Ultra-high resolution, 3-dimensional magnetic resonance imaging of the atherosclerotic vessel wall at clinical 7T.

Accurate quantification and characterization of atherosclerotic plaques with MRI requires high spatial resolution acquisitions with excellent image quality. The intrinsically better signal-to-noise ratio (SNR) at high-field clinical 7T compared to the widely employed lower field strengths of 1.5 and 3T may yield significant improvements to vascular MRI. However, 7T atherosclerosis imaging also presents specific challenges, related to local transmit coils and B1 field inhomogeneities, which may overshadow these theoretical gains. We present the development and evaluation of 3D, black-blood, ultra-high resolution vascular MRI on clinical high-field 7T in comparison lower-field 3T. These protocols were applied for in vivo imaging of atherosclerotic rabbits, which are often used for development, testing, and validation of translatable cardiovascular MR protocols. Eight atherosclerotic New Zealand White rabbits were imaged on clinical 7T and 3T MRI scanners using 3D, isotropic, high (0.63 mm3) and ultra-high (0.43 mm3) spatial resolution, black-blood MR sequences with extensive spatial coverage. Following imaging, rabbits were sacrificed for validation using fluorescence imaging and histology. Image quality parameters such as SNR and contrast-to-noise ratio (CNR), as well as morphological and functional plaque measurements (plaque area and permeability) were evaluated at both field strengths. Using the same or comparable imaging parameters, SNR and CNR were in general higher at 7T compared to 3T, with a median (interquartiles) SNR gain of +40.3 (35.3-80.1)%, and a median CNR gain of +68.1 (38.5-95.2)%. Morphological and functional parameters, such as vessel wall area and permeability, were reliably acquired at 7T and correlated significantly with corresponding, widely validated 3T vessel wall MRI measurements. In conclusion, we successfully developed 3D, black-blood, ultra-high spatial resolution vessel wall MRI protocols on a 7T clinical scanner. 7T imaging was in general superior to 3T with respect to image quality, and comparable in terms of plaque area and permeability measurements.

Imaging-assisted nanoimmunotherapy for atherosclerosis in multiple species.

Nanomedicine research produces hundreds of studies every year, yet very few formulations have been approved for clinical use. This is due in part to a reliance on murine studies, which have limited value in accurately predicting translational efficacy in larger animal models and humans. Here, we report the scale-up of a nanoimmunotherapy from mouse to large rabbit and porcine atherosclerosis models, with an emphasis on the solutions we implemented to overcome production and evaluation challenges. Specifically, we integrated translational imaging readouts within our workflow to both analyze the nanoimmunotherapeutic's in vivo behavior and assess treatment response in larger animals. We observed our nanoimmunotherapeutic's anti-inflammatory efficacy in mice, as well as rabbits and pigs. Nanoimmunotherapy-mediated reduction of inflammation in the large animal models halted plaque progression, supporting the approach's translatability and potential to acutely treat atherosclerosis.

Nanobody-Facilitated Multiparametric PET/MRI Phenotyping of Atherosclerosis.

OBJECTIVES: This study sought to develop an integrative positron emission tomography (PET) with magnetic resonance imaging (MRI) procedure for accurate atherosclerotic plaque phenotyping, facilitated by clinically approved and nanobody radiotracers. BACKGROUND: Noninvasive characterization of atherosclerosis remains a challenge in clinical practice. The limitations of current diagnostic methods demonstrate that, in addition to atherosclerotic plaque morphology and composition, disease activity needs to be evaluated. METHODS: We screened 3 nanobody radiotracers targeted to different biomarkers of atherosclerosis progression, namely vascular cell adhesion molecule (VCAM)-1, lectin-like oxidized low-density lipoprotein receptor (LOX)-1, and macrophage mannose receptor (MMR). The nanobodies, initially radiolabeled with copper-64 (64Cu), were extensively evaluated in Apoe-/- mice and atherosclerotic rabbits using a combination of in vivo PET/MRI readouts and ex vivo radioactivity counting, autoradiography, and histological analyses. RESULTS: The 3 nanobody radiotracers accumulated in atherosclerotic plaques and displayed short circulation times due to fast renal clearance. The MMR nanobody was selected for labeling with gallium-68 (68Ga), a short-lived radioisotope with high clinical relevance, and used in an ensuing atherosclerosis progression PET/MRI study. Macrophage burden was longitudinally studied by 68Ga-MMR-PET, plaque burden by T2-weighted MRI, and neovascularization by dynamic contrast-enhanced (DCE) MRI. Additionally, inflammation and microcalcifications were evaluated by fluorine-18 (18F)-labeled fluorodeoxyglucose (18F-FDG) and 18F-sodium fluoride (18F-NaF) PET, respectively. We observed an increase in all the aforementioned measures as disease progressed, and the imaging signatures correlated with histopathological features. CONCLUSIONS: We have evaluated nanobody-based radiotracers in rabbits and developed an integrative PET/MRI protocol that allows noninvasive assessment of different processes relevant to atherosclerosis progression. This approach allows the multiparametric study of atherosclerosis and can aid in early stage anti-atherosclerosis drug trials.

