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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Circulation. Author manuscript; available in PMC Apr 24, 2009.
Published in final edited form as:
PMCID: PMC2673051
NIHMSID: NIHMS104428

An activatable MR imaging agent reports myeloperoxidase activity in healing infarcts and detects the anti-inflammatory effects of atorvastatin on ischemia-reperfusion injury non-invasively

Matthias Nahrendorf, MD,1,2,3,* David Sosnovik, MD PhD,2,4 John Chen, MD PhD,1,2 Peter Panizzi, PhD,2 Jose-Luiz Figueiredo, MD,1,2 Elena Aikawa, MD PhD,2,3 Peter Libby, MD,3,5 Filip K. Swirski, PhD,2 and Ralph Weissleder, MD PhD1,2,3,6

Abstract

Background:

Ischemic injury of the myocardium causes timed recruitment of neutrophils and monocytes/macrophages, which produce substantial amounts of local myeloperoxidase (MPO). MPO forms reactive chlorinating species capable of inflicting oxidative stress and altering protein function by covalent modification. We have developed a small molecule, gadolinium-based activatable sensor for magnetic resonance imaging (MRI) of MPO activity (MPO-Gd). MPO-Gd is first radicalized by MPO, and then either spontaneously oligomerizes or binds to matrix proteins, all leading to enhanced spin-lattice-relaxivity and delayed wash-out kinetics. We hypothesized that MPO-imaging could be used to locally and non-invasively measure inflammatory responses after myocardial ischemia in a murine model.

Methods and Results:

We injected 0.3 mmol/kg of MPO-Gd (or Gd-DTPA as control) and performed MRI up to 120min later in mice 2 days after MI. Contrast-to-noise ratio (CNR, infarct versus septum) following Gd-DTPA injection peaked at 10 min, and returned to pre-injection values at 60 min. Following injection of MPO-Gd, CNR peaked later and was higher than Gd-DTPA (40.8±10.4 versus 10.5±0.2, p<0.05). MPO-imaging was validated by MRI of MPO−/− mice and correlated well with immunoreactive staining (r2=0.92, p<0.05), tissue activity by guaiacol assay (r2=0.65, p<0.001) and immunoblotting. In time course imaging, activity peaked 2 days after coronary ligation. Flow cytometry of digested infarcts detected MPO in neutrophils and monocytes/macrophages. Furthermore, serial MPO-imaging accurately tracked the anti-inflammatory effects of atorvastatin therapy after ischemia reperfusion injury.

Conclusion:

MPO-Gd enables in-vivo assessment of MPO activity in injured myocardium. This approach allows non-invasive evaluation of the inflammatory response to ischemia and has the potential to guide the development of novel cardioprotective therapies.

Keywords: magnetic resonance imaging, myocardial infarction, inflammation, myeloperoxidase, reperfusion

Introduction

Ischemic injury to the myocardium elicits a strong inflammatory response1-3. During the acute inflammatory phase, cytokine release, adhesion molecule expression, and cell preponderance in the blood pool govern the sequential recruitment of neutrophils and monocytes4, which likely introduce myeloperoxidase (MPO) into the injured myocardial tissue. The extent and profile of the ensuing leukocyte influx regulates consecutive healing stages4, while poor healing may lead to infarct rupture and heart failure5. MPO catalyses chloride oxidation to hypochlorous acid and generates other highly reactive moieties such as chlorine, tyrosyl radicals and aldehydes, all of which contribute to the microbicidal and viricidal properties of phagocytes6. These oxidizing species are cytotoxic7, inhibit enzymes such as ATPase8, and crosslink proteins9. Therefore, while MPO is prominently involved in the first line defense against infections mounted by the innate immune system, it may prove detrimental to the remodeling process after myocardial infarction7. Clinically, high MPO plasma levels predicted an increased 5 year mortality in patients with acute myocardial infarction10.

Gadolinium chelates (e.g., Gd-DTPA), currently the only clinically approved imaging agent in cardiovascular MRI, distribute passively to the extracellular space and do not reflect the degree of active inflammation, as acute and chronic infarction enhance alike11. We have recently developed an activatable and specific MPO sensor (5-hydroxytryptamide, MPO-Gd)12 and use it in the current study to image MPO activity in the heart. We hypothesize that during the inflammatory phase of myocardial ischemic injury, MPO activates the small molecule substrate, which then polymerize and exhibit increased T1 relaxivity, protein binding and “trapping” in areas of high MPO activity, all leading to increased enhancement on T1-weighted MRI. We correlate non-invasive imaging data with ex vivo MPO tissue activity, study MPO activity in wild type and MPO−/− mice with myocardial infarction and ischemia reperfusion injury. We further aimed to use this agent to characterize the time course of post-infarction MPO activity, and demonstrate that the agent possessed adequate dynamic range to image the anti-inflammatory actions of statin therapy in vivo.

