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Originally published online as doi:10.1189/jlb.1107756 on June 11, 2008

Published online before print June 11, 2008
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(Journal of Leukocyte Biology. 2008;84:760-768.)
© 2008 by Society for Leukocyte Biology

Recruitment of the inflammatory subset of monocytes to sites of ischemia induces angiogenesis in a monocyte chemoattractant protein-1-dependent fashion

Benjamin J. Capoccia, Alyssa D. Gregory and Daniel C. Link1

Division of Oncology, Washington University School of Medicine, St. Louis, Missouri, USA

1 Correspondence: Division of Oncology, Washington University School of Medicine, Box 8007, 660 South Euclid Ave., St. Louis, MO 63110, USA. E-mail: dlink{at}im.wustl.edu


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ABSTRACT
 
There is accumulating evidence that delivery of bone marrow cells to sites of ischemia by direct local injection or mobilization into the blood can stimulate angiogenesis. This has stimulated tremendous interest in the translational potential of angiogenic cell population(s) in the bone marrow to mediate therapeutic angiogenesis. However, the mechanisms by which these cells stimulate angiogenesis are unclear. Herein, we show that the inflammatory subset of monocytes is selectively mobilized into blood after surgical induction of hindlimb ischemia in mice and is selectively recruited to ischemic muscle. Adoptive-transfer studies show that delivery of a small number of inflammatory monocytes early (within 48 h) of induction of ischemia results in a marked increase in the local production of MCP-1, which in turn, is associated with a secondary, more robust wave of monocyte recruitment. Studies of mice genetically deficient in MCP-1 or CCR2 indicate that although not required for the early recruitment of monocytes, the secondary wave of monocyte recruitment and subsequent stimulation of angiogenesis are dependent on CCR2 signaling. Collectively, these data suggest a novel role for MCP-1 in the inflammatory, angiogenic response to ischemia.

Key Words: MCP-1 • CCR2


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INTRODUCTION
 
Monocytes play a key role in mediating tissue repair and stimulating angiogenesis in response to ischemia. Within the first 3 days after ischemic injury, local production of specific cytokines and chemokines allows for the recruitment of monocytes from the blood to the damaged tissue [1 2 3 ]. The importance of monocytes in angiogenesis is highlighted by their ability to stimulate endothelial cell proliferation and/or migration through secretion of angiogenic growth factors, degradation of extracellular matrix by the release of proteases, and increases in vascular permeability by the deposition of fibrin [2 3 4 ]. Not surprisingly, inhibition of monocyte recruitment to ischemic tissue is associated with decreased angiogenesis [5 , 6 ]. Conversely, we showed recently that early delivery of monocytes to sites of ischemia markedly enhanced reperfusion in a murine model of acute hindlimb ischemia [7 ]. Moreover, monocyte accumulation in tumors has been shown to promote neoangiogenesis and tumor progression [8 9 10 11 ].

To stimulate angiogenesis, monocytes must home to, and be retained at, sites of ischemia. There is experimental evidence that vascular endothelial growth factor (VEGF), stromal-derived factor-1{alpha} (SDF-1{alpha}; CXCL12), and MCP-1 contribute to monocyte recruitment and/or retention to sites of ischemia [12 13 14 15 ]. For example, Cursiefen et al. [13 ] demonstrated that local administration of VEGF increased monocyte recruitment and angiogenesis in a mouse model of corneal neovascularization. Similarly, perivascular expression of SDF-1 during wound-healing leads to the retention of monocytes within ischemic tissue [16 ]. Finally, local infusion of MCP-1 protein or MCP-1 gene transfer into ischemic tissue increases the recruitment of monocytes and stimulates revascularization [17 18 19 ]. However, there is conflicting data as to the role of these proteins in stimulating angiogenesis, suggesting that the type of ischemic injury and/or tissue type may determine the importance of each chemoattractant. Hence, the signals that allow for the recruitment and activation of monocytes during the early stages of ischemia are not fully understood.

