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First published online May 14, 2007
doi: 10.1242/10.1242/jcs.003533

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Contextual role for angiopoietins and TGF1 in blood vessel stabilization

Schepens Eye Research Institute, Departments of Pathology and Ophthalmology, Harvard Medical School, Boston, MA 02114, USA

View larger version (59K): [in this window] [in a new window]   Fig. 1. VEGF and FGF2 have additive effects on the induction of capillary formation by BREC in a 3D collagen assay. (A) Phase-contrast images of BREC after 4 days in collagen, untreated or treated with 25 ng/ml VEGF, or with 25 ng/ml VEGF plus 2.5 ng/ml FGF2. (B) Total length of CLS after 2 (white bars) and 4 (black bars) days. Treatment with VEGF or VEGF plus FGF2 led to a significant increase in the total length of CLS at both 2 and 4 days (P<0.0006 in all comparisons). Administration of VEGF plus FGF2 for 4 days induced the longest CLS and was significantly different from VEGF (4 days, *P<0.03) and VEGF plus FGF2 (2 days, **P<0.007). Proliferation was assessed on the third day, after exposure to the growth factors for 13 hours (incorporation of BrdU over 12 hours). VEGF plus FGF2 caused a significant increase in the number of BrdU-positive cells vs no treatment or VEGF only (#P<0.0009). (C) Visualization of endothelial nuclei, apoptotic ECs and proliferating ECs on the third day, after treatment with VEGF plus FGF2 and application of BrdU for 12 hours. Bar, 100 µm.

View larger version (67K): [in this window] [in a new window]   Fig. 2. TGF1 induces regression of CLS and apoptosis of BREC. (A) Phase-contrast image of BREC after treatment with VEGF plus FGF2 for 2 days, followed by VEGF, FGF2 plus TGF1 for an additional 2 days. (B) Quantification of the effects of TGF1 on the length of CLS and number of BrdU-positive cells. A pre-established capillary network completely regressed after addition of TGF1 for 2 days (*P<0.0001 vs VEGF plus FGF2) and proliferation was significantly decreased within the 13 hours of the assay (#P<0.002 vs VEGF plus FGF2). (C) Images of the same field of CLS assayed for apoptosis and proliferation after treatment with VEGF (25 ng/ml), FGF2 (2.5 ng/ml) plus TGF1 (1 ng/ml). Bar, 100 µm.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Ang1 rescues CLS from TGF1-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 TGF1 (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 TGF1-induced capillary regression (*P<0.0001), an effect that was antagonized by Ang2. In the absence of TGF1, 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 TGF1 plus Ang1 was not significantly different from TGF1 alone. TGF1 plus Ang2 induced significantly less proliferation than TGF1 plus Ang1 (#P<0.02). In the absence of TGF1, 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 TGF1, 500 ng/ml Ang1 and 500 ng/ml Ang2. Bar, 100 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 4. VEGF is necessary for the Ang1-mediated rescue from TGF1-induced apoptosis. (A) Phase-contrast micrograph of BREC after treatment for 2 days with VEGF plus FGF2 and an additional 2 days with VEGF-Trap, FGF2, TGF1 plus Ang1. (B) Quantification of the effects of VEGF on the length of CLS and number of BrdU-positive cells. Inhibition of VEGF with VEGF-Trap produced a significant decrease in the length of CLS (*P<0.0001 vs VEGF after 4 days). Proliferation within a pre-established network was not significantly influenced by the presence or absence of VEGF. Concentrations of factors used: 25 ng/ml VEGF, 2.5 ng/ml FGF2, 1 ng/ml TGF1, 500 ng/ml Ang1, and 1 µg/ml VEGF-Trap. Bar, 100 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 5. FGF2 and Ang-1 have additive effects on the maintenance of CLS in the absence of VEGF. (A) Phase-contrast micrographs of BREC after treatment for 2 days with VEGF plus FGF2 and an additional 2 days of VEGF neutralization, in the absence or presence of Ang1. (B) Neutralization of VEGF significantly decreased the length of CLS (*P<0.0001 compared to 2 days treatment with VEGF plus FGF2), whereas Ang1 provided partial protection from the regression, an effect not antagonized by Ang2 (**P<0.04). The angiopoietins did not significantly alter proliferation from that with VEGF-Trap plus FGF2 alone. (C) Phase-contrast micrographs of BREC after treatment for 2 days in the presence of VEGF followed by VEGF neutralization for an additional 2 days, in the absence or presence of Ang1. (D) All treatments with VEGF-Trap led to a significant reduction of CLS compared with baseline (VEGF after 2 days, P<0.0001). In combination with VEGF neutralization, Ang1 treatment led to significantly more CLS (*P<0.03 vs no angiopoietins), an effect that was not influenced by Ang2. Addition of FGF2 or angiopoietins did not significantly alter proliferation from that with VEGF-Trap alone. Concentrations of factors used: 25 ng/ml VEGF, 2.5 ng/ml FGF2, 1 ng/ml TGF1, 500 ng/ml Ang1, 500 ng/ml Ang2, and 1 µg/ml VEGF-Trap. Bar, 100 µm.