Protease Imaging of Human Atheromata Captures Molecular Information of Atherosclerosis, Complementing Anatomic Imaging
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.
Conclusion— These results could justify and facilitate clinical trials to evaluate the use of protease-sensing molecular optical imaging in human atherosclerosis patients.
Conventional 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.2–4
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.5–7 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.
We and others2,3,8,9 have developed protease-sensing near-infrared fluorescent (NIRF) molecular imaging agents that are optically silent at injection because of auto-quenching between closely spaced fluorochromes. After enzyme-specific protease-mediated 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 system or fluorescence-sensing catheter-based system,4 as shown by multiple preclinical studies. Because NIR photons may travel up to 5 cm deep into the body, noninvasive fluorescent tomography systems may eventually detect NIRF signals from human carotid atheromata.10,11
However, a wide translational gap12 exists between the laboratory and atherosclerosis clinic. In particular, there is no prospective study comparing NIRF molecular imaging data with clinical data. We undertook the present study to partially bridge this gap and generate data that might be useful in the justification and design of future human trials of cathepsin-B (CatB) and matrix metalloproteinase (MMP)-2/-9 based NIRF imaging. In this prospective study, we estimate the potential clinical efficacy of these agents by cross-correlating molecular imaging data derived from patient tissues of either carotid endarterectomy or stenting with clinical data and histopathology data.
Materials and Methods
This study was approved by the Institutional Review Boards of the Dongguk University Ilsan Hospital and Asan Medical Center, Korea. A full description of the methods used is available (please see http://atvb.ahajournals.org).
From July 2006 to June 2008, 52 patients with carotid stenosis were enrolled in this study (41 men, 11 women; age±SD, 67.0±9.0 years). Thirty-seven patients were treated with carotid endarterectomy (n=41 specimens, 4 bilateral surgeries) in the Division of Vascular Surgery at Asan Medical Center. The other 15 were patients from the Department of Neurology at Dongguk University Ilsan Hospital who underwent carotid angioplasty with stenting and had emboli collected in a vascular protection device during the procedure. All the patients underwent systemic investigations, including assessment of ischemic cerebral events, vascular risk factors, medication history, duplex ultrasonography, MRI with MRA, transfemoral cerebral angiography (13 of the 37 surgical patients), and other routine admission laboratory tests. The Table summarizes demographic and clinical features of the enrolled cases for CatB plaque imaging (n=31), CatB emboli imaging (n=15), MMP-2/-9 plaque imaging (n=6), and control plaque imaging (n=4).
Human Carotid Atheroma and Emboli
Atherosclerotic plaques were obtained at carotid endarterectomy. The fresh carotid specimen were washed with normal saline and tissue-cultured in DMEM (3 mL) with either 2 nmol CatB-activatable probe (ProSense-680; Visen Medical) or 3 nmol MMP-2/-9–activatable probe (synthesized as described previously8,9 with some modification; please see http://atvb.ahajournals.org for further details with probe characterization) in 37°C CO2 incubator. For control imaging, DMEM without the probes was used. Before and 24 hours after the incubation, molecular optical imaging was performed using a NIRF imaging machine with a charge-coupled device camera (CoolSnap-EZ; Roper Scientific).
In the angioplasty group, emboli dislodged from atherosclerotic plaques were obtained from the protection device. They were washed with normal saline and tissue-cultured in DMEM (1 mL) with the CatB probe (0.67 nmol). Before and 2 hours after the incubation, NIRF reflectance imaging was performed, and then the specimens were paraffin-embedded. The emboli imaging was performed to estimate potential clinical usefulness of in vivo human CatB imaging by seeing if the vulnerable portion of a plaque, whose matrix is fragile enough to be easily separated from the plaque body and to be embolized to cause stroke in association with the procedure, would have strong proteolytic enzyme activity.
