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(Circulation. 2001;104:203.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Detection of Monocyte Chemoattractant Protein-1 Receptor Expression in Experimental Atherosclerotic Lesions

An Autoradiographic Study

Katsuichi Ohtsuki, MD; Motoya Hayase, MD; Koichi Akashi, MD; Susan Kopiwoda, MS; H. William Strauss, MD

From the Division of Nuclear Medicine, Department of Radiology (K.O., S.K., H.W.S.), the Division of Cardiovascular Medicine, Department of Medicine (M.H.), and the Departments of Pathology and Developmental Medicine (K.A.), Stanford University School of Medicine, Stanford, Calif.

Correspondence to H. William Strauss, MD, Division of Nuclear Medicine, Stanford University Medical Center, Room H-0101, 300 Pasteur Dr, Stanford, CA 94305. billstra{at}leland.stanford.edu

	Abstract

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Background—— Monocytes, a common component of atheroma, are attracted to the lesion site in response to chemotactic signals, particularly expression of monocyte chemoattractant peptide 1 (MCP-1). This study assessed the feasibility of using radiolabeled MCP-1 to identify monocytes and macrophages that have localized at sites of experimental arterial lesions.

Methods and Results—— The biodistribution of radiolabeled MCP-1 was determined in normal mice, and localization in experimental atheroma was determined in cholesterol-fed rabbits 4 weeks after arterial injury of the iliac artery (9 rabbits) and the abdominal aorta (1 rabbit). Vessels were harvested and autoradiographed after intravenous administration of ¹²⁵I-labeled MCP-1 and Evans blue dye. The arteries were evaluated histologically by hematoxylin and eosin staining and immune staining with a monoclonal antibody specific for rabbit macrophages (RAM-11). ¹²⁵I-MCP-1 has a blood clearance half-time of 10 minutes and circulates in association with cells. The liver, lungs, and kidneys had the highest concentration of ¹²⁵I-MCP-1 at 5 and 30 minutes after tracer administration. Autoradiograms revealed accumulation of ¹²⁵I-MCP-1 in the damaged artery wall, with an average ratio of lesion to normal vessel of 6:1 (maximum 45:1). The accumulation of ¹²⁵I-MCP-1 in the reendothelialized (plaque formation) areas was greater than in the deendothelialized (Evans blue-positive) areas (6.55±2.26 versus 4.34±1.43 counts/pixel, P<0.05). The uptake of ¹²⁵I-MCP-1 correlated with the number of macrophages per unit area (r=0.85, P<0.0001).

Conclusions—— Radiolabeled MCP-1 may be a useful tracer for imaging monocyte/macrophage-rich experimental atherosclerotic lesions.

Key Words: monocyte chemoattractant proteins • atherosclerosis • macrophages • autoradiography

	Introduction

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Coronary atherosclerosis ranges from lesions that are likely to rupture, vulnerable¹ plaque, to stable, calcified lesions. Vulnerable plaque is characterized by a thin cap, a necrotic core (associated with lakes of lipid), and a marked cellular infiltration containing monocytes, macrophages, and foam cells.² Monocytes are attracted to sites of arterial injury by several substances, such as monocyte chemoattractant protein-1 (MCP-1),³ interleukin 1, tumor necrosis factor-, and interferon-,^4,5 that are produced by cells at sites of inflammation. Monocytes migrating to the site of cytokine production enter the lesion, transform into macrophages, and remain in the lesion. In arterial lesions, macrophages may progress to foam cells by accumulating lipoprotein cholesterol through scavenger receptor-mediated endocytosis.⁶

MCP-1, a monomeric polypeptide with a weight of 9 to 15 kDa,^7,8 is produced by endothelial cells,^4,9 smooth muscle cells,^10,11 and monocytes/macrophages.^8,12 The peptide is recognized by CCR-2 receptors expressed on monocytes.¹³ MCP-1 is a potent monocyte chemoattractant and is largely responsible for the recruitment of monocytes/macrophages to the vessel wall in the process of atherogenesis.¹¹

We hypothesized that lesions with high concentrations of macrophages could be identified by labeling of cells that were already attracted to the site of inflammation. Because activated macrophages express a high concentration of chemotactic receptors on their surface, we selected radiolabeled MCP-1 (¹²⁵I-MCP-1) to test this hypothesis.¹ (MCP-1 is available as a research peptide from a number of vendors, including Peprotech. The material can also be obtained as iodinated MCP-1 from the New England Nuclear Co.)

