Published online before print January 2, 2008, doi: 10.1161/CIRCULATIONAHA.107.719765
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(Circulation. 2008;117:388-395.)
© 2008 American Heart Association, Inc.
Imaging |
Noninvasive In Vivo Imaging of Monocyte Trafficking to Atherosclerotic Lesions
From the Center for Molecular Imaging Research (M.F.K., J.G., F.K.S., R.E.G., J.R.A., R.W.), Massachusetts General Hospital and Harvard Medical School, Charlestown, Mass; Center for Systems Biology (R.W.), Massachusetts General Hospital and Harvard Medical School, Boston, Mass; Cardiovascular Division (F.K.S., P.L.), Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass; Cardiovascular Research Center (R.E.G.), Massachusetts General Hospital and Harvard Medical School, Charlestown, Mass; and Donald W. Reynolds Cardiovascular Clinical Research Center on Atherosclerosis at Harvard Medical School (F.K.S., P.L., R.E.G., R.W.), Boston, Mass. Dr Kircher is currently with the Department of Radiology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Mass; Dr Grimm is currently with the Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY; and Dr Allport is currently with the Novartis Institutes for BioMedical Research Inc, Cambridge, Mass.
Correspondence to Ralph Weissleder, MD, PhD, Center for Systems Biology, Massachusetts General Hospital, Richard B. Simches Research Center, 185 Cambridge St, 8th Floor, Boston, MA 02114. E-mail rweissleder{at}mgh.harvard.edu
Received June 11, 2007; accepted October 30, 2007.
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Abstract |
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Background— Monocytes play a key role in atherogenesis, but their participation has been discerned largely via ex vivo analyses of atherosclerotic lesions. We sought to establish a noninvasive technique to determine monocyte trafficking to atherosclerotic lesions in live animals.
Methods and Results— Using a micro–single-photon emission computed tomography small-animal imaging system and a Food and Drug Administration–approved radiotracer ([indium 111] oxyquinoline, 111In-oxine), we demonstrate here that monocyte recruitment to atherosclerotic lesions can be visualized in a noninvasive, dynamic, and 3-dimensional fashion in live animals. We show in vivo that monocytes are recruited avidly to plaques within days of adoptive transfer. Using micro–single-photon emission computed tomography imaging as a screening tool, we were able to investigate modulatory effects on monocyte recruitment in live animals. We found that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors rapidly and substantially reduce monocyte recruitment to existing atherosclerotic lesions, as imaged here in vivo.
Conclusions— This novel approach to track monocytes to atherosclerotic plaques in vivo should have broad applications and create new insights into the pathogenesis of atherosclerosis and other inflammatory diseases.
Key Words: imaging • atherosclerosis • plaque • cells
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Introduction |
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In the past 2 decades, the concept of atherogenesis has changed dramatically from the notion of a lipid storage disease to the recognition that inflammation drives this process.1–5 Substantial evidence identifies monocytes as key cellular participants in the initiation and development of atherosclerotic plaques.5 Adhesion of monocytes to the arterial endothelium and their migration into the intima occur early in atherogenesis.6 Monocyte accumulation characterizes fatty streaks, the earliest grossly detectable lesion of human and experimental atherosclerosis.2 Monocytes differentiate into macrophages7 and secrete inflammatory cytokines that stimulate smooth muscle cell proliferation and migration8 and produce proteolytic enzymes that can degrade collagen and render the growing plaque’s cap thin and susceptible to rupture.1,9
Clinical Perspective p 395
This key role of monocytes in all phases of atherogenesis highlights the need to understand better the dynamics of monocyte recruitment to developing plaques and to establish clinically viable imaging tools to visualize this process in high-risk patient groups or in the development and testing of novel therapies. To date, the data describing monocyte recruitment to atherosclerotic plaques have derived mainly from excised tissue specimens (eg, in experimental animals or endarterectomy or autopsy samples) that have undergone analysis either histologically,10 by polymerase chain reaction methods,11,12 or by flow cytometry.7 Thus, the analysis of monocyte trafficking to lesions has often been restricted to a static view that does not reflect the dynamics of this process. The ability to assess recruitment of monocytes in vivo would be of benefit in advancing our understanding of atherogenesis, evaluating antiinflammatory drugs designed to inhibit monocyte recruitment, and stratifying patients at risk for developing complications of atherosclerosis.
