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Abstract Histological analysis with 2,3,5-triphenyltetrazolium chloride (TTC) staining is the most frequently used tool to detect myocardial ischemia/reperfusion injury. However, its practicality is often challenged by poor image quality in gross histology, leading to an equivocal infarct-boundary delineation and potentially compromised measurement accuracy. Here, we introduce several crucial refinements in staining protocol and sample processing, which enable TTC images to be analyzed with light microscopy. The refined protocol involves a two-step TTC staining process (perfusion and immersion) and subsequent Zamboni fixation to differentiate myocardial viability and necrosis, and use of Coomassie brilliant blue to label area-at-risk. After the duo-staining steps were completed, the heart sample was embedded and sliced transversally by a cryostat into a series of thin sections (50 µm) for microscopic analysis. The refined TTC (redTTC) assay yielded remarkably high-quality images with striking color intensity and sharply defined boundaries, permitting unambiguous and reliable delineation of the infarct and area-at-risk. In the same animals, the redTTC assay showed good agreement with the in-vivo gold standard measurements (LGE and MEMRI). Meanwhile, redTTC imaging allows tracking of viable cardiomyocytes at cellular resolution, and with this enhanced capability, we convincingly demonstrated the pro-survival action of stem cells based-therapy. Therefore, the redTTC assay represents a significant technical advance that permits precise detection of the true extent of cardiac injury and cardiomyocyte viability. This approach is cost-effective and may be adapted for use in diverse applications, making it highly appealing to many laboratories performing ischemia/reperfusion injury experiments.

Abstract People with diabetes have an increased cardiovascular risk and a poorer outcome after myocardial infarction (MI). However, the exact underlying mechanisms are still unclear, as is the question of which non-invasive measures could be used to predict the altered risk for the patient at early stages of the disease and adapt personalized treatment. Here, we used a holistic magnetic resonance approach to monitor longitudinally not only the main target heart, but also liver, peripheral/skeletal muscle, bone marrow, and hematopoiesis during disease development and subsequent MI. In prediabetic mice, we found a strong accumulation of lipids in all organs which preceded even a significant whole-body weight gain. Intramyocellular lipids (IMCLs) were most sensitive to reveal in vivo very early alterations in tissue properties during the prediabetic state. Subsequent induction of MI led to a persistent impairment of contractile function in septal/posterior segments of prediabetic hearts which correlated with their lipid load prior MI. At the same time, prediabetic cardiomyocytes exhibited sarcomere function at its limit resulting in overload and lower compensatory contractility of the healthy myocardium after MI. In summary, we identified IMCLs as very early marker in murine prediabetes and together with the cardiac lipid load as predictive for the functional outcome after MI.

Abstract Aims Cancer therapy-related cardiac dysfunction (CTRCD) is a dreaded complication of anthracycline therapy. CTRCD most frequently appears in patients with cardiovascular risk factors (CVR) or known cardiovascular disease. However, limited data exist on incidence and course of anthracycline-induced CTRCD in patients without preexisting risk factors. We therefore aimed to longitudinally investigate a cohort of young women on anthracycline treatment due to breast cancer without cardiovascular risk factors or known cardiovascular disease (NCT03940625). Methods and results We enrolled 59 women with primary breast cancer and scheduled anthracycline-based therapy, but without CVR or preexisting cardiovascular disease. We conducted a longitudinal assessment before, immediately and 12 months after cancer therapy with general laboratory, electrocardiograms, echocardiography and cardiovascular magnetic resonance (CMR), including myocardial relaxometry with T1, T2 and extracellular volume mapping. Every single patient experienced a drop in CMR-measured left ventricular ejection fraction (LVEF) of 6 ± 3% immediately after cancer therapy. According to the novel definition 32 patients (54.2%) developed CTRCD after 12 months defined by reduction in LVEF, global longitudinal strain (GLS) and/or biomarkers elevation, two of them were symptomatic. Global myocardial T2 relaxation times as well as myocardial mass increased coincidently with a decline in wall-thickening. While T2 values and myocardial mass normalized after 12 months, LVEF and GLS remained impaired. Conclusion In every single patient anthracyclines induce a decline of myocardial contractility, even among patients without pre-existing risk factors for CTRCD. Our data suggest to thoroughly evaluate whether this may lead to an increased risk of future cardiovascular events. Graphical Abstract Reduced myocardial contractility in low-risk patients receiving anthracycline-based cancer therapy. This study included 59 otherwise healthy women with primary breast cancer undergoing anthracycline-based chemotherapy. CMR was performed at baseline, directly and 12 months after cancer therapy. A decline in left ventricular function was observed in every single patient accompanied by transient edema. More than 50% were diagnosed with cancer therapy related cardiovascular dysfunction. LVEF: left ventricular function, CTRCD: cancer therapy related cardiovascular dysfunction, GLS = Global longitudinal strain, hs-TnT = high sensitive Troponin T, NT-pro BNP = NT-pro brain natriuretic peptide

