Vascular endothelial growth-factor receptors (VEGFRs) regulate the cardiovascular system. VEGFR1 is required for the recruitment of haematopoietic precursors and migration of monocytes and macrophages, whereas VEGFR2 and VEGFR3 are essential for the functions of vascular endothelial and lymphendothelial cells, respectively. Recent insights have shed light onto VEGFR signal transduction and the interplay between different VEGFRs and VEGF co-receptors in development, adult physiology and disease.

Tumor vascularization occurs through several distinct biological processes, which not only vary between tumor type and anatomic location, but also occur simultaneously within the same cancer tissue. These processes are orchestrated by a range of secreted factors and signaling pathways and can involve participation of non-endothelial cells, such as progenitors or cancer stem cells. Anti-angiogenic therapies using either antibodies or tyrosine kinase inhibitors have been approved to treat several types of cancer. However, the benefit of treatment has so far been modest, some patients not responding at all and others acquiring resistance. It is becoming increasingly clear that blocking tumors from accessing the circulation is not an easy task to accomplish. Tumor vessel functionality and gene expression often differ vastly when comparing different cancer subtypes, and vessel phenotype can be markedly heterogeneous within a single tumor. Here, we summarize the current understanding of cellular and molecular mechanisms involved in tumor angiogenesis and discuss challenges and opportunities associated with vascular targeting.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Vascular endothelial growth factor receptor-2 (VEGFR-2) activation by VEGF-A is essential in vasculogenesis and angiogenesis. We have generated a pan-phosphorylation site map of VEGFR-2 and identified one major tyrosine phosphorylation site in the kinase insert (Y951), in addition to two major sites in the C-terminal tail (Y1175 and Y1214). In developing vessels, phosphorylation of Y1175 and Y1214 was detected in all VEGFR-2-expressing endothelial cells, whereas phosphorylation of Y951 was identified in a subset of vessels. Phosphorylated Y951 bound the T-cell-specific adapter (TSAd), which was expressed in tumor vessels. Mutation of Y951 to F and introduction of phosphorylated Y951 peptide or TSAd siRNA into endothelial cells blocked VEGF-A-induced actin stress fibers and migration, but not mitogenesis. Tumor vascularization and growth was reduced in TSAd-deficient mice, indicating a critical role of Y951-TSAd signaling in pathological angiogenesis

Cancer produces a variety of collateral effects in patients beyond the malignancy itself, including threats to distal organ functions. However, the basis for such effects, associated with either primary or metastatic tumors, are generally poorly understood. In this study, we show how heart and kidney vascular function is impaired by neutrophils that accumulate in those tissues as a result of tumor formation in two different transgenic mouse models of cancer (RIP1-Tag2 model of insulinoma and MMTV-PyMT model of breast cancer). Neutrophil depletion by systemic administration of an anti-Gr1 antibody improved vascular perfusion and prevented vascular leakage in kidney vessels. We also observed the accumulation of platelet-neutrophil complexes, a signature of neutrophil extracellular traps (NET), in the kidneys of tumor-bearing mice that were completely absent from healthy nontumor-bearing littermates. NET accumulation in the vasculature was associated with upregulation of the proinflammatory adhesion molecules ICAM-1, VCAM-1, and E-selectin, as well as the proinflammatory cytokines IL1b, IL6, and the chemokine CXCL1. Administering DNase I to dissolve NETs, which have a high DNA content, restored perfusion in the kidney and heart to levels seen in nontumor-bearing mice, and also prevented vessel leakage in the blood vasculature of these organs. Taken together, our findings strongly suggest that NETs mediate the negative collateral effects of tumors on distal organs, acting to impair vascular function, and to heighten inflammation at these sites.Cancer Res; 75(13); 2653-62. Ó2015 AACR.