Materials and Methods

Mouse myocardial infarction

This study used fifty-six C57BL6 mice, (Jackson Labs, Bar Harbor, ME; validation experiments, n=19; time course, n= 15, flow cytometry, n=10, ischemia reperfusion experiments, n=12). In addition, we used 4 heterozygous and 3 homozygous MPO deficient mice for validation purposes (Jackson Labs, Bar Harbor, ME). Myocardial infarction was induced by left coronary artery ligation as described previously3. For sham surgery, a thoracotomy was performed and a suture was passed underneath the coronary artery that was not ligated. In animals subjected to ischemia reperfusion injury, the coronary artery was ligated for 30 minutes followed by removal of the ligature. In reperfusion-injury experiments, mice were treated per gavage with control treatment (n=6) or with 100mg/kg atorvastatin (n=6) 24 hours and 1 hour before surgery. Mice were anesthetized for all surgical and imaging procedures by inhalation anesthesia (isoflurane 1-2% v/v + 2L O2). The institutional Subcommittee on Research Animal Care at Massachusetts General Hospital approved all animal studies.

Synthesis of MPO-Gd

The MPO-sensitive imaging agent bis-5-hydroxytryptamide-diethylenetriamine-pentaacetate (bis-5HT-DTPA(Gd), MPO-Gd; MW 866 g/mol) was synthesized as described previously12. Briefly, DTPA-bisanhydride was reacted with serotonin in dimethylformamide in the presence of an excess of triethylamine12. The product bis-5HT-DTPA was isolated by recrystallization from methanol and acetone.

MRI

We performed in vivo MRI after intravenous injection of MPO-Gd or gadopentetate dimeglumine (Gd-DTPA, Berlex Laboratories, Montville, NJ) at a dosage of 0.3 mmol/kg bodyweight. A seven Tesla horizontal bore scanner (Pharmascan, Bruker, Billerica, MA) and a dedicated mouse heart birdcage coil (Rapid Biomedical, Wuerzburg, Germany) were used to obtain delayed hyperenhancement images of the left ventricle in its short axis. We employed ECG and respiratory gating using a T1 weighted gradient echo FLASH-sequence13 with the following parameters: echo time (TE), 2.7 ms; 16 frames per heart cycle (TR 7.0-12.0 ms depending on heart rate); flip angle 60 degrees; in-plane resolution 200×200 μm; slice thickness 1 mm; NEX 8. The images were then analyzed using OsiriX DICOM reader (freeware, Geneva, Switzerland, www.osirix-viewer.com). The signal intensity was measured in the infarcted, akinetic lateral left ventricular wall, the non-infarcted interventricular septum, and a region outside the animal to calculate the contrast-to-noise ratio: CNR = (target signal − septal signal) / (standard deviation of the noise). The area enhanced after injection of MPO-Gd was quantified as a fraction of the entire left ventricular myocardial area at mid-ventricular level for comparison to immunoreactive MPO presence in histological sections. In addition, we quantified the akinetic myocardial area as a percentage of total LV area in the midventricular imaging slice.

MPO Activity Assay

Apical infarcted portions of hearts from various time-points after MI were homogenized (Omni International, Marietta, GA) for 30 seconds on ice in potassium phosphate buffer pH 7.0 with cetyltimethylammonium bromide. Samples were sonicated, freeze-thawed three times, and centrifuged to remove debris and 50-fold dilutions of heart samples were dissolved in potassium phosphate buffer containing 120 μM guaiacol, 900 μM H202. Change of absorption at 470 nm was measured with a Cary 50 spectrophotometer (Varian, Palo Alto, CA). Purified MPO was used to obtain a standard curve. Units of MPO activity were defined as the molar change oxidized guaiacol absorbance (E470 nm = 26.6mM−1 cm−1) with time14. Guaiacol oxidation progress curves were analyzed by least squares fitting of a line equation to the data using Scientist software (MicroMath, St. Louis, MO). Bicinchoninic acid protein assays (Pierce, Rockford, IL) were performed to determine total protein concentration of heart samples and to normalize data.