Here, we present an approach that enables noninvasive, dynamic, quantitative and high-resolution in vivo imaging of monocyte trafficking to atherosclerotic lesions. We demonstrate the feasibility of detection of the recruitment of monocytes labeled with [indium 111] oxyquinoline (111In-oxine) to existing atherosclerotic lesions of apolipoprotein E–deficient (ApoE–/–) mice by micro–single-photon emission computed tomography (micro-SPECT/CT). The long half-life of 111In (2.8 days) enabled the detection of monocytes for up to 7 days after adoptive transfer, and the high-resolution anatomic data derived from CT allowed localization of hotspots of monocyte infiltration in a submillimeter range. Furthermore, we demonstrate that micro-SPECT/CT imaging allows for assessment of the effect of an intervention (statin drugs) on monocyte recruitment to plaques in vivo. This technique may open new avenues to investigate the mechanisms of atherogenesis and other inflammatory diseases noninvasively and to evaluate new antiinflammatory therapies effectively. Importantly, with both human SPECT/CT scanners and the Food and Drug Administration–approved 111In-oxine in clinical use, the results of the present study support the potential to conduct similar studies in humans in the future.
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Methods |
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Materials
RPMI 1640, HBSS, and FBS were purchased from Cellgro (Herndon, Va). PBS was obtained from BioWhittaker (Walkersville, Md). 111In-oxine was purchased from Amersham-GE (Piscataway, NJ). Ultravist iodinated contrast agent for CT imaging was obtained from Berlex (Richmond, Calif).
Antibodies
The primary polyclonal anti-GFP (green fluorescent protein) antibody was purchased from Chemicon International (Temecula, Calif; catalog No. AB3080).
Animals
ApoE–/– mice (B6.129P2-Apoetm1Unc), enhanced GFP (EGFP)-expressing mice [C57BL/6-Tg(ACTB-EGFP)1Osb/J], and C57BL/6 mice (6 to 8 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, Me). The ApoE–/– animals had been backcrossed to the C57BL/6 background for at least 10 generations. Mice were cared for according to the institution’s animal facility guidelines. At 4 weeks of age, ApoE–/– animals were placed on a Western diet (42% adjusted caloric diet, 0.2% cholesterol, Harlan Teklad, Madison, Wis) and fed until used for experiments. The remaining animals remained on a regular chow diet. All protocols were approved by the Animal Review Committee.
Statin Treatment
ApoE–/– animals in the treatment group received simvastatin (oral, 0.57 mg · kg–1 · d–1, Zocor, Merck, Whitehouse Station, NJ) and atorvastatin (oral, 0.57 mg · kg–1 · d–1; Lipitor, Pfizer, New York, NY) dissolved in 100 µL of PBS via intragastric instillation for 3 days by mouth (0.57 mg/kg corresponds to a 40-mg dose used clinically in humans [70 kg body weight]; recommended atorvastatin dose in humans is 10 to 80 mg/d). Mevastatin (Sigma, St Louis, Mo) was administered subcutaneously (20 mg · kg–1 · d–1, according to Budzyn et al13) dissolved in 20 µL of PBS. Control animals received PBS only via intragastric or subcutaneous instillation, respectively.
Measurement of Cholesterol Levels
Total blood cholesterol levels of atorvastatin-treated mice and nontreated control mice were measured with a Raichem (San Diego, Calif) cholesterol reagent kit according to the protocol of the manufacturer.
Monocyte Isolation
We used a 2-step combination of density gradient separation and negative immunomagnetic depletion to isolate murine monocytes from the peripheral blood, as described by Swirski et al,14 with minor modifications. In brief, peripheral blood (1 to 1.3 mL) was drawn from C57BL/6 mice (typically n=15 mice/isolation) via cardiac puncture, diluted 5:1 with HBSS, and subjected to density centrifugation (30 minutes, 1600 rpm, room temperature) over Histopaque-1083 (Sigma). The buffy coat containing peripheral blood mononuclear cells (PBMCs) was aspirated, washed 3 times in HBSS, and subsequently washed once in MACS buffer (PBS containing 0.1% [wt/vol] BSA and 0.5 mmol/L EDTA). T cells, B cells, and natural killer cells were depleted from the PBMC population with a cocktail of immunomagnetic beads (anti-CD90, anti-CD45R, and anti-CD49b beads; Miltenyi Biotec, Auburn, Calif). Monocyte purity was determined by morphological analysis of cells prepared by cytospin and Wright-Giemsa staining, as well as independently via fluorescence-activated cell sorter analysis.
Monocyte Labeling, Viability, and Function
Labeling
Purified monocytes were labeled with 111In-oxine according to the manufacturer’s protocol (Amersham Health Medi-Physics, Arlington Heights, Ill). Briefly, cells were washed with HBSS, spun, and resuspended in 111In-oxine for 20 minutes at 37°C, pH 6.5 to 7.5. The cells were then washed 3 times with HBSS.
Viability
Potential cytotoxic effects of 111In-oxine on monocyte viability were assessed by Trypan blue exclusion staining.
