Edmund J. Keliher

Rapid monocyte kinetics in acute myocardial infarction are sustained by extramedullary monocytopoiesis

IL-1b signaling augments continued splenic monocyte supply during acute inflammation.

18F-4V for PET-CT imaging of VCAM-1 expression in atherosclerosis.

OBJECTIVES The aim of this study was to iteratively develop and validate an (18)F-labeled small vascular cell adhesion molecule (VCAM)-1 affinity ligand and demonstrate the feasibility of imaging VCAM-1 expression by positron emission tomography-computed tomography (PET-CT) in murine atherosclerotic arteries. BACKGROUND Hybrid PET-CT imaging allows simultaneous assessment of atherosclerotic lesion morphology (CT) and may facilitate early risk assessment in individual patients. The early induction, confinement of expression to atherosclerotic lesions, and accessible position in proximity to the blood pool render the adhesion molecule VCAM-1 an attractive imaging biomarker for inflamed atheroma prone to complication. METHODS A cyclic, a linear, and an oligomer affinity peptide, internalized into endothelial cells by VCAM-1-mediated binding, were initially derivatized with DOTA to determine their binding profiles and pharmacokinetics. The lead compound was then (18)F-labeled and tested in atherosclerotic apoE(-/-) mice receiving a high-cholesterol diet as well as wild type murine models of myocardial infarction and heart transplant rejection. RESULTS The tetrameric peptide had the highest affinity and specificity for VCAM-1 (97% inhibition with soluble VCAM-1 in vitro). In vivo PET-CT imaging using (18)F-4V showed 0.31 +/- 0.02 SUV in murine atheroma (ex vivo %IDGT 5.9 +/- 1.5). (18)F-4V uptake colocalized with atherosclerotic plaques on Oil Red O staining and correlated to mRNA levels of VCAM-1 measured by quantitative reverse transcription polymerase chain reaction (R = 0.79, p = 0.03). Atherosclerotic mice receiving an atorvastatin-enriched diet had significantly lower lesional uptake (p < 0.05). Furthermore, (18)F-4V imaging in myocardial ischemia after coronary ligation and in transplanted cardiac allografts undergoing rejection showed high in vivo PET signal in inflamed myocardium and good correlation with ex vivo measurement of VCAM-1 mRNA by quantitative polymerase chain reaction. CONCLUSIONS (18)F-4V allows noninvasive PET-CT imaging of VCAM-1 in inflammatory atherosclerosis, has the dynamic range to quantify treatment effects, and correlates with inflammatory gene expression.

Hybrid PET-optical imaging using targeted probes.

Fusion imaging of radionuclide-based molecular (PET) and structural data [x-ray computed tomography (CT)] has been firmly established. Here we show that optical measurements [fluorescence-mediated tomography (FMT)] show exquisite congruence to radionuclide measurements and that information can be seamlessly integrated and visualized. Using biocompatible nanoparticles as a generic platform (containing a 18F isotope and a far red fluorochrome), we show good correlations between FMT and PET in probe concentration (r2 > 0.99) and spatial signal distribution (r2 > 0.85). Using a mouse model of cancer and different imaging probes to measure tumoral proteases, macrophage content and integrin expression simultaneously, we demonstrate the distinct tumoral locations of probes in multiple channels in vivo. The findings also suggest that FMT can serve as a surrogate modality for the screening and development of radionuclide-based imaging agents.

Reactive polymer enables efficient in vivo bioorthogonal chemistry

There has been intense interest in the development of selective bioorthogonal reactions or “click” chemistry that can proceed in live animals. Until now however, most reactions still require vast surpluses of reactants because of steep temporal and spatial concentration gradients. Using computational modeling and design of pharmacokinetically optimized reactants, we have developed a predictable method for efficient in vivo click reactions. Specifically, we show that polymer modified tetrazines (PMT) are a key enabler for in vivo bioorthogonal chemistry based on the very fast and catalyst-free [4 + 2] tetrazine/trans-cyclooctene cycloaddition. Using fluorescent PMT for cellular resolution and 18F labeled PMT for whole animal imaging, we show that cancer cell epitopes can be easily reacted in vivo. This generic strategy should help guide the design of future chemistries and find widespread use for different in vivo bioorthogonal applications, particularly in the biomedical sciences.

