Detection of Early Endothelial Dysfunction by Optoacoustic Tomography.

Variations in vascular wall shear stress are often presumed to result in the formation of atherosclerotic lesions at specific arterial regions, where continuous laminar flow is disturbed. The influences of altered blood flow dynamics and oscillations on the integrity of endothelial cells and the endothelial layer have been extensively studied in vitro and in vivo. Under pathological conditions, the Arg-Gly-Asp (RGD) motif binding integrin αvß3 has been identified as a relevant target, as it induces endothelial cell activation. Animal models for in vivo imaging of endothelial dysfunction (ED) mainly rely on genetically modified knockout models that develop endothelial damage and atherosclerotic plaques upon hypercholesterolemia (ApoE-/- and LDLR-/-), thereby depicting late-stage pathophysiology. The visualization of early ED, however, remains a challenge. Therefore, a carotid artery cuff model of low and oscillating shear stress was applied in CD-1 wild-type mice, which should be able to show the effects of altered shear stress on a healthy endothelium, thus revealing alterations in early ED. Multispectral optoacoustic tomography (MSOT) was assessed as a non-invasive and highly sensitive imaging technique for the detection of an intravenously injected RGD-mimetic fluorescent probe in a longitudinal (2-12 weeks) study after surgical cuff intervention of the right common carotid artery (RCCA). Images were analyzed concerning the signal distribution upstream and downstream of the implanted cuff, as well as on the contralateral side as a control. Subsequent histological analysis was applied to delineate the distribution of relevant factors within the carotid vessel walls. Analysis revealed a significantly enhanced fluorescent signal intensity in the RCCA upstream of the cuff compared to the contralateral healthy side and the downstream region at all time points post-surgery. The most obvious differences were recorded at 6 and 8 weeks after implantation. Immunohistochemistry revealed a high degree of αv-positivity in this region of the RCCA, but not in the left common carotid artery (LCCA) or downstream of the cuff. In addition, macrophages could be detected by CD68 immunohistochemistry in the RCCA, showing ongoing inflammatory processes. In conclusion, MSOT is capable of delineating alterations in endothelial cell integrity in vivo in the applied model of early ED, where an elevated expression of integrin αvß3 was detected within vascular structures.

A Novel Mouse Model of Staphylococcus aureus Vascular Graft Infection: Noninvasive Imaging of Biofilm Development in Vivo.

Staphylococcus aureus causes very serious infections of vascular grafts. Knowledge of the molecular mechanisms of this disease is largely lacking because of the absence of representable models. Therefore, the aim of this study was to set up a mouse model of vascular graft infections that closely mimics the human situation. A catheter was inserted into the right carotid artery of mice, which acted as a vascular graft. Mice were infected i.v. using 8 different S. aureus strains, and development of the infection was followed up. Although all strains had varying abilities to form biofilm in vitro and different levels of virulence in mice, they all caused biofilm formation on the grafts. This graft infection was monitored using magnetic resonance imaging (MRI) and 18F-fluordeoxyglucose positron emission tomography (FDG-PET). MRI allowed the quantification of blood flow through the arteries, which was decreased in the catheter after infection. FDG-PET revealed high inflammation levels at the site of the catheter after infection. This model closely resembles the situation in patients, which is characterized by a tight interplay between pathogen and host, and can therefore be used for the testing of novel treatment, diagnosis, and prevention strategies. In addition, combining MRI and PET with microscopic techniques provides an appropriate way to characterize the course of these infections and to precisely analyze biofilm development.

6-hydroxydopamine-induced Parkinson's disease-like degeneration generates acute microgliosis and astrogliosis in the nigrostriatal system but no bioluminescence imaging-detectable alteration in adult neurogenesis.

