Abstract
Tissue factor (TF), a transmembrane glycoprotein that acts as an essential cofactor to factor VII/VIIa, initiates the exogenous blood coagulation cascade leading to thrombin generation and subsequent thrombus formation in vivo. TF expression is closely related to plaque vulnerability, and high TF expression is shown in macrophage-rich atheromatous lesions, making TF a potential target for detecting atheromatous lesions in vivo. Thus, we prepared 99mTc-labeled anti-TF-monoclonal antibody (TF-mAb) IgG as a molecular probe and evaluated its usefulness to achieve TF-specific imaging using myocardial infarction–prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. Methods: Anti-TF-mAb was created using a standard hybridoma technique and was labeled by 99mTc with 6-hydrazinonicotinic acid (HYNIC) as a chelating agent to obtain 99mTc-TF-mAb. The immunoreactivity of HYNIC-TF-mAb was estimated by flow cytometry. WHHLMI and control rabbits were injected intravenously with 99mTc-TF-mAb. Twenty-four hours after the injection, the aorta was removed and radioactivity was measured. Autoradiography and histologic studies were performed using serial aorta sections. Subclass matched antibody (IgG1) was used as a negative control. Results: HYNIC-TF-mAb showed 93% immunoreactivity of the anti-TF-mAb. The radioactivity accumulation in WHHLMI aortas was 6.1-fold higher than that of control rabbits. Autoradiograms showed a heterogeneous distribution of radioactivity in the intima of WHHLMI aortas. Regional radioactivity accumulation was positively correlated with TF expression density (R = 0.64, P < 0.0001). The highest radioactivity accumulation in percentage injected dose × body weight/mm2 × 102 was found in atheromatous lesions (5.2 ± 1.9) followed by fibroatheromatous (2.1 ± 0.7), collagen-rich (1.8 ± 0.7), and neointimal lesions (1.8 ± 0.6). In contrast, 99mTc-IgG1 showed low radioactivity accumulation in WHHLMI aortas that was independent of the histologic grade of lesions. Conclusion: The TF-detecting ability and preferential accumulation in atheromatous lesions of 99mTc-TF-mAb were demonstrated, indicating its potential for selective imaging of macrophage-rich atheromatous lesions in vivo.
Thrombus formation triggered by plaque rupture is the most important mechanism leading to the onset of acute arterial disease and ischemic sudden death. Thus, the development of a method for detecting thrombus-forming vulnerable plaques before rupture has been clinically desired to more precisely estimate risk and provide effective treatment. Although several molecular imaging probes have been investigated (1,2), the target molecules of such probes were not directly related to the thrombotic process.
Tissue factor (TF), a transmembrane glycoprotein that acts as an essential cofactor to factor (F) VII/VIIa, initiates the exogenous blood coagulation cascade leading to thrombin generation and subsequent thrombus formation. TF expression was identified in atherosclerotic lesions, including in endothelial cells, smooth muscle cells, monocytes, and, especially, macrophages or foam cells (3). In human pathologic lesions, the TF content of de novo lipid-rich plaques was higher than that of stenotic fibrous plaques (4), and such lipid-rich plaque tissue was 6 times more thrombogenic than fibrous plaques. In addition, our recent study also demonstrated that TF expression was closely related to plaque vulnerability, with high TF expression specifically in macrophage-rich atheromatous lesions among heterogeneous atherosclerotic lesions (5). Given these data, TF is a potential target for probes detecting atheromatous lesions at higher risk for rupture in vivo.
In the present study, we prepared a monoclonal antibody to TF (TF-mAb) and labeled it with 99mTc (99mTc-TF-mAb) as a molecular probe. Using an atherosclerosis model (myocardial infarction–prone Watanabe heritable hyperlipidemic [WHHLMI] rabbits) (6), we investigated the accumulation of 99mTc-TF-mAb in atherosclerotic lesions in comparison with histologic characteristics and evaluated the potential of 99mTc-TF-mAb as a molecular probe for detecting vulnerable atheromatous lesions.
