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           Search results for: GFP Expressing Human Brain Microvascular Endothelial Cells   


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Tumor and endothelial cell hybrids participate in glioblastoma vasculature.

Recently antiangiogenic therapy with bevacizumab has shown a high but transient efficacy in glioblastoma (GBM). Indeed, GBM is one of the most angiogenic human tumors and endothelial proliferation is a hallmark of the disease. We therefore hypothesized that tumor cells may participate in endothelial proliferation of GBM.

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Cell-cell fusion and internalization of the CNS-based, HIV-1 co-receptor, APJ.

APJ, a member of the human G protein-coupled seven-transmembrane receptor family, has been shown to serve as a coreceptor for the entry of human immunodeficiency virus type I (HIV-1) and simian immunodeficiency virus (SIV), and it is dramatically expressed in central nervous system (CNS)-based cells. In this study, expression of APJ tagged with the green fluorescent protein (GFP) and a fluorescent peptide, 5-carboxyfluorescein (5-CF) conjugated Apelin-13, were utilized for studying receptor internalization and recycling, in stably expressing indicator cells, human neurons, primary CNS microvascular endothelial cells (MVECs), and astrocytes. Fusion of the C-terminus of APJ to the N-terminus of GFP did not alter receptor ligand binding and functions, including signaling and internalization. Using 293 cells stably expressing APJ-GFP, we demonstrated that rapid internalization of the APJ receptor was induced by stimulation with Apelin-36 and Apelin-13, in a dose-dependent manner. Furthermore, investigations showed that the internalized APJ was colocalized with transferrin receptors, suggesting that the internalization of APJ induced by Apelin is likely to be via clathrin-coated pits. Interestingly, we found that the internalized APJ molecules were recycled to the cell surface within 60 min after removal of Apelin-13, but most of the internalized APJ still remained in the cytoplasm, even 2 h after washout of Apelin-36. The intact cytoplasmic C-terminal domain was found to be required for ligand-induced APJ internalization. Human neurons were dramatically stained by the APJ-binding fluorescent peptides. Primary human fetal astrocytes were less strongly labeled with 5-CF-Apelin-13, and in primary human CNS MVECs only weak distribution of green fluorescence specific for APJ in the cytoplasm was observed. Apelin-36 blocked cell membrane fusion mostly due to steric interference, with only a very modest effect on receptor internalization. The CNS represents a unique reservoir site for HIV-1. As such, molecular therapeutics and small molecular inhibitors of HIV-1 entry via this unique CNS receptor are now able to be rationally designed.

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