Evaluation of drug effects on cerebral blood flow and glucose uptake in un-anesthetized and un-stimulated rats: application of free-moving apparatus enabling to keep rats free during PET/SPECT tracer injection and uptake.

OBJECTIVES: The purpose of this study is the development of novel fluorine-18-fluorodeoxyglucose (F-FDG)-PET and Tc-hexamethylpropylene amine oxime (HMPAO) SPECT methods with free-moving apparatus on conscious rats to investigate brain activity without the effects of anesthesia and tactual stimulation. We also assessed the sensitivity of the experimental system by an intervention study using fluoxetine as a reference drug. MATERIALS AND METHODS: A catheter was inserted into the femoral vein and connected to a free-moving cannula system. After fluoxetine administration, the rats were given an injection of F-FDG or Tc-HMPAO via the intravenous cannula and released into a free-moving cage. After the tracer was trapped in the brain, the rats were anesthetized and scanned with PET or SPECT scanners. Then a volume of interest analysis and statistical parametric mapping were performed. RESULTS: We could inject the tracer without touching the rats, while keeping them conscious until the tracers were distributed and trapped in the brain using the developed system. The effects of fluoxetine on glucose uptake and cerebral blood flow were perceptively detected by volume of interest and statistical parametric mapping analysis. CONCLUSION: We successfully developed free-moving F-FDG-PET and Tc-HMPAO-SPECT imaging systems and detected detailed glucose uptake and cerebral blood flow changes in the conscious rat brain with fluoxetine administration. This system is expected to be useful to assess brain activity without the effects of anesthesia and tactual stimulation to evaluate drug effect or animal brain function.

Glucose uptake of the muscle and adipose tissues in diabetes and obesity disease models: evaluation of insulin and ß3-adrenergic receptor agonist effects by 18F-FDG.

OBJECTIVE: One of the major causes of diabetes and obesity is abnormality in glucose metabolism and glucose uptake in the muscle and adipose tissue based on an insufficient action of insulin. Therefore, many of the drug discovery programs are based on the concept of stimulating glucose uptake in these tissues. Improvement of glucose metabolism has been assessed based on blood parameters, but these merely reflect the systemic reaction to the drug administered. We have conducted basic studies to investigate the usefulness of glucose uptake measurement in various muscle and adipose tissues in pharmacological tests using disease-model animals. METHODS: A radiotracer for glucose, 18F-2-deoxy-2-fluoro-D-glucose (18F-FDG), was administered to Wistar fatty rats (type 2 diabetes model), DIO mouse (obese model), and the corresponding control animals, and the basal glucose uptake in the muscle and adipose (white and brown) tissues were compared using biodistribution method. Moreover, insulin and a ß3 agonist (CL316,243), which are known to stimulate glucose uptake in the muscle and adipose tissues, were administered to assess their effect. 18F-FDG uptake in each tissue was measured as the radioactivity and the distribution was confirmed by autoradiography. RESULTS: In Wistar fatty rats, all the tissues measured showed a decrease in the basal level of glucose uptake when compared to Wistar lean rats. On the other hand, the same trend was observed only in the white adipose tissue in DIO mice, while brown adipose tissue showed increments in the basal glucose uptake in this model. Insulin administration stimulated glucose uptake in both Wistar lean and fatty rats, although the responses were inhibited in Wistar fatty rats. The same tendency was shown also in control mice, but clear increments in glucose uptake were not observed in the muscle and brown adipose tissue of DIO mice after insulin administration. ß3 agonist administration showed the similar trend in Wistar lean and fatty rats as insulin, while the responses were inhibited in the adipose tissues of Wistar fatty rats. CONCLUSION: A system to monitor tissue glucose uptake with 18F-FDG enabled us to detect clear differences in basal glucose uptake between disease-model animals and their corresponding controls. The responses in the tissues to insulin or ß3 agonist could be identified. Taken as a whole, the biodistribution method with 18F-FDG was confirmed to be useful for pharmacological evaluation of anti-diabetic or anti-obesity drugs using disease-model animals.

