Journal of Vascular Surgery
Volume 45, Issue 6, Supplement , Pages A48-A56, June 2007

The chemokine system in arteriogenesis and hind limb ischemia

  • Paula K. Shireman, MD

      Affiliations

    • Corresponding Author InformationReprint requests: Paula K. Shireman, MD, Associate Professor, Division of Vascular Surgery, MC 7741, 7703 Floyd Curl Dr, San Antonio, TX 78229-3900.

South Texas Veterans Health Care System, Departments of Surgery and Medicine, Sam and Ann Barshop Institute for Longevity and Aging Studies, the University of Texas Health Science Center, San Antonio, Tex.

Received 21 December 2006; accepted 11 February 2007.

Article Outline

Chemokines (chemotactic cytokines) are important in the recruitment of leukocytes to injured tissues and, as such, play a pivotal role in arteriogenesis and the tissue response to ischemia. Hind limb ischemia represents a complex model with arteriogenesis (collateral artery formation) occurring in tissues with normal perfusion while areas exhibiting ischemic necrosis undergo angiogenesis and skeletal muscle regeneration; monocytes and macrophages play an important role in all three of these processes. In addition to leukocyte trafficking, chemokines are produced by and chemokine receptors are present on diverse cell types, including myoblasts, endothelial, and smooth muscle cells. Thus, the chemokine system may have direct effects as well as inflammatory-mediated effects on arteriogenesis, angiogenesis, and skeletal muscle regeneration. This article reviews the complexity of the hind limb ischemia model and the role of the chemokine system in arteriogenesis and the tissue response to ischemia. Special emphasis will be placed on the roles of monocytes/macrophages and CCL2/monocyte chemotactic protein-1 (MCP-1) in these processes.

 

Arteriogenesis (collateral artery formation)1 and tissue regeneration2 have great therapeutic promise for the treatment of a wide variety of medical conditions. For example, extremity injuries compromise 50% to 60% of all combat casualties observed in Iraqi war veterans, resulting in blood vessel injury, large soft tissue defects, and high amputation rates.3, 4 Skeletal muscle is the tissue most vulnerable to ischemic damage in the extremities,5 but it also has an amazing potential to regenerate.6 Myogenic progenitor cells reside in skeletal muscle, proliferate, and fuse together or with damaged muscle fibers to regenerate muscle.

Angiogenesis and inflammation are critical to the process of muscle regeneration, but the complex interactions between these multiple cells types are poorly understood. In addition to inflammatory cell recruitment, chemokines may also contribute to these complex processes by directly acting on cell types other than inflammatory cells.1 A better understanding of the mechanisms of arteriogenesis, skeletal muscle regeneration, angiogenesis, and how inflammatory cells and chemokines influence these processes could lead to new therapies for limb salvage from ischemic and traumatic injury.

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Complexity of the hind limb ischemia model 

Many studies have used animal hind limb ischemia models to delineate the multiple factors that influence arteriogenesis and the tissue response to ischemia. Numerous mouse models have been used, ranging from simple femoral artery ligation to extensive excisions of the femoral artery, vein, and nerve,7 but multiple technical differences can make comparisons difficult. Nevertheless, the complex tissue responses to ischemia in the hind limb are different in anatomically distinct regions. In areas of severe ischemic injury, typically below the knee, muscle necrosis is accompanied by an intense inflammatory process, angiogenesis, and muscle regeneration.8, 9, 10 In contrast, arteriogenesis predominates in areas proximal to the ischemic regions (ie, the thigh).8, 10 Given these diverse pathophysiologic events, it is important to consider each region separately.

In most hind limb ischemia models, the thigh is the main site of arteriogenesis. Arteriogenesis is probably induced by fluid shear stress within the developing collateral arteries and is not initiated by hypoxia.11 Generally, necrosis is not present in the thigh muscles, and ischemia is mild or absent.12, 13 In the absence of hypoxia, capillary density in the thigh does not increase after femoral artery ligation.10

In contrast, an inflammatory infiltrate, angiogenesis, and skeletal muscle regeneration occur when tissue necrosis is present.8, 9, 10 Angiogenesis is induced by hypoxia, and capillary density increases in areas of severe, acute ischemia.10, 14 Tissue necrosis generally occurs in the muscles below the knee, and the extent of necrosis varies in different muscle groups. The tibialis anterior and soleus muscles undergo extensive necrosis with regeneration, whereas the gastrocnemius muscles exhibit variable necrosis after femoral artery excision.8

