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.

The angiopoietins Ang1 (ANGPT1) and Ang2 (ANGPT2) are secreted factors that bind to the endothelial cellspecific receptor tyrosine kinase Tie2 (TEK) and regulate angiogenesis. Ang1 activates Tie2 to promote blood vessel maturation and stabilization. In contrast, Ang2, which is highly expressed by tumor endothelial cells, is thought to inhibit Tie2 activity and destabilize blood vessels, thereby facilitating VEGF-dependent vessel growth. Here, we show that the inhibition of tumor xenograft growth caused by an Ang2-specific antibody (REGN910) is reversed by systemic administration of the Tie2 agonist Ang1. These results indicate that Ang2 blockade inhibits tumor growth by decreasing Tie2 activity, showing that Ang2 is a Tie2 activator. REGN910 treatment of tumors resulted in increased expression of genes that are repressed by Tie2 activation, providing further evidence that REGN910 inhibits Tie2 signaling. Combination treatment with REGN910 plus the VEGF blocker aflibercept reduced tumor vascularity and tumor perfusion more dramatically than either single agent, resulting in more extensive tumor cell death and more potent inhibition of tumor growth. Challenging the prevailing model of Ang2 as a destabilizing factor, our findings indicate that Ang2 plays a protective role in tumor endothelial cells by activating Tie2, thereby limiting the antivascular effects of VEGF inhibition. Thus, blockade of Ang2 might enhance the clinical benefits currently provided by anti-VEGF agents. Cancer Res; 73(1); 108-18. Ó2012 AACR.

Cultured astrocytes are known to possess a range of neurotrophic activities in culture. In order to examine which factors may be responsible for these activities, we have examined the expression of the genes for four known neurotrophic factors-ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3)-in purified astrocyte cultures derived from neonatal rat hippocampus. Hippocampal astrocytes were found to express mRNA for three neurotrophic factors-CNTF, NGF and NT3-at significantly higher levels than other cultured cell types or cell lines examined. BDNF messenger RNA (mRNA), however, was undetectable in these astrocytes. The levels of CNTF, NGF and NT3 mRNA in astrocytes were largely unaffected by their degree of confluency, while serum removal caused only a transient decrease in mRNA levels, which returned to basal levels within 48 h. Astrocyte-derived CNTF was found to comigrate with recombinant rat CNTF at 23 kD on a Western blot. Immunocytochemical analysis revealed strong CNTF immunoreactivity in the cytoplasm of astrocytes, weak staining in the nucleus, but no CNTF at the cell surface. NGF and NT3 were undetectable immunocytochemically. CNTF-like activity, as assessed by bioassay on ciliary ganglion neurons, was found in the extract of cultured astrocytes but not in conditioned medium, whereas astrocyte-conditioned medium supported survival of dorsal root ganglion neurons but not ciliary or nodose ganglion neurons. This conditioned medium activity was neutralized with antibodies to NGF. Astrocyte extract also supported survival of dorsal root ganglion and nodose ganglion neurons, but these activities were not blocked by anti-NGF. Part, but not all, of the activity in astrocyte extracts which sustained nodose ganglion neurons could be attributed to CNTF.

Until recently, astrocytes were not considered as sites for neurotrophic factor action. We show here that, both in vivo and in vitro, astrocytes express receptors for two separate families of neurotrophic factors. In the intact adult rat CNS, astrocytes express the extracellular domain of the neurotrophin receptor TrkB and, in a more restricted population, the low-affinity nerve growth factor receptor p75LNGFR. In the lesioned CNS, expression of the alpha component of the receptor for ciliary neurotrophic factor (CNTFR alpha) switches from a purely neuronal localization to cells in the glial scar at the edge of the wound. Using cultured hippocampal astrocytes as a model to address the functional status of these receptors, we have found only the truncated forms of TrkB and TrkC, which are incapable of signal transduction as measured by protein tyrosine phosphorylation or immediate early gene induction. In contrast, a fully functional CNTF receptor complex capable of signal transduction is present on cultured astrocytes. Thus, the neurotrophin receptors may act primarily to sequester or present the neurotrophins, whereas in the case of CNTF a functional response can be initiated within the astrocyte.