Abstract Vascular inflammation plays a key role in the development of aortic diseases. A potential novel target for treatment might be CD73, an ecto-5′-nucleotidase that generates anti-inflammatory adenosine in the extracellular space. Here, we investigated whether a lack of CD73 results in enhanced aortic inflammation. To this end, angiotensin II was infused into wildtype and CD73−/− mice over 10 days. Before and after infusion, mice were analyzed using magnetic resonance imaging, ultrasound, flow cytometry, and histology. The impact of age and gender was investigated using female and male mice of three and six months of age, respectively. Angiotensin II infusion led to increased immune cell infiltration in both genotypes’ aortae, but depletion of CD73 had no impact on immune cell recruitment. These findings were not modified by age or sex. No substantial difference in morphological or functional characteristics could be detected between wildtype and CD73−/− mice. Interestingly, the expression of CD73 on neutrophils decreased significantly in wildtype mice during treatment. In summary, we have found no evidence that CD73 deficiency affects the onset of aortic inflammation. However, as CD73 expression decreased during disease induction, an increase in CD73 by pharmaceutical intervention might result in lower vascular inflammation and less vascular disease.

Abstract Background Macrophages play a pivotal role in vascular inflammation and predict cardiovascular complications. Fluorine-19 magnetic resonance imaging (19F MRI) with intravenously applied perfluorocarbon allows a background-free direct quantification of macrophage abundance in experimental vascular disease models in mice. Recently, perfluorooctyl bromide-nanoemulsion (PFOB-NE) was applied to effectively image macrophage infiltration in a pig model of myocardial infarction using clinical MRI scanners. In the present proof-of-concept approach, we aimed to non-invasively image monocyte/macrophage infiltration in response to carotid artery angioplasty in pigs using 19F MRI to assess early inflammatory response to mechanical injury. Methods In eight minipigs, two different types of vascular injury were conducted: a mild injury employing balloon oversize angioplasty only (BA, n = 4) and a severe injury provoked by BA in combination with endothelial denudation (BA + ECDN, n = 4). PFOB-NE was administered intravenously three days after injury followed by 1H and 19F MRI to assess vascular inflammatory burden at day six. Vascular response to mechanical injury was validated using X-ray angiography, intravascular ultrasound and immunohistology in at least 10 segments per carotid artery. Results Angioplasty was successfully induced in all eight pigs. Response to injury was characterized by positive remodeling with predominantly adventitial wall thickening and concomitant infiltration of monocytes/macrophages. No severe adverse reactions were observed following PFOB-NE administration. In vivo 19F signals were only detected in the four pigs following BA + ECDN with a robust signal-to-noise ratio (SNR) of 14.7 ± 4.8. Ex vivo analysis revealed a linear correlation of 19F SNR to local monocyte/macrophage cell density. Minimum detection limit of infiltrated monocytes/macrophages was estimated at approximately 410 cells/mm2. Conclusions In this proof-of-concept study, 19F MRI enabled quantification of monocyte/macrophage infiltration after vascular injury with sufficient sensitivity. This may provide the opportunity to non-invasively monitor vascular inflammation with MRI in patients after angioplasty or even in atherosclerotic plaques.

Abstract Polyphosphoesters (PPEs) are used in tissue engineering and drug delivery, as polyelectrolytes, and flame-retardants. Mostly polyphosphates have been investigated but copolymers involving different PPE subclasses have been rarely explored and the reactivity ratios of different cyclic phospholanes have not been reported. We synthesized binary and ternary PPE copolymers using cyclic comonomers, including side-chain phosphonates, phosphates, thiophosphate, and in-chain phosphonates, through organocatalyzed ring-opening copolymerization. Reactivity ratios were determined for all cases, including ternary PPE copolymers, using different nonterminal models. By combining different comonomers and organocatalysts, we created gradient copolymers with adjustable amphiphilicity and microstructure. Reactivity ratios ranging from 0.02 to 44 were observed for different comonomer sets. Statistical ring-opening copolymerization enabled the synthesis of amphiphilic gradient copolymers in a one-pot procedure, exhibiting tunable interfacial and magnetic resonance imaging (MRI) properties. These copolymers self-assembled in aqueous solutions, 31 P MRI imaging confirmed their potential as MRI-traceable nanostructures. This systematic study expands the possibilities of PPE-copolymers for drug delivery and theranostics.