The mouse strain used can also influence extent, location, and severity of necrosis.7, 10, 12 However, depending on the extent of arterial disruption and mouse strain used, necrosis can also occur in the thigh8, 15 and if not directly stated by the authors, can be inferred by the presence of a robust inflammatory infiltrate in the early stages of ischemia and the presence of centrally located nuclei (ie, regenerated muscle fibers)16 after necrosis resolves. Of interest is that chronic ischemia in rats in the below knee muscles, either by placement of ameroid constrictors14 or ligation of the iliac artery,17, 18 results in increased capillary density in the absence of tissue necrosis or inflammation. Given the diversity of hind limb ischemia models, specification of the muscle used, presence of necrosis, extent of arterial disruption and animal strain can assist in resolving seemingly contradictory results.

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Brief overview of chemokines and chemokine receptors 

Chemokines (chemotactic cytokines) are important in leukocyte trafficking and influence a diverse array of normal and pathophysiologic processes, which have been extensively reviewed in previous articles,1, 19, 20, 21, 22 including arteriogenesis, angiogenesis, and skeletal muscle regeneration. The four families of chemokines are determined by the numbers and spacing of cysteine residues adjacent to the amino terminus: CC, CXC, CX3C, and XC (Table I, Table II).19 Redundancy exists within the CC and CCX chemokine families; multiple chemokines may activate several receptors, whereas individual receptors may be activated by several chemokines. In contrast, the CX3C and XC families, so far, consist of only one chemokine receptor in each family.20

Table I. The CC family of chemokines and chemokine receptors
ReceptorChemokine ligandsCell typesDisease connection
CCR1CCL3 (MIP-1α), CCL5 (RANTES), CCL7 (MCP-3), CCL14 (HCC1)T cells, monocytes, eosinophils, basophilsRheumatoid arthritis, multiple sclerosis
CCR2CCL2 (MCP-1), CCL8 (MCP-2), CCL7 (MCP-3), CCL13 (MCP-4), CCL16 (HCC4)Monocytes, dendritic cells (immature), memory T cellsAtherosclerosis, rheumatoid arthritis, multiple sclerosis, resistance to intracellular pathogens, type 2 diabetes mellitus
CCR3CCL11 (eotaxin), CCL24 (eotaxin-2), CCL7 (MCP-3), CCL5 (RANTES), CCL8 (MCP-2), CCL13 (MCP-4)Eosinophils, basophils, mast cells, Th2, plateletsAllergic asthma and rhinitis
CCR4CCL17 (TARC), CCL22 (MDC)T cells (Th2), dendritic cells (mature), basophils, macrophages, plateletsParasitic infection, graft rejection, T-cell homing to skin
CCR5CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CCL11 (eotaxin), CCL14 (HCC1), CCL16 (HCC4)T cells, monocytesHIV-1 coreceptor (T-topic strains), transplant rejection
CCR6CCL20 (MIP-3β), LARC)T cells (T regulatory and memory), B cells, dendritic cellsMucosal humoral immunity, allergic asthma, intestinal T-cell homing
CCR7CCL19 (ELC), CCL21(SLC)T cells, dendritic cells (mature)Transport of T cells and dendritic cells to lymph node, antigen presentation, and cellular immunity
CCR8CCL1 (I309)T cells (Th2), monocytes, dendritic cellsDendritic-cell migration to lymph node, type 2 cellular immunity, granuloma formation
CCR9CCL25 (TECK)T cells, IgA+ plasma cellsHoming to T cells and IgA+ plasma cells to the intestine, inflammatory bowel disease
CCR10CCL27 (CTACK), CCL28 (MEC)T cellsT-cell homing to intestine and skin

MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T-cell expressed and secreted; HIV, human immunodeficiency virus; MCP, monocyte chemotactic protein; HCC, hemofiltrate chemokine; Th2, type 2 helper T cell; TARC, thymus and activation-regulated chemokine; MDC, macrophage-derived chemokine; LARC, liver and activation-regulated chemokine; ELC, Epstein-Barr I1-ligand chemokine; SLC, secondary lymphoid-tissue chemokine; TECK, thymus-expressed chemokine; CTACK, cutaneous T-cell-attracting chemokine; MEC, mammary-enriched chemokine.; IG, immunoglobulin

From Charo and Ransohoff.19 Copyright © 2006 Massachusetts Medical Society. All rights reserved.

