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

BackgroundA function for the microRNA (miRNA) pathway in vascular development and angiogenesis has been firmly established. miRNAs with selective expression in the vasculature are attractive as possible targets in miRNA-based therapies. However, little is known about the expression of miRNAs in microvessels in vivo. Here, we identified candidate microvascular-selective miRNAs by screening public miRNA expression datasets.MethodsBioinformatics predictions of microvascular-selective expression were validated with real-time quantitative reverse transcription PCR on purified microvascular fragments from mouse. Pericyte expression was shown with in situ hybridization on tissue sections. Target sites were identified with 3' UTR luciferase assays, and migration was tested in a microfluid chemotaxis chamber.ResultsmiR-145, miR-126, miR-24, and miR-23a were selectively expressed in microvascular fragments isolated from a range of tissues. In situ hybridization and analysis of Pdgfb retention motif mutant mice demonstrated predominant expression of miR-145 in pericytes. We identified the Ets transcription factor Friend leukemia virus integration 1 (Fli1) as a miR-145 target, and showed that elevated levels of miR-145 reduced migration of microvascular cells in response to growth factor gradients in vitro.ConclusionsmiR-126, miR-24 and miR-23a are selectively expressed in microvascular endothelial cells in vivo, whereas miR-145 is expressed in pericytes. miR-145 targets the hematopoietic transcription factor Fli1 and blocks migration in response to growth factor gradients. Our findings have implications for vascular disease and provide necessary information for future drug design against miRNAs with selective expression in the microvasculature.

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The exocyst is a protein complex that ensures spatial targeting of exocytotic vesicles to the plasma membrane. We present microarray data obtained from differentiating mouse embryonic stem cell cultures that identify an up-regulation of exocyst complex component 3-like 2 (exoc3l2) mRNA in sprouting blood vessels. Vascular expression of exoc3l2 is confirmed by qPCR analysis of different mouse tissues and immunofluorescence analyses of mouse brain sections. We detect an up-regulation of exoc3l2 mRNA synthesis in primary human endothelial cells in response to VEGFA, and this response is enhanced when the cells are grown on a three-dimensional collagen I matrix. Myc-tagged EXOC3L2 co-precipitates with the exocyst protein EXOC4, and immunofluorescence detection of EXOC3L2 shows partial subcellular colocalization with EXOC4 and EXOC7. Finally, we show that exoc3l2 silencing inhibits VEGF receptor 2 phosphorylation and VEGFA-directed migration of cultured endothelial cells.