VE-PTP controls blood vessel development by balancing Tie-2 activity

Abstract

Vascular endothelial protein tyrosine phosphatase (VE-PTP) is an endothelial-specific receptor-type tyrosine phosphatase that associates with Tie-2 and VE-cadherin. VE-PTP gene disruption leads to embryonic lethality, vascular remodeling defects, and enlargement of vascular structures in extraembryonic tissues. We show here that antibodies against the extracellular part of VE-PTP mimic the effects of VE-PTP gene disruption exemplified by vessel enlargement in allantois explants. These effects require the presence of the angiopoietin receptor Tie-2. Analyzing the mechanism we found that anti-VE-PTP antibodies trigger endocytosis and selectively affect Tie-2-associated, but not VE-cadherin-associated VE-PTP. Dissociation of VE-PTP triggers the activation of Tie-2, leading to enhanced endothelial cell proliferation and enlargement of vascular structures through activation of Erk1/2. Importantly, the antibody effect on vessel enlargement is also observed in newborn mice. We conclude that VE-PTP is required to balance Tie-2 activity and endothelial cell proliferation, thereby controlling blood vessel development and vessel size.

Figure 1.

Antibodies against VE-PTP trigger vessel enlargement in allantois explants and down-regulate VE-PTP. (A) Allantois explants from E8.5 wild-type embryos were cultured on gelatin-coated ultrathin glass slides in the presence of polyclonal antibodies against VE-PTP (α-VE-PTP) or preimmune antibodies (control) for 22 h and subsequently stained by indirect immunofluorescence with a monoclonal antibody for VE-cadherin. Bar, 50 µm. (B) Average endothelial cord diameters (top) and branching points (bottom) were determined for 5 control and 5 anti–VE-PTP treated allantois explants, with 30 randomly chosen vessels per explant (as shown in A); ***, P < 0,001. (C) Confluent mouse bEnd.5 cells were treated with polyclonal antibodies against VE-PTP or preimmune antibodies for 1 h either in normal culture medium or 0.45 M sucrose containing medium (as indicated) to block endocytosis. Aliquots of cell lysates with identical protein content were immunoblotted for VE-PTP and equal loading was controlled by blotting for the endothelial antigen ESAM (as indicated on the right). Molecular weight markers are indicated on the left.

Figure 2.

Antibodies against VE-PTP trigger endocytosis and down-regulation of Tie-2–associated but not VE-cadherin–associated VE-PTP. (A) Confluent bEnd.3 cells were treated with polyclonal antibodies against VE-PTP (α-VE-PTP) or preimmune antibodies (control IgG) for 20 min. Subsequently, cells were either fixed and permeabilized (top and bottom) or only fixed (middle) and stained with Alexa 568–conjugated secondary antibodies. Cell nuclei were counterstained with Hoechst. Bar, 20 µm. (B) bEnd.5 cells were treated with polyclonal antibodies against VE-PTP (α-VE-PTP) or preimmune antibodies (control) for 1 h. VE-PTP was immunoprecipitated from endothelial cells and analyzed by immunoblotting for coprecipitated VE-cadherin and Tie-2, respectively, or for VE-PTP (as indicated underneath). Aliquots of cell lysates with identical protein content were directly immunoblotted for VE-cadherin or Tie-2 (bottom). Quantified signal intensities are indicated. Molecular weight markers are indicated. (C) FACS analysis showing the surface expression of Tie-2, VE-cadherin, and VE-PTP of bEnd.5 cells after 1 h pretreatment with monoclonal antibodies against VE-PTP (red) or preimmune antibodies (blue). The mean fluorescence FACS signal for VE-PTP is indicated in percent (bottom).

Figure 3.