MATERIALS AND METHODS
Design and Preparation of 99mTc-TF-mAb and 99mTc-IgG1
A monoclonal antibody (mouse IgG1 subclass) for rabbit TF (193Ser-207Cys, extracellular domain) was established using a standard hybridoma technique. 99mTc-pertechnetate was eluted in saline solution on a daily basis from 99Mo–99mTc generators (Ultra-Techne Kow; FUJIFILM RI Pharma Co., Ltd.).
Anti-TF-mAb was radiolabeled with 99mTc (99mTc-TF-mAb) after derivatization with 6-hydrazinonicotinic acid (HYNIC) (7), as previously reported (8). In brief, HYNIC-N-hydroxysuccinimide was reacted with TF-mAb, and the mixture was purified by size-exclusion filtration with a diafiltration membrane (Amicon Ultra 4 [molecular weight cutoff, 30,000]; Millipore Co.). An equal volume of 99mTc-(tricine)2, prepared by the method of Larsen et al. (9), was added to the purified solution of HYNIC-TF-mAb to obtain 99mTc-TF-mAb. After purification by size-exclusion filtration with a PD-10 column, the radiochemical purity of 99mTc-TF-mAb was more than 95% by another size-exclusion filtration.
For the control study, negative control mouse IgG1 (0102-01; Southern Biotechnology Associates Inc.) was used for the preparation of 99mTc-IgG1. The radiochemical purity of 99mTc-IgG1 was also estimated to be more than 95%.
Animals
All animal procedures were approved by the Kyoto University Animal Care Committee. Three male Japanese White rabbits (age, 3 mo) were used to obtain peritoneal macrophages. For biodistribution studies of 99mTc-TF-mAb, 5 WHHLMI rabbits (4 male, 1 female; age, 12–18 mo; mean weight ± SD, 3.4 ± 0.2 kg; supplied by the Institute for Experimental Animals, Kobe University School of Medicine, Japan) were used. Four male Japanese White rabbits (age, 3 mo; mean weight ± SD, 1.9 ± 0.2 kg) were used for the control study. For 99mTc-IgG1 studies, 3 WHHLMI rabbits (1 male and 2 female; age, 11–12 mo; mean weight ± SD, 3.2 ± 0.1 kg) were used. The animals were fed standard chow and given water ad libitum.
Immunoreactivity of HYNIC-TF-mAb
Rabbit peritoneal macrophages were obtained by the method of Ishii et al. (10), with minor modifications. Cells were suspended at a final concentration of 2.5 × 106 cells/mL in medium A (Dulbecco's modified Eagle's medium containing 1 mM glutamine, 100 U of penicillin per milliliter, 100 mg of streptomycin per milliliter [pH 7.4], and 0.2% lactalbumin hydrolysate). Aliquots of the cell suspension were cultured in plastic petri dishes in a humidified 5% CO2 incubator at 37°C. After 2 h, each dish was washed twice with 10 mL of medium A to remove nonadherent cells. Monolayers were cultured for 18 h at 37°C in 20 mL of medium A, and cells were washed twice with 10 mL of medium A and then used for experiments. More than 95% of the cells were viable, as determined by a trypan blue exclusion test, and almost all of the attached cells showed positive nonspecific esterase staining.
Antibodies (5 μg/mL, 100 μL; TF-mAb, HYNIC-TF-mAb, or negative control IgG1) were added to the cells (106) and incubated for 30 min at 4°C. After cells were washed, Alexa Fluor 488 goat antimouse IgG antibody (x0931; DakoCytomation) (10 μg/mL, 100 μL) was added for 30 min at 4°C. Fluorescence levels were measured using a flow cytometer (Becton Dickinson Inc.). Data were analyzed using BD CellQuest Pro (BD Biosciences), and an immunoreactivity index was calculated as the ratio of the median fluorescence intensity for either TF-mAb or HYNIC-TF-mAb to that of negative-control IgG1. Measurements were performed 3 times per rabbit using 3 Japanese White rabbits, and the ratios were expressed as mean ± SD.