Function
We used a model of thioglycolate-induced sterile peritonitis modified after that of Segal et al.15 Sterile thioglycolate medium (1 mL) was injected intraperitoneally into C57BL/6 mice (n=3). After 24 hours, 3x106 monocytes were isolated as described and divided into 2 batches. Batch 1 was labeled with 111In-oxine (5 pCi/cell) and subsequently colabeled with the indocyanine dye DiO (Molecular Probes, Carlsbad, Calif). Batch 2 was incubated with PBS only and subsequently labeled with the dye DiD (Molecular Probes). Batches 1 and 2 were then mixed together, and 106 cells of the mixture were injected via tail vein into each of the 3 recipient mice with peritonitis. Again 24 hours later, animals were euthanized and peritoneal lavages performed. The recovered cells were analyzed via fluorescence-activated cell sorter analysis in the FL1 channel (to detect DiO+/111In-oxine–labeled monocytes) and the FL4 channel (to detect DiD+/mock-labeled monocytes).
Micro-SPECT/CT Imaging and Postprocessing
For in vivo SPECT/CT imaging, monocytes were isolated as described and labeled with 111In-oxine. For selected experiments, PBMCs or PBMCs without monocytes were also used for experiments. Recipient ApoE–/– or C57BL/6 wild-type mice, respectively, were placed under general isoflurane inhalation anesthesia, and 3x106 cells were injected via tail vein. The total amount of activity injected into each animal was measured with a radioisotope calibrator (Capintec, Ramsey, NJ) immediately after injection and before imaging time points to account for differences in injected/excreted activity. SPECT/CT imaging was performed with an integrated high-resolution micro-SPECT/CT imaging system (X-SPECT; Gamma Medica Inc, Northridge, Calif), which combines SPECT and CT on 1 gantry, allowing for immediate coregistration of the SPECT and CT data sets by the scanner’s software without additional postprocessing. For imaging, mice were anesthetized by isoflurane inhalation, with parameters set so that the animals were breathing shallowly to reduce artifacts from respiratory movements. The animals were scanned repeatedly from 30 minutes after injection to up to 10 days after adoptive transfer. For quantitative analysis, an imaging time point of 5 days was used, because activity in the blood interfering with the results was negligible by this time. Average imaging time was 30 minutes and was adjusted when needed to correct for differences in activity between animals.
Ultravist 300 was injected via pump (60 µL/min) over the 10-minute CT scans for vascular contrast. In select experiments, animals were euthanized, and contrast was infused via syringe into the left ventricle. The ascending aorta, aortic arch, descending (thoracic) aorta, and descending (abdominal) aorta were segmented out along with muscle background by use of the contrast-enhanced CT images with Amira software (Mercury Computer Systems, Inc, Chelmsford, Mass). The regions of interest from the CT images were propagated to the registered SPECT images, and the mean value of each region of interest was tabulated. These mean values of each aortic region of interest were divided by the mean muscle region of interest to give a signal-to-noise ratio. Three-dimensional (3D) rendering was performed with Amira software on a high-end PC workstation.
Autoradiography
Mice were euthanized and aortas excised and exposed on a Phosphorimager SI (Molecular Dynamics, Sunnyvale, Calif) for 24 hours. 111In-oxine standards were exposed together with the specimen on the same plate, which allowed for calibration and quantitative data analysis by region-of-interest analysis with ImageJ software (National Institutes of Health, Bethesda, Md) as described previously.16 Background activity measured in C57BL/6 mice was subtracted from activity measured in ApoE–/– mice.
Immunohistochemistry
Monocytes were isolated from EGFP-expressing mice (The Jackson Laboratory) and adoptively transferred into 20-week-old ApoE–/– mice. EGFP was used for its antigenic rather than its fluorescent properties. Five days after transfer, animals were euthanized and aortas excised, frozen in OCT compound, and cut into 5-µm sections in a cryostat. After sections were dried at room temperature for 20 minutes, they were treated with 0.3% hydrogen peroxide to inhibit endogenous peroxidase activity and were incubated for 30 minutes in blocking solution. Adjacent sections were then incubated with primary polyclonal anti-GFP (Chemicon, catalog No. AB3080) or rat anti-mouse Mac-3 antibodies diluted in PBS supplemented with 4% of the species-respective normal serum (GFP 1:25; Mac-3 1:50). After sections were washed with PBS, species-appropriate secondary antibodies were applied, followed by avidin-peroxidase complex (Vectastain ABC kit; Vector Laboratories, Burlingame, Calif). Slides were rinsed in PBS after each incubation step. The reaction was visualized with 3-amino-9-ethyl carbazole substrate (AEC; Sigma). Sections were counterstained with Gill’s hematoxylin solution (Sigma) and mounted.
Statistical Methods
To test for differences between treatment groups, 1-way ANOVA with Tukey multiple-comparison test was computed with GraphPad Prism software (GraphPad Software, San Diego, Calif). With small sample sizes and an unbalanced design, the data may not meet ANOVA assumptions, and therefore, caution is required in interpretation. To test for correlation between monocyte numbers and SPECT signal, Pearson’s product-moment correlation coefficient was calculated with Excel (Microsoft, Redmond, Wash).