Abstract In vivo monitoring of polymers is crucial for drug delivery and tissue regeneration. Magnetic resonance imaging (MRI) is a whole-body imaging technique, and heteronuclear MRI allows quantitative imaging. However, MRI agents can result in environmental pollution and organ accumulation. To address this, we introduce biocompatible and biodegradable polyphosphoesters, as MRI-traceable polymers using the 31P centers in the polymer backbone. We overcome challenges in 31P MRI, including background interference and low sensitivity, by modifying the molecular environment of 31P, assembling polymers into colloids, and tailoring the polymers’ microstructure to adjust MRI-relaxation times. Specifically, gradient-type polyphosphonate-copolymers demonstrate improved MRI-relaxation times compared to homo- and block copolymers, making them suitable for imaging. We validate background-free imaging and biodegradation in vivo using Manduca sexta. Furthermore, encapsulating the potent drug PROTAC allows using these amphiphilic copolymers to simultaneously deliver drugs, enabling theranostics. This first report paves the way for polyphosphoesters as background-free MRI-traceable polymers for theranostic applications.

Abstract Background ST-segment elevation myocardial infarction (STEMI) still causes significant mortality and morbidity despite best-practice revascularization and adjunct medical strategies. Within the STEMI population, there is a spectrum of higher and lower risk patients with respect to major adverse cardiovascular and cerebral events (MACCE) or re-hospitalization due to heart failure. Myocardial and systemic metabolic disorders modulate patient risk in STEMI. Systematic cardiocirculatory and metabolic phenotyping to assess the bidirectional interaction of cardiac and systemic metabolism in myocardial ischemia is lacking. Methods Systemic organ communication in STEMI (SYSTEMI) is an all-comer open-end prospective study in STEMI patients > 18 years of age to assess the interaction of cardiac and systemic metabolism in STEMI by systematically collecting data on a regional and systemic level. Primary endpoint will be myocardial function, left ventricular remodelling, myocardial texture and coronary patency at 6 month after STEMI. Secondary endpoint will be all-cause death, MACCE, and re-hospitalisation due to heart failure or revascularisation assessed 12 month after STEMI. The objective of SYSTEMI is to identify metabolic systemic and myocardial master switches that determine primary and secondary endpoints. In SYSTEMI 150–200 patients are expected to be recruited per year. Patient data will be collected at the index event, within 24 h, 5 days as well as 6 and 12 months after STEMI. Data acquisition will be performed in multilayer approaches. Myocardial function will be assessed by using serial cardiac imaging with cineventriculography, echocardiography and cardiovascular magnetic resonance. Myocardial metabolism will be analysed by multi-nuclei magnetic resonance spectroscopy. Systemic metabolism will be approached by serial liquid biopsies and analysed with respect to glucose and lipid metabolism as well as oxygen transport. In summary, SYSTEMI enables a comprehensive data analysis on the levels of organ structure and function alongside hemodynamic, genomic and transcriptomic information to assess cardiac and systemic metabolism. Discussion SYSTEMI aims to identify novel metabolic patterns and master-switches in the interaction of cardiac and systemic metabolism to improve diagnostic and therapeutic algorithms in myocardial ischemia for patient-risk assessment and tailored therapy. Trial registration Trial Registration Number: NCT03539133

BackgroundInflammation and metabolism exhibit a complex interplay, where inflammation influences metabolic pathways, and in turn, metabolism shapes the quality of immune responses. Here, glucose turnover is of special interest, as proinflammatory immune cells mainly utilize glycolysis to meet their energy needs. Noninvasive approaches to monitor both processes would help elucidate this interwoven relationship to identify new therapeutic targets and diagnostic opportunities.MethodsFor induction of defined inflammatory hotspots, LPS-doped Matrigel plugs were implanted into the neck of C57BL/6J mice. Subsequently, 1H/19F magnetic resonance imaging (MRI) was used to track the recruitment of 19F-loaded immune cells to the inflammatory focus and deuterium (2H) magnetic resonance spectroscopy (MRS) was used to monitor the metabolic fate of [6,6-2H2]glucose within the affected tissue. Histology and flow cytometry were used to validate the in vivo data.ResultsAfter plug implantation and intravenous administration of the 19F-containing contrast agent, 1H/19F MRI confirmed the infiltration of 19F-labeled immune cells into LPS-doped plugs while no 19F signal was observed in PBS-containing control plugs. Identification of the inflammatory focus was followed by i.p. bolus injection of deuterated glucose and continuous 2H MRS. Inflammation-induced alterations in metabolic fluxes could be tracked with an excellent temporal resolution of 2 min up to approximately 60 min after injection and demonstrated a more anaerobic glucose utilization in the initial phase of immune cell recruitment.Conclusion1H/2H/19F MRI/MRS was successfully employed for noninvasive monitoring of metabolic alterations in an inflammatory environment, paving the way for simultaneous in vivo registration of immunometabolic data in basic research and patients.

