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...
M atrix-disorganizing proteases, such as cathepsins or matrix metalloproteinases from macrophages, can destabilize atheromata, followed by plaque rupture to cause thromboembolic stroke or myocardial infarction. [1][2][3][4] We previously showed that molecular imaging of cathepsin-B (CatB) or matrix metalloproteinase-2/9 protease activity reflected the inflammatory component of atherosclerotic pathology in mice 5 and human atheromata. 6 We also showed that the protease imaging could quantitatively demonstrate plaque-stabilizing effects of antiatherosclerotic drugs 5 and exercise training 7 in mice. The principle of photodynamic therapy (PDT) is to kill unwanted cells by using a combination of photosensitizers and light illumination to generate highly reactive oxygen species that locally destroys cells over short diffusion distances.8-10 A recent PDT study using macrophage-targeted photosensitizers preliminarily demonstrated the viability of near-infrared lightactivated therapeutic nanoagents in the treatment of atherosclerotic vascular disease by showing preferential destruction of macrophages in vitro and in mouse atheromata. Objective-To investigate whether an intravenously injected cathepsin-B activatable theranostic agent (L-SR15) would be cleaved in and release a fluorescent agent (chlorin-e6) in mouse atheromata, allowing both the diagnostic visualization and therapeutic application of these fluorophores as photosensitizers during photodynamic therapy to attenuate plaquedestabilizing cathepsin-B activity by selectively eliminating macrophages. Approach and Results-Thirty-week-old apolipoprotein E knock-out mice (n=15) received intravenous injection of L-SR15 theranostic agent, control agent D-SR16, or saline 3× (D0, D7, D14). Twenty-four hours after each injection, the bilateral carotid arteries were exposed, and Cy5.5 near-infrared fluorescent imaging was performed. Fluorescent signal progressively accumulated in the atheromata of the L-SR15 group animals only, indicating that photosensitizers had been released from the theranostic agent and were accumulating in the plaque. After each imaging session, photodynamic therapy was applied with a continuous-wave diode-laser. Additional near-infrared fluorescent imaging at a longer wavelength (Cy7) with a cathepsin-B-sensing activatable molecular imaging agent showed attenuation of cathepsin-B-related signal in the L-SR15 group. Histological studies demonstrated that L-SR15-based photodynamic therapy decreased macrophage infiltration by inducing apoptosis without significantly affecting plaque size or smooth muscle cell numbers. Toxicity studies (n=24) showed that marked erythematous skin lesion was generated in C57/BL6 mice at 24 hours after intravenous injection of free chlorin-e6 and ultraviolet light irradiation; however, L-SR15 or saline did not cause cutaneous phototoxicity beyond that expected of ultraviolet irradiation alone, neither did we observe systemic toxicity or neurobehavioral changes. Conclusions-This is the first study showing that macrophage-se...
Inflammation in atherosclerotic plaques causes plaque vulnerability and rupture, leading to thromboembolic complications. Cathepsin B (CatB) proteases secreted by macrophages play a major role in plaque inflammation. We used a CatB-activatable near-infrared fluorescence (NIRF) imaging agent to demonstrate the inflammatory component in mice atheromata and the atherosclerosis- modulating effects of atorvastatin or glucosamine treatments. Apolipoprotein E knockout mice (n = 35) were fed normal chow, a Western diet, a Western diet + atorvastatin, a Western diet + glucosamine, or a Western diet + atorvastatin + glucosamine for 14 weeks. Twenty-four hours after the intravenous injection of a CatB-activatable probe, ex vivo NIRF imaging of the aortas and brains was performed, followed by histology. The CatB-related signal, observed in the aortas but not in the cerebral arteries, correlated very well with protease activity and the presence of macrophages on histology. Animals on Western diets could be distinguished from animals on a normal diet. The antiatherosclerotic effects of atorvastatin and glucosamine could be demonstrated, with reduced CatB-related signal compared with untreated animals. Plaque populations were heterogeneous within individuals, with some plaques showing a high and others a lower CatB-related signal. These differences in signal intensity could not be predicted by visual inspection of the plaques but did correlate with histologic evidence of inflammation in every case. This suggests that vulnerable inflamed plaques can be identified by optical molecular imaging.
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