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First published online March 12, 2004
doi: 10.1242/10.1242/jcs.00999

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Deregulation of Flk-1/vascular endothelial growth factor receptor-2 in fibroblast growth factor receptor-1-deficient vascular stem cell development

¹ Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, Dag Hammarskjöldsv. 20, 751 85 Uppsala, Sweden
² ImClone Systems Incorporated, 180 Varick Street, New York, NY 10014, USA

View larger version (72K): [in a new window]   Fig. 1. Expression of endothelial-specific markers during vascular development in EBs. (A) Immunoblotting was performed on total cell lysates of ES cells and EBs cultured for the indicated number of days, using antibodies against VEGFR-2, VE-cadherin and CD31. ß-catenin expression was analyzed as a loading control. (B) Immunohistochemical staining for VEGFR-2, VE-cadherin and CD31 in EBs cultured for the indicated number of days. One representative EB of eight for each condition is shown. Each condition was examined on two or more separate occasions. Arrows indicate blood islands visualized by VEGFR-2 and CD31 staining in day 4 EBs. Scale bars: 100 µm day 4 and 6; 200 µm day 8. (C) VE-cadherin was expressed in a 10-day old EB at the cell-cell contacts of the endothelial cells and the expression overlapped with CD31 expression. Scale bars: 100 µm.

View larger version (85K): [in a new window]   Fig. 2. Hallmarks of vascular development in the EBs. (A) Co-localization of TAL-1/SCL and VEGFR-2 at day 6 and 8. Note that more mature vessels did not stain positive for TAL-1. (B) At day 16, sprouting angiogenesis could be readily identified morphologically in the EBs, as fine vessels ending in a brush of lamellopodia from the endothelial cell in the distal tip of the vessel (arrow). (C) Benzidine-stained erythroblasts visualized as dark clusters (arrows) inside the CD31-positive vessels. (D) While CD31 expression was seen in sprouting vessels (arrow), vWF staining appeared in larger vessels (arrowhead). Scale bars: 100 µm.

View larger version (75K): [in a new window]   Fig. 3. Distinct effect of exogenous FGF-2 and VEGF-A on vascular development. EBs were cultured in the absence (basal) or presence of 20 ng/ml VEGF-A or FGF-2 from day 0 to day 8 and stained immunohistochemically or by fluorescence using antibodies against CD31 (A) or -smooth muscle actin (ASMA) (B) as indicated. Each growth factor produced distinct morphologies of the endothelial and smooth muscle cell pools. The bottom panels in A and B are higher magnification images of part of the boxed regions in the upper panels. Scale bars: A, 300 µm; B, upper panel 200 µm, lower panel 100 µm. (B) Co-localization (arrowheads) of CD31-positive cells (red) with ASMA cells (green) is shown in the far right image. (C) Quantification of CD31 staining as area (i.e. area without holes) in 8-day EBs (mean±s.d., n=6). *P<0.0001 FGF-2 compared to basal and FGF-2 compared to VEGF-A. (D) Quantification of CD31 staining as vessel length in 8-day EBs (mean±s.d., n=6). *P<0.0001 FGF-2 compared to basal, **P=0.0002 VEGF-A compared to basal.

View larger version (82K): [in a new window]   Fig. 4. Combined effects of FGF-2 and VEGF-A. (A) EBs were cultured in the presence of individual factors VEGF-A (80 ng/ml) or FGF-2 (80 ng/ml) or a combination of the two (20 ng/ml FGF-2 and 30 ng/ml VEGF-A) as indicated from day 0 to day 8, followed by immunohistochemical anti-CD31 staining. Scale bars: 100 µm (upper) and 200 µm (lower). Boxed regions are shown at a higher magnification in the lower panels. (B) Western blotting of total lysates of 8-day EBs showed growth factor-regulated expression of VEGFR-2, CD31 and VE-cadherin. ß-catenin was used as loading control. B, basal; F20, FGF-2 at 20 ng/ml; V30, VEGF-A at 30 ng/ml; F20+V30, FGF-2 at 20 ng/ml and VEGF-A at 30 ng/ml). (C) Quantification of protein bands in western blot shown in Fig. 4B.

View larger version (93K): [in a new window]   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.

View larger version (68K): [in a new window]   Fig. 6. Expression of endothelial cell markers in FGFR-1 -/- EBs. (A) Real-time PCR to determine CD31, VEGF-C, Ephrin B2 and VEGFR-2, VEGFR-1, VEGF-A transcription levels in 8-day-old FGFR+/- (+/-) or FGFR-/- (-/-) EBs cultured in basal conditions (i.e. without addition of exogenous growth factors). Changes are relative to ß-actin transcript levels. (B; right) Immunohistochemical staining for VEGFR-2 in FGFR-1-/- EBs cultured for 8 days under basal condition. Scale bars: 200 µm. (Left) Expression of VEGFR-2 protein in total cell lysates of basal treated FGFR-1+/- (+/-) and FGFR-1-/- (-/-) 8-day-old EBs. ß-catenin was used as loading control. (C) Immunocomplex kinase assay of VEGFR-2 on day-8 FGFR-1+/- (+/-) or FGFR-1-/- (-/-) EBs cultured under basal conditions (without exogenous growth factors) and unstimulated (-) or stimulated (+) with 100 ng/ml of VEGF-A for 10 minutes. Slower migrating, kinase active VEGFR-2 appeared in the FGFR-1-/- EBs (arrowhead). (D) CD31 staining of FGFR-1-/- EBs in basal conditions treated with either control anti-rat IgG, neutralizing anti-VEGFR-2 (30 µg/ml), or anti-VEGF-A (5 µg/ml) antibodies from day 6-8. Scale bars: 200 µm. (E) Quantification of CD31 staining as area (i.e. area without holes) in 8-day FGFR-1-/- EBs treated with anti-VEGFR-2 or anti-VEGF-A antibodies from day 6-8. Results are expressed as percentage of FGFR-1-/- EBs treated with control IgG (mean±s.d., n=4). *P=0.002 for anti-VEGFR-2 treatment compared to control. *P=0.0306 for anti-VEGF-A treatment compared to control.

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