Michael T. Kuhlmann

Up-regulation of nestin in the infarcted myocardium potentially indicates differentiation of resident cardiac stem cells into various lineages including cardiomyocytes.

To identify proteins involved in cardiac regeneration, a proteomics approach was applied. A total of 26 proteins, which displayed aberrant expression in mouse hearts infarcted through ligation of the left anterior descending coronary artery, were identified. These included the intermediate filament protein nestin, which was up‐regulated in the infarct border zone. Corresponding changes were observed for its mRNA. Nestin mRNA was also up‐regulated in hearts from 17 of 19 patients with end‐stage heart failure, including 4 with acute myocardial infarction in comparison with 8 donor hearts. Immunofluorescence confo‐cal laser scanning microscopy revealed that nestin is expressed, on the one hand, in small proportions of cardiomyocytes, endothelial cells, smooth muscle cells, neuronal cells, and fibroblasts. On the other hand, it was found to be coexpressed with the stem cell markers c‐kit, Sca‐1, Mdr‐1, and Abcg2 in small interstitial cells. In infarcted hearts from chimeric mice transplanted with bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice, less than 1% of nestin‐positive cells coexpressed EGFP, although EGFP‐positive cells were abundant in these. Consequently, enhanced expression of nestin in the injured myocardium might reflect spontaneous regenerative processes supposedly based on the differentiation of resident cardiac stem cells into diverse cardiac cell types. Scobioala, S., Klocke, R., Kuhlmann, M., Tian, W., Hasib, L., Milting, H., Koenig, S., Stelljes, M., El‐Banayosy, A., Tenderich, G., Michel, G., Breithardt, G., Nikol, S. Up‐regulation of nestin in the infarcted myocardium potentially indicates differentiation of resident cardiac stem cells into various lineages including cardiomyo‐cytes. FASEB J. 22, 1021–1031 (2008)

Isochronous Assessment of Cardiac Metabolism and Function in Mice Using Hybrid PET/MRI

Recently, integrated small-animal PET/MRI prototypes that provide isochronous and coregistered datasets of morphology and function through the simultaneous acquisition of PET and MRI data have been developed. However, the need for MRI compatibility can constrain the technical design of the PET components and may lead to a lower sensitivity and lower spatial and temporal resolutions. The aim of this study was to evaluate the suitability of a prototype preclinical PET/MRI system for the simultaneous assessment of cardiac metabolism and function in mice. A stand-alone high-resolution small-animal PET scanner using the same evaluation protocols was used as a reference. Methods: Simultaneous PET/MR images of an infarct mouse model (21 animals plus 3 controls) were acquired. The imaging performance of the MRI-compatible PET insert was evaluated with respect to count sensitivity; myocardium-to-background contrast; suitability for the analysis of global left ventricular function; and uptake difference in scar, border-zone, and remote regions. The radiotracer 18F-FDG was used to acquire cardiac gated PET data, applying retrospective coincidence sorting. The PET insert data were coregistered to the MR images by determination of the appropriate transformation matrix. Results: An optimal registration of PET and MR images from the integrated system was achieved, and the reconstructed images showed a good visual correspondence in infarct areas between PET and MRI data. As expected, the PET insert showed a poorer performance with respect to counting rate and myocardium-to-background ratio than did the high-resolution PET. Assessment of left ventricular volumes was possible with the current PET/MRI prototype. A good correlation was found between PET and MRI (R > 0.95). Local PET uptake was successfully determined for different tissue, and a differentiation among remote, border-zone, and scar tissue was possible. However, the uptake difference for the PET/MRI prototype was lower than that for the high-resolution stand-alone PET system. Conclusion: A hybrid PET/MRI prototype was successfully used to assess cardiac parameters in an infarct mouse model, although performance was reduced when compared with a high-resolution animal PET scanner. Future technical improvements are expected to result in comparable performance while providing higher registration accuracy.

Metabolite identification of a radiotracer by electrochemistry coupled to liquid chromatography with mass spectrometric and radioactivity detection.

Radioligands, which specifically bind to a receptor or enzyme (target), enable molecular imaging of the target expression by positron emission tomography (PET). One very promising PET tracer is (S)-1-(4-(2-[(18)F]-fluoroethoxy)benzyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (isatin), a caspase-3 inhibitor, which has been developed at the University Hospital of Münster to image cell death (apoptosis). The translation of this novel tracer from preclinical evaluation to clinical examinations requires biodistribution studies, which characterize the pharmakodynamics and metabolic fate of the compound. This information is used to further optimize the radioligands and to interpret radioactive signals from tissues upon injection of the radioligand in vivo with respect to their specificity. The analysis of the metabolism of radioligands is hampered by the low amount of the compound being typically injected (nano/picomolar amount per injection). In the present study, electrochemistry (EC) is applied to elucidate the oxidative metabolism pathway of the radiotracer. Previous studies have demonstrated that EC can be utilized as a complementary tool to conventional in vitro approaches in drug metabolism studies. Thereby, potential oxidative metabolites of the isatin are determined by EC coupled to electrospray ionization mass spectrometry (EC/ESI-MS). Moreover, using EC/liquid chromatography (LC) and ESI-ion trap MS(n), structural elucidation of the oxidation products is performed. Comparatively to EC, in vitro metabolism studies with rat liver microsomes are conducted. Finally, the developed LC/ESI-MS method is applied to determine metabolites in body fluids and cell extracts from in vivo studies with the nonradioactive ((19)F) and radioactive isatin ((18)F). On the basis of the electrochemically generated oxidation products of the radioligand, the major radioactive metabolite occurring in vivo was successfully identified.

