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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

VEGFR2 stalk cell activity in ve-ptp−/− EBs.WT and ve-ptp−/− EBs cultured in 3D collagen gels with VEGF (20 ng ml−1) until day 14, unless otherwise indicated. (a) Immunostaining of ECs (CD31; green) and pericytes (NG2; red) in WT and ve-ptp−/− EBs. Scale bars; 100μm. (b) Quantification of CD31-positive sprout area per EB. Mean±s.d., n=6 EBs per genotype. *P<0.05, t-test. (c) Quantification of sprout length, from core to sprout tip. Mean±s.d., n=100 sprouts per genotype. (d) Flow sorting of dissociated WT and ve-ptp−/− EBs (day 9; 2D culture) identified CD31 and VE-cadherin double-positive ECs. Mean±s.d., n=150 EBs per genotype. *P<0.05, t-test. (e) Detection of CD31-positive long (> 3 μm) filopodia (asterisk) per 100 μm EB sprout length. Mean±s.d., n=20 sprouts per genotype. ***P<0.001, t-test. (f) Representation of pY1175 VEGFR2 (upper) and VEGFR2 (middle) immunofluorescent staining using a 16-colour intensity scale. Merged image (bottom panel) shows pVEGFR2 (green), VEGFR2 (red) and CD31 (white). Scale bar; 20 μm. High-magnification insets show pVEGFR2; scale bar; 5 μm. (g) Ratio pVEGFR2/VEGFR2 fluorescent intensities per total CD31-area. Mean±s.d., n= 7 sprouts per genotype. The mean pVEGFR2/VEGFR2 ratio differed significantly between WT and ve-ptp−/− stalk cell region of the sprout; P<0.001 as determined using paired two-tailed t-test. (h) Vertical-view image of WT and ve-ptp−/− sprouts immunostained for pY1175 VEGFR2 (green), VEGFR2 (red) and CD31 (white). Arrows indicate pVEGFR2. Scale bars; 5 μm. (i) Immunostaining for VE-PTP (green) and CD31 (blue) in WT sprouts, show localization of VE-PTP in junctions (arrowheads) in the stalk region. Broken lines in the left, larger panel indicate position of vertical-view images shown in panels to the right. Scale bar; 20 μm. All analyses were repeated at least three times.
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f1: VEGFR2 stalk cell activity in ve-ptp−/− EBs.WT and ve-ptp−/− EBs cultured in 3D collagen gels with VEGF (20 ng ml−1) until day 14, unless otherwise indicated. (a) Immunostaining of ECs (CD31; green) and pericytes (NG2; red) in WT and ve-ptp−/− EBs. Scale bars; 100μm. (b) Quantification of CD31-positive sprout area per EB. Mean±s.d., n=6 EBs per genotype. *P<0.05, t-test. (c) Quantification of sprout length, from core to sprout tip. Mean±s.d., n=100 sprouts per genotype. (d) Flow sorting of dissociated WT and ve-ptp−/− EBs (day 9; 2D culture) identified CD31 and VE-cadherin double-positive ECs. Mean±s.d., n=150 EBs per genotype. *P<0.05, t-test. (e) Detection of CD31-positive long (> 3 μm) filopodia (asterisk) per 100 μm EB sprout length. Mean±s.d., n=20 sprouts per genotype. ***P<0.001, t-test. (f) Representation of pY1175 VEGFR2 (upper) and VEGFR2 (middle) immunofluorescent staining using a 16-colour intensity scale. Merged image (bottom panel) shows pVEGFR2 (green), VEGFR2 (red) and CD31 (white). Scale bar; 20 μm. High-magnification insets show pVEGFR2; scale bar; 5 μm. (g) Ratio pVEGFR2/VEGFR2 fluorescent intensities per total CD31-area. Mean±s.d., n= 7 sprouts per genotype. The mean pVEGFR2/VEGFR2 ratio differed significantly between WT and ve-ptp−/− stalk cell region of the sprout; P<0.001 as determined using paired two-tailed t-test. (h) Vertical-view image of WT and ve-ptp−/− sprouts immunostained for pY1175 VEGFR2 (green), VEGFR2 (red) and CD31 (white). Arrows indicate pVEGFR2. Scale bars; 5 μm. (i) Immunostaining for VE-PTP (green) and CD31 (blue) in WT sprouts, show localization of VE-PTP in junctions (arrowheads) in the stalk region. Broken lines in the left, larger panel indicate position of vertical-view images shown in panels to the right. Scale bar; 20 μm. All analyses were repeated at least three times.

Mentions: We analysed sprouting angiogenesis in mouse embryoid bodies (EBs) from wild-type (WT) and ve-ptp−/− embryonic stem cells (ESCs)821. Ve-ptp−/− EBs formed a denser network of vessel sprouts with similar length but with increased area compared with WT EBs (Fig. 1a–c). There was a tenfold increase in CD31/VE-cadherin double-positive ECs in VEGF-treated ve-ptp−/− EBs compared with VEGF-treated wild-type EBs (Fig. 1d). Moreover, the ve-ptp−/− ECs extended numerous long filopodia throughout the sprout, while most WT stalk cells did not (Fig. 1e). We hypothesized that increased EC proliferation and filopodia formation might be due to elevated VEGFR2 activity.