Biodistribution Studies
A simple schematic of our experimental protocol is shown in Figure 1. After 12 h of fasting, rabbits were initially anesthetized with ketamine (intramuscularly, 35 mg/kg) and xylazine (intramuscularly, 5 mg/kg). Either 99mTc-TF-mAb (547–1,024 MBq, 300 μg) or 99mTc-IgG1 (848–1,038 MBq, 300 μg) was injected into a marginal ear vein (5 WHHLMI rabbits and 4 control rabbits for the 99mTc-TF-mAb study, 3 WHHLMI rabbits for the 99mTc-IgG1 study). Twenty-four hours after the injection, animals were sacrificed by pentobarbital overdose. The ascending-arch, thoracic, and abdominal aortas, blood, and other tissues (muscle, lung, liver, kidneys, and heart) were removed. The ascending-arch aortas were divided into 6 segments, and the thoracic and abdominal aortas were divided into 9 segments. Each segment was weighed and immediately fixed in a solution containing L-(+)-lysine hydrochloride (75 mM) and 4% paraformaldehyde in phosphate buffer (37.5 mM, pH 7.4) (11). The radioactivity of each sample was measured with a well-type γ-counter (1480 Wizard 3″; PerkinElmer Japan Co.). The results were expressed as the differential uptake ratio (DUR), calculated as (tissue activity/tissue weight)/(injected radiotracer activity/animal body weight), with activities given in becquerels and weights in grams. The aorta-to-blood (A/B) ratio and the aorta-to-muscle (A/M) ratio were calculated from the DUR for each tissue sample.
Simple schematic of this study. ARG = autoradiography.
Autoradiography
Eight segments, the second and fifth segments of the ascending aortic arch and the second, fifth, and eighth segments from the thoracic and the abdominal aortas, from each animal were used for autoradiography studies. These segments were frozen and cut into 20-μm-thick slices with a cryomicrotome. The sections were thawed and mounted on silane-coated slides, which were then placed on a phosphor image plate (Fuji Imaging Plate BAS-MS; Fuji Photo Film) for 24 h together with a calibrated standard (99mTcO4− solution). The autoradiography images were analyzed with a computerized imaging analysis system (Bio Imaging Analyzer BAS2500 and Image Gauge Software; Fuji Photo Film). The radioactivity in each region of interest was expressed as percentage injected dose × body weight/mm2, calculated as (radioactivity in the region of interest)/(injected radioactivity/animal body weight).
Histologic Analysis
The tissue sections used for autoradiography studies were also subjected to Azan–Mallory and hematoxylin and eosin staining. Serial sections of the slices from the autoradiography studies were subjected to immunohistochemical staining (for TF, macrophages, and smooth muscle cells) using specific antibodies and an Envision+ kit (Dako) with hematoxylin counterstaining. The antibodies used were TF-mAb (4510; American Diagnostica Inc.), rabbit macrophage–specific mAb RAM-11 (Dako), and human smooth-muscle actin–specific mAb 1A4 (Dako). Immunostaining with subclass-matched irrelevant IgG served as a negative control. Azan–Mallory and hematoxylin and eosin staining were performed by standard procedures. TF expression density was determined as a percentage of the positively stained region using a VHX digital microscope (Keyence Corp.).
Classification of Atherosclerotic Lesions
We divided atherosclerotic lesions in WHHLMI rabbits into the following 4 categories, using a classification scheme based on the recommendations of the American Heart Association (12,13) and Azan–Mallory and hematoxylin and eosin staining, as previously described (14–17): neointimal (types I–III), atheromatous (type IV), fibroatheromatous (types Va and Vb), and collagen-rich (type Vc). Supplemental Figures 1A–1P (supplemental materials are available online only at http://jnm.snmjournals.org) show representative photomicrographs of the histologic features of each atherosclerotic lesion category in WHHLMI rabbits.
Regions of interest were placed to cover each atherosclerotic lesion in the aortic section of the WHHLMI rabbit and then transferred to the corresponding autoradiography images (Supplemental Figs. 1Q–1S).