Non-FDG imaging of atherosclerosis: Will imaging of MMPs assess plaque vulnerability?

Acute ruptures of atherosclerotic plaques with subsequent occlusion account for the vast majority of clinical events such as myocardial infarction or stroke. New imaging approaches focusing on the visualization of inflammation in the vessel wall could emerge as tools for individualized risk assessment and prevention of events. To this end, PET employing 18F-fluorodeoxyglucose (FDG) has recently been introduced for the first clinical trials. Although this approach nicely visualizes plaques inflammation questions remain with respect to if and how this inflammatory signal can be employed for predicting individual plaque rupture. Molecular imaging of proteases such as matrix-metalloproteinases (MMPs) involved in several steps in plaque progression driving plaques into vulnerable, rupture-prone states seems a promising alternative approach. This review introduces and discusses the vulnerable plaque concept, animal models with human-like plaque ruptures and the potential of a FDG versus a non-FDG MMP-targeted strategy to image rupture-prone plaques.

Multimodal Imaging Reveals Temporal and Spatial Microglia and Matrix Metalloproteinase Activity after Experimental Stroke

Stroke is the most common cause of death and disability from neurologic disease in humans. Activation of microglia and matrix metalloproteinases (MMPs) is involved in positively and negatively affecting stroke outcome. Novel, noninvasive, multimodal imaging methods visualizing microglial and MMP alterations were employed. The spatio-temporal dynamics of these parameters were studied in relation to blood flow changes. Micro positron emission tomography (μPET) using [18F]BR-351 showed MMP activity within the first days after transient middle cerebral artery occlusion (tMCAo), followed by increased [18F]DPA-714 uptake as a marker for microglia activation with a maximum at 14 days after tMCAo. The inflammatory response was spatially located in the infarct core and in adjacent (penumbral) tissue. For the first time, multimodal imaging based on PET, single photon emission computed tomography, and magnetic resonance imaging revealed insight into the spatio-temporal distribution of critical parameters of poststroke inflammation. This allows further evaluation of novel treatment paradigms targeting the postischemic inflammation.

Distribution of lipid-based nanoparticles to infarcted myocardium with potential application for MRI-monitored drug delivery.

Adverse cardiac remodeling after myocardial infarction ultimately causes heart failure. To stimulate reparative processes in the infarct, efficient delivery and retention of therapeutic agents is desired. This might be achieved by encapsulation of drugs in nanoparticles. The goal of this study was to characterize the distribution pattern of differently sized long-circulating lipid-based nanoparticles, namely micelles (~15 nm) and liposomes (~100 nm), in a mouse model of myocardial infarction (MI). MI was induced in mice (n=38) by permanent occlusion of the left coronary artery. Nanoparticle accumulation following intravenous administration was examined one day and one week after surgery, representing the acute and chronic phase of MI, respectively. In vivo magnetic resonance imaging of paramagnetic lipids in the micelles and liposomes was employed to monitor the trafficking of nanoparticles to the infarcted myocardium. Ex vivo high-resolution fluorescence microscopy of fluorescent lipids was used to determine the exact location of the nanoparticles in the myocardium. In both acute and chronic MI, micelles permeated the entire infarct area, which renders them very suited for the local delivery of cardioprotective or anti-remodeling drugs. Liposomes displayed slower and more restricted extravasation from the vasculature and are therefore an attractive vehicle for the delivery of pro-angiogenic drugs. Importantly, the ability to non-invasively visualize both micelles and liposomes with MRI creates a versatile approach for the development of effective cardioprotective therapeutic interventions.

Variability of Proliferation and Diffusion in Different Lung Cancer Models as Measured by 3′-Deoxy-3′-18F-Fluorothymidine PET and Diffusion-Weighted MR Imaging

