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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 is required for EC polarization and lumen formation.Panels a, c, f, g and k show immunostaining of WT and ve-ptp−/− EBs at day 14 or 21, treated with 20 ng ml−1 of VEGF or, when indicated, 3 ng ml−1. Hoechst 33342 (blue) staining shows nuclei. Dashed lines show position of parallel vertical-view images. (a) Podocalyxin (Pdx; red), moesin (Msn; white), CD31 (green). Scale bars, 20 μm. Vertical-view images represent sprout tip, stalk and root; scale bars, 10 μm. Asterisk, lumen; arrowhead, abnormal podocalyxin distribution. (b) Quantification of ECs with abnormal podocalyxin per total ECs in a. Mean±s.d., n= 25 sprouts per genotype. ***P<0.001, t-test. (c) CD31 (green) and podocalyxin (red), show lumen in WT (asterisk) but cup-shaped ve-ptp−/− sprouts. Scale bars, 20 μm. (d) Quantification of abnormal podocalyxin/total CD31+ ECs in day 21 EBs. Mean±s.d.; n= 6 sprouts per genotype. *P<0.05, t-test. (e) Quantification of sprouts with continuous or discontinuous lumen and cup-shaped sprouts in the WT and ve-ptp−/− EBs. Mean±s.d.; n=10 sprouts/WT EBs and 20/ve-ptp−/− EBs. (f) CD31 (green) and podocalyxin (red) in EBs treated with different concentrations of VEGF. Arrowheads, abnormal podocalyxin distribution. Scale bars, 20 μm. (g) pY658 VE-cadherin (green) and VE-cadherin (red) in EBs treated with different concentrations of VEGF. Scale bars, 20 μm. (h) Quantification of ECs with abnormally localized podocalyxin in f. Mean±s.d.; n= 14–15 sprouts for each condition, *P<0.05, **P<0.01, t-test. (i) Quantification of pVE-cadherin/VE-cadherin in g. Mean±s.d.; n= 5–8 sprouts for each condition, *P<0.05, t-test. (j) HUVECs treated individually with VEGF, Ang1 or VEGF+Ang1, immunoprecipitation (IP) of VE-cadherin and VEGFR2, and immunoblotting for pY658 VE-cadherin, VE-cadherin, pY1175 VEGFR2 and VEGFR2. (k) WT and ve-ptp−/− EBs at day 14 represented by 3D Imaris images show VE-cadherin (VE-cad; green) and ZO1 (red). Arrows, fragmented VE-cadherin. Scale bars, 20 μm. (l) Quantification of VE-cadherin colocalized with ZO1 per 100 μm sprout length. Mean±s.d. n=4 sprouts for each condition. *P<0.05, t-test.
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f5: VE-PTP is required for EC polarization and lumen formation.Panels a, c, f, g and k show immunostaining of WT and ve-ptp−/− EBs at day 14 or 21, treated with 20 ng ml−1 of VEGF or, when indicated, 3 ng ml−1. Hoechst 33342 (blue) staining shows nuclei. Dashed lines show position of parallel vertical-view images. (a) Podocalyxin (Pdx; red), moesin (Msn; white), CD31 (green). Scale bars, 20 μm. Vertical-view images represent sprout tip, stalk and root; scale bars, 10 μm. Asterisk, lumen; arrowhead, abnormal podocalyxin distribution. (b) Quantification of ECs with abnormal podocalyxin per total ECs in a. Mean±s.d., n= 25 sprouts per genotype. ***P<0.001, t-test. (c) CD31 (green) and podocalyxin (red), show lumen in WT (asterisk) but cup-shaped ve-ptp−/− sprouts. Scale bars, 20 μm. (d) Quantification of abnormal podocalyxin/total CD31+ ECs in day 21 EBs. Mean±s.d.; n= 6 sprouts per genotype. *P<0.05, t-test. (e) Quantification of sprouts with continuous or discontinuous lumen and cup-shaped sprouts in the WT and ve-ptp−/− EBs. Mean±s.d.; n=10 sprouts/WT EBs and 20/ve-ptp−/− EBs. (f) CD31 (green) and podocalyxin (red) in EBs treated with different concentrations of VEGF. Arrowheads, abnormal podocalyxin distribution. Scale bars, 20 μm. (g) pY658 VE-cadherin (green) and VE-cadherin (red) in EBs treated with different concentrations of VEGF. Scale bars, 20 μm. (h) Quantification of ECs with abnormally localized podocalyxin in f. Mean±s.d.; n= 14–15 sprouts for each condition, *P<0.05, **P<0.01, t-test. (i) Quantification of pVE-cadherin/VE-cadherin in g. Mean±s.d.; n= 5–8 sprouts for each condition, *P<0.05, t-test. (j) HUVECs treated individually with VEGF, Ang1 or VEGF+Ang1, immunoprecipitation (IP) of VE-cadherin and VEGFR2, and immunoblotting for pY658 VE-cadherin, VE-cadherin, pY1175 VEGFR2 and VEGFR2. (k) WT and ve-ptp−/− EBs at day 14 represented by 3D Imaris images show VE-cadherin (VE-cad; green) and ZO1 (red). Arrows, fragmented VE-cadherin. Scale bars, 20 μm. (l) Quantification of VE-cadherin colocalized with ZO1 per 100 μm sprout length. Mean±s.d. n=4 sprouts for each condition. *P<0.05, t-test.

