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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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Tie2 is required in VE-PTP-mediated VEGFR2 dephosphorylation.(a) WT EBs in 3D collagen with VEGF (20 ng ml−1) in the presence or absence of Tie2/Fc protein (4 μg ml−1) show CD31 (green), podocalyxin (red) and Hoechst 33342 (blue). Dashed lines show position of vertical-view images displayed in panels labelled tip, stalk and root. Scale bars, 10 μm. Asterisk, lumen; arrowhead, abnormal podocalyxin distribution. (b) Quantification of ECs with abnormal podocalyxin distribution. Mean±s.d., n= 20 sprouts per condition. ***P<0.001, t-test. (c) pVEGFR2 (green), VEGFR2 (red) and CD31 (white) at day 14 in WT EBs treated or not with Tie2/Fc protein (4 μg ml−1). Arrows indicate pVEGFR2. Scale bars; 20 μm. (d) Ratio of pVEGFR2/VEGFR2 intensities from sprout tip to stalk. Data show the mean; n= 8 sprouts/condition. (e) Ang1 transcript expression/HPRT expression in WT and ve-ptp−/− EBs in 2D collagen with VEGF (20 ng ml−1) at day 14. Mean±s.d. Statistics are based on two independent experiments with n=150 EBs per experiment.
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f6: Tie2 is required in VE-PTP-mediated VEGFR2 dephosphorylation.(a) WT EBs in 3D collagen with VEGF (20 ng ml−1) in the presence or absence of Tie2/Fc protein (4 μg ml−1) show CD31 (green), podocalyxin (red) and Hoechst 33342 (blue). Dashed lines show position of vertical-view images displayed in panels labelled tip, stalk and root. Scale bars, 10 μm. Asterisk, lumen; arrowhead, abnormal podocalyxin distribution. (b) Quantification of ECs with abnormal podocalyxin distribution. Mean±s.d., n= 20 sprouts per condition. ***P<0.001, t-test. (c) pVEGFR2 (green), VEGFR2 (red) and CD31 (white) at day 14 in WT EBs treated or not with Tie2/Fc protein (4 μg ml−1). Arrows indicate pVEGFR2. Scale bars; 20 μm. (d) Ratio of pVEGFR2/VEGFR2 intensities from sprout tip to stalk. Data show the mean; n= 8 sprouts/condition. (e) Ang1 transcript expression/HPRT expression in WT and ve-ptp−/− EBs in 2D collagen with VEGF (20 ng ml−1) at day 14. Mean±s.d. Statistics are based on two independent experiments with n=150 EBs per experiment.

Mentions: To further define the role of Ang1/Tie2 in EC polarization and lumen formation, we treated WT EBs with a Tie2-extracellular domain soluble protein (sTie2/Fc) to neutralize endogenous Ang1 produced by the cultures. We hypothesized that blocking Ang1 using sTie2/Fc would suppress the capacity of VE-PTP to dephosphorylate VEGFR2. Indeed, podocalyxin was broadly distributed and surrounded ECs in sTie2/Fc-treated EBs, whereas untreated EB sprouts displayed polarized, apical distribution of podocalyxin (Fig. 6a). Furthermore, the sTie2/Fc-treated vessel sprouts showed reduced capacity to lumenize. In agreement with a role for VEGFR2 in this process, WT EBs treated with sTie2/Fc showed increased levels of both VEGFR2 and pVEGFR2, and the pVEGFR2/VEGFR2 ratio was elevated in the stalk region (Fig. 6c). These data show that neutralization of endogenously expressed Ang1 (Fig. 6e) by the sTie2/Fc phenocopied the effect of ve-ptp targeting, resulting in elevated pVEGFR2 activity and unpolarized vessels with lumen defects. Therefore, even though VEGF and Ang1 did not regulate the extent of formation of the trimeric complex (Fig. 2i), Ang1 appeared critical in junctional translocation of the complex.


