TY - JOUR
T1 - Biological action of angiopoietin-2 in a fibrin matrix model of angiogenesis is associated with activation of Tie2
AU - Teichert-Kuliszewska, Krystyna
AU - Maisonpierre, Peter C.
AU - Jones, Nina
AU - Campbell, Andrew I.M.
AU - Master, Zubin
AU - Bendeck, Michelle P.
AU - Alitalo, Kari
AU - Dumont, Daniel J.
AU - Yancopoulos, George D.
AU - Stewart, Duncan J.
N1 - Funding Information:
This study was supported by a grant from the Medical Research Council of Canada ([ 10960). Dr Stewart is the Dexter Hung-Cho Man Chair of Cardiology, University of Toronto. The authors thank Lee Roth for his valuable help in performing morphometric analysis.
PY - 2001/2/16
Y1 - 2001/2/16
N2 - The endothelial cell (EC) specific tyrosine kinase receptor, Tie2, interacts with at least two ligands, angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang1 stimulates Tie2 receptor autophosphorylation, while Ang2 has been reported to inhibit Ang1-induced Tie2 receptor autophosphorylation. We studied the effects of Ang1 and Ang2 in an in vitro model of angiogenesis. Human ECs (HUVEC), cultured on 3-D fibrin matrices, were treated with conditioned media (CM) from stably transfected cells expressing human Ang1 or Ang2, or with purified recombinant proteins. EC tube formation was measured as a differentiation index (DI), calculated as the ratio of total tube length over residual of EC monolayer. CM from Ang1 overexpressing A10 SMC or HEK293T cells induced profound HUVEC differentiation, resulting in the formation of extensive capillary-like tubes within 48 h (DI: 24.58±5.91 and 19.13±7.86, respectively) vs. control (DI: 2.73±1.68 and 2.15±1.45, respectively, both P<0.001). Interestingly, CM from two independent cell lines overexpressing Ang2 also produced a significant increase in EC differentiation (DI: 9.22±3.00 and 9.72±4.84, both P<0.005 vs. control) although the degree of angiogenesis was significantly less then that seen with Ang1. Addition of Ang1* (a genetically engineered variant of naturally occurring Ang1) or Ang2 also resulted in dose dependent increases in DI, which were blocked by an excess of soluble Tie2 receptor (20 μg/ml). Both Ang1* and Ang2 induced modest increases in [3H]thymidine incorporation into HUVECs (20 and 26%, respectively), which were inhibited by excess soluble Tie2. Although Ang2 was unable to induce significant Tie2 receptor phosphorylation during a 5-min exposure, a 24-h pretreatment with Ang2, followed by brief re-exposure, produced Tie2 phosphorylation in HUVEC comparable to that produced by Ang1*. These results demonstrate for the first time that Ang2 may have a direct role in stimulating Tie2 receptor signaling and inducing in vitro angiogenesis. Our findings suggest that the physiological role of Ang2 is more complex than previously recognized: acting alternately to promote or blunt Tie2 receptor signaling in endothelial cells, depending on local conditions.
AB - The endothelial cell (EC) specific tyrosine kinase receptor, Tie2, interacts with at least two ligands, angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2). Ang1 stimulates Tie2 receptor autophosphorylation, while Ang2 has been reported to inhibit Ang1-induced Tie2 receptor autophosphorylation. We studied the effects of Ang1 and Ang2 in an in vitro model of angiogenesis. Human ECs (HUVEC), cultured on 3-D fibrin matrices, were treated with conditioned media (CM) from stably transfected cells expressing human Ang1 or Ang2, or with purified recombinant proteins. EC tube formation was measured as a differentiation index (DI), calculated as the ratio of total tube length over residual of EC monolayer. CM from Ang1 overexpressing A10 SMC or HEK293T cells induced profound HUVEC differentiation, resulting in the formation of extensive capillary-like tubes within 48 h (DI: 24.58±5.91 and 19.13±7.86, respectively) vs. control (DI: 2.73±1.68 and 2.15±1.45, respectively, both P<0.001). Interestingly, CM from two independent cell lines overexpressing Ang2 also produced a significant increase in EC differentiation (DI: 9.22±3.00 and 9.72±4.84, both P<0.005 vs. control) although the degree of angiogenesis was significantly less then that seen with Ang1. Addition of Ang1* (a genetically engineered variant of naturally occurring Ang1) or Ang2 also resulted in dose dependent increases in DI, which were blocked by an excess of soluble Tie2 receptor (20 μg/ml). Both Ang1* and Ang2 induced modest increases in [3H]thymidine incorporation into HUVECs (20 and 26%, respectively), which were inhibited by excess soluble Tie2. Although Ang2 was unable to induce significant Tie2 receptor phosphorylation during a 5-min exposure, a 24-h pretreatment with Ang2, followed by brief re-exposure, produced Tie2 phosphorylation in HUVEC comparable to that produced by Ang1*. These results demonstrate for the first time that Ang2 may have a direct role in stimulating Tie2 receptor signaling and inducing in vitro angiogenesis. Our findings suggest that the physiological role of Ang2 is more complex than previously recognized: acting alternately to promote or blunt Tie2 receptor signaling in endothelial cells, depending on local conditions.
KW - Angiogenesis
KW - Cell culture/isolation
KW - Endothelial receptors
KW - Extracellular matrix
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U2 - 10.1016/S0008-6363(00)00231-5
DO - 10.1016/S0008-6363(00)00231-5
M3 - Article
C2 - 11166279
AN - SCOPUS:0035895712
SN - 0008-6363
VL - 49
SP - 659
EP - 670
JO - Cardiovascular Research
JF - Cardiovascular Research
IS - 3
ER -