Near-infrared fluorescent imaging of matrix metalloproteinase activity after myocardial infarction

Abstract

Background: We used a molecular probe activated by protease cleavage to image expression of matrix metalloproteinases (MMPs) in the heart after myocardial infarction.

Methods and results: We synthesized and characterized a near-infrared fluorescent (NIRF) probe that is activated by proteolytic cleavage by MMP2 and MMP9. The NIRF probe was injected into mice at various time points up to 4 weeks after myocardial infarction induced by ligation of the left anterior descending coronary artery. NIRF imaging of MMP activity increased in the infarct region, with maximal expression at 1 to 2 weeks, persisting to 4 weeks. Zymography and real-time polymerase chain reaction analysis showed that MMP9 expression is increased at 2 to 4 days, and MMP2 expression is increased at 1 to 2 weeks. Dual-label confocal microscopy showed colocalization of NIRF imaging with neutrophils on day 2, and flow cytometric analysis confirmed that NIRF signal is associated with leukocytes in the infarct zone.

Conclusions: This study demonstrates that the activity of MMPs in the myocardium may be imaged by use of specific activity-dependent molecular probes.

Figure 1. Activation of NIRF probe by MMP2 and MMP9

NIRF probe (0.2 μM), in 50 mM Tris HCl pH 7.5, 10 mM CaCl₂, 100 mM NaCl, 0.005% Brij-35, was incubated with 5 μg of active MMP2 or MMP9 (Oncogene, CA). Fluorescence was measured at various time points. Error bars show SEM.

Figure 2

NIRF imaging of MMP activated probe in the mouse heart after MI. Top row, probe was injected at the time of MI and imaged at 2 days. Bottom row, probe was injected at 5 days and imaged at 7 days. Left column, brightfield photograph; middle column, NIFR imaging; right column, hematoxylin and eosin staining.

Figure 3

Time course of MIRF signal following MI. A. NIRF signal intensity in infarct region (closed circles) and remote myocardium (open circles) is shown over time. Error bars show SEM.

Figure 4

Zymography of MMP2 and MMP9 activity in heart following MI. A. Infarct zone. B. Remote zone. Gelatin in the polyacrylamide gel is hydrolyzed by MMP2 and MMP9 activity, resulting in clear bands.

Figure 5

Real time PCR analysis of MMP2 and MMP9 mRNA levels in infarct regions following MI. RNA levels are expressed as a ratio normalized to beta-actin mRNA.

Figure 5

Real time PCR analysis of MMP2 and MMP9 mRNA levels in infarct regions following MI. RNA levels are expressed as a ratio normalized to beta-actin mRNA.

Figure 6. Microscopic NIRF imaging

A microscopic NIRF image and an adjacent section stained with H and E. Magnification is 10×.

Figure 7

Dual label confocal staining for MMP9, macrophages, and neutrophils, MMP9 is stained with Texas Red, while macrophages (top row) and neutrophils (bottom row) are stained with FITC conjugated antibody. MMP9 activity colocalizes strongly with neutrophils at 1 day.

Figure 8

Flow cytometry of gelatinase probe activation and CD45, Infarcted and non-infarcted regions of the heart were dissected, minced, and digested with collagenase. Cells were stained with CD45-FITC conjugated antibody, and analyzed for CD45 and the NIRF signal intensity.