Conventional Imaging-Based and Molecular Imaging-Based Plaque Characterization
Sonographic plaque characterization13 was performed to obtain plaque stenosis estimates (percentage reduction of diameter), classify the degree of carotid stenosis (normal, <50%, 50%–69%, ≥70% stenosis, or near occlusion), measure overall plaque echodensity with computer assistance, and type carotid plaque based on ultrasonic heterogeneity or echolucency. Mechanism of cerebral infarction was determined as previously described.14 Then, plaques that had caused a recent ipsilateral embolic stroke within 1 month were classified as symptomatic plaques.
NIRF reflectance imaging and lesion quantification were performed as published before,3 with some modification. After the normalization, median NIRF signal intensities (grayscale pixel intensities from 0–255) from each entire carotid endarterectomy specimen and subregions were calculated. The subregions included bulb, proximal portion of the proximal internal carotid artery (ICA), distal portion of the proximal ICA, common carotid artery, and external carotid artery (Figure 1A). To analyze the anatomic distribution of the protease-related signal, the mean CatB signal or mean MMP signal was mapped on a template (averaged accumulation map). Median NIRF signal intensities were calculated for CatB emboli images, too.
Each carotid specimen was examined grossly for macroscopic plaque ulceration and surface thrombus. Fresh-frozen microsections (5-μm thickness) were used for fluorescence microscopy imaging, hematoxylin and eosin staining, Masson-trichrome staining, and immunohistochemical staining (CatB, MMP-2, MMP-9, macrophages). Immunohistochemistry was performed using the avidin-biotin-peroxidase method. A vascular pathologist who was blinded to the clinical and imaging data examined all the gross/micropathology data. Semiquantitative pathological examination was performed to identify “definitely unstable plaques”15 (AHA grade VI16), which were defined as having rupture as well as thrombus, large hemorrhage, and thin inflamed cap.
NIRF CatB or MMP Imaging Senses the Activity of Inflammatory Proteases From Macrophages In or Around the Bulb and Complicated Areas of Human Carotid Plaques
NIRF CatB or MMP imaging reflected the proteolytic enzyme activities from macrophages in complicated human atheromata (Figures 1 and 2⇓). Ulcerated, hemorrhagic lesions were present at pathology in 18 of 41 endarterectomy specimens and showed strong signal in or around the ulcerations in 14 (77%) cases (11 of 13 lesions imaged with CatB, and 4 of 6 lesions imaged with MMP). Negligible NIRF signal was detected in the control imaging without the probes (Figure I).
In the mean CatB images or MMP images, strong protease-related signal was localized to the carotid bifurcation area (Figure 3). Quantitative data corroborated this (Table I). Briefly, strong CatB signal clusters (the highest or second-highest intensity lesions in the 5 subregions of each plaque) were observed mostly in the carotid bulb (18 of 23 cases) or in the proximal portion of the proximal ICA (19 of 23 cases). Likewise, most of strong MMP signal clusters were located in the bulb or the proximal portion of the proximal ICA (5 of 6 cases).
CatB activities did not differ between the carotid plaques from the patients with recent-onset ipsilateral embolic strokes within 1 month (n=11) and those with no such stroke in the past month (n=20; Mann–Whitney test; P=0.95). The mean CatB images of the 2 groups, however, revealed a qualitative difference (Figure 3). In the symptomatic plaques, a bigger and stronger signal was located in the bulb area, whereas the signals from asymptomatic ones were scattered over somewhat larger areas.
NIRF CatB Imaging of Emboli Dislodged During Carotid Stenting Reveals Strong Protease-Related Signal Corresponding to Inflammatory Changes
We performed CatB imaging of emboli that had been dislodged from the carotid atheromata during angioplasty and stenting procedure (Figure 4). All the emboli collected in the protection device had strong CatB-related activity relating to macrophages. The CatB activities tend to be higher in the cases complicated with stenting-associated acute embolic cerebral infarctions (n=11; median=66 arbitrary units [AU]) than in those without such lesions (n=4; median=53 AU) on diffusion-weighted MRI performed at 24 hours after the intervention. However, this association was not statistically significant (P=0.09; Mann–Whitney test).