Parkinson's disease is a slowly progressing neurodegenerative disorder caused by loss of dopaminergic neurons in the substantia nigra (SN), leading to severe impairment in motor and non-motor functions. Endogenous subventricular zone (SVZ) neural stem cells constantly give birth to new cells that might serve as a possible source for regeneration in the adult brain. However, neurodegeneration is accompanied by neuroinflammation and dopamine depletion, potentially compromising regeneration. We therefore employed in vivo imaging methods to study striatal deafferentation (N-ω-fluoropropyl-2ß-carbomethoxy-3ß-(4-[(123) I]iodophenyl)nortropane single photon emission computed tomography, DaTscan(™) ) and neuroinflammation in the SN and striatum (N,N-diethyl-2-(2-(4-(2-[(18) F]fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide positron emission tomography, [(18) F]DPA-714 PET) in the intranigral 6-hydroxydopamine Parkinson's disease mouse model. Additionally, we transduced cells in the SVZ with a lentivirus encoding firefly luciferase and followed migration of progenitor cells in the SVZ-olfactory bulb axis via bioluminescence imaging under disease and control conditions. We found that activation of microglia in the SN is an acute process accompanying the degeneration of dopaminergic cell bodies in the SN. Dopaminergic deafferentation of the striatum does not influence the generation of doublecortin-positive neuroblasts in the SVZ, but generates chronic astrogliosis in the nigrostriatal system.

Velocity mapping of the aortic flow at 9.4 T in healthy mice and mice with induced heart failure using time-resolved three-dimensional phase-contrast MRI (4D PC MRI).

OBJECTIVES: In this study, we established and validated a time-resolved three-dimensional phase-contrast magnetic resonance imaging method (4D PC MRI) on a 9.4 T small-animal MRI system. Herein we present the feasibility of 4D PC MRI in terms of qualitative and quantitative flow pattern analysis in mice with transverse aortic constriction (TAC). MATERIALS AND METHODS: 4D PC FLASH images of a flow phantom and mouse heart were acquired at 9.4 T using a four-point phase-encoding scheme. The method was compared with slice-selective PC FLASH and ultrasound using Bland-Altman analysis. Advanced 3D streamlines were visualized utilizing Voreen volume-rendering software. RESULTS: In vitro, 4D PC MRI flow profiles showed the transition between laminar and turbulent flow with increasing velocities. In vivo, 4D PC MRI data of the ascending aorta and the pulmonary artery were confirmed by ultrasound, resulting in linear regressions of R (2) > 0.93. Magnitude- and direction-encoded streamlines differed substantially pre- and post-TAC surgery. CONCLUSIONS: 4D PC MRI is a feasible tool for in vivo velocity measurements on high-field small-animal scanners. Similar to clinical measurement, this method provides a complete spatially and temporally resolved dataset of the murine cardiovascular blood flow and allows for three-dimensional flow pattern analysis.

Imaging of MMP activity in postischemic cardiac remodeling using radiolabeled MMP-2/9 activatable peptide probes.

The noninvasive imaging of matrix metalloproteinases (MMPs) activity in postischemic myocardial tissue holds great promise to predict cardiac function post-myocardial infarction. Consequently, development of MMP specific molecular imaging probes for noninvasive visualization and quantification of MMP activity is of great interest. A novel MMP imaging strategy is based on activatable cell-penetrating peptide probes (ACPP) that are sensitive to the proteolytic activity of MMP-2 and -9. The MMP-mediated activation of these ACPPs drives probe accumulation at the target site. The aim of this study was the development and characterization of radiolabeled MMP-2/9 sensitive ACPPs to assess MMP activity in myocardial remodeling in vivo. Specifically, a short and long-circulating MMP activatable cell-penetrating imaging probe (ACPP and Alb-ACPP, respectively; the latter is an ACPP modified with an albumin binding ligand that prolongs blood clearance) were successfully synthesized and radiolabeled. Subsequently, their biodistributions were determined in vivo in a Swiss mouse model of myocardial infarction. Both peptide probes showed a significantly higher uptake in infarcted myocardium compared to remote myocardium. The biodistribution for dual-isotope radiolabeled probes, which allowed us to discriminate between uncleaved ACPP and activated ACPP, showed increased retention of activated ACPP and activated Alb-ACPP in infarcted myocardium compared to remote myocardium. The enhanced retention correlated to gelatinase levels determined by gelatin zymography, whereas no correlation was found for the negative control: an MMP-2/9 insensitive non-ACPP. In conclusion, radiolabeled MMP sensitive ACPP probes enable to assess MMP activity in the course of remodeling post-myocardial infarction in vivo. Future research should evaluate the feasibility and the predictive value of the ACPP strategy for assessing MMP activity as a main player in postinfarction myocardial remodeling in vivo.

