|
|
Blood, Vol. 111, Issue 7, 3479-3488, April 1, 2008
A proangiogenic peptide derived from vascular endothelial growth factor receptor-1 acts through  5β1 integrin
Blood Soro et al.
111: 3479
Supplemental materials for: Soro et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 71.8 KB)
- Figure S1. HUVEC were pre-incubated with peptide 12 or peptide 1, as a negative control, and then loaded into Boyden chambers to analyze migration towards VEGFR-1/Fc or fibronectin (FN) (JPG, 22.2 KB)
-
Data represent the mean of twelve microscopic fields and are indicated as percentage of inhibition with respect to migration observed in the absence of the peptides ± s.e.m.
- Figure S2. HUVEC were incubated on 24-well plates pre-coated with a type I collagen gel mixture (JPG, 132 KB)
-
In the presence of VEGF-A (panels A, B, and F) and of a function blocking antibody against VEGFR-1 (AF321, panel (A), an antibody raised against the VEGFR-1 amino acid sequence including that of peptide 12 (panel B), or VEGFR-1/Fc (panel F). The two antibodies and VEGFR-1/Fc were also used in the assay in the absence of VEGF-A (panels C, D, and E, respectively). To analyze the role of growth factors in peptide 12-mediated tubule formation, the function blocking antibody against VEGFR-1 or VEGFR-1/Fc were used in the presence of peptide 12 (panels G and H, respectively). Results are representative of three independent experiments. Bars: 50 µm.
- Figure S3 (JPG, 81 KB)
-
In the left panels, representative histological pictures taken at day 17 of the neovascular growth induced by peptide 12, Y220A peptide (100 µg/pellet each), and VEGF-A (400 ng/pellet). In the right panels, the same samples were immunostained with an antibody against PECAM/CD31 to visualize vessels. EP: corneal epithelium. Arrows indicate newly formed vessels, 18× original magnification.
- Figure S4. Peptide 12 (100 µg) and VEGF-A (300 ng) were co-released from adjacent pellets in the corneal stroma (JPG, 26 KB)
-
Data are reported as angiogenic score calculated at day 17. Numbers are means ± s.e.m. of 6 implants for each experimental point. *P<0.05 peptide 12 + VEGF-A versus VEGF-A or peptide 12 alone (Student-Newman-Keuls multiple comparison test after ANOVA analysis).
- Figure S5 (JPG, 146 KB)
-
(A) Animals were implanted with 100 µg/pellet of peptide 12 in one eye and with 300 ng/pellet of VEGF-A in the other one. The angiogenic effect of peptide 12 (panel a) was blocked by Bevacizumab (panel b, 4 negative implants out of 4 performed, day 10), and significantly inhibited by Sunitinib (panel c, 1 positive implant out of 4 performed, day 10). Overlapping results were obtained with VEGF-A with 4 negative implants out of 4 performed, at day 10 in the Bevacizumab group (compare panel d and e), and 1 positive implants out of 4 performed, at day 10 in the Sunitinib group (compare panel d and f). Data are reported in (B) for peptide 12 and in (C) for VEGF-A as angiogenic score (means ± SEM) during time (days). P<0.01 peptide 12 alone versus Bevacizumab or Sunitinib treatment at all time points (B), and VEGF-A alone versus Bevacizumab treatment at day 6 and 10 (C); P<0.05 VEGF-A alone versus Sunitinib treatment at day 10 (C) (Student-Newman-Keuls multiple comparison test after ANOVA analysis).
- Figure S6 (JPG, 29.6 KB)
-
(A) K562 cells or HUVEC were cultured in the presence of fetal calf serum (lane 1 and 4, respectively). K562 were also activated with 5 mM MnCl2 and let adhere on fibronectin-coated dishes (lane 2), or incubated with 20 µg/ml fibronectin in suspension (lane 3). HUVEC were also let to adhere on fibronectin (lane 5). Cell lysates from were immunoprecipitated with an anti-VEGFR-1 antibody directed against the intracellular region of the receptor, and blots were probed with an anti- 1 integrin antibody (clone JB1A, Chemicon, upper panel10). Data show the results of a representative experiment. (B) RT-PCR analysis of human ERG (top panel) and -actin (bottom panel) transcripts in K562 cell lines and HUVEC.
|
|
