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VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation.

Hayashi M, Majumdar A, Li X, Adler J, Sun Z, Vertuani S, Hellberg C, Mellberg S, Koch S, Dimberg A, Koh GY, Dejana E, Belting HG, Affolter M, Thurston G, Holmgren L, Vestweber D, Claesson-Welsh L - Nat Commun (2013)

Bottom Line: Vessels in ve-ptp(-/-) teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization.Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels.We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor-2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation.

View Article: PubMed Central - PubMed

Affiliation: Uppsala University, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Dag Hammarskjölds v. 20, 751 85 Uppsala, Sweden.

ABSTRACT
Vascular endothelial growth factor (VEGF) guides the path of new vessel sprouts by inducing VEGF receptor-2 activity in the sprout tip. In the stalk cells of the sprout, VEGF receptor-2 activity is downregulated. Here, we show that VEGF receptor-2 in stalk cells is dephosphorylated by the endothelium-specific vascular endothelial-phosphotyrosine phosphatase (VE-PTP). VE-PTP acts on VEGF receptor-2 located in endothelial junctions indirectly, via the Angiopoietin-1 receptor Tie2. VE-PTP inactivation in mouse embryoid bodies leads to excess VEGF receptor-2 activity in stalk cells, increased tyrosine phosphorylation of VE-cadherin and loss of cell polarity and lumen formation. Vessels in ve-ptp(-/-) teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization. Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels. We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor-2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation.

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Related in: MedlinePlus

Dephosphorylation of VEGFR2 at junctions.(a) HUVECs transfected with control or ve-ptp siRNA were stimulated with VEGF (20 ng ml−1: 60 min), and with Ang1 (200 ng ml−1; 60 min) individually or in combination, followed by PLA for pVEGFR2/VEGFR2 complexes and immunostaining with anti-ZO1 antibodies (green) to visualize cell–cell junctions. Red dots represent PLA products. Scale bars, 10 μm. High-magnification insets show PLA products at junctions; scale bars; 5 μm. (b) Quantification of number of pVEGFR2/VEGFR2 PLA products per cell. Mean±s.d., n=45 cells per condition repeated twice. ***P<0.001, t-test. (c) Quantification of number of pVEGFR2/VEGFR2 PLA products at junctions per cell. Mean±s.d., n= 45 cells per condition repeated twice. *P<0.05, ***P<0.001, t-test. (d) HUVECs were treated with 200 ng ml−1 of Ang1 (30 min), followed by addition of 20 ng ml−1 of VEGF (5 min), alternatively the two ligands were used individually (Ang1 for 30 min and VEGF for 5 min), processed and subjected to PLA using antibodies against VEGFR2 and VE-PTP and immunostaining for ZO1 (green). Scale bars, 20 μm. High-magnification insets show VEGFR2/VE-PTP PLA spots at cell–cell junctions; scale bars, 10 μm. (e) Quantification of VEGFR2/VE-PTP PLA complexes per cell. Mean±s.d., n= 30 cells per condition. *P<0.05, t-test. (f) Quantification of VEGFR2/VE-PTP PLA complexes at junctions per cell. Mean±s.d., n=45 cells per condition repeated twice. **P<0.01, ***P<0.001, t-test. (g) VEGFR2/VE-PTP PLA was performed in HUVECs transfected with control and tie2 siRNA and treated with VEGF and Ang1 as in d, followed by PLA for VEGFR2/VE-PTP complexes, and immunostaining with anti-ZO1 antibodies. Scale bars, 20 μm. High-magnification insets show PLA products at junction; scale bars, 10 μm. (h) Quantification of VEGFR2/VE-PTP PLA complexes per cell. Mean±s.d., n= 100 cells per condition, repeated twice. **P<0.01, t-test.
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f4: Dephosphorylation of VEGFR2 at junctions.(a) HUVECs transfected with control or ve-ptp siRNA were stimulated with VEGF (20 ng ml−1: 60 min), and with Ang1 (200 ng ml−1; 60 min) individually or in combination, followed by PLA for pVEGFR2/VEGFR2 complexes and immunostaining with anti-ZO1 antibodies (green) to visualize cell–cell junctions. Red dots represent PLA products. Scale bars, 10 μm. High-magnification insets show PLA products at junctions; scale bars; 5 μm. (b) Quantification of number of pVEGFR2/VEGFR2 PLA products per cell. Mean±s.d., n=45 cells per condition repeated twice. ***P<0.001, t-test. (c) Quantification of number of pVEGFR2/VEGFR2 PLA products at junctions per cell. Mean±s.d., n= 45 cells per condition repeated twice. *P<0.05, ***P<0.001, t-test. (d) HUVECs were treated with 200 ng ml−1 of Ang1 (30 min), followed by addition of 20 ng ml−1 of VEGF (5 min), alternatively the two ligands were used individually (Ang1 for 30 min and VEGF for 5 min), processed and subjected to PLA using antibodies against VEGFR2 and VE-PTP and immunostaining for ZO1 (green). Scale bars, 20 μm. High-magnification insets show VEGFR2/VE-PTP PLA spots at cell–cell junctions; scale bars, 10 μm. (e) Quantification of VEGFR2/VE-PTP PLA complexes per cell. Mean±s.d., n= 30 cells per condition. *P<0.05, t-test. (f) Quantification of VEGFR2/VE-PTP PLA complexes at junctions per cell. Mean±s.d., n=45 cells per condition repeated twice. **P<0.01, ***P<0.001, t-test. (g) VEGFR2/VE-PTP PLA was performed in HUVECs transfected with control and tie2 siRNA and treated with VEGF and Ang1 as in d, followed by PLA for VEGFR2/VE-PTP complexes, and immunostaining with anti-ZO1 antibodies. Scale bars, 20 μm. High-magnification insets show PLA products at junction; scale bars, 10 μm. (h) Quantification of VEGFR2/VE-PTP PLA complexes per cell. Mean±s.d., n= 100 cells per condition, repeated twice. **P<0.01, t-test.

Mentions: To further identify the subcellular localization of VE-PTP-regulated VEGFR2, we performed in situ proximity ligation assays (PLA) using antibodies against pVEGFR2 and VEGFR2 and oligonucleotide-ligated secondary antibodies. PLA products representing phosphorylated VEGFR2 increased with VEGF and decreased with VEGF+Ang1 treatment (quantification in Fig. 4b). Silencing of ve-ptp augmented PLA-detection of pVEGFR2 in response to VEGF; importantly, inclusion of Ang1 was without effect in VE-PTP-deficient cells. The pVEGFR2/VEGFR2 PLA spots were localized preferentially at or close to junctions in ve-ptp-silenced cells (Fig. 4a–c). Quantification of junctional localization showed fourfold induction of pVEGFR2/VEGFR2 in WT cells, compared with tenfold induction in ve-ptp-silenced cells. Controls for the PLA reactions (Supplementary Fig. S5) by omitting primary antibodies showed a high degree of specificity.


VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation.

Hayashi M, Majumdar A, Li X, Adler J, Sun Z, Vertuani S, Hellberg C, Mellberg S, Koch S, Dimberg A, Koh GY, Dejana E, Belting HG, Affolter M, Thurston G, Holmgren L, Vestweber D, Claesson-Welsh L - Nat Commun (2013)

