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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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VE-PTP regulates VEGFR2 activity and polarization in vivo.(a) WT and ve-ptp−/− teratoma sections show CD31 (green), podocalyxin (red) and Hoechst 33342 (blue). Asterisk, lumen. Scale bars, 20 μm. (b) Podocalyxin intensity/CD31-positive area (mm3) in a. Mean±s.d.; n= 15 CD31-positive vessels of 50 μm length/3 teratomas/genotype. *P<0.05, t-test. (c) WT and ve-ptp−/− teratoma sections. Left: 16-colour intensity scale representation of pVEGFR2 immunostaining. Right: Hoechst 33342 (blue), pY1175 VEGFR2 (green), and CD31 (grey). Scale bars, 20 μm. (d) pY1175 VEGFR2 intensity/CD31-positive area (mm2) in c. Mean±s.d.; n= 12 (WT) and 15 (ve-ptp−/−) blood vessels of 30 μm length from 3 teratomas per genotype. **P<0.01, t-test. (e) Lectin-perfused WT and ve-ptp−/− teratomas. Hoechst 33342 (blue), lectin-FITC (green), pY1175 VEGFR2 (red) and VE-PTP (white). Right: control immunostaining without primary antibodies. Asterisk, lumen. Arrows show VE-PTP expression in WT teratomas. Arrowheads show pVEGFR2 in ve-ptp−/− teratomas. Scale bars, 20 μm. (f) Lectin-perfused WT and ve-ptp−/− teratomas show lectin-FITC (green), VE-cadherin (red) and VE-PTP (white). Asterisk, lumen. Arrowheads in the lower ve-ptp−/− panel indicate fragmented VE-cadherin immunostaining. Scale bars, 20 μm. (g) Lectin-perfused B16 F10 mouse melanomas show lectin-FITC (green), VE-cadherin (VE-cad; red) and VE-PTP (white). Asterisk, lumen. Arrowheads in the lower panel indicate fragmented VE-cadherin immunostaining. Scale bars, 20 μm. (h) Model illustrating the contribution of VE-PTP in silencing VEGFR2 and Tie2 at junctions to support proper EC polarity and vessel morphogenesis. VE-PTP exists in complex with VEGFR2 and Tie2 in the WT condition (left). VEGF induces activation of VEGFR2 and in parallel, dissociation from VE-PTP. VEGF and Ang1 induce translocation of the trimeric complex to junctions where the activated receptors are silenced by VE-PTP. This is compatible with formation of polarized and lumenized vessels. In the VE-PTP-deficient condition (ve-ptp−/−; right), VEGF and Ang1 induce activation and translocation of receptors to junctions, where excess activity leads to VE-cadherin phosphorylation and formation of unpolarized and lumen-less pathological vasculature.
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f7: VE-PTP regulates VEGFR2 activity and polarization in vivo.(a) WT and ve-ptp−/− teratoma sections show CD31 (green), podocalyxin (red) and Hoechst 33342 (blue). Asterisk, lumen. Scale bars, 20 μm. (b) Podocalyxin intensity/CD31-positive area (mm3) in a. Mean±s.d.; n= 15 CD31-positive vessels of 50 μm length/3 teratomas/genotype. *P<0.05, t-test. (c) WT and ve-ptp−/− teratoma sections. Left: 16-colour intensity scale representation of pVEGFR2 immunostaining. Right: Hoechst 33342 (blue), pY1175 VEGFR2 (green), and CD31 (grey). Scale bars, 20 μm. (d) pY1175 VEGFR2 intensity/CD31-positive area (mm2) in c. Mean±s.d.; n= 12 (WT) and 15 (ve-ptp−/−) blood vessels of 30 μm length from 3 teratomas per genotype. **P<0.01, t-test. (e) Lectin-perfused WT and ve-ptp−/− teratomas. Hoechst 33342 (blue), lectin-FITC (green), pY1175 VEGFR2 (red) and VE-PTP (white). Right: control immunostaining without primary antibodies. Asterisk, lumen. Arrows show VE-PTP expression in WT teratomas. Arrowheads show pVEGFR2 in ve-ptp−/− teratomas. Scale bars, 20 μm. (f) Lectin-perfused WT and ve-ptp−/− teratomas show lectin-FITC (green), VE-cadherin (red) and VE-PTP (white). Asterisk, lumen. Arrowheads in the lower ve-ptp−/− panel indicate fragmented VE-cadherin immunostaining. Scale bars, 20 μm. (g) Lectin-perfused B16 F10 mouse melanomas show lectin-FITC (green), VE-cadherin (VE-cad; red) and VE-PTP (white). Asterisk, lumen. Arrowheads in the lower panel indicate fragmented VE-cadherin immunostaining. Scale bars, 20 μm. (h) Model illustrating the contribution of VE-PTP in silencing VEGFR2 and Tie2 at junctions to support proper EC polarity and vessel morphogenesis. VE-PTP exists in complex with VEGFR2 and Tie2 in the WT condition (left). VEGF induces activation of VEGFR2 and in parallel, dissociation from VE-PTP. VEGF and Ang1 induce translocation of the trimeric complex to junctions where the activated receptors are silenced by VE-PTP. This is compatible with formation of polarized and lumenized vessels. In the VE-PTP-deficient condition (ve-ptp−/−; right), VEGF and Ang1 induce activation and translocation of receptors to junctions, where excess activity leads to VE-cadherin phosphorylation and formation of unpolarized and lumen-less pathological vasculature.

Mentions: To provide in vivo confirmation of a role for VE-PTP in vessel lumen formation, we used WT and ve-ptp−/− ESCs to establish teratomas in severe combined immunodeficient (SCID) mice. Teratomas become vascularized in part from the host and in part through differentiation of ECs from the ESCs34. As shown in Fig. 7a, vessels in ve-ptp−/− teratomas displayed disorganized ECs with broadly distributed podocalyxin expression (Fig. 7a). Immunostaining for pVEGFR2 moreover demonstrated significant induction of VEGFR2 phosphorylation in CD31-positive ECs in ve-ptp−/− teratomas (Fig. 7c). Co-immunostaining for pVEGFR2 and VE-PTP showed that ECs in perfused, lumenized vessels in WT teratomas expressed VE-PTP, but displayed very low levels of pVEGFR2. In contrast, disorganized and poorly perfused vessels in ve-ptp−/− teratomas, which we confirmed lacked expression of VE-PTP, displayed prominent pVEGFR2 immunostaining (Fig. 7e). Omitting the primary antibody demonstrated the specificity of the immunostainings (Fig. 7e). There was furthermore a correlation between the extent of perfusion, the expression of VE-PTP and the ordered arrangement of VE-cadherin in the WT teratomas. In non-perfused vascular structures in the ve-ptp−/− teratomas, the VE-cadherin pattern appeared fragmented (Fig. 7f). We also analysed VE-PTP expression in B16 F10 melanoma and identified a correlation between perfusion (functionality), VE-PTP expression and continuous VE-cadherin immunostaining on the one hand, and lack of perfusion, lack of VE-PTP expression and fragmented VE-cadherin on the other hand (Fig. 7g). Our data support a model in which Tie2-mediated suppression of VEGFR2 activity at EC junctions through VE-PTP is essential for formation of functional vessels. In pathological conditions characterized by excess VEGF production and faulty expression VE-PTP, ECs fail to polarize, which leads to a poorly functional vasculature with partially or completely collapsed lumen (Fig. 7h).


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)

VE-PTP regulates VEGFR2 activity and polarization in vivo.(a) WT and ve-ptp−/− teratoma sections show CD31 (green), podocalyxin (red) and Hoechst 33342 (blue). Asterisk, lumen. Scale bars, 20 μm. (b) Podocalyxin intensity/CD31-positive area (mm3) in a. Mean±s.d.; n= 15 CD31-positive vessels of 50 μm length/3 teratomas/genotype. *P<0.05, t-test. (c) WT and ve-ptp−/− teratoma sections. Left: 16-colour intensity scale representation of pVEGFR2 immunostaining. Right: Hoechst 33342 (blue), pY1175 VEGFR2 (green), and CD31 (grey). Scale bars, 20 μm. (d) pY1175 VEGFR2 intensity/CD31-positive area (mm2) in c. Mean±s.d.; n= 12 (WT) and 15 (ve-ptp−/−) blood vessels of 30 μm length from 3 teratomas per genotype. **P<0.01, t-test. (e) Lectin-perfused WT and ve-ptp−/− teratomas. Hoechst 33342 (blue), lectin-FITC (green), pY1175 VEGFR2 (red) and VE-PTP (white). Right: control immunostaining without primary antibodies. Asterisk, lumen. Arrows show VE-PTP expression in WT teratomas. Arrowheads show pVEGFR2 in ve-ptp−/− teratomas. Scale bars, 20 μm. (f) Lectin-perfused WT and ve-ptp−/− teratomas show lectin-FITC (green), VE-cadherin (red) and VE-PTP (white). Asterisk, lumen. Arrowheads in the lower ve-ptp−/− panel indicate fragmented VE-cadherin immunostaining. Scale bars, 20 μm. (g) Lectin-perfused B16 F10 mouse melanomas show lectin-FITC (green), VE-cadherin (VE-cad; red) and VE-PTP (white). Asterisk, lumen. Arrowheads in the lower panel indicate fragmented VE-cadherin immunostaining. Scale bars, 20 μm. (h) Model illustrating the contribution of VE-PTP in silencing VEGFR2 and Tie2 at junctions to support proper EC polarity and vessel morphogenesis. VE-PTP exists in complex with VEGFR2 and Tie2 in the WT condition (left). VEGF induces activation of VEGFR2 and in parallel, dissociation from VE-PTP. VEGF and Ang1 induce translocation of the trimeric complex to junctions where the activated receptors are silenced by VE-PTP. This is compatible with formation of polarized and lumenized vessels. In the VE-PTP-deficient condition (ve-ptp−/−; right), VEGF and Ang1 induce activation and translocation of receptors to junctions, where excess activity leads to VE-cadherin phosphorylation and formation of unpolarized and lumen-less pathological vasculature.
