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. 2009 Oct;29(10):1444-51.
doi: 10.1161/ATVBAHA.109.193086. Epub 2009 Jul 16.

Hybrid in vivo FMT-CT imaging of protease activity in atherosclerosis with customized nanosensors

Matthias Nahrendorf  1 , Peter Waterman, Greg Thurber, Kevin Groves, Milind Rajopadhye, Peter Panizzi, Brett Marinelli, Elena Aikawa, Mikael J Pittet, Filip K Swirski, Ralph Weissleder
Affiliations

Hybrid in vivo FMT-CT imaging of protease activity in atherosclerosis with customized nanosensors

Matthias Nahrendorf et al. Arterioscler Thromb Vasc Biol. 2009 Oct.

Abstract

Objective: Proteases are emerging biomarkers of inflammatory diseases. In atherosclerosis, these enzymes are often secreted by inflammatory macrophages, digest the extracellular matrix of the fibrous cap, and destabilize atheromata. Protease function can be monitored with protease activatable imaging probes and quantitated in vivo by fluorescence molecular tomography (FMT). To address 2 major constraints currently associated with imaging of murine atherosclerosis (lack of highly sensitive probes and absence of anatomic information), we compared protease sensors (PS) of variable size and pharmacokinetics and coregistered FMT datasets with computed tomography (FMT-CT).

Methods and results: Coregistration of FMT and CT was achieved with a multimodal imaging cartridge containing fiducial markers detectable by both modalities. A high-resolution CT angiography protocol accurately localized fluorescence to the aortic root of atherosclerotic apoE(-/-) mice. To identify suitable sensors, we first modeled signal kinetics in-silico and then compared 3 probes with oligo-L-lysine cleavage sequences: PS-5, 5 nm in diameter containing 2 fluorochromes, PS-25, a 25-nm version with an elongated lysine chain and PS-40, a polymeric nanoparticle. Serial FMT-CT showed fastest kinetics for PS-5 but, surprisingly, highest fluorescence in lesions of the aortic root for PS-40. PS-40 robustly reported therapeutic effects of atorvastatin, corroborated by ex vivo imaging and qPCR for the model protease cathepsin B.

Conclusions: FMT-CT is a robust and observer-independent tool for noninvasive assessment of inflammatory murine atherosclerosis. Reporter-containing nanomaterials may have unique advantages over small molecule agents for in vivo imaging.

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Conflict of interest statement

Conflict of Interest Disclosures

Kevin Groves and Millind Rajopadhye are employees of VisEn Medical. Ralph Weissleder is a consultant to VisEn Medical. Ralph Weissleder and Peter Waterman hold VisEn shares.

