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Purpose: The tumor microenvironment presents with altered extracellular matrix (ECM) and stroma composition, which may affect treatment efficacy and contribute to tissue stiffness. Ultrasound (US) elastography can visualize and quantify tissue stiffness noninvasively. However, the contributions of ECM and stromal components to stiffness are poorly understood. We therefore set out to quantify ECM and stroma density and their relation to tumor stiffness.Experimental Design: A modified clinical ultrasound system was used to measure tumor stiffness and perfusion during tumor growth in preclinical tumor models. In vivo measurements were compared with collagen mass spectroscopy and automatic analysis of matrix and stromal markers derived from immunofluorescence images.Results: US elastography estimates of tumor stiffness were positively correlated with tumor volume in collagen and myofibroblast-rich tumors, while no correlations were found for tumors with low collagen and myofibroblast content. US elastography measurements were strongly correlated with ex vivo mechanical testing and mass spectroscopy–based measurements of total collagen and immature collagen crosslinks. Registration of ultrasound and confocal microscopy data showed strong correlations between blood vessel density and T-cell density in syngeneic tumors, while no correlations were found for genetic tumor models. In contrast to collagen density, which was positively correlated with stiffness, no significant correlations were observed for hyaluronic acid density. Finally, localized delivery of collagenase led to a significant reduction in tumor stiffness without changes in perfusion 24 hours after treatment.Conclusions: US elastography can be used as a potential biomarker to assess changes in the tumor microenvironment, particularly changes affecting the ECM. Clin Cancer Res; 24(18); 4455–67. ©2018 AACR.

The prevalence of cardiovascular disease increases exponentially with age, highlighting the contribution of aging mechanisms to cardiac diseases. Although model organisms which share human disease pathologies can elucidate mechanisms driving disease, they do not provide us with innate examples how cardiac aging might be slowed or attenuated. The identification of animal models that preserve cardiac function throughout most of life offers an alternative approach to study mechanisms which might slow cardiac aging. One such species may be the naked mole-rat (NMR), a mouse-sized (40 g) rodent with extraordinary longevity (> 37 years), and constant mortality hazard over its four decades of life. We used a cross-sectional study design to measure a range of physiological parameters in NMRs between 2 and 34 years of age and compared these findings with those of mice aged between 3 months and 2.5 years. We observed a rapid decline in body fat content and bone mineral density in old mice, but no changes in NMRs. Similarly, rhythm disorders (premature atrial and ventricular complexes) occurred in aged mice but not in NMRs. Magnetic resonance and ultrasound imaging showed age-dependent increases in cardiac hypertrophy and diastolic dysfunction in mice which were absent in NMRs. Finally, cardiac stress tests showed an age-dependent decline in normalized cardiac output in mice, which was absent in NMRs. Unlike mice, that manifest several aspects of human cardiac aging, NMRs maintain cardiac function and reserve capacity throughout their long lives and may offer insights on how to delay or prevent cardiac aging.

First release; read the publication at andrewgyork.github.io/relaxation_sensors

Dietary interventions can dramatically affect physiological health and organismal lifespan. The degree to which organismal health is improved depends upon genotype and the severity of dietary intervention, but neither the effects of these factors, nor their interaction, have been quantified in an outbred population. Moreover, it is not well understood what physiological changes occur shortly after dietary change and how these may affect the health of an adult population. In this article, we investigated the effect of 6-month exposure of either caloric restriction (CR) or intermittent fasting (IF) on a broad range of physiological traits in 960 1-year old Diversity Outbred mice. We found CR and IF affected distinct aspects of physiology and neither the magnitude nor the direction (beneficial or detrimental) of effects were concordant with the severity of the intervention. In addition to the effects of diet, genetic variation significantly affected 31 of 36 traits (heritabilities ranged from 0.04 to 0.65). We observed significant covariation between many traits that was due to both diet and genetics and quantified these effects with phenotypic and genetic correlations. We genetically mapped 16 diet-independent and 2 diet-dependent significant quantitative trait loci, both of which were associated with cardiac physiology. Collectively, these results demonstrate the degree to which diet and genetics interact to shape the physiological health of adult mice following 6 months of dietary intervention.