Optimizing glioblastoma temozolomide chemotherapy employing lentiviral-based anti-MGMT shRNA technology.

Despite treatments combining surgery, radiation-, and chemotherapy, patients affected by glioblastoma (GBM) have a limited prognosis. Addition of temozolomide (TMZ) to radiation therapy is the standard therapy in clinical application, but effectiveness of TMZ is limited by the tumor's overexpression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). The goal of this study was to use the highly specific and efficient RNA interference (RNAi) pathway to modulate MGMT expression to increase TMZ efficiency in chemotherapy resistant GBM. Using lentiviral-based anti-MGMT small hairpin RNA (shRNA) technology we observed a specific inhibition of the MGMT expression in GBM cell lines as well as in subcutaneous tumors. Tumor growth inhibition was observed following TMZ treatment of xenografts with low MGMT expression in contrast to xenografts with high MGMT expression. Bioluminescence imaging (BLI) measurements indicated that luciferase and shRNA-expressing lentiviruses were able to efficiently transduce the GBM xenografts in vivo. Treatment combining injection of a lentivirus expressing an anti-MGMT shRNA and TMZ induced a reduction of the size of the tumors, in contrast with treatment combining the lentivirus expressing the control shRNA and TMZ. Our data suggest that anti-MGMT shRNA therapy could be used in combination with TMZ chemotherapy in order to improve the treatment of resistant GBM.

Cardiac-respiratory self-gated cine ultra-short echo time (UTE) cardiovascular magnetic resonance for assessment of functional cardiac parameters at high magnetic fields.

BACKGROUND: To overcome flow and electrocardiogram-trigger artifacts in cardiovascular magnetic resonance (CMR), we have implemented a cardiac and respiratory self-gated cine ultra-short echo time (UTE) sequence. We have assessed its performance in healthy mice by comparing the results with those obtained with a self-gated cine fast low angle shot (FLASH) sequence and with echocardiography. METHODS: 2D self-gated cine UTE (TE/TR = 314 µs/6.2 ms, resolution: 129 × 129 µm, scan time per slice: 5 min 5 sec) and self-gated cine FLASH (TE/TR = 3 ms/6.2 ms, resolution: 129 × 129 µm, scan time per slice: 4 min 49 sec) images were acquired at 9.4 T. Volume of the left and right ventricular (LV, RV) myocardium as well as the end-diastolic and -systolic volume was segmented manually in MR images and myocardial mass, stroke volume (SV), ejection fraction (EF) and cardiac output (CO) were determined. Statistical differences were analyzed by using Student t test and Bland-Altman analyses. RESULTS: Self-gated cine UTE provided high quality images with high contrast-to-noise ratio (CNR) also for the RV myocardium (CNRblood-myocardium = 25.5 ± 7.8). Compared to cine FLASH, susceptibility, motion, and flow artifacts were considerably reduced due to the short TE of 314 µs. The aortic valve was clearly discernible over the entire cardiac cycle. Myocardial mass, SV, EF and CO determined by self-gated UTE were identical to the values measured with self-gated FLASH and showed good agreement to the results obtained by echocardiography. CONCLUSIONS: Self-gated UTE allows for robust measurement of cardiac parameters of diagnostic interest. Image quality is superior to self-gated FLASH, rendering the method a powerful alternative for the assessment of cardiac function at high magnetic fields.

G-CSF/SCF reduces inducible arrhythmias in the infarcted heart potentially via increased connexin43 expression and arteriogenesis.

