QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online August 16, 2005
doi: 10.1242/10.1242/jcs.02483

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kano, M. R. Articles by Miyazawa, K. Search for Related Content PubMed PubMed Citation Articles by Kano, M. R. Articles by Miyazawa, K. Social Bookmarking                         What's this?

VEGF-A and FGF-2 synergistically promote neoangiogenesis through enhancement of endogenous PDGF-B–PDGFRß signaling

¹ Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
² Department of Geriatric Medicine, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

View larger version (86K): [in a new window]   Fig. 1. Comparison of effects of VEGF-A/FGF-2 co-stimulation with those of VEGF-A or FGF-2 stimulation alone in a Matrigel plug assay in vivo. (A-C) Matrigel plugs mixed with 1 µg/ml FGF-2 and 200 ng/ml VEGF-A (A), 1 µg/ml VEGF-A alone (B), or 1 µg/ml FGF-2 alone (C) were subcutaneously injected into the abdomen of mice. After 7 days, gels were extracted and examined after Hematoxylin and Eosin (H & E) staining (A-C upper rows; Scale bars: 60 µm), and by immunohistochemistry with anti-PECAM1 or CD34, SMA, or by intravenously administrated FITC-conjugated dextran, as indicated in the figure (lower rows; scale bars: 20 µm). (D,E) Quantification of the formation of new vessels in Matrigel plugs by their lengths is shown. Vessels and vessel-like structures were classified into three categories as follows: mature blood vessels containing red blood cells (RBCs) (red bars), cavities surrounded by cells but without RBCs (light blue), and cells arraying in line but without cavities (blue). *P<0.05, **P<0.01 by Student's t-test. Corresponding figures for NG2 and desmin staining, and F4/80 staining under the VEGF-A-stimulated condition are shown in Figs S1 and S2, respectively.

View larger version (34K): [in a new window]   Fig. 2. Effects of growth factors on ESC-derived VEGFR2+ cells. (A-D) ESC-derived VEGFR2+ cells were treated with growth factors for 1.5 days, followed by immunostaining with anti-PECAM1 (green) and anti-SMA (red). Cells were treated with (A) a combination of VEGF-A (30 ng/ml) and FGF-2 (10 ng/ml; represented by a gradation of green and light blue in this and subsequent figures), (B) VEGF-A (green), (C) FGF-2 (light blue), or (D) PDGF-BB (20 ng/ml; red). Scale bars: 100 µm. (E) Rates (%) of appearance of PECAM1+ cells (green bars) and SMA+ cells (red bars). Error bars represent the standard deviation (s.d.). Corresponding video microscopy recordings taken for a half-day prior to fixation can be seen in Movies 1-4 in supplementary material.

View larger version (65K): [in a new window]   Fig. 3. Expression of markers and PDGF-B/PDGFRß in ESC-derived VEGFR2+ cells treated with various growth factors. (A) Expression of PECAM1, SMA, PDGF-B and PDGFRß was determined by quantitative RT-PCR; VEGFR2+ cells treated with VEGF-A (V), VEGF-A and FGF-2 (V/F), FGF-2 (F) or PDGF-BB (P) for 1.5 days. Vertical axes show relative expression. All expression data were normalized to GAPDH. Results were classified into two groups according to the pattern of expression. Expression of SMA and PDGFRß in FGF-2-treated (F; blue bars) and PDGF-BB-treated (P; red bars) cells, and that of PECAM1 and PDGF-B in VEGF-treated cells (V and V/F; green and blue/green bars, respectively). FGF-2- and PDGF-BB-treated cells (labelled F and P, respectively) in the two images on the left, and VEGF-A- and VEGF-A–FGF-2-treated cells (labelled V and V/F, respectively) in the two images on the right are shown as black bars because expression levels of corresponding genes were not markedly induced. (B,C) Expression of PDGF-B (B) and PDGFRß (C) was immunohistochemically determined in cells treated for 1.5 days. Distribution of SMA is shown for comparison in the lower panels. While PDGF-BB is expressed more in endothelium than in mural cells in the V/F treatment (red arrow), PDGFRß was present in SMA+ cells (yellow arrows) as well as in PECAM1–/SMA– cells (yellow arrowheads), but not in PECAM1+ cells. Bright-field images have been merged in all pictures to demonstrate the presence of endothelial sheets. Scale bars: 20 µm (B) and 50 µm (C).