Molecular imaging allows the noninvasive assessment of cancer progression and response to therapy. The aim of this study was to investigate molecular and cellular determinants of 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) PET and diffusion-weighted (DW) MR imaging in lung carcinoma xenografts. Methods: Four lung cancer cell lines (A549, HTB56, EBC1, and H1975) were subcutaneously implanted in nude mice, and growth was followed by caliper measurements. Glucose uptake and tumor proliferation were determined by 18F-FDG and 18F-FLT PET, respectively. T2-weighted MR imaging was performed, and the apparent diffusion coefficient (ADC) was determined by DW MR imaging as an indicator of cell death. Imaging findings were correlated to histology with markers for tumor proliferation (Ki67, 5-bromo-2′-deoxyuridine [BrdU]) and cell death (caspase-3, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling). The expression of human equilibrative nucleoside transporter 1 (hENT1), thymidine kinase 1 (TK1), thymidylate synthase, and thymidine phosphorylase (TP) were analyzed by Western blot and immunohistochemistry. Thymidine levels were determined by liquid chromatography–mass spectrometry. Results: Xenografts varied with respect to in vivo growth rates. MR imaging and PET revealed intratumoral heterogeneities, which were confirmed by histology. 18F-FLT uptake differed significantly between tumor lines, with A549 and H1975 demonstrating the highest radiotracer accumulation (A549, 8.5 ± 3.2; HTB56, 4.4 ± 0.7; EBC1, 4.4 ± 1.2; and H1975, 12.1 ± 3.5 maximal percentage injected dose per milliliter). In contrast, differences in 18F-FDG uptake were only marginal. No clear relationship between 18F-FLT accumulation and immunohistochemical markers for tumor proliferation (Ki67, BrdU) as well as hENT1, TK1, or TS expression was detected. However, TP was highly expressed in A549 and H1975 xenografts, which was accompanied by low tumor thymidine concentrations, suggesting that tumor thymidine levels influence 18F-FLT uptake in the tumor models investigated. MR imaging revealed higher ADC values within proliferative regions of H1975 and A549 tumors than in HTB56 and EBC1. These ADC values were negatively correlated with cell density but not directly related to cell death. Conclusion: A direct relationship of 18F-FLT with proliferation or ADC with cell death might be complicated by the interplay of multiple processes at the cellular and physiologic levels in untreated tumors. This issue must be considered when using these imaging modalities in preclinical or clinical settings.

Early assessment of the efficacy of temozolomide chemotherapy in experimental glioblastoma using [18F]FLT-PET imaging.

Addition of temozolomide (TMZ) to radiation therapy is the standard treatment for patients with glioblastoma (GBM). However, there is uncertainty regarding the effectiveness of TMZ. Considering the rapid evolution of the disease, methods to assess TMZ efficacy early during treatment would be of great benefit. Our aim was to monitor early effects of TMZ in a mouse model of GBM using positron emission tomography (PET) with 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT). Methods Human glioma cells sensitive to TMZ (Gli36dEGFR-1) were treated with sub-lethal doses of TMZ to obtain cells with lower sensitivity to TMZ (Gli36dEGFR-2), as measured by growth and clonogenic assays. Gli36dEGFR-1 and Gli36dEGFR-2 cells were subcutaneously (s.c.) or intracranially (i.c.) xenografted into nude mice. Mice were treated for 7 days with daily injection of 25 or 50 mg/kg TMZ. Treatment efficacy was measured using [18F]FLT-PET before treatment and after 2 days. Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) were used to determine tumor volumes before treatment and after 7 days. Results A significant difference was observed between TMZ and DMSO treated tumors in terms of variations of [18F]FLT T/B ratio as soon as day 2 in the i.c. as well as in the s.c. mouse model. Variations of [18F]FLT T/B uptake ratio between days 0 and 2 correlated with variations of tumor size between days 0 and 7 (s.c. model: ntumor = 17 in nmice = 11, P<0.01; i.c. model: ntumor/mice = 9, P<0.01). Conclusions Our results indicate that [18F]FLT-PET may be useful for an early evaluation of the response of GBM to TMZ chemotherapy in patients with glioma.

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in (1) and (2)). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells and in vivo in larger animals and humans. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation. In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics as confirmed by in vivo Magnetic Resonance Imaging (MRI): Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed. In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

Combined PET Imaging of the Inflammatory Tumor Microenvironment Identifies Margins of Unique Radiotracer Uptake

The tumor microenvironment is highly heterogeneous. For gliomas, the tumor-associated inflammatory response is pivotal to support growth and invasion. Factors of glioma growth, inflammation, and invasion, such as the translocator protein (TSPO) and matrix metalloproteinases (MMP), may serve as specific imaging biomarkers of the glioma microenvironment. In this study, noninvasive imaging by PET with [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) was used for the assessment of localization and quantification of the expression of TSPO and MMP. Imaging was performed in addition to established clinical imaging biomarker of active tumor volume ([18F]FET) in conjunction with MRI. We hypothesized that each imaging biomarker revealed distinct areas of the heterogeneous glioma tissue in a mouse model of human glioma. Tracers were found to be increased 1.4- to 1.7-fold, with [18F]FET showing the biggest volume as depicted by a thresholding-based, volumes of interest analysis. Tumor areas, which could not be detected by a single tracer and/or MRI parameter alone, were measured. Specific compartments of [18F]DPA-714 (14%) and [18F]BR-351 (11%) volumes along the tumor rim could be identified. [18F]DPA-714 (TSPO) and [18F]BR-351 (MMP) matched with histology. Glioma-associated microglia/macrophages (GAM) were identified as TSPO and MMP sources. Multitracer and multimodal molecular imaging approaches may allow us to gain important insights into glioma-associated inflammation (GAM, MMP). Moreover, this noninvasive technique enables characterization of the glioma microenvironment with respect to the disease-driving cellular compartments at the various disease stages. Cancer Res; 77(8); 1831-41. ©2017 AACR.