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Published online before print May 9, 2006
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,1
* Research Group for Experimental and Clinical Arteriogenesis, Freiburg, Germany;
Department of Experimental Cardiology, UMC Utrecht, Netherlands;
Phoenix X-ray Systems
+ Services GmbH, Stuttgart, Germany;
Department of Cardiology, AMC Amsterdam, Netherlands;
¶ Institute of Anatomy, Department of Pathobiology, University of Veterinary Medicine Vienna, Austria;
|| Center of Anatomy and Cellbiology, Medical University of Vienna, Austria; and
** Charité, Department of Cardiology, Center for Cardiovascular Research CCR, Berlin, Germany
1 Correspondence: Department of Experimental Cardiology, UMC Utrecht, G02.523, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail: i.hoefer{at}umcutrecht.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: collateral artery growth • peripheral vascular disease
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and matrix metalloproteinases (MMPs) [12
13
14
]. The remodeling of the arterial wall is initiated by proteases released by macrophages (i.e., MMPs) and soon followed by mitosis of endothelial cells and vascular smooth muscle cells [15
]. Once functionally mature, collateral arteries cannot be distinguished microanatomically from "normal" arteries [10
]. Enhancing the attraction of monocytes to the site of collateral artery growth (e.g., by MCP-1 infusion) promotes and accelerates this process significantly [16 , 17 ]. Recent experimental studies suggest a direct, functional correlation between collateral artery growth after occlusion of a main artery and the concentration of circulating monocytes [18 ]. Yet, effects on progenitor cells and other leukocyte populations (e.g., granulocytes, lymphocytes) and thus, their influence cannot be excluded by pharmacological alteration of monocyte concentration. Previous reports suggested that progenitor cells could positively influence arteriogenesis [19 ]. However, it was also shown that in adult organisms, bone marrow (BM)-derived cells do not incorporate into growing arteries [20 ]. To further elucidate the influence of monocytes/macrophages on this process, we used osteopetrotic (op/op) B6C3Fe a/a-Csf1–/– mutant mice, deficient in BM macrophages, blood monocytes, and serosal cavity macrophages [21 ]. As a result of an insertional point mutation in the CSF-1 (M-CSF) [22 , 23 ], these mice only produce biologically inactive CSF-1 [23 , 24 ]. In addition, op/op mice suffer from severe osteopetrosis as a result of impaired bone resorption, caused by a lack of functional osteoclasts [25 , 26 ]. This results in distinct bone formation, a domed skull, and impaired tooth eruption. These phenotypical changes can be recognized from Day 10 after birth, whereas heterozygous (+/op) and wild-type (+/+) mice are phenotypically indifferent. Administration of recombinant human CSF-1 to op/op animals in vivo reverses the op/op phenotype by normalizing osteoclastogenesis and monocyte/macrophage concentrations [27 , 28 ].
CSF-1 is a hematopoietic growth factor stimulating the proliferation and differentiation of cells of the monocyte-macrophage lineage [29 , 30 ]. Its cellular effects are mediated via a high-affinity cell surface receptor, which is encoded by the proto-oncogene c-fms [31 ], considered as a marker for cells of the mononuclear phagocyte system [32 ]. Osteoclasts are tissue-specific polykarionic macrophages, created by fusion of promonocytes and monoblasts [33 ], and thus, belong to the monocyte/macrophage lineage.
We therefore hypothesized that in op/op mice, collateral artery growth is impaired as a consequence of the deficiency for monocytes/macrophages. This was tested in a microsphere-based perfusion model using fluorescent microspheres injected under maximal vasodilation with a novel computer-controlled perfusion system. In addition, an innovative technique of high-resolution angiographs was applied to further elucidate the indispensable role of monocytes in the process of arteriogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All animals were kept in Makrolon cages, Type IIL, filled with soft wood bedding. A standard rodent diet was available ad libitum. Wild-type mice (+/+) were discriminated from heterozygous (+/op) mice using capillary electrophoresis by the size difference of the polymerase chain reaction-amplified MCSF fragments [34 ]. op/op mice were distinguished 10 days postpartum by their distinct skull formation and the failure of tooth eruption and were weaned off at 4–5 weeks and kept on a wet-mash diet consisting of normal chow macerated in drinking water ad libitum.