View larger version (49K): [in a new window]   Fig. 4. Detachment of mural cells from endothelial sheets by exogenous PDGF-BB or anti-PDGFRß neutralizing antibody (APB5). (A-C) PDGF-BB (20 ng/ml; A) or anti-PDGFRß neutralizing antibody (APB5, 50 µg/ml; C) was added to cells cultured with VEGF-A and FGF-2 (B). Green: expression of PECAM1; red: SMA; purple: nuclear counterstain. Arrows in A and C indicate detached mural cells; arrowheads in B indicate attached mural cells. Scale bars: 100 µm. (D) Bar chart of distances between the nearest edges of SMA+ mural and PECAM1+ endothelial sheets. Frequency is shown as the percentage of SMA+ cell present in each distance class. (E) Comparison of the rates of appearance of PECAM1+ and SMA+ cells.

View larger version (81K): [in a new window]   Fig. 5. Effects of exogenous PDGF-BB and anti-PDGFRß neutralizing antibody (APB5) in the Matrigel plug assay in vivo. (A-C) PDGF-BB (1 µg/ml; A) or the anti-PDGFRß neutralizing antibody (APB5, 100 µg/ml; C) was mixed with to VEGF-A (200 ng/ml) and FGF-2 (1 µg/ml) in the Matrigel plug assay. (B) V/F treatment only. (Top row) H & E staining. Scale bars: 30 µm. Red arrows show mature blood vessels containing RBCs; orange arrowheads indicate vessels with extravascular RBCs. (Second and third rows) Immunohistochemistry. SMA staining (red) is shown in the second row, and merged images with PECAM1staining (green) are shown in the third row. White arrowheads indicate detached SMA+ cells, and arrows indicate PECAM1+ cells not covered by SMA+ cells. (Fourth row) Permeability assay using intravenously administrated dextran (green), merged with SMA (red) and PECAM1 (blue) images. Arrows indicate leakage of dextran. Scale bars: 20 µm. (Fifth row) Electron micrographs. Scales are indicated in each photograph. MC, mural cells; EC, endothelial cells; RBC, red blood cells; yellow arrows, tight junctions formed between endothelial cells. (D,E) Induction of new vessels and vessel-like structures in Matrigel plugs was quantified as in Fig. 1 but with a new category (orange bars) representing vessels with extravascular RBCs, which indicate hemorrhage. *P<0.05, **P<0.01 by Student's t-test. Corresponding figures for NG2 and desmin staining are shown in Fig. S3 in supplementary material.

View larger version (76K): [in a new window]   Fig. 6. Matrigel plug assay with other types of combined stimulation. (A,B) Combined stimulation with VEGF-A (200 ng/ml) plus PDGF-BB (1 µg/ml) (A, V/P treatment) and FGF-2 (1 µg/ml) plus PDGF-BB (1 µg/ml) (B, F/P treatment). Red arrows and orange arrowheads in H and E stained section (first row) show mature blood vessels and vessels with extravascular RBCs, respectively. White arrowheads and yellow arrows in immunohistochemically stained sections (second and third row) indicate detached SMA+ cells and PECAM1+ cells not covered by SMA+ cells, respectively. Arrows in the permeability-assay section (fourth row) indicate leakage of dextran. Scale bars: 60 µm in H and E stained sections: 20 µm in immunohistochemically stained sections. (C,D) Newly formed vessels and vessel-like structures were quantified using the same method as in Fig. 5. *P<0.05, **P<0.01. Corresponding figures for NG2 and desmin staining are shown in Fig. S4 in supplementary material.

View larger version (98K): [in a new window]   Fig. 7. A scheme of synergistic effects of VEGF-A and FGF-2 in neovasculature formation through enhancement of endogenous PDGF-B–PDGFRß signaling. VEGF-A induces secretion of PDGF-BB from endothelial cells in addition to its mitogenic effects on endothelium. FGF-2 enhances PDGFRß expression in mural cells, in addition to its mitogenic effects on both endothelial and mural cells. Combined stimulation with these two angiogenic molecules thus synergistically enhances intercellular communication through PDGF-B–PDGFRß signaling and causes mural cell recruitment and formation of mature vessels. These effects are blocked by either addition of exogenous PDGF-BB, which could overwhelm the local distribution of endogenous PDGF-BB, or inhibition of PDGFRß by specific antibodies, leading to formation of leaky and hemorrhaging vessels.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter