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Comparative Study
. 2009 Nov;29(11):1794-801.
doi: 10.1161/ATVBAHA.109.194019. Epub 2009 Sep 4.

Bone Marrow-Derived Cell-Specific Chemokine (C-C Motif) receptor-2 Expression Is Required for Arteriolar Remodeling

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Free PMC article
Comparative Study

Bone Marrow-Derived Cell-Specific Chemokine (C-C Motif) receptor-2 Expression Is Required for Arteriolar Remodeling

Meghan M Nickerson et al. Arterioscler Thromb Vasc Biol. .
Free PMC article

Abstract

Objective: Bone marrow-derived cells (BMCs) and inflammatory chemokine receptors regulate arteriogenesis and angiogenesis. Here, we tested whether arteriolar remodeling in response to an inflammatory stimulus is dependent on BMC-specific chemokine (C-C motif) receptor 2 (CCR2) expression and whether this response involves BMC transdifferentiation into smooth muscle.

Methods and results: Dorsal skinfold window chambers were implanted into C57Bl/6 wild-type (WT) mice, as well as the following bone marrow chimeras (donor-host): WT-WT, CCR2(-/-)-WT, WT-CCR2(-/-), and EGFP(+)-WT. One day after implantation, tissue MCP-1 levels rose from "undetectable" to 463 pg/mg, and the number of EGFP(+) cells increased more than 4-fold, indicating marked inflammation. A 66% (28 microm) increase in maximum arteriolar diameter was observed over 7 days in WT-WT mice. This arteriolar remodeling response was completely abolished in CCR2(-/-)-WT mice but largely rescued in WT-CCR2(-/-) mice. EGFP(+) BMCs were numerous throughout the tissue, but we found no evidence that EGFP(+) BMCs transdifferentiate into smooth muscle, based on examination of >800 arterioles and venules.

Conclusions: BMC-specific CCR2 expression is required for injury/inflammation-associated arteriolar remodeling, but this response is not characterized by the differentiation of BMCs into smooth muscle.

Figures

Figure 1
Figure 1
Window chamber implantation elicits inflammation. A: MCP-1 expression in WT mice as determined by ELISA at Days 0 (n=8), 1 (n=8), 6 (n=6), and 13 (n=7). Values are means ± SD. *Significantly different than all other groups at P<0.01. +Significantly different than Days 0 and 1 at P<0.01 and Day 13 at P<0.05. B: Number of discrete EGFP+ BMCs per unit tissue area of window chamber (n=4 per timepoint). *Significantly different than all other groups at P<0.05. **Significantly different than Hours 0, 1, 2, and 12 at P<0.05. +Significantly different than Hours 0, 4, 8, and 12 at P<0.05. C: High endothelial venule imaged with transmitted light (left) and epifluorescence (middle) immediately after window chamber implantation. Merged image (right) shows EGFP+ BMCs in relation to the venule. EGFP+ BMCs that are rolling and/or adhering to the venule are denoted with white arrows, while an extravasated EGFP+ BMC is denoted with a black arrow. Bar=50 μm. D: Time lapse sequence of low magnification transmitted light/epifluorescence merge images taken from an EGFP+-WT chimeric mouse window chamber within the first 8 hours after implantation. EGFP+ BMCs are evident as white spots in the images. Bar = 100μm.
Figure 2
Figure 2
Window chamber implantation induces arteriolar remodeling in WT mice. A: Representative window chamber images under conditions of maximal dilation at Days 6 and 13. Arrowheads denote an arteriole, which is shown at high magnification in the insets, undergoing lumenal diameter expansion. Bar = 400μm. B: Maximum arteriolar diameters at Days 6, 9, and 13 after implantation (n=6 per timepoint). C,D: Changes in maximum arteriolar diameter from Day 6 to 13 (n=6). Values are means ± SEM. *Significantly different (P<0.05) than Day 6. E: Flourescence micrographs from a Day 13 window chamber cross-section immunolabeled for SM α-actin and counterstained for nuclei with DAPI. Arrows in the merge panel indicate smooth muscle nuclei, which are defined as being completely within the SM α-actin staining region. Bar indicates 50 μm. F: Bar graph of the ratio of wall area to lumen area at Days 1 (n=4), 6 (n=3), and 13 (n=3). *Significantly different than Day 1. **Significantly different than Days 1 and 6 (P<0.05). G: Bar graph of nuclei per unit wall area at Days 1 (n=4), 6 (n=3), and 13 (n=3).
Figure 3
Figure 3
The deletion of CCR2 from BMCs significantly reduces the density of F4/80+ monocytes/macrophages in the dorsal skinfold window chamber. A: F4/80 expression in cross-sections taken from Day 13 window chambers implanted on WT-WT (n=4), CCR2−/−-WT (n=3), and WT-CCR2−/− (n=4) mice. Arrowheads denote discrete monocytes, which are shown at high magnification in the insets. Bar = 100μm. B: Bar graph of F4/80+ area as a percentage of total area. C: Bar graph of F4/80+ area per length of section. Values are means ± SD. *Significantly different (P<0.05) than WT-CCR2−/− and WT-WT.
Figure 4
Figure 4
The deletion of CCR2 from BMCs abolishes arteriolar remodeling. A: Representative images of microvessels in dorsal skinfold window chambers of WT-WT, CCR2−/−-WT, and WT-CCR2−/− mice at Days 6 and 13. Arrows indicate arterioles exhibiting diameter enlargement in WT-WT mice and WT-CCR2−/− mice, but no response in CCR2−/−-WT mice. Insets show the arterioles that are denoted with arrows at higher magnification. Bar = 400μm. B: Maximum arteriolar diameters for WT-WT (n=7), CCR2−/−-WT (n=6), and WT-CCR2−/− (n=6) mice. *Significantly different than same group at Day 6 (P<0.05). C, D: Changes in maximum arteriolar diameter from Day 6 to Day 13 for WT-WT, CCR2−/−-WT, and WT-CCR2−/− mice. *Significantly different than all other groups (P<0.05).
Figure 5
Figure 5
BMCs are recruited to the window chamber, but do not differentiate into smooth muscle. Cross-sections from EGFP+-WT mice (n=3 at each timepoint) show spatial locations of EGFP+ BMCs (green) with respect to arterioles and venules, which are denoted by SM α-actin staining (red). While EGFP+ cells were abundant in window chamber cross-sections over 13 days, the co-localization of EGFP and SM α-actin was never observed. Arrows indicate spherical EFGP+ cells in vessel lumens. Bar = 50μm.

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