CSF-1 immunoassay
Whole blood samples were obtained before the initial operation from five op/op, five +/+, and five –/+ to measure CSF-1 concentration with a quantitative sandwich enzyme immunoassay kit in the peripheral blood, according to protocols supplied with the kit (R&D Systems, Minneaspolis, MN).
Femoral artery occlusion
Unilateral ligation of the right femoral artery proximal to the caudal femoral artery was performed under general anesthesia as described previously [13
]. A skin incision on the medial aspect of the thigh was performed. The right femoral artery was exposed and ligated proximal to the caudal femoral artery, using nonresorbable material (silk, 6–0). The skin was closed surgically with intradermal sutures to minimize the risk of skin irritation and track infections. As a result of the high mortality rate of op/op mice under common anesthesia, we established a special anesthetic protocol. Vital parameters were closely monitored at all times. Mice were preoxygenated by placing them in a special designed chamber and sedated 1 h before surgery with acepromacin (1 mg/kg) and buprenorphin (0.03 mg/kg) intraperitoneally (i.p.) to provide sufficient analgesia at all times. Enrofloxacin [85 mg/kg, subcutaneously (s.c.)] was applied preoperatively to all animals, and treatment was continued twice daily for 2 days. Ketamin (25 mg/kg) and xylazine (3 mg/kg) were administered i.p. Isoflurane was used for anesthesia induction and maintenance via a specially designed facemask. As a result of the sufficient sedation and analgesia, a low-dose anesthesia protocol could be run. All surgical procedures were performed under sterile conditions. Application of buprenorphin was continued for 3 days (0.03 mg/kg twice daily, s.c.) postoperatively.
Peripheral blood analysis
EDTA-anticoagulated peripheral blood of 16 animals (n=8 op/op; n=8 +/+) was obtained by puncturing the coccygeal artery for flow cytometry analysis of circulating leukocytes (Epics-XL, Beckman-Coulter, Miami, FL). Labeling for flow cytometry was performed using conjugated rat anti-mouse antibodies. Leukocytes were stained with a phycoerythrin (PE)-conjugated anti-CD45 (leukocyte common antigen) antibody, T lymphocytes with a fluorescein isothiocyanate (FITC)-conjugated anti-CD3, thymocytes with a PE-Texas Red anti-CD4, and monocytes with a PE-Cy5-conjugated anti-F4/80 antibody (all Caltag Laboratories, S. San Francisco, CA). After securing proper compensation, tubes were analyzed for the different subpopulations. The number of granulocytes, lymphocytes, and monocytes was expressed as percentage of the total leukocyte count. Total number of leukocytes/µl was measured after addition of fluorescent beads in a defined concentration as an internal standard.
Immunohistochemistry examination
A further six animals (n=3 op/op; n=3 +/+) were operated as described above. Animals were killed 7 days after femoral artery ligation, and muscle tissue was harvested from the hind limb (Mm. adductores, M. quadriceps) for immunohistological examinations. Samples were embedded in tissue-freezing medium (Tissue-Tec), snap-frozen at –150°C in 2-methylbutan, and stored at –80°C until further processing. Samples were cryo-cut in sections of 5 µm, fixed in acetone, and incubated overnight with the primary antibody. Macrophages and monocytes were detected using a mouse monocyte/macrophage-specific rat monoclonal antibody against MOMA-2 (1:250, BMA Biomedicals, Switzerland). Cy3-labeled anti-rat immunoglobulin G1 antibody was used as secondary antibody (1:100, goat anti-rat, Abcam, UK). Nuclear staining was performed with Hoechst 33342 (Molecular Probes, Eugene, OR). In addition, tissue was stained with FITC-labeled monoclonal anti- smooth muscle actin (1:100, Sigma Chemical Co., St. Louis, MO) to stain for arterial vessels. Hematoxylin and eosin (HE) staining was performed to exclude tissue damage. Negative controls were performed for all immunological stainings by omission of the primary antibody. There was no sign of ischemic cell damage. Macrophages were counted around collateral arteries with a 200-fold magnification in 20 tissue samples in the area of collateral artery growth (confirmed by angiograms). Macrophage accumulation was expressed as the number of MOMA-2-positive cells in the perivascular tissue of proliferating collateral arteries per mm2.