There is accumulating evidence that the monocytes represent a heterogeneous population, containing subsets with distinct functional properties. Relevant to this discussion, the Tie2+ subset of blood monocytes has been implicated in tumor angiogenesis [20 , 21 ]. In the present study, we show that the angiogenic capacity of monocytes resides within the inflammatory subset. Our data suggest a model in which the early recruitment of a relatively small number of inflammatory monocytes to sites of ischemia induces the local production of chemokines, which in turn, induces a secondary wave of monocyte recruitment and ultimately stimulates angiogenesis. Data are provided, suggesting a novel role for MCP-1 in this process.


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MATERIALS AND METHODS
 
Mice
MCP-1–/– and CCR2–/– mice on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). CX3CR1GFP/+ mice on a C57BL/6 background were a generous gift from Dr. Dan Littman (Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA). Mice were housed in a specific pathogen-free environment. The Washington University Animal Studies Committee (St. Louis, MO, USA) approved all experiments.

Adoptive transfer of CFSE-labeled cells
Bone marrow was harvested from the femurs of donor mice, and mononuclear cells were isolated by centrifugation across a 1.011 density gradient (Histopaque, Sigma-Aldrich, St. Louis, MO, USA) at 1700 g for 30 min. Mononuclear cells were then incubated with 2.5µM CFSE in PBS for 10 min at 37°C. CFSE-labeled mononuclear cells (1x106) were administered i.v. into recipient mice 24 h after the induction of hindlimb ischemia; this cell dose is the minimum number that consistently stimulated angiogenesis in the hindlimb ischemia model [7 ].

Murine hindlimb ischemia model
The hindlimb ischemia surgical procedure was performed as described previously [22 ]. In brief, an incision was made in the skin at the mid-portion of the right hindlimb overlying the femoral artery, and the femoral artery and vein were then dissected free from the nerve, and the proximal portion of the femoral artery and vein ligated with 6-0 silk sutures. The distal portion of the saphenous artery and vein and remaining arterial and venous side branches were ligated, followed by their complete excision from the hindlimb. The overlying skin was then closed using Nexaband veterinary glue (Abbott Animal Health, Abbott Park, IL, USA).

Laser Doppler perfusion imaging
Blood perfusion in the hindlimb was monitored by laser Doppler imaging (MoorLDI-2, Moor Instruments, UK). Before initiating scanning, mice were anesthetized and placed on a heating plate at 37°C to minimize temperature variations. For each time-point, the laser Doppler image obtained was analyzed by averaging the perfusion, expressed as the relative unit of flux as determined by Moor Instruments, over the surface of the ischemic and nonischemic foot. To control for ambient light and temperature, calculated perfusion was expressed as the flux ratio between the ischemic and nonischemic limbs.

Flow cytometry
The adductor muscle from ischemic and nonischemic hindlimbs was surgically isolated after hindlimb ischemia and then treated with 3 mg/ml Type I collagenase (Worthington Biomedical Corp., Lakewood, NJ, USA) for 40 min at 37°C. After filtering through a 50-µm cell strainer (Partec, Munster, Germany), cells were incubated with Fc block (Miltenyi Biotec, Auburn, CA, USA) for 10 min at 4°C, followed by incubation with PE-conjugated antibodies to Gr-1, F4/80, CD3, B220, or NK1.1 (PharMingen, San Diego, CA, USA). Cells were analyzed on a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Data are reported as the total number of the indicated cell type recovered from an entire adductor muscle.

ELISA
Following digestion of adductor muscle with collagenase to generate a single-cell suspension, cells and debris were removed by centrifugation at 500 g for 5 min. The cell-free tissue supernatant was recovered and analyzed using commercial ELISA kits for murine MCP-1, VEGFA, and SDF-1, according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

Inflammatory and resident monocyte subset isolation
Bone marrow mononuclear cells from CX3CR1GFP/+ mice were incubated at 4°C with PE-conjugated Gr-1 antibody (PharMingen). CX3CR1loGr-1+ and CX3CR1hiGr-1– monocytes were isolated using a MoFlo high-speed flow cytometer (Dako Cytomation, Fort Collins, CO, USA).

