Dual optical and nuclear imaging in human melanoma xenografts using a single targeted imaging probe

INTRODUCTION: Dual-labeled imaging agents that allow both nuclear and optical imaging after a single injection would be advantageous in certain applications. In this study, we synthesized and characterized a dual-labeled RGD (Arg-Gly-Asp) peptide and compared nuclear and optical images obtained with this agent. METHODS: 111In-DTPA-Lys(IRDye800)-c(KRGDf) composed of both the 111In chelator diethylenetriaminepentaacetic acid (DTPA) and the near-infrared (NIR) fluorescent dye IRDye800 (excitation/emission, 765/792 nm) was synthesized. The probe was characterized with regard to in vitro biological activity and in vivo pharmacokinetics and the ability to target integrin alphavbeta3. Tumors of mice injected with the dual-labeled probe were imaged both by gamma scintigraphy and NIR fluorescence optical camera. RESULTS: DTPA-Lys(IRDye800)-c(KRGDf), DTPA-Lys-c(KRGDf) and c(KRGDf) inhibited the adhesion of melanoma M21 cells to vitronectin-coated surface with the similar biological activity. Both 111In-DTPA-Lys(IRDye800)-c(KRGDf) and 111In-DTPA-Lys-c(KRGDf) had significantly higher uptakes in alphavbeta3-positive M21 melanoma than in alphavbeta3-negative M21-L melanoma at 4-48 h after their injection. Side-by-side comparison of images obtained using 111In-DTPA-Lys(IRDye800)-c(KRGDf) revealed that in living mice, both optical imaging and gamma scintigraphy enabled noninvasive detection of the bound probe to alphavbeta3-positive tumors, with optical images providing improved resolution and sensitive detection of the superficial lesions and gamma images providing sensitive detection of deeper structures. CONCLUSION: The dual-labeled imaging probe 111In-DTPA-Lys(IRDye800)-c(KRGDf) was found to specifically bind to alphavbeta3 in melanoma tumor cells. Employing both nuclear and optical imaging with a single imaging probe may facilitate translation of NIR fluorescence optical imaging into clinical applications.     

Gene expression levels of matrix metalloproteinases in human atherosclerotic plaques and evaluation of radiolabeled inhibitors as imaging agents for plaque vulnerability

IntroductionAtherosclerotic plaque rupture is the primary cause for myocardial infarction and stroke. During plaque progression macrophages and mast cells secrete matrix-degrading proteolytic enzymes, such as matrix metalloproteinases (MMPs). We studied levels of MMPs and tissue inhibitor of metalloproteinases-3 (TIMP-3) in relation to the characteristics of carotid plaques. We evaluated in vitro two radiolabeled probes targeting active MMPs towards non-invasive imaging of rupture-prone plaques.MethodsHuman carotid plaques obtained from endarterectomy were classified into stable and vulnerable by visual and histological analysis. MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MMP-14, TIMP-3, and CD68 levels were investigated by quantitative polymerase chain reaction. Immunohistochemistry was used to localize MMP-2 and MMP-9 with respect to CD68-expressing macrophages. Western blotting was applied to detect their active forms. A fluorine-18-labeled MMP-2/MMP-9 inhibitor and a tritiated selective MMP-9 inhibitor were evaluated by in vitro autoradiography as potential lead structures for non-invasive imaging.ResultsGene expression levels of all MMPs and CD68 were elevated in plaques. MMP-1, MMP-9, MMP-12 and MMP-14 were significantly higher in vulnerable than stable plaques. TIMP-3 expression was highest in stable and low in vulnerable plaques. Immunohistochemistry revealed intensive staining of MMP-9 in vulnerable plaques. Western blotting confirmed presence of the active form in plaque lysates. In vitro autoradiography showed binding of both inhibitors to stable and vulnerable plaques.ConclusionsMMPs differed in their expression patterns among plaque phenotypes, providing possible imaging targets. The two tested MMP-2/MMP-9 and MMP-9 inhibitors may be useful to detect atherosclerotic plaques, but not the vulnerable lesions selectively.     

