1. In vivo administration of vascular endothelial growth factor (VEGF) and its antagonist, soluble neuropilin-1, predicts a role of VEGF in the progression of acute myeloid leukemia in vivo
by Gunter Schuch, Marcelle Machluf, Georg Bartsch, Masashi Nomi, Henri Richard, Anthony Atala, and Shay Soker
Blood
Volume 100(13):
December 15, 2002
©2002 by American Society of Hematology

2. Expression of VEGF and VEGF receptors in leukemic cells
Expression of VEGF and VEGF receptors in leukemic cells.RNA was isolated from leukemic cell lines grown in culture (A) and freshly isolated human leukemia cells (B) as described in “Materials and methods.” RT-PCR was performed on the RNA, using primers corr...
Expression of VEGF and VEGF receptors in leukemic cells.RNA was isolated from leukemic cell lines grown in culture (A) and freshly isolated human leukemia cells (B) as described in “Materials and methods.” RT-PCR was performed on the RNA, using primers corresponding for human VEGF, KDR, NRP-1, and GAPDH cDNA sequences and murine Flk-1 (KDR) and NRP-1 cDNA sequences, as indicated. PCR products were resolved on a 2% agarose gel, stained with ethidium bromide, and visualized under UV light.
Gunter Schuch et al. Blood 2002;100:
©2002 by American Society of Hematology

3. The leukemic cell lines M1 and HEL do not respond to exogenous VEGF
The leukemic cell lines M1 and HEL do not respond to exogenous VEGF.(A) KDR phosphorylation.
The leukemic cell lines M1 and HEL do not respond to exogenous VEGF.(A) KDR phosphorylation. PAE, PAE-KDR, M1, HEL, and U937 were incubated in the presence (+) or absence (−) of VEGF165 (20 ng/mL) for 30 minutes on ice and then shifted to 37°C for 7 minutes, as described in “Materials and methods.” Cells were lysed and proteins were absorbed on ConA Sepharose and resolved by a 6% SDS-PAGE. Proteins were blotted onto polyvinylidenefluoride (PVDF) membrane, which was subsequently probed with antiphosphotyrosine antibodies. Phosphorylated KDR (Phos KDR) was detected as a band with a molecular weight of approximately 220 kd. (B) Cell proliferation. M1 and HEL cells were incubated in the presence (VEGF) or the absence (control) of VEGF (10 ng/mL) in serum-free medium, as described in “Materials and methods.” Cell numbers were obtained after 24 and 48 hours. Cell numbers represent the average of 3 individual wells.
Gunter Schuch et al. Blood 2002;100:
©2002 by American Society of Hematology

4. The effects of VEGF and sNRP-1 supplementation on chloroma growth in vivo.(A) Tumor volume curves.
The effects of VEGF and sNRP-1 supplementation on chloroma growth in vivo.(A) Tumor volume curves. M1 cells (2 × 106 per mouse) were injected subcutaneously into SCID mice. NMuMG-encapsulated (○), NMuMG/VEGF-encapsulated (●), and NMuMG/sNRP-1–encapsulated (▴) cells (5 × 105 cells per mouse) were injected at the site of M1 cell injection, 72 hours later. The mice (10 per group) were followed for tumor volumes at the indicated times. (B) Final tumor weights. Tumors were harvested 21 days after M1 cell injection and weighed. The average weight and standard deviations were calculated for NMuMG (control), NMuMG/VEGF (VEGF), and NMuMG/sNRP-1 (sNRP-1) groups.
Gunter Schuch et al. Blood 2002;100:
©2002 by American Society of Hematology

5. Opposite effects of VEGF and sNRP-1 on chloroma neovascularization
Opposite effects of VEGF and sNRP-1 on chloroma neovascularization.(A) In vivo secretion of VEGF. Tumors from mice injected with encapsulated NMuMG/VEGF cells were harvested after 21 days and processed for histologic examination.
Opposite effects of VEGF and sNRP-1 on chloroma neovascularization.(A) In vivo secretion of VEGF. Tumors from mice injected with encapsulated NMuMG/VEGF cells were harvested after 21 days and processed for histologic examination. Tissue sections were stained with anti–human VEGF antibodies and photographed under × 100 (left) and × 400 (right) original magnification. A cross-sectioned capsule and surrounding tumor tissue is shown. Some VEGF-positive stained cells can be seen within the capsule. (B) Tumor neovascularization. Tumors from NMuMG (control), NMuMG/VEGF (VEGF), and NMuMG/sNRP-1 (sNRP-1) groups, as indicated, were harvested and processed for histologic examination. Tumor sections were stained with H&E (top panels) and with anti–mouse CD31 antibodies (bottom panels) and photographed under × 400. (C) Microvessel density (MVD) was determined by counting 10 high-power fields (HPFs) from the CD31 stained sections in panel B. The numbers represent the MVD average, and standard deviations were calculated.
Gunter Schuch et al. Blood 2002;100:
©2002 by American Society of Hematology

6. Soluble NRP-1 inhibits liver, spleen, and bone infiltration of leukemic cells.M1 cells (2 × 106 cells per mouse) were injected intravenously into SCID mice.
Soluble NRP-1 inhibits liver, spleen, and bone infiltration of leukemic cells.M1 cells (2 × 106 cells per mouse) were injected intravenously into SCID mice. After 7 days, adenoviral vectors encoding for Fc-sNRP-1 (Avii-ix and Biii-iv), VEGF (Aiv,vi and Bii,v), and lacZ (Ai-iii and Bi,iv) were injected intravenously, as described in “Materials and methods.” On day 28, 2 mice from each group were killed and their bones, livers, and spleens were processed for histologic examination. (A) Liver tissues were processed for LacZ staining (Ai,iv,vii), as described in “Materials and methods.” Liver and spleen tissues were also stained with H&E (ii,v,viii and iii,vi,ix, respectively). Stained sections were examined microscopically and representative images were recorded (original magnification × 200). (B) Bone sections were stained with H&E (i-iii) and with anti–mouse CD31 antibodies (iv-vi). Stained sections were examined microscopically and representative images were recorded (original magnification × 200).
Gunter Schuch et al. Blood 2002;100:
©2002 by American Society of Hematology

7. Soluble NRP-1 prolonged the survival of leukemic mice
Soluble NRP-1 prolonged the survival of leukemic mice.SCID mice were injected with M1 cells followed by intravenous injection of adenoviral vectors encoding for Fc-sNRP-1 (Ad.Fc-sNRP-1; ■), VEGF (Ad.VEGF; ●), and lacZ (Ad.LacZ; ▵), as described in the Figur...
Soluble NRP-1 prolonged the survival of leukemic mice.SCID mice were injected with M1 cells followed by intravenous injection of adenoviral vectors encoding for Fc-sNRP-1 (Ad.Fc-sNRP-1; ■), VEGF (Ad.VEGF; ●), and lacZ (Ad.LacZ; ▵), as described in the Figure 5legend. Mice (10 per group) were followed for 45 days after viral injection and a survival curve was generated. Mice injected with Ad.LacZ or Ad.VEGF had a mean survival of 28 days and mice injected with Ad.Fc-sNRP-1 had a mean survival of 35 days.
Gunter Schuch et al. Blood 2002;100:
©2002 by American Society of Hematology