Mentions: We observed a reduced number of pericytes associated with angiogenic sprouts in ve-ptp−/− EBs (Supplementary Fig. S1a,b); a hallmark of vessel immaturity. We therefore asked whether excess VEGFR2 activity in the absence of VE-PTP would interfere with vessel maturation. In WT sprouts at day 14 of differentiation, the apical/lumen markers podocalyxin and moesin3233 were detected at stalk EC junctions, which contained a clearly distinguishable lumen (Fig. 5a, left). In ve-ptp−/− sprouts, podocalyxin and moesin were disorganized, surrounding certain ECs cells and missing from others (Fig. 5a, right). Strikingly, ve-ptp−/− sprouts did not enclose a lumen but often presented a cup-shaped structure (see stalk cell image in Fig. 5a, right). About 25% of ve-ptp−/− ECs showed abnormally located podocalyxin (Fig. 5b) as determined in a double-blind assessment.


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 is required for EC polarization and lumen formation.Panels a, c, f, g and k show immunostaining of WT and ve-ptp−/− EBs at day 14 or 21, treated with 20 ng ml−1 of VEGF or, when indicated, 3 ng ml−1. Hoechst 33342 (blue) staining shows nuclei. Dashed lines show position of parallel vertical-view images. (a) Podocalyxin (Pdx; red), moesin (Msn; white), CD31 (green). Scale bars, 20 μm. Vertical-view images represent sprout tip, stalk and root; scale bars, 10 μm. Asterisk, lumen; arrowhead, abnormal podocalyxin distribution. (b) Quantification of ECs with abnormal podocalyxin per total ECs in a. Mean±s.d., n= 25 sprouts per genotype. ***P<0.001, t-test. (c) CD31 (green) and podocalyxin (red), show lumen in WT (asterisk) but cup-shaped ve-ptp−/− sprouts. Scale bars, 20 μm. (d) Quantification of abnormal podocalyxin/total CD31+ ECs in day 21 EBs. Mean±s.d.; n= 6 sprouts per genotype. *P<0.05, t-test. (e) Quantification of sprouts with continuous or discontinuous lumen and cup-shaped sprouts in the WT and ve-ptp−/− EBs. Mean±s.d.; n=10 sprouts/WT EBs and 20/ve-ptp−/− EBs. (f) CD31 (green) and podocalyxin (red) in EBs treated with different concentrations of VEGF. Arrowheads, abnormal podocalyxin distribution. Scale bars, 20 μm. (g) pY658 VE-cadherin (green) and VE-cadherin (red) in EBs treated with different concentrations of VEGF. Scale bars, 20 μm. (h) Quantification of ECs with abnormally localized podocalyxin in f. Mean±s.d.; n= 14–15 sprouts for each condition, *P<0.05, **P<0.01, t-test. (i) Quantification of pVE-cadherin/VE-cadherin in g. Mean±s.d.; n= 5–8 sprouts for each condition, *P<0.05, t-test. (j) HUVECs treated individually with VEGF, Ang1 or VEGF+Ang1, immunoprecipitation (IP) of VE-cadherin and VEGFR2, and immunoblotting for pY658 VE-cadherin, VE-cadherin, pY1175 VEGFR2 and VEGFR2. (k) WT and ve-ptp−/− EBs at day 14 represented by 3D Imaris images show VE-cadherin (VE-cad; green) and ZO1 (red). Arrows, fragmented VE-cadherin. Scale bars, 20 μm. (l) Quantification of VE-cadherin colocalized with ZO1 per 100 μm sprout length. Mean±s.d. n=4 sprouts for each condition. *P<0.05, t-test.