Intravascular ultrasound imaging in the assessment of atherosclerotic plaques in rabbit abdominal aorta: comparison with histologic findings

RATIONALE AND OBJECTIVES: To examine the correlation between the echogenicity and the components of atherosclerotic plaques in rabbit. METHODS: The atherosclerotic plaque formation in the abdominal aortas of hyperlipidemic or normolipidemic rabbits was stimulated by inserting polyethylene tubing. Intravascular ultrasound (30-MHz, 4.5 F catheter) investigation was performed at locations in the vessel. The intravascular ultrasound images of the plaques were evaluated and compared with the histologic findings. RESULTS: Ultrasound images delineated areas showing hyperechoic or hypoechoic ultrasound beams in the plaques. Histologic studies revealed that the hyperechoic areas were closely associated with a dense fibrous extracellular matrix, whereas the hypoechoic areas corresponded to lesions showing a marked accumulation of foamy macrophages or proteoglycan-rich loose myxoid extracellular matrix with smooth muscle cell proliferation. CONCLUSION: A good correlation between ultrasound images and histologic features was observed. These results suggest that intravascular ultrasound imaging could provide useful information for assessing the tissue characteristics of atherosclerotic lesions.     

(18)F-FDG accumulation in atherosclerotic plaques: immunohistochemical and PET imaging study.

The rupture of atherosclerotic plaques and the subsequent formation of thrombi are the main factors responsible for myocardial and cerebral infarctions. Thus, the detection of vulnerable plaques in atherosclerotic lesions is a desirable goal, and attempts to image these plaques with (18)F-FDG have been made. In the present study, the relationship between the accumulation of (18)F-FDG and the biologic characteristics of atherosclerotic lesions was investigated. Furthermore, PET imaging of vulnerable plaques was performed with an animal model of atherosclerosis, Watanabe heritable hyperlipidemic (WHHL) rabbits. METHODS: WHHL (n = 11) and control (n = 3) rabbits were injected intravenously with (18)F-FDG, and the thoracic and abdominal aortas were removed 4 h after injection. The accumulated radioactivity was measured, and the number of macrophages and the intimal area were investigated by examination of stained sections. PET and CT images were also acquired at 210 min after injection of the radiotracer. RESULTS: (18)F-FDG accumulated to a significantly higher level in the aortas of the WHHL rabbits (mean +/- SD differential uptake ratio [DUR], 1.47 +/- 0.90) than in those of the control rabbits (DUR, 0.44 +/- 0.15); DUR was calculated as (tissue activity/tissue weight)/(injected radiotracer activity/animal body weight), with activities given in becquerels and weights given in kilograms. (18)F-FDG uptake and the number of macrophages were strongly correlated in the atherosclerotic lesions of the WHHL rabbits (R = 0.81). In the PET analysis, intense (18)F-FDG radioactivity was detected in the aortas of the WHHL rabbits, whereas little radioactivity was seen in the control rabbits. CONCLUSION: The results suggest that macrophages are responsible for the accumulation of (18)F-FDG in atherosclerotic lesions. Because vulnerable plaques are rich in macrophages, (18)F-FDG imaging should be useful for the selective detection of such plaques.     

Radiolabeled Monocyte Chemotactic Protein 1 for the detection of inflammation in experimental atherosclerosis.

Chemotactic peptides, such as Monocyte Chemotactic Protein 1 (MCP-1), play a key role in transendothelial migration of mononuclear cells during the development and progression of atherosclerotic disease. Because atherosclerotic plaques that are precursors of acute coronary events harbor abundant macrophage infiltration, we hypothesized that the detection of a high concentration of MCP-1 receptors on inflammatory cells should noninvasively identify vulnerable plaques. METHODS: Atherosclerotic lesions were induced by balloon deendothelialization of the abdominal aorta, which was followed by a 0.5% cholesterol diet for 16 wk in 7 New Zealand White rabbits; 5 unmanipulated rabbits, fed normal chow for 16 wk, were used as controls. Radionuclide imaging was performed immediately after intravenous (99m)Tc-labeled MCP-1 administration and 3 h later. At the end of imaging session, aortas were explanted and submitted for estimation of quantitative MCP-1 uptake (in percentage injected dose per gram, %ID/g) and pathologic characterization. RESULTS: Atherosclerotic lesions were clearly visible in all hyperlipidemic animal gamma-imaging. No tracer uptake was seen in the control rabbits. The mean quantitative MCP-1 uptake in atherosclerotic lesions was 4-fold higher than that of the aortic specimens from the control rabbits (0.065 +/- 0.005 vs. 0.016 +/- 0.006; P < 0.0001). Histology confirmed a strong correlation between MCP-1 uptake and the number of macrophages in American Heart Association type II-IV lesions (r = 0.87, P < 0.0001). CONCLUSION: Noninvasive radionuclide imaging of inflammation is feasible by MCP-1 in experimentally induced atherosclerosis. It is proposed that detection of the extent of inflammation in advanced atherosclerotic plaques may allow identification of unstable plaques.     

