Olivier Gheysens

Noninvasive molecular imaging of small living subjects using Raman spectroscopy

Molecular imaging of living subjects continues to rapidly evolve with bioluminescence and fluorescence strategies, in particular being frequently used for small-animal models. This article presents noninvasive deep-tissue molecular images in a living subject with the use of Raman spectroscopy. We describe a strategy for small-animal optical imaging based on Raman spectroscopy and Raman nanoparticles. Surface-enhanced Raman scattering nanoparticles and single-wall carbon nanotubes were used to demonstrate whole-body Raman imaging, nanoparticle pharmacokinetics, multiplexing, and in vivo tumor targeting, using an imaging system adapted for small-animal Raman imaging. The imaging modality reported here holds significant potential as a strategy for biomedical imaging of living subjects.

US Imaging of Tumor Angiogenesis with Microbubbles Targeted to Vascular Endothelial Growth Factor Receptor Type 2 in Mice

PURPOSE To prospectively evaluate contrast material-enhanced ultrasonography (US) with microbubbles targeted to vascular endothelial growth factor receptor type 2 (VEGFR2) for imaging tumor angiogenesis in two murine tumor models. MATERIALS AND METHODS Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. A US contrast agent, consisting of encapsulated gaseous microbubbles, was developed specifically to bind to VEGFR2 (by using anti-VEGFR2 antibodies and biotin-streptavidin interaction) which is up-regulated on endothelial cells of tumor blood vessels. VEGFR2-targeted microbubbles (MB(V)), control microbubbles (MB(C)), and nonlabeled microbubbles (MB(N)) were tested for binding specificity on cells expressing VEGFR2 (mouse angiosarcoma SVR cells) and control cells (mouse skeletal myoblast C2C12 cells). Expression of mouse VEGFR2 in culture cells was tested with immunocytochemical and Western blot analysis. Contrast-enhanced US imaging with MB(V) and MB(C) was performed in 28 tumor-bearing nude mice (mouse angiosarcoma, n = 18; rat malignant glioma, n = 10). Differences were calculated by using analysis of variance. RESULTS In cell culture, adherence of MB(V) on SVR cells (2.1 microbubbles per SVR cell) was significantly higher than adherence of control microbubbles (0.01-0.10 microbubble per SVR cell; P < .001) and significantly more MB(V) attached to SVR cells than to C2C12 cells (0.15 microbubble per C2C12 cell; P < .001). In vivo, contrast-enhanced US imaging showed significantly higher average video intensity when using MB(V) compared with MB(C) for angiosarcoma and malignant glioma tumors (P < .001). Results of immunohistochemical analysis confirmed VEGFR2 expression on vascular endothelial cells of both tumor types. CONCLUSION US imaging with contrast microbubbles targeted to VEGFR2 allows noninvasive visualization of VEGFR2 expression in tumor vessels in mice.

Ventricular Phosphodiesterase-5 Expression Is Increased in Patients With Advanced Heart Failure and Contributes to Adverse Ventricular Remodeling After Myocardial Infarction in Mice

Background— Ventricular expression of phosphodiesterase-5 (PDE5), an enzyme responsible for cGMP catabolism, is increased in human right ventricular hypertrophy, but its role in left ventricular (LV) failure remains incompletely understood. We therefore measured LV PDE5 expression in patients with advanced systolic heart failure and characterized LV remodeling after myocardial infarction in transgenic mice with cardiomyocyte-specific overexpression of PDE5 (PDE5-TG). Methods and Results— Immunoblot and immunohistochemistry techniques revealed that PDE5 expression was greater in explanted LVs from patients with dilated and ischemic cardiomyopathy than in control hearts. To evaluate the impact of increased ventricular PDE5 levels on cardiac function, PDE5-TG mice were generated. Confocal and immunoelectron microscopy revealed increased PDE5 expression in cardiomyocytes, predominantly localized to Z-bands. At baseline, myocardial cGMP levels, cell shortening, and calcium handling in isolated cardiomyocytes and LV hemodynamic measurements were similar in PDE5-TG and wild-type littermates. Ten days after myocardial infarction, LV cGMP levels had increased to a greater extent in wild-type mice than in PDE5-TG mice (P<0.05). Ten weeks after myocardial infarction, LV end-systolic and end-diastolic volumes were larger in PDE5-TG than in wild-type mice (57±5 versus 39±4 and 65±6 versus 48±4 &mgr;L, respectively; P<0.01 for both). LV systolic dysfunction and diastolic dysfunction were more marked in PDE5-TG than in wild-type mice, associated with enhanced hypertrophy and reduced contractile function in isolated cardiomyocytes from remote myocardium. Conclusions— Increased PDE5 expression predisposes mice to adverse LV remodeling after myocardial infarction. Increased myocardial PDE5 expression in patients with advanced cardiomyopathy may contribute to the development of heart failure and represents an important therapeutic target.