Immunofluorescence
Muscle sections were fixed in a lysine-based fixative (0.02 M NaH2PO4, 0.08 M Na2HPO4, 0.1 M lysine, 0.01 M sodium metaperiodate, in PBS, mixed 3:1 with 4% paraformaldehyde) for 15 min at room temperature, washed three times with 5% sucrose in PBS, and then flash-frozen in optimum cutting temperature compound (Sakura, Torrance, CA, USA). Frozen tissue sections were incubated at 4°C overnight with primary antibodies to biotinylated F4/80 (Clone BM8, Caltag Labs, Burlingame, CA, USA) and FITC-conjugated CD31 (Clone 390, eBiosciences, San Diego, CA, USA), followed by incubation with streptavidin-PE (eBiosciences).

Generation of CCR2–/– bone marrow chimeras
Bone marrow cells (2x106) were harvested from CCR2–/– mice (homozygous for the Ly5.2 allele) and transplanted into lethally irradiated, congenic (Ly5.1), wild-type mice (The Jackson Laboratory), as described previously [23 ]. Hematopoietic reconstitution with CCR2–/– hematopoietic cells was confirmed 5–6 weeks after transplantation by flow cytometry of blood leukocytes for Ly5.2 expression. Two independent groups of mice received transplants; mice were analyzed separately and the results pooled.

Statistical analysis
Statistical significance was determined by a two-way ANOVA analysis or by a two-sided Student’s t-test.


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RESULTS
 
Monocytes are rapidly and specifically recruited to ischemic tissue
We showed previously that increasing the number of circulating monocytes by adoptive transfer or mobilization with cytokines stimulated angiogenesis following surgical induction of hindlimb ischemia [7 ]. To elucidate mechanisms for this response, we first characterized the recruitment of donor monocytes to ischemic tissue. Donor bone marrow mononuclear cells were labeled with CFSE and infused i.v. into mice 24 h after the induction of hindlimb ischemia; of note, monocytes represented 12 ± 5% of the total bone marrow mononuclear cell population (data not shown). At specific times after adoptive transfer, the ischemic and nonischemic adductor muscles were harvested, and the number of CFSE+ (F4/80+) monocytes within the muscle was determined by flow cytometry. Adoptively transferred monocytes could be detected within the ischemic muscle as early as 4 h after adoptive transfer (0.5±0.07x104 cells), reached peak levels at 24 h (2.8±0.69x104 cells), and were cleared from the muscle by Day 7 post-surgery (Fig. 1A ). CFSE+ neutrophil recruitment to the ischemic muscle showed similar kinetics to monocytes (Fig. 1B) , and adoptively transferred T lymphocytes, B lymphocytes, and NK cells could not be detected in the ischemic muscle (data not shown). Of note, CFSE+ monocytes were not detected in nonischemic muscle, suggesting that adoptively transferred monocytes are recruited specifically from the blood to the ischemic hindlimb (Fig. 1A) .


Figure 1
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  		Figure 1. Recruitment of adoptively transferred bone marrow mononuclear cells to the ischemic hindlimb. CFSE-labeled bone marrow mononuclear cells (1x106) were infused i.v. into C57BL/6 recipient mice 24 h after the induction of unilateral hindlimb ischemia. The adductor muscle from the ischemic and nonischemic hindlimb was isolated at the indicated times after adoptive transfer and was digested with collagenase to generate a single-cell suspension for flow cytometry. (A) Number of CFSE+ F4/80+ monocytes recruited to the ischemic and nonischemic adductor muscle over time (n=3–6 per time-point). (B) Number of CFSE+ Gr-1+ neutrophils recruited to the ischemic and nonischemic adductor muscle (n=3 per time-point). (C) Number of endogenous (non-CFSE-labeled) F4/80+ monocytes recruited to the ischemic adductor muscle (n=3–6 per time-point; *, P<0.001, compared with no adoptive-transfer control at the same time-point). (D) Number of endogenous (non-CFSE-labeled), Gr-1+ neutrophils recruited to the ischemic adductor muscle (n=3–6 per time-point). Data represent the mean ± SEM.