	Methods

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Three sets of experiments were performed: First, the biodistribution of radioiodinated MCP-1 was determined in normal mice. Second, the time course of blood clearance and the partition of activity between cells and plasma was determined in a rabbit after intravenous injection of MCP-1. Third, localization of radiolabeled MCP-1 was determined by autoradiography in rabbits fed an atherogenic diet who had sustained a balloon deendothelialization injury.

Mouse Biodistribution
One microcurie (37 kBq) of ¹²⁵I-labeled recombinant human MCP-1 (New England Nuclear Co, 1 pmol [13 ng]) of peptide was injected intravenously into 20-g male BALB/c mice. Mice were killed at 5, 30, 60, and 180 minutes after administration (n=6 per time point). Samples of blood, thymus, lungs, heart, thyroid, lymph node, abdominal aorta, vena cava, liver, spleen, kidneys, and stomach were harvested, rinsed in saline, weighed, and counted in a well-type scintillation counter (Packard Cobra II, baseline 15 keV, upper level 75 keV). The results were expressed as percent injected dose per gram tissue (% ID/g) and percent injected dose per organ (% ID/organ).

Blood Clearance and Plasma/Whole Blood Partition of ¹²⁵I-MCP-1 in a Rabbit
The blood clearance and plasma/cell partition of MCP-1 was determined to define an optimal time to sample the vessels in the lipid-fed rabbits. Thirty microcuries of ¹²⁵I-labeled MCP-1 was injected intravenously into an anesthetized rabbit, and blood samples (0.2 mL) were collected from the contralateral ear over a period of 3 hours. Radioactivity of whole blood and plasma was measured with a gamma-well counter and expressed as percent injected dose per gram.

Experimental Atherosclerotic Lesions
Nine male New Zealand White rabbits weighing 3.0 to 3.4 kg (Charles River Breeding Laboratories, Wilmington, Mass) were maintained on a high-cholesterol diet (1% cholesterol, Dyets Inc) for 5 weeks. After 1 week on the diet, 1 iliac artery of all 9 rabbits and the aorta of 1 (to determine whether the pattern of localization would be similar in vessels of different caliber) were injured.¹⁴ Animals were anesthetized with ketamine (3 mg/kg) and xylazine (5 mg/kg), a 5F sheath was inserted under fluoroscopy into the right carotid artery, and a guidewire was passed to the iliac artery. An angioplasty balloon (3 mm; Advanced Cardiovascular Systems) was advanced into the iliac artery, inflated to 8 atm, and pulled back to the abdominal aorta 3 times. In addition to the iliac artery, the aorta of 1 rabbit was injured in a similar fashion, with a balloon inflated to 10 atm in the proximal iliac vessel and pulled back to the diaphragm. The carotid artery was ligated, and the wound was closed. The experimental protocols were approved by the Administrative Panel on Laboratory Animal Care of Stanford University.

After 4 additional weeks of diet, 45 µCi (1.67 MBq) of ¹²⁵I-labeled MCP-1 was injected intravenously, followed 2 hours later by 4 mL of a 0.5% solution of Evans blue dye (Sigma) to stain the deendothelialized aorta. One hour later (3 hours after ¹²⁵I-MCP-1), the animals were killed with an overdose of sodium pentobarbital. The distal aorta and both iliac arteries were removed, perfused with 10% buffered formalin (Sigma) under a pressure of 100 cm H₂O, opened longitudinally, and mounted on Styrofoam blocks. The vessels were photographed for subsequent analysis of the sites of deendothelialization and plaque formation.

At the time of death, samples of blood, heart, lung, liver, spleen, and kidney were obtained for scintillation well counting. All samples were weighed and counted in a gamma counter. The data were expressed as ID/g.

Morphometric Analysis (Intima/Media Ratio)
Samples of deendothelialized (Evans blue-positive), reendothelialized (appear lighter than normal vessel, plaque formation), and uninjured areas were biopsied (3 samples from each artery), embedded in paraffin, sectioned into 5-µm-thick slices, and stained with hematoxylin and eosin for light microscopy and histomorphometry. The sections were scanned with x4 magnification and digitized with the Image Analyst Program (Automatix). The external and internal elastic lamina and lumen/intima border were identified, and an intima/media ratio was calculated. The intima was defined as the vessel layer between the internal elastic lamina and the intima/lumen border, and the media was defined as the area between the internal and external elastic laminae. Three regions were measured for each sample.

