Dual Modality Activity Based Probes as Molecular Imaging Agents for Vascular Inflammation

Abstract

Macrophages are cellular mediators of vascular inflammation and are involved in the formation of atherosclerotic plaques. These immune cells secrete proteases such as matrix metalloproteinases and cathepsins that contribute to disease formation and progression. Here, we demonstrate that activity-based probes (ABPs) targeting cysteine cathepsins can be used in murine models of atherosclerosis to non-invasively image activated macrophage populations using both optical and PET/CT methods. The probes can also be used to topically label human carotid plaques demonstrating similar specific labeling of activated macrophage populations. Methods: Macrophage-rich carotid lesions were induced in FVB mice fed on a high-fat diet by streptozotocin injection followed by ligation of the left common carotid artery. Mice with carotid atherosclerotic plaques were injected with the optical or dual modality probes, BMV109 and BMV101 respectively, via the tail vein and non-invasively imaged by optical and small-animal PET/CT at different time points. After non-invasive imaging, the murine carotid arteries were imaged in situ and ex vivo followed by immunofluorescence staining to confirm target labeling. Additionally, human carotid plaques were topically labeled with the probe and analyzed by both SDS-PAGE and immunofluorescence staining to confirm the primary targets of the probe. Results: Quantitative analysis of the signal intensity from both optical and PET/CT imaging showed significantly higher levels of accumulation of BMV109 and BMV101 (p<0.005 and p<0.05 respectively) in the ligated left carotid arteries compared to the right carotid or healthy arteries. Immunofluorescence staining for macrophages in cross-sectional slices of the murine artery demonstrated substantial infiltration of macrophages in the neo-intima and adventitia of the ligated left carotid arteries compared to the right. Analysis of the human plaque tissues by SDS-PAGE confirmed that the primary targets of the probe were cathepsins X, B, S and L. Immunofluorescence labeling of the human tissue with the probe demonstrated co-localization of the probe with CD68, elastin and cathepsin S, similar to that observed in the experimental carotid inflammation murine model. Conclusion: We demonstrate that ABPs targeting the cysteine cathepsins can be used in murine models of atherosclerosis to non-invasively image activated macrophage populations using both optical and PET/CT methods. The probes could also be used to topically label human carotid plaques demonstrating similar specific labeling of activated macrophage populations. Therefore, ABPs targeting the cysteine cathepsins are potentially valuable new reagents for rapid and non-invasive imaging of atherosclerotic disease progression and plaque vulnerability.