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Although T cell checkpoint inhibitors have transformed the treatment of cancer, the molecular determinants of tumor cell sensitivity to T cell–mediated killing need further elucidation. Here, we describe a mouse genome–scale CRISPR knockout screen that identifies tumor cell TNFα signaling as an important component of T cell–induced apoptosis, with NF-κB signaling and autophagy as major protective mechanisms. Knockout of individual autophagy genes sensitized tumor cells to killing by T cells that were activated via specific TCR or by a CD3 bispecific antibody. Conversely, inhibition of mTOR signaling, which results in increased autophagic activity, protected tumor cells from T cell killing. Autophagy functions at a relatively early step in the TNFα signaling pathway, limiting FADD-dependent caspase-8 activation. Genetic inactivation of tumor cell autophagy enhanced the efficacy of immune checkpoint blockade in mouse tumor models. Thus, targeting the protective autophagy pathway might sensitize tumors to T cell–engaging immunotherapies in the clinic.

Angiopoietin-1 (Ang1) and Angiopoietin-2 (Ang2) are ligands for Tie2, an endothelial-specific receptor tyrosine kinase that is an essential regulator of angiogenesis. Here we report the identification, via expression cloning, of thrombomodulin (TM) as another receptor for Ang1 and Ang2. Thrombomodulin is an endothelial cell surface molecule that plays an essential role as a coagulation inhibitor via its function as a cofactor in the thrombin-mediated activation of protein C, an anticoagulant protein, as well as thrombin-activatable fibrinolysis inhibitor (TAFI). Ang1 and Ang2 inhibited the thrombin/TM-mediated generation of activated protein C and TAFI in cultured endothelial cells, and inhibited the binding of thrombin to TM in vitro. Ang2 appears to bind TM with higher affinity than Ang1 and is a more potent inhibitor of TM function. Consistent with a potential role for angiopoietins in coagulation, administration of thrombin to mice rapidly increased plasma Ang1 levels, presumably reflecting release from activated platelets (previously shown to contain high levels of Ang1). In addition, Ang1 levels were significantly elevated in plasma prepared from wound blood, suggesting that Ang1 is released from activated platelets at sites of vessel injury. Our results imply a previously undescribed role for angiopoietins in the regulation of hemostasis.

Ciliary neurotrophic factor (CNTF) is a pleiotropic cytokine which is detectable only at very low levels in the intact adult rat CNS, but following an aspirative lesion to the dorsal hippocampus and overlying cortex, CNTF mRNA levels are dramatically up-regulated in reactive astrocytes. In cultured rat hippocampal astrocytes, CNTF mRNA levels are high, similar to the levels in reactive astrocytes in vivo, but are strongly suppressed after administration of isoproterenol and forskolin, which stimulate the production of intracellular cyclic AMP, induced marked morphological change in the astrocytes and up-regulate glial fibrillary acidic protein mRNA and nerve growth factor mRNA in these cells. Following a single administration of forskolin to cultured astrocytes, suppression of CNTF mRNA was sustained for up to 7 days. A similar down-regulation was observed with the endogenous adrenergic agonists noradrenaline and adrenaline as well as, to a lesser extent, dopamine and adenosine. Down-regulation of CNTF mRNA resulted in a gradual reduction in the level of CNTF protein within the astrocytes. A single addition of forskolin or isoproterenol resulted in a drop in CNTF protein levels to 29 and 52% of control levels respectively after 9 days in vitro, although the rate of turnover of CNTF remained the same. Down-regulation of CNTF mRNA in cultured hippocampal astrocytes by adenylyl cyclase activation was quite specific, as a wide range of growth factors, cytokines and neurotransmitters had little or no effect upon CNTF mRNA levels.