Superparamagnetic iron oxide-enhanced MRI of atherosclerotic plaques in Watanabe hereditable hyperlipidemic rabbits

RATIONALE AND OBJECTIVES: Inflammatory atherosclerotic plaques are characterized by increased endothelial permeability and multiple macrophages. Blood-pool MRI contrast agents like superparamagnetic iron oxide (SPIO) have an affinity for the monocyte-macrophage system and thus, may label inflammatory plaques. The objective was to demonstrate SPIO uptake in plaques of atherosclerotic rabbits by MRI and histology. METHODS: Aortas of anesthetized Watanabe hereditable hyperlipidemic rabbits were studied with a moderately T2*-weighted gradient-echo sequence at 1.5 T. Four groups of five animals each were studied: (1) without ultrasmall SPIO (carboxydextran coating; particle size, 25 nm; estimated plasma half-life, 6 hours) or with imaging after intravenous injection of SPIO at a dose (micromol Fe/kg) and postcontrast time delay (hours) of 50/8 (2), 50/24 (3), or 200/48 (4). In vivo MRI was compared with corresponding ex vivo histological iron stains. RESULTS: Animals receiving 200 micromol Fe/kg demonstrated areas of focal signal loss clearly confined to the aortic wall on a mean of 24 +/- 9 (31% +/- 11%) of 76 +/- 5 images compared with 0 +/- 0 of 76 +/- 5 images in controls (P = 0.009). The number of images with this finding in groups 2 and 3 was not significantly different compared with controls. By microscopy, SPIO-iron was seen in the endothelial cells and subendothelial intimal macrophages of atherosclerosis-prone aortic wall segments. Atherosclerotic lesions demonstrating iron uptake also showed a high macrophage content. CONCLUSIONS: SPIO accumulates in aortic plaques of atherosclerotic rabbits, producing a characteristic MRI finding. As SPIO accumulates in plaques with increased endothelial permeability and a high macrophage content, two established features of plaque inflammation, it may have a potential for noninvasive assessment of inflammatory atherosclerotic plaques.     

Differences in binding of (99m)Tc-disintegrins to integrin alphavbeta3 on tumor and vascular cells

Disintegrins, which contain an Arg-Gly-Asp sequence in their binding domains are antagonists of integrins such as alphavbeta3. The purpose of this study was to compare a range of disintegrins with different integrin selectivities for their binding behavior in vitro to vascular endothelial cells bearing alphavbeta3 and to cultured tumor cells which express alphavbeta3. METHODS: Five disintegrins (bitistatin, kistrin, flavoridin, VLO4 and echistatin) and a cyclic pentapeptide, c[RGDyK], were radiolabeled with (99m)Tc and tested for binding to cells in vitro. RESULTS: (99m)Tc-Kistrin, flavoridin and VLO4 had the highest binding, (99m)Tc-echistatin had moderate binding, and (99m)Tc-bitistatin and (99m)Tc-c[RGDyK] had low binding to cells. The observed binding was attributed to alphavbeta3 to various extents: echistatin, bitistatin>kistrin>flavoridin>VLO4. Cancer cells internalized bound disintegrins after binding, but endothelial cells did not. After binding to endothelial cells, (99m)Tc-kistrin was not displaced by competing peptide or plasma proteins. CONCLUSIONS: These data suggest that radiolabeled kistrin, flavoridin and VLO4 may have advantages over labeled bitistatin and small cyclic peptides for targeting alphavbeta3 in vivo. Since receptor-bound radioligand is not internalized by endothelial cells, disintegrins may provide an advantage for targeting alphavbeta3 on vasculature because they bind strongly to surface receptors and are not readily displaced.     