CXC chemokine ligand 12 (CXCL12; stromal cell-derived factor 1 [SDF-1]), primarily known for its role in embryogenesis and hematopoiesis, has also been implicated in tumor biology and neovascularization. However, its specific role and mechanism of action remain poorly understood. We previously demonstrated that CXCL12 expression is Hypoxia-Inducible Factor (HIF)-1 responsive. Here we use a conditional CXCL12 knockout mouse to show that endothelial-specific deletion of CXCL12 (eKO) does not affect embryogenesis, but reduces the survival of ischemic tissue, altering tissue repair and tumor progression. Loss of vascular endothelial CXCL12 disrupts endothelial – fibroblast crosstalk necessary for stromal growth and vascularization. Single-cell gene expression analysis in combination with a parabiosis model reveals a specific population of non-inflammatory circulating cells, defined by genes regulating neovascularization, which is recruited by endothelial CXCL12. These findings indicate an essential role for endothelial CXCL12 expression during the adult neovascular response in tissue injury and tumor progression.

CXC chemokine ligand 12 (CXCL12; stromal cell-derived factor 1 [SDF-1]), primarily known for its role in embryogenesis and hematopoiesis, has also been implicated in tumor biology and neovascularization. However, its specific role and mechanism of action remain poorly understood. We previously demonstrated that CXCL12 expression is Hypoxia-Inducible Factor (HIF)-1 responsive. Here we use a conditional CXCL12 knockout mouse to show that endothelial-specific deletion of CXCL12 (eKO) does not affect embryogenesis, but reduces the survival of ischemic tissue, altering tissue repair and tumor progression. Loss of vascular endothelial CXCL12 disrupts endothelial – fibroblast crosstalk necessary for stromal growth and vascularization. Single-cell gene expression analysis in combination with a parabiosis model reveals a specific population of non-inflammatory circulating cells, defined by genes regulating neovascularization, which is recruited by endothelial CXCL12. These findings indicate an essential role for endothelial CXCL12 expression during the adult neovascular response in tissue injury and tumor progression.

Purpose: The tumor microenvironment presents with altered extracellular matrix (ECM) and stroma composition, which may affect treatment efficacy and contribute to tissue stiffness. Ultrasound (US) elastography can visualize and quantify tissue stiffness noninvasively. However, the contributions of ECM and stromal components to stiffness are poorly understood. We therefore set out to quantify ECM and stroma density and their relation to tumor stiffness. Experimental Design: A modified clinical ultrasound system was used to measure tumor stiffness and perfusion during tumor growth in preclinical tumor models. In vivo measurements were compared with collagen mass spectroscopy and automatic analysis of matrix and stromal markers derived from immunofluorescence images. Results: US elastography estimates of tumor stiffness were positively correlated with tumor volume in collagen and myofibroblast-rich tumors, while no correlations were found for tumors with low collagen and myofibroblast content. US elastography measurements were strongly correlated with ex vivo mechanical testing and mass spectroscopy–based measurements of total collagen and immature collagen crosslinks. Registration of ultrasound and confocal microscopy data showed strong correlations between blood vessel density and T-cell density in syngeneic tumors, while no correlations were found for genetic tumor models. In contrast to collagen density, which was positively correlated with stiffness, no significant correlations were observed for hyaluronic acid density. Finally, localized delivery of collagenase led to a significant reduction in tumor stiffness without changes in perfusion 24 hours after treatment. Conclusions: US elastography can be used as a potential biomarker to assess changes in the tumor microenvironment, particularly changes affecting the ECM. Clin Cancer Res; 24(18); 4455–67. ©2018 AACR.