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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Monocyte Isolation and Effect of 111In-Oxine Labeling on Monocyte Function
We isolated total murine monocytes using a recently described 2-step negative-depletion method.14 Figure 1A shows representative morphological examples of eluates containing monocytes and the fraction depleted during magnetic separation. Monocyte purity was typically >90% (90.8±3.0%, mean±SD), with most contaminating cells being lymphocytes (7.4±0.9%). Correspondingly, the fraction containing the magnetically depleted cells contained mostly lymphocytes (92.2±4.9%; Figure 1B).
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Labeling of monocytes with 111In-oxine did not affect their overall viability, as shown by Trypan blue exclusion staining (Figure 1C). Cell migration to the peritoneal cavity was used to determine a potential effect of 111In-oxine on the migratory capability of monocytes in vivo. We tagged 111In-oxine–labeled versus nonlabeled monocytes with different cell-tracker dyes, injected a 1:1 mixture intravenously to mice with thioglycolate-induced peritonitis, and assessed the number of monocytes that accumulated in the peritoneal cavity. Monocytes accumulated in the peritoneum, and no statistical difference was present between 111In-oxine–labeled and nonlabeled cells (Figure 1D).
Noninvasive Imaging of Monocyte Trafficking to Atherosclerotic Plaques by Micro-SPECT/CT
Initial in vivo experiments used monocytes isolated from the peripheral blood (n=15) of C57BL/6 wild-type donor mice and adoptively transferred 3x106 monocytes to recipient mice. We serially imaged the recipient animals from 30 minutes until 10 days after the adoptive transfer to determine the timeline for redistribution of monocytes (Figure 1E, and data not shown). Within the first day (0 to 24 hours), a positive signal, which corresponded to labeled monocytes, was detected in heart and lungs. After 36 hours, monocyte distribution had shifted toward liver and spleen, with less activity seen in the heart and lungs. At the 5-day time point, no residual activity was present in the heart and lungs, and a focal hotspot in the ascending aorta now appeared. Histological analysis showed that areas of SPECT/CT activity corresponded to areas of abundant monocyte recruitment. Because murine monocytes do not express a distinct marker that allows for specific antibody staining, GFP-expressing monocytes were transferred in these experiments to allow for detection with anti-GFP antibodies (Figure 1F). The 5-day time point was used in the remaining experiments, because it allows for detection of sufficient remaining radioactive signal while minimizing background signal from monocytes in the circulation. We next tested whether the signal detected in aortas was specific for monocytes (Figure 2). We transferred either monocytes or PBMCs that did not contain monocytes (depleted fraction during negative isolation) into ApoE–/– mice. A second control used injection of monocytes into C57/BL6 wild-type mice. As shown in the CT (Figure 2A) and SPECT/CT overlays (Figure 2B), 111In-oxine–labeled monocytes were recruited to the ascending part of the aorta of ApoE–/– mice. Other parts of the central arterial tree did not show substantial monocyte recruitment. Autoradiography of excised aortas verified the localized recruitment to the ascending aorta (Figure 2C). In contrast, neither PBMCs depleted of monocytes (which consisted mainly of T and B lymphocytes) injected into ApoE–/– mice nor monocytes injected into C57BL/6 wild-type mice demonstrated substantial recruitment to the aorta. Magnified views and a 3D rendering of the 2D SPECT/CT data set of monocyte transfer into ApoE–/– mice are shown in Figure 3 and in Movie I in the online-only Data Supplement.
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To investigate the relationship between the in vivo SPECT signal and the actual numbers of 111In-labeled monocytes in the aortas, we estimated monocyte numbers on the basis of autoradiography data of excised aortas obtained after performance of in vivo SPECT imaging. The analysis showed a strong linear correlation between the in vivo SPECT signal and monocyte content, with an R2 of 0.87 (online-only Data Supplement Figure I).
Statin Treatment of ApoE–/– Mice Rapidly Reduces Acute Monocyte Recruitment to Existing Plaque
Having established SPECT/CT imaging as a tool to assess cellular recruitment to atherosclerotic lesions, we extended our studies to investigate whether a therapeutic intervention could modulate the imaging signal in vivo. Statin treatment can reduce the total number of macrophages in murine plaques after 6 months of treatment17; however, the static nature of the study did not address the question of mechanism of action and whether the statin directly affected monocyte recruitment. Thus, further experiments evaluated in vivo the effect of mevastatin, simvastatin, or atorvastatin on monocyte recruitment to atheromata. We assessed whether statins affected monocyte recruitment and therefore chose to treat recipient animals for only 3 days (1 dose/day) before cells were injected. In vivo micro-SPECT/CT imaging revealed an 
5-fold reduction in monocyte recruitment to plaques compared with placebo-treated mice (P<0.001; Figure 4). All 3 statins examined had similar effects on the reduction of monocyte recruitment (Figure 4). Cholesterol levels between untreated and atorvastatin-treated animals showed no statistically significant difference (data not shown).