动脉粥样硬化斑块MRI和近红外分子影像的实验研究

目的 探讨7.0 T MRI和近红外荧光成像(NIRF)检测动脉粥样硬化(AS)斑块的可行性.方法 对14周龄ApoE-/-小鼠按高脂饮食喂养20周,建立AS模型,以正常C57BL/6小鼠作为对照.MRI实验中,5只ApoE-/-小鼠及5只C57小鼠经尾静脉注入超微超顺磁性氧化铁颗粒(USPIO)前及36 h后分别行7.0 T MRI.NIRF实验中,10只ApoE-/-小鼠和4只C57小鼠经尾静脉注入抗氧化修饰的低密度脂蛋白(oxLDL)抗体-NIR 797(抗-oxLDL-抗体-NIR 797)近红外探针,4只ApoE-/-小鼠经尾静脉注入非特异性IgG-NIR 797,另4只ApoE-/-小鼠注入PBS,24h后分别行NIRF.用SPSS17.0软件对计量数据行独立样本t检验和单因素方差分析.结果 ApoE-/-小鼠注入USPIO 36 h后,在T2WI上腹主动脉斑块信号较注射前减低,相对信号强度分别为0.70±0.04和1.28±0.06,差异有统计学意义(t =3.376,P＜O.05),信号改变率达(-56.58±4.25)％;普鲁士蓝染色证实斑块内有铁沉积.注入抗-oxLDL-抗体-NIR 797 24 h后,ApoE-/-小鼠主动脉离体NIRF示强荧光信号(SNR为42.51 ±5.24)聚集于主动脉根、主动脉弓及降主动脉起始段,而非特异性IgG-NIR 797组(19.58±3.06)、PBS组(4.19±0.82)及对照C57小鼠(2.29±1.11)仅见较弱荧光信号,与靶向探针组比较差异有统计学意义(F =25.104,P＜0.05).斑块油红O染色与NIRF阳性面积分别为(41.69 ±5.29)％和(39.45±5.35)％,两者呈线性相关(r=0.738,P＜0.05,n=8),免疫荧光证实斑块内oxLDL的表达与巨噬细胞共区域.结论 应用新型分子影像探针在7.0 T MRI和NIRF上可有效检测AS斑块,有助于鉴别高危斑块,可为AS多模式成像提供依据.     

In vivo and in vitro evaluation of Cy5.5 conjugated epidermal growth factor receptor binding peptide

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor and plays an important role in carcinogenesis. In this study, the epidermal growth factor receptor binding peptide (EGBP) was identified using a phage display method and evaluated in U87MG cells in order to investigate the possibility to target the EGFR using an optical imaging system. Cyanine dye 5.5 (Cy5.5) was conjugated with EGBP-GGG-SC, EGBP-AOC-SC, and EGBP-AM2BA-SC. Cellular binding study of EGBP-Linker-Cy5.5 conjugates or ¹²⁵I-EGBP-Linker compounds was performed in U87MG cells. Optical imaging studies were performed in U87MG bearing mice. Three of seven clones from the 12-mer peptide library showed a specific binding affinity to rhEGFR, and they encoded the same 12 amino acid peptide sequence, FPMFNHWEQWPP. Confocal images show that the fluorescent signal of EGBP-Linker-Cy5.5 conjugates was decreased in the order: EGBP-AOC-Cy5.5?EGBP-AM2BA-Cy5.5>EGBP-GGG-Cy5.5. EGBP-AOC-Cy5.5 appeared in cell cytoplasm and surface, and it was inhibited by free EGBP apparently. The cellular binding of EGBP-AOC-Cy5.5 exhibited a higher average radiance value than EGBP-GGG-Cy5.5 and EGBP-AM2BA-Cy5.5. Among various ¹²⁵I-EGBP-Linker compounds, EGBP-GGG showed a higher binding than other compounds. However, uptake of ¹²⁵I-EGBP-AOC was clearly inhibited by free EGBP in inhibition study. In an in vivo study, the fluorescent signal of EGBP-AOC-Cy5.5 treated mouse was mainly detected in the tumor and kidney. Among the three derivatives, EGBP-AOC-Cy5.5 was the optimized optical imaging agent for U87MG EGFR positive tumors in the animal model. This study demonstrated the EGBP-Linker-Cy5.5 conjugates may be useful as a potential EGFR target optical probe.     