Flow cytometry

After sacrifice, hearts were excised and the tissue was prepared as described previously.3 To visualize monocytes/macrophages and neutrophils, the suspension was incubated with a mixture of monoclonal antibodies. The following antibodies were used: anti-CD90-PE, 53-2.1, anti-B220-PE, RA3-6B2, anti-CD49b-PE, DX5, anti-NK1.1-PE, PK136, anti-Ly-6G-PE, 1A8, CD11bAPC-Cy7, M1/70, anti-CD31-FITC, 390 (all BD Biosciences, San Jose, CA), anti-MPO, 8F4 (Hycult Biotechnology, Canton, MA). Monocytes/macrophages were identified as CD11bhi (CD90/B220/CD49b/NK1.1/Ly-6G)lo. Neutrophils were identified as CD11bhi (CD90/B220/CD49b/NK1.1/Ly-6G)hi. For intracellular staining of MPO, cells were permeabilized and fixed with a Cytofix/Cytoperm Kit (BD Biosciences, San Jose, CA). Flow cytometry was performed on an LSRII (BD Biosciences, San Jose, CA).

MPO genotyping

MPO deficient mice were obtained from Jackson Labs (Bar Harbour, ME). Genomic DNA was isolated from overnight proteinase K (50 ug) digestion of tail-vein clips at 55°C. Primers flanking the insertion site of the neomycin cassette were used to generate a 155 bp product band corresponding to the wild-type allele in identical Taq polymerase reactions containing the same concentration of genomic DNA (conditions and primer sequences were provided by Jackson Labs). To verify the MPO−/− genotype, western blotting for MPO protein was performed as described below for heart homogenates 2 days after MI.

Western blot analysis

Samples of heart homogenates were subjected to SDS gel (4-15%, BioRad, Hercules, CA) electrophoresis. Blots were incubated with rabbit primary antibodies for MPO (Millipore, Billerica, MA), and ICAM-1, ICAM-2 and VCAM-1 (Santa Cruz, Santa Cruz, CA), washed with PBS/0.5% Tween20, and visualized with Western Lightning (PerkinElmer, Waltham, MA) oxidation by horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA). To verify that similar amounts of protein were loaded, blots were stripped with Restore solution (Pierce, Rockford, IL) and the procedure was repeated with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rockland, Gilbertsville, PA). Densitometry was performed using ImageJ with a custom set of macros that quantified signal intensity in a standardized fashion.

Histopathology

Hearts were excised and rinsed in PBS and embedded in OCT (Sakura Finetek, Torrance, Ca). Serial 6 μm thick sections were collected in the midventricular level and used for immunohistochemical staining for neutrophils (NIMP-R14, Abcam, Cambridge, MA), monocytes/macrophages (Mac-3, M3/84, BD Pharmingen), and MPO (NeoMarkers, Freemont, CA). The reaction was visualized as a three-step-staining procedure using biotinylated secondary antibodies (BA4001, Vector Laboratories, Burlingame, CA) and AEC Substrate Kit (Vector Laboratories). MPO stained area was quantified as a fraction of the entire short axis ring at 2x magnification using OsiriX software and then correlated to the MPO-Gd enhanced region in MRI of the same animal.

Statistics

Results are expressed as mean±SD. The data sets were tested for normality using the Kolmogorov-Smirnov test with the Dallal-Wilkinson-Lilliefors correction, and for equality of variances using the F test. Data were compared using the unpaired two-sided t-test. If either normality or equality of variances were rejected, the nonparametric Mann-Whitney test was used. For multiple comparisons, we used ANOVA followed by Bonferroni post test. The significance level in all tests was 0.05. We used Graphpad Prism 4.0c for Macintosh (GraphPad Software, Inc., San Diego, CA) for statistical analysis.

RESULTS

MPO-Gd accumulates in acutely infarcted myocardium and exhibits delayed wash out kinetics when compared to Gd-DTPA

To study the enhancement pattern and kinetics after injection of MPO-Gd, we imaged 4 mice per group two days after coronary ligation, before and up to 2 hours after injection of equal doses of MPO-Gd and Gd-DTPA. Delayed enhancement was observed in the akinetic infarct at 10 and 30 minutes after injection of the conventional gadolinium chelate, with complete return of CNR to baseline at 60 minutes (Fig. 1). Peak CNR after injection of Gd-DTPA was observed at 10 minutes (10.5±0.2). After injection of MPO-Gd, the peak CNR was significantly higher (CNR 60 minutes, 40.8±10.4), and enhancement was observed even at 120 minutes after injection (Fig. 1). Comparison of both curves using a two-tailed Kolmogorov-Smirnov test showed significantly brighter enhancement for MPO-Gd (p=0.026).

