Platelet-derived growth factor inhibits basic fibroblast growth factor angiogenic properties in vitro and in vivo through its α receptor by Francesco De Marchis, Domenico Ribatti, Claudia Giampietri, Alessandro Lentini, Debora Faraone, Marco Scoccianti, Maurizio C. Capogrossi, and Antonio Facchiano Blood Volume 99(6):2045-2053 March 15, 2002 ©2002 by American Society of Hematology

BAEC migration in the presence of bFGF and PDGF-BB BAEC migration in the presence of bFGF and PDGF-BB.(A) BAEC migration was examined in response to increasing concentrations of PDGF-BB alone (dashed line) or increasing concentrations of PDGF-BB in the presence of 10 ng/mL bFGF (continuous line). BAEC migration in the presence of bFGF and PDGF-BB.(A) BAEC migration was examined in response to increasing concentrations of PDGF-BB alone (dashed line) or increasing concentrations of PDGF-BB in the presence of 10 ng/mL bFGF (continuous line). The bFGF effect was strongly inhibited by PDGF-BB in a dose-dependent manner. (inset) Denatured PDGF-BB did not show any inhibitory effect. (B) BAEC migration was examined in response to increasing concentrations of bFGF alone (dashed line) or increasing concentrations of bFGF in the presence of 10 ng/mL PDGF-BB (continuous line). The bFGF effect was strongly inhibited by PDGF-BB at all concentrations. Data are expressed as average ± SD of 4 experiments carried out in duplicate. Francesco De Marchis et al. Blood 2002;99:2045-2053 ©2002 by American Society of Hematology

BAEC proliferation in the presence of bFGF and PDGF-BB BAEC proliferation in the presence of bFGF and PDGF-BB.BAEC proliferation was evaluated in response to 0.1% BSA, bFGF alone (10 ng/mL), PDGF-BB alone (10 ng/mL), or bFGF/PDGF-BB (10 ng/mL each). BAEC proliferation in the presence of bFGF and PDGF-BB.BAEC proliferation was evaluated in response to 0.1% BSA, bFGF alone (10 ng/mL), PDGF-BB alone (10 ng/mL), or bFGF/PDGF-BB (10 ng/mL each). The bFGF mitogenic effect was lowered to control levels in the presence of PDGF-BB. Data are expressed as average ± SD of 4 experiments carried out in duplicate. Francesco De Marchis et al. Blood 2002;99:2045-2053 ©2002 by American Society of Hematology

BAEC differentiation into tubular structures on Matrigel BAEC differentiation into tubular structures on Matrigel.BAEC differentiation on Matrigel-coated wells in the presence of (A) 0.1% BSA, (B) 1% FCS, (C) 10 ng/mL bFGF, (D) 10 ng/mL PDGF-BB, or (E) 10 ng/mL bFGF/PDGF-BB. BAEC differentiation into tubular structures on Matrigel.BAEC differentiation on Matrigel-coated wells in the presence of (A) 0.1% BSA, (B) 1% FCS, (C) 10 ng/mL bFGF, (D) 10 ng/mL PDGF-BB, or (E) 10 ng/mL bFGF/PDGF-BB. (F) Quantification of branching points was carried out on 10 fields. The differentiation effect of bFGF was lowered to control levels in the presence of PDGF-BB. This effect was evident at 3, 6, 8, and 24 hours. Figures and the bar graph refer to the effect at 3 hours. Experiments were carried out 3 times in duplicate. Panels A to E refer to a representative experiment. Francesco De Marchis et al. Blood 2002;99:2045-2053 ©2002 by American Society of Hematology

BAEC migration in the presence of PDGF-AA BAEC migration in the presence of PDGF-AA.BAEC migration in response to PDGF-BB or bFGF, alone or in combination (10 ng/mL), was examined in the absence and in the presence of PDGF-AA (10 ng/mL). BAEC migration in the presence of PDGF-AA.BAEC migration in response to PDGF-BB or bFGF, alone or in combination (10 ng/mL), was examined in the absence and in the presence of PDGF-AA (10 ng/mL). PDGF-AA showed no chemotactic effect on BAEC, but it inhibited bFGF-induced migration, and the magnitude of this effect was comparable to that of an equal concentration of PDGF-BB. Data are expressed as average ± SD of 3 experiments carried out in duplicate. Francesco De Marchis et al. Blood 2002;99:2045-2053 ©2002 by American Society of Hematology