Evaluation of anatomy based reconstruction for partial volume correction in brain FDG-PET

UNLABELLED FDG-PET contributes to the diagnosis and management of neurological diseases. In some of these diseases, pathological gray matter (GM) areas may have a reduced FDG uptake. Detection of these regions can be difficult and some remain undiscovered using visual assessment. The main reason for this detection problem is the relatively small thickness of GM compared to the spatial resolution of PET, known as the partial volume effect. We have developed an anatomy-based maximum-a-posteriori reconstruction algorithm (A-MAP) which corrects for this effect during the reconstruction using segmented magnetic resonance (MR) data. Monte-Carlo based 3-D brain software phantom simulations were used to investigate the influence of the strength of anatomy-based smoothing in GM, the influence of misaligned MR data, and the effect of local segmentation errors. A human observer study was designed to assess the detection performance of A-MAP versus post-smoothed maximum-likelihood (ML) reconstruction. We demonstrated the applicability of A-MAP using real patient data. The results for A-MAP showed improved recovery values and robustness for local segmentation errors. Misaligned MR data reduced the recovery values towards those obtained by post-smoothed ML, for small registration errors. In the human observer study, detection accuracy of hypometabolic regions was significantly improved using A-MAP, compared to post-smoothed ML (P < 0.004). The patient study confirmed the applicability of A-MAP in clinical practice. CONCLUSION A-MAP is a promising technique for voxel-based partial volume correction of FDG-PET of the human brain.

(18)F-FDG PET/CT for early detection of embolism and metastatic infection in patients with infective endocarditis.

PurposeIn the acute setting of endocarditis it is very important to assess both the vegetation itself, as well as potential life-threatening complications, in order to decide whether antibiotic therapy will be sufficient or urgent surgery is indicated. A single whole-body scan investigating inflammatory changes could be very helpful to achieve a swift and efficient assessment.MethodsIn this study we assessed whether 18F-FDG can be used to detect and localize peripheral embolism or distant infection. Twenty-four patients with 25 episodes of endocarditis, enrolled between March 2006 and February 2008, underwent 18F-FDG PET/CT imaging on a dedicated PET/CT scanner.ResultsPET/CT imaging revealed a focus of peripheral embolization and/or metastatic infection in 11 episodes (44%). One episode had a positive PET/CT scan result for both embolism and metastatic infection. PET/CT detected seven positive cases (28%) in which there was no clinical suspicion. Valve involvement of endocarditis was seen only in three patients (12%).ConclusionPET/CT may be an important diagnostic tool for tracing peripheral embolism and metastatic infection in the acute setting of infective endocarditis, since a PET/CT scan detected a clinically occult focus in nearly one third of episodes.

Differential Effects of Progenitor Cell Populations on Left Ventricular Remodeling and Myocardial Neovascularization After Myocardial Infarction

