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First published online 2 January 2008
doi: 10.1242/dev.002071

Development 135, 523-532 (2008)
Published by The Company of Biologists 2008
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FGF2-dependent neovascularization of subcutaneous Matrigel plugs is initiated by bone marrow-derived pericytes and macrophages

Ulrich Tigges*, Elizabeth Gore Hyer, Jeffrey Scharf and William B. Stallcup

Burnham Institute for Medical Research, Cancer Research Center, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA.


Figure 1
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  		Fig. 1. Early presence of pericytes in Matrigel vessels. Matrigel plugs were removed for analysis after 9 days (A-C) or 5 days (D-I). In A-F, endothelial cells were labeled for CD31 (green), while pericytes were labeled for NG2 (magenta). In G-I, pericytes were labeled for both NG2 (magenta) and PDGFβR (green). Cell nuclei were stained using DAPI (blue). At 5 days, the vessel-like networks contain NG2+PDGFβR+ pericytes in the absence of CD31-positive endothelial cells. At 9 days, both types of cells are present in the structures. All panels represent z-stacks of confocal images. Scale bar: 40 µm.

 

Figure 2
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  		Fig. 2. Functional perfusion of FGF2-induced Matrigel vessels. Mice containing Matrigel plugs were utilized on day 5 (A-D) and day 9 (E-H) for FITC-dextran (FITC-dx; green) perfusion to identify vessels connected to the circulation. Sections prepared from the plugs were stained for DAPI (blue), CD31 (red) and NG2 (magenta). At day 5, prior to the appearance of CD31+ endothelial cells, FITC-dextran is not present in the cellular networks. At day 9, FITC-dextran perfusion is apparent in vessels containing both pericytes and endothelial cells. All panels represent z-stacks of confocal images. Scale bars: 40 µm.

 

Figure 3
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  		Fig. 3. CD34 and Sca-1-positive cells in Matrigel vessels. Sections from 5 day (A-H) and 9 day (I-L) Matrigel plugs were stained for DAPI (blue), NG2 (magenta), CD34 (red), Sca-1 (red) and CD31 (green). Prior to the appearance of CD31+ endothelial cells, many cells in the vessel-like networks co-express NG2 and the markers CD34 and Sca-1. In older plugs, CD34 is co-localized with CD31 on endothelial cells. A CD31- CD34- pericyte is marked with an arrowhead. All panels are z-stacks of confocal images. Scale bars: 40 µm.

 

Figure 4
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  		Fig. 4. Flow cytometric analysis of NG2-positive, CD34-positive populations. (A,B) Flow cytometry was used to examine NG2 and CD34 expression by (A) cells recovered from dissociated 5-day Matrigel plugs and (B) cells in total adult bone marrow. In A, 8.2% of the Matrigel population expressed NG2 alone, 6.6% expressed CD34 alone and 74.2% expressed both markers. In B, 5.3% of the total bone marrow population expressed NG2 alone, 8.1% expressed CD34 alone and 1.7% expressed both markers. In control experiments with second antibodies alone, fewer than 0.5% of cells were scored as positive.

 

Figure 5
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  		Fig. 5. Contribution of macrophages to Matrigel vascularization. Sections of 5 day (A-D) and 9 day (E-H) Matrigel plugs were stained for DAPI (blue), NG2 (magenta), CD31 (green) and F4/80 (red) to investigate the presence of macrophages in developing vessels. At day 5, NG2+F4/80- pericytes are present (arrowhead). Some F4/80+ macrophages also express NG2 (arrow), while others are NG2- (asterisk). At day 9, NG2 and F4/80 are expressed on non-overlapping populations of pericytes and macrophages, respectively. At this stage, pericytes (arrowhead) exhibit a more intimate physical relationship with endothelial cells than macrophages do (arrows). All panels represent z-stacks of confocal images. Sale bars: 40 µm.

 

Figure 6
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  		Fig. 6. Bone marrow origin of vascular cells. Matrigel plugs were established in irradiated mice that had been reconstituted using bone marrow cells from an EGFP+ donor. At day 5 (A-D) sections were analyzed by staining for NG2 (magenta), CD34 (red) and DAPI (blue). The EGFP marker (green) is seen in cells that co-express NG2 and CD34 (arrows). Also at day 5 (E-H), NG2 (magenta), F4/80 (red), EGFP (green) and DAPI (blue) labeling were compared. The EGFP label is apparent in both populations of macrophages: F4/80+NG2+ (arrows) and F4/80+NG2- (arrowheads). At day 9 (I-L), sections were analyzed for NG2 (magenta), CD31 (red), EGFP (green) and DAPI (blue). The EGFP marker is frequently found in NG2+ cells (arrowheads), and more rarely in CD31+ cells (arrows). All panels represent z-stacks of confocal images. Scale bars: 40 µm.

 

Figure 7
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  		Fig. 7. Additional details concerning pericyte origin. (A-D) Expression of CD45 by pericytes and macrophages. Sections of 5 day Matrigel plugs were stained for DAPI (blue), CD45 (red), F4/80 (green) and NG2 (magenta). Although many NG2+ cells express both CD45 and F4/80, others express only CD45 (arrow) and still others lack both CD45 and F4/80 (arrowheads). All panels represent z-stacks of confocal images. Scale bar: 40 µm. (E-H) Mature Matrigel vessels are derived from immature cellular networks. Five-day Matrigel plugs were transplanted into EGFP+ recipients and allowed to continue their vascularization for 14 additional days. Sections were analyzed by staining for NG2 (magenta), CD31 (red) and DAPI (blue). The majority of vessels in the transplanted plugs were negative for EGFP (green), demonstrating that the cellular components of the vessels were already present prior to transplantation. In other vessels such as the one shown here, the EGFP label is absent from NG2+ pericytes (arrowheads) and present in some but not all CD31+ endothelial cells (arrows). Thus, only a subpopulation of the endothelial cells are recruited subsequent to transplantation. All panels represent z-stacks of confocal images. Scale bar: 40 µm.

 

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