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f7: VE-PTP regulates VEGFR2 activity and polarization in vivo.(a) WT and ve-ptp−/− teratoma sections show CD31 (green), podocalyxin (red) and Hoechst 33342 (blue). Asterisk, lumen. Scale bars, 20 μm. (b) Podocalyxin intensity/CD31-positive area (mm3) in a. Mean±s.d.; n= 15 CD31-positive vessels of 50 μm length/3 teratomas/genotype. *P<0.05, t-test. (c) WT and ve-ptp−/− teratoma sections. Left: 16-colour intensity scale representation of pVEGFR2 immunostaining. Right: Hoechst 33342 (blue), pY1175 VEGFR2 (green), and CD31 (grey). Scale bars, 20 μm. (d) pY1175 VEGFR2 intensity/CD31-positive area (mm2) in c. Mean±s.d.; n= 12 (WT) and 15 (ve-ptp−/−) blood vessels of 30 μm length from 3 teratomas per genotype. **P<0.01, t-test. (e) Lectin-perfused WT and ve-ptp−/− teratomas. Hoechst 33342 (blue), lectin-FITC (green), pY1175 VEGFR2 (red) and VE-PTP (white). Right: control immunostaining without primary antibodies. Asterisk, lumen. Arrows show VE-PTP expression in WT teratomas. Arrowheads show pVEGFR2 in ve-ptp−/− teratomas. Scale bars, 20 μm. (f) Lectin-perfused WT and ve-ptp−/− teratomas show lectin-FITC (green), VE-cadherin (red) and VE-PTP (white). Asterisk, lumen. Arrowheads in the lower ve-ptp−/− panel indicate fragmented VE-cadherin immunostaining. Scale bars, 20 μm. (g) Lectin-perfused B16 F10 mouse melanomas show lectin-FITC (green), VE-cadherin (VE-cad; red) and VE-PTP (white). Asterisk, lumen. Arrowheads in the lower panel indicate fragmented VE-cadherin immunostaining. Scale bars, 20 μm. (h) Model illustrating the contribution of VE-PTP in silencing VEGFR2 and Tie2 at junctions to support proper EC polarity and vessel morphogenesis. VE-PTP exists in complex with VEGFR2 and Tie2 in the WT condition (left). VEGF induces activation of VEGFR2 and in parallel, dissociation from VE-PTP. VEGF and Ang1 induce translocation of the trimeric complex to junctions where the activated receptors are silenced by VE-PTP. This is compatible with formation of polarized and lumenized vessels. In the VE-PTP-deficient condition (ve-ptp−/−; right), VEGF and Ang1 induce activation and translocation of receptors to junctions, where excess activity leads to VE-cadherin phosphorylation and formation of unpolarized and lumen-less pathological vasculature.
Mentions: To provide in vivo confirmation of a role for VE-PTP in vessel lumen formation, we used WT and ve-ptp−/− ESCs to establish teratomas in severe combined immunodeficient (SCID) mice. Teratomas become vascularized in part from the host and in part through differentiation of ECs from the ESCs34. As shown in Fig. 7a, vessels in ve-ptp−/− teratomas displayed disorganized ECs with broadly distributed podocalyxin expression (Fig. 7a). Immunostaining for pVEGFR2 moreover demonstrated significant induction of VEGFR2 phosphorylation in CD31-positive ECs in ve-ptp−/− teratomas (Fig. 7c). Co-immunostaining for pVEGFR2 and VE-PTP showed that ECs in perfused, lumenized vessels in WT teratomas expressed VE-PTP, but displayed very low levels of pVEGFR2. In contrast, disorganized and poorly perfused vessels in ve-ptp−/− teratomas, which we confirmed lacked expression of VE-PTP, displayed prominent pVEGFR2 immunostaining (Fig. 7e). Omitting the primary antibody demonstrated the specificity of the immunostainings (Fig. 7e). There was furthermore a correlation between the extent of perfusion, the expression of VE-PTP and the ordered arrangement of VE-cadherin in the WT teratomas. In non-perfused vascular structures in the ve-ptp−/− teratomas, the VE-cadherin pattern appeared fragmented (Fig. 7f). We also analysed VE-PTP expression in B16 F10 melanoma and identified a correlation between perfusion (functionality), VE-PTP expression and continuous VE-cadherin immunostaining on the one hand, and lack of perfusion, lack of VE-PTP expression and fragmented VE-cadherin on the other hand (Fig. 7g). Our data support a model in which Tie2-mediated suppression of VEGFR2 activity at EC junctions through VE-PTP is essential for formation of functional vessels. In pathological conditions characterized by excess VEGF production and faulty expression VE-PTP, ECs fail to polarize, which leads to a poorly functional vasculature with partially or completely collapsed lumen (Fig. 7h).

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