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)

VEGFR2 stalk cell activity in ve-ptp−/− EBs.WT and ve-ptp−/− EBs cultured in 3D collagen gels with VEGF (20 ng ml−1) until day 14, unless otherwise indicated. (a) Immunostaining of ECs (CD31; green) and pericytes (NG2; red) in WT and ve-ptp−/− EBs. Scale bars; 100μm. (b) Quantification of CD31-positive sprout area per EB. Mean±s.d., n=6 EBs per genotype. *P<0.05, t-test. (c) Quantification of sprout length, from core to sprout tip. Mean±s.d., n=100 sprouts per genotype. (d) Flow sorting of dissociated WT and ve-ptp−/− EBs (day 9; 2D culture) identified CD31 and VE-cadherin double-positive ECs. Mean±s.d., n=150 EBs per genotype. *P<0.05, t-test. (e) Detection of CD31-positive long (> 3 μm) filopodia (asterisk) per 100 μm EB sprout length. Mean±s.d., n=20 sprouts per genotype. ***P<0.001, t-test. (f) Representation of pY1175 VEGFR2 (upper) and VEGFR2 (middle) immunofluorescent staining using a 16-colour intensity scale. Merged image (bottom panel) shows pVEGFR2 (green), VEGFR2 (red) and CD31 (white). Scale bar; 20 μm. High-magnification insets show pVEGFR2; scale bar; 5 μm. (g) Ratio pVEGFR2/VEGFR2 fluorescent intensities per total CD31-area. Mean±s.d., n= 7 sprouts per genotype. The mean pVEGFR2/VEGFR2 ratio differed significantly between WT and ve-ptp−/− stalk cell region of the sprout; P<0.001 as determined using paired two-tailed t-test. (h) Vertical-view image of WT and ve-ptp−/− sprouts immunostained for pY1175 VEGFR2 (green), VEGFR2 (red) and CD31 (white). Arrows indicate pVEGFR2. Scale bars; 5 μm. (i) Immunostaining for VE-PTP (green) and CD31 (blue) in WT sprouts, show localization of VE-PTP in junctions (arrowheads) in the stalk region. Broken lines in the left, larger panel indicate position of vertical-view images shown in panels to the right. Scale bar; 20 μm. All analyses were repeated at least three times.
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f1: VEGFR2 stalk cell activity in ve-ptp−/− EBs.WT and ve-ptp−/− EBs cultured in 3D collagen gels with VEGF (20 ng ml−1) until day 14, unless otherwise indicated. (a) Immunostaining of ECs (CD31; green) and pericytes (NG2; red) in WT and ve-ptp−/− EBs. Scale bars; 100μm. (b) Quantification of CD31-positive sprout area per EB. Mean±s.d., n=6 EBs per genotype. *P<0.05, t-test. (c) Quantification of sprout length, from core to sprout tip. Mean±s.d., n=100 sprouts per genotype. (d) Flow sorting of dissociated WT and ve-ptp−/− EBs (day 9; 2D culture) identified CD31 and VE-cadherin double-positive ECs. Mean±s.d., n=150 EBs per genotype. *P<0.05, t-test. (e) Detection of CD31-positive long (> 3 μm) filopodia (asterisk) per 100 μm EB sprout length. Mean±s.d., n=20 sprouts per genotype. ***P<0.001, t-test. (f) Representation of pY1175 VEGFR2 (upper) and VEGFR2 (middle) immunofluorescent staining using a 16-colour intensity scale. Merged image (bottom panel) shows pVEGFR2 (green), VEGFR2 (red) and CD31 (white). Scale bar; 20 μm. High-magnification insets show pVEGFR2; scale bar; 5 μm. (g) Ratio pVEGFR2/VEGFR2 fluorescent intensities per total CD31-area. Mean±s.d., n= 7 sprouts per genotype. The mean pVEGFR2/VEGFR2 ratio differed significantly between WT and ve-ptp−/− stalk cell region of the sprout; P<0.001 as determined using paired two-tailed t-test. (h) Vertical-view image of WT and ve-ptp−/− sprouts immunostained for pY1175 VEGFR2 (green), VEGFR2 (red) and CD31 (white). Arrows indicate pVEGFR2. Scale bars; 5 μm. (i) Immunostaining for VE-PTP (green) and CD31 (blue) in WT sprouts, show localization of VE-PTP in junctions (arrowheads) in the stalk region. Broken lines in the left, larger panel indicate position of vertical-view images shown in panels to the right. Scale bar; 20 μm. All analyses were repeated at least three times.
Mentions: We analysed sprouting angiogenesis in mouse embryoid bodies (EBs) from wild-type (WT) and ve-ptp−/− embryonic stem cells (ESCs)821. Ve-ptp−/− EBs formed a denser network of vessel sprouts with similar length but with increased area compared with WT EBs (Fig. 1a–c). There was a tenfold increase in CD31/VE-cadherin double-positive ECs in VEGF-treated ve-ptp−/− EBs compared with VEGF-treated wild-type EBs (Fig. 1d). Moreover, the ve-ptp−/− ECs extended numerous long filopodia throughout the sprout, while most WT stalk cells did not (Fig. 1e). We hypothesized that increased EC proliferation and filopodia formation might be due to elevated VEGFR2 activity.

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