The Degree of Carotid Stenosis or Ultrasonographic Echolucency Is Weakly Correlated With the CatB NIRF Signal Intensity
CatB NIRF signal intensity correlated with the degree of carotid stenosis (Figure 5A), diameter reduction measured on longitudinal section images of the duplex ultrasonography (r=0.51; P=0.005). MMP imaging data showed a similar trend (Figure 5B). Ten of the 15 (67%) cases with 80% to 99% stenosis had CatB activities higher than the median value of 87.8 AU (Figure 5C). Only 5 of the 15 (33.3%) cases with <80% stenosis had CatB activities higher than the median activity. However, it should be noted that all but 2 cases (6/8) in the 70% to 79% range of stenosis, in which carotid endarterectomy is indicated according to the current practice guidelines,1 had CatB activities lower than the median value.
In the CatB cases, duplex ultrasonography and angiography showed that the most stenotic portions were in the distal portion of the proximal ICA (n=12), proximal portion of the proximal ICA (n=6), or carotid bulb (n=5). In the MMP cases, the most stenotic portions were in the distal portion of the proximal ICA (n=2), proximal portion of the proximal ICA (n=3), or carotid bulb (n=1). CatB signal intensity in the most stenotic portion of each plaque was strong in 14 of 23 cases. MMP signal intensity in the most stenotic portion of each plaque was strong in 4 of 6 cases. It is notable that not infrequently (9 of 23 in CatB cases and 2 of 6 MMP cases), the most stenotic portions had only weak signals.
Grayscale median values of echodensities measured from 65 regions of interest on the duplex ultrasonography tended to be inversely and weakly correlated with the median CatB signal intensities from corresponding areas on the molecular optical imaging (Figure 5D), which showed a marginal significance (r=0.24; P=0.06). In addition, echolucent or heterogeneous plaques were non-significantly associated with high protease activities (please see http://atvb.ahajournals.org for further details).
CatB NIRF Signal Intensity Is Lower in the Plaques From Patients With Statin Medication
CatB activities tended to be lower in the statin users than in the nonusers (median=88.2 vs 93.3 AU; P=0.12; Mann–Whitney test). When the CatB activities were dichotomized, the lower CatB group was more frequently associated with statin use (10/15) than the higher CatB group was (5/16; P=0.049; χ2 test; Table II).
Pathology-Proven Definitely Unstable Plaques Are Relatively Frequent in the Higher CatB Group Compared With the Lower CatB Group
Semiquantitative pathological examination could be performed in selected cases (22 of the 31 CatB plaque imaging) because tissue status of some carotid specimens was not optimal for histopathologic examinations, probably because of the long incubation time. When CatB activities were dichotomized based on the median value of the 22 cases (86.6 AU), in the higher CatB group 5 of 11 (45%) carotid plaques were classified as definitely unstable. In the lower CatB group, 1 out of 11 (9%) carotid plaques were classified as definitely unstable. Such an association was not observed when the cases were dichotomized based on the median echodensities (22.0 AU); there were 3 definitely unstable plaques (27%) in both the higher (n=11) and lower (n=11) echodensity groups.
Conventional imaging approaches including angiography, ultrasound, and MRI/MRA offer primarily structural information, whereas molecular imaging can provide underlying molecular information on pathological processes such as inflammatory protease activities.2–4 We present observational evidence that plaque vulnerability can be assessed by means of molecular imaging, which complements and adds to traditional anatomic information.
