Tumour growth requires accompanying expansion of the host vasculature, with tumour progression often correlated with vascular density. Vascular endothelial growth factor (VEGF) is the best-characterized inducer of tumour angiogenesis. We report that VEGF dynamically regulates tumour endothelial expression of Delta-like ligand 4 (Dll4), which was previously shown to be absolutely required for normal embryonic vascular development. To define Dll4 function in tumour angiogenesis, we manipulated this pathway in murine tumour models using several approaches. Here we show that blockade resulted in markedly increased tumour vascularity, associated with enhanced angiogenic sprouting and branching. Paradoxically, this increased vascularity was non-productive-as shown by poor perfusion and increased hypoxia, and most importantly, by decreased tumour growth-even for tumours resistant to anti-VEGF therapy. Thus, VEGF-induced Dll4 acts as a negative regulator of tumour angiogenesis; its blockade results in a striking uncoupling of tumour growth from vessel density, presenting a novel therapeutic approach even for tumours resistant to anti-VEGF therapies.

Intercellular junctions mediate adhesion and communication between adjoining cells. Although formed by different molecules, tight junctions (TJs) and adherens junctions (AJs) are functionally and structurally linked, but the signalling pathways behind this interaction are unknown. Here we describe a cell-specific mechanism of crosstalk between these two types of structure. We show that endothelial VE-cadherin at AJs upregulates the gene encoding the TJ adhesive protein claudin-5. This effect requires the release of the inhibitory activity of forkhead box factor FoxO1 and the Tcf-4-beta-catenin transcriptional repressor complex. Vascular endothelial (VE)-cadherin acts by inducing the phosphorylation of FoxO1 through Akt activation and by limiting the translocation of beta-catenin to the nucleus. These results offer a molecular basis for the link between AJs and TJs and explain why VE-cadherin inhibition may cause a marked increase in permeability.

No abstract

We report the cloning of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-binding protein (ABP), a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP) with two sets of three PDZ domains, which binds the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. We show that the protein interactions of the ABP/GRIP family differ from the PSD-95 family, which binds N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and heteromultimers through PDZ-PDZ interactions but do not bind PSD-95. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors.

Despite genetic evidence establishing angiopoietin-1 (Ang-1) as an essential regulator of vascular development, the molecular mechanisms underlying Ang-1 function are almost completely uncharacterized. In this report, we demonstrate that Ang-1, via Akt activation, is a potent inhibitor of the forkhead transcription factor FKHR (FOXO1), identifying for the first time a nuclear signaling pathway through which Ang-1 modulates gene expression. We use microarray analysis to show that FKHR, whose function in endothelial cells has not previously been elucidated, regulates many genes associated with vascular destabilization and remodeling (including angiopoietin-2, an Ang-1 antagonist) and endothelial cell apoptosis (e.g., survivin, TRAIL). Ang-1 inhibits FKHR-mediated changes in gene expression and FKHR-induced apoptosis. Analysis of gene expression changes induced by an activated version of Akt confirms that FKHR is a major target through which Akt regulates transcription in endothelial cells. We use RNA interference to demonstrate that FKHR is required for the expression of genes (including Ang-2) that have important vascular functions. Our data suggest a novel, tissue-specific role for the Akt/FKHR pathway in the vasculature and suggest a mechanistic basis for the previously described actions of Ang-1 as a regulator of endothelial cell survival and blood vessel stability.[Keywords: Ang-1; FKHR; Akt; endothelial cell] Supplemental material is available at http://www.genesdev.org.

We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, Myc-GluR2delta1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate.

Angiopoietin (Ang)-2, a context-dependent agonist͞antagonist for the vascular-specific Tie2 receptor, is highly expressed by endothelial cells at sites of normal and pathologic angiogenesis. One prevailing model suggests that in these settings, Ang-2 acts as an autocrine Tie2 blocker, inhibiting the stabilizing influence of the Tie2 activator Ang-1, thereby promoting vascular remodeling. However, the effects of endogenous Ang-2 on cells that are actively producing it have not been studied in detail. Here, we demonstrate that Ang-2 expression is rapidly induced in endothelial cells by the transcription factor FOXO1 after inhibition of the phosphatidylinositol 3-kinase͞Akt pathway. We employ RNAi and blocking antibodies to show that in this setting, Ang-2 unexpectedly functions as a Tie2 agonist, bolstering Akt activity so as to provide negative feedback on FOXO1-regulated transcription and apoptosis. In addition, we show that Ang-2, like Ang-1, activates Tie2͞Akt signaling in vivo, thereby inhibiting the expression of FOXO1 target genes. Consistent with a role for Ang-2 as a Tie2 activator, we demonstrate that Ang-2 inhibits vascular leak. Our data suggests a model in which Ang-2 expression is induced in stressed endothelial cells, where it acts as an autocrine Tie2 agonist and protective factor.

tributed to the design of experiments, data analysis, and interpretation of results. PB provided transgenic mice. DMM contributed to the design of experiments, data analysis, interpretation of results, and writing of the manuscript.