Effect of Exercise and Calorie Restriction on Tissue Acylcarnitines, Tissue Desaturase Indices, and Fat Accumulation in Diet-Induced Obese Rats.

Both exercise and calorie restriction interventions have been recommended for inducing weight-loss in obese states. However, there is conflicting evidence on their relative benefits for metabolic health and insulin sensitivity. This study seeks to evaluate the differential effects of the two interventions on fat mobilization, fat metabolism, and insulin sensitivity in diet-induced obese animal models. After 4 months of ad libitum high fat diet feeding, 35 male Fischer F344 rats were grouped (n = 7 per cohort) into sedentary control (CON), exercise once a day (EX1), exercise twice a day (EX2), 15% calorie restriction (CR1) and 30% calorie restriction (CR2) cohorts. Interventions were carried out over a 4-week period. We found elevated hepatic and muscle long chain acylcarnitines with both exercise and calorie restriction, and a positive association between hepatic long chain acylcarnitines and insulin sensitivity in the pooled cohort. Our result suggests that long chain acylcarnitines may not indicate incomplete fat oxidation in weight loss interventions. Calorie restriction was found to be more effective than exercise in reducing body weight. Exercise, on the other hand, was more effective in reducing adipose depots and muscle triglycerides, favorably altering muscle/liver desaturase activity and improving insulin sensitivity.

In Vivo PET Imaging of HDL in Multiple Atherosclerosis Models.

OBJECTIVES: The goal of this study was to develop and validate a noninvasive imaging tool to visualize the in vivo behavior of high-density lipoprotein (HDL) by using positron emission tomography (PET), with an emphasis on its plaque-targeting abilities. BACKGROUND: HDL is a natural nanoparticle that interacts with atherosclerotic plaque macrophages to facilitate reverse cholesterol transport. HDL-cholesterol concentration in blood is inversely associated with risk of coronary heart disease and remains one of the strongest independent predictors of incident cardiovascular events. METHODS: Discoidal HDL nanoparticles were prepared by reconstitution of its components apolipoprotein A-I (apo A-I) and the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine. For radiolabeling with zirconium-89 ((89)Zr), the chelator deferoxamine B was introduced by conjugation to apo A-I or as a phospholipid-chelator (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-deferoxamine B). Biodistribution and plaque targeting of radiolabeled HDL were studied in established murine, rabbit, and porcine atherosclerosis models by using PET combined with computed tomography (PET/CT) imaging or PET combined with magnetic resonance imaging. Ex vivo validation was conducted by radioactivity counting, autoradiography, and near-infrared fluorescence imaging. Flow cytometric assessment of cellular specificity in different tissues was performed in the murine model. RESULTS: We observed distinct pharmacokinetic profiles for the two (89)Zr-HDL nanoparticles. Both apo A-I- and phospholipid-labeled HDL mainly accumulated in the kidneys, liver, and spleen, with some marked quantitative differences in radioactivity uptake values. Radioactivity concentrations in rabbit atherosclerotic aortas were 3- to 4-fold higher than in control animals at 5 days' post-injection for both (89)Zr-HDL nanoparticles. In the porcine model, increased accumulation of radioactivity was observed in lesions by using in vivo PET imaging. Irrespective of the radiolabel's location, HDL nanoparticles were able to preferentially target plaque macrophages and monocytes. CONCLUSIONS: (89)Zr labeling of HDL allows study of its in vivo behavior by using noninvasive PET imaging, including visualization of its accumulation in advanced atherosclerotic lesions. The different labeling strategies provide insight on the pharmacokinetics and biodistribution of HDL's main components (i.e., phospholipids, apo A-I).

Imaging Macrophage and Hematopoietic Progenitor Proliferation in Atherosclerosis.