A similar approach was used to assess the recruitment of endogenous (non-CFSE-labeled) monocytes to the ischemic adductor muscle. At baseline (prior to surgery), few monocytes were present in the adductor muscle (Fig. 1C) . In control mice receiving saline alone, recruitment of endogenous monocytes to the ischemic muscle was observed at 24 h after the induction of hindlimb ischemia but did not reach maximal levels until Day 4. In mice that received adoptively transferred bone marrow mononuclear cells, the recruitment of endogenous monocytes was markedly accelerated (Fig. 1C) . By 48 h post-surgery (24 h after adoptive transfer), the number of endogenous monocytes present in the ischemic adductor muscle was increased approximately fivefold compared with saline-treated mice (5.6±0.9x104 vs. 1.2±0.2, P<0.001). Interestingly, the number of endogenous monocytes recruited to the ischemic tissue greatly exceeded the number of CFSE-labeled monocytes. At 48 h after surgery, CFSE-labeled monocytes represent less than 1% of the total monocytes present in the ischemic tissue (Fig. 1A and 1C) . Histological studies confirmed these findings, showing that the number of monocytes present in ischemic adductor muscle was markedly increased in mice that had received bone marrow mononuclear cells compared with saline-treated mice (Fig. 2 ). In both cases, monocytes appeared to be localized primarily in perivascular regions. Of note, no difference in the number of endogenous neutrophils, B and T lymphocytes, or NK cells recruited to the ischemic muscle was observed between saline-treated and adoptive-transfer mice (Fig. 1D , and data not shown). Collectively, these data suggest that recruitment of a relatively small number of bone marrow monocytes to the ischemic tissue leads to a rapid and selective recruitment of endogenous monocytes.


Figure 2
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  		Figure 2. Histological assessment of monocyte recruitment to ischemic muscle. Representative photomicrographs showing monocytes (F4/80+, red) and endothelial cells (CD31+, green) in ischemic adductor muscle 24 h after adoptive transfer of bone marrow mononuclear cells (A) or saline alone (B). Sections were counterstained with 4',6-diamidino-2-phenylindole to identify nuclei. (C). Isotype control for F4/80. Data are representative of four independent experiments.

The inflammatory subset of monocytes improves reperfusion after hindlimb ischemia
Recent data suggest that monocytes can be divided into two distinct subsets based on the expression of the fractalkine receptor CX3CR1 [24 ]. The inflammatory subset of monocytes expresses low levels of CX3CR1 but high levels of CCR2 and Gr-1. In contrast, the resident subset of monocytes expresses high levels of CX3CR1 but low levels of CCR2 or Gr-1 and is recruited in a CX3CR1-dependent manner to noninflamed tissues [24 ]. Using transgenic mice expressing GFP under the control of the 2.1-kb CX3CR1 promoter [25 ], the ability of inflammatory and resident monocytes to be mobilized from the bone marrow to blood and to home to ischemic tissue was determined following induction of hindlimb ischemia. The number of inflammatory but not resident monocytes in the blood increased after the induction of hindlimb ischemia (Fig. 3A ). This increase appears to be secondary to enhanced mobilization, as only a modest increase in the number of bone marrow inflammatory monocytes was observed (Fig. 3B) . We next assessed the recruitment of these monocyte subsets to the ischemic tissue. Consistent with their ability to emigrate to sites of inflammation, a massive influx of inflammatory monocytes to the ischemic hindlimb was observed (Fig. 3C) . In contrast, little recruitment of resident monocytes was detected. To directly assess angiogenic capacity, inflammatory and resident monocytes were sorted and adoptively transferred into mice 24 h after induction of hindlimb ischemia. Whereas inflammatory monocytes improved reperfusion of the ischemic hindlimb, no effect was observed with resident monocytes (Fig. 3D) . These data suggest that following ischemic injury, inflammatory monocytes are mobilized into the blood and actively recruited to ischemic tissue, where they stimulate angiogenesis.