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)

Tie2 is required in VE-PTP-mediated VEGFR2 dephosphorylation.(a) WT EBs in 3D collagen with VEGF (20 ng ml−1) in the presence or absence of Tie2/Fc protein (4 μg ml−1) show CD31 (green), podocalyxin (red) and Hoechst 33342 (blue). Dashed lines show position of vertical-view images displayed in panels labelled tip, stalk and root. Scale bars, 10 μm. Asterisk, lumen; arrowhead, abnormal podocalyxin distribution. (b) Quantification of ECs with abnormal podocalyxin distribution. Mean±s.d., n= 20 sprouts per condition. ***P<0.001, t-test. (c) pVEGFR2 (green), VEGFR2 (red) and CD31 (white) at day 14 in WT EBs treated or not with Tie2/Fc protein (4 μg ml−1). Arrows indicate pVEGFR2. Scale bars; 20 μm. (d) Ratio of pVEGFR2/VEGFR2 intensities from sprout tip to stalk. Data show the mean; n= 8 sprouts/condition. (e) Ang1 transcript expression/HPRT expression in WT and ve-ptp−/− EBs in 2D collagen with VEGF (20 ng ml−1) at day 14. Mean±s.d. Statistics are based on two independent experiments with n=150 EBs per experiment.
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f6: Tie2 is required in VE-PTP-mediated VEGFR2 dephosphorylation.(a) WT EBs in 3D collagen with VEGF (20 ng ml−1) in the presence or absence of Tie2/Fc protein (4 μg ml−1) show CD31 (green), podocalyxin (red) and Hoechst 33342 (blue). Dashed lines show position of vertical-view images displayed in panels labelled tip, stalk and root. Scale bars, 10 μm. Asterisk, lumen; arrowhead, abnormal podocalyxin distribution. (b) Quantification of ECs with abnormal podocalyxin distribution. Mean±s.d., n= 20 sprouts per condition. ***P<0.001, t-test. (c) pVEGFR2 (green), VEGFR2 (red) and CD31 (white) at day 14 in WT EBs treated or not with Tie2/Fc protein (4 μg ml−1). Arrows indicate pVEGFR2. Scale bars; 20 μm. (d) Ratio of pVEGFR2/VEGFR2 intensities from sprout tip to stalk. Data show the mean; n= 8 sprouts/condition. (e) Ang1 transcript expression/HPRT expression in WT and ve-ptp−/− EBs in 2D collagen with VEGF (20 ng ml−1) at day 14. Mean±s.d. Statistics are based on two independent experiments with n=150 EBs per experiment.
Mentions: To further define the role of Ang1/Tie2 in EC polarization and lumen formation, we treated WT EBs with a Tie2-extracellular domain soluble protein (sTie2/Fc) to neutralize endogenous Ang1 produced by the cultures. We hypothesized that blocking Ang1 using sTie2/Fc would suppress the capacity of VE-PTP to dephosphorylate VEGFR2. Indeed, podocalyxin was broadly distributed and surrounded ECs in sTie2/Fc-treated EBs, whereas untreated EB sprouts displayed polarized, apical distribution of podocalyxin (Fig. 6a). Furthermore, the sTie2/Fc-treated vessel sprouts showed reduced capacity to lumenize. In agreement with a role for VEGFR2 in this process, WT EBs treated with sTie2/Fc showed increased levels of both VEGFR2 and pVEGFR2, and the pVEGFR2/VEGFR2 ratio was elevated in the stalk region (Fig. 6c). These data show that neutralization of endogenously expressed Ang1 (Fig. 6e) by the sTie2/Fc phenocopied the effect of ve-ptp targeting, resulting in elevated pVEGFR2 activity and unpolarized vessels with lumen defects. Therefore, even though VEGF and Ang1 did not regulate the extent of formation of the trimeric complex (Fig. 2i), Ang1 appeared critical in junctional translocation of the complex.

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