Imaging system
In this study, a novel angiography method using an industrial microfocus X-ray system was applied. Six animals (n=3 op/op; n=3 +/+) were anesthetized, and a catheter was inserted into the distal abdominal aorta. Hind limbs were perfused using 20 ml 0.9% NaCl (39°C) at 100 mmHg. A bismuth gelatine-based contrast agent adapted from a formula developed by Fulton [35
] was injected via the catheter (at 100 mmHg) under visual control until the distal arteries (plantar medial and lateral artery) were filled completely. Adenosine (6 mg/100 ml) was added to NaCl and the contrast medium to ensure maximal vasodilatation at all times. Animals were killed and immediately placed on crushed ice to allow the contrast agent to gel. Digital images were taken by means of an industrial microfocus X-ray system (portable clinical blood analyzer 160 with NanofocusTM tube, Phoenix X-ray Systems + Services GmbH, Wunstorf, Germany), which uses the principle of the X-ray shadow microscope [36
]. The system comprises a transmission-type open X-ray tube and an image chain with a 6-inch dual-field image intensifier and a 12-bit charged-coupled device camera (1000x850 pixels). The tube was operated at a voltage of 55 kV and a target current of 30 µA; the focal spot size of the tube was adjusted to 1.7 µm. The live image was averaged over 100 frames and electronically processed to optimize the contrast for the used contrast agent. As a reference for the artery diameters, a wire pentrameter W 13 CU (EN 462-1) was attached to the object, and direct measurements were performed by the motor increments of the computer numeric control-driven precision object stage of the system.
Hemodynamic measurements
Seven days after the initial surgery, animals (n=6 op/op; n=9 +/+) were reanesthetized, heparinized (500 IE/mouse), and placed in dorsal recumbency. Perfusion with fluorescent microspheres was performed as described previously [13
]. Modifications to optimize pressure and constant flow rate at all times were performed using a novel, computer-controlled system. Briefly, the abdominal aorta was cannulated cranially to the bifurcation with a polyethylene catheter (inner diameter: 0.58 mm; outer diameter: 0.96 mm). Both hind limbs were perfused with 0.9% NaCl, Tween 20 (0.1%), and adenosine (1 mg/kg body weight/min), using six differently labeled, 15-µm fluorescent microspheres (red, blue-green, orange, yellow-green, crimson, and scarlet, Molecular Probes). The perfusion buffer was heated to body temperature (39°C) and infused at six different systemic pressure levels (60, 70, 80, 90, 100, and 110 mmHg). Clotting of microspheres was prevented by an integrated mixing system. Calf muscles from both hind limbs were dissected carefully, homogenized, and digested. The number of microspheres in both hind limbs was counted using flow cytometry after addition of an internal standard microsphere label (Epics XL-MCL, Beckman-Coulter). Perfusion was expressed as percentage of perfusion restoration of the ligated compared with the unligated (normal) extremity, which served as an internal control.
