Cathepsin Activity-Based Probes and Inhibitor for Preclinical Atherosclerosis Imaging and Macrophage Depletion

<div><p>Background and Purpose</p><p>Cardiovascular disease is the leading cause of death worldwide, mainly due to an increasing prevalence of atherosclerosis characterized by inflammatory plaques. Plaques with high levels of macrophage infiltration are considered “vulnerable” while those that do not have significant inflammation are considered stable; cathepsin protease activity is highly elevated in macrophages of vulnerable plaques and contributes to plaque instability. Establishing novel tools for non-invasive molecular imaging of macrophages in plaques could aid in preclinical studies and evaluation of therapeutics. Furthermore, compounds that reduce the macrophage content within plaques should ultimately impact care for this disease.</p><p>Methods</p><p>We have applied quenched fluorescent cathepsin activity-based probes (ABPs) to a murine atherosclerosis model and evaluated their use for <i>in vivo</i> imaging using fluorescent molecular tomography (FMT), as well as <i>ex vivo</i> fluorescence imaging and fluorescent microscopy. Additionally, freshly dissected human carotid plaques were treated with our potent cathepsin inhibitor and macrophage apoptosis was evaluated by fluorescent microscopy.</p><p>Results</p><p>We demonstrate that our ABPs accurately detect murine atherosclerotic plaques non-invasively, identifying cathepsin activity within plaque macrophages. In addition, our cathepsin inhibitor selectively induced cell apoptosis of 55%±10% of the macrophage within excised human atherosclerotic plaques.</p><p>Conclusions</p><p>Cathepsin ABPs present a rapid diagnostic tool for macrophage detection in atherosclerotic plaque. Our inhibitor confirms cathepsin-targeting as a promising approach to treat atherosclerotic plaque inflammation.</p></div

Non-invasive imaging of plaques in murine atherosclerosis.

<p>Diabetic, fat-fed mice with a ligated carotid artery were injected with non-quenched probe GB123 or quenched probe GB137 as indicated. Fluorescent molecular tomography (FMT) was used to monitor and follow the pharmacokinetics and signal accumulation in plaques. <b>(a, b)</b> Left images: front overlay of fluorescence and bright field. Middle images: side view of fluorescence alone. These images show strong fluorescence signal (arrows) (GB123 at 4 hours and GB137 at 2 hours post probe injection) around the ligated left carotid artery. Right images show <i>ex vivo</i> fluorescent image of excised heart and carotid arteries (ligated artery is marked).</p

Macrophage labeling with fluorescent activity based probe.

<p>Ligated and control carotid arteries from mice treated with GB123 (<b>a</b>) or GB137 (<b>b</b>) (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160522#pone.0160522.g001" target="_blank">Fig 1</a>) were embedded in OCT and serial sectioned. Samples were stained for F4/80, a macrophage marker, and scanned by a confocal microscope: DAPI (blue), Cy5 labeled by probe (red), F4/80 (green), yellow color is overlay of red and green fluorescence. Cathepsin probes were found to co-localize with F4/80 macrophages.</p

Cathepsin inhibitor induces specific macrophage apoptosis.

<p>Freshly excised human atherosclerotic tissue samples were treated with the cathepsin inhibitor GB111-NH₂ for 24 hours. Serial frozen sections were stained for CD68 and cleaved caspase-3 and visualized by a confocal microscope: DAPI (blue), cleaved caspase-3 (green), CD68 (red), yellow color is overlay of red and green fluorescence. GB111-NH₂ was found to induce specific macrophage cell death (a). Co-localization analysis of CD68 and cleaved Caspase 3 positive cells. Bar graphs present the fraction of apoptotic macrophages out of total CD68 population (b) and the fraction of macrophages out of total apoptotic cells is shown in (c). Data is mean ± SEM (n = 3).</p