| 11. |
- Sun, Zuyue, et al.
(författare)
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VEGFR2 induces c-Src signaling and vascular permeability in vivo via the adaptor protein TSAd
- 2012
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 209:7, s. 1363-1377
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Tidskriftsartikel (refereegranskat)abstract
- Regulation of vascular endothelial (VE) growth factor (VEGF)-induced permeability is critical in physiological and pathological processes. We show that tyrosine phosphorylation of VEGF receptor 2 (VEGFR2) at Y951 facilitates binding of VEGFR2 to the Rous sarcoma (Src) homology 2-domain of T cell-specific adaptor (TSAd), which in turn regulates VEGF-induced activation of the c-Src tyrosine kinase and vascular permeability. c-Src was activated in vivo and in vitro in a VEGF/TSAd-dependent manner, and was regulated via increased phosphorylation at pY418 and reduced phosphorylation at pY527. Tsad silencing blocked VEGF-induced c-Src activation, but did not affect pathways involving phospholipase C gamma, extracellular regulated kinase, and endothelial nitric oxide. VEGF-induced rearrangement of VE-cadherin-positive junctions in endothelial cells isolated from mouse lungs, or in mouse cremaster vessels, was dependent on TSAd expression, and TSAd formed a complex with VE-cadherin, VEGFR2, and c-Src at endothelial junctions. Vessels in tsad(-/-) mice showed undisturbed flow and pressure, but impaired VEGF-induced permeability, as measured by extravasation of Evans blue, dextran, and microspheres in the skin and the trachea. Histamine-induced extravasation was not affected by TSAd deficiency. We conclude that TSAd is required for VEGF-induced, c-Src-mediated regulation of endothelial cell junctions and for vascular permeability.
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| 12. |
- Tugues, Sònia, et al.
(författare)
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Histidine-Rich Glycoprotein Uptake and Turnover Is Mediated by Mononuclear Phagocytes.
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:9, s. e107483-
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Tidskriftsartikel (refereegranskat)abstract
- Histidine-rich glycoprotein (HRG) is implicated in tumor growth and metastasis by regulation of angiogenesis and inflammation. HRG is produced by hepatocytes and carried to tissues via the circulation. We hypothesized that HRG's tissue distribution and turnover may be mediated by inflammatory cells. Biodistribution parameters were analyzed by injection of radiolabeled, bioactive HRG in the circulation of healthy and tumor-bearing mice. 125I-HRG was cleared rapidly from the blood and taken up in tissues of healthy and tumor-bearing mice, followed by degradation, to an increased extent in the tumor-bearing mice. Steady state levels of HRG in the circulation were unaffected by the tumor disease both in murine tumor models and in colorectal cancer (CRC) patients. Importantly, stromal pools of HRG, detected in human CRC microarrays, were associated with inflammatory cells. In agreement, microautoradiography identified 125I-HRG in blood vessels and on CD45-positive leukocytes in mouse tissues. Moreover, radiolabeled HRG bound in a specific, heparan sulfate-independent manner, to differentiated human monocytic U937 cells in vitro. Suppression of monocyte differentiation by systemic treatment of mice with anti-colony stimulating factor-1 neutralizing antibodies led to reduced blood clearance of radiolabeled HRG and to accumulation of endogenous HRG in the blood. Combined, our data show that mononuclear phagocytes have specific binding sites for HRG and that these cells are essential for uptake of HRG from blood and distribution of HRG in tissues. Thereby, we confirm and extend our previous report that inflammatory cells mediate the effect of HRG on tumor growth and metastatic spread.
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| 13. |
- Tugues, Sonia, et al.
(författare)
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Tetraspanin CD63 Promotes Vascular Endothelial Growth Factor Receptor 2-beta 1 Integrin Complex Formation, Thereby Regulating Activation and Downstream Signaling in Endothelial Cells in Vitro and in Vivo
- 2013
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:26, s. 19060-19071
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Tidskriftsartikel (refereegranskat)abstract
- CD63 is a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) implicated in the regulation of membrane protein trafficking, leukocyte recruitment, and adhesion processes. We have investigated the involvement of CD63 in endothelial cell (EC) signaling downstream of beta 1 integrin and VEGF. We report that silencing of CD63 in primary ECs arrested capillary sprouting and tube formation in vitro because of impaired adhesion and migration of ECs. Mechanistically, CD63 associated with both beta 1 integrin and the main VEGF receptor on ECs, VEGFR2. Our data suggest that CD63 serves to bridge between beta 1 integrin and VEGFR2 because CD63 silencing disrupted VEGFR2-beta 1 integrin complex formation identified using proximity ligation assays. Signaling downstream of beta 1 integrin and VEGFR2 was attenuated in CD63-silenced cells, although their cell surface expression levels remained unaffected. CD63 was furthermore required for efficient internalization of VEGFR2 in response to VEGF. Importantly, systemic delivery of VEGF failed to potently induce VEGFR2 phosphorylation and downstream signaling in CD63-deficient mouse lungs. Taken together, our findings demonstrate a previously unrecognized role for CD63 in coordinated integrin and receptor tyrosine kinase signaling in vitro and in vivo.
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| 14. |
- Yan, Junhong, et al.
(författare)
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Highly sensitive and specific protein detection via proximity ligation in capillary westerns
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Detection and quantification of proteins and their post-translational modifications are crucial todecipher functions of complex networks in cell biology and medicine. Affinity-based protein analyseshave an important role in proteomic research, and western blotting is one of the most-widely usedtechniques. The NanoPro 1000 system developed by ProteinSimple is an instrument that can resolveand identify proteins and their isoforms through capillary isoelectric focusing with antibody baseddetection directly in the capillaries, in analogy to conventional western blotting. The in situ proximityligation assay (PLA) serves to detect the location of proteins using pairs of oligonucleotide-conjugatedantibodies that mediate formation of rolling circle amplification (RCA) products upon recognition ofthe target protein, or single or dual primary antibodies binding the target. PLA can offer improvedspecificity of detection, and increased sensitivity via, RCA initiated by oligonucleotides attached tothe antibody pairs. Here we have used in situ PLA to detect single or pairs of primary antibodiesbinding proteins separated in the NanoPro 1000 system for highly sensitive and specific detection ofproteins and their isoforms. We achieved at least 10-fold improved detection limits for Erk protein inHDMEC cell lysates, compared with the standard NanoPro 1000 assays, and we demonstrated greatlyenhanced specificity of detection by combining pairs of antibodies, each of which exhibited crossreactivesignals when used on their own.
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