Fig. 3. Ang1 rescues CLS from TGFβ1-induced apoptosis. (A) Phase-contrast micrograph of BREC after 2 days treatment with VEGF plus FGF2 and an additional 2 days either with VEGF, FGF2 plus TGFβ1 (top panel) or with VEGF plus FGF2 (bottom panel) in various combinations with Ang1 and Ang2. (B) Total length of CLS after 2 days (white bars) and 4 days (black bars). After 4 days all treatments are significantly different compared to baseline (VEGF plus FGF2 after 2 days, P<0.0001). The addition of Ang1 rescued 60% of a pre-established network from TGFβ1-induced capillary regression (^*P<0.0001), an effect that was antagonized by Ang2. In the absence of TGFβ1, Ang1 or Ang2 had no effect on the length of CLS after 4 days. Proliferation was measured on the third day by incorporation of BrdU over 12 hours. The number of BrdU-positive cells after addition of TGFβ1 plus Ang1 was not significantly different from TGFβ1 alone. TGFβ1 plus Ang2 induced significantly less proliferation than TGFβ1 plus Ang1 (^#P<0.02). In the absence of TGFβ1, addition of Ang1 or Ang2 did not cause significantly different proliferation than with VEGF plus FGF2. Concentrations of factors used: 25 ng/ml VEGF, 2.5 ng/ml FGF2, 1 ng/ml TGFβ1, 500 ng/ml Ang1 and 500 ng/ml Ang2. Bar, 100 µm.