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 noninvasively 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 noninvasively imaged by optical and small-animal PET/CT at different time points. After noninvasive 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 sodium dodecyl sulfate polyacrylamide gel electrophoresis 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 than 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 neointima and adventitia of the ligated left carotid arteries compared with the right. Analysis of the human plaque tissues by sodium dodecyl sulfate polyacrylamide gel electrophoresis 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 colocalization 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 noninvasively 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 noninvasive imaging of atherosclerotic disease progression and plaque vulnerability.

Cysteine proteases in atherosclerosis

“…took a major step towards translating molecular imaging into clinical practice by developing a dual‐modality ABP for positron emission tomography–computed tomography and optical analysis. That study showed that the novel molecular agent can accurately discriminate between inflamed and normal arteries in vivo , and ex vivo sections from these animals further supported their findings by showing that the fluorescence signal colocalized with activated macrophages . The authors also demonstrated encouraging data using this imaging modality for idiopathic pulmonary fibrosis in human subjects , which further support their utility in the clinical arena.…”

Optical Nanoparticles for Cardiovascular Imaging

“…Hence, it is possible to find other examples of activatable optical probes for plaque imaging based on the selective detection of Cathepsin B. This is the case for the work published by Withana et al who developed a molecular activatable probe sensitive to Cathepsin B whose structure is schematically drawn in Figure a . This molecular fluorescent structure showed a great intensity enhancement in presence of Cathepsin B and the authors used it for in vivo plaque imaging in an experimental atherosclerotic murine model.…”

“…c) Corresponding in situ fluorescence imaging of the activatable fluorescent probe in murine carotid arteries and control healthy mouse. Reproduced with permission . Copyright 2016, Society of Nuclear Medicine and Molecular Imaging.…”

Molecular Imaging of Hydrolytic Enzymes Using PET and SPECT

“… 102 The PET/SPECT probes for cathepsins have been recently reviewed, 103 and only a few articles on nuclear imaging of cathepsins have appeared since. 104 - 106 …”