Dephosphorylation of VEGFR2 at junctions.(a) HUVECs transfected with control or ve-ptp siRNA were stimulated with VEGF (20 ng ml−1: 60 min), and with Ang1 (200 ng ml−1; 60 min) individually or in combination, followed by PLA for pVEGFR2/VEGFR2 complexes and immunostaining with anti-ZO1 antibodies (green) to visualize cell–cell junctions. Red dots represent PLA products. Scale bars, 10 μm. High-magnification insets show PLA products at junctions; scale bars; 5 μm. (b) Quantification of number of pVEGFR2/VEGFR2 PLA products per cell. Mean±s.d., n=45 cells per condition repeated twice. ***P<0.001, t-test. (c) Quantification of number of pVEGFR2/VEGFR2 PLA products at junctions per cell. Mean±s.d., n= 45 cells per condition repeated twice. *P<0.05, ***P<0.001, t-test. (d) HUVECs were treated with 200 ng ml−1 of Ang1 (30 min), followed by addition of 20 ng ml−1 of VEGF (5 min), alternatively the two ligands were used individually (Ang1 for 30 min and VEGF for 5 min), processed and subjected to PLA using antibodies against VEGFR2 and VE-PTP and immunostaining for ZO1 (green). Scale bars, 20 μm. High-magnification insets show VEGFR2/VE-PTP PLA spots at cell–cell junctions; scale bars, 10 μm. (e) Quantification of VEGFR2/VE-PTP PLA complexes per cell. Mean±s.d., n= 30 cells per condition. *P<0.05, t-test. (f) Quantification of VEGFR2/VE-PTP PLA complexes at junctions per cell. Mean±s.d., n=45 cells per condition repeated twice. **P<0.01, ***P<0.001, t-test. (g) VEGFR2/VE-PTP PLA was performed in HUVECs transfected with control and tie2 siRNA and treated with VEGF and Ang1 as in d, followed by PLA for VEGFR2/VE-PTP complexes, and immunostaining with anti-ZO1 antibodies. Scale bars, 20 μm. High-magnification insets show PLA products at junction; scale bars, 10 μm. (h) Quantification of VEGFR2/VE-PTP PLA complexes per cell. Mean±s.d., n= 100 cells per condition, repeated twice. **P<0.01, t-test.
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f4: Dephosphorylation of VEGFR2 at junctions.(a) HUVECs transfected with control or ve-ptp siRNA were stimulated with VEGF (20 ng ml−1: 60 min), and with Ang1 (200 ng ml−1; 60 min) individually or in combination, followed by PLA for pVEGFR2/VEGFR2 complexes and immunostaining with anti-ZO1 antibodies (green) to visualize cell–cell junctions. Red dots represent PLA products. Scale bars, 10 μm. High-magnification insets show PLA products at junctions; scale bars; 5 μm. (b) Quantification of number of pVEGFR2/VEGFR2 PLA products per cell. Mean±s.d., n=45 cells per condition repeated twice. ***P<0.001, t-test. (c) Quantification of number of pVEGFR2/VEGFR2 PLA products at junctions per cell. Mean±s.d., n= 45 cells per condition repeated twice. *P<0.05, ***P<0.001, t-test. (d) HUVECs were treated with 200 ng ml−1 of Ang1 (30 min), followed by addition of 20 ng ml−1 of VEGF (5 min), alternatively the two ligands were used individually (Ang1 for 30 min and VEGF for 5 min), processed and subjected to PLA using antibodies against VEGFR2 and VE-PTP and immunostaining for ZO1 (green). Scale bars, 20 μm. High-magnification insets show VEGFR2/VE-PTP PLA spots at cell–cell junctions; scale bars, 10 μm. (e) Quantification of VEGFR2/VE-PTP PLA complexes per cell. Mean±s.d., n= 30 cells per condition. *P<0.05, t-test. (f) Quantification of VEGFR2/VE-PTP PLA complexes at junctions per cell. Mean±s.d., n=45 cells per condition repeated twice. **P<0.01, ***P<0.001, t-test. (g) VEGFR2/VE-PTP PLA was performed in HUVECs transfected with control and tie2 siRNA and treated with VEGF and Ang1 as in d, followed by PLA for VEGFR2/VE-PTP complexes, and immunostaining with anti-ZO1 antibodies. Scale bars, 20 μm. High-magnification insets show PLA products at junction; scale bars, 10 μm. (h) Quantification of VEGFR2/VE-PTP PLA complexes per cell. Mean±s.d., n= 100 cells per condition, repeated twice. **P<0.01, t-test.
Mentions: To further identify the subcellular localization of VE-PTP-regulated VEGFR2, we performed in situ proximity ligation assays (PLA) using antibodies against pVEGFR2 and VEGFR2 and oligonucleotide-ligated secondary antibodies. PLA products representing phosphorylated VEGFR2 increased with VEGF and decreased with VEGF+Ang1 treatment (quantification in Fig. 4b). Silencing of ve-ptp augmented PLA-detection of pVEGFR2 in response to VEGF; importantly, inclusion of Ang1 was without effect in VE-PTP-deficient cells. The pVEGFR2/VEGFR2 PLA spots were localized preferentially at or close to junctions in ve-ptp-silenced cells (Fig. 4a–c). Quantification of junctional localization showed fourfold induction of pVEGFR2/VEGFR2 in WT cells, compared with tenfold induction in ve-ptp-silenced cells. Controls for the PLA reactions (Supplementary Fig. S5) by omitting primary antibodies showed a high degree of specificity.

Bottom Line: Vessels in ve-ptp(-/-) teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization.Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels.We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor-2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation.

View Article: PubMed Central - PubMed

Affiliation: Uppsala University, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Dag Hammarskjölds v. 20, 751 85 Uppsala, Sweden.

ABSTRACT
Vascular endothelial growth factor (VEGF) guides the path of new vessel sprouts by inducing VEGF receptor-2 activity in the sprout tip. In the stalk cells of the sprout, VEGF receptor-2 activity is downregulated. Here, we show that VEGF receptor-2 in stalk cells is dephosphorylated by the endothelium-specific vascular endothelial-phosphotyrosine phosphatase (VE-PTP). VE-PTP acts on VEGF receptor-2 located in endothelial junctions indirectly, via the Angiopoietin-1 receptor Tie2. VE-PTP inactivation in mouse embryoid bodies leads to excess VEGF receptor-2 activity in stalk cells, increased tyrosine phosphorylation of VE-cadherin and loss of cell polarity and lumen formation. Vessels in ve-ptp(-/-) teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization. Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels. We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor-2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation.

Show MeSH
Related in: MedlinePlus