RATIONALE: Local plaque macrophage proliferation and monocyte production in hematopoietic organs promote progression of atherosclerosis. Therefore, noninvasive imaging of proliferation could serve as a biomarker and monitor therapeutic intervention. OBJECTIVE: To explore (18)F-FLT positron emission tomography-computed tomography imaging of cell proliferation in atherosclerosis. METHODS AND RESULTS: (18)F-FLT positron emission tomography-computed tomography was performed in mice, rabbits, and humans with atherosclerosis. In apolipoprotein E knock out mice, increased (18)F-FLT signal was observed in atherosclerotic lesions, spleen, and bone marrow (standardized uptake values wild-type versus apolipoprotein E knock out mice, 0.05 ± 0.01 versus 0.17 ± 0.01, P<0.05 in aorta; 0.13 ± 0.01 versus 0.28 ± 0.02, P<0.05 in bone marrow; 0.06 ± 0.01 versus 0.22 ± 0.01, P<0.05 in spleen), corroborated by ex vivo scintillation counting and autoradiography. Flow cytometry confirmed significantly higher proliferation of macrophages in aortic lesions and hematopoietic stem and progenitor cells in the spleen and bone marrow in these mice. In addition, (18)F-FLT plaque signal correlated with the duration of high cholesterol diet (r(2)=0.33, P<0.05). Aortic (18)F-FLT uptake was reduced when cell proliferation was suppressed with fluorouracil in apolipoprotein E knock out mice (P<0.05). In rabbits, inflamed atherosclerotic vasculature with the highest (18)F-fluorodeoxyglucose uptake enriched (18)F-FLT. In patients with atherosclerosis, (18)F-FLT signal significantly increased in the inflamed carotid artery and in the aorta. CONCLUSIONS: (18)F-FLT positron emission tomography imaging may serve as an imaging biomarker for cell proliferation in plaque and hematopoietic activity in individuals with atherosclerosis.

Evaluation of the therapeutic efficacy of a MEK inhibitor (TAK-733) using ¹8F-fluorodeoxyglucose-positron emission tomography in the human lung xenograft model A549.

OBJECTIVE: The aim of this study was to evaluate the potential of (18)F-fluorodeoxyglucose-positron emission tomography ((18)F-FDG-PET) for monitoring the therapeutic efficacy of TAK-733, an inhibitor of mitogen-activated protein kinase kinase, in nude rats bearing A549 (human lung carcinoma) xenografts. METHODS: TAK-733 was administered orally by gavage to nude xenograft rats for 2 weeks, at dosage levels of 0 (0.5% w/v methylcellulose solution), 1, 3, and 10 mg/kg/day (n = 8/dose). Tumor size was measured before treatment (day 0), and on days 1, 3, 7, 9, 11, and 14. PET scans were performed pretreatment (day 0), and on days 2, 4, 7, 10, and 14. Tracer accumulations in tumor tissue were quantified as the mean standard uptake value (SUVmean). RESULTS: No deaths or treatment-related body weight losses occurred during the study period. TAK-733 showed dose-dependent inhibition of tumor growth and (18)F-FDG uptake in tumor tissue. At a dosage of 10 mg/kg, TAK-733 treatment produced a statistically significant reduction in tumor weight from day 11 compared with the vehicle group (P < 0.05). Tumor growth was inhibited in the 10 mg/kg group with a treated/control value of 31% on day 14. The SUVmean on day 2 in this dosage group was statistically lower than that observed on day 0, and that seen in the vehicle group on day 2 (P < 0.05 for both comparisons). Furthermore, this reduction in SUVmean at 10 mg/kg was maintained over time. In the two lower dosage groups (1 and 3 mg/kg), SUVmean gradually increased over time. CONCLUSIONS: (18)F-FDG-PET enabled early determination of late anti-tumor activity in response to TAK-733 treatment.

18F-FDG PET and intravascular ultrasonography (IVUS) images compared with histology of atherosclerotic plaques: 18F-FDG accumulates in foamy macrophages.

PURPOSE: Intravascular ultrasonography (IVUS) and (18)F-FDG PET have been used to evaluate the efficacy of antiatherosclerosis drugs. These two modalities image different characteristics of atherosclerotic plaques, and a comparison of IVUS and PET images with histology has not been performed. The aim of this study was to align IVUS and PET images using anatomic landmarks in Watanabe heritable hyperlipidaemic (WHHL) rabbits, enabling comparison of their depiction of aortic atherosclerosis. Cellular (18)F-FDG localization was evaluated by (3)H-FDG microautoradiography (micro-ARG). METHODS: A total of 19 WHHL rabbits (7 months of age) were divided into three groups: baseline (n = 6), 3 months (n = 4), and 6 months (n = 9). PET, IVUS and histological images of the same aortic segments were analysed. Infiltration by foamy macrophages was scored from 0 to IV using haematoxylin and eosin (H&E) and antimacrophage immunohistochemical staining, and compared with (3)H-FDG micro-ARG findings in two additional WHHL rabbits. RESULTS: IVUS images did not identify foamy macrophage deposition but revealed the area of intimal lesions (r = 0.87). (18)F-FDG PET revealed foamy macrophage distribution in the plaques. The intensity of (18)F-FDG uptake was correlated positively with the degree of foamy macrophage infiltration. Micro-ARG showed identical (3)H-FDG accumulation in the foamy macrophages surrounding the lipid core of the plaques. CONCLUSION: F-FDG PET localized and quantified the degree of infiltration of foamy macrophages in atherosclerotic lesions. IVUS defined the size of lesions. (18)F-FDG PET is a promising imaging technique for evaluating atherosclerosis and for monitoring changes in the composition of atherosclerotic plaques affecting their stability.