Migration of BAECs transfected with dominant-negative receptors Migration of BAECs transfected with dominant-negative receptors.BAECs were transfected either with a dominant-negative PDGF-Rα vector (DN-PDGF-Rα), with a dominant-negative PDGF-Rβ vector (DN-PDGF-Rβ, or with a PcDNA3 empty vector and were cotransfected wit... Migration of BAECs transfected with dominant-negative receptors.BAECs were transfected either with a dominant-negative PDGF-Rα vector (DN-PDGF-Rα), with a dominant-negative PDGF-Rβ vector (DN-PDGF-Rβ, or with a PcDNA3 empty vector and were cotransfected with pEGFP-N1 reporter vector. A dominant-negative versus reporter-vector molar ratio of 4:1 was used. Under these conditions both plasmids were internalized in the same cell; when migrated cells were examined, only GFP-positive cells were counted. Neither the empty vector nor the dominant-negative vectors affected bFGF-induced migration. PDGF-BB inhibitory effect on bFGF-induced migration was present in the empty-vector–transfected cells and in the DN-PDGF-Rβ–transfected cells, whereas it was absent in DN-PDGF-Rα–transfected cells. In the latter, BAEC migration was increased in the presence of PDGF-BB and bFGF compared with bFGF alone. *Statistically significant difference (P < .01). Data are expressed as average ± SD of 4 experiments carried out in duplicate. Francesco De Marchis et al. Blood 2002;99:2045-2053 ©2002 by American Society of Hematology

PDGF receptor phosphorylation and MAP kinase activation PDGF receptor phosphorylation and MAP kinase activation.(A) PDGF receptor phosphorylation was investigated at 5-minute PDGF-BB exposure (10 ng/mL), with or without bFGF. PDGF receptor phosphorylation and MAP kinase activation.(A) PDGF receptor phosphorylation was investigated at 5-minute PDGF-BB exposure (10 ng/mL), with or without bFGF. Increased phosphorylation of PDGF-Rα and PDGF-Rβ was found in the presence of bFGF/PDGF-BB compared with PDGF-BB alone (average increase, 52% ± 18%). Anti–PDGF-BB neutralizing antibody (Ab-PDGF-BB) markedly inhibited PDGF receptor phosphorylation. This experiment was carried out 3 times with similar results. One representative experiment is shown. (B) Phosphorylation of PDGF-Rα was investigated in BAECs exposed to PDGF-AA, which is a PDGF-Rα–specific agonist, in the absence and in the presence of bFGF. Under these conditions, bFGF/PDGF-AA–treated cells showed increased phosphorylation compared with PDGF-AA alone. Receptor phosphorylation was markedly reduced in DN-PDGF-Rα–transfected cells. This experiment was carried out 3 times, with similar results. One representative experiment is shown. (C) ERK1/2 phosphorylation induced by bFGF alone, PDGF-BB alone, and bFGF/PDGF-BB was detected with an antibody specifically recognizing the activated forms at 44 kd and 42 kd, respectively. A marked phosphorylation decrease was found in the presence of bFGF/ PDGF-BB compared with bFGF alone. This experiment was carried out 5 times with similar results. One representative experiment is shown. Francesco De Marchis et al. Blood 2002;99:2045-2053 ©2002 by American Society of Hematology

Macroscopic observations of the PDGF-AA and PGDF-BB effect on bFGF-induced neovascularization in the CAM assay.Gelatin sponge adsorbed with bFGF (500 μg) is surrounded by allantoic vessels that develop radially toward the implant in a spoked-wheel pattern (... Macroscopic observations of the PDGF-AA and PGDF-BB effect on bFGF-induced neovascularization in the CAM assay.Gelatin sponge adsorbed with bFGF (500 μg) is surrounded by allantoic vessels that develop radially toward the implant in a spoked-wheel pattern (A), whereas no vascular reaction is detectable around PBS-treated (B) and PDGF-AA–treated (C) sponges. Allantoic vessels were less numerous in the specimens treated with bFGF added to PDGF-AA and PDGF-BB (500 μg each) (D and F, respectively), whereas PDGF-BB alone induced a moderate angiogenic response (E). Asterisks indicate sponges. Panels refer to a representative experiment. Original magnification, × 50. Francesco De Marchis et al. Blood 2002;99:2045-2053 ©2002 by American Society of Hematology

Angiogenesis in Matrigel assay Angiogenesis in Matrigel assay.Angiogenesis was measured in Matrigel plugs injected subcutaneously in CD1 mice. Angiogenesis in Matrigel assay.Angiogenesis was measured in Matrigel plugs injected subcutaneously in CD1 mice. Matrigel was added with bFGF (150 ng/mL), PDGF-BB (150 ng/mL), or both. The bFGF-induced vessel formation was significantly reduced in the presence of PDGF-BB. Data were collected on 8 animals for bFGF alone, 6 animals for PDGF-BB alone, 11 animals for bFGF/PDGF-BB treatment, and 8 animals for negative controls. Data are expressed as average ± SD. Francesco De Marchis et al. Blood 2002;99:2045-2053 ©2002 by American Society of Hematology