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VEGFR2 induces c-Src signaling and vascular permeability in vivo via the adaptor protein TSAd.

Sun Z, Li X, Massena S, Kutschera S, Padhan N, Gualandi L, Sundvold-Gjerstad V, Gustafsson K, Choy WW, Zang G, Quach M, Jansson L, Phillipson M, Abid MR, Spurkland A, Claesson-Welsh L - J. Exp. Med. (2012)

Bottom Line: Tsad silencing blocked VEGF-induced c-Src activation, but did not affect pathways involving phospholipase Cγ, extracellular regulated kinase, and endothelial nitric oxide.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, 751 85 Uppsala, Sweden.

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 Cγ, 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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Impaired VEGF-induced extravasation of Evans blue and FITC-dextran in TSAd-deficient mice. (A) Systemic (i.v.) administration of Evans blue in WT and tsad−/− mice was followed 2 h later by intradermal injection of vehicle or VEGF (100 ng) on the back. Evans blue dye leakage in dorsal skin was assessed after 30 min. Representative images of three independent experiments using two to four mice/genotype each time with similar result. Bar, 5 mm. (B) Quantification of extravasated Evans blue by formamide extraction from skin samples in A, corrected for tissue weight. n = 5 (WT) and 6 (tsad−/−) mice/genotype from 2 independent experiments. *, P < 0.05; **, P < 0.01. (C) Basal extravasation of systemically delivered Evans blue in kidney, lung, and skin as quantified by formamide extraction. n = 3 mice/genotype. Experiment was performed once. (D) Intradermal injection of vehicle or VEGF in the ear was followed by systemic administration (i.v.) of 70-kD FITC-dextran and leakage was assessed 30 min later. Representative images of three separate experiments using three to four mice/genotype each time. Bar, 100 µm. (E) Quantification of FITC-dextran extravasation by determination of fluorescence in the tissues shown in C. n = 4 mice/genotype from 1 representative experiment.*, P < 0.05; **, P < 0.01.
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fig2: Impaired VEGF-induced extravasation of Evans blue and FITC-dextran in TSAd-deficient mice. (A) Systemic (i.v.) administration of Evans blue in WT and tsad−/− mice was followed 2 h later by intradermal injection of vehicle or VEGF (100 ng) on the back. Evans blue dye leakage in dorsal skin was assessed after 30 min. Representative images of three independent experiments using two to four mice/genotype each time with similar result. Bar, 5 mm. (B) Quantification of extravasated Evans blue by formamide extraction from skin samples in A, corrected for tissue weight. n = 5 (WT) and 6 (tsad−/−) mice/genotype from 2 independent experiments. *, P < 0.05; **, P < 0.01. (C) Basal extravasation of systemically delivered Evans blue in kidney, lung, and skin as quantified by formamide extraction. n = 3 mice/genotype. Experiment was performed once. (D) Intradermal injection of vehicle or VEGF in the ear was followed by systemic administration (i.v.) of 70-kD FITC-dextran and leakage was assessed 30 min later. Representative images of three separate experiments using three to four mice/genotype each time. Bar, 100 µm. (E) Quantification of FITC-dextran extravasation by determination of fluorescence in the tissues shown in C. n = 4 mice/genotype from 1 representative experiment.*, P < 0.05; **, P < 0.01.

Mentions: Because c-Src has been implicated in the regulation of vascular permeability (Weis and Cheresh, 2005), we analyzed tsad−/− mice, which are fertile and apparently healthy, for their ability to respond to VEGF in a classical Miles assay. Evans blue was administered by tail vein injection in mice that received an intradermal bolus of 100 ng VEGF or vehicle on the back (Fig. 2 A). Permeability was induced by VEGF in WT mice, but there was a significant decrease in VEGF-induced Evans blue extravasation in tsad−/− skin (Fig. 2 B). There was no change in basal leakage of Evans blue in the kidney, lung, or skin, between WT and tsad−/− mice (Fig. 2 C).