Vulnerability Index
An index of morphologic destabilization characteristics, the vulnerability index, was calculated for each lesion in the WHHLMI rabbits by the method of Shiomi et al. (18). The vulnerability index was defined as the ratio of the lipid component area (macrophages and extracellular lipid deposits) to the fibromuscular component area (smooth muscle cells and collagen fibers). Collagen fibers and extracellular lipid deposits (extracellular vacuoles and lacunae) were determined with Azan–Mallory staining. Macrophages and smooth muscle cells were determined with immunohistochemical staining (17).
Statistical Analysis
Data are presented as mean ± SD. Statistical analysis was performed with the Mann–Whitney U test to compare aortic segments of WHHLMI and control rabbits (Table 1). Radioactivity that accumulated in nontargeted organs among antibodies and animals was compared using 1-way ANOVA, with post hoc analysis by the Holm test (Fig. 2). Correlation coefficients were assessed by Spearman rank correlation coefficients (Fig. 3). Lesion types were compared using the Kruskal–Wallis test, with post hoc analysis by the Scheffé test (Fig. 4). A 2-tailed value of P less than 0.05 was considered statistically significant.
Radioactivity distribution in lung, liver, kidneys, and heart. Data are mean ± SD. †P < 0.0001. ‡P < 0.001. $P < 0.01. #P < 0.05.
Regression analyses of TF expression density with 99mTc-TF-mAb (A) and 99mTc-IgG1 (B) accumulation. ■ = neointimal lesion;●= atheromatous lesion; × = fibroatheromatous lesion; ▲ = collagen-rich lesion.
Distribution profiles of radioactivity accumulation, TF expression, macrophage density, and vulnerability index in atherosclerotic lesions in 99mTc-TF-mAb (A) and 99mTc-IgG1 (B) study. A = atheromatous lesions; C = collagen-rich lesions; F = fibroatheromatous lesions; N = neointimal lesions. Data are represented as mean ± SD.
Accumulation Levels of 99mTc-TF-mAb and 99mTc-IgG1 in Aortic Segments of Control and WHHLMI Rabbits at 24 Hours After Injection
RESULTS
Immunoreactivity of HYNIC-TF-mAb
Using fluorescent-activated cell sorter analysis of rabbit peritoneal macrophages, we could clearly distinguish the signals of TF-mAb and HYNIC-TF-mAb from that of the negative control IgG1. The median fluorescence intensity ratios of TF-mAb and HYNIC-TF-mAb to control IgG1 were 2.90 ± 0.06 and 2.69 ± 0.11, respectively, and the difference between the labeled and unlabeled TF antibodies was not statistically significant.
Biodistribution Studies
Accumulation levels of 99mTc-TF-mAb and 99mTc-IgG1 in the aortic segments of WHHLMI and control rabbits are summarized in Table 1. The accumulation level of 99mTc-TF-mAb in each aortic segment of WHHLMI rabbits (ascending arch, 3.08 ± 0.57 DUR; thoracic, 3.07 ± 1.44 DUR; and abdominal, 2.49 ± 0.64 DUR) was 5.1- to 7.1-fold higher than that of control rabbits (ascending arch, 0.60 ± 0.05 DUR; thoracic, 0.51 ± 0.11 DUR; and abdominal, 0.35 ± 0.06 DUR), and the differences were significant in each case. Blood-pool radioactivity levels of 99mTc-TF-mAb at 24 h were 7.5 ± 0.0 and 4.0 ± 0.6 DUR in WHHLMI and control rabbits, respectively. A/B and A/M ratios were significantly higher in WHHLMI rabbits than in control rabbits (A/B, 0.38 ± 0.09 in WHHLMI and 0.12 ± 0.02 in control rabbits; A/M, 19.3 ± 19.1 in WHHLMI and 1.0 ± 0.6 in control rabbits). In addition, the level of 99mTc-TF-mAb accumulation in WHHLMI rabbit aortas was 1.5- to 3.3-fold higher than the level of 99mTc-IgG1 accumulation, and the differences were significant.
