|
|
Circulation. Author manuscript; available in PMC 2009 April 24. Published in final edited form as: Published online 2008 February 11. doi: 10.1161/CIRCULATIONAHA.107.756510. | 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 1Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Simches Research Building, 185 Cambridge St., Boston, MA 02114 2Center for Molecular Imaging Research, Massachusetts General Hospital and Harvard Medical School, Building 149, 13th St., Charlestown, MA 02129 3Donald W. Reynolds Cardiovascular Clinical Research Center on Atherosclerosis at Harvard Medical School, 75 Francis Street, Boston, MA 02115 4Department of Cardiology, Massachusetts General Hospital, 75 Francis Street, Boston, MA 02115 5Cardiovascular Division, Department of Medicine, Brigham & Women's Hospital, 75 Francis Street, Boston, MA 02115 6Department of Systems Biology, Harvard Medical School, Boston 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 Ischemic injury to the myocardium elicits a strong inflammatory response 1-3. During the acute inflammatory phase, cytokine release, adhesion molecule expression, and cell preponderance in the blood pool govern the sequential recruitment of neutrophils and monocytes 4, which likely introduce myeloperoxidase (MPO) into the injured myocardial tissue. The extent and profile of the ensuing leukocyte influx regulates consecutive healing stages 4, while poor healing may lead to infarct rupture and heart failure 5. 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 phagocytes 6. These oxidizing species are cytotoxic 7, inhibit enzymes such as ATPase 8, and crosslink proteins 9. 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 infarction 7. Clinically, high MPO plasma levels predicted an increased 5 year mortality in patients with acute myocardial infarction 10. 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 alike 11. 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. 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 previously 3. 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 O 2). 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 previously 12. Briefly, DTPA-bisanhydride was reacted with serotonin in dimethylformamide in the presence of an excess of triethylamine 12. 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-sequence 13 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 H 20 2. 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 (E 470 nm = 26.6mM −1 cm −1) with time 14. 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 CD11b hi (CD90/B220/CD49b/NK1.1/Ly-6G) lo. Neutrophils were identified as CD11b hi (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. 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 (). 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 (). Comparison of both curves using a two-tailed Kolmogorov-Smirnov test showed significantly brighter enhancement for MPO-Gd (p=0.026). 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 (). 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 (). These experiments established that enhancement after MPO-Gd injection correlates closely with myeloperoxidase activity. 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 infarct 4, contributed predominantly to local MPO activity, followed by monocytes/macrophages. All other cell types, such as lymphocytes contributed negligibly to the MPO signal (). 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 (). 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 (). 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 () and yielded a significant correlation (r2=0.65, p<0.001, ). 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. 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 (), 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 (). 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 (). 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 () and in western blotting (). 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 results 15.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 chelate 16. 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 agent 16. 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 kinetics 16. 