VEGFR2 induces c-Src signaling and vascular permeability in vivo via the adaptor protein TSAd.

Sun Z, Li X, Massena S, Kutschera S, Padhan N, Gualandi L, Sundvold-Gjerstad V, Gustafsson K, Choy WW, Zang G, Quach M, Jansson L, Phillipson M, Abid MR, Spurkland A, Claesson-Welsh L - J. Exp. Med. (2012)

Impaired VEGF-induced extravasation of Evans blue and FITC-dextran in TSAd-deficient mice. (A) Systemic (i.v.) administration of Evans blue in WT and tsad−/− mice was followed 2 h later by intradermal injection of vehicle or VEGF (100 ng) on the back. Evans blue dye leakage in dorsal skin was assessed after 30 min. Representative images of three independent experiments using two to four mice/genotype each time with similar result. Bar, 5 mm. (B) Quantification of extravasated Evans blue by formamide extraction from skin samples in A, corrected for tissue weight. n = 5 (WT) and 6 (tsad−/−) mice/genotype from 2 independent experiments. *, P < 0.05; **, P < 0.01. (C) Basal extravasation of systemically delivered Evans blue in kidney, lung, and skin as quantified by formamide extraction. n = 3 mice/genotype. Experiment was performed once. (D) Intradermal injection of vehicle or VEGF in the ear was followed by systemic administration (i.v.) of 70-kD FITC-dextran and leakage was assessed 30 min later. Representative images of three separate experiments using three to four mice/genotype each time. Bar, 100 µm. (E) Quantification of FITC-dextran extravasation by determination of fluorescence in the tissues shown in C. n = 4 mice/genotype from 1 representative experiment.*, P < 0.05; **, P < 0.01.
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fig2: Impaired VEGF-induced extravasation of Evans blue and FITC-dextran in TSAd-deficient mice. (A) Systemic (i.v.) administration of Evans blue in WT and tsad−/− mice was followed 2 h later by intradermal injection of vehicle or VEGF (100 ng) on the back. Evans blue dye leakage in dorsal skin was assessed after 30 min. Representative images of three independent experiments using two to four mice/genotype each time with similar result. Bar, 5 mm. (B) Quantification of extravasated Evans blue by formamide extraction from skin samples in A, corrected for tissue weight. n = 5 (WT) and 6 (tsad−/−) mice/genotype from 2 independent experiments. *, P < 0.05; **, P < 0.01. (C) Basal extravasation of systemically delivered Evans blue in kidney, lung, and skin as quantified by formamide extraction. n = 3 mice/genotype. Experiment was performed once. (D) Intradermal injection of vehicle or VEGF in the ear was followed by systemic administration (i.v.) of 70-kD FITC-dextran and leakage was assessed 30 min later. Representative images of three separate experiments using three to four mice/genotype each time. Bar, 100 µm. (E) Quantification of FITC-dextran extravasation by determination of fluorescence in the tissues shown in C. n = 4 mice/genotype from 1 representative experiment.*, P < 0.05; **, P < 0.01.
Mentions: Because c-Src has been implicated in the regulation of vascular permeability (Weis and Cheresh, 2005), we analyzed tsad−/− mice, which are fertile and apparently healthy, for their ability to respond to VEGF in a classical Miles assay. Evans blue was administered by tail vein injection in mice that received an intradermal bolus of 100 ng VEGF or vehicle on the back (Fig. 2 A). Permeability was induced by VEGF in WT mice, but there was a significant decrease in VEGF-induced Evans blue extravasation in tsad−/− skin (Fig. 2 B). There was no change in basal leakage of Evans blue in the kidney, lung, or skin, between WT and tsad−/− mice (Fig. 2 C).

Bottom Line: Tsad silencing blocked VEGF-induced c-Src activation, but did not affect pathways involving phospholipase Cγ, extracellular regulated kinase, and endothelial nitric oxide.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, 751 85 Uppsala, Sweden.

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 Cγ, 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.

Show MeSH
Related in: MedlinePlus