Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in physiologic and pathologic angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by endocytosis and secretory transport through the Golgi apparatus. To date, the mechanism whereby the VEGFR2 traffics through the Golgi apparatus remains incompletely characterized. We show in human endothelial cells that binding of VEGF to the cell surface localized VEGFR2 stimulates exit of intracellular VEGFR2 from the Golgi apparatus. Brefeldin A treatment reduced the level of surface VEGFR2, confirming that VEGFR2 traffics through the Golgi apparatus en route to the PM. Mechanistically, we show that inhibition of syntaxin 6, a Golgilocalized target membrane-soluble Nethylmaleimide attachment protein receptor (t-SNARE) protein, interferes with VEGFR2 trafficking to the PM and facilitates lysosomal degradation of the VEGFR2. In cell culture, inhibition of syntaxin 6 also reduced VEGF-induced cell proliferation, cell migration, and vascular tube formation. Furthermore, in a mouse ear model of angiogenesis, an inhibitory form of syntaxin 6 reduced VEGFinduced neovascularization and permeability. Our data demonstrate the importance of syntaxin 6 in the maintenance of cellular VEGFR2 levels, and suggest that the inhibitory form of syntaxin 6 has good potential as an antiangiogenic agent. IntroductionMembers of the vascular endothelial growth factor (VEGF) family bind to cell-surface receptors to regulate both physiologic and pathologic angiogenesis. 1 The activities of the VEGF-A isoform are mediated primarily through its interactions with 2 high-affinity receptor tyrosine kinases expressed on the vascular endothelium: VEGF receptor 2 (VEGFR2, KDR, Flk-1) and VEGFR1 (Flt-1). 2 VEGF-mediated angiogenic signaling has been attributed primarily to the signal transduction processes that are initiated by VEGFR2. 3 Cell-surface VEGFR2 internalized by the clathrin-dependent endocytic process is constitutively recycled back to the plasma membrane (PM). [4][5][6][7] Upon VEGF stimulation a fraction of the internalized endocytic pool of VEGFR2 is sorted toward late endosomes and lysosomes for degradation, whereas the remainder is recycled to the PM. 4,5,[8][9][10] Antiangiogenic approaches currently in use work by blocking VEGF binding to VEGFR2 and/or signaling by the receptor tyrosine kinases. 11 These strategies highlight the importance of intracellular transport mechanisms that coordinate the expression of VEGFR2 at distinct subcellular locations.The Golgi apparatus is a central hub for membrane trafficking across the mammalian cell. It receives newly synthesized proteins and lipids from the endoplasmic reticulum (ER), modifies many of these cargoes as they pass through, and finally sorts them to various destinations as they exit. [12][13][14][15] Thus the Golgi apparatus is a likely candidate for regulating VEGFR2 trafficking. Unraveling the precise transport pathway and the molecular players may offer better understanding of regulation of the VEGFR2 f...

To identify DNA copy number changes that had a direct influence on mRNA expression in gastric cancer, cDNA microarray-based comparative genomic hybridization (aCGH) and gene expression profiling were performed using 17 K cDNA microarrays. A set of 158 genes showing Pearson correlation coefficients over 0.6 between DNA copy number changes and mRNA expression level variations was selected. In an independent gene expression profiling of 60 tissue samples, the 158 genes were able to distinguish most of the normal and tumor tissues in an unsupervised hierarchical clustering, suggesting that the differential expression patterns displayed by this specific group of genes are most likely based on the gene copy number changes. Furthermore, 43 statistically significant (P<0.01) genes were selected that correctly distinguished all of the tissue samples. The copy number changes detected by aCGH can be verified by fluorescence in situ hybridization and real-time polymerase chain reaction. The selected genes include those that were previously identified as being tumor suppressors or deleted in various tumors, including GATA binding protein 4 (GATA4), monoamine oxidase A (MAOA), cyclin C (CCNC), and oncogenes including malignant fibrous histiocytoma amplified sequence 1 (MFHAS1/MASL1), high mobility group AT-hook 2 (HMGA2), PPAR binding protein (PPARBP), growth factor receptor-bound protein 7 (GRB7), and TBC1 (tre-2, BUB2, cdc16) domain family, member 1 (TBC1D1).

Background Abdominal aortic aneurysm (AAA) rupture risk is currently determined based on size and symptoms. This approach does not address the rupture risk associated with small aneurysms. Given the role of matrix metalloproteinases (MMPs) in AAA weakening and rupture, we investigated the potential of MMP-targeted imaging for detection of aneurysm biology and prediction of outcome in a mouse model of AAA with spontaneous rupture. Methods and Results Fifteen week-old mice (n=66) were infused with angiotensin II for four weeks to induce AAA. Saline-infused mice (n=16) served as control. The surviving animals underwent in vivo MMP-targeted microSPECT/CT imaging, using RP805, a 99mTc-labeled MMP-specific tracer, followed by ex vivo planar imaging, morphometry and gene expression analysis. RP805 uptake in suprarenal aorta on microSPECT images was significantly higher in animals with AAA, as compared with angiotensin II-infused animals without AAA or control animals. CD68 expression and MMP activity were increased in AAA and significant correlations were noted between RP805 uptake and CD68 expression or MMP activity, but not aortic diameter. A group of angiotensin II-infused animals (n=24) were imaged at 1 week and were followed for an additional three weeks. RP805 uptake in suprarenal aorta at 1 week was significantly higher in mice that later developed rupture/AAA. Furthermore, tracer uptake at 1 week correlated with aortic diameter at 4 weeks. Conclusions MMP-targeted imaging reflects vessel wall inflammation and can predict future aortic expansion or rupture in murine AAA. If confirmed in humans, this may provide a new paradigm for AAA risk stratification.