Table II. The CXC, CX3C, and XC families of chemokines and chemokine receptors
ReceptorChemokine ligandsCell typesDisease connection
CXCR1CXCL8 (interleukin-8), CXCL6 (GCP2)Neutrophils, monocytesInflammatory lung disease, COPD
CXCR2CXCL8, CXCL1 (GROα), CXCL2 (GROβ), CXL3 (GROγ), CXCL5 (ENA-78), CXCL6Neutrophils, monocytes, microvascular endothelial cellsInflammatory lung disease, COPD, angiogenic for tumor growth
CXCR3-ACXCL9 (MIG), CXCL10 (IP-10), CXCL11 (I-TAC)Type 1 helper cells, mast cells, mesangial cellsInflammatory skin disease, multiple sclerosis, transplant rejection
CXCR3-BCXCL4 (PF4), CXCL9 (MIG), CXCL10 (IP-10), CXCL11Microvascular endothelial cells, neoplastic cellsAngiostatic for tumor growth
CXCR4CXCL12 (SDF-1)Widely expressedHIV-1 coreceptor (T-cell-tropic), tumor metastases, hematopoiesis
CXCR5CXCL13 (BCA-1)B cells, follicular helper T cellsFormation of B-cell follicles
CXCR6CXCL16 (SR-PSOX)CD8+ T cells, natural killer cells, and memory CD4+ T cellsInflammatory liver disease, atherosclerosis (CXCL16)
CX3CR1CX3CL1 (fractalkine)Macrophages, endothelial cells, smooth muscle cellsAtherosclerosis
XCR1XCL1 (lymphotactin), XCL2T cells, natural killer cellsRheumatoid arthritis, IgA nephropathy, tumor response

GCP, Granulocyte chemotactic protein; COPD, chronic obstructive pulmonary disease; GRO, growth-regulated oncogene; ENA, epithelial-cell-derived neutrophil-activating peptide; MIG, monokine induced by interferon-γ; IP-10. interferon-inducible protein 10; I-TAC, interferon-inducible T-cell α chemoattractant; PF, platelet factor; SDF, stromal-cell-derived factor; HIV, human immunodeficiency virus; BCA-1, B-cell chemoattractant 1;SR-PSOX, scavenger receptor for phosphatidylserine-containing oxidized lipids.

From Charo and Ransohoff.19 Copyright © 2006 Massachusetts Medical Society. All rights reserved.

The systematic nomenclature refers only to human chemokines, although many human chemokines have mouse orthologs. Mouse and human synonyms for chemokine orthologs may differ, however, such as CXCL1; growth-regulated oncogene-α (GROα) for human and keratinocyte-derived chemokine (KC) in mouse. Furthermore, human orthologs have not yet been identified for some mouse chemokines, such as CCL6/multidrug resistance-associated protein-1 (MRP-1) and CCL12/monocytes chemotactic protein-5 (MCP-5).23 In this report, chemokines have been referred to as the human synonym, unless otherwise specified by “mouse.”

The CC chemokines (Table I) primarily attract mononuclear cells, including monocytes, eosinophils, basophils, dendritic cells, and T lymphocytes.19 CCL2/monocyte chemotactic protein-1 (MCP-1) is one of the most extensively studied chemokines in hind limb ischemia models.1 CXC chemokines (Table II) primarily attract neutrophils (CXCL1-3 and 5-8) or lymphocytes (CXCL4 and 9-16).21

Monocytes/macrophages are important in arteriogenesis,11 angiogenesis,24, 25 and muscle regeneration,26, 27 and multiple chemokines/chemokine receptors induce monocyte/macrophage recruitment.19 Monocytes are circulating cells that are recruited to sites of inflammation and differentiate into macrophages when activated in tissues.20 Peripheral blood monocytes express CCR1, CCR2, CCR3, CCR5, and CXCR4.28, 29, 30 CCR2 and CCR3 expression are decreased during differentiation to macrophages, whereas CCR1, CCR4, CCR5 and CXCR4 expression remain high,29 possibly to keep activated macrophages at the site of inflammation.20 CCR2 is the only known receptor for CCL2. Of interest is that CCL2 has a nonredundant role in regulating monocyte infiltration during inflammation,19 as demonstrated by CCL2–/– and CCR2–/– mice that exhibit deficient monocyte recruitment in essentially every tissue under a broad range of inflammatory-inducing conditions.31 Thus, a large array of chemokines can affect monocytes/macrophage recruitment and thereby influence arteriogenesis and the tissue response to ischemia.

