth cones extending on high levels of laminin-1 (LN) but not growth cones extending on low levels of LN or on fibronectin. Blocking integrin function significantly decreases netrin-induced growth cone collapse on high LN. Netrin-1 and integrins interact on growth cones; netrin-1 causes integrin activation, a conformational shift to a high ligand-affinity state. Netrin-1 directly binds to integrin α3 and α6 peptides, further suggesting a netrin-integrin interaction. Interestingly, our data reveal that netrin-1 increases growth cone levels of cAMP in a substratum-specific manner and that netrin-induced growth cone collapse requires increased cAMP in combination with integrin activation. Manipulations that either decrease cAMP levels or integrin activation block netrin-induced collapse. These results imply a common mechanism for growth cone collapse and novel interactions between integrins, netrin-1 and cAMP that contribute to growth cone guidance.

Severe skin loss constitutes a major unsolved clinical problem worldwide. For this reason, in the last decades there has been a major push towards the development of novel therapeutic approaches to enhance skin wound healing. Neo-vessel formation through angiogenesis is a critical step during the wound healing process. Besides the contribution of pre-existing endothelial cells (EC), endothelial progenitor cells (EPCs) have also been implicated in wound healing acting either by differentiating into EC that incorporate the neo-vessels, or via the production of paracrine factors that improve angiogenesis. Here we tested the importance of different extracellular matrices (ECM) in regulating the angiogenic and wound healing potential of cord blood-derived EPC (CB-EPC). We compared the properties of several ECM and particularly of fibrin fragment E (FbnE) in regulating EPC adhesion, proliferation, differentiation and healing-promotion in vitro and in vivo. Our results show that CB-EPCs have increased adhesion and endothelial differentiation when plated on FbnE compared to collagens, fibronectin or fibrin. Using integrin neutralizing antibodies, we show that CB-EPC adhesion to FbnE is mediated by integrin ?5?1. Gene expression analysis of CB-EPCs plated on different substrates revealed that CB-EPC grown on FbnE shows increased expression of paracrine factors such as VEGF-A, TGF-?1, SDF-1, IL-8 and MIP-1?. Accordingly, conditioned media from CB-EPC grown on FbnE induced EC tube formation and monocyte migration in vitro. To test the wound healing effects of FbnE in vivo we used an FbnE enriched scaffold in a cutaneous wound healing mouse model. In accordance with our in vitro data, co-administration of the FbnE enriched scaffold with CB-EPC significantly accelerated wound closure and wound vascularization, compared FbnE enriched scaffold alone or to using collagen-based scaffolds. Our results show that FbnE modulates several CB-EPC properties in vivo and in vitro, and as such promotes wound healing. We suggest the use of FbnE-based scaffolds represents a promising approach to resolve wound healing complications arising from different pathologies.

Here we show that endothelial cells (EC) require matrix type 1-metalloproteinase (MT1-MMP) for the formation of lumens and tube networks in 3-dimensional (3D) collagen matrices. A fundamental consequence of EC lumen formation is the generation of vascular guidance tunnels within collagen matrices through an MT1-MMP-dependent proteolytic process. Vascular guidance tunnels represent a conduit for EC motility within these spaces (a newly remodeled 2D matrix surface) to both assemble and remodel tube structures. Interestingly, it appears that twice as many tunnel spaces are created than are occupied by tube networks after several days of culture. After tunnel formation, these spaces represent a 2D migratory surface within 3D collagen matrices allowing for EC migration in an MMP-independent fashion. Blockade of EC lumenogenesis using inhibitors that interfere with the process (eg, integrin, MMP, PKC, Src) completely abrogates the formation of vascular guidance tunnels. Thus, the MT1-MMP-dependent proteolytic process that creates tunnel spaces is directly and functionally coupled to the signaling mechanisms required for EC lumen and tube network formation. In summary, a fundamental and previously unrecognized purpose of EC tube morphogenesis is to create networks of matrix conduits that are necessary for EC migration and tube remodeling events critical to blood vessel assembly.

Host interactions with tumor cells contribute to tumor progression by several means. This study was done to determine whether mammary epithelium could interact with breast carcinoma by producing substances capable of inducing motility in the cancer cells. Conditioned medium of immortalized 184A1 mammary epithelium collected in serum-free conditions induced dose-dependent motility in the MCF-7 breast carcinoma cell line by both a semiquantitative scattering assay and a Boyden chamber assay. Purification of the motility factor revealed that it was laminin 332 (formerly laminin 5) by mass spectroscopy. A Western blot of the 184A1 conditioned medium using a polyclonal antibody confirmed the presence of laminin 332 in the conditioned medium. Blockage of the motility with antibodies to the laminin 332 and its receptor components, alpha(3) and beta(1) integrins, provided further evidence that tumor cell motility was caused by the laminin 332 in the conditioned medium. Invasion of MCF-7, BT-20, and MDA-MB-435 S was induced by purified laminin 332 and 184A1 conditioned medium and blocked by an anti-alpha(3) integrin antibody. Staining of carcinoma in situ from breast cancer specimens revealed that laminin 332 in the myoepithelium adjacent to the preinvasive cells provided a source of laminin 332 that could potentially encourage the earliest steps of stromal invasion. In metaplastic breast carcinomas, the presence of laminin 332-producing cells coexpressing alpha(3) integrin and the greater metastatic potential of tumors with higher laminin 332 levels suggest that laminin 332 expression is associated with aggressive features in these human breast cancers.