Abstract Aims Therapeutic options targeting post‐ischaemic cardiac remodelling are sparse. The bioactive sphingolipid sphingosine‐1‐phosphate (S1P) reduces ischaemia/reperfusion injury. However, its impact on post‐ischaemic remodelling independently of its infarct size (IS)‐reducing effect is yet unknown and was addressed in this study. Methods and results Acute myocardial infarction (AMI) in mice was induced by permanent ligation of the left anterior descending artery (LAD). C57Bl6 were treated with the S1P lyase inhibitor 4‐deoxypyridoxine (DOP) starting 7 days prior to AMI to increase endogenous S1P concentrations. Cardiac function and myocardial healing were assessed by cardiovascular magnetic resonance imaging (cMRI), murine echocardiography, histomorphology, and gene expression analysis. DOP effects were investigated in cardiomyocyte‐specific S1P receptor 1 deficient (S1PR1 Cardio Cre+) and Cre− control mice and S1P concentrations measured by LC‐MS/MS. IS and cardiac function did not differ between control and DOP‐treated groups on day one after LAD‐ligation despite fourfold increase in plasma S1P. In contrast, cardiac function was clearly improved and myocardial scar size reduced, respectively, on Day 21 in DOP‐treated mice. The latter also exhibited smaller cardiomyocyte size and reduced embryonic gene expression. The benefit of DOP treatment was abolished in S1PR1 Cardio Cre+. Conclusions S1P improves cardiac function and myocardial healing post AMI independently of initial infarct size and accomplishes this via the cardiomyocyte S1PR1. Hence, in addition to its beneficial effects on I/R injury, S1PR1 may be a promising target in post‐infarction myocardial remodelling as adjunctive therapy to revascularization as well as in patients not eligible for standard interventional procedures.

Here, combining diagnostic imaging modalities and in vivo assays, Windfelder and colleagues established tobacco hornworm larvae Manduca sexta as an alternative high-throughput platform to study the innate immunity of the gut and host-pathogen interactions. Using the platform, the authors identify mediators of gut inflammation, differentiate pathogens from gut mutualist bacteria, and demonstrate pharmacological interventions.

IntroductionGraves’ disease (GD) is an autoimmune disorder caused by autoantibodies against the thyroid stimulating hormone receptor (TSHR) leading to overstimulation of the thyroid gland. Thyroid eye disease (TED) is the most common extra thyroidal manifestation of GD. Therapeutic options to treat TED are very limited and novel treatments need to be developed. In the present study we investigated the effect of linsitinib, a dual small-molecule kinase inhibitor of the insulin-like growth factor 1 receptor (IGF-1R) and the Insulin receptor (IR) on the disease outcome of GD and TED.MethodsLinsitinib was administered orally for four weeks with therapy initiating in either the early (“active”) or the late (“chronic”) phases of the disease. In the thyroid and the orbit, autoimmune hyperthyroidism and orbitopathy were analyzed serologically (total anti-TSHR binding antibodies, stimulating anti TSHR antibodies, total T4 levels), immunohistochemically (H&E-, CD3-, TNFa- and Sirius red staining) and with immunofluorescence (F4/80 staining). An MRI was performed to quantify in vivo tissue remodeling inside the orbit.ResultsLinsitinib prevented autoimmune hyperthyroidism in the early state of the disease, by reducing morphological changes indicative for hyperthyroidism and blocking T-cell infiltration, visualized by CD3 staining. In the late state of the disease linsitinib had its main effect in the orbit. Linsitinib reduced immune infiltration of T-cells (CD3 staining) and macrophages (F4/80 and TNFa staining) in the orbita in experimental GD suggesting an additional, direct effect of linsitinib on the autoimmune response. In addition, treatment with linsitinib normalized the amount of brown adipose tissue in both the early and late group. An in vivo MRI of the late group was performed and revealed a marked decrease of inflammation, visualized by 19F MR imaging, significant reduction of existing muscle edema and formation of brown adipose tissue.ConclusionHere, we demonstrate that linsitinib effectively prevents development and progression of thyroid eye disease in an experimental murine model for Graves’ disease. Linsitinib improved the total disease outcome, indicating the clinical significance of the findings and providing a path to therapeutic intervention of Graves’ Disease. Our data support the use of linsitinib as a novel treatment for thyroid eye disease.

Summary: The tobacco hornworm is used extensively as a model system for ecotoxicology, immunology and gut physiology. Here, we established a micro-computed tomography approach based on the oral application of the clinical contrast agent iodixanol, allowing for a high-resolution quantitative analysis of the Manduca sexta gut. This technique permitted the identification of previously unknown and understudied structures, such as the crop or gastric ceca, and revealed the underlying complexity of the hindgut folding pattern, which is involved in fecal pellet formation. The acquired data enabled the volume rendering of all gut parts, the reliable calculation of their volumes, and the virtual endoscopy of the entire alimentary tract. It can provide information for accurate orientation in histology uses, enable quantitative anatomical phenotyping in three dimensions, and allow the calculation of locally effective midgut concentrations of applied chemicals. This atlas will provide critical insights into the evolution of the alimentary tract in lepidopterans.