Granulocyte colony-stimulating factor (G-CSF), alone or in combination with stem cell factor (SCF), can improve hemodynamic cardiac function after myocardial infarction. Apart from impairing the pump function, myocardial infarction causes an enhanced vulnerability to ventricular arrhythmias. Therefore, we investigated the electrophysiological effects of G-CSF/SCF and the underlying cellular events in a murine infarction model. G-CSF/SCF improved cardiac output after myocardial infarction. Although G-CSF/SCF led to a twofold increased, potentially proarrhythmic homing of bone marrow (BM)-derived cells to the area of infarction, <1% of these cells adopted a cardial phenotype. Inducibility of ventricular tachycardias during programmed stimulation was reduced 5 wk after G-CSF/SCF treatment. G-CSF/SCF increased cardiomyocyte diameter, arteriogenesis, and expression of connexin43 in the border zone of the infarction. An enhanced expression of the G-CSF receptor demonstrated in cardiomyocytes and other cell types of the infarcted myocardium indicates a sensitization of the heart to direct influences of this cytokine. In addition to paracrine effects potentially caused by the increased homing of BM-derived cells, these might contribute to the therapeutic effects of G-CSF.

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.

Monitoring Endothelin-A Receptor Expression during the Progression of Atherosclerosis.

Cardiovascular disease remains the most frequent cause of death worldwide. Atherosclerosis, an underlying cause of cardiovascular disease, is an inflammatory disorder associated with endothelial dysfunction. The endothelin system plays a crucial role in the pathogenesis of endothelial dysfunction and is involved in the development of atherosclerosis. We aimed to reveal the expression levels of the endothelin-A receptor (ETAR) in the course of atherogenesis to reveal possible time frames for targeted imaging and interventions. We used the ApoE-/- mice model and human specimens and evaluated ETAR expression by quantitative rtPCR (qPCR), histology and fluorescence molecular imaging. We found a significant upregulation of ETAR after 22 weeks of high-fat diet in the aortae of ApoE-/- mice. With regard to translation to human disease, we applied the fluorescent probe to fresh explants of human carotid and femoral artery specimens. The findings were correlated with qPCR and histology. While ETAR is upregulated during the progression of early atherosclerosis in the ApoE-/- mouse model, we found that ETAR expression is substantially reduced in advanced human atherosclerotic plaques. Moreover, those expression changes were clearly depicted by fluorescence imaging using our in-house designed ETAR-Cy 5.5 probe confirming its specificity and potential use in future studies.

Isolating Crucial Steps in Induction of Infective Endocarditis With Preclinical Modeling of Host Pathogen Interaction.

Animal models of Staphylococcus aureus infective endocarditis (IE), especially in rodents, are commonly used to investigate the underlying pathogenesis, disease progression, potential diagnostic approaches, and therapeutic treatment. All these models are based on surgical interventions, and imply valve trauma by placing a polyurethane catheter at the aortic root. While the influence of endothelial damage and inflammation on the induction of IE has been studied intensively, the role of the catheter, as permanent source of bacteremia, and the interplay with bacterial virulence factors during the formation of IE is poorly understood. In our study, we aimed at identifying which set of preconditions is required for induction and formation of IE: (1) tissue injury, (2) permanent presence of bacteria, and (3) presence of the full bacterial repertoire of adhesion proteins. We investigated the manifestation of the disease in different modifications of the animal model, considering different degrees of endothelial damage and the presence or absence of the catheter. In four infection models the induction of IE was assessed by using two bacterial strains with different expression patterns of virulence factors - S. aureus 6850 and Newman. In vivo magnetic resonance imaging showed conspicuous morphological structures on the aortic valves, when an endothelial damage and a continuous bacterial source were present simultaneously. Cellular and inflammatory pathophysiology were characterized additionally by histology, real-time quantitative polymerase chain reaction analysis, and bacterial counts, revealing strain-specific pathogenesis and manifestation of IE, crucially influenced by bacterial adherence and toxicity. The severity of IE was dependent on the degree of endothelial irritation. However, even severe endothelial damage in the absence of a permanent bacterial source resulted in reduced valve infection. The spread of bacteria to other organs was also dependent on the pathogenic profile of the infectious agent.

