Activatable magnetic resonance imaging agent reports myeloperoxidase activity in healing infarcts and noninvasively detects the antiinflammatory effects of atorvastatin on ischemia-reperfusion injury

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 used a small-molecule, gadolinium-based activatable sensor for magnetic resonance imaging 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 washout kinetics. We hypothesized that MPO imaging could be used to measure inflammatory responses after myocardial ischemia locally and noninvasively in a murine model.

Methods and results: We injected 0.3 mmol/kg MPO-Gd (or Gd-DTPA as control) and performed magnetic resonance imaging up to 120 minutes later in mice 2 days after myocardial infarction. The contrast-to-noise ratio (infarct versus septum) after Gd-DTPA injection peaked at 10 minutes and returned to preinjection values at 60 minutes. After injection of MPO-Gd, the contrast-to-noise ratio 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 magnetic resonance imaging 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 antiinflammatory effects of atorvastatin therapy after ischemia-reperfusion injury.

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

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

1A: Strong and persistent hyperenhancement is seen in the infarct zone in mice injected with MPO-Gd. Two hours after injection, the infarcted left ventricular free wall is brightly enhanced, at a time when conventional Gd-DTPA has been washed out completely (1B). 1C: The contrast to noise ratio shows higher values and delayed wash-out after injection of MPO-Gd when compared to conventional Gd-DTPA.

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

2A: The infarct of a wild type mouse 2 hours after injection of MPO-Gd is brightly enhanced. Imaging was performed on day 2 after MI in all mice. 2B: Intermediate-level enhancement is observed in heterozygous MPO deficient mice, corresponding to 50% enzyme activity levels. 2C: In homozygous MPO deficient mice, enhancement after injection of MPO-Gd is significantly reduced, establishing the specificity of MPO-Gd. 2D: Contrast-to-noise ratio in respective genotypes show significantly diminished enhancement in MPO deficient mice. * p<0.05. 2E: PCR and Western blotting of representative genotypes shown in 2A-C.

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

Intracellular MPO levels 2 days after MI. Single-cell suspensions of infarcts were prepared and stained with antibodies to detect lymphocytes (Gate i: (CD90/B220/CD49b/NK1.1/Ly-6G)^hi CD11b^lo), monocytes/macrophages (Gate ii: (CD90/B220/CD49b/NK1.1/Ly-6G)^lo CD11b^hi), and neutrophils (Gate iii: (CD90/B220/CD49b/NK1.1/Ly-6G)^hi CD11b^hi). Intracellular staining was conducted to detect MPO levels and is represented as mean fluorescent intensities (MFI). Mean and SEM are shown, n=3. * P < 0.001. On day 2 after MI, neutrophils are the main contributors to MPO activity in the infarct, followed by monocyte/macrophages.

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

4A: Low resolution Hematoxilin/Eosin stain of a 2-day-old infarct. Inset depicts location of high magnification histology shown in 4B-D. Magnification 40x, scale bar 500μm. 4B: Immunoreactive staining for MPO shows the presence of the enzyme in 2-day-old infarction and co-localization with macrophages (4C) and neutrophils (4D). Magnification 200X, scale bar 100μm.

Figure 5. Time course of MPO activity after myocardial infarction

5A-B: Short axis MRI of representative mice on day 2 (5A) and day 8 (5B) after coronary ligation. The insets demonstrate geometry of the short axis view (dotted line). On day 2, bright enhancement is observed in the infarcted, akinetic left ventricular free wall (arrows). The enhancement is significantly diminished 8 days after MI, consistent with dampened inflammation. 5C: Time course of contrast-to-noise ratio after coronary ligation. Three to four mice were imaged per time point. The peak enhancement is observed on day 2. 5D: MPO tissue activity assessed by guaiacol assay in the same animals shows a similar time course as MPO-imaging, with the peak activity occurring on day 2. 5E: Western blots show no MPO in the hearts of sham operated mice, strong staining on day 1-4 post MI, and low signal on day 8 after coronary ligation, corroborating the in vivo MRI and tissue activity assay data. 5F: Correlation of contrast to noise ratio obtained by MRI with tissue activity levels measured by guaiacol assay.

Figure 6. Monitoring of therapy by serial imaging of MPO activity in ischemia reperfusion injury

6A and B: MRI in mice that were subjected to 30 minutes of myocardial ischemia, followed by 4 hours of reperfusion. After injection of MPO-Gd, the lateral wall enhances in both treated and untreated groups. 6C and D: 24 hours later, mice were reinjected with MPO-Gd and imaged again. Treatment with atorvastatin results in significantly diminished enhancement (6E).

Figure 7. Atorvastatin decreases adhesion molecule expression and cell recruitment

7A: Western blots for VCAM-1, ICAM-1 and ICAM-2 show decreased levels of these adhesion molecules after ischemia reperfusion injury in mice treated with atorvastatin. Consequently, MPO expression is reduced in atorvastatin treated mice. * p < 0.05 7B: Flow cytometry of cell suspensions dislodged from the injured lateral wall shows significantly diminished recruitment of neutrophils as well as monocyte/macrophages in mice treated with atorvastatin.