Relatively high radioactivity accumulations were found in the liver and kidneys of all 3 groups (Fig. 2). We observed that the 99mTc-TF-mAb cleared rather more slowly from the bodies of WHHLMI rabbits than from control rabbits.
Regional Distribution of 99mTc-TF-mAb, in Comparison with TF Expression
In the autoradiography study, heterogeneous 99mTc-TF-mAb accumulation was observed in the intima of WHHLMI rabbit aortas (Fig. 5B), whereas no marked accumulation was found in the aortas of control rabbits (Fig. 5A). Variable TF expression was detected in the intimal regions of the WHHLMI rabbit aorta (Figs. 5D, 5F, and 5G). Higher accumulation levels of 99mTc-TF-mAb were found in regions with high TF expression, whereas lower accumulation was observed in regions with low TF expression (Fig. 5, compare 5B with 5F and 5G). Consequently, regional 99mTc-TF-mAb accumulation levels in the aorta section were positively correlated with TF expression density in WHHLMI rabbits (R = 0.64, P < 0.0001) (Fig. 3A). No obvious TF expression was observed in the aorta of control rabbits (Figs. 5C, 5E, and 3B).
Regional distribution of 99mTc-TF-mAb and TF expression in aortic sections. Autoradiogram (A and B) and TF immunohistochemical staining (C–G) of control (C and E) and WHHLMI rabbits (D, F, and G). (E–G) High-magnification images of TF immunohistochemical staining in regions depicted in C and D. Identical color window was applied to both autoradiographic images (A and B). Bar = 1 mm (A–D) and 100 μm (E–G).
Relationship Between 99mTc-TF-mAb Accumulation and Histologic Characteristics
The plaques were categorized according to histopathologic classification criteria as follows: neointimal (n = 12 for 99mTc-TF-mAb study and n = 7 for 99mTc-IgG1 study), atheromatous (n = 40 for 99mTc-TF-mAb study and n = 20 for 99mTc-IgG1 study), fibroatheromatous (n = 43 for 99mTc-TF-mAb study and n = 21 for 99mTc-IgG1 study), and collagen-rich (n = 62 for 99mTc-TF-mAb study and n = 36 for 99mTc-IgG1 study). No lesions showed hemorrhage, plaque rupture, or thrombi (type VI). The level of 99mTc-TF-mAb accumulation was dependent on the histologic grade of the lesions (Fig. 4A) and was prominently and significantly the highest (P < 0.0001) in atheromatous lesions (type IV), compared with other lesions. The accumulation level of 99mTc-TF-mAb was 3.0-, 2.4-, and 2.9-fold higher in atheromatous lesions than in neointimal, fibroatheromatous, and collagen-rich lesions, respectively. The vulnerability index was also the highest in atheromatous lesions, followed in decreasing order by fibroatheromatous, neointimal, and collagen-rich lesions. Consequently, the highest level of 99mTc-TF-mAb accumulation and the highest vulnerability index were both observed in atheromatous lesions. In contrast, 99mTc-IgG1 accumulation in lesions was low and did not correlate with the histologic grade of lesions, with no significant differences among the lesion types (Fig. 4B).
DISCUSSION
In the present study, we designed a new imaging agent, 99mTc-TF-mAb, for the purpose of discriminating atherosclerotic lesions at higher risk for rupture (thrombogenic atheromatous lesions) from more stable lesions and evaluated the potential of 99mTc-TF-mAb using an atherosclerotic rabbit model. Our major findings are that a positive correlation was demonstrated between regional 99mTc-TF-mAb accumulation and TF expression density in atherosclerotic lesions of WHHLMI rabbits but not with 99mTc-IgG1 and that significantly higher 99mTc-TF-mAb accumulation was found in grade IV, more vulnerable atheromatous lesions, than in neointimal lesions or other more stable lesions. Thus, we demonstrate the potential of 99mTc-TF-mAb for molecular imaging of TF expression and selectively detecting atheromatous plaques at higher risk for rupture.
