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 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.

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