Figure 1
In vivo imaging of MPO activity 2 days after myocardial infarction

MPO-Gd specifically targets myeloperoxidase activity

Experiments using transgenic mice evaluated the specificity of MPO-Gd for MPO activity. We compared the CNR one hour after injection in 4 wild-type mice, 4 heterozygous and 3 homozygous mice deficient for MPO, two days after coronary ligation. MPO−/− mice exhibited significantly diminished enhancement (ANOVA, p = 0.02 for MPO−/− versus MPO+/+), and an intermediate CNR was observed in MPO+/− mice (Fig. 2A-D). The akinetic LV wall area was not different between homozygous and heterozygous mice deficient for MPO and wild type mice (MPO+/+, 50±4%, MPO+/− 49±4%, MPO−/− 49±6%, p = 0.9). Therefore, it is unlikely that differences in enhancement observed between genotypes are caused by varying infarct size. Genotypes of mice were confirmed by PCR, and the absence of MPO protein in mice that were homozygous-deficient for the MPO gene was observed by immunoblotting (Fig. 2E). These experiments established that enhancement after MPO-Gd injection correlates closely with myeloperoxidase activity.

Figure 2
Imaging of MPO deficient mice establishes specificity of MPO-Gd for myeloperoxidase

Cellular MPO studies

We next investigated the individual cellular contributions to MPO activity in the healing infarct by flow cytometry. Flow cytometry of single cell suspension obtained from digested infarcts revealed that neutrophils, the most numerous cell type in a 2-day old mouse myocardial infarct4, contributed predominantly to local MPO activity, followed by monocytes/macrophages. All other cell types, such as lymphocytes contributed negligibly to the MPO signal (Fig. 3).

Figure 3
Cellular contribution to MPO activity in 2-day-old infarcts

MRI enhancement of MPO-Gd corresponds to immunoreactive MPO protein

To further investigate the specificity of MPO-Gd, we performed immunoreactive staining of hearts after MR imaging. The enhanced fraction of the LV myocardium visualized by MRI was not significantly different from and correlated well with the MPO positive fraction quantified by immunostaining for the enzyme (MRI, 45±10%; histology, 44±12%; p=0.9; r2=0.92, p<0.05). Adjacent sections were stained for the presence of neutrophils and macrophages, and both cell types co-localized with MPO (Fig. 4).

Figure 4
Immunoreactive staining reveals distribution of MPO in the infarct and co-localization with neutrophils and macrophages

Time course of MPO activity during infarct healing

We next followed the time course of MPO activity in healing myocardial infarcts. Three to four mice per day were imaged on days 1-8 after coronary ligation, and sacrificed after MRI to correlate ex vivo tissue activities to in vivo MRI data. The peak enhancement occured on day 2 after coronary ligation. This observation was corroborated by tissue activity measurements and immunoblotting, which also showed that MPO activity peaked on day 2 after infarction (Fig. 5A-C). Very little enhancement remained by day 8, consistent with decreasing cellularity and inflammatory activity in the infarcted myocardium at this later time point. Comparison of MRI-derived CNR to ex vivo tissue activity and immunoblotting corroborated the time course (Fig. 5D-E) and yielded a significant correlation (r2=0.65, p<0.001, Fig. 5F). The peak MRI-derived CNR on day 2 was 6.1-times higher than on day 8, comparable to the 10-fold difference detected in vitro. The moderate value of the correlation coefficient most likely results from CNR being measured in the midventricular slice, while the whole apical portion of the LV was used for the guaiacol assay, possibly also reflecting differences in individual infarct sizes.