Figure 3
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  		Figure 3. Contribution of the inflammatory subset of monocytes to reperfusion. Hindlimb ischemia was surgically induced in CX3CR1GFP/+ mice, and bone marrow, peripheral blood, and adductor muscle were harvested at baseline, 24 h, and 48 h after surgery. Inflammatory and resident monocytes were quantified in each tissue by flow cytometry. (A) Peripheral blood; (B) bone marrow; (C) ischemic adductor muscle. (D) 2.5 x 105 CX3CR1hi Gr-1– resident or CX3CR1lo Gr-1+ inflammatory monocytes were adoptively transferred into wild-type recipient mice 24 h after induction of hindlimb ischemia. Blood flow was measured by laser Doppler perfusion imaging. *, P < 0.05, relative to Time 0 for the same genotype; **, P < 0.01, relative to Time 0 for the same genotype. Data represent the mean ± SEM.

Local production of MCP-1 in ischemic muscle is increased after adoptive transfer of bone marrow mononuclear cells
The ability of a relatively small number of adoptively transferred monocytes to trigger a massive influx of endogenous monocytes suggested that the local production of one or more monocyte chemoattractants is being induced. To test this possibility, we measured the local concentration in ischemic muscle of three proteins implicated in the recruitment of monocytes to sites of ischemia: namely, MCP-1, VEGFA, and SDF-1 [12 13 14 15 ]. The most dramatic increase was observed with MCP-1. In control and adoptively transferred mice, the local production of MCP-1 increased after induction of hindlimb ischemia (Fig. 4A ). However, 24 h after adoptive transfer of bone marrow mononuclear cells (48 h after the surgical induction of hindlimb ischemia), the level of MCP-1 was increased 4.6-fold from control mice (173.5±24.6 pg/ml vs. 37.9±8.2, respectively). Of note, peak levels of MCP-1 in control mice were achieved on Day 7 after surgery and remained elevated at least through Day 14 after surgery (Fig. 4A , and data not shown). Local production of SDF-1 but not VEGFA in ischemic muscle was also increased significantly in mice adoptively transferred with bone marrow mononuclear cells (Fig. 4B and 4C) . These data suggest the hypothesis that increased local production of MCP-1 and/or SDF-1 plays an important role in the recruitment of endogenous monocytes and subsequent stimulation of angiogenesis.


Figure 4
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  		Figure 4. Local production of MCP-1, VEGFA, and SDF-1 in ischemic adductor muscle. Ischemic adductor muscle was harvested at the indicated time, and the tissue was digested with collagenase to generate a single-cell suspension. Cells and debris were removed by centrifugation, and the protein levels of MCP-1 (A), SDF-1 (B), and VEGFA (C) in the tissue supernatant were measured by ELISA (n=3–6 per time-point). *, P < 0.05; **, P < 0.001, compared with no adoptive transfer). Data represent the mean ± SEM. WT, Wild-type.

Enhanced reperfusion following adoptive transfer of monocytes is dependent on CCR2 signaling
To examine the contribution of MCP-1 to angiogenesis, restoration of blood flow was characterized in MCP-1–/– mice. The magnitude and kinetics of reperfusion in MCP-1–/– mice were comparable with strain-matched controls (Fig. 5A ). We next asked whether the enhanced angiogenesis observed after adoptive transfer of bone marrow mononuclear cells is dependent on MCP-1. Wild-type bone marrow mononuclear cells were infused i.v. into MCP-1–/– recipient mice 24 h after induction of hindlimb ischemia. Strikingly, the improvement in hindlimb reperfusion observed after adoptive transfer of wild-type mononuclear cells was completely abrogated in MCP-1–/– mice (Fig. 5B) . These data suggest that the angiogenic response elicited by the adoptive transfer of mononuclear cells is dependent on MCP-1.


