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. 2004 Jun 21;101(26):9833–9838. doi: 10.1073/pnas.0400337101

Fig. 1.

Fig. 1.

IGF-I promotes brain angiogenesis. (A) IGF-I (10-7 M) stimulates proliferation of cultured brain endothelial cells as determined with the 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay. VEGF (10-7 M) was used as a positive control [n = 3 independent experiments (nine replicates per experiment)]. (B) In the presence of IGF-I (10-7 M) endothelial cells plated on collagen form tubular structures, a prerequisite in the angiogenic process. Endothelial cells were labeled with tomato lectin (red). Note the absence of tubes under control conditions. (C) IGF-I stimulates both VEGF (upper gel) and VEGFR1 (lower gel) in endothelial cell cultures. Representative Western blots are shown. Lower gels show blots that were re-assayed with anti-PI3K antibody to control for protein load. Histograms show densitometric quantitation of the blots. **, P < 0.01; ***, P < 0.001 vs. control, n = 5. (D) The transcriptional modulator HIF-1α is required for IGF-I-induced activation of VEGF synthesis. (Top) Representative microphotograph of an endothelial cell monolayer transfected with HA-tagged DN HIF-1α. Cells were stained with anti-lectin (green) and anti-HA tag (red). Note the nuclear location of mutant HIF-1α.(Middle) Representative VEGF blot of cultures transfected with empty pcDNA3 vector (control) and DN-HIF-1α. IGF-I increases VEGF only in mock-transfected cultures. Re-blotting with anti-PI3K shows an equal protein load in all lanes. (Bottom) Densitometric quantitation of five independent experiments. ***, P < 0.001 vs. all other groups. (E) A 2-week s.c. infusion of IGF-I (50 μg/kg per day) elicits a significant increase in vascularization of the adult mouse brain. (Top) Vessels were identified with biotynilated-tomato lectin (arrow) and the percent surface of brain parenchyma covered with vessels estimated by scoring vessel contacts with the points of the grid (a 36-point grid is shown). (Scale bar = 25 μm.) (Middle) The histograms indicate vessel density in the cerebellum and the hippocampus as percent of control (100%). *, P < 0.05 vs. saline-treated controls (n = 5 animals per group). (Bottom) Representative cerebellar section with lectin-positive vessels of saline and IGF-I-treated mice. Vessel density is increased after IGF-I treatment. ML, molecular layer; GL, granule cell layer. (Scale bar = 50 μm.)