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 atorvastatin 17-19 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 myocardium 3, 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 Ly6C hi monocytes, the inflammatory monocyte subtype that accumulates in the first phase of leukocyte recruitment after MI 4. Both of these cell types are first line responders that express high levels of MPO. Systemic neutrophilia 24, 25 as well as monocytosis 26, 27 after acute MI associate with graver prognosis in patients. In addition, MPO deficiency alleviated the evolution of heart failure in mice after coronary ligation 7. 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 size 28, 29. In patients with coronary artery disease, atorvastatin reduced myeloperoxidase-derived oxidants independent of changes in lipid parameters 30 and statins attenuated myocardial ischemic injury in patients with acute coronary syndromes 31. 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 neutrophils 32. As a part of the leukocyte adhesion cascade, these adhesion proteins regulate inflammatory cell recruitment 32. Therefore, atorvastatin treatment likely reduced leukocyte recruitment via reduced expression of adhesion molecules, analogous to the situation in atherosclerosis 33, 34. In addition, statin therapy may also decrease MPO expression in macrophages 35. Our findings are consistent with reports of reduced infarct size in mice after HMG-CoA reductase inhibition 28, 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 healing 36. 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 remodeling 1, 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. 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). 1. Blankesteijn WM, Creemers E, Lutgens E, Cleutjens JP, Daemen MJ, Smits JF. Dynamics of cardiac wound healing following myocardial infarction: observations in genetically altered mice. Acta Physiol Scand. 2001;173:75–82. [PubMed] 2. Frangogiannis NG, Smith CW, Entman ML. The inflammatory response in myocardial infarction. Cardiovasc Res. 2002;53:31–47. [PubMed] 3. Nahrendorf M, Sosnovik DE, Waterman P, Swirski FK, Pande AN, Aikawa E, Figueiredo JL, Pittet MJ, Weissleder R. Dual channel optical tomographic imaging of leukocyte recruitment and protease activity in the healing myocardial infarct. Circ Res. 2007;100:1218–1225. [PubMed] 4. Nahrendorf M, Swirski FK, Aikawa E, Stangenberg L, Wurdinger T, Figueiredo JL, Libby P, Weissleder R, Pittet MJ. The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions. J Exp Med. 2007;204:3037–47. [PubMed] 5. Sutton MG, Sharpe N. Left ventricular remodeling after myocardial infarction: pathophysiology and therapy. Circulation. 2000;101:2981–2988. [PubMed] 6. Rosen H, Crowley JR, Heinecke JW. Human neutrophils use the myeloperoxidase-hydrogen peroxide-chloride system to chlorinate but not nitrate bacterial proteins during phagocytosis. J Biol Chem. 2002;277:30463–30468. [PubMed] 7. Vasilyev N, Williams T, Brennan ML, Unzek S, Zhou X, Heinecke JW, Spitz DR, Topol EJ, Hazen SL, Penn MS. Myeloperoxidase-generated oxidants modulate left ventricular remodeling but not infarct size after myocardial infarction. Circulation. 2005;112:2812–2820. [PubMed] 8. Eley DW, Eley JM, Korecky B, Fliss H. Impairment of cardiac contractility and sarcoplasmic reticulum Ca2+ ATPase activity by hypochlorous acid: reversal by dithiothreitol. Can J Physiol Pharmacol. 1991;69:1677–1685. [PubMed] 9. Heinecke JW. Mechanisms of oxidative damage by myeloperoxidase in atherosclerosis and other inflammatory disorders. J Lab Clin Med. 1999;133:321–325. [PubMed] 10. Mocatta TJ, Pilbrow AP, Cameron VA, Senthilmohan R, Frampton CM, Richards AM, Winterbourn CC. Plasma concentrations of myeloperoxidase predict mortality after myocardial infarction. J Am Coll Cardiol. 2007;49:1993–2000. [PubMed] 11. Fuster V, Kim RJ. Frontiers in cardiovascular magnetic resonance. Circulation. 2005;112:135–144. [PubMed] 12. Querol M, Chen JW, Weissleder R, Bogdanov A., Jr. DTPA-bisamide-based MR sensor agents for peroxidase imaging. Org Lett. 2005;7:1719–1722. [PubMed] 13. Yang Z, Berr SS, Gilson WD, Toufektsian MC, French BA. Simultaneous evaluation of infarct size and cardiac function in intact mice by contrast-enhanced cardiac magnetic resonance imaging reveals contractile dysfunction in noninfarcted regions early after myocardial infarction. Circulation. 2004;109:1161–1167. [PubMed] 14. Klebanoff SJ, Waltersdorph AM, Rosen H. Antimicrobial activity of myeloperoxidase. Methods Enzymol. 1984;105:399–403. [PubMed] 15. Yellon DM, Hausenloy DJ. Myocardial reperfusion injury. N Engl J Med. 2007;357:1121–1135. [PubMed] 16. Chen JW, Querol Sans M, Bogdanov A, Jr., Weissleder R. Imaging of myeloperoxidase in mice by using novel amplifiable paramagnetic substrates. Radiology. 