Investigation of Fat Metabolism during Antiobesity Interventions by Magnetic Resonance Imaging and Spectroscopy.

The focus of current treatments for obesity is to reduce the body weight or visceral fat, which requires longer duration to show effect. In this study, we investigated the short-term changes in fat metabolism in liver, abdomen, and skeletal muscle during antiobesity interventions including Sibutra mine treatment and diet restriction in obese rats using magnetic resonance imaging, magnetic resonance spectroscopy, and blood chemistry. Sibutramine is an antiobesity drug that results in weight loss by increasing satiety and energy expenditure. The Sibutramine-treated rats showed reduction of liver fat and intramyocellular lipids on day 3. The triglycerides (TG) decreased on day 1 and 3 compared to baseline (day 0). The early response/nonresponse in different fat depots will permit optimization of treatment for better clinical outcome rather than staying with a drug for longer periods.

Comparison of contrast agents for atherosclerosis imaging using cultured macrophages: FDG versus ultrasmall superparamagnetic iron oxide.

UNLABELLED: Various noninvasive imaging methods have been developed to evaluate atherosclerotic plaques. Among them, (18)F-FDG PET and MR imaging with ultrasmall superparamagnetic iron oxide particles (USPIO) have been used to quantify plaque inflammation. Both methods are based on the efficient uptake of FDG and USPIO by macrophages in atherosclerotic lesions. Differently polarized macrophages have been reported to have different characteristics that are involved in the pathologic development of atherosclerosis. M1 polarized macrophages are considered the more proatherogenic phenotype than M2 polarized macrophages. However, little is known regarding the association between macrophage polarization and FDG or USPIO accumulation. In this study, we investigated intracellular FDG and USPIO accumulation in M1 and M2 polarized macrophages. METHODS: THP-1 macrophages were differentiated into M1 and M2 polarized macrophages. Under optimal glucose conditions, we investigated the (3)H-labeled FDG uptake in M1 and M2 polarized macrophages. We then investigated intracellular USPIO uptake by M1 and M2 macrophages. RESULTS: We found that M1 polarization, compared with M2 polarization, results in increased intracellular accumulation of FDG. To elucidate the mechanism by which FDG was preferentially accumulated in M1 macrophages, we examined messenger RNA expressions of glucose transporters (GLUTs) and hexokinases, which have pivotal roles in glucose uptake, and glucose-6-phosphatase (G6Pase), which catalyzes the reverse reaction of hexokinase. In M1 macrophages, GLUT-1, GLUT-3, hexokinase 1, and hexokinase 2 were upregulated and G6Pase was downregulated. In contrast to FDG, M1 polarization resulted in decreased intracellular accumulation of USPIO. We found that scavenger receptor A and CD11b, which are involved in USPIO binding and uptake, were significantly downregulated by M1 polarization. CONCLUSION: Compared with M2, proatherogenic M1 macrophages preferentially accumulated FDG but not USPIO, suggesting that FDG PET is a useful method for the detection of proinflammatory M1 macrophages.

Biological characterization of TAK-901, an investigational, novel, multitargeted Aurora B kinase inhibitor.

Protein kinases Aurora A, B, and C play essential roles during mitosis and cell division, are frequently elevated in cancer, and represent attractive targets for therapeutic intervention. TAK-901 is an investigational, multitargeted Aurora B kinase inhibitor derived from a novel azacarboline kinase hinge-binder chemotype. TAK-901 exhibited time-dependent, tight-binding inhibition of Aurora B, but not Aurora A. Consistent with Aurora B inhibition, TAK-901 suppressed cellular histone H3 phosphorylation and induced polyploidy. In various human cancer cell lines, TAK-901 inhibited cell proliferation with effective concentration values from 40 to 500 nmol/L. Examination of a broad panel of kinases in biochemical assays revealed inhibition of multiple kinases. However, TAK-901 potently inhibited only a few kinases other than Aurora B in intact cells, including FLT3 and FGFR2. In rodent xenografts, TAK-901 exhibited potent activity against multiple human solid tumor types, and complete regression was observed in the ovarian cancer A2780 model. TAK-901 also displayed potent activity against several leukemia models. In vivo biomarker studies showed that TAK-901 induced pharmacodynamic responses consistent with Aurora B inhibition and correlating with retention of TAK-901 in tumor tissue. These preclinical data highlight the therapeutic potential of TAK-901, which has entered phase I clinical trials in patients within a diverse range of cancers.

