Fig 1: Angiogenic protein profiles of HMEC-1 cells treated with IGF-2 or Des(1-6)IGF-2 alone or in combination with IGFBP-6. A screening for 43 human angiogenic factors was performed on conditioned medium collected from HMEC-1 cells treated with medium, 100 ng/mL IGF-2, 100 ng/mL Des(1-6)IGF-2, 100 ng/mL IGF-2 + 1,000 ng/mL IGFBP-6, or 100 ng/mL Des(1-6)IGF-2 + 1,000 ng/mL IGFBP-6. (a) Antibody blots for each condition is shown with the corresponding protein location in the layout (n = 1). The 43 proteins were divided over two separate blots. (b) Heatmap displaying pixel density is expressed for all detected proteins. (c) Pixel densities of three selected targets—uPAR, MCP-1 and IL-6 are shown. (d–i) Elisa validation and quantification (pg/ml) of uPAR (n = 9) (d,e), MCP-1 (n = 8-9) (f,g) and IL-6 (n = 8-9) (h,i) in conditioned medium. Concentration of all targets increase upon IGF-2 or Des(1-6)IGF-2 stimulation. IGFBP-6 inhibits effects of IGF-2 but not Des(1-6)IGF-2. Statistical analysis for ELISA: comparisons between negative control and IGF-2 or Des(1-6)IGF-2 were performed using repeated measures ANOVA with Holm-Sidak multiple comparisons test, while comparisons between IGF-2 vs. IGF-2 + IGFBP-6 and Des(1-6)IGF-2 vs. Des(1-6)IGF-2 + IGFBP-6 were analyzed using repeated measures ANOVA with Sidak’s multiple comparisons test. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. HMEC-1: human microvascular endothelial cells; NC, negative control; PC, positive control; IGF-2, insulin-like growth factor 2; IGFBP-6, IGF-binding protein 6.
Fig 2: IGF-2 and its variants enhance HMEC-1 migration. (a,b) Chemotactic migration was assessed using a transwell migration assay with the IncuCyte® S3 Live-Cell Analysis System. (a) At 24 h, all variants significantly increased HMEC-1 migration compared to the negative control (n = 7-12, one-way ANOVA with Dunnett’s multiple comparisons test). (b) The addition of IGFBP-6 to IGF-2 significantly reduced IGF-2-induced migration, while IGFBP-6 had no effect on the migratory response to Des(1-6)IGF-2 (n = 7, one-way ANOVA with Sidak’s multiple comparisons test). (c–e) Results of the scratch wound healing assay, as monitored using the IncuCyte® system. (c) Representative images of the wound area (white) over time. Scale bar: 500 µm. (d) Migration is expressed as relative wound density (cell density in the wound area relative to the cell density outside the wound area). Cells were treated with 100 ng/mL IGF-2 or Des(1-6)IGF-2, which significantly enhanced wound closure at 24 h, while Leu27IGF-2 had no significant effect (n = 16, mixed-effects analysis with Dunnett’s multiple comparisons test). (e) IGFBP-6 showed an inhibitory effect on IGF-2-induced migration, while Des(1-6)IGF-2-induced migration remained unaffected (n = 14-16, mixed-effects analysis with Sidak’s multiple comparisons test). Data are presented as mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001. HMEC-1, human microvascular endothelial cells; IGF-2, insulin-like growth factor 2; IGFBP-6, IGF-binding protein 6.
Fig 3: IGF-2 and Des(1-6)IGF-2 promote endothelial tube formation. HMEC-1were seeded on Matrigel-coated angiogenesis μ-slides and treated with 100 ng/mL IGF-2, Des(1-6)IGF-2, or Leu27IGF-2. IGF-2 and Des(1-6)IGF-2 were also tested combined with 1,000 ng/mL IGFBP-6. After 8 h, tube formation was quantified and two key parameters were analyzed: number of nodes, indicating network complexity, and total tube length (in pixel, measuring overall network size. (a) Representative images of tube formation at 8 h for all conditions. Scale bar: 500 µm. (b,d) IGF-2 and Des(1-6)IGF-2 significantly increased node formation (b) and total tube length (d), while Leu27IGF-2 had no effect (n = 15–20). (c,e) IGFBP-6 inhibited IGF-2-induced increases in all parameters but did not affect Des(1-6)IGF-2 (n = 15–20). Mixed-effects analysis with Dunnett’s multiple comparisons test was used to compare IGF-2 and variants to the negative control. For comparing IGF-2 and Des(1-6)IGF-2 with their combination with IGFP-6, repeated measures ANOVA with Sidak’s multiple comparisons test was used. Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001. HMEC-1: human microvascular endothelial cells; IGF-2, insulin-like growth factor 2; IGFBP-6, IGF-binding protein 6.
Fig 4: In ovo angiogenic effect of IGF-2 and Des(1-6)IGF-2 on the chorioallantoic membrane (CAM). Sponges containing vehicle, 5 ng of IGF-2, 5 Des(1-6)IGF-2, or Leu27IGF-2 were placed on the CAM of chicken embryos at embryonic day 9. After 48 h, CAMs were photographed and the number of blood vessels intersecting a 3 mm radius around each sponge was manually counted (n = 13–15 eggs for each treatment, from 3 independent CAM experiments). (a) Representative images of the CAM with the analyzed area indicated by a dashed black circle. Scale bars represent 3 mm. (b) Quantification of the number of blood vessels intersecting the 3 mm radius, normalized to the negative control. Data are expressed as mean ± S.E.M. *p < 0.05 compared to the negative control, as determined by one-way ANOVA followed by Dunnett’s post-hoc test. CAM, chicken chorioallantoic membrane; IGF-2, insulin-like growth factor 2; IGFBP-6, IGF-binding protein 6.
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