Antibodies against VE-PTP do not trigger endocytosis of Tie-2 and leave VE-cadherin and VE-PTP at endothelial cell contacts unaffected. (A) Confluent bEnd.3 cells were treated with polyclonal antibodies against VE-PTP (α-VE-PTP) or preimmune antibodies (control IgG) for 20 min. Subsequently, fixed and permeabilized cells were stained with Alexa 568–conjugated secondary antibodies (internalized VE-PTP, internalized control) and for Tie-2 (Tie-2). An anti–Tie-2 staining control is shown in Fig. S5. Cell nuclei were counterstained with Hoechst. Bar, 25 µm. (B) Confluent bEnd.3 cells were treated with monoclonal antibodies against VE-PTP (α-VE-PTP) or control antibodies (control IgG) for 30 min. Subsequently, fixed and permeabilized cells were stained with Alexa 568–conjugated secondary antibodies (internalized VE-PTP, internalized control) and for VE-cadherin and VE-PTP. Cell nuclei were counterstained with Hoechst. Internalized VE-PTP was not detected with new antibodies against VE-PTP, probably because epitopes were masked by the antibodies that had triggered endocytosis. Bar, 10 µm.

Figure 4.

VE-PTP expression inhibited by either antibodies, siRNA, or gene disruption triggers Tie-2 tyrosine phosphorylation in endothelial cells. (A) bEnd.5 cells were treated with polyclonal antibodies against VE-PTP or preimmune antibodies for 1 h and subsequently immunoprecipitated for Tie-2, followed by immunoblotting with anti-phosphotyrosine antibodies (pTyr) and antibodies against Tie-2. Aliquots of cell lysates with identical protein content were directly immunoblotted for VE-PTP and Tie-2 (bottom). (B) Similar as in A, except that antibodies were only incubated for 3 min (C) bEnd.5 cells were either transfected with control siRNA or with siRNA directed against VE-PTP. 24 h later, Tie-2 was immunoprecipitated and immunocomplexes (top) or cell lysates (bottom) were analyzed by immunoblotting with antibodies against phosphotyrosine (pTyr), Tie-2, VE-PTP, and plakoglobin (as indicated). (D) HUVECs instead of bEnd.5 cells were analyzed as in C. (E) Embryonic endothelioma cells established either from wild-type (+/+), heterozygous (+/mut), or homozygous (mut/mut) VE-PTP mutant embryos were subjected to immunoprecipitations with antibodies against Tie-2. Immunocomplexes were analyzed by immunoblotting with antibodies against phosphotyrosine or Tie-2 (as indicated). (F) Quantification of Tie-2 tyrosine phosphorylation (±SD) in wild-type (+/+; n = 2 cell lines), heterozygous (+/mut; n = 3 cell lines), or homozygous (mut/mut; n = 3 cell lines) VE-PTP mutant endothelioma, analyzed as in E. **, P < 0,01.

Figure 5.

Down-regulation of VE-PTP by siRNA, but not by anti–VE-PTP antibodies, enhances tyrosine phosphorylation of VE-cadherin–associated plakoglobin. (A) bEnd.5 cells were treated with polyclonal antibodies against VE-PTP (α-VE-PTP) or preimmune antibodies (control) for 1 h and subsequently immunoprecipitated for VE-cadherin, followed by immunoblotting with antibodies against phosphotyrosine (pTyr), VE-cadherin, or plakoglobin. Aliquots of cell lysates with identical protein content were directly immunoblotted for VE-PTP and VE-cadherin (bottom). (B) Similar as in A, except that antibody preincubation of cells was replaced by transfection with either control siRNA or VE-PTP siRNA.

Figure 6.

VE-PTP regulates Tie-2 phosphorylation via its active phosphatase. (A) HUVECs either untransfected, or expressing GFP or expressing mouse Flag-VE-PTP (as indicated above) were either untreated or treated with polyclonal antibodies against mouse VE-PTP or preimmune antibodies (as indicated above) for 1 h and subsequently immunoprecipitated for Tie-2, followed by immunoblotting with anti-phosphotyrosine antibodies (pTyr) and antibodies against Tie-2. Aliquots of cell lysates with identical protein content were directly immunoblotted for Flag-VE-PTP and hVE-PTP (bottom). Quantified signal intensities are indicated. (B) As in A with mouse Flag-VE-PTP being replaced by the corresponding inactive phosphatase mutant Flag-VE-PTP-C/S. White lines indicate that intervening lanes have been spliced out.