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f5: VE-PTP is required for EC polarization and lumen formation.Panels a, c, f, g and k show immunostaining of WT and ve-ptp−/− EBs at day 14 or 21, treated with 20 ng ml−1 of VEGF or, when indicated, 3 ng ml−1. Hoechst 33342 (blue) staining shows nuclei. Dashed lines show position of parallel vertical-view images. (a) Podocalyxin (Pdx; red), moesin (Msn; white), CD31 (green). Scale bars, 20 μm. Vertical-view images represent sprout tip, stalk and root; scale bars, 10 μm. Asterisk, lumen; arrowhead, abnormal podocalyxin distribution. (b) Quantification of ECs with abnormal podocalyxin per total ECs in a. Mean±s.d., n= 25 sprouts per genotype. ***P<0.001, t-test. (c) CD31 (green) and podocalyxin (red), show lumen in WT (asterisk) but cup-shaped ve-ptp−/− sprouts. Scale bars, 20 μm. (d) Quantification of abnormal podocalyxin/total CD31+ ECs in day 21 EBs. Mean±s.d.; n= 6 sprouts per genotype. *P<0.05, t-test. (e) Quantification of sprouts with continuous or discontinuous lumen and cup-shaped sprouts in the WT and ve-ptp−/− EBs. Mean±s.d.; n=10 sprouts/WT EBs and 20/ve-ptp−/− EBs. (f) CD31 (green) and podocalyxin (red) in EBs treated with different concentrations of VEGF. Arrowheads, abnormal podocalyxin distribution. Scale bars, 20 μm. (g) pY658 VE-cadherin (green) and VE-cadherin (red) in EBs treated with different concentrations of VEGF. Scale bars, 20 μm. (h) Quantification of ECs with abnormally localized podocalyxin in f. Mean±s.d.; n= 14–15 sprouts for each condition, *P<0.05, **P<0.01, t-test. (i) Quantification of pVE-cadherin/VE-cadherin in g. Mean±s.d.; n= 5–8 sprouts for each condition, *P<0.05, t-test. (j) HUVECs treated individually with VEGF, Ang1 or VEGF+Ang1, immunoprecipitation (IP) of VE-cadherin and VEGFR2, and immunoblotting for pY658 VE-cadherin, VE-cadherin, pY1175 VEGFR2 and VEGFR2. (k) WT and ve-ptp−/− EBs at day 14 represented by 3D Imaris images show VE-cadherin (VE-cad; green) and ZO1 (red). Arrows, fragmented VE-cadherin. Scale bars, 20 μm. (l) Quantification of VE-cadherin colocalized with ZO1 per 100 μm sprout length. Mean±s.d. n=4 sprouts for each condition. *P<0.05, t-test.
Mentions: We observed a reduced number of pericytes associated with angiogenic sprouts in ve-ptp−/− EBs (Supplementary Fig. S1a,b); a hallmark of vessel immaturity. We therefore asked whether excess VEGFR2 activity in the absence of VE-PTP would interfere with vessel maturation. In WT sprouts at day 14 of differentiation, the apical/lumen markers podocalyxin and moesin3233 were detected at stalk EC junctions, which contained a clearly distinguishable lumen (Fig. 5a, left). In ve-ptp−/− sprouts, podocalyxin and moesin were disorganized, surrounding certain ECs cells and missing from others (Fig. 5a, right). Strikingly, ve-ptp−/− sprouts did not enclose a lumen but often presented a cup-shaped structure (see stalk cell image in Fig. 5a, right). About 25% of ve-ptp−/− ECs showed abnormally located podocalyxin (Fig. 5b) as determined in a double-blind assessment.

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