Human antibody against C domain of tenascin-C visualizes murine atherosclerotic plaques ex vivo.

Targeting proteins that are overexpressed in atherosclerotic plaques may open novel diagnostic applications. The C domain of tenascin-C is absent from normal adult tissues but can be inserted during tumor progression or tissue repair into the molecule by alternative splicing. We tested the ability of the human antibody G11, specific to this antigen, to reveal murine atherosclerotic plaques ex vivo. The antibody directed against the extra domain B of fibronectin (L19) was used as a reference. METHODS: We intravenously injected (125)I-labeled G11 or L19 antibodies into apolipoprotein E-deficient (ApoE(-/-)) mice and harvested the aortae 4 or 24 h later. En face analyses of distal aortae and longitudinal sections of the aortic arch were performed to compare antibody uptake using autoradiography with plaque staining using oil red O. Plaque macrophages were detected by immunohistochemistry (anti-CD68 staining). Biodistribution of injected antibodies was investigated in aortae and blood at 4 and 24 h. RESULTS: En face analyses revealed a significant correlation between radiolabeled G11 and fat-stained areas, increasing from 4 to 24 h, with a correlation coefficient of 0.92 (P < 0.0001) and an average signal-to-noise ratio of 104:1 at 24 h. Plaque imaging using L19 showed similar results (r = 0.86; P < 0.0001; signal-to-noise ratio, 72:1 at 24 h). Uptake of radiolabeled antibodies in histologic sections colocalized with fat staining and activated macrophages in aortic plaques. Biodistribution analyses confirmed specific accumulation in aortic plaques as well as rapid blood pool clearance of the antibodies 24 h after injection. Immunofluorescence analyses revealed increased expression of tenascin and fibronectin isoforms in macrophage-rich plaques. CONCLUSION: The antibody G11, specific to the C domain of tenascin-C, visualizes murine atherosclerotic plaques ex vivo. In conjunction with the increased expression of the C domain of tenascin-C in macrophage-rich plaques, the colocalization of G11 uptake with activated macrophages, and the favorable target-to-blood ratio at 24 h, this antibody may be useful for molecular imaging of advanced atherosclerotic plaques in the intact organism.     

Uptake of inflammatory cell marker [<Superscript>11</Superscript>C]PK11195 into mouse atherosclerotic plaques

Purpose  The ligand [¹¹C]PK11195 binds with high affinity and selectivity to peripheral benzodiazepine receptor, expressed in high amounts in macrophages. In humans, [¹¹C]PK11195 has been used successfully for the in vivo imaging of inflammatory processes of brain tissue. The purpose of this study was to explore the feasibility of [¹¹C]PK11195 in imaging inflammation in the atherosclerotic plaques. Methods  The presence of PK11195 binding sites in the atherosclerotic plaques was verified by examining the in vitro binding of [³H]PK11195 onto mouse aortic sections. Uptake of intravenously administered [¹¹C]PK11195 was studied ex vivo in excised tissue samples and aortic sections of a LDLR/ApoB48 atherosclerotic mice. Accumulation of the tracer was compared between the atherosclerotic plaques and non-atherosclerotic arterial sites by autoradiography and histological analyses. Results  The [³H]PK11195 was found to bind to both the atherosclerotic plaques and the healthy wall. The autoradiography analysis revealed that the uptake of [¹¹C]PK11195 to inflamed regions in plaques was more prominent (p = 0.011) than to non-inflamed plaque regions, but overall it was not higher than the uptake to the healthy vessel wall. Also, the accumulation of ¹¹C radioactivity into the aorta of the atherosclerotic mice was not increased compared to the healthy control mice. Conclusions  Our results indicate that the uptake of [¹¹C]PK11195 is higher in inflamed atherosclerotic plaques containing a large number of inflammatory cells than in the non-inflamed plaques. However, the tracer uptake to other structures of the artery wall was also prominent and may limit the use of [¹¹C]PK11195 in clinical imaging of atherosclerotic plaques.     