Acquisition of immunological memory is an important evolutionary strategy that evolved to protect the host from repetitive challenges from infectious agents. It was believed for a long time that memory formation exclusively occurs in the adaptive part of the immune system with the formation of highly specific memory T cells and B cells. In the past 10–15 years, it has become clear that innate immune cells, such as monocytes, natural killer cells, or neutrophil granulocytes, also have the ability to generate some kind of memory. After the exposure of innate immune cells to certain stimuli, these cells develop an enhanced secondary response with increased cytokine secretion even after an encounter with an unrelated stimulus. This phenomenon has been termed trained innate immunity (TI) and is associated with epigenetic modifications (histone methylation, acetylation) and metabolic alterations (elevated glycolysis, lactate production). TI has been observed in tissue-resident or circulating immune cells but also in bone marrow progenitors. Risk-factors for cardiovascular diseases (CVDs) which are associated with low-grade inflammation, such as hyperglycemia, obesity, or high salt, can also induce TI with a profound impact on the development and progression of CVDs. In this review, we briefly describe basic mechanisms of TI and summarize animal studies which specifically focus on TI in the context of CVDs.

Abstract Magnetic resonance imaging (MRI) is the key whole-body imaging technology for observing processes within a living object providing excellent resolution and contrast between soft tissues. In the present work, we exploited the non-destructive properties of MRI to track longitudinally the dynamic changes that take place in developing pupae of the Emperor Moth (Saturnia pavonia) during the last days before eclosion. While in diapause pupae, body fluid was almost homogeneously distributed over the internal compartments, as soon as wings, legs, flight muscles and the head region were fully developed, a significant redistribution of water levels occurred between thoracic and abdominal regions. During the last two days before eclosion, the developing moths transferred substantial amounts of liquid into the gut and the labial gland, and in case of females, into developing eggs. Concomitantly, the volume of the air sacs increased drastically and their expansion/compression became clearly visible in time-resolved MR images. Furthermore, besides ventilation of the tracheal system, air sacs are likely to serve as volume reservoir for liquid transfer during development of the moths inside their pupal case. In parallel, we were able to monitor noninvasively lipid consumption, cardiac activity and haemolymph circulation during final metamorphosis.

This article contains raw and processed data related to research published by Vega et al. (2022). This complementary dataset provides further insight into the experimental validation of a single common carotid artery occlusion (CCAO) model upon pretreatment with pertussis toxin (PTX). We present data showing the extent of different PTX concentrations on neurological severity measured by Bederson score following CCAO. In addition, data indicate a protective effect of isoflurane on cerebral infarction and neurological deficits, as well as the consequences of PTX pretreatment on reperfusion after occlusion using time-of-flight magnetic resonance angiography. With these data, we aim to provide detailed experimental settings of this newly described model.

Abstract This study investigates whether a chronotherapeutic treatment of hepatocellular carcinoma (HCC) may improve treatment efficacy and mitigate side effects on non‐tumoral liver (NTL). HCC was induced in Per2::luc mice which were irradiated at four time points of the day. Proliferation and DNA‐double strand breaks were analyzed in irradiated and nonirradiated animals by detection of Ki67 and γ‐H2AX. Prior to whole animal experiments, organotypic slice cultures were investigated to determine the dosage to be used in whole animal experiments. Irradiation was most effective at the proliferation peaks in HCC at ZT02 (early inactivity phase) and ZT20 (late activity phase). Irradiation effects on NTL were minimal at ZT20. As compared with NTL, nonirradiated HCC revealed disruption in daily variation and downregulation of all investigated clock genes except Per1. Irradiation affected rhythmic clock gene expression in NTL and HCC at all ZTs except at ZT20 (late activity phase). Irradiation at ZT20 had no effect on total leukocyte numbers. Our results indicate ZT20 as the optimal time point for irradiation of HCC in mice at which the ratio between efficacy of tumor treatment and toxic side effects was maximal. Translational studies are now needed to evaluate whether the late activity phase is the optimal time point for irradiation of HCC in man.

The prediction of major cardiovascular events is still an unsolved problem. Here, the authors present a multi-color, multi-targeted non-invasive imaging technology that allows reliable in vivo identification of silent but prognostically highly relevant danger patterns prior to myocardial infarction in mice.