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To corroborate the effect observed during in vivo screening, we then examined the effect of statins on monocyte recruitment by an independent method. Animals were euthanized, aortas were excised, and recruitment was quantified by autoradiography. This approach demonstrated an 10-fold reduction in monocyte recruitment in statin-treated mice (P<0.001; Figure 5). To test whether the statin effect would act on the site of the vessel wall or on the monocytes themselves, we treated the donor mice, whereas the recipient mice were left untreated. The untreated and treated groups did not differ significantly, which suggests that statin does not act on the monocytes but rather on the vessel wall to reduce trafficking under the conditions of these experiments (Figure 5).
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Discussion |
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We report here that monocyte recruitment to plaques can be assessed by micro-SPECT/CT imaging and thus can be visualized in a noninvasive, dynamic, 3D, and quantitative fashion. The long half-life of radiotracer isotopes available for SPECT imaging enables repetitive imaging of monocyte biodistribution in the same animal, tracking its migration from circulation to atherosclerotic lesions.
Previous reports have indicated that lesion macrophage content may be gauged by magnetic resonance imaging,18,19 optical coherence tomography,20 or CT21; however, these methods can only describe the total number of resident macrophages or overall metabolic activity, respectively, in a plaque at a given time. Because atherogenesis is a chronic and dynamic process, the number of macrophages present in a plaque at a given time is the combined result of monocyte recruitment, differentiation, death, and efflux from the plaque. The same limitation holds true for positron-emission tomography studies with fluorodeoxyglucose, which have shown promise in estimating the overall degree of vascular inflammation.22,23 In contrast to these techniques, in vivo SPECT/CT imaging now enables assessment of monocyte accumulation serially in the same animal, which allows a much more direct study of factors that modulate recruitment.
The method we describe here has several limitations. The resolution of the present approach is lower than that of certain high-resolution modalities such as magnetic resonance imaging. Coregistration with CT (as in the present study) and, in the future, with magnetic resonance imaging can reduce this shortcoming by providing greater anatomic detail and hence better spatial localization of the tracer signal.
SPECT/CT imaging of adoptively transferred cells measures the total number of 
-photons in a defined 3D space and assumes an equal number of photons emitted from each cell. The method may underestimate the number of recruited cells as they undergo apoptosis, which may lead to loss of tracer. The known progressive loss of 111In over time in healthy cells is relatively constant, although some variations may occur in vivo in different disease models. Indium that has lost its association with cells does not significantly influence the local cell-bound activity, because our prior studies have shown that free 111In is excreted rapidly through the kidneys.14
A confounding variable in the interpretation of the results is the possibility that monocytes turn over in atherosclerotic plaques because of continued recruitment and emigration. However, data from Llodra et al24 have shown that although this is true for wild-type mice, only minimal emigration of monocytes from plaques in ApoE–/– mice occurs. On the basis of these data, we believe that the signal detected in lesions in the present study reflects the accumulation of monocytes over the 5-day period from adoptive transfer until the imaging end point. We understand that the number of adoptively transferred monocytes (3x106) exceeded the number of circulating monocytes in the recipient animal, which may affect the rate of monocyte trafficking, particularly in the initial hours or days.
Several technical factors were crucial to the development of a robust imaging technique. Because monocytes occur in relatively small numbers in the peripheral blood of mice and do not express a known unique surface marker, their isolation represents a challenge. Here, we used a 2-step method, recently described and characterized in more detail,14 that allows high-purity isolation while preserving cell viability and functionality.
Prerequisites for a suitable radioactive tracer used to label monocytes include ready availability, negligible cytotoxic effects, sufficient labeling efficiency, and a long half-life. The radiotracer compound 111In-oxine used here is available in a form ready for coincubation with cells and is approved by the Food and Drug Administration for labeling autologous leukocytes. Despite extensive testing in vitro and in vivo, we did not find adverse effects on viability or function of monocytes.
111In-oxine has a physical half-life (t1/2=2.8 days) that is significantly longer than that of isotopes available for positron emission tomography imaging, such as 64Cu (12.7 hours) or 18F (110 minutes).25 This translated to our ability to visualize monocyte trafficking by micro-SPECT/CT for up to 7 to 10 days, whereas the longest cell-tracking periods possible with state-of-the-art positron emission tomography imaging have been reported to be 24 to 36 hours,25 except for herpes simplex virus thymidine kinase (HSV-tk) techniques, which require genetic modification of cells. This property has particular importance to the present study, because we were able to use a delayed imaging time point (5 days) for the readout, thus avoiding detection of false-positive signal derived from circulating monocytes (we had recently determined the half-life of circulating blood monocytes in mice to be 43.5±7.9 hours, which corresponds to 95% monocyte clearance from the circulation at 5 days after injection14).
