Figure 7
Figure 7. Blockade of GM-CSF inhibits MALP-2-induced angiogenesis. (A) GM-CSF levels in supernatants of HUVECs after siRNA application (10 nM) and MALP-2 (1 μg/mL) stimulation for 24 hours determined by ELISA (n = 3). *P < .05 vs control. #P < .05 vs MALP-2. (B) Quantification of the numbers (#) of tube-like structures of HUVEC after application of siRNA (10 nM) and MALP-2 (1 μg/mL) for 24 hours. bFGF (100 ng/mL) was used as positive control (n = 4). *P < .05 vs control. #P < .05 vs MALP-2 plus control siRNA. (C) Quantification of the number (#) of tube-like structures of HUVEC after application of GM-CSF antibody and MALP-2 (1 μg/mL) for 24 hours. AB indicates antibody (10 μg/mL). Unspecific IgG (10 μg/mL) was used as antibody control (n = 4). **P < .01 vs control. ##P < .05 vs MALP-2. (D) Proliferation of HUVECs was determined by BrdU incorporation after application of GM-CSF antibody and MALP-2 (1 μg/mL) for 16 hours. AB indicates antibody (10 μg/mL). Unspecific IgG (10 μg/mL) was used as antibody control (n = 4). **P < .01 vs control. ##P < .01 vs MALP-2. (E) Migration of HUVEC (1 × 105) was carried out in transwell cell-culture inserts for 24 hours. As chemoattractant, supernatants from unstimulated HUVEC or after 24-hour stimulation with MALP-2 (1 μg/mL) alone or pretreated with GM-CSF antibody were used (n = 4). AB indicates antibody (10 μg/mL). Unspecific IgG (10 μg/mL) was used as antibody control (n = 4). *P < .05 vs control. ##P < .01 vs MALP-2. (F) Representative pictures of hematoxylin and eosin–stained sections of Matrigel plugs 6 days after the administration of GM-CSF (100 ng/mL) and MALP-2 (1 μg/mL). AB indicates antibody (10 μg/mL each). Insets represent the total Matrigel plug. Vascular structures are filled with erythrocytes. Image acquisition: Leica DM 4000B microscope, 20×/0.50 NA dry objective, Leica DFC 320 camera, Leica QWin Version 3 software. (G) Hemoglobin content is given as micrograms per milligram of Matrigel. (H) Vascular structures are expressed as percentage vascular area per field. Unspecific IgG (10 μg/mL) was used as antibody control (n = 4-8). **P < .01 vs control. ##P < .01 vs MALP-2. (A-E,G,H) Error bars represent mean ± SEM.

Blockade of GM-CSF inhibits MALP-2-induced angiogenesis. (A) GM-CSF levels in supernatants of HUVECs after siRNA application (10 nM) and MALP-2 (1 μg/mL) stimulation for 24 hours determined by ELISA (n = 3). *P < .05 vs control. #P < .05 vs MALP-2. (B) Quantification of the numbers (#) of tube-like structures of HUVEC after application of siRNA (10 nM) and MALP-2 (1 μg/mL) for 24 hours. bFGF (100 ng/mL) was used as positive control (n = 4). *P < .05 vs control. #P < .05 vs MALP-2 plus control siRNA. (C) Quantification of the number (#) of tube-like structures of HUVEC after application of GM-CSF antibody and MALP-2 (1 μg/mL) for 24 hours. AB indicates antibody (10 μg/mL). Unspecific IgG (10 μg/mL) was used as antibody control (n = 4). **P < .01 vs control. ##P < .05 vs MALP-2. (D) Proliferation of HUVECs was determined by BrdU incorporation after application of GM-CSF antibody and MALP-2 (1 μg/mL) for 16 hours. AB indicates antibody (10 μg/mL). Unspecific IgG (10 μg/mL) was used as antibody control (n = 4). **P < .01 vs control. ##P < .01 vs MALP-2. (E) Migration of HUVEC (1 × 105) was carried out in transwell cell-culture inserts for 24 hours. As chemoattractant, supernatants from unstimulated HUVEC or after 24-hour stimulation with MALP-2 (1 μg/mL) alone or pretreated with GM-CSF antibody were used (n = 4). AB indicates antibody (10 μg/mL). Unspecific IgG (10 μg/mL) was used as antibody control (n = 4). *P < .05 vs control. ##P < .01 vs MALP-2. (F) Representative pictures of hematoxylin and eosin–stained sections of Matrigel plugs 6 days after the administration of GM-CSF (100 ng/mL) and MALP-2 (1 μg/mL). AB indicates antibody (10 μg/mL each). Insets represent the total Matrigel plug. Vascular structures are filled with erythrocytes. Image acquisition: Leica DM 4000B microscope, 20×/0.50 NA dry objective, Leica DFC 320 camera, Leica QWin Version 3 software. (G) Hemoglobin content is given as micrograms per milligram of Matrigel. (H) Vascular structures are expressed as percentage vascular area per field. Unspecific IgG (10 μg/mL) was used as antibody control (n = 4-8). **P < .01 vs control. ##P < .01 vs MALP-2. (A-E,G,H) Error bars represent mean ± SEM.

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