Detection of Macrophages in Aortic Aneurysms by Nanoparticle Positron Emission Tomography–Computed Tomography

Objective—Current management of aortic aneurysms (AAs) relies primarily on size criteria to determine whether invasive repair is indicated to preempt rupture. We hypothesized that emerging molecular imaging tools could be used to more sensitively gauge local inflammation. Because macrophages are key effector cells that destabilize the extracellular matrix in the arterial wall, it seemed likely that they would represent suitable imaging targets. We here aimed to develop and validate macrophage-targeted nanoparticles labeled with fluorine-18 (18F) for positron emission tomography–computed tomography (PET-CT) detection of inflammation in AAs. Methods and Results—Aneurysms were induced in apolipoprotein E−/− mice via systemic administration of angiotensin II. Mice were imaged using PET-CT and a monocyte/macrophage–targeted nanoparticle. AAs were detected by contrast-enhanced micro-CT and had a mean diameter of 1.85±0.08 mm, whereas normal aortas measured 1.07±0.03 (P<0.05). The in vivo PET signal was significantly higher in aneurysms (standard uptake value, 2.46±0.48) compared with wild-type aorta (0.82±0.05, P<0.05). Validation with scintillation counting, autoradiography, fluorescence, and immunoreactive histology and flow cytometry demonstrated that nanoparticles localized predominantly to monocytes and macrophages within the aneurysmatic wall. Conclusion—PET-CT imaging with 18F-labeled nanoparticles allows quantitation of macrophage content in a mouse model of AA.

In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation

Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where, together with fibrin and platelets, the bacteria initiate the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin could be used to detect S. aureus in endocarditic vegetations via noninvasive fluorescence or positron emission tomography (PET) imaging. These prothrombin derivatives bound staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.

Synthesis of [18F]BODIPY: Bifunctional Reporter for Hybrid Optical/Positron Emission Tomography Imaging

Fluorescence-based intravital imaging approaches have been essential for studying the function and distribution of biologically active small molecules and their molecular targets in vivo.[1] While optical imaging enables visualization over a broad scope of spatial resolution ranging from subcellular to whole body anatomical imaging, its application is constrained by the optical transparency of tissues. Optical imaging modalities are thus better suited for small animal imaging rather than for translational applications.[2] In contrast, Positron Emission Tomography (PET) is used routinely for clinical applications but its use is limited primarily to macroscopic spatial and temporal imaging. A strategy that would enable seamless switching between fluorescence and PET imaging would thus be highly desirable for several reasons. First, the ability to visualize PET probes at the microscopic level would allow more rapid validation of the former in vivo and consequently expedite the development of novel PET probes. Second, for macroscopic hybrid imaging[3], it would be possible for optical and PET signals to be superimposed onto structural information derived from magnetic resonance imaging (MRI) or computed tomography (CT). Likewise, the ability to image in more than one or two channels would be useful for visualizing underlying physiological events. Third, hits from microscopic screens of small molecule fluorochrome-adducts could be more rapidly translated into clinically relevant applications.

Synthesis and In Vivo Imaging of a 18F‐Labeled PARP1 Inhibitor Using a Chemically Orthogonal Scavenger‐Assisted High‐Performance Method

Pedal to the metal: Using inverse Diels-Alder catalyst free TCO/Tz cycloadditions, we were able to quickly and selectively generate an 18F-labeled AZD2281-derivative from multiple different scaffolds (A–E). Excess cold material was removed within minutes using a TCO scavenger resin. This protocol allows the parallel synthesis of a library of potential PET imaging agents in a short time, increasing the efficiency of lead compound detection. The novel PET probe was successfully tested in biological assays and its potency and targeted accumulation was confirmed in vivo.

89Zr-Labeled Dextran Nanoparticles Allow in Vivo Macrophage Imaging

Tissue macrophages play a critical role both in normal physiology and in disease states. However, because of a lack of specific imaging agents, we continue to have a poor understanding of their absolute numbers, flux rates, and functional states in different tissues. Here, we describe a new macrophage specific positron emission tomography imaging agent, labeled with zirconium-89 ((89)Zr), that was based on a cross-linked, short chain dextran nanoparticle (13 nm). Following systemic administration, the particle demonstrated a vascular half-life of 3.9 h and was found to be located primarily in tissue resident macrophages rather than other white blood cells. Subsequent imaging of the probe using a xenograft mouse model of cancer allowed for quantitation of tumor-associated macrophage numbers, which are of major interest in emerging molecular targeting strategies. It is likely that the material described, which allows the visualization of macrophage biology in vivo, will likewise be useful for a multitude of human applications.

High‐Yielding, Two‐Step 18F Labeling Strategy for 18F‐PARP1 Inhibitors

Positron emission tomography (PET) of labeled metabolites, drugs, proteins and nanomaterials[1-3] is rapidly emerging as a powerful imaging tool to detect and stage disease, to study human biology, to investigate pharmacokinetics and pharmacodynamics of new drugs, or to measure treatment efficacy in clinical trials.[4-8] 18F is one of the most commonly used isotopes for clinical imaging given its half-life, ease of production, wide availability, and compatibility with microfluidics syntheses.[9] Despite extensive use and well established procedures of labeling some small molecules, facile 18F platform-type universally adaptable labeling strategies are still largely missing. This is especially true for rapid labeling of small molecules that emerge from high throughput screens or for optimizing hybrid and modular imaging agents. Bioorthogonal chemistries represent one avenue to develop such generic labeling platforms.