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Chemokines and arteriogenesis 

Brief review of arteriogenesis 

Arteriogenesis is defined as the structural enlargement by growth of pre-existing arteriolar connections into true collateral arteries.11, 32 Collateral arteries can increase their lumen size by active proliferation and remodeling, thus increasing the capacity to carry blood to ischemic regions.33 Under normal circumstances, small amounts of blood flow may occur within a pre-existing network of arteriolar connections. With sudden arterial occlusion secondary to an embolus or a slowly progressive stenosis, a pressure gradient develops across the arteriolar network causing increased blood flow velocity with a concomitant change in fluid shear stress. Increased fluid shear stress results in increased production of endothelial adhesion molecules34, 35 and production of CCL2.36

Inflammatory cells, especially monocytes/macrophages, are attracted to the collateral artery by CCL2 and traverse the vessel wall by binding to the adhesion molecules. Monocytes/macrophages can produce large amounts of growth factors24 and, in turn, may stimulate endothelial and smooth muscle cell proliferation, both of which are necessary for collateral artery growth.11 As the collateral artery grows, the disturbed flow that creates different regions of high and low fluid shear stress may normalize, possibly signaling to the collateral artery to cease growing, despite the persistence ischemia in distal tissue beds.

Chemokines important in arteriogenesis 

Transcriptional profiling of the nonischemic adductor muscle in the thigh after femoral artery excision revealed increased expression of CC chemokines CCL2, CCL3, and CCL7, as well as CXC chemokines murine macrophage inflammatory protein-2 (MIP-2), CXCL9, and CXCL10.13 Although these data are suggestive that these factors are important in collateral artery formation, involvement in other processes occurring simultaneously in the adductor muscle cannot be excluded.13

The most extensively studied chemokine contributing to arteriogenesis is CCL2.32 In extensive studies of a rabbit hind limb model, Schaper et al37 infused CCL2 into the proximal end of ligated femoral arteries. After 7 days, angiograms demonstrated an increase in collateral artery formation in the CCL2-treated animals and hemodynamic measurements revealed an increase in collateral conductance.37 Increased monocyte accumulation in the walls of collateral arteries in the CCL2-treated animals compared with controls was also observed. Infusion of intercellular adhesion molecule-1 (ICAM-1) antibody diminished the CCL2-induced increase in collateral artery formation, suggesting the CCL2 mechanism of action was recruitment of inflammatory cells.38 Maximal endothelial and smooth muscle cell proliferation occurred at 3 days after femoral artery ligation and corresponded to monocyte adherence and migration through the collateral artery wall.39 Monocyte accumulation was greatest at 3 days, and CCL2 infusion augmented this response. After 7 days, monocytes were rarely observed in collateral arteries.40

Hind limb ischemia studies using CCL2–/– mice have consistently documented decreased restoration of perfusion.41, 42, 43 One curious finding is that results in studies of restoration of perfusion using CCR2–/– mice, the main receptor for CCL2,1 have been variable: one study exhibited decreased restoration of perfusion in CCR2–/– mice44 and two reports demonstrated similar perfusion45, 46 as control mice. Several possibilities may explain these seemingly contradictory results:

First, CCL2 may function through receptors other than CCR2,47 and the loss of CCL2 signaling through these undefined receptors may be responsible for the delayed restoration of perfusion in CCL2–/– mice.

Alternatively, CCR2–/– mice exhibit increased tissue CCL2 compared with control mice in response to hind limb ischemia.45 Increased CCL2 may allow binding to alternate receptors that are not normally activated at physiologic levels of CCL2 and may compensate for the loss of CCR2 receptors.