Abstract Placental hypoperfusion and hypoxia are key drivers in complications during fetal development such as fetal growth restriction and preeclampsia. In order to study the mechanisms of disease in mouse models, the development of quantitative biomarkers of placental hypoxia is a prerequisite. The goal of this exploratory study was to establish a technique to noninvasively characterize placental partial pressure of oxygen (PO2) in vivo in the Lgals1 (lectin, galactoside-binding, soluble, 1) deficient mouse model of preeclampsia using fluorine magnetic resonance imaging. We hypothesized a decrease in placental oxygenation in knockout mice. Wildtype and knockout animals received fluorescently labeled perfluoro-5-crown-15-ether nanoemulsion i.v. on day E14-15 during pregnancy. Placental PO2 was assessed via calibrated 19F MRI saturation recovery T1 mapping. A gas challenge with varying levels of oxygen in breathing air (30%, 60% and 100% O2) was used to validate that changes in oxygenation can be detected in freely breathing, anesthetized animals. At the end of the experiment, fluorophore-coupled lectin was injected i.v. to label the vasculature for histology. Differences in PO2 between breathing conditions and genotype were statistically analyzed with linear mixed-effects modeling. As expected, a significant increase in PO2 with increasing oxygen in breathing air was found. PO2 in Lgals1 knockout animals was decreased but this effect was only present at 30% oxygen in breathing air, not at 60% and 100%. Histological examinations showed crossing of the perfluorocarbon nanoemulsion to the fetal blood pool but the dominating contribution of 19F MR signal is estimated at > 70% from maternal plasma based on volume fraction measurements of previous studies. These results show for the first time that 19F MRI can characterize oxygenation in mouse models of placental malfunction.

Abstract After myocardial infarction (MI), epicardial cells reactivate their embryonic program, proliferate and migrate into the damaged tissue to differentiate into fibroblasts, endothelial cells and, if adequately stimulated, to cardiomyocytes. Targeting epicardium-derived stromal cells (EpiSC) by specific ligands might enable the direct imaging of EpiSCs after MI to better understand their biology, but also may permit the cell-specific delivery of small molecules to improve the post-MI healing process. Therefore, the aim of this study was to identify specific peptides by phage display screening to enable EpiSC specific cargo delivery by active targeting. To this end, we utilized a sequential panning of a phage library on cultured rat EpiSCs and then subtracted phage that nonspecifically bound blood immune cells. EpiSC specific phage were analyzed by deep sequencing and bioinformatics analysis to identify a total of 78 300 ± 31 900 different, EpiSC-specific, peptide insertion sequences. Flow cytometry of the five most highly abundant peptides (EP1, -2, –3, -7 or EP9) showed strong binding to EpiSCs but not to blood immune cells. The best binding properties were found for EP9 which was further studied by surface plasmon resonance (SPR). SPR revealed rapid and stable association of EpiSCs with EP9. As a negative control, THP-1 monocytes did not associate with EP9. Coupling of EP9 to perfluorocarbon nanoemulsions (PFCs) resulted in the efficient delivery of 19F cargo to EpiSCs and enabled their visualization by 19F MRI. Moreover, active targeting of EpiSCs by EP9-labelled PFCs was able to outcompete the strong phagocytic uptake of PFCs by circulating monocytes. In summary, we have identified a 7-mer peptide, (EP9) that binds to EpiSCs with high affinity and specificity. This peptide can be used to deliver small molecule cargos such as contrast agents to permit future in vivo tracking of EpiSCs by molecular imaging and to transfer small pharmaceutical molecules to modulate the biological activity of EpiSCs.

Impaired oxygen utilization is the underlying pathophysiological process in different shock states. Clinically most important are septic and hemorrhagic shock, which comprise more than 75% of all clinical cases of shock. Both forms lead to severe dysfunction of the microcirculation and the mitochondria that can cause or further aggravate tissue damage and inflammation. However, the detailed mechanisms of acute and long-term effects of impaired oxygen utilization are still elusive. Importantly, a defective oxygen exploitation can impact multiple organs simultaneously and organ damage can be aggravated due to intense organ cross-talk or the presence of a systemic inflammatory response. Complexity is further increased through a large heterogeneity in the human population, differences in genetics, age and gender, comorbidities or disease history. To gain a deeper understanding of the principles, mechanisms, interconnections and consequences of impaired oxygen delivery and utilization, interdisciplinary preclinical as well as clinical research is required. In this review, we provide a “tool-box” that covers widely used animal disease models for septic and hemorrhagic shock and methods to determine the structure and function of the microcirculation as well as mitochondrial function. Furthermore, we suggest magnetic resonance imaging as a multimodal imaging platform to noninvasively assess the consequences of impaired oxygen delivery on organ function, cell metabolism, alterations in tissue textures or inflammation. Combining structural and functional analyses of oxygen delivery and utilization in animal models with additional data obtained by multiparametric MRI-based techniques can help to unravel mechanisms underlying immediate effects as well as long-term consequences of impaired oxygen delivery on multiple organs and may narrow the gap between experimental preclinical research and the human patient.

