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First published online 1 February 2005
doi: 10.1242/jcs.01653

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The Tie-2 ligand Angiopoietin-2 destabilizes quiescent endothelium through an internal autocrine loop mechanism

Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, 79106 Freiburg, Germany

View larger version (95K): [in a new window]   Fig. 1. Induction of endothelial-cell detachment from endothelial-cell/smooth-muscle-cell co-culture spheroids by pharmacological Tie-2 inhibition. Co-culture spheroids of HUVECs and HUASMCs were treated for 4 hours with 10 nM A-422885.66 and, after fixation, whole-mount stained for CD31. Control co-culture spheroids form an intact monolayer of CD31-positive endothelial cells (A). Inhibition of Tie-2 phosphorylation by A-422885.66 leads to detachment of endothelial cells from the endothelial-cell monolayer, as indicated by holes in the monolayer (B, arrowheads). This process cannot be antagonized by exogenous recombinant Ang-1 (C, arrowheads).

View larger version (74K): [in a new window]   Fig. 2. Destabilization of endothelial-cell-monolayer integrity in co-culture spheroids of HUVECs and HUASMCs by exogenous Ang-2. Control co-culture spheroids have an intact CD31-positive endothelial-cell monolayer (A). Treatment of co-culture spheroids with Ang-1 (B) or VEGF (E) has no effect on the integrity of the surface endothelial-cell monolayer. Treatment of co-culture spheroids with Ang-2 leads to destabilization of the endothelial-cell monolayer within 4 hours, as evidenced by intense detachment of individual and groups of endothelial cells (C; high magnification in D, arrowheads). Monolayer destabilization elicited by exogenous Ang-2 is rescued by Ang-1 (F), sTie-2 (G) and VEGF (H). (I) Quantitative assessment of the Ang-2-mediated denudation by image analysis of the denuded spheroid surface area. Results are expressed as means ± s.d. of three independent experiments quantifying ten spheroids per experiment. **, P<0.001 compared with all other treatment groups.

View larger version (136K): [in a new window]   Fig. 3. Time course of Ang-2-mediated endothelial-cell-monolayer destabilization in HUVEC/HUASMC co-culture spheroids. Gaps in the endothelial-cell monolayer are first observed after 30 minutes. Individual endothelial cells have detached from the monolayer within 60 minutes of Ang-2 exposure (arrowheads).

View larger version (112K): [in a new window]   Fig. 4. Ang-2-mediated endothelial-cell-monolayer destabilization in explanted fragments of human umbilical vein. Fragments of umbilical vein were cultured overnight and exposed to different combinations of cytokines for 4 hours. Samples were then fixed, embedded and processed for CD34 immunohistochemistry. Control umbilical-cord fragments have an intact CD34-positive surface monolayer (A). Treatment with exogenous Ang-2 leads to vascular destabilization, as evidenced by detachment of the endothelial cells (B, higher magnification in D). Ang-1 (C), sTie-2 (E) and VEGF (F) are all able to rescue vascular destabilization elicited by exogenous Ang-2.

View larger version (50K): [in a new window]   Fig. 5. Rescue of PMA-mediated disintegration of surface endothelial cells co-cultured with smooth-muscle cells in three-dimensional spheroids. Co-culture spheroids of HUVECs and HUASMCs were incubated in spinner culture with the indicated combinations of reagents for 4 hours and endothelial-cell detachment was quantified by automated image analysis of CD31 whole-mount-stained spheroids. PMA treatment leads to massive denudation of the surface endothelial-cell monolayer within 4 hours (A,C). Both Ang-1 (A,E) and VEGF (A,F) inhibit the PMA-mediated destabilization of the endothelial-cell monolayer. In turn, sTie-2 cannot block PMA-mediated perturbation of endothelial-cell-monolayer integrity (A,D). **, P<0.001 compared with control; #, P<0.001 compared with PMA treatment.

View larger version (45K): [in a new window]   Fig. 6. Ang-2-dependent PMA-mediated destabilization of co-culture spheroids. Co-culture spheroids of siRNA-treated or untreated HUVECs with HUASMCs were incubated in spinner culture with the indicated combination of reagents for 4 hours and endothelial-cell detachment was quantified by automated image analysis of CD31 whole-mount-stained spheroids. PMA treatment leads to massive denudation of the surface monolayer of the untreated endothelial cells within 4 hours (A,C) but not of the monolayer of Ang-2-siRNA-silenced endothelial cells (A,E). **, P<0.001 compared with control.

View larger version (42K): [in a new window]   Fig. 7. Perturbation of endothelial-cell-monolayer integrity in co-culture spheroids of Ang-1 or Ang-2 overexpressing HUVECs and HUASMCs. Control HUVECs (B) or HUVECs retrovirally transduced with Ang-1 (C, endo. Ang-1) or Ang-2 (D, endo. Ang-2) were mixed with HUASMCs and allowed to form co-culture spheroids for 48 hours. Established co-culture spheroids were then kept in spinner culture for 4 hours and endothelial-cell surface coverage was quantified by automated image analysis following whole-mount staining for CD31 using Alexa Fluor 546 as fluorescent dye. Control HUVECs (B) and Ang-1 expressing HUVECs (C) form stable, differentiated co-culture spheroids with HUASMCs. By contrast, Ang-2 overexpressing HUVECs form co-culture spheroids with HUASMCs, with endothelial cells detaching upon mechanical challenge in spinner culture (A,D). Co-culture spheroids of Ang-2-overexpressing HUVECs and HUASMCs can be stabilized by exogenous Ang-1 (F) or VEGF (G) but not by sTie-2 (A,E). **, P<0.01 compared with control.