Atherosclerosis imaging on the molecular level

On the basis of clinical observations that acute coronary events often result from rupture of atherosclerotic plaques at sites with no or minor luminal narrowing, the search for techniques by which to identify vulnerable, rupture-prone lesions has developed into a quest for the holy grail of cardiovascular medicine. Vulnerable plaques may show characteristic morphologic features, but they may still differ in their biology and their activity, which ultimately leads to rupture. As a consequence, considerable efforts have been undertaken to identify biologic mechanisms of atherosclerotic lesions by use of molecular-targeted radiolabeled probes. A variety of approaches aiming at plaque inflammation, apoptosis, smooth muscle cell proliferation, extracellular matrix activation, or platelet binding have been introduced. Nevertheless, molecular imaging of atherosclerosis is still a work in progress. Challenges related to the best targeting approach, to translation of animal model results to the clinical setting, to adequate imaging methodology for visualization of coronary artery biology, and to a suitable target patient population will need to be overcome. But the field is steadily moving ahead and getting closer to the ultimate goal of an improved clinical risk assessment through in vivo assessment of vascular biology.     

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 ¹⁸F-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 ¹⁸F-FDG localization was evaluated by ³H-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 ³H-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). ¹⁸F-FDG PET revealed foamy macrophage distribution in the plaques. The intensity of ¹⁸F-FDG uptake was correlated positively with the degree of foamy macrophage infiltration. Micro-ARG showed identical ³H-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. ¹⁸F-FDG PET is a promising imaging technique for evaluating atherosclerosis and for monitoring changes in the composition of atherosclerotic plaques affecting their stability.     

Diagnosis of arthritis using near-infrared fluorochrome Cy5.5

PURPOSE: Near-infrared range fluorescence (NIRF) imaging is a potential tool to diagnose biologic processes in vivo. This applicability study sought to define whether imaging with fluorochrome Cy5.5 can identify arthritis in murine antigen-induced arthritis (AIA). MATERIALS AND METHODS: On day 7 of AIA (n = 9 mice), fluorescence intensities in inflamed and contralateral knee joints (the latter as internal control) were measured before and after intravenous injection of Cy5.5 (until 72 hours). Cy5.5 joint deposition was verified by confocal laser-scanning microscopy. Dye phagocytosis was evaluated in cultured macrophages (cell line PMJ2-R) by FACS analysis. Cy5.5 binding to serum protein was tested by NIRF scanning and gel electrophoresis. RESULTS: Between 2 and 72 hours, the arthritic knee joints showed significantly higher fluorescence intensities compared with contralateral joints. Microscopy confirmed Cy5.5 deposition in the synovial membrane. Cultured macrophages actively phagocytosed Cy5.5. Cy5.5 bound mainly to albumin as the main serum protein. CONCLUSION: NIRF imaging with Cy5.5 can identify arthritic joints in vivo, likely due to nonspecific deposition.     

Extracellular Traps in Lipid-Rich Lesions of Carotid Atherosclerotic Plaques: Implications for Lipoprotein Retention and Lesion Progression

PurposeTo investigate the presence and location of extracellular traps (ETs) in atherosclerotic plaques and to determine whether they are spatially associated with inflammatory cells and the lipid core.Materials and MethodsHuman carotid atherosclerotic plaques were collected from seven patients after surgical endarterectomy. Sequential tissue sections were stained with hematoxylin-eosin or subjected to immunohistochemistry to detect ETs, neutrophils and macrophages or apolipoprotein B (ApoB). To demonstrate the specificity of the antibody used to detect ETs, the adjacent tissue section was pretreated with deoxyribonuclease-1 (DNase-1) before immunostaining for ETs.ResultsAll seven carotid plaques demonstrated advanced atherosclerotic lesions. Extensive ET and ApoB immunostaining was detected predominantly within the acellular lipid core. Along the edges of the lipid core, confocal microscopy revealed areas suggestive of active release of ETs from MPO-positive cells. Pretreatment of tissue sections with DNase-1 abolished ET signal in the extracellular matrix, but not the signal within the cells along the margins of the core.ConclusionsThe localization of ETs to the lipid core suggests a possible binding site for lipoproteins, which may further promote lesion progression and inflammation.     