The ability to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes (CMs) makes them an attractive source for repairing injured myocardium, disease modeling, and drug testing. Although current differentiation protocols yield hPSC-CMs to >90% efficiency, hPSC-CMs exhibit immature characteristics. With the goal of overcoming this limitation, we tested the effects of varying passive stretch on engineered heart muscle (EHM) structural and functional maturation, guided by computational modeling. Human embryonic stem cells (hESCs, H7 line) or human induced pluripotent stem cells (IMR-90 line) were differentiated to hPSC-derived cardiomyocytes (hPSC-CMs) in vitro using a small molecule based protocol. hPSC-CMs were characterized by troponin+ flow cytometry as well as electrophysiological measurements. Afterwards, 1.2 × 106 hPSC-CMs were mixed with 0.4 × 106 human fibroblasts (IMR-90 line) (3:1 ratio) and type-I collagen. The blend was cast into custom-made 12-mm long polydimethylsiloxane reservoirs to vary nominal passive stretch of EHMs to 5, 7, or 9 mm. EHM characteristics were monitored for up to 50 days, with EHMs having a passive stretch of 7 mm giving the most consistent formation. Based on our initial macroscopic observations of EHM formation, we created a computational model that predicts the stress distribution throughout EHMs, which is a function of cellular composition, cellular ratio, and geometry. Based on this predictive modeling, we show cell alignment by immunohistochemistry and coordinated calcium waves by calcium imaging. Furthermore, coordinated calcium waves and mechanical contractions were apparent throughout entire EHMs. The stiffness and active forces of hPSC-derived EHMs are comparable with rat neonatal cardiomyocyte-derived EHMs. Three-dimensional EHMs display increased expression of mature cardiomyocyte genes including sarcomeric protein troponin-T, calcium and potassium ion channels, β-adrenergic receptors, and t-tubule protein caveolin-3. Passive stretch affects the structural and functional maturation of EHMs. Based on our predictive computational modeling, we show how to optimize cell alignment and calcium dynamics within EHMs. These findings provide a basis for the rational design of EHMs, which enables future scale-up productions for clinical use in cardiovascular tissue engineering.

Cancer immunotherapies have demonstrated durable responses in a range of different cancers. However, only a subset of patients responds to these therapies. We set out to test if non-invasive imaging of tumor perfusion and vascular inflammation may be able to explain differences in T-cell infiltration in pre-clinical tumor models, relevant for treatment outcomes. Tumor perfusion and vascular cell adhesion molecule (VCAM-1) density were quantified using magnetic resonance imaging (MRI) and correlated with infiltration of adoptively transferred and endogenous T-cells. MRI biomarkers were evaluated for their ability to detect tumor rejection 3 days after T-cell transfer. Baseline levels of these markers were used to assess their ability to predict PD-L1 treatment response. We found correlations between MRI-derived VCAM-1 density and infiltration of endogenous or adoptively transferred T-cells in some preclinical tumor models. Blocking T-cell binding to endothelial cell adhesion molecules (VCAM-1/ICAM) prevented T-cell mediated tumor rejection. Tumor rejection could be detected 3 days after adoptive T-cell transfer prior to tumor volume changes by monitoring the extracellular extravascular volume fraction. Imaging tumor perfusion and VCAM-1 density before treatment initiation was able to predict the response of MC38 tumors to PD-L1 blockade. These results indicate that MRI based assessment of tumor perfusion and VCAM-1 density can inform about the permissibility of the tumor vasculature for T-cell infiltration which may explain some of the observed variance in treatment response for cancer immunotherapies.

Emerging research suggests that multiple tumor compartments can influence treatment responsiveness and relapse, yet the search for therapeutic resistance mechanisms remains largely focused on acquired genomic alterations in cancer cells. Here we show how treatment-induced changes occur in multiple tumor compartments during tumor relapse and can reduce benefit of follow-on therapies. By using serial biopsies, next-generation sequencing, and single-cell transcriptomics, we tracked the evolution of multiple cellular compartments within individual lesions during first-line treatment response, relapse, and second-line therapeutic interventions in an autochthonous model of melanoma. We discovered that although treatment-relapsed tumors remained genetically stable, they converged on a shared resistance phenotype characterized by dramatic changes in tumor cell differentiation state, immune infiltration, and extracellular matrix (ECM) composition. Similar alterations in tumor cell differentiation were also observed in more than half of our treatment-relapsed patient tumors. Tumor cell-state changes were coincident with ECM remodeling and increased tumor stiffness, which alone was sufficient to alter tumor cell fate and reduce treatment responses in melanoma cell lines in vitro. Despite the absence of acquired mutations in the targeted pathway, resistant tumors showed significantly decreased responsiveness to second-line therapy intervention within the same pathway. The ability to preclinically model relapse and refractory settings—while capturing dynamics within and crosstalk between all relevant tumor compartments—provides a unique opportunity to better design and sequence appropriate clinical interventions.

Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor β (TGFβ) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+ T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFβ-blocking and anti-PD-L1 antibodies reduced TGFβ signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFβ shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.

Vascular occlusion can result in fatal myocardial infarction, stroke or loss of limb in peripheral arterial disease. Interventional balloon angioplasty is a common first line procedure for vascular disease treatment, but long term success is limited by restenosis and neointimal hyperplasia. Cellular therapies have been proposed to mitigate these issues; however efficacy is low, in part due to poor cell retention. We show that magnetic targeting of mesenchymal stem cells gives rise to a 6-fold increase in cell retention following balloon angioplasty in a rabbit model using a clinically applicable permanent magnet. Cells labelled with superparamagnetic iron oxide nanoparticles exhibit no negative effects on cell viability, differentiation or secretion patterns. The increase in stem cell retention leads to a reduction in restenosis three weeks after cell delivery.

Background— The use of tissue engineering approaches in combination with exogenously produced cardiomyocytes offers the potential to restore contractile function after myocardial injury. However, current techniques assessing changes in global cardiac performance after such treatments are plagued by relatively low detection ability. Since the treatment is locally performed, this detection could be improved by myocardial strain imaging that measures regional contractility. Methods and Results— Tissue engineered heart muscles (EHMs) were generated by casting human embryonic stem cell–derived cardiomyocytes with collagen in preformed molds. EHMs were transplanted (n=12) to cover infarct and border zones of recipient rat hearts 1 month after ischemia reperfusion injury. A control group (n=10) received only sham placement of sutures without EHMs. To assess the efficacy of EHMs, magnetic resonance imaging and ultrasound-based strain imaging were performed before and 4 weeks after transplantation. In addition to strain imaging, global cardiac performance was estimated from cardiac magnetic resonance imaging. Although no significant differences were found for global changes in left ventricular ejection fraction (control −9.6±1.3% versus EHM −6.2±1.9%; P =0.17), regional myocardial strain from tagged magnetic resonance imaging was able to detect preserved systolic function in EHM-treated animals compared with control (control 4.4±1.0% versus EHM 1.0±0.6%; P =0.04). However, ultrasound-based strain failed to detect any significant change (control 2.1±3.0% versus EHM 6.3±2.9%; P =0.46). Conclusions— This study highlights the feasibility of using cardiac strain from tagged magnetic resonance imaging to assess functional changes in rat models following localized regenerative therapies, which may not be detected by conventional measures of global systolic performance.

Sarcomere assembly is a highly orchestrated and dynamic process which adapts, during perinatal development, to accommodate growth of the heart. Sarcomeric components, including titin, undergo an isoform transition to adjust ventricular filling. Many sarcomeric genes have been implicated in congenital cardiomyopathies, such that understanding developmental sarcomere transitions will inform the aetiology and treatment. We sought to determine whether Thymosin β4 (Tβ4), a peptide that regulates the availability of actin monomers for polymerization in non-muscle cells, plays a role in sarcomere assembly during cardiac morphogenesis and influences adult cardiac function. In Tβ4 null mice, immunofluorescence-based sarcomere analyses revealed shortened thin filament, sarcomere and titin spring length in cardiomyocytes, associated with precocious up-regulation of the short titin isoforms during the postnatal splicing transition. By magnetic resonance imaging, this manifested as diminished stroke volume and limited contractile reserve in adult mice. Extrapolating to an in vitro cardiomyocyte model, the altered postnatal splicing was corrected with addition of synthetic Tβ4, whereby normal sarcomere length was restored. Our data suggest that Tβ4 is required for setting correct sarcomere length and for appropriate splicing of titin, not only in the heart but also in skeletal muscle. Distinguishing between thin filament extension and titin splicing as the primary defect is challenging, as these events are intimately linked. The regulation of titin splicing is a previously unrecognised role of Tβ4 and gives preliminary insight into a mechanism by which titin isoforms may be manipulated to correct cardiac dysfunction., Highlights • Thymosin β4 (Tβ4) regulates sarcomere length in the postnatal heart. • Τβ4 KO mice have shorter thin filaments and express shorter titin isoforms. • Τβ4 KO mice possess limited contractile reserve, in response to dobutamine stress. • Splicing may be a novel role for Tβ4 and a target to correct cardiac dysfunction.