Immunohistochemistry
Tissue slices adjacent to those used for hematoxylin and eosin staining were stained with an antibody specific for rabbit macrophages (RAM-11, Dako). Sections were incubated with primary antibody, anti-rabbit IgG secondary antibody (biotin conjugate), and avidin peroxidase for 20 minutes. Peroxidase was visualized with chromagen (Zymed Laboratories Inc). Three cross sections were immunostained for each tissue. RAM-11-positive cells in the intima and media were counted under x100 magnification in 4 to 6 regions and expressed as the number per 0.25 mm².

Autoradiography
The vessels were covered with 1 layer of plastic (Saran) wrap, placed on x-ray film (Kodak Diagnostic Film Min-R), and stored for 10 weeks in a Kodak Min-R2 cassette. The autoradiographs were developed, scanned (300 DPI, 256 gray shades), and quantified by use of the public domain NIH Image program. Regions of interest (4 to 6 mm²) were set on the denuded, plaque, and uninjured areas and background (6 regions each) as for morphometric and immunohistochemical analysis. The relative density of the injured vessel area and uninjured area (counts/pixel) was obtained after background correction. The accumulation ratio of tracer in each injured area was defined as the ratio of its regional density to the mean density of 4 uninjured regions.

Statistical Analysis
Results were expressed as mean±SD. Comparisons between 2 groups were made with a 2-tailed Student’s t test for paired data. Variables among 3 groups were compared by ANOVA with Scheffé’s post hoc teSt. The accumulation ratio of ¹²⁵I-MCP-1 was correlated with the number of macrophages by linear regression. A value of P<0.05 was considered statistically significant.

	Results

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Biodistribution of ¹²⁵I-Labeled MCP-1 in Mice
The tissue distribution of ¹²⁵I-labeled MCP-1 is summarized in Table 1 (% ID/g) and Table 2 (% ID/organ). The blood clearance half-life is 10 minutes (% ID/organ). Blood activity decreased to 9.70% ID/g at 60 minutes and 2.86% ID/g at 180 minutes. On the basis of ID/organ, liver, lungs, and kidneys had the highest concentration at 5 and 30 minutes after tracer administration. At 60 minutes, lymph nodes (% ID/g) had the 3 higheSt. At 180 minutes, the blood and small intestine had the highest concentration (% ID/organ).

View this table: [in this window] [in a new window]   Table 1. Biodistribution (% ID/g) of 125I in Mice

View this table: [in this window] [in a new window]   Table 2. Biodistribution (% ID/Organ) of 125I MCP-1 in Mice

Blood Clearance of ¹²⁵I-MCP-1 in a Rabbit
Blood clearance of ¹²⁵I-labeled MCP-1 in the rabbit is similar to that in the mouse, with a half-life of <20 minutes. The concentration of radioactivity (% ID/g) in the blood was 0.39% at 1 minute after the administration of ¹²⁵I-MCP-1 and decreased to 0.10% at 60 minutes and 0.05% at 180 minutes. The plasma concentration was 0.05% at 1 minute after injection and decreased to 0.02% at 60 minutes and 0.01% at 180 minutes. The concentration of ¹²⁵I-MCP-1 in whole blood ranged from 4 to 7 times greater than that in plasma (Figure 1), suggesting that ¹²⁵I-MCP-1 is bound to cells.

View larger version (40K): [in this window] [in a new window]   Figure 1. Blood clearance of 125I-labeled MCP-1. Whole-blood counts per gram (solid bars), and plasma counts per gram (open bars) were plotted vs time after injection. Error bars are 1 SD. Hematocrit was 45%. Concentration of radioactivity (% ID/g) in blood was 0.39% at 1 minute after administration of 125I-MCP-1 and decreased to 0.10% at 60 minutes and 0.05% at 180 minutes.