Statistical analysis
All data are presented as mean ± SEM. Intergroup comparisons were performed using an unpaired Student’s t-test (SigmaStat). Probability values (P) of <0.05 were required for assumption of statistic significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.001 pm/ml) compared with wild-type mice, whereas heterozygote (op/+) mice showed no significant reduction of CSF-1 (140.07±9.0 pg/ml) compared with wild-type mice (140.9±10.71 pg/ml) as a result of the recessive mutation of the CSF-1-coding gene [22
, 23
].
op/op phenotype
Differences between op/op mice and wild-type controls during anesthesia were noted concerning higher sensitivity to toxic effects of isoflurane (respiratory depression) and to injectable agents (2 agonists, morphins, ketamin) and a longer recovery period. No delay in wound healing was noted, and clinical examination of op/op versus wild-type controls showed no significant difference concerning neurologic function between groups on Day 7. No animals were lost during or after femoral ligation. We also did not observe any gangrene or tissue necrosis after femoral artery occlusion. op/op mice, although subjected to decreased circulating, mononuclear cells, did not suffer from wound infection or opportunistic infects.
Peripheral blood count
Total leukocyte count revealed no significant differences between op/op and wild-type mice (leukocytes/µl; op/op: 5104±228; +/+: 5328±272). Further differentiation of the leukocyte subpopulations showed a significant reduction of circulating monocytes in op/op mice, 0.67% ± 0.38%, versus controls, 1.53% ± 0.32%. We could also observe a significant increase of granulocytes, 33.66% ± 6.67%, versus 22.83 ± 7.47% in wild-type mice, as well as a decrease of lymphocytes in op/op mice, 65.67% ± 6.58%, versus 75.65% ± 7.31% in control animals, and there was no significant difference in the percentage of T lymphocytes or thymocytes. This lymphopenia conforms to previous studies [21
, 28
]. Nevertheless, this is, to our knowledge, the first study to quantify accurately and specifically the number of the different leukocyte population by flow cytometry analysis in op/op mice (Fig. 1
).
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Because of various transgenic, mutant, and knockout strains, the mouse hind-limb model became an indispensable part of cardiovascular research. As a result of its small size, evaluation of collateral flow after femoral artery ligation is limited. In this study, we present two improved techniques to morphologically and functionally evaluate collateral flow. Angiography with conventional X-ray systems has considerable limitations in providing detailed images of small vessels with a diameter of less than 200 µm [40 , 41 ]. The use of an industrial nanofocus X-ray system was adjusted to 1.7 µm and allows precise identification of pre-existent anastomoses and developed collateral arteries in a two-dimensional (2D) and 3D view. As a result of the differences in the vascular network between ligated and control hind limb, pressure gradients and vascular resistance vary significantly. Therefore, the amount of infused contrast agent differs, making an impeccable assessment of collateral artery diameter impossible. Measurements of the arterial diameter thus have to be evaluated individually and in relation to the diameter of the femoral artery distal to the site of ligation.
Angiographically, op/op mice showed no difference in the total number of arteries, confirming our histological examinations. However, the number of collateral anastomoses showing the typical corkscrew appearance of growing collateral arteries indicating adaptive proliferation was significantly lower in the mutant mice (+/+: 6.33±1.15; op/op: 3.67±1.15; P<0.05). In addition, the diameter of the recruited collateral arteries was considerably smaller as compared with the control mice (Fig. 3 ).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CSF-1 levels in the serum of op/op mice were not detectable, whereas heterozygous mice (+/–) showed comparable levels as homozygous +/+ mice. This resulted in a significant reduction of circulating monocytes in op/op mice without affecting the total leukocyte count. As a consequence, monocyte invasion and subsequent perivascular macrophage accumulation were considerably lower in op/op mice. This was reflected by a reduced total number of tissue macrophages in general (data not shown) and the amount of macrophages in the perivascular tissue of growing collateral arteries in particular. Noteworthy is the fact that the ability to recruit monocytes/macrophages per se is not hampered significantly by the mutation, thus indicating a normal cell function [43 ] apart from the decreased production.