A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction. A therapeutic ideal--relative to cell transplantation--would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm's tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.

Background: Myocardial infarction (MI) leads to gradually decreased cardiac contractility that can ultimately cause heart failure or sudden death. One potential therapy to recover this MI-impaired contractile function is the regenerative approach that integrates both tissue engineering and exogenously produced cardiomyocytes. Although this contractile recovery has been assessed by different imaging methods in previous studies, these assessments in global cardiac performance are plagued by their relatively low sensitivity. As this treatment is locally performed, the sensitivity of its therapeutic assessment can be improved by myocardial strain imaging that assesses regional contractility. Methods: We first made tissue engineered heart muscles (EHMs) by casting human embryonic stem cell-derived cardiomyocytes (ESC-CMs) with collagen in preformed molds. Then we transplanted these EHMs (n=12) into the post-MI rat hearts one month after this injure. We also performed sham surgeries on a control group (n=9) without EHM transplantation. Meanwhile, strain imaging from cardiac magnetic resonance imaging (MRI) and ultrasound were performed prior to and four weeks after EHM transplantations. In addition, global cardiac performance such as left ventricular ejection fraction (EF) was estimated from cardiac MRI. Results: Based on the measurements, regional myocardial strain measured from tagged MRI detected the preserved systolic function in the EHM-treated group compared to control group (P=0.04). In contrary, the global function EF was unable to detect this significant difference between both groups (P=0.38). Similarly, myocardial strain measured from cardiac ultrasound also failed to detect the significance between both treatment and control groups (P=0.24). Conclusions: This study for the first time confirmed that regional assessment of contractile function offered higher sensitivity compared to the conventional measure of global systolic performance. This finding highlights the feasibility and sensitivity of cardiac strain derived from tagged MRI as a sensitive method to assess the efficacy of regenerative therapies in both preclinical and clinical studies.

Background Bone marrow mesenchymal stromal cells ( BMMSC s) are cardioprotective in acute myocardial infarction ( AMI ) because of release of paracrine angiogenic and prosurvival factors. Hypoxia‐inducible factor 1‐α ( HIF 1‐α), rapidly degraded during normoxia, is stabilized during ischemia and upregulates various cardioprotective genes. We hypothesized that BMMSC s engineered to overexpress mutant, oxygen‐resistant HIF 1‐α would confer greater cardioprotection than nontransfected BMMSC s in sheep with AMI . Methods and Results Allogeneic BMMSC s transfected with a minicircle vector encoding mutant HIF 1‐α ( BMMSC ‐ HIF ) were injected in the peri‐infarct of sheep (n=6) undergoing coronary occlusion. Over 2 months, infarct volume measured by cardiac magnetic resonance ( CMR ) imaging decreased by 71.7±1.3% ( P LV ) percent ejection fraction (% EF ) increased near 2‐fold ( P BMMSC s ( BMMSC ; n=6) displayed less infarct size limitation and percent LVEF improvement, whereas in placebo‐treated animals (n=6), neither parameters changed over time. HIF 1‐α‐transfected BMMSC s ( BMMSC ‐ HIF ) induced angio‐/arteriogenesis and decreased apoptosis by HIF 1‐mediated overexpression of erythropoietin, inducible nitrous oxide synthase, vascular endothelial growth factor, and angiopoietin‐1. Cell tracking using paramagnetic iron nanoparticles in 12 additional sheep revealed enhanced long‐term retention of BMMSC ‐ HIF . Conclusions Intramyocardial delivery of BMMSC ‐ HIF reduced infarct size and improved LV systolic performance compared to BMMSC , attributed to increased neovascularization and cardioprotective effects induced by HIF 1‐mediated overexpression of paracrine factors and enhanced retention of injected cells. Given the safety of the minicircle vector and the feasibility of BMMSC s for allogeneic application, this treatment may be potentially useful in the clinic.