To investigate the relationship between structural and molecular information related to the carotid atheromata, we correlated protease-related signal levels with the degree of stenosis and ultrasonographic appearance. We also compared protease signal intensity profiles to the site of maximum anatomic stenosis. We showed that the carotid plaques from the patients with higher-degree stenosis were more inflammatory, as judged by higher CatB or MMP activity, than those with lower-degree stenosis were. However, the correlations were rather weak, and not infrequently the most stenotic portion of a plaque had relatively weak protease activity. Thus, there is correspondence but also some informative divergence between anatomic and molecular imaging. On ultrasound, vulnerable plaques are thought to be echolucent and heterogeneous.17,18 We showed that CatB protease activity tended to be stronger in the heterogeneous echolucent plaques; the correlation, however, was not strong. This again suggests that molecular imaging provides new data not accessed by anatomy-based imaging.
Histological composition of the carotid plaque has been associated with plaque instability and presenting vascular symptoms,19 influencing the prognosis and therefore the indication for carotid intervention.17 We demonstrated that the molecular optical imaging could sense atherosclerosis pathophysiology. Geometrically, higher CatB or MMP-related signals were observed in the bifurcation area than in the straight portions, including the common carotid artery or distal part of the proximal ICA. Unlike straight arteries, the regions of stenosed or branched arteries being exposed to disturbed flow conditions are prone to atheromata development with high protease activities.20,21 These proteases go on to fragment elastins and probably break-up the fibrous plaque.21 In our study, strong protease activity was frequently observed in or around the unstable ulcerated and hemorrhagic areas of the carotid atheromata. This is in agreement with a recent immunohistochemical study22 and an in situ hybridization study.23 However, it is important to note that currently available antibodies do not distinguish active proteases from inactive precursors that lack proteolytic capacity, and thus our study based on the activatable NIRF probes is likely yielding information more applicable to the pathophysiology of plaque rupture.3
Previously, carotid endarterectomy specimens excised as intact cylinders were subjected to a standardized angioplasty procedure under radiological guidance in an ex vivo pulsatile flow model.24 Macrophage infiltration within the plaques correlated with emboli number and the plasma MMP-9 level.24 In the actual clinical setting, we demonstrated that all the emboli from carotid plaques, which were collected in each patient’s blood stream in vivo, had strong CatB-related imaging signal. These emboli are likely fragile portions of plaques, vulnerable to dislodgement and embolization during the catheterization, and having high proteolytic enzyme activity. We therefore believe that protease-sensing molecular imaging has potential to provide in vivo histopathology data reflecting the vulnerability of atherosclerotic plaques.
The ideal tool to identify vulnerable plaques would allow detection of lesions at high risk for vascular events and would allow the assessment of risk-altering treatments so that the success of therapy can be judged in a timely manner. In fact, pathology-proven definitely unstable plaques were more frequently found in the higher CatB group than in the lower CatB group. Moreover, therapeutic effect of statin could be reflected by the molecular optical imaging, although the CatB-lowering effect of statin was not accompanied by lower serum cholesterol levels.25 Contrary to our expectations, total CatB-related fluorescence did not differ in the plaques from the patients with recent-onset ipsilateral embolic strokes within 1 month and those without such a stroke. This might be partly explained by a selection bias; patients with definitely “stable” plaques, optimal negative controls, would have a relatively low chance of getting the surgery and being enrolled in the study. Nevertheless, CatB molecular imaging did show a difference in the distribution pattern of the enzyme activity signal. In the symptomatic plaques, stronger signal was densely concentrated in the bulb area, whereas the signal from asymptomatic plaques was more diffuse. Hypothetically, if strong CatB activity is focally localized to a critical structure such as a thin fibrous cap overlying a necrotic core, total protease activity may not represent actual vulnerability of the plaque. In this context, molecular imaging and structural imaging might have to complement each other, rather than each serving as a stand-alone technique, for the identification of vulnerable plaques. In conclusion, our study suggests that future prospective clinical trials to evaluate the use of protease-based molecular imaging technologies in human atherosclerosis patients are warranted.
The authors thank Hyunjin Chung for the NIRF microscopy imaging, and Ju Hee Ryu for helping in vitro characterization of the MMP probe.
Sources of Funding
Supported by a grant (A08020) of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea.
Received July 17, 2009; revision accepted November 4, 2009.
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