Tissue factor detection for selectively discriminating unstable plaques in an atherosclerotic rabbit model.

UNLABELLED: 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 (99m)Tc-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 (99m)Tc with 6-hydrazinonicotinic acid (HYNIC) as a chelating agent to obtain (99m)Tc-TF-mAb. The immunoreactivity of HYNIC-TF-mAb was estimated by flow cytometry. WHHLMI and control rabbits were injected intravenously with (99m)Tc-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 (IgG(1)) 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/mm(2) × 10(2) 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, (99m)Tc-IgG(1) 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 (99m)Tc-TF-mAb were demonstrated, indicating its potential for selective imaging of macrophage-rich atheromatous lesions in vivo.

Imaging with radiolabelled anti-membrane type 1 matrix metalloproteinase (MT1-MMP) antibody: potentials for characterizing atherosclerotic plaques.

PURPOSE: Membrane type 1 matrix metalloproteinase (MT1-MMP) activates pro-MMP-2 and pro-MMP-13 to their active forms and plays important roles in the destabilization of atherosclerotic plaques. This study sought to determine the usefulness of (99m)Tc-labelled monoclonal antibody (mAb), recognizing MT1-MMP, for imaging atherosclerosis in a rabbit model (WHHLMI rabbits). METHODS: Anti-MT1-MMP monoclonal IgG(3) and negative control IgG(3) were radiolabelled with (99m)Tc after derivatization with 6-hydrazinonicotinic acid (HYNIC) to yield (99m)Tc-MT1-MMP mAb and (99m)Tc-IgG(3), respectively. WHHLMI and control rabbits were injected with these radio-probes. The aorta was removed and radioactivity was measured at 24 h after the injection. Autoradiography and histological studies were performed. RESULTS: (99m)Tc-MT1-MMP mAb accumulation in WHHLMI rabbit aortas was 5.4-fold higher than that of control rabbits. Regional (99m)Tc-MT1-MMP mAb accumulation was positively correlated with MT1-MMP expression (r = 0.59, p < 0.0001), while (99m)Tc-IgG(3) accumulation was independent of MT1-MMP expression (r = 0.03, p = NS). The highest (99m)Tc-MT1-MMP mAb accumulation was found in atheromatous lesions (4.8 ± 1.9, %ID×BW/mm(2) × 10(2)), followed in decreasing order by fibroatheromatous (1.8 ± 1.3), collagen-rich (1.6 ± 1.0) and neointimal lesions (1.5 ± 1.5). In contrast, (99m)Tc-IgG(3) accumulation was almost independent of the histological grade of lesions. CONCLUSION: Higher (99m)Tc-MT1-MMP mAb accumulation in grade IV atheroma was shown in comparison with neointimal lesions or other more stable lesions. Nuclear imaging with (99m)Tc-MT1-MMP mAb, in combination with CT and MRI, could provide new diagnostic imaging capabilities for detecting vulnerable plaques, although further investigations to improve target to blood ratios are strongly required.

Targeting of lectinlike oxidized low-density lipoprotein receptor 1 (LOX-1) with 99mTc-labeled anti-LOX-1 antibody: potential agent for imaging of vulnerable plaque.