Figure 5
Time course of MPO activity after myocardial infarction

In-vivo imaging of the action of an anti-inflammatory intervention

Detection of moderately expressed targets, serial imaging and monitoring of therapy effects are benchmarks for any new molecular imaging technology. We therefore employed MPO-Gd to follow the development of ischemia reperfusion injury. Four hours after onset of reperfusion, control mice exhibited a patchy enhancement pattern in the hypokinetic left ventricular free wall, and the signal consolidated further at 24 hours (Fig. 6), at which time the peak CNR was comparable to permant ligation (24.3±4.5 vs. 26.0±6.4, p=0.65). In mice treated with atorvastatin, similarly increased CNR values were observed at 4 hours, however the signal was significantly attenuated at the 24 hour time point (Fig. 6). Importantly, the pre-injection scan at the second time point did not show enhancement in either group. Therefore, complete wash-out of MPO-Gd was achieved within 24 hours. Flow cytometry of cells harvested from infarcts revealed that the absolute number of neutrophils and monocyte/macrophages per mg infarct tissue diminished in mice treated with atorvastatin, providing an explanation for lower MPO activity in this group (Fig. 7). Immunoblotting of the adhesion molecules VCAM-1, ICAM-1 and ICAM-2 showed decreased levels in atorvastatin-treated mice, thus likely leading to decreased cell recruitment and lower MPO activity observed by in vivo MRI (Fig. 6) and in western blotting (Fig. 7).

Figure 6
Monitoring of therapy by serial imaging of MPO activity in ischemia reperfusion injury
Figure 7
Atorvastatin decreases adhesion molecule expression and cell recruitment

DISCUSSION

Although a wealth of preclinical data has implicated oxidative stress in the pathogenesis of reperfusion injury, clinical trials of free radical scavengers have yielded conflicting results15.We show here that an MPO-activatable gadolinium chelate can be used to directly image MPO activity in the heart non-invasively by MRI. This agent might improve the understanding of the pathophysiology of oxidative injury in acute myocardial ischemia and may help to implement successful clinical strategies. The described imaging technology has high inherent sensitivity, since it not only reported massive oxidative stress related to inflammatory cell recruiment on day 2 after MI, but also detected subtle increases in MPO activity as early as 4 hours after onset of reperfusion injury, even with agent concentrations in the clinically plausible range. Studies in MPO−/− mice and correlation of MPO-imaging with biochemical and morphological assays provide strong evidence supporting the specificity of the imaging signal as a reporter for MPO activity.

MPO-Gd is a small molecule, with a size comparable to that of clinically used gadolinium chelates. This property facilitates delivery of the molecule to the target area in the injured myocardium. Myeloperoxidase-derived hypochlorous acid activates the probe through the oxidation of the hydroxytryptamide moieties on the chelate16. The ligands then react with each other, leading to polymerization of the agent into dimers, tetramers and occasionally even pentamers. This polymerization decreases the tumbling rate of gadolinium, activates the probe, and enhances the T1-shortening effect of the imaging agent16. Furthermore, the increased size of the polymer and cross linking to surrounding matrix proteins promote the retention of the probe in areas of high MPO activity, which results in substantially decelerated wash-out kinetics16. Therefore, we found very bright enhancement at one hour after injection, at a time when conventional Gd-DTPA and non-activated MPO-Gd have been washed out of the myocardium completely. Nevertheless, as demonstrated in serial imaging of reperfusion injury, the wash-out is also rapid enough to facilitate frequent serial imaging. Of note, this study also shows that MPO-Gd derived signal exhibits an adequate dynamic range to detect changes in MPO activity. MPO expression was modulated in several ways. Wildtype, heterozygous and homozygous MPO-deficient mice were imaged and a strong linear relationship was seen between the MR signal intensity and the genotype of the mouse imaged. In addition, imaging with MPO-Gd visualized anti-inflammatory effects of atorvastatin17-19 after myocardial reperfusion injury in mice in vivo. In our study, we used MPO-Gd in a murine model and at high field strength. Since the longitudinal relaxivity (R1) of gadolinium chelates increases at lower field strength,20-22 lower doses of MPO-Gd will likely be detectable at 1.5 Tesla. It is quite possible that the dose of 0.3 mmol/kg used in our study at 7 Tesla would be able to be reduced to the clinically approved dose of 0.1 mmol/kg at 1.5 Tesla. This prediction, however, will need to be confirmed by imaging larger animals at clinical field strengths.

Recent studies have used magnetofluorescent nanoparticles to image inflammation after ischemic injury of the myocardium3, 23. The biological target of this strategy is different, however. While nanoparticles are ingested by phagocytes and therefore report their presence, MPO-Gd is a functional reporter that probes activity of a pro-oxidant enzyme. Combined strategies using both magnetic nanoparticles and the MPO-Gd chelate could provide novel and complementary information, a conjecture that will require further study.