Granulocyte colony-stimulating factor (G-CSF) for cardio- and cerebrovascular regenerative applications.

The cytokine granulocyte colony-stimulating factor (G-CSF) is produced by numerous cell types including immune and endothelial cells. G-CSF binding to its receptor G-CSF-R which belongs to the cytokine receptor type I family depends on the interaction of alpha-helical motifs of the former and two fibronectin type III as well as an immunoglobulin-like domain of the latter. It activates several signalling transduction pathways including PI3K/Akt, Jak/Stat and MAP kinase, thereby promoting survival, proliferation, differentiation and mobilisation of haematopoietic stem and progenitor cells. Accordingly, recombinant human (rh)G-CSF has been extensively used in clinical haematology and oncology to enable bone marrow transplantation or to treat chemotherapy-associated neutropenia. Using animal models it has been recently shown that G-CSF, alone or in combination with other cytokines such as stem cell factor (SCF), causes an accumulation of bone marrow-derived cells in the infarcted heart which, however, do not differentiate into cardiac cells. Nevertheless, since beneficial effects on structural and functional properties were observed in animal models of cardiac, brain and hindlimb ischaemia other mechanisms of G-CSF action must be operative. Recent evidence suggests paracrine effects mediated by the immigrated bone marrow-derived cells and/or direct effects of the cytokine on resident G-CSF-R expressing cells. In both cases these may include promotion of cellular survival, proliferation and differentiation. First clinical studies in patients with myocardial infarction, heart failure and stroke have been accomplished and are reviewed in this paper.

Surgical animal models of heart failure related to coronary heart disease.

Coronary heart disease is caused by atherosclerotic narrowing of coronary arteries. It accounts for about two-thirds of heart failure cases, which are frequently secondary to myocardial infarction. Despite considerable progress in the understanding and management of heart failure, its incidence, prevalence and economic burden are steadily increasing. Therefore, efficient preventive and therapeutic measures are urgently needed. In order to investigate the mechanisms involved in the pathogenesis of coronary heart disease-related heart failure and to develop therapies, appropriate animal models are indispensable. According to the aetiology of this disorder, surgical models are based on various methods allowing for the narrowing or occlusion of coronary arteries. Depending on the duration and extent of the impairment of coronary blood flow and its consequences for cardiac tissue, these are classified as models of myocardial infarction, cardiac ischemia/reperfusion injury, or chronic cardiac ischemia. In addition, factors such as species, strain, and gender of the laboratory animals also significantly contribute to the pathophysiology of the induced disorder and, therefore, have to be taken into consideration thoroughly when an animal model is to be established.

Molecular imaging of MMP activity discriminates unstable from stable plaque phenotypes in shear-stress induced murine atherosclerosis.

PURPOSE: As atherosclerotic plaque ruptures are the primary cause of ischaemic events, their preventive identification by imaging remains a clinical challenge. Matrix metalloproteinases (MMP) are involved in plaque progression and destabilisation and are therefore promising targets to characterize rupture-prone unstable plaques. This study aims at evaluating MMP imaging to discriminate unstable from stable plaque phenotypes. METHODS: ApoE deficient mice (ApoE-/-) on a high cholesterol diet underwent implantation of a tapered cuff around the right common carotid artery (CCA) inducing a highly inflamed atherosclerotic plaque upstream (US) and a more stable plaque phenotype downstream (DS) of the cuff. 8 weeks after surgery, the MMP inhibitor-based photoprobe Cy5.5-AF443 was administered i.v. 3h prior to in situ and ex vivo fluorescence reflectance imaging of the CCAs. Thereafter, CCAs were analysed regarding plaque size, presence of macrophages, and MMP-2 and MMP-9 concentrations by immunohistochemistry and ELISA. RESULTS: We found a significantly higher uptake of Cy5.5-AF443 in US as compared to DS plaques in situ (1.29 vs. 1.06 plaque-to-background ratio; p<0.001), which was confirmed by ex vivo measurements. Immunohistochemistry revealed a higher presence of macrophages, MMP-2 and MMP-9 in US compared to DS plaques. Accordingly, MMP-2 concentrations were significantly higher in US plaques (47.2±7.6 vs. 29.6±4.6 ng/mg; p<0.05). CONCLUSIONS: In the ApoE-/- cuff model MMP-2 and MMP-9 activities are significantly higher in upstream low shear stress-induced unstable atherosclerotic plaques as compared to downstream more stable plaque phenotypes. MMP inhibitor-based fluorescence molecular imaging allows visualization of these differences in shear stress-induced atherosclerosis.