PET/MRI dual-modality tumor imaging using arginine-glycine-aspartic (RGD)-conjugated radiolabeled iron oxide nanoparticles

The purpose of this study was to develop a bifunctional iron oxide (IO) nanoparticle probe for PET and MRI scans of tumor integrin alphavbeta3 expression. METHODS: Polyaspartic acid (PASP)-coated IO (PASP-IO) nanoparticles were synthesized using a coprecipitation method, and particle size and magnetic properties were measured. A phantom study was used to assess the efficacy of PASP-IO as a T2-weighted MRI contrast agent. PASP-IO nanoparticles with surface amino groups were coupled to cyclic arginine-glycine-aspartic (RGD) peptides for integrin alphavbeta3 targeting and macrocyclic 1,4,7,10-tetraazacyclododecane-N,N',N',N',-tetraacetic acid (DOTA) chelators for PET after labeling with 64Cu. IO nanoparticle conjugates were further tested in vitro and in vivo to determine receptor targeting efficacy and feasibility for dual PET/MRI. RESULTS: PASP-IO nanoparticles made by single-step reaction have a core size of 5 nm with a hydrodynamic diameter of 45 +/- 10 nm. The saturation magnetization of PASP-IO nanoparticles is about 117 emu/g of iron, and the measured r2 and r2* are 105.5 and 165.5 (s.mM)(-1), respectively. A displacement competitive binding assay indicates that DOTA-IO-RGD conjugates bound specifically to integrin alphavbeta3 in vitro. Both small-animal PET and T2-weighted MRI show integrin-specific delivery of conjugated RGD-PASP-IO nanoparticles and prominent reticuloendothelial system uptake. CONCLUSION: We have successfully developed an IO-based nanoprobe for simultaneous dual PET and MRI of tumor integrin expression. The success of this bifunctional imaging approach may allow for earlier tumor detection with a high degree of accuracy and provide further insight into the molecular mechanisms of cancer.     

Optical imaging of spontaneous breast tumors using protease sensing 'smart' optical probes

OBJECTIVE: The objective of this study was to determine if spontaneous breast cancer lesions can be detected by fluorescence reflectance imaging (FRI) and fluorescence mediated tomography (FMT) using protease-sensing optical probes. MATERIALS AND METHODS: Transgenic (FVB/N-TgN (WapHRAS)69Lin Y)) mice, which spontaneously develop breast cancer, were injected intravenously with a cathepsin-sensing fluorescent imaging probe. FRI and FMT were performed 24 hours after probe injection and region of interest (ROI) analysis was performed. Magnetic resonance images were acquired for anatomic coregistration with the FMT data. Moreover, correlative immunohistochemistry and fluorescence microscopy were performed. RESULTS: All tumor nodules were clearly delineated by FRI showing an average signal intensity of 380 +/- 106 AU. Similarly, tumors were clearly detected by FMT imaging. Immunohistochemistry confirmed cathepsin-B expression of primary tumors and fluorescence microscopy revealed a strong Cy 5.5 deposition in the tissue. CONCLUSIONS: FRI and FMT using "smart" protease sensing probes permits detection of experimental spontaneous breast cancers. Because the expression levels of various proteases correlate with patient outcome, this technique may not only help to detect, but also to differentiate breast cancers noninvasively.     