The SPECT signal was localized predominantly in regions of the ascending aorta, the site of the largest plaques in the majority of animals, whereas certain small lesions did not emit detectable signal. This observation may result from lesion heterogeneity, ie, some lesions may be more active than others. This method may therefore be used to identify lesions that are recruiting more monocytes. Alternatively, the total number of cells in smaller lesions is below the SPECT detection threshold. Further improvement of labeling efficiency and improvement in SPECT technology should increase sensitivity in the future. With an in vivo imaging system at hand, we could then use it to efficiently assess the effect of modulating factors on monocyte recruitment, thus avoiding labor-intensive ex vivo procedures. All 3 tested statins had an immediate effect on monocyte recruitment that was rapid and independent of significant effects on plasma cholesterol levels. To the best of our knowledge, this is the first time that such immediate statin effects have been reported. Prospective clinical trials have convincingly demonstrated that statins can effectively lower the incidence of cardiovascular events in primary and secondary prevention.26 An increasing body of evidence suggests that statins cause these clinical benefits in part through antiinflammatory effects not directly related to the lowering of low-density lipoprotein cholesterol, a hypothesis supported by the present observations. Statin-induced alterations in arterial biology that may not depend on cholesterol lowering include reduction in the expression of factors involved in the recruitment of inflammatory cells, such as monocyte chemoattractant protein-1,17,27 intercellular adhesion molecule-1,26 interleukin-6 and -8,28 tumor necrosis factor-
,17 nuclear factor-
B activity27 and monocytosis.7 Several reviews point to uncertainty about the in vivo relevance of such "pleiotropic" effects.26,29 Recent analyses of clinical trials, however, support the concept that an important component of reduction by statins of recurrent cardiovascular events does not depend on low-density lipoprotein lowering.30,31 The relevance of the low-density lipoprotein–independent effects of statins also remains unclear, because many in vitro studies used statin concentrations too high to have clinical relevance.29 Most of the studies in mice used dosages between 10 and 30 mg/kg,32,33 which is at least an order of magnitude higher than the clinically prescribed range of 10 to 80 mg per person per day (0.14 to 1.14 mg/kg for a standard weight of 70 kg). The atorvastatin dosage used in the present study (0.57 mg/kg) corresponds to a 40-mg dose in humans. The present study, using a low dose, showed that atorvastatin causes an immediate and substantial reduction in monocyte recruitment to atherosclerotic plaques. After treatment of ApoE–/– mice with only 3 statin doses, monocyte recruitment to plaques fell 5-fold in vivo. This finding has considerable importance because it highlights the potential of statins to suppress a key step in atherogenesis via an effect independent of low-density lipoprotein cholesterol lowering. Furthermore, it supports the relevance of previous in vitro reports on the effect of statins on mediators of monocyte attachment to and transmigration through the vascular endothelium. Reciprocal experiments treated the donor mice (and therefore exposed the monocytes to atorvastatin but not the vessel wall of the recipients) and showed no significant reduction in monocyte recruitment. This finding suggests that atorvastatin reduces recruitment by acting at the level of the arterial wall rather than on the monocytes themselves. However, the possibility remains that the statin effect on monocytes in vivo may have been lost during monocyte isolation and no longer operates on adoptive transfer. Therefore, we cannot conclude that statins affect the vascular wall exclusively under these conditions.
In summary, we present novel mechanistic insight into monocyte accumulation in atheromata and the antiinflammatory action of statins using a new tool that permits noninvasive tracking of cells to the vascular wall in vivo. Using monocytes in the context of atherogenesis, we exemplify how this technique can be applied to elucidate important biological events. Because the key components of the technique are in clinical use, its application to human patients may be within reach.
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Acknowledgments |
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We thank Gregory Wojtkiewicz and Pratik Patel for assistance with data analysis and image postprocessing, Dr Elena Aikawa for performing immunohistochemistry, and Dr Emerson Liu for technical advice. We also acknowledge Dr Mikael Pittet for critical review of the manuscript and Drs Farouc Jaffer and Matthias Nahrendorf for helpful discussions.
Sources of Funding
This work was supported in part by National Institutes of Health grants U01 HL080731 and R24 CA92782 (both to Dr Weissleder), the Donald W. Reynolds Cardiovascular Clinical Research Center on Atherosclerosis at Harvard Medical School (to Drs Libby and Weissleder), the American Heart Association (both Dr Kircher and Dr Swirski are recipients of American Heart Association Postdoctoral Fellowship Awards), and a seed grant from the Radiological Society of North America (to Dr Grimm).
Disclosures
Dr Libby is the recipient of a research grant from the National Heart, Lung, and Blood Institute and serves as a consultant/advisory board member of GE Medical Systems. Dr Allport has an ownership interest in Novartis Pharmaceuticals. Dr Weissleder has an ownership interest in VisEn Medical. The remaining authors report no conflicts.
