Protease Imaging of Human Atheromata Captures Molecular Information of Atherosclerosis, Complementing Anatomic Imaging

Abstract

Objective-There is hope that molecular imaging can identify vulnerable atherosclerotic plaques. However, there is a paucity of clinical translational data to guide the future development of this field. Here, we cross-correlate cathepsin-B or matrix metalloproteinase-2/-9 molecular optical imaging data of human atheromata or emboli with conventional imaging data, clinical data, and histopathologic data. Methods and Results-Fifty-two patients undergoing carotid endarterectomy (41 atheromata) or carotid stenting (15 captured emboli) were studied with protease-activatable imaging probes. We show that protease-related fluorescent signal in carotid atheromata or in emboli closely reflects the pathophysiologic alterations of plaque inflammation and statin-mediated therapeutic effects on plaque inflammation. Inflammation-related fluorescent signal was observed in the carotid bifurcation area and around ulcero-hemorrhagic lesions. Pathologically proven unstable plaques had high cathepsin-B-related fluorescent signal. The distribution patterns of the mean cathepsin-B imaging signals showed a difference between the symptomatic vs asymptomatic plaque groups. However, the degree of carotid stenosis or ultrasonographic echodensity was weakly correlated with the inflammatory proteolytic enzyme-related signal, suggesting that molecular imaging yields complimentary new information not available to conventional imaging. Key Words: atherosclerosis Ⅲ cathepsin-B Ⅲ molecular imaging Ⅲ protease Ⅲ structural imaging C onventional imaging approaches such as angiography and ultrasound offer primarily structural information and yields limited data on plaque stability. The degree of carotid stenosis, as determined by structural imaging, is currently the most important therapeutic parameter in deciding on vascular intervention in addition to medical treatment. 1 There is hope that the emerging technologies of molecular imaging could provide a window of insight into the underlying molecular processes that give rise to plaque rupture. [2][3][4] Vulnerable plaques are characterized by the presence of inflammatory mediators and proteolytic enzymes, such as cathepsins and matrix metalloproteinases, which disturb the structural integrity of atheromatous plaques and consequently provoke plaque rupture to expose the lipid-rich plaque interior to thrombin-activating blood cascades. [5][6][7] It is the presence of these molecular species that distinguishes stable atheromatous lesions from unstable ones. We chose to leverage these molecular differences by devising imaging agents to probe for these enzymes. Conclusion-TheseWe and others 2,3,8,9 have developed protease-sensing nearinfrared fluorescent (NIRF) molecular imaging agents that are optically silent at injection because of auto-quenching between closely spaced fluorochromes. After enzyme-specific proteasemediated cleavage, fluorochromes are dequenced and become brightly fluorescent. 2,3,8,9 This technology has potential clinical applicability when combined with an intraoperative NIRF imaging...

Possible Pivotal Role of Latent Chronic T. gondii Infection in the Pathogenesis of Atherosclerosis

“…Lipoprotein modification and uptake by atherosclerotic lesion cells, mainly macrophages and VSMCs, are important pathological steps in the formation of atherosclerotic plaques [669,673,675,677,[680][681][682][683] . CatL [680,684,685] and CatB [686,687] expression is enhanced in human coronary atherosclerotic lesions, carotid lesions, and in abdominal aortic aneurysms [676] . CatB, CatL and CatD, were found in macrophage-derived foam cells in lipid-rich plaque areas [688,689] .…”

A New Micro–Computed Tomography–Based High-Resolution Blood–Brain Barrier Imaging Technique to Study Ischemic Stroke

“…The amount of iopromide-leakage was calculated as 'area x mean mCT density'. Furthermore, using ImageJ, color-coded maps for groups of animals were produced by summation of multiple maps with color coding to indicate the amount of lesion overlap: mean lesion maps 11 (for NIRF images and mCT images) and accumulation lesion (frequency) maps 10 (for digital images captured from the TTC-stained slices).…”

Direct Thrombus Imaging as a Means to Control the Variability of Mouse Embolic Infarct Models

“…Twenty-four hours later, the animals were euthanized, and the brains were removed and imaged ex vivo using a NIRF imaging machine 11,12,18 (Supplemental Methods). Fresh frozen sections of the brains were used for NIRF microscopic imaging (10-m-thick sections) or immediate triphenyl tetrazolium chloride staining (2-mm-thick sections) to delineate the infarct area, whereas some specimens were paraffin-embedded for histology.…”

“…3 Factors, such as spontaneous lysis 8 or distal embolization of thrombi after injection into the MCA, as well as anatomic variations in the circle of Willis, could have major and variable impacts on the resulting infarct. 10 This variability could mask and confound the potential therapeutic effects 3 of neuroprotective drugs, and by adding to experimental noise, might mask or heighten the effects of experimental treatments, leading to hard-to-interpret data.What is needed is an imaging methodology that would allow the visualization of thrombi [11][12][13][14] and their characterization after injection into the cerebral vasculature; this would control for at least some of the experimental variability related to clot lysis, migration, and fragmentation.To address this issue, we labeled thrombi optically with a molecular imaging thrombus marker-a 15-amino acid pep- When inducing embolic strokes in mice, researchers monitor the decrease of cerebral blood flow (CBF) in the MCA with a laser-Doppler flowmeter. We hypothesized that not only CBF decrease, 17 but also thrombus location and status, would affect the induced infarct territory or size, which is one of the most important outcomes in stroke research.…”

“…What is needed is an imaging methodology that would allow the visualization of thrombi [11][12][13][14] and their characterization after injection into the cerebral vasculature; this would control for at least some of the experimental variability related to clot lysis, migration, and fragmentation.…”