After femoral artery occlusion, perfusion restoration, depending on collateral artery growth, was inhibited significantly in op/op mice, correlating directly with the significantly reduced macrophage accumulation in the perivascular tissue of the mutant mice as compared with the genetic background. High-resolution angiography confirmed these results. As op/op mice still show a certain level of monocytopoiesis, the arteriogenic response was not abolished completely. Thus, this study provides additional evidence for the key role of monocytes/macrophages during early arteriogenesis, while angiogenesis has been described as not to be reduced in op/op mice [44 ]. This indicates that potential future therapies should focus on and account for the pivotal role of monocytes in the context of arteriogenesis.
In addition to the inability to maintain normal levels of circulating monocytes, we were also able to demonstrate a relative lymphopenia in favor of a relative granulocytosis, which is most likely a result of the approximately twofold, increased level of GM-CSF in op/op mice [23 ]. This, furthermore, indicates that increased GM-CSF levels are insufficient to induce an appropriate arteriogenic response if the target cell itself (i.e., the macrophage) is missing. Another conclusion that can be drawn from this observation is that granulocytes apparently do not contribute significantly to collateral artery growth, as their number was increased significantly in op/op mice. Conversely, one could speculate that the observed effects might be partly attributable to the concomitant lymphopenia, which is supported by previous reports investigating angiogenesis in a murine hind-limb ischemia model [45 , 46 ]. The model used in this study does not lead to ischemia, and as described previously, T lymphocyte-deficient, nude mice do not show a reduction of collateral artery growth [13 ]. Further, lymphocyte attraction did not improve arteriogenesis in a comparable rabbit hind-limb model, confirming that the observed inhibition of arteriogenesis was not a result of the lymphopenia in the op/op mice [47 ].
In this study, we used a novel technique to visualize arteriogenesis in the mouse hind limb. Applying the microfocus X-ray system with a spatial resolution of 1.7 µm, there was no difference in the total amount of arterial anastomoses between op/op and wild-type mice. This, once again, highlights that arteriogenesis is not a de novo formation of arteries but merely the enlargement of pre-existing connections. However, the diameter and tortuosity of collateral arteries were apparently lower in op/op mice as compared with wild-types. In particular, the number of arteries with the typical corkscrew appearance indicating the active proliferation of a collateral artery was considerably lower in the mutant mice. Although this technique allows a detailed look at the development of the collateral network, it is important to note that it cannot be an appropriate substitute for functional measurements but rather, visualizing morphological alterations and anatomical variations.
The small diameter of vessels in the mouse model (15–100 µm), the high number of collateral anastomoses, minimal deviations from symmetrical positioning, as well as different peripheral resistance (ligated vs. unligated extremity) result in different contrast agent-filling, which makes an intraindividual comparison impossible. It also has to be noted that according to the law of Hagen-Poiseuille, the vessel diameter contributes to blood flow in the fourth power. Even if it were possible to accurately measure the diameter of the complete collateral network, which is per se virtually impossible, the length of the vessel, blood viscosity, and the pressure gradient could not be assessed by this method, no matter how sensitive it is. When assessing an "angiographic score" by counting pixels of contrast-opacified vessels, as commonly used with standard techniques (spatial resolution of 200 µm) [48 ], arterial and venous filling cannot be distinguished. The latter occurs in the unligated extremity as a result of the higher resistance of the contralateral (ligated) side and because arteriovenous anastomoses are filled before small arteriolar vessels.
Thus, this method cannot replace functional parameters; however, when documenting collateral artery growth, more than one method is needed to assess morphological and functional parameters in all species (including humans). High-resolution angiographs under maximum vasodilatation can therefore be regarded as a useful, supportive method.
Previous studies have indicated that monocytes/macrophages represent the cellular key mediator in the process of collateral artery growth (arteriogenesis) in different organs. In this study, we show for the first time that arteriogenesis is reduced significantly in a nonpharmacologically induced setting of monocyte deficiency. Our results thus provide evidence for a direct correlation between collateral artery growth and the number of circulating monocytes.
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ACKNOWLEDGEMENTS |
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Received February 7, 2006; revised March 20, 2006; accepted March 28, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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