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References |
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|---|
1. Li AC, Glass CK. The macrophage foam cell as a target for therapeutic intervention. Nat Med. 2002; 8: 1235–1242.[CrossRef][Medline] [Order article via Infotrieve]
2. Libby P. Inflammation in atherosclerosis. Nature. 2002; 420: 868–874.[CrossRef][Medline] [Order article via Infotrieve]
3. Binder CJ, Chang MK, Shaw PX, Miller YI, Hartvigsen K, Dewan A, Witztum JL. Innate and acquired immunity in atherogenesis. Nat Med. 2002; 8: 1218–1226.[CrossRef][Medline] [Order article via Infotrieve]
4. Libby P, Ridker PM. Inflammation and atherosclerosis: role of C-reactive protein in risk assessment. Am J Med. 2004; 116 (suppl 6A): 9S–16S.[CrossRef][Medline] [Order article via Infotrieve]
5. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med. 1999; 340: 115–126.
6. Sanz J, Moreno PR, Fuster V. Update on advances in atherothrombosis. Nat Clin Pract Cardiovasc Med. 2007; 4: 78–89.[CrossRef][Medline] [Order article via Infotrieve]
7. Swirski FK, Libby P, Aikawa E, Alcaide P, Luscinskas FW, Weissleder R, Pittet MJ. Ly-6C monocytes dominate hypercholesterolemia-associated monocytosis and give rise to macrophages in atheromata. J Clin Invest. 2007; 117: 195–205.[CrossRef][Medline] [Order article via Infotrieve]
8. Libby P, Ridker PM, Maseri A. Inflammation and atherosclerosis. Circulation. 2002; 105: 1135–1143.
9. Libby P. Current concepts of the pathogenesis of the acute coronary syndromes. Circulation. 2001; 104: 365–372.
10. Lessner SM, Prado HL, Waller EK, Galis ZS. Atherosclerotic lesions grow through recruitment and proliferation of circulating monocytes in a murine model. Am J Pathol. 2002; 160: 2145–2155.
11. Kim CJ, Khoo JC, Gillotte-Taylor K, Li A, Palinski W, Glass CK, Steinberg D. Polymerase chain reaction-based method for quantifying recruitment of monocytes to mouse atherosclerotic lesions in vivo: enhancement by tumor necrosis factor-alpha and interleukin-1 beta. Arterioscler Thromb Vasc Biol. 2000; 20: 1976–1982.
12. Steinberg D, Khoo JC, Glass CK, Palinski W, Almazan F. A new approach to determining the rates of recruitment of circulating leukocytes into tissues: application to the measurement of leukocyte recruitment into atherosclerotic lesions. Proc Natl Acad Sci U S A. 1997; 94: 4040–4044.
13. Budzyn K, Marley PD, Sobey CG. Chronic mevastatin modulates receptor-dependent vascular contraction in eNOS-deficient mice. Am J Physiol Regul Integr Comp Physiol. 2004; 287: R342–R348.
14. Swirski FK, Pittet MJ, Kircher MF, Aikawa E, Jaffer FA, Libby P, Weissleder R. Monocyte accumulation in mouse atherogenesis is progressive and proportional to extent of disease. Proc Natl Acad Sci U S A. 2006; 103: 10340–10345.
15. Segal BH, Kuhns DB, Ding L, Gallin JI, Holland SM. Thioglycollate peritonitis in mice lacking C5, 5-lipoxygenase, or p47(phox): complement, leukotrienes, and reactive oxidants in acute inflammation. J Leukoc Biol. 2002; 71: 410–416.
16. Kircher MF, Allport JR, Graves EE, Love V, Josephson L, Lichtman AH, Weissleder R. In vivo high resolution three-dimensional imaging of antigen-specific cytotoxic T-lymphocyte trafficking to tumors. Cancer Res. 2003; 63: 6838–6846.
17. Kleemann R, Princen HM, Emeis JJ, Jukema JW, Fontijn RD, Horrevoets AJ, Kooistra T, Havekes LM. Rosuvastatin reduces atherosclerosis development beyond and independent of its plasma cholesterol-lowering effect in APOE*3-Leiden transgenic mice: evidence for antiinflammatory effects of rosuvastatin. Circulation. 2003; 108: 1368–1374.
18. Amirbekian V, Lipinski MJ, Briley-Saebo KC, Amirbekian S, Aguinaldo JG, Weinreb DB, Vucic E, Frias JC, Hyafil F, Mani V, Fisher EA, Fayad ZA. Detecting and assessing macrophages in vivo to evaluate atherosclerosis noninvasively using molecular MRI. Proc Natl Acad Sci U S A. 2007; 104: 961–966.