Distribution of ¹²⁵I-MCP-1 in the Injured Arteries of Rabbits
Plaque areas had significantly higher ID/g than uninjured areas of the vessel (0.12±0.04 versus 0.02±0.006, P=0.0002) (Figure 2, bottom left) and blood (0.04±0.01% ID/g, P=0.0005). The maximal ratio of plaque to blood radioactivity was 7.08. The distribution of ¹²⁵I-labeled MCP-1 in rabbit tissue 3 hours after administration is summarized in Table 3. The kidneys had the highest concentration of radiolabel (0.94±0.13% ID/g), significantly higher than that of plaque (P<0.0001). The spleen had the second highest concentration (0.04±0.007% ID/g), which was lower than that of plaque (P=0.0005).

View larger version (52K): [in this window] [in a new window]   Figure 2. Photo compares iliac arteries stained with Evans blue dye (A) with corresponding autoradiograph (B) and immune-stained sections (C, center column) and hematoxylin-eosin sections (right column) taken from a control site (1) and a denuded area (2) and plaque area (3). I indicates intima; M, media. Most pronounced areas of radioactivity correspond to reendothelialized (plaque) region (white area; 3), which had highest concentration of RAM-11-stained cells. Middle level of radioactivity corresponded to deendothelialized area (blue area; 2). Areas of lowest activity corresponded to noninjured areas (white area; 1). Bottom left, Activity per gram in uninjured and plaque region of artery. Error bars are 1 SD. Bottom center, Average number of macrophages per 0.25 mm2 in denuded and plaque regions. Accumulation of 125I-MCP-1 in reendothelialized areas was greater than that in deendothelialized area (6.55±2.26 vs 4.34±1.43 counts/pixel, P<0.05). Error bars are 1 SD. Bottom right, Relationship of 125I-MCP-1 lesion/normal accumulation ratio to number of macrophages per unit area (r=0.85, P<0.0001).

View this table: [in this window] [in a new window]   Table 3. Biodistribution of 125I MCP-1 at 3 Hours After Administration

Intima/Media Ratio
The medial layer was similar in the intact, deendothelialized, and reendothelialized lesions. Plaque had higher intima/media ratios (2.92±1.42, n=8) than denuded (1.51±0.33, n=8) and uninjured (0.03±0.06, n=8) areas (P<0.02 and P<0.0001, respectively). The intima/media ratio in the denuded areas was significantly higher than that in the intact areas (P<0.02) (Figure 3).

View larger version (29K): [in this window] [in a new window]   Figure 3. Plot of intima/media ratio in uninjured regions, zones of denuded endothelium, and regions of plaque (reendothelialized regions). Reendothelialized lesions showed higher intima/media ratio (2.92±1.42) than deendothelialized lesions (1.51±0.33) and intact areas (0.03±0.06). *P<0.02, P<0.0001. Error bars are 1 SD.

Immunohiostochemistry
Cells staining with RAM-11 were significantly greater in plaque (n=8) than denuded (n=8) areas (29.81±10.59 versus 17.03±10.10, P<0.05) (Figure 2, photo, center column and graph, center); few RAM-11-positive cells were observed in intact areas. RAM-11-positive cells were detected in both the media and intima of plaque regions.

Autoradiography
In the injured regions, 3 levels of radioactivity were seen (Figure 2A, and autoradiograph, B): (1) areas of regenerating endothelium, which did not stain with Evans blue dye (appear pale, marked as 3 on the photo and autoradiograph), had the most intense accumulation of ¹²⁵I-MCP-1 (regions of plaque); (2) deendothelialized regions, which stain with Evans blue, corresponded to areas of moderate radioactivity (denuded areas, marked as 2 on the photo and autoradiograph); and (3) noninjured areas, which did not stain with Evans blue (marked as 1 on the photo and autoradiograph), had faint accumulation of radioactivity. Although there was some variation in the intensity of activity among different animals or different reendothelialized or deendothelialized regions, a similar pattern of ¹²⁵I-MCP-1 distribution was seen in all animals.

The maximum lesion:normal vessel ratio of ¹²⁵I-MCP-1 was 44.8:1. The accumulation of ¹²⁵I-MCP-1 in the reendothelialized areas was greater than in the deendothelialized area (6.55±2.26 versus 4.34±1.43 density per pixel, P<0.05). The accumulation of ¹²⁵I-MCP-1 correlated (r=0.85, P<0.0001) with the number of macrophages per unit area (Figure 2, graph, right).