2006;240:473–481. [PubMed] 17. Mital S, Liao JK. Statins and the myocardium. Semin Vasc Med. 2004;4:377–384. [PubMed] 18. Schonbeck U, Libby P. Inflammation, immunity, and HMG-CoA reductase inhibitors: statins as antiinflammatory agents? Circulation. 2004 Jun;109:II18–26. [PubMed] 19. Wolfrum S, Grimm M, Heidbreder M, Dendorfer A, Katus HA, Liao JK, Richardt G. Acute reduction of myocardial infarct size by a hydroxymethyl glutaryl coenzyme A reductase inhibitor is mediated by endothelial nitric oxide synthase. J Cardiovasc Pharmacol. 2003;41:474–480. [PubMed] 20. Ghaghada KB, Bockhorst KH, Mukundan S, Jr., Annapragada AV, Narayana PA. High-resolution vascular imaging of the rat spine using liposomal blood pool MR agent. AJNR Am J Neuroradiol. 2007;28:48–53. [PubMed] 21. Pintaske J, Martirosian P, Graf H, Erb G, Lodemann KP, Claussen CD, Schick F. Relaxivity of Gadopentetate Dimeglumine (Magnevist), Gadobutrol (Gadovist), and Gadobenate Dimeglumine (MultiHance) in human blood plasma at 0.2, 1.5, and 3 Tesla. Invest Radiol. 2006;41:213–221. [PubMed] 22. Morawski AM, Winter PM, Crowder KC, Caruthers SD, Fuhrhop RW, Scott MJ, Robertson JD, Abendschein DR, Lanza GM, Wickline SA. Targeted nanoparticles for quantitative imaging of sparse molecular epitopes with MRI. Magn Reson Med. 2004;51:480–486. [PubMed] 23. Sosnovik DE, Nahrendorf M, Deliolanis N, Novikov M, Aikawa E, Josephson L, Rosenzweig A, Weissleder R, Ntziachristos V. Fluorescence tomography and magnetic resonance imaging of myocardial macrophage infiltration in infarcted myocardium in vivo. Circulation. 2007;115:1384–1391. [PubMed] 24. Kirtane AJ, Bui A, Murphy SA, Barron HV, Gibson CM. Association of peripheral neutrophilia with adverse angiographic outcomes in ST-elevation myocardial infarction. Am J Cardiol. 2004;93:532–536. [PubMed] 25. Kyne L, Hausdorff JM, Knight E, Dukas L, Azhar G, Wei JY. Neutrophilia and congestive heart failure after acute myocardial infarction. Am Heart J. 2000;139:94–100. [PubMed] 26. Maekawa Y, Anzai T, Yoshikawa T, Asakura Y, Takahashi T, Ishikawa S, Mitamura H, Ogawa S. Prognostic significance of peripheral monocytosis after reperfused acute myocardial infarction:a possible role for left ventricular remodeling. J Am Coll Cardiol. 2002;39:241–246. [PubMed] 27. Mariani M, Fetiveau R, Rossetti E, Poli A, Poletti F, Vandoni P, D'Urbano M, Cafiero F, Mariani G, Klersy C, De Servi S. Significance of total and differential leucocyte count in patients with acute myocardial infarction treated with primary coronary angioplasty. Eur Heart J. 2006;27:2511–2515. [PubMed] 28. Jones SP, Trocha SD, Lefer DJ. Pretreatment with simvastatin attenuates myocardial dysfunction after ischemia and chronic reperfusion. Arterioscler Thromb Vasc Biol. 2001;21:2059–2064. [PubMed] 29. Lefer DJ, Scalia R, Jones SP, Sharp BR, Hoffmeyer MR, Farvid AR, Gibson MF, Lefer AM. HMG-CoA reductase inhibition protects the diabetic myocardium from ischemiareperfusion injury. Faseb J. 2001;15:1454–1456. [PubMed] 30. Shishehbor MH, Brennan ML, Aviles RJ, Fu X, Penn MS, Sprecher DL, Hazen SL. Statins promote potent systemic antioxidant effects through specific inflammatory pathways. Circulation. 2003;108:426–431. [PubMed] 31. Patti G, Pasceri V, Colonna G, Miglionico M, Fischetti D, Sardella G, Montinaro A, Di Sciascio G. Atorvastatin pretreatment improves outcomes in patients with acute coronary syndromes undergoing early percutaneous coronary intervention: results of the ARMYDA-ACS randomized trial. J Am Coll Cardiol. 2007;49:1272–1278. [PubMed] 32. Ley K, Laudanna C, Cybulsky MI, Nourshargh S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol. 2007;7:678–689. [PubMed] 33. Sukhova GK, Williams JK, Libby P. Statins reduce inflammation in atheroma of nonhuman primates independent of effects on serum cholesterol. Arterioscler Thromb Vasc Biol. 2002;22:1452–1458. [PubMed] 34. Nahrendorf M, Jaffer FA, Kelly KA, Sosnovik DE, Aikawa E, Libby P, Weissleder R. Noninvasive vascular cell adhesion molecule-1 imaging identifies inflammatory activation of cells in atherosclerosis. Circulation. 2006;114:1504–1511. [PubMed] 35. Kumar AP, Reynolds WF. Statins downregulate myeloperoxidase gene expression in macrophages. Biochem Biophys Res Commun. 2005;331:442–451. [PubMed] 36. Roberts R, DeMello V, Sobel BE. Deleterious effects of methylprednisolone in patients with myocardial infarction. Circulation. 1976;53:I204–206. [PubMed]
|
The publisher's final edited version of this article is available free at 
