Fig. 3.
Fig. 3. Effects of VEGF-A and VEGF-C on vasculoangiogenesis. / Representative vascular beds and networks stained with the anti-CD31 antibody in the para-aortic splanchnopleural mesoderm (P-Sp) coculturing system. Culture conditions are described in “Materials and methods.” Vascular bed formation (V.B.) proliferated around the P-Sp explant (arrowhead). Subsequently, endothelial cells sprouted on OP9 cells to form a vascular network (V.N.) after 14 days of culturing. (B) High magnification shows that vascular networks formed around vascular beds. (C) RNA was isolated from P-Sp explant, OP9 stromal cells, and whole embryos at E9.5, and the indicated transcripts were detected by RT-PCR using gene-specific primers described in “Materials and methods.” The PCR products were electrophoresed on 1.5% agarose gels. Bands were visualized by ethidium bromide staining. Messenger RNA from whole embryos was used as a positive control. (D) Staining profile of the cultured P-Sp explant. Plates were fixed after 10 days of culturing and then stained with anti–VEGFR-2 antibody (i). VEGFR-3 expression was visualized by LacZ staining using VEGFR-3+/LacZ P-Sp (ii). VEGFR-2 was expressed in vascular beds and networks. VEGFR-3 expression was only in vascular beds. (E) VEGF-A (100 ng/mL) or VEGF-C (100 ng/mL) was added to the culture of P-Sp explants. After 14 days of coculturing, the vascular cells were stained with the anti-CD31 antibody. Enhanced vascular bed formation is seen in the presence of VEGF-A (Eii) and VEGF-C (Eiii) compared with no factor (Ei) (low). Vascular network formation is not changed in the presence of VEGF-A and VEGF-C at higher magnification (high). (F) Quantitative analysis of vascular bed areas was performed with the NIH image computer analyzing system. The vascular bed area from P-Sp explants was measured. Error bars indicate SEM (n = 4). *P < .001, **P < .01. (G) The development of hematopoietic cells in the presence of VEGF-A and VEGF-C. The number of cells in the presence of VEGF-A and VEGF-C were reduced in a dose-dependent manner. Error bars indicate SEM (n = 4). Scale bar indicates 1 mm (A,E; low magnification,), 0.5 mm (B,D,E; high magnification).

Effects of VEGF-A and VEGF-C on vasculoangiogenesis.

Representative vascular beds and networks stained with the anti-CD31 antibody in the para-aortic splanchnopleural mesoderm (P-Sp) coculturing system. Culture conditions are described in “Materials and methods.” Vascular bed formation (V.B.) proliferated around the P-Sp explant (arrowhead). Subsequently, endothelial cells sprouted on OP9 cells to form a vascular network (V.N.) after 14 days of culturing. (B) High magnification shows that vascular networks formed around vascular beds. (C) RNA was isolated from P-Sp explant, OP9 stromal cells, and whole embryos at E9.5, and the indicated transcripts were detected by RT-PCR using gene-specific primers described in “Materials and methods.” The PCR products were electrophoresed on 1.5% agarose gels. Bands were visualized by ethidium bromide staining. Messenger RNA from whole embryos was used as a positive control. (D) Staining profile of the cultured P-Sp explant. Plates were fixed after 10 days of culturing and then stained with anti–VEGFR-2 antibody (i). VEGFR-3 expression was visualized by LacZ staining using VEGFR-3+/LacZ P-Sp (ii). VEGFR-2 was expressed in vascular beds and networks. VEGFR-3 expression was only in vascular beds. (E) VEGF-A (100 ng/mL) or VEGF-C (100 ng/mL) was added to the culture of P-Sp explants. After 14 days of coculturing, the vascular cells were stained with the anti-CD31 antibody. Enhanced vascular bed formation is seen in the presence of VEGF-A (Eii) and VEGF-C (Eiii) compared with no factor (Ei) (low). Vascular network formation is not changed in the presence of VEGF-A and VEGF-C at higher magnification (high). (F) Quantitative analysis of vascular bed areas was performed with the NIH image computer analyzing system. The vascular bed area from P-Sp explants was measured. Error bars indicate SEM (n = 4). *P < .001, **P < .01. (G) The development of hematopoietic cells in the presence of VEGF-A and VEGF-C. The number of cells in the presence of VEGF-A and VEGF-C were reduced in a dose-dependent manner. Error bars indicate SEM (n = 4). Scale bar indicates 1 mm (A,E; low magnification,), 0.5 mm (B,D,E; high magnification).

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