Finally, CXCR3 is another receptor that maybe important in arteriogenesis.41 CXCR3–/– mice demonstrated decreased collateral artery formation and impaired restoration of perfusion after femoral artery ligation. Of interest was that the infusion of bone marrow mononuclear cells from wild-type, but not CXCR3–/– mice, resulted in normal restoration of perfusion and collateral artery density in CXCR3–/– mice, suggesting that bone marrow–derived cell CXCR3 expression was essential for arteriogenesis.41

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Chemokines and angiogenesis 

Brief review of angiogenesis: role of vascular endothelial cell growth factor and monocytes/macrophages 

Vascular endothelial growth factor (VEGF) is one of the most important factors in angiogenesis, and hypoxia is a potent inducer of VEGF48, 49 The three VEGF receptors are VEGFR-1 (Flt-1), VEGFR-2 (KDR or Flk-1), and VEGFR-3 (Flt-4). VEGFR-2 mediates the angiogenic effects of VEGF after embryogenesis. The role of VEGFR-1 in angiogenesis is more controversial, but it is thought to act as a “decoy” receptor and may negatively impact angiogenesis by binding, and thereby decreasing, the availability of VEGF. VEGFR-3 activation mediates the lymphangiogenic effects of VEGF.48

Angiogenesis proceeds by a carefully orchestrated series of events, many of which are regulated by VEGF. First, endothelial cells become activated by inflammation or growth factors and proliferation ensues. Proteolysis of the basement membrane, controlled mainly by the matrix metalloproteinases, allows for endothelial cell migration, lumen formation, and anastomoses with other capillaries.49 VEGF promotes endothelial cell survival, proliferation, migration, and lumen formation.50, 51

Although VEGF was originally thought to act mainly on endothelial cells, VEGF receptors are also present on bone marrow-derived hematopoietic stem cells and inflammatory cells. Thus, VEGF induces the mobilization of endothelial progenitor cells and recruits inflammatory cells, including monocytes/macrophages.51 Of interest is that VEGF-mediated actions on monocytes/macrophages are accomplished by the VEGFR-1 receptor.52 In turn, macrophages can produce VEGF and assist in proteolysis of the extracellular matrix, thereby potentially amplifying the angiogenesis cascade.48 VEGF has a heparin-binding domain that allows for VEGF incorporation into the extracellular matrix. Macrophage degradation of the extracellular matrix can release VEGF to further augment angiogenesis.53

Although monocytes/macrophages contribute angiogenic and angiostatic factors, the overall effect of monocytes/macrophages is thought to promote angiogenesis by three potential mechanisms24, 25:

First, monocytes/macrophages secrete many angiogenic factors such as VEGF, CXCL8 (interleukin-8), granulocyte colony stimulating factor, transforming growth factor-α and β, platelet-derived growth factor, tumor necrosis factor-α, and prostaglandins.24, 25 Many of these factors act by promoting endothelial cell proliferation, migration, or tube formation, or a combination of these. Communication between endothelial cells and monocytes/macrophages appears to be bidirectional, because endothelial cell–secreted factors also induce chemotaxis and increased angiogenic activity in monocytes/macrophages, thus initiating a positive feedback cycle. For example, endothelial cells produce VEGF and CCL2, and both are chemoattractants for monocytes/macrophages.25 Monocytes/macrophages must be activated to promote angiogenesis. Activation does not lead to enhancement of all monocyte/macrophage activities, however, but selectively increases a subset of monocytes/macrophage activities in a stimulus-dependent manner.24

Second, macrophages digest the extracellular matrix, making it more prone to endothelial cell penetration. Endothelial cells migrate from established vessels to form new capillaries; thus, a path through the extracellular matrix must be created. Macrophages are efficient in the phagocytosis of extracellular matrix to create tunnels for endothelial cell migration. Macrophages are rich sources of matrix metalloproteinases, the proteolytic enzymes that are essential for extracellular matrix breakdown.25 Of interest is that CCL2-directed macrophage chemotaxis further amplifies this tunnel-drilling process.54, 55

Third, monocytes/macrophages may provide cellular components of the vessel wall by transdifferentiation.25, 55, 56 Monocytes/macrophages exhibit a phenotypic overlap of cell surface markers with sinusoidal and microvascular endothelial cells. Under angiogenic growth conditions, monocytes/macrophage precursors can differentiate into endothelial-like cells.57, 58 In vitro, peritoneal macrophages formed tunnels in matrigel and three-dimensional structures resembling microvessels.55 Thus, factors that affect monocytes/macrophage recruitment, such as chemokines, can influence angiogenesis.