Efficient cardiac regeneration postinfarction (MI) requires the replacement of lost cardiomyocytes, formation of new coronary vessels and appropriate modulation of the inflammatory response. However, insight into how to stimulate repair of the human heart is currently limited. Using the embryonic paradigm of regeneration, we demonstrated that the actin-binding peptide thymosin β4 (Tβ4), required for epicardium-derived coronary vasculogenesis, can recapitulate its embryonic role and activate quiescent adult epicardial cells (EPDCs). Once stimulated, EPDCs facilitate neovascularization of the ischemic adult heart and, moreover, contribute bona fide cardiomyocytes. EPDC-derived cardiomyocytes structurally and functionally integrate with resident muscle to regenerate functional myocardium, limiting pathological remodeling, and effecting an improvement in cardiac function. Alongside pro-survival and anti-inflammatory properties, these regenerative roles, via EPDCs, markedly expand the range of therapeutic benefits of Tβ4 to sustain and repair the myocardium after ischemic damage.

Delivery of cells into tubular tissue constructs with large diameters poses significant spatial and temporal challenges. This study describes preliminary findings for a novel process for rapid and uniform seeding of cells onto the luminal surface of large tubular constructs. Fibroblasts, tagged with superparamagnetic iron oxide nanoparticles (SPION), were directed onto the luminal surface of tubular constructs by a magnetic field generated by a k4-type Halbach cylinder device. The spatial distribution of attached cells, as measured by the mean number of cells, was compared with a conventional, dynamic, rotational cell-delivery technique. Cell loading onto the constructs was measured by microscopy and magnetic resonance imaging. The different seeding techniques employed had a significant effect on the spatial distribution of the cells ( p < 0.0001). The number of attached cells at defined positions within the same construct was significantly different for the dynamic rotation technique ( p < 0.05). In contrast, no significant differences in the number of cells attached to the luminal surface were found between the defined positions on the construct loaded with the Halbach cylinder. The technique described overcomes limitations associated with existing cell-delivery techniques and is amenable to a variety of tubular organs where rapid loading and uniform distribution of cells for therapeutic applications are required.

Purpose: Our aim was to compare different magnet arrangements for magnetic cell delivery to human lower leg arteries and investigate the theoretical targeting efficiency under realistic flow conditions as a possible treatment after angioplasty. Additionally the potential of scaling down or translating the magnetic actuation device for preclinical studies was explored.Methods: Using finite element methods, the magnetic field distribution was calculated in 3D for the optimization of magnet arrangements. Computational fluid dynamics simulations were performed for the human posterior tibial artery with the geometry and boundary condition data derived from magnetic resonance imaging(MRI) studies. These simulations were used to trace the trajectories of cells for an optimized magnet arrangement. Additionally the behavior of cells close to the vessel wall was investigated using a fluid-structure interaction model.Results: The optimal magnet for the lower leg arteries was a Halbach cylinder k3 variety (12 elements with 90° rotation steps for the magnetization orientation). With this magnet, numerical simulations predict a targeting efficiency of 6.25% could be achieved in the posterior tibial artery for cells containing 150 pg iron. Similar simulations, which were scaled down to rabbit dimensions while keeping the forces acting on a cell constant, lead to similar predicted targeting efficiencies. Fluid dynamic and fluid-structure interaction simulations predict that magnetically labeled cells within a 0.5% radii distance to the vessel wall would be attracted and remain at the wall under physiological flow conditions.Conclusions: First pass capture of magnetically labeled cells under pulsatile flow conditions in human lower leg arteries leads to low targeting efficiencies. However, this can be increased to almost 100% by stopping the blood flow for 5 min. A magnetic actuation device can be designed for animal models that generate magnetic forces achievable for cells in human leg arteries.

Cardiovascular magnetic resonance (CMR) imaging is the modality of choice for clinical studies of the heart and vasculature, offering detailed images of both structure and function with high temporal resolution. Small animals are increasingly used for genetic and translational research, in conjunction with models of common pathologies such as myocardial infarction. In all cases, effective methods for characterising a wide range of functional and anatomical parameters are crucial for robust studies. CMR is the gold-standard for the non-invasive examination of these models, although physiological differences, such as rapid heart rate, make this a greater challenge than conventional clinical imaging. However, with the help of specialised magnetic resonance (MR) systems, novel gating strategies and optimised pulse sequences, high-quality images can be obtained in these animals despite their small size. In this review, we provide an overview of the principal CMR techniques for small animals for example cine, angiography and perfusion imaging, which can provide measures such as ejection fraction, vessel anatomy and local blood flow, respectively. In combination with MR contrast agents, regional dysfunction in the heart can also be identified and assessed. We also discuss optimal methods for analysing CMR data, particularly the use of semi-automated tools for parameter measurement to reduce analysis time. Finally, we describe current and emerging methods for imaging the developing heart, aiding characterisation of congenital cardiovascular defects. Advanced small animal CMR now offers an unparalleled range of cardiovascular assessments. Employing these methods should allow new insights into the structural, functional and molecular basis of the cardiovascular system.

