EGFL7 ligates αvß3 integrin to enhance vessel formation.

Angiogenesis, defined as blood vessel formation from a preexisting vasculature, is governed by multiple signal cascades including integrin receptors, in particular integrin αVß3. Here we identify the endothelial cell (EC)-secreted factor epidermal growth factor-like protein 7 (EGFL7) as a novel specific ligand of integrin αVß3, thus providing mechanistic insight into its proangiogenic actions in vitro and in vivo. Specifically, EGFL7 attaches to the extracellular matrix and by its interaction with integrin αVß3 increases the motility of EC, which allows EC to move on a sticky underground during vessel remodeling. We provide evidence that the deregulation of EGFL7 in zebrafish embryos leads to a severe integrin-dependent malformation of the caudal venous plexus, pointing toward the significance of EGFL7 in vessel development. In biopsy specimens of patients with neurologic diseases, vascular EGFL7 expression rose with increasing EC proliferation. Further, EGFL7 became upregulated in vessels of the stroke penumbra using a mouse model of reversible middle cerebral artery occlusion. Our data suggest that EGFL7 expression depends on the remodeling state of the existing vasculature rather than on the phenotype of neurologic disease analyzed. In sum, our work sheds a novel light on the molecular mechanism EGFL7 engages to govern physiological and pathological angiogenesis.

The polarity protein Scrib is essential for directed endothelial cell migration.

RATIONALE: Polarity proteins are involved in the apico-basal orientation of epithelial cells, but relatively little is known regarding their function in mesenchymal cells. OBJECTIVE: We hypothesized that polarity proteins also contribute to endothelial processes like angiogenesis. METHODS AND RESULTS: Screening of endothelial cells revealed high expression of the polarity protein Scribble (Scrib). On fibronectin-coated carriers Scrib siRNA (siScrib) blocked directed but not random migration of human umbilical vein endothelial cells and led to an increased number and disturbed orientation of cellular lamellipodia. Coimmunoprecipitation/mass spectrometry and glutathione S-transferase (GST) pulldown assays identified integrin α5 as a novel Scrib interacting protein. By total internal reflection fluorescence (TIRF) microscopy, Scrib and integrin α5 colocalize at the basal plasma membrane of endothelial cells. Western blot and fluorescence activated cell sorting (FACS) analysis revealed that silencing of Scrib reduced the protein amount and surface expression of integrin α5 whereas surface expression of integrin αV was unaffected. Moreover, in contrast to fibronectin, the ligand of integrin α5, directional migration on collagen mediated by collagen-binding integrins was unaffected by siScrib. Mechanistically, Scrib supported integrin α5 recycling and protein stability by blocking its interaction with Rab7a, its translocation into lysosomes, and its subsequent degradation by pepstatin-sensitive proteases. In siScrib-treated cells, reinduction of the wild-type protein but not of PSD95, Dlg, ZO-1 (PDZ), or leucine rich repeat domain mutants restored integrin α5 abundance and directional cell migration. The downregulation of Scrib function in Tg(kdrl:EGFP)(s843) transgenic zebrafish embryos delayed the angiogenesis of intersegmental vessels. CONCLUSIONS: Scrib is a novel regulator of integrin α5 turnover and sorting, which is required for oriented cell migration and sprouting angiogenesis.

The F-BAR protein NOSTRIN participates in FGF signal transduction and vascular development.

F-BAR proteins are multivalent adaptors that link plasma membrane and cytoskeleton and coordinate cellular processes such as membrane protrusion and migration. Yet, little is known about the function of F-BAR proteins in vivo. Here we report, that the F-BAR protein NOSTRIN is necessary for proper vascular development in zebrafish and postnatal retinal angiogenesis in mice. The loss of NOSTRIN impacts on the migration of endothelial tip cells and leads to a reduction of tip cell filopodia number and length. NOSTRIN forms a complex with the GTPase Rac1 and its exchange factor Sos1 and overexpression of NOSTRIN in cells induces Rac1 activation. Furthermore, NOSTRIN is required for fibroblast growth factor 2 dependent activation of Rac1 in primary endothelial cells and the angiogenic response to fibroblast growth factor 2 in the in vivo matrigel plug assay. We propose a novel regulatory circuit, in which NOSTRIN assembles a signalling complex containing FGFR1, Rac1 and Sos1 thereby facilitating the activation of Rac1 in endothelial cells during developmental angiogenesis.