UNLABELLED: Lectinlike oxidized low-density lipoprotein (LDL) receptor 1 (LOX-1), a cell surface receptor for oxidized LDL, has been implicated in vascular cell dysfunction related to plaque instability, which could be a potential target for an atherosclerosis imaging tracer. In this study, we designed and prepared (99m)Tc-labeled anti-LOX-1 monoclonal IgG and investigated its usefulness as an atherosclerosis imaging agent. METHODS: Anti-LOX-1 monoclonal IgG and control mouse IgG2a were labeled with (99m)Tc after derivatization with 6-hydrazinonicotinic acid to yield (99m)Tc-LOX-1-mAb and (99m)Tc-IgG2a, respectively. Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits (atherosclerosis model) and control rabbits were injected intravenously with these probes, and in vivo planar imaging was performed. At 24 h after the injection, the aortas were removed, and radioactivity was measured. Autoradiography and histologic studies were performed with serial aortic sections. RESULTS: The level of (99m)Tc-LOX-1-mAb accumulation was 2.0-fold higher than the level of (99m)Tc-IgG2a accumulation in WHHLMI rabbit aortas, and the level of (99m)Tc-LOX-1-mAb accumulation in WHHLMI rabbit aortas was 10.0-fold higher than the level of (99m)Tc-LOX-1-mAb accumulation in control rabbit aortas. In vivo imaging clearly visualized the atherosclerotic aortas of WHHLMI rabbits. Autoradiography and histologic studies revealed that regional (99m)Tc-IgG2a accumulation was independent of the histologic grade of the lesions; however, regional (99m)Tc-LOX-1-mAb accumulation was significantly correlated with LOX-1 expression density and the vulnerability index. The highest level of (99m)Tc-LOX-1-mAb accumulation, expressed as {radioactivity in region of interest (Bq/mm(2))/[injected radioactivity (Bq)/animal body weight (g)]} x 10(2), was found in atheromatous lesions (3.8 +/- 1.1 [mean +/- SD]), followed in decreasing order by fibroatheromatous lesions (2.0 +/- 1.0), collagen-rich lesions (1.6 +/- 0.8), and neointimal lesions (1.4 +/- 0.7). CONCLUSION: The level of (99m)Tc-LOX-1-mAb accumulation in grade IV atheroma was higher than that in neointimal lesions or other, more stable lesions. Nuclear imaging of LOX-1 expression with (99m)Tc-LOX-1-mAb may be a useful means for predicting atheroma at high risk for rupture.

Prominent lectin-like oxidized low density lipoprotein (LDL) receptor-1 (LOX-1) expression in atherosclerotic lesions is associated with tissue factor expression and apoptosis in hypercholesterolemic rabbits.

BACKGROUND: Despite increasing in vitro evidence that lectin-like oxidized low density lipoprotein (LDL) receptor-1 (LOX-1), a cell-surface receptor for oxidized LDL, is implicated in the atherogenesis and thrombus formation, its in vivo participation to the atherosclerotic plaque destabilization, rupture and thrombus formation remains unclear. Here, we compared the in vivo expression of LOX-1, with tissue factor (TF) expression and cell apoptosis, in atherosclerotic lesions of myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits. METHODS AND RESULTS: We prepared sixty series of cross sections in the aortic arch and the thoracic aorta from four WHHLMI rabbits. LOX-1 and TF expression, as well as apoptotic events were determined by immunohistochemical staining and TUNEL methods, respectively. LOX-1 expression was mainly observed in the macrophage-rich lipid areas of vulnerable plaque-like atheromatous lesions where TF expression and apoptotic events were prominent. LOX-1 expression was positively correlated with TF expression (r=0.53, p<0.0001), apoptotic events (r=0.52, p<0.0001) and morphological vulnerability (r=0.63, p<0.0001). CONCLUSIONS: LOX-1 expression appears to be closely associated with TF expression, apoptotic events and the morphological vulnerability, suggesting the in vivo involvement of LOX-1 in the destabilization and rupture of atherosclerotic lesions and the subsequent thrombus formation. The present findings in hypercholesterolemic rabbits should help advance our understanding of the pathophysiology of atherosclerosis.

Distribution profiles of membrane Type-1 matrix metalloproteinase (MT1-MMP), matrix metalloproteinase-2 (MMP-2) and cyclooxygenase-2 (COX-2) in rabbit atherosclerosis: comparison with plaque instability analysis.