Figure 7.

VE-PTP counterbalances Ang1 activation of Tie-2 and regulates Tie-1 tyrosine phosphorylation dependent on Tie-2. (A) bEnd.5 cells were either transfected with control siRNAs or with siRNAs directed against VE-PTP. 24 h later, cells were stimulated either with 600 ng/ml Ang1 or 600 ng/ml Ang2 for 10 min, or unstimulated (unstim). Immunoprecipitates of Tie-2 (top two panels) or cell lysates (bottom two panels) were immunoblotted for the indicated antigens. (B) HUVECs were either transfected with control siRNAs or with siRNAs directed against hVE-PTP. 24 h later, siRNA-transfected cells (left two lanes) and untransfected cells, stimulated either with 600 ng/ml Ang1 for 10 min or unstimulated (right two lanes), were subjected to immunoprecipitations for Tie-2, followed by immunoblotting with antibodies against the phosphorylated tyrosine 992 in the active loop of the kinase (pTyr 992) and Tie-2. Aliquots of cell lysates were immunoblotted directly for hVE-PTP and Tie-2 (bottom two panels). White line indicates that intervening lanes have been spliced out. (C) bEnd.5 cells were treated either with polyclonal antibodies against VE-PTP (α-VE-PTP) or preimmune antibodies (control) for 1 h, followed by either Ang1 stimulation for 10 min (Ang1) or no stimulation (unstim). Phosphorylation of tyrosine 992 was analyzed as described for B. Quantified signal intensities are indicated. (D) bEnd.5 cells were stimulated with 200 ng/ml COMP-Ang1 for 10 min or left untreated. VE-PTP immunoprecipitates (top three panels) and cell lysates (bottom two panels) were analyzed by immunoblotting for Tie-2, VE-cadherin, and VE-PTP as indicated on the right. (E) Embryonic endothelioma cells established either from wild-type (+/+) or Tie-2–deficient (−/−) embryos were antibody pretreated as indicated and subjected to immunoprecipitations with antibodies against Tie-1. Immunocomplexes were analyzed by immunoblotting with antibodies against phosphotyrosine or Tie-1 (as indicated). Aliquots of cell lysates were immunoblotted directly for Tie-1 and Tie-2 (bottom two panels).

Figure 8.

Vessel enlargement induced by anti–VE-PTP antibodies requires Tie-2. (A) Allantois explants from E8.5 Tie-2 +/+, Tie-2 +/−, or Tie-2 −/− embryos (as indicated) were cultured on gelatin-coated ultrathin glass slides in the presence of polyclonal antibodies against VE-PTP for 22 h. (B) Allantois explants from E8.5 embryos were cultured in medium (untreated) or in the presence of 600 ng/ml Ang1. Subsequently, endothelium was visualized in all samples in A and B by indirect immunofluorescence staining for VE-cadherin. Bars, 50 µm.

Figure 9.