Angiogenic and angiostatic properties of CXC chemokines 

The CXC chemokine family members exhibit both angiogenic properties, mediated by CXCR1, CXCR2, and CXCR4, and angiostatic properties, mediated by CXCR3 and CXCR5 (Table III).59, 60, 61 Although chemokines can exert a proangiogenic effect by recruitment of inflammatory cells, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8 can mediate angiogenesis in the absence of preceding inflammation through activation of CXCR2.62 Angiostatic chemokines are generally thought to inhibit the actions of proangiogenic growth factors, such as VEGF, on endothelial cell proliferation, chemotaxis, and other processes.59 Conflicting data of proangiogenic vs angiostatic properties of individual chemokines are common, however.

Table III. Angiogenic and angiostatic chemokines and chemokine receptors
ReceptorChemokine Ligands
Angiogenic
CCR1CCL15 (leukotactin-1)
CCR2CCL2 (MCP-1)
CCR3CCL11 (eotaxin); CCL15
CCR8CCL1 (I309)
CXCR1CXCL8 (interleukin-8); CXCL6 (GCP2)
CXCR2CXCL8; CXCL1 (GROα), CXCL2 (GROβ), CXL3 (GROγ), CXCL5 (ENA-78), CXCL6, CXCL7 (NAP-2)
CXCR4CXCL12 (SDF-1)
CX3CR1CX3CL1 (fractalkine)
Angiostatic
CXCR3CXCL4 (PF4), CXCL9 (MIG), CXCL10 (IP-10), CXCL11 (I-TAC)
CXCR5CXCL13 (BCA-1)

MCP, Monocyte chemotactic protein; GCP, granulocyte chemotactic protein; GRO, growth-regulated oncogene; ENA, epithelial-cell-derived neutrophil-activating peptide; NAP, neutrophil-activating peptide; SDF, stromal-cell-derived factor; PF, platelet factor; MIG, monokine induced by interferon-γ; IP-10, interferon-inducible protein 10; I-TAC, interferon-inducible T-cell α chemoattractant; BCA-1, B-cell chemoattractant 1.

Table derived from Bernardini et al,59 Rosenkilde and Schwartz,60 and Hwang et al.1

Angiogenesis is typically studied in animal models by using a variety of in vitro endothelial cell assays. Endothelial cells isolated from various sources express different chemokine receptors and may, in part, reconcile conflicting reports present in this area. In addition, limited antibodies are available that are specific for each chemokine and chemokine receptor, making difficult the determination of specific chemokine/chemokine receptor pair effects on the different aspects of angiogenesis.59 For example, although chemokines that activate CXCR3 inhibit in vitro endothelial cell proliferation and chemotaxis (ie, angiostatic),59 decreased capillary density was observed in ischemic calf muscles of CXCR3–/– mice after femoral artery ligation, suggesting impairments in angiogenesis in the absence of CXCR3.41

Angiogenic properties of CXCL12/stromal-cell-derived factor-1 (SDF-1) may be mediated by several mechanisms. CXCR4, the receptor for CXCL12, is widely expressed on many cell types, including inflammatory and endothelial cells. CXCL12 induces endothelial cell proliferation, chemotaxis, and tube formation. Furthermore, CXCL12 increases VEGF production, whereas VEGF can upregulate CXCR4 expression on endothelial cells; thus, VEGF and CXCL12 may act in a synergistic fashion to promote angiogenesis.60 In addition, CXCL12 is a potent chemotactic agent for endothelial progenitor cells that promote angiogenesis by incorporation into capillary networks or by paracrine effects from endothelial progenitor cell–secreted factors, or both.63, 64, 65

Angiogenic properties of CC chemokines 

Perhaps the most studied CC chemokine (Table III) in angiogenesis is CCL2. In addition to monocyte/macrophage recruitment, vascular cells are also influenced by the CCL2/CCR2 axis. Endothelial cells express CCR2, which is up-regulated by inflammatory cytokines. CCL2 induces endothelial cell migration, and production of CCL2 was increased after endothelial cell injury. After mechanical injury to endothelial cell monolayers, wound repair was delayed by inhibition of CCL2 and was improved by addition of exogenous CCL2.66 Furthermore, CCL2 directly induces vascular smooth muscle cell proliferation67 and migration,68 whereas CCR2 is expressed on vascular smooth muscle cells.69