Figure 5
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  		Figure 5. Reperfusion in MCP-1–/– mice. (A) Restoration of blood flow in wild-type (C57BL/6) or MCP-1–/– mice following induction of hindlimb ischemia. (B) Restoration of blood flow after adoptive transfer of 1 x 106 wild-type bone marrow mononuclear cells (BM-MNC; or saline) into wild-type or MCP-1–/– mice 24 h after induction of hindlimb ischemia. (C and D) CFSE-labeled bone marrow mononuclear cells (1x106) were infused i.v. into wild-type or MCP-1–/– recipient mice 24 h after the induction of hindlimb ischemia. (C) Number of CFSE+, F4/80+ adoptively transferred monocytes recruited to the ischemic adductor muscle 4 h after adoptive transfer (n=6 for each genotype). NS, Not significant. (D) Number of endogenous (non-CFSE-labeled) F4/80+ monocytes recruited to the ischemic adductor muscle 28, 48, and 72 h after the induction of hindlimb ischemia (n=3–6 per time-point; **, P<0.001). Data represent the mean ± SEM.

To characterize the mechanism by which MCP-1 contributes to reperfusion, we assessed the recruitment of adoptively transferred bone marrow mononuclear cells to the ischemic tissue in MCP-1–/– mice. Surprisingly, labeled donor monocytes were recruited to the ischemic muscle of MCP-1–/– mice with the same efficiency as seen in strain-matched, wild-type controls (P=not significant; Fig. 5C ). However, the secondary wave of endogenous monocyte recruitment was markedly suppressed in MCP-1–/– mice (Fig. 5D) . Thus, despite normal recruitment to ischemic muscle, wild-type, adoptively transferred monocytes are unable to initiate and/or propagate the signals required for optimal endogenous monocyte recruitment in MCP-1-deficient hosts.

To begin to characterize these signals, we measured the level of MCP-1, SDF-1, and VEGF in the ischemic muscle of MCP-1–/– mice following adoptive transfer of wild-type bone marrow mononuclear cells. No increase in MCP-1 was observed, suggesting that adoptively transferred monocytes are not a major source of MCP-1 in ischemic muscle (Fig. 6A ). However, the magnitude of the induction of SDF-1 and VEGF in MCP-1–/– mice was comparable with levels seen in wild-type recipient mice after adoptive transfer (Fig. 6B and 6C) . These data suggest that expression of SDF-1 and VEGF in the ischemic tissue is not sufficient to induce maximal monocyte recruitment or angiogenesis.


Figure 6
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  		Figure 6. MCP-1, SDF-1, and VEGFA levels in ischemic muscle from MCP-1–/– mice. The level of MCP-1 (A), SDF-1 (B), and VEGFA (C) in muscle supernatant was measured, as described in Figure 4 . *, P < 0.05; **, P < 0.01; ***, P < 0.001, relative to no adoptive transfer. Data represent the mean ± SEM.

The surge in local MCP-1 production after the adoptive transfer of monocytes suggested the possibility that MCP-1 may be directly responsible for the recruitment of the secondary wave of endogenous monocytes. To test this possibility, we generated CCR2–/– bone marrow chimeras, in which the hematopoietic cells were CCR2–/–, and nonhematopoietic tissues (including endothelium and smooth muscle cells) were CCR2+/+. Hindlimb ischemia was induced in these chimeras, and 24 h later, wild-type bone marrow mononuclear cells were infused i.v. As expected, early recruitment of donor monocytes to the hindlimb was comparable with control mice (Fig. 7A ). However, recruitment of the secondary wave of endogenous (CCR2–/–) monocytes was markedly suppressed (Fig. 7B) . Furthermore, despite normal recruitment of donor monocytes, adoptive transfer of wild-type bone marrow cells failed to enhance reperfusion in CCR2–/– chimeras (Fig. 7C) . Collectively, these data show that CCR2 signals, although dispensable for the early recruitment of donor monocytes, play a key role in the secondary wave on endogenous monocyte recruitment and subsequent stimulation of angiogenesis.