19. Ruehm SG, Corot C, Vogt P, Kolb S, Debatin JF. Magnetic resonance imaging of atherosclerotic plaque with ultrasmall superparamagnetic particles of iron oxide in hyperlipidemic rabbits. Circulation. 2001; 103: 415–422.
20. Tearney GJ, Yabushita H, Houser SL, Aretz HT, Jang IK, Schlendorf KH, Kauffman CR, Shishkov M, Halpern EF, Bouma BE. Quantification of macrophage content in atherosclerotic plaques by optical coherence tomography. Circulation. 2003; 107: 113–119.
21. Hyafil F, Cornily JC, Feig JE, Gordon R, Vucic E, Amirbekian V, Fisher EA, Fuster V, Feldman LJ, Fayad ZA. Noninvasive detection of macrophages using a nanoparticulate contrast agent for computed tomography. Nat Med. 2007; 13: 636–641.[CrossRef][Medline] [Order article via Infotrieve]
22. Bural GG, Torigian DA, Chamroonrat W, Alkhawaldeh K, Houseni M, El-Haddad G, Alavi A. Quantitative assessment of the atherosclerotic burden of the aorta by combined FDG-PET and CT image analysis: a new concept. Nucl Med Biol. 2006; 33: 1037–1043.[CrossRef][Medline] [Order article via Infotrieve]
23. Elmaleh DR, Fischman AJ, Tawakol A, Zhu A, Shoup TM, Hoffmann U, Brownell AL, Zamecnik PC. Detection of inflamed atherosclerotic lesions with diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) and positron-emission tomography. Proc Natl Acad Sci U S A. 2006; 103: 15992–15996.
24. Llodra J, Angeli V, Liu J, Trogan E, Fisher EA, Randolph GJ. Emigration of monocyte-derived cells from atherosclerotic lesions characterizes regressive, but not progressive, plaques. Proc Natl Acad Sci U S A. 2004; 101: 11779–11784.
25. Adonai N, Nguyen KN, Walsh J, Iyer M, Toyokuni T, Phelps ME, McCarthy T, McCarthy DW, Gambhir SS. Ex vivo cell labeling with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography. Proc Natl Acad Sci U S A. 2002; 99: 3030–3035.
26. Wolfrum S, Jensen KS, Liao JK. Endothelium-dependent effects of statins. Arterioscler Thromb Vasc Biol. 2003; 23: 729–736.
27. Bustos C, Hernandez-Presa MA, Ortego M, Tunon J, Ortega L, Perez F, Diaz C, Hernandez G, Egido J. HMG-CoA reductase inhibition by atorvastatin reduces neointimal inflammation in a rabbit model of atherosclerosis. J Am Coll Cardiol. 1998; 32: 2057–2064.
28. Rezaie-Majd A, Maca T, Bucek RA, Valent P, Muller MR, Husslein P, Kashanipour A, Minar E, Baghestanian M. Simvastatin reduces expression of cytokines interleukin-6, interleukin-8, and monocyte chemoattractant protein-1 in circulating monocytes from hypercholesterolemic patients. Arterioscler Thromb Vasc Biol. 2002; 22: 1194–1199.
29. Libby P, Aikawa M. Mechanisms of plaque stabilization with statins. Am J Cardiol. 2003; 91: 4B–8B.[Medline] [Order article via Infotrieve]
30. Morrow DA, de Lemos JA, Sabatine MS, Wiviott SD, Blazing MA, Shui A, Rifai N, Califf RM, Braunwald E. Clinical relevance of C-reactive protein during follow-up of patients with acute coronary syndromes in the Aggrastat-to-Zocor Trial. Circulation. 2006; 114: 281–288.
31. Ridker PM, Cannon CP, Morrow D, Rifai N, Rose LM, McCabe CH, Pfeffer MA, Braunwald E. C-reactive protein levels and outcomes after statin therapy. N Engl J Med. 2005; 352: 20–28.
32. Nachtigal P, Jamborova G, Pospisilova N, Pospechova K, Solichova D, Zdansky P, Semecky V. Atorvastatin has distinct effects on endothelial markers in different mouse models of atherosclerosis. J Pharm Pharm Sci. 2006; 9: 222–230.[Medline] [Order article via Infotrieve]
33. Suzuki M, Kakuta H, Takahashi A, Shimano H, Tada-Iida K, Yokoo T, Kihara R, Yamada N. Effects of atorvastatin on glucose metabolism and insulin resistance in KK/Ay mice. J Atheroscler Thromb. 2005; 12: 77–84.[Medline] [Order article via Infotrieve]
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Footnotes |
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The online-only Data Supplement, consisting of a movie and a figure, is available with this article at http://circ.ahajournals.org/cgi/content/full/ CIRCULATIONAHA.107.719765/DC1.
Guest Editor for this article was Kenneth Walsh, PhD.
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Circulation 2008 117: 331-332.
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