Several studies have suggested a direct effect of CCL2 on angiogenesis in the absence of inflammation. Using the ex vivo rat thoracic aortic ring assay, the addition of CCL2 to the media resulted in increased angiogenesis.70, 71, 72 The CCL2-induced angiogenesis was mediated by up-regulation of hypoxia-inducible factor-1α, which in turn induced VEGF-A expression. VEGF-A–activated RhoA small G protein increased endothelial cell migration and proliferation. Inhibition of RhoA small G protein abrogated the CCL2-induced angiogenesis without affecting the increased VEGF production. This suggested that CCL2-induced angiogenesis was composed of two sequential steps: induction of VEGF-A expression by CCL2 with subsequent VEGF-A–induced angiogenesis.70

Although CCL2 can induce VEGF production, VEGF can also induce CCL2 production by the activated protein-1 binding site of the CCL2 promoter region. VEGF-induced tubule formation in angiogenesis and vascular permeability in the Miles assay was inhibited by anti-CCL2 antibody.73 Endothelial cells exposed to VEGF74 or brief ischemia75 produced CCL2. Thus, endothelial cells secrete CCL2 and VEGF, both of which are chemotactic for monocytes/macrophages. Monocytes/macrophages also secrete CCL2 and VEGF, which in turn, affect endothelial cell functions, further amplifying the angiogenesis cascade.48

CCL2 has also induced angiogenesis in a variety of in vivo models. CCL2 increased angiogenesis in both the matrigel plug and chick chorioallantoic membrane assays.71 CCL2 and VEGF induced similar increases in angiogenesis in the rabbit cornea assay. CCL2 angiogenesis was associated with macrophage recruitment, however, which was absent with VEGF. Of interest was that CCL2 induced a more rapid angiogenic response than VEGF with later vessel regression.76

In vivo measurements of capillary density in CCL2–/– mice have been contradictory, depending on the model. CCL2–/– mice exhibited decreased capillary density compared with wild-type mice in dermal wounds77 and in ischemic muscle after femoral artery ligation.41 In contrast, CCL2–/– mice exhibited similar capillary density compared with wild-type mice in a myocardial ischemia model.78 Decreased capillary density was observed in CCR2–/– mice in association with decreased monocyte/macrophage recruitment and delayed restoration of tissue VEGF after toxic muscle injury (unpublished observation, PK Shireman, San Antonio, Tex, 2006).

Additional CC chemokines also exhibit proangiogenic effects (Table III). CCL1/I309,79 CCL11/eotaxin,80 and CCL15/leukotactin-1,61 all directly induce endothelial cell migration as well as stimulate in vivo angiogenesis. Finally, CC chemokine receptors CCR2 and CCR5 were expressed on endothelial progenitor cells and may be critical for endothelial progenitor cell homing to regions of active angiogenesis.81

Angiogenic properties of the CXC3 chemokine family 

CX3CL1/fractalkine, the only chemokine in the CXC3 family, induced endothelial cell proliferation, migration, and tube formation as well as promoted angiogenesis in vivo.82, 83 The receptor for CX3CL1, CX3CR1, was also expressed on endothelial cells.82

Finally, a novel, three-dimensional in vitro model of angiogenesis used oligonucleotide arrays to evaluate gene expression in endothelial cells during three phases of angiogenesis: sprouting, branching, and network formation. Multiple chemokines and chemokine receptors were expressed differentially during the various phases of angiogenesis. Interestingly, CCL2, CCL5/regulated on activation normal T-cell expressed and secreted (RANTES) and CX3CL1 were maximally increased during the sprouting phase.84 This report highlights the delicate balance of chemokines necessary for normal angiogenesis.

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Chemokines and skeletal muscle regeneration 

Brief review of skeletal muscle regeneration 

The three central components in skeletal muscle regeneration are perfusion, inflammation, and satellite cells/myogenic progenitor cells. First, perfusion is essential to muscle regeneration. Regeneration was dependent on the invasion of blood vessels into the transplanted muscle before regeneration occurred.85 In vivo, muscle regeneration occurred from the outside of the muscle bundle toward the inner regions,86 possibly secondary to the need for capillaries to form before muscle regeneration could proceed. Thus, angiogenesis was critical to muscle regeneration, and impairments in angiogenesis could lead to altered muscle regeneration.