Peak MPO activity occurred in the infarct on day 2, during the initial pro-inflammatory phase after infarction. This time point coincides with dominant presence of neutrophils and Ly6Chi monocytes, the inflammatory monocyte subtype that accumulates in the first phase of leukocyte recruitment after MI4. Both of these cell types are first line responders that express high levels of MPO. Systemic neutrophilia24, 25 as well as monocytosis26, 27 after acute MI associate with graver prognosis in patients. In addition, MPO deficiency alleviated the evolution of heart failure in mice after coronary ligation7. The ability to image MPO activity in vivo could provide novel insights into the functional status of inflammatory cells and thus facilitate the development of novel therapies to optimize infarct healing.

Using serial non-invasive MPO-imaging, we followed leukocyte recruitment, and monitored a significant attenuation of MPO activity by atorvastatin therapy. This may reflect the ability of statins to reduce infarct size28, 29. In patients with coronary artery disease, atorvastatin reduced myeloperoxidase-derived oxidants independent of changes in lipid parameters30 and statins attenuated myocardial ischemic injury in patients with acute coronary syndromes31. We used immunoblotting and flow cytometry to investigate the underlying mechanism of the treatment effect observed by MPO-imaging in the present study. Atorvastatin decreased expression of VCAM-1, ICAM-1 and ICAM-2, the endothelial binding sites for the integrins VLA-4 on monocytes and LFA-1 on neutrophils32. As a part of the leukocyte adhesion cascade, these adhesion proteins regulate inflammatory cell recruitment32. Therefore, atorvastatin treatment likely reduced leukocyte recruitment via reduced expression of adhesion molecules, analogous to the situation in atherosclerosis33, 34. In addition, statin therapy may also decrease MPO expression in macrophages35. Our findings are consistent with reports of reduced infarct size in mice after HMG-CoA reductase inhibition28, 29 and indicate how MPO-imaging might facilitate the discovery process for novel therapy targeting ischemia reperfusion injury.

This study shows that a novel activatable MRI probe can image MPO activity in the myocardium in vivo. We further demonstrate that MPO-Gd accurately reported MPO activity in vivo and possessed adequate sensitivity and dynamic range to detect treatment effects. Inflammation after ischemia may vary in the clinical setting, impressively shown in clinical trials demonstrating severe adverse effects of steroids on infarct healing36. MPO-Gd gauges activity of myeloperoxidase in vivo, which can serve as a surrogate for the intensity of leukocyte influx. Preclinical data support that inflammation impacts the extend of post MI remodeling1, 2, 4, and therefore prognosis. Molecular MRI of MPO activity in animals, and ultimately in humans, could facilitate non-invasive imaging of the natural history of inflammation and its impact on myocardial healing/remodeling. This would allow the role of inflammation in animal models and humans to be compared directly and the efficacy of various immune modulators to be better understood. MPO imaging could ultimately be used as part of a “personalized” regimen in those patients at highest risk of remodeling (large anterior infarction) to guide novel therapeutic strategies. Furthermore, leukocyte recruitment is a general component of inflammation. This molecular imaging sensor thus could play an important translational role in not only ischemic heart disease but also other inflammatory cardiovascular conditions such as atherosclerosis, myocarditis, and transplant rejection.

ACKNOWLEDGEMENTS

The authors would like to thank Lee Josephson, PhD, Fred Reynolds, PhD, Elisenda Rodriguez, PhD, and Micheal Breckwoldt for chemical synthesis, the CMIR Mouse Imaging Program (Carlos Rangel, BS; Anne Yu, BS) for assistance with MR imaging, the CMIR Pathology Core for assistance with histology (Vincent Lok, BS; Yoshiko Iwamoto, BS), Jennifer Panizzi, PhD, and Martin Etzrodt, MS, for assistance with immunoblotting and densitometry. This work was funded in part by the D.W. Reynolds Foundation, UO1-HL080731 (RW), RO1-HL078641 (RW), and R24-CA92782 (RW).

Footnotes

Publisher's Disclaimer: Disclaimer: The manuscript and its contents are confidential, intended for journal review purposes only, and not to be further disclosed.

Author Disclosures

Matthias Nahrendorf: No disclosures

David Sosnovik: No disclosures

John Chen: No disclosures

Peter Panizzi: No disclosures

Jose-Luiz Figueiredo: No disclosures

Elena Aikawa: No disclosures

Peter Libby: No disclosures

Filip Swirski: No disclosures

Ralph Weissleder: No disclosures

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