Figures

Figure 1
Figure 1. Probe design and characterization
A: scheme of three different probe designs (PS: protease sensor). White and red stars depict quenched and unquenched fluorochromes, respectively. B: Structure of protease cleavage site on respective PS. C: Size exclusion HPLC (SEC) of PS-5, PS-25 and PS-40 and the used fluorochrome as a low molecular weight (1 kDa) reference reflects differences in size. The largest sensor, PS-40, is eluted first, and the smallest last. D: Fluorescence of inactive (blue) and activated sensors shows the emission peak and the activation after incubation with trypsin. Sufficient quenching is achieved for all three sensors, however, PS-40 is most silent in its inactive form and has therefore the highest increase in fluorescence when activated. E: Comparison of fluorescence intensity of trypsin activated probes at equal concentrations in PBS. F: In vitro activation of PS-25 with different enzymes. The highest activation efficacy was found for incubation with cathepsin B.
Figure 2
Figure 2. Pharmacokinetics and ex vivo imaging of activation
A: Blood half life of protease sensors. B: Modeling results for protease sensors of different size and design. The fluorescence inside inflamed atherosclerotic plaques was simulated taking into account pharmacokinetics, cellular uptake and wash in/out of target tissue, and suggests that PS-40 reaches the highest fluorescence intensity inside plaques and has slowest wash out kinetics. C–E: Fluorescence reflectance imaging (FRI) of excised aortas from apoE−/− mice and wild type mice (insets in panel C) 24 hours after injection of respective protease sensor. The signal intensity was significantly higher in plaque (Plq, arrow) when compared to vascular territories without lesions (background, BG, arrowhead) and highest for PS-40 (D). However, the target to background ratio, which normalizes fluorescence in plaques to signal in non-diseased vascular territory, is sufficiently higher in apoE−/− than in wild type mice on normal and on high fat diet for all three sensors (E). * p < 0.05. F and G: Dual-channel FRI of aortas from apoE−/− mice that were co-injected with spectrally resolved protease sensors. For this experiment, a higher wavelength version of PS-25 was used (excitation/emission 750/780). Overall similar activation mapping was observed, with small regional differences between respective sensors.
Figure 3
Figure 3. In vivo FMT-CT
In vivo Fluorescence Molecular Tomography-Computed Tomography imaging (FMT-CT). A–C: Image co-registration is based on fiducial landmarks (arrows) that are incorporated into the animal holder and are identifiable on CT (A) and FMT (B). The software co-aligns these fiducials to create a hybrid data set (C). Fluorescence signal in the aortic root of an apoE−/−mouse is encircled. D–F: 2-dimensional FMT-CT long axis views of apoE−/− mice injected with respective protease sensors. Fluorescence signal is observed in the aortic root and arch, regions with high plaque load and high ex vivo fluorescence signal (as shown in Figure 2). G–I: CT only views of D–F. Arrow heads depict vascular calcification, likely colocalizing with plaques. J–R: 3-dimensional maximum intensity projection of hybrid data sets show skeletal and vascular anatomy and the distribution of fluorescence signal. Most signal is observed in the root and arch, however, Q and R show additional activation of the protease sensor in the carotid artery, also a region predisposed to atheroma build-up in this model. S-U: FMT-CT after injection of respective sensor into wild type mice. V: Fluorochrome concentration reflecting protease activity plotted over time. W: Protease activity 24 hours after injection of sensors. *p<0.05.
Figure 4
Figure 4. Microscopic mapping of probe activation
Immunofluorescence microscopy of cathepsin B presence (green, emission 620nm), DAPI-stained DNA in cell nuclei (blue, emission 460nm) and protease sensor activation (red, emission 700nm) shows colocalisation in atherosclerotic plaques (yellow, arrows). Left panel shows histology from apoE−/− mouse injected with PS-5, middle PS-25, and right PS-40. The error bar denotes 50µm.
Figure 5
Figure 5. Flow cytometric profiling of cellular signal contributions
Flow cytometric profiling of cellular signal contributions in liquified aortas and background fluorescence in the blood 24 hours after injection of protease sensors into 3 age-matched apoE−/− mice. A: Incubation of cell suspensions with an antibody cocktail enabled multicolor flow cytometric analysis of lymphoid cells, myeloid cells including monocytes/macrophages and neutrophils, and other cells, including stromal cell of the aortic wall. Histograms for these cell types show the signal intensity in the protease sensor channel, which is dominated by myeloid cells in the aorta for all sensors. In the blood, marked differences can be observed. While there is hardly any blood signal after injection of PS-5, myeloid cell signal is observed after injection of PS-25 and even more so for PS-40. B: Mean fluorescence intensity per cell type. Monocyte/macrophages are the dominant cellular signal source for all 3 sensors. The highest mean fluorescence intensity per cell is observed after injection of PS-40.
Figure 6
Figure 6. FMT-CT detection of therapy with PS-40
The protease sensor with the highest sensitivity, PS-40, was used in a therapy trial to investigate its potential as a non-invasive imaging biomarker in drug development. A–B: FMT-CT data sets in respective treatment groups. C: Fluorescence activity in the aortic root measured by FMT, 24 hours after injection of PS-40. The protease sensor detected effects of atorvastatin treatment. *p<0.05. D–E: Ex vivo fluorescence reflectance imaging of excised aortas corroborated in vivo FMT data. *p<0.05. F: Quantitative PCR analysis showed lower translation of cathepsin B in apoE−/− treated with atorvastatin, confirming the imaging data. *p<0.05. G: Plasma cholesterol levels in respective trial groups.
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