OBJECTIVES We compared biological repair after acute myocardial infarction (AMI) with selected porcine progenitor cell populations. BACKGROUND Cell types and mechanisms responsible for myocardial repair after AMI remain uncertain. METHODS In a blinded, randomized study, we infused autologous late-outgrowth endothelial progenitor cells (EPC) (n = 10, 34 +/- 22 x 10(6) CD29-31-positive, capable of tube formation), allogeneic green fluorescent peptide-labeled mesenchymal stem cells (MSC) (n = 11, 10 +/- 2 x 10(6) CD29-44-90-positive, capable of adipogenic and osteogenic differentiation), or vehicle (CON) (n = 12) in the circumflex artery 1 week after AMI. Systolic function (ejection fraction), left ventricular (LV) end-diastolic and end-systolic volumes, and infarct size were assessed with magnetic resonance imaging at 1 week and 7 weeks. Cell engraftment and vascular density were evaluated on postmortem sections. RESULTS Recovery of LV ejection fraction from 1 to 7 weeks was similar between groups, but LV remodeling markedly differed with a greater increase of LV end-systolic volume in MSC and CON (+11 +/- 12 ml/m(2) and +7 +/- 8 ml/m(2) vs. -3 +/- 11 ml/m(2) in EPC, respectively, p = 0.04), and a similar trend was noted for LV end-diastolic volume (p = 0.09). After EPC, infarct size decreased more in segments with >50% infarct transmurality (p = 0.02 vs. MSC and CON) and was associated with a greater vascular density (p = 0.01). Late outgrowth EPCs secrete higher levels of the pro-angiogenic placental growth factor (733 [277 to 1,214] pg/10(6) vs. 59 [34 to 88] pg/10(6) cells in MSC, p = 0.03) and incorporate in neovessels in vivo. CONCLUSIONS Infusion of late-outgrowth EPCs after AMI improves myocardial infarction remodeling via enhanced neovascularization but does not mediate cardiomyogenesis. Endothelial progenitor cell transfer might hold promise for heart failure prevention via pro-angiogenic or paracrine matrix-modulating effects.

Imaging of VEGF receptor in a rat myocardial infarction model using PET.

Myocardial infarction (MI) leads to left ventricular (LV) remodeling, which leads to the activation of growth factors such as vascular endothelial growth factor (VEGF). However, the kinetics of a growth factors receptor expression, such as VEGF, in the living subject has not yet been described. We have developed a PET tracer (64Cu-DOTA-VEGF121 [DOTA is 1,4,7,10-tetraazadodecane-N,N′,N″,N‴-tetraacetic acid]) to image VEGF receptor (VEGFR) expression after MI in the living subject. Methods: In Sprague–Dawley rats, MI was induced by ligation of the left coronary artery and confirmed by ultrasound (n = 8). To image and study the kinetics of VEGFRs, 64Cu-DOTA-VEGF121 PET scans were performed before MI induction (baseline) and on days 3, 10, 17, and 24 after MI. Sham-operated animals served as controls (n = 3). Results: Myocardial origin of the 64Cu-DOTA-VEGF121 signal was confirmed by CT coregistration and autoradiography. VEGFR specificity of the 64Cu-DOTA-VEGF121 probe was confirmed by in vivo use of a 64Cu-DOTA-VEGFmutant. Baseline myocardial uptake of 64Cu-DOTA-VEGF121 was minimal (0.30 ± 0.07 %ID/g [percentage injected dose per gram of tissue]); it increased significantly after MI (day 3, 0.97 ± 0.05 %ID/g; P < 0.05 vs. baseline) and remained elevated for 2 wk (up to day 17 after MI), after which time it returned to baseline levels. Conclusion: We demonstrate the feasibility of imaging VEGFRs in the myocardium. In summary, we imaged and described the kinetics of 64Cu-DOTA-VEGF121 uptake in a rat model of MI. Studies such as the one presented here will likely play a major role when studying pathophysiology and assessing therapies in different animal models of disease and, potentially, in patients.

Monitoring of the Biological Response to Murine Hindlimb Ischemia With 64Cu-Labeled Vascular Endothelial Growth Factor-121 Positron Emission Tomography