Anti–VE-PTP antibody treatment of allantois explants stimulates endothelial cell proliferation and enlargement of endothelial cords through activation of Erk1/2. (A) Allantois explants of E8.5 embryos from knock-in mice expressing VE-cadherin-GFP from the VE-cadherin genetic locus were cultured on gelatin-coated ultrathin glass slides for 12 h and were then analyzed by live imaging during the next 12 h (see Video 2), while they were cultured in the presence of polyclonal antibodies against VE-PTP. Subsequently, allantoides were fixed and double stained for PECAM-1 and phospho-Histone 3. Arrows indicate proliferating endothelial cells with a characteristic round cell shape. Bar, 20 µm. (B) Same as in A, depicting larger areas of the explants. Bar, 100 µm. (C) Percentage of phospho-Histone 3–positive endothelial cells per volume tissue in E8.5 allantois explants cultured with polyclonal antibodies against VE-PTP or preimmune antibodies for 12 h. *, P < 0,05. (D) bEnd.5 cells were treated with mAb against ESAM (control) or for indicated time periods with a mAb against VE-PTP. Cell lysates were analyzed by immunoblotting with anti–phospho-Erk1/2–specific antibodies (pT202/pY204) and antibodies against Erk1/2. Treatment with mAb against ESAM gave a similar result as in the absence of antibodies (not depicted). (E) Endothelioma cells of wild-type genotype (Tie-2 +/+) or deficient for Tie-2 (Tie-2 −/−) were treated with monoclonal antibodies against ESAM (control) or against VE-PTP for 1 h, followed by immunoblotting cell lysates with anti–phospho-Erk1/2–specific antibodies (pT202/pY204) and antibodies against Erk1/2, as indicated on the right. (F) bEnd.5 cells were treated with 50 µM of the Erk1/2 inhibitor PD98059 (PD 98059) or DMSO only (control) for 30 min, followed by incubation with a control mAb (control) or mAb against VE-PTP (α-VE-PTP) in the presence of the inhibitor or DMSO for 20 min. Cell lysates were analyzed by immunoblotting with anti–phospho-Erk1/2–specific antibodies (pT202/pY204) and antibodies against Erk1/2. (G) Mouse Flag-VE-PTP expressing HUVECs were treated with polyclonal antibodies against VE-PTP or preimmune antibodies for 20 min and subsequently cell lysates were analyzed by immunoblotting with anti–phospho-Erk1/2–specific antibodies (pT202/pY204), anti–phospho-Akt–specific antibodies (Ser473) and antibodies against Erk1/2 and Akt. (H) Allantois explants from E8.5 wild-type embryos were cultured on gelatin-coated glass slides either in the presence of Erk1/2 inhibitor PD 98059 (PD 98059), polyclonal antibodies against VE-PTP (α-VE-PTP), or the combination of both (α-VE-PTP + PD 98059) or left untreated for 22 h. Endothelial structures were stained with a mAb against VE-cadherin by indirect immunofluorescence Bar, 100 µm. (I) Quantification of the experiment illustrated in H. Average endothelial cord diameters were determined for allantois explants that were left untreated (untreated, n = 3), cultured in the presence of Erk1/2 inhibitor PD 98059 (PD 98059, n = 3), polyclonal antibodies against VE-PTP (α-VE-PTP, n = 11), or the combination of both (α-VE-PTP + PD 98059, n = 7) for 22 h; **, P < 0,01.

Figure 10.

Antibodies against VE-PTP induce blood vessel enlargement and endothelial proliferation in juvenile mice and lead to activation of Tie-2. (A) 7-d-old wild-type mice were injected daily i.p. with 100 µg antibodies against VE-PTP (α-VE-PTP) or preimmune antibodies (control) for a period of 7 d. 100-µm vibratome sections of tongues were immunostained for PECAM-1. Bar, 60 µm. (B) Average vessel diameter in the tongue of wild-type mice injected daily i.p. with 100 µg polyclonal (α-VE-PTP pAb), or monoclonal (a-VE-PTP mAb) antibodies against VE-PTP, preimmune antibodies (control pAb), or monoclonal antibodies against ESAM (control mAb), respectively, for a period of 7 d. In each case three animals were analyzed. (C) 7-d-old wild-type mice were treated as described for A. Lungs of mice were lysed and subjected to immunoprecipitation for Tie-2 and subsequent immunoblotting with antibodies either against phosphorylated tyrosine or against Tie-2 (as indicated). White line indicates that intervening lanes have been spliced out. (D) Mice were treated as in A and trachea whole mounts were immunostained for PECAM-1. Arrowheads indicate enlarged blood vessels. L, lymphatic vessels. Bar, 50 µm. (E) Mice were treated for 4 d as in A and vibratome sections of tongues were immunostained for PECAM-1 and phospho-Histone 3. Asterisk, nonendothelial nucleus; arrowheads, endothelial nuclei. Bar, 50 µm. (F) Quantification of the experiment illustrated in E with phospho-Histone 3–positive endothelial nuclei counted per volume tissue (38 × 200 × 500 µm). ***, P < 0,001.