Apelin Controls Angiogenesis-Dependent Glioblastoma Growth.

Glioblastoma (GBM) present with an abundant and aberrant tumor neo-vasculature. While rapid growth of solid tumors depends on the initiation of tumor angiogenesis, GBM also progress by infiltrative growth and vascular co-option. The angiogenic factor apelin (APLN) and its receptor (APLNR) are upregulated in GBM patient samples as compared to normal brain tissue. Here, we studied the role of apelin/APLNR signaling in GBM angiogenesis and growth. By functional analysis of apelin in orthotopic GBM mouse models, we found that apelin/APLNR signaling is required for in vivo tumor angiogenesis. Knockdown of tumor cell-derived APLN massively reduced the tumor vasculature. Additional loss of the apelin signal in endothelial tip cells using the APLN-knockout (KO) mouse led to a further reduction of GBM angiogenesis. Direct infusion of the bioactive peptide apelin-13 rescued the vascular loss-of-function phenotype specifically. In addition, APLN depletion massively reduced angiogenesis-dependent tumor growth. Consequently, survival of GBM-bearing mice was significantly increased when APLN expression was missing in the brain tumor microenvironment. Thus, we suggest that targeting vascular apelin may serve as an alternative strategy for anti-angiogenesis in GBM.

Production, characterization, and efficient transfection of highly pure oligodendrocyte precursor cultures from mouse embryonic neural progenitors.

Much current knowledge of oligodendrocyte biology, the myelin-forming cells in the central nervous system, comes from cell culture studies mainly from postnatal rat tissue but mouse cells have been much more difficult to produce in large quantities. We have developed a high yield protocol for production of oligodendrocyte precursor cells from mouse embryonic neural progenitors grown as neurospheres. Neurospheres can be maintained and expanded for long periods in culture in the presence of epidermal growth factor (EGF). When floating neurospheres were plated on substrate-coated dishes in media supplemented with platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), the spheres attached and generated migrating cells that were predominantly oligodendrocyte-lineage cells. Furthermore, cells in spheres could be shifted to the oligodendrocyte phenotype prior to plating on substrate, by incubation in suspension with PDGF/bFGF. Single cell suspensions plated after dissociation of either EGF-treated neurospheres or PDGF/bFGF-treated oligospheres had the bipolar, elongated morphology characteristic of oligodendrocyte precursor cells. mRNA and protein expression analysis of the cells generated by this method confirmed their oligodendrocyte lineage. Oligodendrocyte precursors generated by this method matured in response to ciliary neurotrophic factor treatment, producing cells with multiple processes and myelin-like membranes. The most important aspect of this protocol is the ability to generate very high numbers of relatively pure mouse oligodendrocyte progenitor cells, which can be easily transfected. These studies open up many kinds of investigations on transgenic and mutant mouse oligodendrocytes, thereby providing a valuable tool to study oligodendrocyte biology and development.

Interaction of fascin and protein kinase Calpha: a novel intersection in cell adhesion and motility.

Coordination of protrusive and contractile cell-matrix contacts is important for cell adhesion and migration, but the mechanisms involved are not well understood. We report an unexpected direct association between fascin, an actin-bundling component of filopodia, microspikes and lamellipodial ribs, and protein kinase Calpha (PKCalpha), a regulator of focal adhesions. The association is detectable by protein-protein binding in vitro, by coimmunoprecipitation from cell extracts, and in live cells as fluorescence resonance energy transfer detected by fluorescence imaging lifetime microscopy. The interaction is physiologically regulated by the extracellular matrix context of cells, depends on activation of PKCalpha and is mediated by the C1B domain of PKCalpha. Strikingly, a fascin mutant, fascin S39D, associates constitutively with PKCalpha. Through use of a newly developed set of membrane-permeable peptides that separately inhibit either fascin/PKCalpha or fascin/actin binding, we have uncovered that specific blockade of the fascin/PKCalpha interaction increases cell migration on fibronectin in conjunction with increased fascin protrusions and remodeling of focal adhesions. These results identify the fascin-PKCalpha interaction as an important novel intersection in the regulation and networking of cell-matrix contacts.

Characterisation of Drosophila thrombospondin defines an early origin of pentameric thrombospondins.

Thrombospondins (TSPs) are multidomain oligomers that have complex roles in cell interactions and tissue organisation. The five vertebrate TSPs comprise two subgroups, A and B, that are assembled as trimers or pentamers, respectively. An invertebrate TSP was recently discovered in Drosophila melanogaster, but there is no knowledge of the oligomerisation status or properties of this molecule. We developed by bioinformatics a new dataset containing the single TSP of Drosophila melanogaster and four other newly identified invertebrate TSPs to examine the phylogenetic relationships of TSPs. These analyses clearly indicate pentamerisation as an early attribute of TSPs. We demonstrate experimentally that D.melanogaster TSP is assembled as a pentamer, has heparin-binding activity and is a component of extracellular matrix (ECM). During embryogenesis, the TSP transcript is concentrated at muscle attachment sites and is expressed by a subset of myoblasts and in imaginal discs. These novel results establish TSPs as highly conserved ECM components in both invertebrates and vertebrates and open fresh perspectives on the conservation of structure and biological function within this family.