Glioblastoma are aggressive astrocytic brain tumours characterized by microvascular proliferation and an abnormal vasculature, giving rise to brain oedema and increased patient morbidity. Here, we have characterized the transcriptome of tumour-associated blood vessels and describe a gene signature clearly associated with pleomorphic, pathologically altered vessels in human glioblastoma (grade IV glioma). We identified 95 genes differentially expressed in glioblastoma vessels, while no significant differences in gene expression were detected between vessels in non-malignant brain and grade II glioma. Differential vascular expression of ANGPT2, CD93, ESM1, ELTD1, FILIP1L and TENC1 in human glioblastoma was validated by immunohistochemistry, using a tissue microarray. Through qPCR analysis of gene induction in primary endothelial cells, we provide evidence that increased VEGF-A and TGFβ2 signalling in the tumour microenvironment is sufficient to invoke many of the changes in gene expression noted in glioblastoma vessels. Notably, we found an enrichment of Smad target genes within the distinct gene signature of glioblastoma vessels and a significant increase of Smad signalling complexes in the vasculature of human glioblastoma in situ. This indicates a key role of TGFβ signalling in regulating vascular phenotype and suggests that, in addition to VEGF-A, TGFβ2 may represent a new target for vascular normalization therapy.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) signaling pathway is in clinical use, but its effect on vascular function and the tumor microenvironment is poorly understood. Here, we investigate cross‐talk between VEGF and proinflammatory TNF‐α signaling in endothelial cells and its impact on leukocyte recruitment. We found that cotreatment with VEGF decreased TNF‐α‐induced Jurkat cell adhesion to human microvascular endothelial cells by 40%. This was associated with inhibition of TNF‐α‐mediated regulation of 86 genes, including 2 T‐lymphocyte‐attracting chemokines, CXCL10 and CXCL11 [TNF‐α concentration 1 ng/ml; 50% inhibition/inhibitory concentration (IC50) VEGF, 3 ng/ml]. Notably, VEGF directly suppressed TNF‐α‐induced gene expression through negative cross‐talk with the NF‐κB‐signaling pathway, leading to an early decrease in IFN regulatory factor 1 (IRF‐1) expression and reduced phosphorylation of signal transducer and activator of transcription 1 (p‐Stat1) at later times. Inhibition of VEGF signaling in B16 melanoma tumor‐bearing mice by sunitinib treatment resulted in up‐regulation of CXCL10 and CXCL11 in tumor vessels, accompanied by up to 18‐fold increased infiltration of CD3+ T‐lymphocytes in B16 tumors. Our results demonstrate a novel role of VEGF in negative regulation of NF‐κB signaling and endothelial activation in the tumor microenvironment and provide evidence that pharmacological inhibition of VEGF signaling enhances T‐lymphocyte recruitment through up‐regulation of chemokines CXCL10 and CXCL11.—Huang, H., Langenkamp, E., Georganaki, M., Loskog, A., Fuchs, P. F., Dieterich, L. C., Kreuger, J., Dimberg, A., VEGF suppresses T‐lymphocyte infiltration in the tumor microenvironment through inhibition of NF‐κB‐induced endothelial activation. FASEB J. 29, 227–238 (2015). http://www.fasebj.org

Tumor angiogenesis occurs through regulation of genes that orchestrate endothelial sprouting and vessel maturation, including deposition of a vessel-associated extracellular matrix. CD93 is a transmembrane receptor that is upregulated in tumor vessels in many cancers, including high-grade glioma. Here, we demonstrate that CD93 regulates β1 integrin signaling and organization of fibronectin fibrillogenesis during tumor vascularization. In endothelial cells and mouse retina, CD93 was found to be expressed in endothelial filopodia and to promote filopodia formation. The CD93 localization to endothelial filopodia was stabilized by interaction with multimerin-2 (MMRN2), which inhibited its proteolytic cleavage. The CD93-MMRN2 complex was required for activation of β1 integrin, phosphorylation of focal adhesion kinase (FAK), and fibronectin fibrillogenesis in endothelial cells. Consequently, tumor vessels in gliomas implanted orthotopically in CD93-deficient mice showed diminished activation of β1 integrin and lacked organization of fibronectin into fibrillar structures. These findings demonstrate a key role of CD93 in vascular maturation and organization of the extracellular matrix in tumors, identifying it as a potential target for therapy.