BACKGROUND: Despite increasing evidence that membrane type 1 matrix metalloproteinase (MT1-MMP), matrix metalloproteinase-2 (MMP-2), and cyclooxygenase-2 (COX-2) are involved in the pathogenesis of atherosclerosis, the possible links among these enzymes remain unclear. Accordingly, we investigated the distribution of MT1-MMP, MMP-2, and COX-2 immunohistologically in the atherosclerotic lesions of hypercholesterolemic (WHHLMI) rabbits. METHODS AND RESULTS: Distribution of MT1-MMP, MMP-2, and COX-2 was examined by immunohistochemical staining using sixty cross sections of the ascending-arch and thoracic aortas prepared from 4 WHHLMI rabbits. MT1-MMP and MMP-2 staining was prominently observed in the macrophage-rich regions of the atheromatous lesions, and was positively correlated with morphological vulnerability (r=0.63 for MT1-MMP; r=0.60 for MMP-2; p<0.0001). MT1-MMP staining was positively correlated with MMP-2 staining (r=0.61, p<0.0001). COX-2 staining was also the highest in the macrophage-rich regions of the atheromatous lesions, with relatively high staining levels in other more stable lesions. CONCLUSIONS: Co-distribution of MT1-MMP, MMP-2, and COX-2 was demonstrated in grade IV atheroma, indicating a possible link among these enzymes in the destabilization of atherosclerotic plaques. The relatively high COX-2 distribution in other more stable lesions may indicate its additional roles in the stabilization of atherosclerotic lesions. The present findings in hypercholesterolemic rabbits should help advance our understanding of the pathophysiology of atherosclerosis and provide useful information for the development of new therapeutic and diagnostic (imaging) agents that target MMPs and COX-2 in atherosclerosis.

99mTc-Annexin A5 for noninvasive characterization of atherosclerotic lesions: imaging and histological studies in myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits.

PURPOSE: Apoptosis is commonly observed in advanced atherosclerotic lesions. 99mTc-annexin A5 (99mTc-annexin V) has been proposed as a potential tracer for imaging apoptosis in atherosclerotic plaques. Accordingly, we determined the usefulness of 99mTc-annexin A5 as an atherosclerosis imaging tracer in a rabbit model (myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits; WHHLMI rabbits) of spontaneous atherosclerosis. METHODS: The WHHLMI and control rabbits were injected intravenously with 99mTc-annexin A5. After in vivo planar imaging, the radioactivity in the aorta was measured. Autoradiography, TUNEL staining, Azan-Mallory staining and immunohistological studies were performed serially throughout the aorta. RESULTS: 99mTc-Annexin A5 accumulation in the aorta of the WHHLMI rabbits was 5.6-fold higher than in that of control rabbits. Autoradiography showed heterogeneous multifocal accumulation of 99mTc-annexin A5 in WHHLMI rabbits. 99mTc-Annexin A5 accumulation was highest in the atheromatous lesions (6.2+/-2.5, %IDxBW/mm2x10(3)), followed in decreasing order by neointimal (4.9+/-1.3), fibroatheromatous (4.5+/-1.9), and collagen-rich lesions (3.3+/-1.4). The regional 99mTc-annexin A5 accumulation was significantly correlated with the TUNEL-positive cell density, macrophage density and "vulnerability index," an index of the morphological destabilized characteristics. The in vivo imaging clearly visualized the atherosclerotic lesions in WHHLMI rabbits. CONCLUSION: The present study in WHHLMI rabbits showed higher 99mTc-annexin A5 accumulation in grade IV atheroma than in other more stable lesions. 99mTc-Annexin A5 may be useful in identifying atheroma that is at higher risk for rupture and possibly in assessing the response to anti-atherosclerotic therapy.

Lectin-like oxidized LDL receptor-1 (LOX-1) expression is associated with atherosclerotic plaque instability--analysis in hypercholesterolemic rabbits.

Lectin-like oxidized LDL receptor-1 (LOX-1), a cell-surface receptor for oxidized LDL (Ox-LDL), has been implicated in vascular cell dysfunction related to atherosclerotic plaque instability, according to cell culture experiments. In the present study, we investigated the relationship between LOX-1 expression and plaque instability in hypercholesterolemic rabbits by immunohistological analyses in vivo. We prepared thirty series of cross sections of the thoracic aorta from six myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits (12-24 months), in which seventy atherosclerotic plaques were observed. LOX-1, matrix metalloproteinase-9 (MMP-9), monocyte chemoattractant protein-1 (MCP-1) expression, apoptotic events, plaque instability index (an index of the morphological destabilization of atherosclerotic plaques) and fibromuscular cap thickness in each atherosclerotic plaque were determined by immunohistochemical staining, TUNEL staining and Azan-Mallory staining. LOX-1 expression was positively correlated with the plaque instability index and MMP-9 expression. LOX-1 expression was more prominent in atherosclerotic plaques with thinner fibromuscular cap (<100 microm). Furthermore, LOX-1 expression was shown in the macrophage-rich lipid core area where MCP-1 expression and apoptotic events were prominent. These results indicate that enhanced LOX-1 expression was associated with histologically unstable atherosclerotic plaques in hypercholesterolemic rabbits, suggesting the involvement of LOX-1 in the destabilization of atherosclerotic plaques in vivo.