Figure 7
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  		Figure 7. Reperfusion in CCR2–/– chimeric mice. CCR2–/– bone marrow chimeras were established by transplanting CCR2–/– bone marrow cells into irradiated wild-type mice. Following hematopoietic reconstitution (5–6 weeks), hindlimb ischemia was induced in these chimeras (or wild-type C57BL/6 mice), and 24 h later, CFSE-labeled, wild-type bone marrow cells were infused i.v. (A) Number of CFSE+, F4/80+ monocytes recruited to the ischemic adductor muscle 4 h after adoptive transfer (n=3–6 for each genotype). (B) Number of endogenous (non-CFSE-labeled) F4/80+ monocytes recruited to the ischemic adductor muscle 48 h after induction of hindlimb ischemia (n=3–6 for each genotype; ***, P<0.0001). (C) Restoration of blood flow in wild-type mice (C57BL/6) or CCR2–/– chimeras after adoptive transfer of 1 x 106 wild-type bone marrow cells or saline alone (PBS). Data represent the mean ± SEM.


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DISCUSSION
 
In this study, we show that adoptive transfer of the inflammatory subset of monocytes early after induction of hindlimb ischemia markedly enhances reperfusion following acute ischemia. Recruitment of inflammatory monocytes to the ischemic hindlimb results in increased local expression of MCP-1 and is associated with a massive secondary wave of endogenous monocyte recruitment. Interestingly, although the initial recruitment of adoptively transferred monocytes is independent of MCP-1, the secondary wave of monocyte recruitment and subsequent enhanced reperfusion is dependent on CCR2 signaling. These data add to accumulating evidence showing that monocytes play a central role in regulating angiogenesis at sites of ischemia and implicate a novel role for MCP-1 in this pathway.

There is evidence that monocytes are a heterogeneous cell population, containing subsets with distinct biological functions. Indeed, recent data suggest that Tie2 expression identifies a subset of monocytes in humans and mice that has angiogenic capacity [20 , 21 ]. Tie2+ monocytes are selectively recruited to tumors and stimulate angiogenesis in a paracrine manner [20 ]. Furthermore, depletion of Tie2+ monocytes inhibited tumor-associated neovascularization [20 ]. Romagnani et al. [26 ] identified a subset of circulating CD14+ monocytes that expressed low levels of CD34. These CD34+ CD14+ cells had the capacity for multilineage differentiation, including the ability to form endothelial cells. In the present study, we show that the inflammatory subset of monocytes, as defined by low CX3CR1 and high Gr-1 expression, is specifically recruited to sites of ischemia and able to promote revascularization. Of note, the bone marrow is a rich reservoir for inflammatory monocytes that are readily mobilizable by ischemia (Fig. 3) or cytokines (data not shown). In contrast, resident monocytes had no angiogenic capacity in our hindlimb ischemia model. Interestingly, Venneri et al. [21 ] reported that Tie2+ monocytes were mainly contained with the resident subset of human monocytes. Whether this apparent discrepancy is secondary to species-specific differences in Tie2 expression or reflects the presence of multiple angiogenic monocyte subsets remains to be determined.