Cutaneous melanoma represents the leading cause of skin cancer deaths. The prognosis of highly aggressive, metastatic melanoma is still very poor, due to the resistance of the disseminated tumor to existing therapies. The clarification of the molecular mechanisms regulating melanoma growth and progression might help identify novel molecular targets for the development of new therapeutic interventions. We previously showed that gonadotropin-releasing hormone (GnRH) receptors are expressed in melanoma cells; activation of these receptors by means of GnRH agonists significantly reduces cell proliferation. In the current study, we first showed that GnRH agonists significantly reduced the metastatic behavior of melanoma cells in terms of both cell motility (haptotactic assay using laminin as the chemoattractant) and invasiveness (cell invasion assay evaluating the capacity of the cells to invade a reconstituted extracellular matrix barrier). On the basis of this observation, we then investigated the molecular mechanisms underlying the antimetastatic activity of GnRH agonists. We found that, in melanoma cells, a) the activity of the alpha3 integrin subunit is crucial for the migratory behavior of the cells; b) GnRH agonists significantly reduced alpha3 integrin expression (Western blotting and immunofluorescence studies); c) GnRH agonists significantly reduced MMP-2 expression (comparative RT-PCR) and activity (zymographic analysis performed on cell culture media). These data indicate that GnRH agonists, in addition to the previously reported antiproliferative effect, elicit a strong inhibitory activity on the migratory/invasive behavior of melanoma cells expressing GnRH receptors. These compounds reduce the metastatic potential of melanoma cells by interfering with the expression/activity of cell adhesion molecules (alpha3 integrin) and matrix metalloproteinase (MMP-2).

Gonadotropin-releasing hormone (GnRH) neurons, a small number of cells scattered in the hypothalamic region of the basal forebrain, play an important role in reproductive function. These cells originate in the olfactory placode and migrate into the basal forebrain in late embryonic life. Here, we show that reelin, which is expressed along the route of the migrating cells, has an inhibitory role in guiding GnRH neurons to the basal forebrain. Only a small (approximately 5%) subpopulation of these neurons expresses one of the reelin receptors (ApoER2/Lrp8), and all GnRH neurons appear to lack the intracellular adaptor protein Dab1, suggesting that the function of reelin is not mediated by the conventional signal transduction pathway. The importance of reelin in the establishment of GnRH neurons in the hypothalamus was confirmed by our finding that the brains of developing and adult reeler mice of both sexes contained a markedly reduced number of these neuroendocrine neurons. Furthermore, the testes of adult males showed dilation of seminiferous tubules and reduction in their density when compared with controls. Mutants lacking the reelin receptors ApoER2 and Vldlr, and scrambler mice lacking Dab1, showed a normal complement of GnRH neurons in the hypothalamus, confirming that the effect of reelin in their migration is independent of Dab1.

Collagens have been shown to influence the survival and function of cultured beta-cells; however, the utilization and function of individual collagen receptors in beta-cells is largely unknown. The integrin superfamily contains up to five collagen receptors, but we have determined that alpha(1)beta(1) is the primary receptor utilized by both fetal and adult beta-cells. Cultured beta-cells adhered to and migrated on collagen type IV (Col-IV), and these responses were mediated almost exclusively by alpha(1)beta(1). The migration of cultured beta-cells to Col-IV significantly exceeded that to other matrix components suggesting that this substrate is of unique importance for beta-cell motility. The interaction of alpha(1)beta(1) with Col-IV also resulted in significant insulin secretion at basal glucose concentrations. A subset of beta-cells in developing islets was confirmed to express alpha(1)beta(1), and this expression co-localized with Col-IV in the basal membranes of juxtaposed endothelial cells. Our findings indicate that alpha(1)beta(1) and Col-IV contribute to beta-cell functions known to be important for islet morphogenesis and glucose homeostasis.

Reelin is a secreted glycoprotein that regulates neuronal positioning in cortical brain structures through the VLDLR and ApoER2 receptors and the adaptor protein Dab1. In addition to cellular disorganization, dendrite abnormalities are present in the brain of reeler mice lacking Reelin. It is unclear whether these defects are due primarily to cellular ectopia or the absence of Reelin. Here we examined dendrite development in the hippocampus of normal and mutant mice and in dissociated cultures. We found that dendrite complexity is severely reduced in homozygous mice deficient in Reelin signaling both in vivo and in vitro, and it is also reduced in heterozygous mice in the absence of cellular ectopia. Addition of Reelin interfering antibodies, receptor antagonists, and Dab1 phosphorylation inhibitors prevented dendrite outgrowth from normal neurons, whereas addition of recombinant Reelin rescued the deficit in reeler cultures. Thus, the same signaling pathway controls both neuronal migration and dendrite maturation.