Patients with acute ischemic stroke (AIS) present an increased incidence of systemic inflammatory response syndrome and release of Troponin T coinciding with cardiac dysfunction. The nature of the cardiocirculatory alterations remains obscure as models to investigate systemic interferences of the brain-heart-axis following AIS are sparse. Thus, this study aims to investigate acute cardiocirculatory dysfunction and myocardial injury in mice after reperfused AIS. Ischemic stroke was induced in mice by transient right-sided middle cerebral artery occlusion (tMCAO). Cardiac effects were investigated by electrocardiograms, 3D-echocardiography, magnetic resonance imaging (MRI), invasive conductance catheter measurements, histology, flow-cytometry, and determination of high-sensitive Troponin T (hsTnT). Systemic hemodynamics were recorded and catecholamines and inflammatory markers in circulating blood and myocardial tissue were determined by immuno-assay and flow-cytometry. Twenty-four hours following tMCAO hsTnT was elevated 4-fold compared to controls and predicted long-term survival. In parallel, systolic left ventricular dysfunction occurred with impaired global longitudinal strain, lower blood pressure, reduced stroke volume, and severe bradycardia leading to reduced cardiac output. This was accompanied by a systemic inflammatory response characterized by granulocytosis, lymphopenia, and increased levels of serum-amyloid P and interleukin-6. Within myocardial tissue, MRI relaxometry indicated expansion of extracellular space, most likely due to inflammatory edema and a reduced fluid volume. Accordingly, we found an increased abundance of granulocytes, apoptotic cells, and upregulation of pro-inflammatory cytokines within myocardial tissue following tMCAO. Therefore, reperfused ischemic stroke leads to specific cardiocirculatory alterations that are characterized by acute heart failure with reduced stroke volume, bradycardia, and changes in cardiac tissue and accompanied by systemic and local inflammatory responses.

The pathophysiology of the initiation and progression of abdominal aortic aneurysms (AAAs) and aortic dissections (AADs) is still unclear. However, there is strong evidence that monocytes and macrophages are of crucial importance in these processes. Here, we utilized a molecular imaging approach based on background-free 19F MRI and employed perfluorocarbon nanoemulsions (PFCs) for in situ19F labeling of monocytes/macrophages to monitor vascular inflammation and AAA/AAD formation in angiotensin II (angII)-treated apolipoproteinE-deficient (apoE−/−) mice. In parallel, we used conventional 1H MRI for the characterization of aortic flow patterns and morphology. AngII (1 μg/kg/min) was infused into apoE−/− mice via osmotic minipumps for 10 days and mice were monitored by multiparametric 1H/19F MRI. PFCs were intravenously injected directly after pump implantation followed by additional applications on day 2 and 4 to allow an efficient 19F loading of circulating monocytes. The combination of angiographic, hemodynamic, and anatomical measurements allowed an unequivocal classification of mice in groups with developing AAAs, AADs or without any obvious aortic vessel alterations despite the exposure to angII. Maximal luminal and external diameters of the aorta were enlarged in AAAs, whereas AADs showed either a slight decrease of the luminal diameter or no alteration. 1H/19F MRI after intravenous PFC application demonstrated significantly higher 19F signals in aortae of mice that developed AAAs or AADs as compared to mice in which no aortic disorders were detected. High resolution 1H/19F MRI of excised aortae revealed a patchy pattern of the 19F signals predominantly in the adventitia of the aorta. Histological analysis confirmed the presence of macrophages in this area and flow cytometry revealed higher numbers of immune cells in aortae of mice that have developed AAA/AAD. Importantly, there was a linear correlation of the 19F signal with the total number of infiltrated macrophages. In conclusion, our approach enables a precise differentiation between AAA and AAD as well as visualization and quantitative assessment of inflammatory active vascular lesions, and therefore may help to unravel the complex interplay between macrophage accumulation, vascular inflammation, and the development and progression of AAAs and AADs.

Fluorine-19 magnetic resonance imaging (19F MRI) with intravenously applied perfluorooctyl bromide-nanoemulsions (PFOB-NE) has proven its feasibility to visualize inflammatory processes in experimental disease models. This approach is based on the properties of monocytes/macrophages to ingest PFOB-NE particles enabling specific cell tracking in vivo. However, information on safety (cellular function and viability), mechanism of ingestion and impact of specific disease environment on PFOB-NE uptake is lacking. This information is, however, crucial for the interpretation of 19F MRI signals and a possible translation to clinical application. To address these issues, whole blood samples were collected from patients with acute ST-elevation myocardial infarction (STEMI), stable coronary artery disease (SCAD) and healthy volunteers. Samples were exposed to fluorescently-labeled PFOB-NE and particle uptake, cell viability and migration activity was evaluated by flow cytometry and MRI. We were able to show that PFOB-NE is ingested by human monocytes in a time- and subset-dependent manner via active phagocytosis. Monocyte function (migration, phagocytosis) and viability was maintained after PFOB-NE uptake. Monocytes of STEMI and SCAD patients did not differ in their maximal PFOB-NE uptake compared to healthy controls. In sum, our study provides further evidence for a safe translation of PFOB-NE for imaging purposes in humans.