Soluble epoxide hydrolase regulates hematopoietic progenitor cell function via generation of fatty acid diols.

Fatty acid epoxides are important lipid signaling molecules involved in the regulation of vascular tone and homeostasis. Tissue and plasma levels of these mediators are determined by the activity of cytochrome P450 epoxygenases and the soluble epoxide hydrolase (sEH), and targeting the latter is an effective way of manipulating epoxide levels in vivo. We investigated the role of the sEH in regulating the mobilization and proliferation of progenitor cells with vasculogenic/reparative potential. Our studies revealed that sEH down-regulation/inhibition impaired the development of the caudal vein plexus in zebrafish, and decreased the numbers of lmo2/cmyb-positive progenitor cells therein. In mice sEH inactivation attenuated progenitor cell proliferation (spleen colony formation), but the sEH products 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME) and 11,12- dihydroxyeicosatrienoic acid stimulated canonical Wnt signaling and rescued the effects of sEH inhibition. In murine bone marrow, the epoxide/diol content increased during G-CSF-induced progenitor cell expansion and mobilization, and both mobilization and spleen colony formation were reduced in sEH(-/-) mice. Similarly, sEH(-/-) mice showed impaired functional recovery following hindlimb ischemia, which was rescued following either the restoration of bone marrow sEH activity or treatment with 12,13-DiHOME. Thus, sEH activity is required for optimal progenitor cell proliferation, whereas long-term sEH inhibition is detrimental to progenitor cell proliferation, mobilization, and vascular repair.

Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish.

The extracellular matrix is crucial for organogenesis. It is a complex and dynamic component that regulates cell behavior by modulating the activity, bioavailability and presentation of growth factors to cell surface receptors. Here, we determined the role of the extracellular matrix protein Nephronectin (Npnt) in heart development using the zebrafish model system. The vertebrate heart is formed as a linear tube in which myocardium and endocardium are separated by a layer of extracellular matrix termed the cardiac jelly. During heart development, the cardiac jelly swells at the atrioventricular (AV) canal, which precedes valve formation. Here, we show that Npnt expression correlates with this process. Morpholino-mediated knockdown of Npnt prevents proper valve leaflet formation and trabeculation and results in greater than 85% lethality at 7 days post-fertilization. The earliest observed phenotype is an extended tube-like structure at the AV boundary. In addition, the expression of myocardial genes involved in cardiac valve formation (cspg2, fibulin 1, tbx2b, bmp4) is expanded and endocardial cells along the extended tube-like structure exhibit characteristics of AV cells (has2, notch1b and Alcam expression, cuboidal cell shape). Inhibition of has2 in npnt morphants rescues the endocardial, but not the myocardial, expansion. By contrast, reduction of BMP signaling in npnt morphants reduces the ectopic expression of myocardial and endocardial AV markers. Taken together, our results identify Npnt as a novel upstream regulator of Bmp4-Has2 signaling that plays a crucial role in AV canal differentiation.

A mutation in zebrafish hmgcr1b reveals a role for isoprenoids in vertebrate heart-tube formation.

In vertebrates, the morphogenetic assembly of the primitive heart tube requires the medial migration and midline fusion of the bilateral myocardial epithelia. Several mutations that result in abnormal heart-tube formation have been studied; however, an understanding of the underlying molecular and cellular mechanisms of the migration and fusion of these epithelial sheets is far from complete. In a forward genetic screen to identify genes regulating early zebrafish heart development, we identified a mutation in the 3-hydroxy-3-methylglutaryl-Coenzyme A reductase 1b (hmgcr1b) gene that affects myocardial migration to the midline and subsequent heart-tube morphogenesis. The mutant phenotype can be rescued with injections of mevalonate, the direct product of HMGCR activity. Furthermore, treatment of embryos with pharmacological inhibitors of isoprenoid synthesis, which occurs downstream of mevalonate production, resulted in defective heart-tube formation. Interestingly, in hmgcr1b mutant embryos and embryos treated with HMGCR inhibitors, both RasCT20-eGFP and RhoaCT32-eGFP fusion proteins were mislocalized away from the plasma membrane in embryonic myocardial cells. We conclude that protein prenylation, acting downstream of Hmgcr1b and possibly through Ras and, or, Rho signaling, is required for the morphogenesis of the myocardial sheets for formation of the primitive heart tube.