The role of MCP-1 in angiogenesis is controversial. Local expression of MCP-1 has been linked to increased macrophage recruitment and angiogenesis at tumor sites [27 28 29 ]. Voskuil et al. [30 ] reported that MCP-1–/– mice had impaired monocyte recruitment and blood flow recovery after hindlimb ischemia, which could be reversed by local treatment with purified MCP-1 protein. Consistent with this finding, Heil and colleagues [31 ] showed that mice lacking CCR2 had impaired collateral artery formation after induction of hindlimb ischemia. In contrast, two recent studies showed that revascularization following induction of the hindlimb ischemia model was comparable between CCR2–/– mice and strain-matched controls [32 , 33 ]; these authors suggested that differences in the strain of mice used might account for the discrepant findings. Recently, Nahrendorf and colleagues [34 ] showed that monocyte recruitment after myocardial infarction takes place in two distinct phases. They showed that early recruitment of monocytes (Days 0–3 after infarction) but not late recruitment (Days 4–7) requires CCR2 signaling [34 ]. Collectively, these observations suggest that the contribution of CCR2 signaling to angiogenesis may depend on the site and type of ischemic injury and phase of recovery from ischemia.

In the present study, we show that revascularization after surgical induction of hindlimb ischemia in MCP-1–/– mice on a C57BL/6 background is comparable with strain-matched, wild-type mice. However, the enhanced revascularization induced by adoptive transfer of monocytes is completely dependent on MCP-1. Surprisingly, our data suggest that the initial recruitment of monocytes to sites of ischemia is not dependent on MCP-1. Rather, the surge in local MCP-1 production induced after the adoptive transfer of monocytes appears to be necessary to trigger the secondary (and much greater) wave of monocyte recruitment. Consistent with this conclusion, we show that in CCR2–/– bone marrow chimeras that lack CCR2 expression on monocytes, the secondary wave of monocyte recruitment is markedly attenuated, and the reperfusion response to the adoptive transfer of monocytes is abrogated. Of note, MCP-1 also has been shown to directly stimulate endothelial cell proliferation and smooth muscle migration [35 36 37 38 39 40 ]. However, our studies of the CCR2–/– chimeras suggest that CCR2 signaling on endothelial cells and smooth muscle cells is not sufficient to stimulate angiogenesis in this model. Finally, MCP-1 also has been shown to directly induce VEGFA expression from endothelial cells [39 ]. However, we show that local production of VEGFA (and SDF-1) in ischemic muscle is comparable in MCP-1–/– and wild-type mice. Thus, in this model of acute ischemia, production of VEGFA and SDF-1 is not dependent on MCP-1.

The mechanism by which adoptively transferred monocytes stimulate the local surge in MCP-1 production remains unclear. Previous studies have shown that along with monocytes/macrophages, MCP-1 is secreted from smooth muscle cells and endothelial cells within ischemic tissue [29 , 41 , 42 ]. The failure of wild-type monocytes to induce MCP-1 expression after adoptive transfer into MCP-1–/– recipients shows that donor monocytes are not the primary source of MCP-1 and support an indirect mechanism. Of interest, VEGF production by monocytes has been shown to stimulate MCP-1 expression from endothelial cells [38 , 41 ]. Unfortunately, we were unable to reproducibly identify MCP-1-expressing cells in tissue sections; thus, the cellular source of MCP-1 in our model is unclear.

In summary, we provide novel evidence supporting a key role for MCP-1 in angiogenesis. Although not required for the initial recruitment of monocytes or the local production of SDF-1 or VEGFA, MCP-1 is required for the secondary recruitment of monocytes and maximal stimulation of angiogenesis. We show that the angiogenic capacity of circulating monocytes is contained within the inflammatory subset. The ability of this subset of monocytes to stimulate therapeutic angiogenesis in the clinical setting merits further study.


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ACKNOWLEDGEMENTS
 
This work was supported by a grant from the National Institutes of Health, R01 HL073762 (D. C. L.). B. J. C. designed the research, analyzed the data, and wrote the paper. A. D. G. performed research and analyzed data. D. C. L. designed the research. We thank Dr. Dan Littman for providing the CX3CR1GFP/+ mice. We also thank Amgen (Thousand Oaks, CA, USA) for its generous gift of G-CSF.

Received November 15, 2007; revised May 9, 2008; accepted May 11, 2008.


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