To establish the accuracy, intra- and inter-observer variabilities of four different segmentation methods for measuring cardiac functional parameters in healthy and infarcted rat hearts.Six Wistar rats were imaged before and after myocardial infarction using an electrocardiogram and respiratory-gated spoiled gradient echo sequence. Blinded and randomized datasets were analyzed by various semi-automatic and manual segmentation methods to compare their measurement bias and variability. In addition, the accuracy of these methods was assessed by comparison with reference measurements acquired from high-resolution three-dimensional (3D) datasets of a heart phantom.Relative inter- and intra-observer variability were found to be similar for all four methods. Semi-automatic segmentation methods reduced analysis time by up to 70%, while yielding similar measurement bias and variability compared with manual segmentation. Semi-automatic methods were found to underestimate the ejection fraction for healthy hearts compared with manual segmentation while overestimating them in infarcted hearts. However, semi-automatic segmentation of short axis slices agreed better with 3D reference scans of a heart phantom compared with manual segmentation.Semi-automatic segmentation methods are faster than manual segmentation, while offering a similar intra- and inter-observer variability. However, a potential bias has been observed between healthy and infarcted hearts for different methods, which should also be considered when selecting the most appropriate analysis technique.

Rationale: Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell–derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure. Objective: Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model. Methods and Results: We constructed EHMs from human embryonic stem cell–derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P =0.83, P =0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P =0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, −6.7±1.4% versus control, −10.9±1.5%; n>12; P =0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P =0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring. Conclusions: EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.

Summary Animals have evolved homeostatic responses to changes in oxygen availability that act on different time scales. Although the hypoxia-inducible factor (HIF) transcriptional pathway that controls long term responses to low oxygen (hypoxia) has been established1, the pathway that mediates acute responses to hypoxia in mammals is not well understood. Here we show that the olfactory receptor Olfr78 is highly and selectively expressed in oxygen-sensitive glomus cells of the carotid body, a chemosensory organ at the carotid artery bifurcation that monitors blood oxygen and stimulates breathing within seconds when oxygen declines2. Olfr78 mutants fail to increase ventilation in hypoxia but respond normally to hypercapnia. Glomus cells are present in normal numbers and appear structurally intact, but hypoxia-induced carotid body activity is diminished. Lactate, a metabolite that rapidly accumulates in hypoxia and induces hyperventilation3–6, activates Olfr78 in heterologous expression experiments, induces calcium transients in glomus cells, and stimulates carotid sinus nerve activity through Olfr78. We propose that in addition to its role in olfaction, Olfr78 acts as a hypoxia sensor in the breathing circuit by sensing lactate produced when oxygen levels decline.

Introduction: Both human embryonic stem cell-derived cardiomyocytes (ESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) can serve as an unlimited cell source for cardiac regenerative therapy. However, the functional equivalency of both approaches has not been previously reported. Here we performed head-to-head comparison on the beneficial effects of ESC-CM and iPSC-CMs in restoring cardiac function in a rat myocardial infarction (MI) model. Methods & Results: Human ESCs and iPSCs were differentiated into cardiomyocytes using small molecules. FACS analysis confirmed ~85% and ~83% of cells differentiated from ESCs and iPSCs, respectively, were positive for cardiac troponin T, and immunofluorescence staining demonstrated that ESC-CMs and iPSC-CMs have striated sarcomeric structure (Figure A-B). Both ESC-CMs and iPSC-CMs displayed similar maturity for calcium handling (transient amplitude: ΔF/F 0 = 3.8±0.3; time to peak: ~200 ms; 50% transient duration: ~400 ms). qRT-PCR showed that ESC-CMs and iPSC-CMs expressed CASQ2, GJA5, KCNJ2, KCNJ5, MYH6, MYH7, and SCN5A at comparable levels to human fetal heart tissue. Next, ESC-CMs and iPSC-CMs were injected into the left ventricular free wall of infarcted hearts (adult nude rats; n=14, 10, respectively). Cardiac function was assessed by MRI one month post cell injection and the hearts were harvested and stained for human cardiac markers. Both ESC-CMs and iPSC-CMs could engraft in ischemic rat hearts (Figure C). Comprehensive functional analysis with small animal magnetic resonance imaging (MRI), echocardiography, and pressure-volume loop analysis are underway. Conclusion: We set out to perform head to head comparison for the first time that iPSC-CMs may facilitate cardiac repair at comparable levels to ESC-CMs. Unlike allogeneic ESC-CM therapy, autologous iPSC-CMs could be used to overcome immune rejection for cardiac cell transplantation in the future.