The mouse abdominal heart transplantation model is a basic and important immunological research model. We developed a technique for placing entire everting sutures instead of half inverting and half everting sutures for anastomosis between donor and recipients' caval veins. The purpose of this study was to evaluate this modified method. Methods: Each technique was used in 25 mice subjected to isogenic abdominal heart transplantation. Recipient operation time, graft warm ischaemia time, time of caval anastomosis, and re-beating time were recorded. After transplantation, the heartbeat was palpated through the abdominal wall once a day for 100 days. Results: Recipient operation time (40.7 ± 2.5 min vs. 44.3 ± 2.3 min, p < 0.01), cava-caval anastomosis time (8.4 ± 1.3 min vs. 12.1 ± 1.2 min, p < 0.01), and warm ischaemia time were significantly shorter (23.4 ± 1.7 min vs. 27.2 ± 1.6 min, p < 0.01) with the modified technique. Re-beating time was 1.2 ± 0.4 minutes with the modified technique vs. 1.5 ± 0.5 minutes (p = 0.04). There was a tendency for less surgical complications in the modified group, but there were no differences in survival rates. Conclusion: The new suturing technique for mouse cardiac transplantation facilitates easier anastomosis of the outflow tract, thereby reducing operation, warm ischaemia, and re-beating times.

Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.

PURPOSE: To implement and characterize a fluorine-19 ((19)F) magnetic resonance imaging (MRI) technique and to test the hypothesis that the (19)F MRI signal in steady state after intravenous injection of a perfluoro-15-crown-5 ether (PCE) emulsion may be exploited for angiography in a pre-clinical in vivo animal study. MATERIALS AND METHODS: In vitro at 9.4T, the detection limit of the PCE emulsion at a scan time of 10 min/slice was determined, after which the T(1) and T(2) of PCE in venous blood were measured. Permission from the local animal use committee was obtained for all animal experiments. 12 µl/g of PCE emulsion was intravenously injected in 11 mice. Gradient echo (1)H and (19)F images were obtained at identical anatomical levels. Signal-to-noise (SNR) and contrast-to-noise (CNR) ratios were determined for 33 vessels in both the (19)F and (1)H images, which was followed by vessel tracking to determine the vessel conspicuity for both modalities. RESULTS: In vitro, the detection limit was ∼400 µM, while the (19)F T(1) and T(2) were 1350±40 and 25±2 ms. The (19)F MR angiograms selectively visualized the vasculature (and the liver parenchyma over time) while precisely coregistering with the (1)H images. Due to the lower SNR of (19)F compared to (1)H (17±8 vs. 83±49, p

: Aims Prostaglandin E2 (PGE2) has been shown to mediate anti-ischaemic effects and cardiomyocyte hypertrophy and there is evidence for an involvement of the prostaglandin EP3-receptor subtype. This study focuses on the EP3-mediated hypertrophic action and investigates intracellular signalling pathways of the EP3-receptor subtype in the murine heart. : Methods and results Cardiac function was analyzed in vivo by magnetic resonance imaging (MRI) in transgenic (tg) mice with cardio-specific overexpression of the EP3 receptor in comparison with wild-type (wt) mice. Left ventricular (LV) function was determined in isolated perfused hearts subjected to 60 min of zero-flow ischaemia and 45 min of reperfusion. Calcineurin activity and nuclear activity of nuclear factor of activated T-cells (NFAT) were determined by a modified malachite green assay and ELISA, respectively. Extracellular matrix compounds were analyzed by RT–PCR and histology. MRI indicated a significant increase in end-diastolic and end-systolic volume in tg hearts. LV ejection fraction was severely decreased in tg hearts while the relative LV mass was significantly increased. In Langendorff perfused hearts, EP3-receptor overexpression resulted in a marked blunting of the ischaemia-induced increase in LV end-diastolic pressure and creatine kinase release. Analysis of EP3-receptor-mediated signalling revealed significantly increased calcineurin activity and nuclear activity of NFAT in tg hearts. Moreover, elevated mRNA levels of collagen types I and III as well as the collagen-binding proteoglycans biglycan and decorin were detected in tg hearts. : Conclusion EP3-receptor-mediated signalling results in a significant anti-ischaemic action and activation of the pro-hypertrophic calcineurin signalling pathway, suggesting the involvement of the EP3 subtype in both PGE2-mediated cardioprotection as well as cardiac hypertrophy. [ABSTRACT FROM AUTHOR]