Immunoreactivity and Specificity of 99mTc-TF-mAb
Immunoreactivity, specificity, and detectable but functionally silent labeling are indispensable prerequisites of in vivo molecular imaging probes using immunodetection. In this study, flow cytometric analyses indicated that modification of TF-mAb with HYNIC did not significantly affect the immunoreactivity of the original TF-mAb. In addition, autoradiography and immunohistochemical studies showed that 99mTc-TF-mAb accumulation in atherosclerotic lesions correlated well with TF expression density, which was higher in atheromatous lesions, as expected (Figs. 3A and 4A). Further, contrary to the results with 99mTc-TF-mAb (Fig. 4A), the results with 99mTc-IgG1 (Fig. 4B) showed that accumulation of 99mTc-labeled nonspecific IgG in atheromatous lesions was not significantly different from that in other types of lesions (i.e., neointimal, fibroatheromatous, and collagen-rich lesions). These findings strongly suggest the potential of 99mTc-TF-mAb to specifically recognize TF in vivo.
TF as a Target Molecule for Plaque Imaging
TF, selected as a target molecule for molecular imaging in this study, initiates the exogenous blood coagulation cascade leading to thrombus formation in vivo and represents a good marker for late-stage vulnerable lesions. TF in atherosclerotic lesions was identified in several cell types, such as endothelial cells, smooth muscle cells, monocytes, macrophages, and foam cells (3), similar to lectinlike oxidized low-density lipoprotein receptor 1 (LOX-1). TF expression is reported to be increased in the later stages of atheromatous progression and thus was selectively detected in atheromatous lesions in this report (Fig. 4). These findings are comparable to those of our previous immunohistochemical study (5) and another human study (4). On these bases, TF should be a potential target for detecting atheromatous plaques at higher risk for rupture in vivo. To our knowledge, this is the first report of the development of an in vivo TF imaging probe.
On the other hand, a series of imaging agents has targeted fibrin and factor XIII in thrombi using antibodies or peptides (1), with at least partial success. In the blood-coagulation cascade, TF initiates the system, and factor XIII covalently cross-links fibrin polymers and renders the thrombus more resistant to lysis. Therefore, 99mTc-TF-mAb will be useful for the early detection of the cascade, and fibrin and factor XIII imaging probes can detect later stages and thrombi themselves. In this study, 99mTc-TF-mAb corresponded with TF expression and showed preferential accumulation in atheromatous lesions and in lesions with increased vulnerability. Although further studies are required to investigate which target molecules in the cascade are most appropriate to estimate how unstable or vulnerable a plaque is in vivo, TF is a potential target. Furthermore, because great efforts have been made in the development of anticoagulation and antiplatelet pharmaceuticals for the treatment of atherosclerosis and hyperlipidemia, effective imaging probes to target blood-coagulation cascades are also required for efficient drug development.
Limitations of 99mTc-TF-mAb
One drawback of 99mTc-TF-mAb is its relatively slow clearance from the blood, which is an intrinsic problem of molecular probes using antibodies. Recent advances in antibody engineering, however, should provide a promising solution for this issue. Radioprobes derived from low-molecular-weight polypeptides or compounds, small recombinant antibody fragments (Fab, scFv), engineered variants (diabodies, triabodies, minibodies, and single-domain antibodies), or pretargeting antibody methods show rapid clearance of radioactivity from the circulation (19–21). Image-subtraction techniques (22–24) or kinetic model analysis (25,26) may also help solve this issue. Accordingly, 99mTc-TF-mAb or its derivatives have great potential as in vivo molecular imaging probes and deserve further investigation.