	Discussion

Top Abstract Introduction Methods Results Discussion Conclusions References

 
These data suggest that radiolabeled MCP-1, a cytokine for monocytes and macrophages, localizes in experimental arterial lesions induced by balloon deendothelialization and a 1% cholesterol diet in the rabbit. Sites of regenerating endothelium correlated with the highest ¹²⁵I-MCP-1 and macrophage localization. MCP-1 activity in the lesions was greater than that in the blood and far greater than that in the normal arterial wall.

The ratio of whole blood to plasma suggests that the majority of circulating peptide is associated with cells. Cellular association may also explain the transient pulmonary uptake of ¹²⁵I-MCP-1. The "first-pass" presentation of MCP-1 to the alveolar macrophages is at a relatively higher concentration than after hemodilution. These cells express receptors for this peptide that bind transiently because the number of receptors is not upregulated. The peptide clears slowly from the uninflamed lungs. But in atheroma, the number of receptors is markedly upregulated, causing both a high initial uptake and prolonged retention of the peptide at the lesion site.

Many radionuclide imaging approaches have been advocated to detect atherosclerotic lesions. Previous publications describe the use of radiolabeling: autologous lipoproteins^15–18; platelets and platelet-specific antibodies^19–21; agents that bind to activated glycoprotein IIb/IIIa receptors expressed on activated platelets²²; an adenine nucleotide analogue, Ap₄A, directed at ADP association sites on platelets²³; fibrinogen^24,25; and an IgM antibody^26,27 that recognizes proliferating arterial smooth muscle cells found in active plaque. Some of these agents, such as LDL, platelets, and the antibody recognizing proliferating smooth muscle (Z2D3), have been tested in humans. The agents had some success identifying lesions in the carotid arteries but have not identified atheroma with a sufficient sensitivity to be clinically useful. None of these agents have demonstrated disease in coronary vessels. These studies demonstrate the remarkably dynamic nature of atheroma, however, because autologous LDL localized in human carotid lesions within hours of administration.¹⁷ This finding is confirmed by the localization of MCP-1 in the lesion within 180 minutes of injection.

It is not clear that 180 minutes is an optimum time to detect atheroma, however, because lesion kinetics were not evaluated in this study. A short interval between injection and sampling minimizes the opportunity for dissociation and metabolism of the peptide at the receptor site, whereas a longer interval allows more clearance of peptide from background structures, such as the lungs and blood. On the basis of these factors, the 3-hour interval was selected.

An average ratio of lesion to normal vessel of >6:1 was observed, which was substantially greater than residual activity in the blood. These data suggest that external radionuclide imaging of macrophage-rich arterial lesions may be feasible with radiolabeled MCP-1 when the agent is labeled with either ¹²³I or ^99mTc.

In the present study, external imaging with a gamma camera was not attempted because of the low energy of ¹²⁵I and the extremely low dose of ¹²⁵I-MCP-1.

Identifying vulnerable plaque is clinically important because these lesions are more likely to erode or rupture the fibrous cap, resulting in acute coronary occlusion.^28–31 Although myocardial perfusion imaging is widely used to detect ischemic lesions, the location of ischemia on a stress perfusion scan has a limited correlation with the location of a subsequent infarction.^32,33 Similarly, serial coronary arteriograms demonstrate populations of lesions that undergo gradual but continuous progression over time³⁴ and lesions that may remain stable for long intervals. Progression of atheroma often occurs in lesions with a high concentration of monocytes and macrophages.

These observations suggest that quantitative evaluation of macrophages in lesions may be useful to identify those lesions with a high risk of plaque rupture. The present study suggests radiolabeled MCP-1 as a candidate radiotracer to assess plaque vulnerability on the basis of the expression of MCP-1 receptors on macrophages that have localized at the lesion site. Large lesions located in superficial vessels may be detectable by external radionuclide imaging. The small size of many coronary plaques, the depth of the heart, and the fact that it is in motion, however, make external imaging of these lesions challenging with today’s technology. To detect small atheroma, it may be necessary to place the detector close to the plaque,³⁵ which will require an intravascular radionuclide detection device.

	Conclusions

Top Abstract Introduction Methods Results Discussion Conclusions References

 
¹²⁵I-MCP-1 localizes in macrophage-rich experimental atherosclerotic lesions in vivo.

	Acknowledgments

 
Dr Ohtsuki was supported in part by a generous grant from the Uehara Memorial Foundation.

Received October 30, 2000; revision received March 14, 2001; accepted March 15, 2001.

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Top Abstract Introduction Methods Results Discussion Conclusions References

 

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