Gadofluorine-enhanced magnetic resonance imaging of carotid atherosclerosis in Yucatan miniswine

OBJECTIVE: The aim of this study was to determine whether gadofluorine, a paramagnetic magnetic resonance imaging (MRI) contrast agent, selectively enhances carotid atherosclerotic plaques in Yucatan miniswine. METHODS: Atherosclerotic plaques were induced in the left carotid arteries (LCA) of Yucatan miniswine (n=3) by balloon denudation and high cholesterol diet. T1-weighted MRI was performed before and 24 hours after gadofluorine injection (at a dose of 100 micromol/kg) to assess the enhancement of the balloon-injured LCA wall relative to healthy, uninjured right carotid artery (RCA) wall. Histopathology was performed to verify the presence and composition of the atherosclerotic plaques imaged with MRI. RESULTS: Gadofluorine was found to enhance LCA atherosclerotic lesions relative to RCA wall by 21% (P<0.025) 24 hours after contrast injection. Enhancement of healthy LCA wall relative to healthy RCA wall was not observed. CONCLUSION: Gadofluorine selectively enhances carotid atherosclerotic plaques in Yucatan miniswine. Gadofluorine appears to be a promising MR contrast agent for detection of atherosclerotic plaques in vivo.     

Molecular imaging of atherosclerotic plaques with technetium-99m-labelled antisense oligonucleotides

Purpose The purpose of this study was to visualise experimental atherosclerotic lesions using radiolabelled antisense oligonucleotides (ASONs).Methods Atherosclerosis was induced in New Zealand White rabbits fed 1% cholesterol for approximately 60 days. In vivo and ex vivo imaging was performed in atherosclerotic rabbits and normal control rabbits after i.v. injection of 92.5±18.5 MBq ^99mTc-labelled ASON or ^99mTc-labelled sense oligonucleotides. Immediately after the in vivo imaging, the animals were sacrificed and ex vivo imaging of the aortic specimens was performed. Biodistribution of radiolabelled c-myc ASON was evaluated in vivo in atherosclerotic rabbits.Results Planar imaging revealed accumulation of ^99mTc-labelled c-myc ASON in atherosclerotic lesions along the artery wall. Ex vivo imaging further demonstrated that the area of activity accumulation matched the area of atherosclerotic lesions. In contrast, no atherosclerotic lesions were found in the vessel wall and no positive imaging results were obtained in animals of the control group.Conclusion This molecular imaging approach has potential for non-invasive imaging of atherosclerotic plaques at an early stage.     

In vivo imaging of integrin ανβ<Subscript>3</Subscript> expression using fluorescence-mediated tomography

Purpose Optical imaging would be desirable for cancer diagnostics since it can potentially resolve relevant oncological target structures in vivo. We therefore synthesised an αvβ₃ targeted fluorochrome and imaged tumour xenografts with different αvβ₃ expression levels using both planar and tomographic optical imaging methods. Methods An αvβ₃-targeted RGD peptide was labelled with a cyanine dye (Cy 5.5). Binding of the optical tracer was tested on M21 melanoma (n = 5), HT-1080 fibrosarcoma (n = 6) and MCF-7 adenocarcinoma (n = 5) cells and their tumour xenografts. All optical imaging studies were performed using two-dimensional planar fluorescence reflectance imaging (FRI) technology and three-dimensional fluorescence-mediated tomography (FMT). Results In vitro, the peptide-dye conjugate showed a clear binding affinity to αvβ₃-positive M21 and HT-1080 cells while αvβ₃-negative MCF-7 cells and pre-dosing with the free RGD peptide revealed little to no fluorescence. In vivo, tumour xenografts were clearly visualised by FRI and FMT up to 24 h post injection. FMT allowed quantification of the fluorochrome distribution in deeper tissue sections showing an average fluorochrome concentration of 417.61 ± 105.82 nM Cy 5.5 (M21), 353.68 ± 54.02 nM Cy 5.5 (HT-1080) and 262.83 ± 155.36 nM Cy 5.5 (MCF-7) in the target tissue 60 min after tracer administration. Competition with the free RGD peptide resulted in a reduction in the fluorochrome concentration in M21 tumour tissue (294.35 ± 84.27 nM). Conclusion RGD-Cy 5.5 combined with novel tomographic optical imaging methods allows non-invasive imaging of tumour-associated αvβ₃ expression and may thus be a promising strategy for sensitive evaluation of tumour target expression.