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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 2, 2003 as doi:10.1096/fj.03-0071fje. |
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* Research Group for Experimental and Clinical Arteriogenesis at the Department for Internal Medicine III, Albert-Ludwigs University of Freiburg, 79106 Freiburg; and
Max-Planck Institute of Immunobiology, 79108 Freiburg, Germany
3Correspondence: Research Group for Experimental and Clinical Arteriogenesis at the Department for Internal Medicine III, Albert-Ludwigs University Freiburg, Breisacher Strasse 66 (ZKF) D-79106 Freiburg im Breisgau, Germany. E-mail: meyer{at}med1.ukl.uni-freiburg.de
SPECIFIC AIMS
Previous studies have shown that granulocyte macrophage–colony-stimulating factor (GM-CSF) and transforming growth factor-ß1 (TGFß1) as well as bone marrow cells and peripheral blood mononuclear cells are able to stimulate collateral growth in animal models or human subjects. We therefore characterized the expression of several genes and proteins (e.g., monocyte chemoattractive protein-1, MCP-1; matrix metalloproteinase-12, MMP-12) known to be involved in arteriogenesis in murine bone marrow-derived macrophages (BMDM) under different concentrations of GM-CSF and TGFß1 to verify the arteriogenic potential of these cells for therapeutic applications and further detected the expression of MCP-1, MMP-12, and arginase in proliferating collateral arteries to determine the role of these factors in vivo.
PRINCIPAL FINDINGS
1. BMDM express genes typical for macrophages and stem cells
The expression of genes typical for tissue resident macrophages like CD68 (light density lipoprotein receptor), CD14, Toll-like 4 (lipopolysaccharide receptor), and 
M-integrin was confirmed. c-kit and the VEGF-receptor flt-1 as marker for hematopoietic stem cells were detectable on the level of gene expression. VEGF as wells as Flt-1 were found to be secreted by BMDM (Fig. 1
C, D).
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2. GM-CSF and TGFß1 differentially alter expression of MCP in BMDM
GM-CSF dose-dependently (5 and 50 ng/mL for 10 h, n=4) led to an up-regulation of monocyte chemoattractive protein gene expression (MCP-1, -3, and -5, P<0.05), which was confirmed for MCP-1 and -5 on the protein level after incubation for 15 h (P<0.05, Fig. 1A
, B). TGFß1, in contrast, did not significantly influence the expression of MCP-1 and -3 but reduced MCP-5 gene and protein expression at all tested concentrations (0.05, 0.5, and 5 ng/mL, P<0.05; n=3; Fig. 1A, B
).
3. GM-CSF stimulates the expression of MMP-12 in BMDM
GM-CSF (5 and 50 ng/mL) led to a 5- to 6-fold rise in the gene expression of MMP-12 (P<0.05); at a dosage of 50 ng/mL, MMP-14 was found to be 5- to 11-fold (P<0.05) up-regulated. TGFß1 treatment did not alter the gene expression of MMP-12 or -14. Immunofluorescent staining revealed a dose-dependent augmentation in the expression of MMP-12 by GM-CSF compared with untreated control cells (Fig. 1E-H
).
4. Arginase-1 gene expression and activity is enhanced by GM-CSF in BMDM
Arginase-1 gene expression showed a 26-fold increase compared with control cells when stimulated with GM-CSF (5 ng/mL, P<0.05). At higher GM-CSF concentrations (50 ng/mL) the increase was only 13-fold (P<0.05). TGFß1 did not significantly alter the gene expression level of arginase-1. The increase observed in arginase-1 gene expression by GM-CSF (5 ng/mL) was paralleled by a 2.5-fold increase in arginase-1 activity (52.6±1.1 vs. 134.1±2.9 mU/106 cells), whereas TGFß1 did not enhance arginase-1 activity. When both cytokines were added simultaneously, the increase in activity was not further augmented. This was reflected on the level of protein expression, where highest levels of arginase-1 were detectable via immunofluorescence at 5 ng/mL GM-CSF vs. control cells. However, arginase-1 staining revealed that the enzyme was not expressed uniformly in all cells. Thus, GM-CSF but not TGFß1 is an inducer of arginase-1 gene expression and activity in BMDM.
5. GM-CSF stimulates macrophage accumulation around proliferating collaterals: in situ expression of MMP-12, MCP-1 and arginase
Balb/c mice were treated s.c. for 3 days with GM-CSF (20 µg·day–1·kg–1, n=3) or PBS (n=3) after ligation of the femoral artery. Staining of hindlimb tissue using a mouse-specific marker for monocytes/macrophages (MOMA-2) showed an 
2.5-fold increase in the number of macrophages around proliferating collateral arteries in the GM-CSF group (P<0.05) (Fig. 2
A). Surprisingly, expression of MCP-1 was found to be nearly restricted to cells constituting the vessel wall (media) at this time (Fig. 2B, C
) whereas a strong expression of MMP-12 was detectable in macrophages localized in the adventitia (Fig. 2D, E
), lending strength to the important role of this metalloproteinase for the arteriogenic remodeling process. In contrast, arginase expression was detectable in macrophages as well as in the media (Fig. 2F, G
).
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CONCLUSIONS AND SIGNIFICANCE
We present here the first comprehensive in vitro analysis of BMDM to elucidate their putative arteriogenic potential for therapeutic applications. Our results show that treatment of BMDM with GM-CSF/TGFß1 modulates the expression of several factors that influence arteriogenesis on the level of cytokine expression, matrix degradation, and collagen synthesis (Fig. 3
). We provide novel data showing that GM-CSF in vivo stimulates the arteriogenic process by increasing the number of monocytes/macrophages around proliferating collaterals. Increased stimulation in the expression of MCP in BMDM indicates that the arteriogenic effect of GM-CSF but not of TGFß1 might be mediated at least partially by an augmented secretion of macrophage chemokines that attract mononuclear cells to the site of collateral remodeling. The low amount of macrophage MCP-1 detectable in vivo in hindlimb tissue suggests that after 3 days of collateral artery remodeling, MCP-1 expression becomes silenced in this cell population but more pronounced in smooth muscle cells. This might indicate a time- and cell type-specific regulation of MCP-1 during the arteriogenic remodeling process. Furthermore, GM-CSF could facilitate the expansion of growing blood vessels as indicated by the up-regulation of MMP-12 gene and protein expression in BMDM. Our in vivo data show that this protease is expressed mainly in macrophages localized in the adventitia during the arteriogenic process, thus lending strength to the important role of MMP for tissue remodeling a hallmark of the arteriogenic process. We show that GM-CSF but not TGFß1 stimulates the activity of arginase-1 in BMDM, an enzyme critically linked with cell growth and connective tissue production. Indeed, in vivo, this enzyme was found to be expressed highly in macrophages and the vessel wall (media) during collateral proliferation, a situation where an increased amount of collagen is needed. Altogether, this study supports the hypothesis that arteriogenesis is a multistage mechanism including monocyte/macrophage adhesion and transmigration, proarteriogenic cytokine expression, degradation of connective tissue, and collagen synthesis regulation. Selective modulation of these mechanisms as well as cell-based therapies supplying arteriogenic factors in vivo point to new strategies to influence collateral artery growth.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0071fje 
2 The first and second authors contributed equally to this work.
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posthoc test). MMP-12 expression was detectable using a mouse specific polyclonal antibody (red) and nuclear staining with Hoechst 33342 (blue) after stimulation with GM-CSF (5 and 50 ng/mL, F, G) vs. untreated control cells (E). A negative control was performed by omission of the primary antibody (H).
