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Fig. 5. Effects of angiogenic growth factors on vascular development in FGFR-1-/- and VEGFR-2-/- EBs. (A) EBs lacking expression of FGFR-1 or VEGFR-2 (indicated by -/-) and control FGFR-1+/- EBs were treated with VEGF-A or FGF-2 at indicated concentrations from day 0 to day 8 and stained for CD31. FGFR-1-/- showed a marked increase in basal vascularization compared to FGFR-1+/- control EBs. Treatment with FGF-2 had no additional effect on vascularization in the FGFR-1-/- EBs, whereas VEGF-A at 80 ng/ml stimulated formation of a typical peripheral capillary plexus. In the VEGFR-2-/- EBs, FGF-2 at different concentrations stimulated CD31 positive cells to migrate and form clusters in the periphery (arrows). VEGF-A was essentially without effect. Scale bars: 200 µm. (B) Quantification of area of CD31 staining (i.e. area without holes) in 8-day FGFR-1+/- (+/-) and FGFR-1-/- (-/-) EBs. Results are expressed as percentage of control FGFR-1+/- EBs (mean±s.d., n=5). *P=0.0438 relative to control. (C) Quantification of CD31 staining as vessel length in 8 day FGFR-1+/- (+/-) and FGFR-1-/- (-/-) EBs. Results are expressed as percentage of control FGFR-1+/- EBs (mean±s.d., n=5). *P=0.0019 FGFR-1-/- compared with FGFR-1+/-. (D) Immunofluorescence staining for 
-smooth muscle actin in FGFR-1-/- EBs. Treatment with FGF-2 resulted in stress fiber formation and lining up of cells (arrow) in agreement with the effect of FGF-2 in the R1 EBs (cf. Fig. 3B). Scale bars: 100 µm. (E) Semi-quantitative PCR on cDNA derived from 8-day-old R1 and FGFR-1-/- EBs using FGFR-1 specific primers; as a control, ß-actin transcript levels were analyzed. (F) Immunoblotting for VEGFR-2 on total cell lysates from R1 or VEGFR-2-/- EBs cultured for 8 days. ß-catenin was used as a loading control.
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