Imaging, myeloid precursor immortalization, and genome editing for defining mechanisms of leukocyte recruitment in vivo.

Recruitment of leukocytes from the blood to sites of inflammation poses a promising target for new diagnostic and therapeutic approaches. We aimed to develop a novel method to non-invasively analyze molecular mechanisms of leukocyte migration in pre-clinical models of inflammation in vivo. Methods: We used the ER-HoxB8 system to transiently immortalize murine myeloid precursors from wildtype and CD18- as well as MRP14-deficient mice. A VLA4α-/- cell line was generated by CRISPR/Cas9-mediated gene editing. We analyzed the migration of wildtype and knockout leukocytes in vivo by optical and nuclear imaging in mice with irritant contact dermatitis, cutaneous granuloma, experimental arthritis and myocardial infarction. Results: Transient immortalization, gene editing and in vivo imaging can be combined to analyze migratory mechanisms of murine leukocytes, even for gene deletions resulting in lethal phenotypes in mice. We reliably confirmed known migratory defects of leukocytes deficient for the adhesion molecules CD18 or VLA4α. Also, using our new method we identified a new role of the most abundant calcium-binding proteins in phagocytes and major alarmins in many inflammatory diseases, MRP8 and MRP14, for transmigration in vivo. Conclusion: We provide a combinatorial approach to rapidly characterize molecular mechanisms of leukocyte recruitment in vivo, with the potential to aid in identification of diagnostic and therapeutic targets in inflammatory pathologies.

A specific dietary intervention to restore brain structure and function after ischemic stroke.

Occlusion of the middle cerebral artery (MCAo) is among the most common causes of ischemic stroke in humans. Cerebral ischemia leads to brain lesions existing of an irreversibly injured core and an ischemic boundary zone, the penumbra, containing damaged but potentially salvageable tissue. Using a transient occlusion (30 min) of the middle cerebral artery (tMCAo) mouse model in this cross-institutional study we investigated the neurorestorative efficacy of a dietary approach (Fortasyn) comprising docosahexaenoic acid, eicosapentaenoic acid, uridine, choline, phospholipids, folic acid, vitamins B12, B6, C, and E, and selenium as therapeutic approach to counteract neuroinflammation and impairments of cerebral (structural+functional) connectivity, cerebral blood flow (CBF), and motor function. Male adult C57BL/6j mice were subjected to right tMCAo using the intraluminal filament model. Following tMCAo, animals were either maintained on Control diet or switched to the multicomponent Fortasyn diet. At several time points after tMCAo, behavioral tests, and MRI and PET scanning were conducted to identify the impact of the multicomponent diet on the elicited neuroinflammatory response, loss of cerebral connectivity, and the resulting impairment of motor function after experimental stroke. Mice on the multicomponent diet showed decreased neuroinflammation, improved functional and structural connectivity, beneficial effect on CBF, and also improved motor function after tMCAo. Our present data show that this specific dietary intervention may have beneficial effects on structural and functional recovery and therefore therapeutic potential after ischemic stroke.

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.

Diabetic db/db mice do not develop heart failure upon pressure overload: a longitudinal in vivo PET, MRI, and MRS study on cardiac metabolic, structural, and functional adaptations.