Background— Vascular endothelial growth factor-121 (VEGF121), an angiogenic protein secreted in response to hypoxic stress, binds to VEGF receptors (VEGFRs) overexpressed on vessels of ischemic tissue. The purpose of this study was to evaluate 64Cu-VEGF121 positron emission tomography for noninvasive spatial, temporal, and quantitative monitoring of VEGFR2 expression in a murine model of hindlimb ischemia with and without treadmill exercise training. Methods and Results— 64Cu-labeled VEGF121 and a VEGF mutant were tested for VEGFR2 binding specificity in cell culture. Mice (n=58) underwent unilateral ligation of the femoral artery, and postoperative tissue ischemia was assessed with laser Doppler imaging. Longitudinal VEGFR2 expression in exercised and nonexercised mice was quantified with 64Cu-VEGF121 positron emission tomography at postoperative day 8, 15, 22, and 29 and correlated with postmortem &ggr;-counting. Hindlimbs were excised for immunohistochemistry, Western blotting, and microvessel density measurements. Compared with the VEGF mutant, VEGF121 showed specific binding to VEGFR2. Perfusion in ischemic hindlimbs fell to 9% of contralateral hindlimb on postoperative day 1 and recovered to 82% on day 29. 64Cu-VEGF121 uptake in ischemic hindlimbs increased significantly (P<0.001) from a control level of 0.61±0.17% ID/g (percentage of injected dose per gram) to 1.62±0.35% ID/g at postoperative day 8, gradually decreased over the following 3 weeks (0.59±0.14% ID/g at day 29), and correlated with &ggr;-counting (R2=0.99). Compared with nonexercised mice, 64Cu-VEGF121 uptake was increased significantly (P≤0.0001) in exercised mice (at day 15, 22, and 29) and correlated with VEGFR2 levels as obtained by Western blotting (R2=0.76). Ischemic hindlimb tissue stained positively for VEGFR2. In exercised mice, microvessel density was increased significantly (P<0.001) compared with nonexercised mice. Conclusions— 64Cu-VEGF121 positron emission tomography allows longitudinal spatial and quantitative monitoring of VEGFR2 expression in murine hindlimb ischemia and indirectly visualizes enhanced angiogenesis stimulated by treadmill exercise training.

A Comparison Between a Time Domain and Continuous Wave Small Animal Optical Imaging System

We present a phantom study to evaluate the performance of the eXplore Optix (Advanced Research Technologies-GE Healthcare), the first commercially available time-domain tomography system for small animal fluorescence imaging, and compare its capabilities with the widely used IVIS 200 (Xenogen Corporation-Caliper) continuous wave planar imaging system. The eXplore Optix, based on point-wise illumination and collection scheme, is found to be a log order more sensitive with significantly higher detection depth and spatial resolution as compared with the wide-area illumination IVIS 200 under the conditions tested. A time-resolved detection system allows the eXplore Optix to measure the arrival time distribution of fluorescence photons. This enables fluorescence lifetime measurement, absorption mapping, and estimation of fluorescent inclusion depth, which in turn is used by a reconstruction algorithm to calculate the volumetric distribution of the fluorophore concentration. An increased acquisition time and lack of ability to image multiple animals simultaneously are the main drawbacks of the eXplore Optix as compared with the IVIS 200.

Molecular Imaging of the Efficacy of Heat Shock Protein 90 Inhibitors in Living Subjects

Heat shock protein 90 alpha (Hsp90 alpha)/p23 and Hsp90 beta/p23 interactions are crucial for proper folding of proteins involved in cancer and neurodegenerative diseases. Small molecule Hsp90 inhibitors block Hsp90 alpha/p23 and Hsp90 beta/p23 interactions in part by preventing ATP binding to Hsp90. The importance of isoform-selective Hsp90 alpha/p23 and Hsp90 beta/p23 interactions in determining the sensitivity to Hsp90 was examined using 293T human kidney cancer cells stably expressing split Renilla luciferase (RL) reporters. Interactions between Hsp90 alpha/p23 and Hsp90 beta/p23 in the split RL reporters led to complementation of RL activity, which was determined by bioluminescence imaging of intact cells in cell culture and living mice using a cooled charge-coupled device camera. The three geldanamycin-based and seven purine-scaffold Hsp90 inhibitors led to different levels of inhibition of complemented RL activities (10-70%). However, there was no isoform selectivity to both classes of Hsp90 inhibitors in cell culture conditions. The most potent Hsp90 inhibitor, PU-H71, however, led to a 60% and 30% decrease in RL activity (14 hr) in 293T xenografts expressing Hsp90 alpha/p23 and Hsp90 beta/p23 split reporters respectively, relative to carrier control-treated mice. Molecular imaging of isoform-specific Hsp90 alpha/p23 and Hsp90 beta/p23 interactions and efficacy of different classes of Hsp90 inhibitors in living subjects have been achieved with a novel genetically encoded reporter gene strategy that should help in accelerating development of potent and isoform-selective Hsp90 inhibitors.