A higher renal accumulation of 99mTc-IgG1 than of 99mTc-TF-mAb was observed in WHHLMI rabbits. Although an exact mechanistic explanation for this significant difference is not clear, several other investigators have also reported a relatively high renal accumulation after the injection of radiolabeled mAbs (27–29). Because we evaluated the biodistribution 24 h after the injection (relatively late phase), renal accumulation may be ascribed to metabolic or degradation products of 99mTc-labeled antibodies (30). Thus, further ex vivo metabolite analysis studies could help to clarify the mechanism. In addition, it is known that the excretion system of WHHLMI rabbits is compromised (31), which could alter the renal accumulation of tracers. On the other hand, although a certain degree of TF expression was observed in glomeruli (32), this could not be a reason for the higher renal accumulation of 99mTc-IgG1.
Recently, the focus of anticoagulant research has turned to inhibition of the TF-FVIIa complex, and many pharmaceutical industry research programs have attempted to discover TF-FVIIa complex inhibitors (33). Studies in monkeys have indicated that inhibition of the TF-FVIIa complex, compared with other anticoagulants that inhibit thrombin or FXa, results in an improved profile . It is well known that the pathways for blood coagulation are interdependent, and the initiation, amplification, and propagation stages are closely regulated by positive and negative feedback loops. Thus, repeated doses of anticoagulants might increase the expression of ineffective (silent) TF complex in plaques because of such feedback processes independent of the antiatherosclerotic effect, although a lowering of net TF expression would be expected. The TF antibody we established in this study recognizes 193Ser-207Cys in the extracellular domain, which is distant from the protein sites related to complex formation with FVIIa. Therefore, the 99mTc-TF-mAb we developed can estimate the net TF expression in plaques, providing a useful tool to investigate the effect of such anticoagulants in vivo.
Comparison with Other Imaging Probes
In the search for suitable molecular probes to assess atherosclerotic lesion characteristics, many targets, including macrophage activity, angiogenesis, apoptosis, and cell tracking (monocyte, stem cell, lymphocyte), have been assessed (1,2,34–36). However, the usefulness of these probes is still under preliminary investigation, except for 18F-FDG, a marker of inflammation, and 99mTc-annexin A5, a marker of ongoing apoptotic cell death, which are currently in clinical studies. In previous studies, we evaluated macrophage imaging using 18F-FDG (11) and also 99mTc-LOX-1-mAb (17), which targets a scavenger receptor highly expressed on macrophages and foam cells and showed the usefulness for detection of atherosclerotic lesions. However, 18F-FDG accumulated in relatively stable lesions because of the presence of macrophages in such lesions, as also seen in this report (Fig. 4). We also previously showed a certain degree of LOX-1 expression in relatively stable lesions with 99mTc-LOX-1-mAb. As for 99mTc-annexin A5, the accumulation ratios of atheromatous lesions to other lesions of 99mTc-TF-mAb (atheromatous to neointimal, 3.0; atheromatous to fibroatheromatous, 2.4; and atheromatous to collagen-rich, 2.9) were markedly higher than those of 99mTc-annexin A5 (atheromatous to neointimal,1.3; atheromatous to fibroatheromatous, 1.3; atheromatous to collagen-rich, 1.8) (15). Our previous study in apolipoprotein E–null mice also showed relatively high 18F-FDG accumulation levels in early lesions, resulting in lower accumulation ratios for advanced to early lesions in comparison with 99mTc-annexin A5 (37). Thus, the desirable features of 99mTc-TF-mAb further confirm its potential as a molecular probe for detecting atheromatous lesions at higher risk for rupture.
CONCLUSION
In this study, we succeeded in determining TF expression using 99mTc-TF-mAb in WHHLMI rabbits. Consequently, we demonstrated prominently higher accumulation of 99mTc-TF-mAb in grade IV atheroma. These findings strongly indicate that molecular imaging of TF should provide clinically useful information on the thrombogenicity of atherosclerotic plaques.
Acknowledgments
This work was partly supported by a grant-in-aid for general scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and from the Japan Society for the Promotion of Science and by a research grant from the Association for Nuclear Technology in Medicine and Takeda Science Foundation.
- © 2010 by Society of Nuclear Medicine
REFERENCES
- Received for publication July 13, 2010.
- Accepted for publication September 8, 2010.