Glioblastoma multiforme (GBM) is treated by surgical resection followed by radiochemotherapy. Bevacizumab is commonly deployed for anti‐angiogenic therapy of recurrent GBM; however, innate immune cells have been identified as instigators of resistance to bevacizumab treatment. We identified angiopoietin‐2 (Ang‐2) as a potential target in both naive and bevacizumab‐treated glioblastoma. Ang‐2 expression was absent in normal human brain endothelium, while the highest Ang‐2 levels were observed in bevacizumab‐treated GBM. In a murine GBM model, VEGF blockade resulted in endothelial upregulation of Ang‐2, whereas the combined inhibition of VEGF and Ang‐2 leads to extended survival, decreased vascular permeability, depletion of tumor‐associated macrophages, improved pericyte coverage, and increased numbers of intratumoral T lymphocytes. CD206+ (M2‐like) macrophages were identified as potential novel targets following anti‐angiogenic therapy. Our findings imply a novel role for endothelial cells in therapy resistance and identify endothelial cell/myeloid cell crosstalk mediated by Ang‐2 as a potential resistance mechanism. Therefore, combining VEGF blockade with inhibition of Ang‐2 may potentially overcome resistance to bevacizumab therapy.

The homeostasis of the central nervous system is maintained by the blood–brain barrier (BBB). Angiopoietins (Ang-1/Ang-2) act as antagonizing molecules to regulate angiogenesis, vascular stability, vascular permeability and lymphatic integrity. However, the precise role of angiopoietin/Tie2 signaling at the BBB remains unclear. We investigated the influence of Ang-2 on BBB permeability in wild-type and gain-of-function (GOF) mice and demonstrated an increase in permeability by Ang-2, both in vitro and in vivo. Expression analysis of brain endothelial cells from Ang-2 GOF mice showed a downregulation of tight/adherens junction molecules and increased caveolin-1, a vesicular permeability-related molecule. Immunohistochemistry revealed reduced pericyte coverage in Ang-2 GOF mice that was supported by electron microscopy analyses, which demonstrated defective intra-endothelial junctions with increased vesicles and decreased/disrupted glycocalyx. These results demonstrate that Ang-2 mediates permeability via paracellular and transcellular routes. In patients suffering from stroke, a cerebrovascular disorder associated with BBB disruption, Ang-2 levels were upregulated. In mice, Ang-2 GOF resulted in increased infarct sizes and vessel permeability upon experimental stroke, implicating a role of Ang-2 in stroke pathophysiology. Increased permeability and stroke size were rescued by activation of Tie2 signaling using a vascular endothelial protein tyrosine phosphatase inhibitor and were independent of VE-cadherin phosphorylation. We thus identified Ang-2 as an endothelial cell-derived regulator of BBB permeability. We postulate that novel therapeutics targeting Tie2 signaling could be of potential use for opening the BBB for increased CNS drug delivery or tighten it in neurological disorders associated with cerebrovascular leakage and brain edema.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1551-3) contains supplementary material, which is available to authorized users.

Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor (VEGF) and plays an important role in mediating cell motility. However, the NRP1 signaling pathways important for cell motility are poorly understood. Here we report that p130Cas tyrosine phosphorylation is stimulated by hepatocyte growth factor and platelet-derived growth factor in U87MG glioma cells and VEGF in endothelial cells and is dependent on NRP1 via its intracellular domain. In endothelial cells, NRP1 silencing reduced, but did not prevent, VEGF receptor 2 (VEGFR2) phosphorylation, while expression of a mutant form of NRP1 lacking the intracellular domain (NRP1⌬C) did not affect receptor phosphorylation in U87MG cells or human umbilical vein endothelial cells (HUVECs). In HUVECs, NRP1 was also required for VEGF-induced phosphorylation of proline-rich tyrosine kinase 2, which was necessary for p130Cas phosphorylation. Importantly, knockdown of NRP1 or p130Cas or expression of either NRP1⌬C or a nontyrosine-phosphorylatable substrate domain mutant protein (p130 Cas15F ) was sufficient to inhibit growth factor-mediated migration of glioma and endothelial cells. These data demonstrate for the first time the importance of the NRP1 intracellular domain in mediating a specific signaling pathway downstream of several receptor tyrosine kinases and identify a critical role for a novel NRP1-p130Cas pathway in the regulation of chemotaxis.Neuropilin-1 (NRP1) is a coreceptor for vascular endothelial growth factor (VEGF) in endothelial cells and is essential for embryonic angiogenesis and vascular development (10,29). Though the precise cellular functions of NRP1 have yet to be elucidated, there is a growing body of evidence supporting a key role for NRP1 in the migration of both endothelial and tumor cells (9,11,15,19). NRP1 is thought to act as a coreceptor for VEGF by forming complexes with the VEGF receptor tyrosine kinase (RTK) VEGFR2. Complexation between NRP1 and VEGFR2 enhances VEGF binding, and inhibition of complex formation is associated with reduced VEGFR2 phosphorylation, intracellular signaling, mitogenesis, cell migration, and angiogenesis (16,18,28,34,35). However, the precise role of NRP1 in VEGF signaling remains unclear. Recent evidence indicates that NRP1 also regulates tumor and vascular cell functions stimulated by other growth factors, such as hepatocyte growth factor (HGF) and plateletderived growth factor (PDGF). Overexpression of NRP1 promotes tumor progression by potentiating the effect of the HGF/c-Met pathway, and tumor cell invasion mediated by the HGF/c-Met pathway is dependent on NRP1 through an association with c-Met (11, 15). Furthermore, NRP1 and NRP2 can bind HGF and mediate HGF stimulation of endothelial cell migration and proliferation (30). A recent report showed that NRP1 is also required for tumor cell-derived PDGF-mediated migration of smooth muscle cells (2). While these results indicate that NRP1 is required for optimal growth factor signaling important for cell motility, it remains unclear whether NR...

Neuropilin 1 (NRP1) is a receptor for class 3 semaphorins and vascular endothelial growth factor (VEGF) A and is essential for cardiovascular development. Biochemical evidence supports a model for NRP1 function in which VEGF binding induces complex formation between NRP1 and VEGFR2 to enhance endothelial VEGF signalling. However, the relevance of VEGF binding to NRP1 for angiogenesis in vivo has not yet been examined. We therefore generated knock-in mice expressing Nrp1 with a mutation of tyrosine (Y) 297 in the VEGF binding pocket of the NRP1 b1 domain, as this residue was previously shown to be important for high affinity VEGF binding and NRP1-VEGFR2 complex formation. Unexpectedly, this targeting strategy also severely reduced NRP1 expression and therefore generated a NRP1 hypomorph. Despite the loss of VEGF binding and attenuated NRP1 expression, homozygous Nrp1 Y297A/Y297A mice were born at normal Mendelian ratios, arguing against NRP1 functioning exclusively as a VEGF 164 receptor in embryonic angiogenesis. By overcoming the mid-gestation lethality of full Nrp1-null mice, homozygous Nrp1 Y297A/Y297A mice revealed essential roles for NRP1 in postnatal angiogenesis and arteriogenesis in the heart and retina, pathological neovascularisation of the retina and angiogenesis-dependent tumour growth. KEY WORDS: NRP1, VEGF, Angiogenesis, Arteriogenesis, Retina, Hindbrain INTRODUCTIONNRP1 is a transmembrane receptor for the VEGF 165 isoform (VEGF 164 in mice) and the neuronal guidance cue SEMA3A, with essential roles in both vascular and neuronal development (reviewed by Pellet-Many et al., 2008;Raimondi and Ruhrberg, 2013). Accordingly, Nrp1-null mice die before birth with severe cardiovascular and neuronal defects (Kitsukawa et al., 1997;Kawasaki et al., 1999 Centre for Cardiovascular Biology and Medicine, BHF Laboratories, Division of Medicine, University College London, 5 University Street, London WC1E 6JJ, UK. *These authors contributed equally to this work ‡ These authors contributed equally to this work § Authors for correspondence (I.Zachary@ucl.ac.uk; c.ruhrberg@ucl.ac.uk) This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.Received 25 August 2013; Accepted 3 November 2013 whereas mice carrying a mutated extracellular domain that abolishes SEMA3A, not VEGF 164 , binding show defective nerve, but not blood vessel, patterning (Gu et al., 2003;Vieira et al., 2007). These and other genetic, biochemical and cell biological data support a model in which VEGF 165 binding induces complex formation between NRP1 and VEGFR2 (KDR -Mouse Genome Informatics) to enhance VEGFR2 signalling during EC migration in vitro (e.g. Soker et al., 2002;Wang et al., 2003;Evans et al., 2011) and arteriogenesis in vivo (Lanahan et al., 2013).The extracellular NRP1 a1/a2 and b1/b2 domains are crucial f...

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