AIMS: Heart failure is associated with altered myocardial substrate metabolism and impaired cardiac energetics. Comorbidities like diabetes may influence the metabolic adaptations during heart failure development. We quantified to what extent changes in substrate preference, lipid accumulation, and energy status predict the longitudinal development of hypertrophy and failure in the non-diabetic and the diabetic heart. METHODS AND RESULTS: Transverse aortic constriction (TAC) was performed in non-diabetic (db/+) and diabetic (db/db) mice to induce pressure overload. Magnetic resonance imaging, 31P magnetic resonance spectroscopy (MRS), 1H MRS, and 18F-fluorodeoxyglucose-positron emission tomography (PET) were applied to measure cardiac function, energy status, lipid content, and glucose uptake, respectively. In vivo measurements were complemented with ex vivo techniques of high-resolution respirometry, proteomics, and western blotting to elucidate the underlying molecular pathways. In non-diabetic mice, TAC induced progressive cardiac hypertrophy and dysfunction, which correlated with increased protein kinase D-1 (PKD1) phosphorylation and increased glucose uptake. These changes in glucose utilization preceded a reduction in cardiac energy status. At baseline, compared with non-diabetic mice, diabetic mice showed normal cardiac function, higher lipid content and mitochondrial capacity for fatty acid oxidation, and lower PKD1 phosphorylation, glucose uptake, and energetics. Interestingly, TAC affected cardiac function only mildly in diabetic mice, which was accompanied by normalization of phosphorylated PKD1, glucose uptake, and cardiac energy status. CONCLUSION: The cardiac metabolic adaptations in diabetic mice seem to prevent the heart from failing upon pressure overload, suggesting that restoring the balance between glucose and fatty acid utilization is beneficial for cardiac function.

Imaging Reveals the Connection Between Spontaneous Coronary Plaque Ruptures, Atherothrombosis, and Myocardial Infarctions in HypoE/SRBI-/- Mice.

UNLABELLED: The hyperlipidemic mouse model HypoE/SRBI(-/-) has been shown to develop occlusive coronary atherosclerosis followed by myocardial infarctions and premature deaths in response to high-fat, high-cholesterol diet (HFC). However, the causal connection between myocardial infarctions and atherosclerotic plaque rupture events in the coronary arteries has not been investigated so far. The objective of this study was to assess whether diet-induced coronary plaque ruptures trigger atherothrombotic occlusions, resulting in myocardial infarctions in HFC-fed HypoE/SRBI(-/-) mice. METHODS: HypoE/SRBI(-/-) mice were characterized with respect to the individual dynamics of myocardial infarctions and features of infarct-related coronary atherosclerosis by serial noninvasive molecular and functional imaging, histopathology, and a pharmaceutical intervention. Detailed histologic analysis of whole mouse hearts was performed when spontaneously occurring acute myocardial infarctions were diagnosed by imaging. RESULTS: Using the imaging-triggered approach, we discovered thrombi in 32 (10.8%) of all 296 atherosclerotic coronary plaques in 14 HFC-fed HypoE/SRBI(-/-) mice. These thrombi typically were found in arteries presenting with inflammatory plaque phenotypes. Acetylsalicylic acid treatment did not attenuate the development of atherosclerotic coronary plaques but profoundly reduced the incidence of premature deaths, the number of thrombi (7 in 249 plaques), and also the degree of inflammation in the culprit lesions. CONCLUSION: HFC-induced ruptures of coronary plaques trigger atherothrombosis, vessel occlusions, myocardial infarctions, and sudden death in these mice. Thus, the HypoE/SRBI(-/-) mouse model mimics major features of human coronary heart disease and might therefore be a valuable model for the investigation of molecular and cellular parameters driving plaque rupture-related events and the development of new interventional approaches.

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 [(18)F]BR-351 showed MMP activity within the first days after transient middle cerebral artery occlusion (tMCAo), followed by increased [(18)F]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.