Second, inflammatory cells infiltrate the area after muscle damage, and neutrophils predominate initially. Neutrophils are key players in early inflammation associated with muscle injury, but these cells can also exaggerate muscle damage.26, 87 Although some reports claim a beneficial or neutral role of neutrophils in muscle regeneration,88, 89 most suggest that neutrophils increase muscle injury by generation of oxidant stress.26, 87, 90 In fact, myeloperoxidase may be a major factor in neutrophil-mediated damage; for example, myeloperoxidase–/– mice had less muscle membrane injury induced by mechanical loading than wild-type mice despite similar levels of inflammation.90

Neutrophils also secrete substances that attract and activate macrophages.91 This is important because macrophages replace neutrophils to facilitate the resolution of inflammation.92 Thus, the initial neutrophil inflammatory infiltrate was replaced by macrophages. Within 3 days after injury, macrophages predominated during skeletal muscle regeneration and were essential for the removal of necrotic tissue. Macrophages also produce a vast array of growth factors and enzymes that influence many aspects of the regenerative process, including angiogenesis and the chemotaxis, proliferation, and differentiation of myoblasts.93

Third, satellite cell activation is required to repair or replace the injured muscle. Satellite cells are a small population of resident muscle progenitor cells that are multipotent. Satellite cells are quiescent in normal muscle, but become activated and proliferate in response to muscle damage and express myogenic markers (now termed myoblasts). Myoblasts ultimately fuse to existing muscle or fuse together to form new myofibers during regeneration of damaged skeletal muscle tissue.6 Satellite cells may arise from several different sources and controversy exists about their origin.6

Importance chemokines in muscle regeneration 

Transcriptional profiling of injured skeletal muscle undergoing regeneration demonstrated increases in a variety of chemokines and chemokine receptors, especially in the CC family: CCL2, CCL3, CCL4, CCL6, CCL7, CCL9, and CCL12 as well as the CXC chemokine MIP-2 (mouse) were elevated after toxin-induced94, 95 and freeze-induced96 muscle injury. Chemokine receptors CXCR4, CCR1, CCR2, and CCR5 were also elevated.95 Similar results were obtained from ischemic posterior calf muscles after femoral artery ligation.97 Of note, the histologic appearance of regenerating muscle was similar after toxic, freeze, and ischemic injury,95, 96, 97, 98 suggesting that the muscle regeneration process was similar regardless of the inciting injury.

Skeletal muscle is able to regenerate after injury because of myogenic progenitor cells that are able to proliferate and repair/replace damaged muscle.6 Proliferating myoblasts secrete the neutrophil chemoattractant factor CXCL5/LPS-inducible CXC chemokine (LIX, mouse)99 as well as macrophage chemotactic factors CCL2 and CX3CL1.100 The presence of macrophages enhanced myoblast proliferation while reducing myoblast apoptosis, potentially facilitating muscle regeneration.100 Myoblasts also express chemokine receptors CCR2101 and CXCR4,102 suggesting that myoblast chemotaxis into injured tissue may be directed by chemokines. Thus, myoblasts are able to secrete and respond to chemokines.

In necrotic muscle after femoral artery excision, muscle regeneration was impaired in CCL2–/– mice43 and CCR2–/– mice45 in association with decreased macrophage recruitment. Because differences in arteriogenesis could potentially lead to a more prolonged and severe ischemic insult, thereby impairing skeletal muscle regeneration, toxic muscle injury was also performed and exhibited similar impairments in muscle regeneration and macrophage recruitment (unpublished observation, PK Shireman, San Antonio, Tex, 2006). Impaired muscle regeneration in CCR2–/– mice was also observed after freeze injury,103 whereas CCR5–/– mice exhibited normal muscle regeneration.104 Of interest was that partial macrophage depletion also resulted in similar impairments in muscle regeneration,105 suggesting that macrophage recruitment by the CCL2/CCR2 axis may be crucial to normal muscle regeneration.

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Conclusions 

Arteriogenesis, angiogenesis, and muscle regeneration are complex, interrelated processes that involve multiple cell types. Chemokines by direct and inflammatory-mediated mechanisms play an integral role in all three of these processes. A better understanding of the cellular interactions necessary for the successful formation of collateral arteries and recovery of injured tissues may lead to more successful strategies for tissue regeneration and engineering.

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 Competition of interest: none.Supported by grants from the National Institutes of Health (HL070158, HL074236) and the Veterans Administration.

PII: S0741-5214(07)00316-3

doi:10.1016/j.jvs.2007.02.030

Journal of Vascular Surgery
Volume 45, Issue 6, Supplement , Pages A48-A56, June 2007

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