Introduction: Recent advances in regenerative medicine for cardiovascular disease (CVD) therapy focus on delivery of autologous induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to recover heart function without immunosuppression. Here, we perform a direct comparison of iPSC-CMs from a healthy individual, a DCM patient, and following genome-correction with transcription activator-like effector nucleases (TALENs) both in vitro and in vivo. Methods & Results: We established patient-specific iPSC-CMs that recapitulate DCM disease-phenotypes in-vitro, including disrupted sarcomeres and impaired Ca2+ handling (p Conclusions: We generated TALEN-corrected iPSC-CMs from a DCM patient, which recover in vitro functional parameters of healthy control iPSC-CMs. We transplanted for the first time patient-specific and TALEN-corrected iPSC-CMs into a rodent MI model. This approach may represent a viable option for mechanistic analysis and regenerative medicine in CVD patients. Figure 1

Nearly 8% of the human population carries an inactivating point mutation in the gene that encodes the cardioprotective enzyme aldehyde dehydrogenase 2 (ALDH2). This genetic polymorphism (ALDH2*2) is linked to more severe outcomes from ischemic heart damage and an increased risk of coronary artery disease (CAD), but the underlying molecular bases are unknown. We investigated the ALDH2*2 mechanisms in a human model system of induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) generated from individuals carrying the most common heterozygous form of the ALDH2*2 genotype. We showed that the ALDH2*2 mutation gave rise to elevated amounts of reactive oxygen species and toxic aldehydes, thereby inducing cell cycle arrest and activation of apoptotic signaling pathways, especially during ischemic injury. We established that ALDH2 controls cell survival decisions by modulating oxidative stress levels and that this regulatory circuitry was dysfunctional in the loss-of-function ALDH2*2 genotype, causing up-regulation of apoptosis in cardiomyocytes after ischemic insult. These results reveal a new function for the metabolic enzyme ALDH2 in modulation of cell survival decisions. Insight into the molecular mechanisms that mediate ALDH2*2-related increased ischemic damage is important for the development of specific diagnostic methods and improved risk management of CAD and may lead to patient-specific cardiac therapies.

Background— Cell therapies offer the potential to improve cardiac function after myocardial infarction. Although injection of single-cell suspensions has proven safe, cell retention and survival rates are low. Tissue-engineered grafts allow cell delivery with minimal initial cell loss and mechanical support to the heart. However, graft performance cannot be easily compared, and optimal construct thickness, vascularization, and survival kinetics are unknown. Methods and Results— Cardiac tissue slices (CTS) were generated by sectioning mouse hearts (n=40) expressing firefly luciferase and green fluorescent protein into slices of defined size and thickness using a vibrating blade microtome. Bioluminescence imaging of CTS transplanted onto hearts of immunodeficient mice demonstrated survival of ≤30% of transplanted cells. Cardiac slice perfusion was re-established within 3 days, likely through anastomosis of pre-existing vessels with the host vasculature and invasion of vessels from the host. Immunofluorescence showed a peak in cell death 3 days after transplantation and a gradual decline thereafter. MRI revealed preservation of contractile function and an improved ejection fraction 1 month after transplantation of CTS (28±2% CTS versus 22±2% control; P =0.05). Importantly, this effect was specific to CTS because transplantation of skeletal muscle tissue slices led to faster dilative remodeling and higher animal mortality. Conclusions— In summary, this is the first study to use CTS as a benchmark to validate and model tissue-engineered graft studies. CTS transplantation improved cell survival, established reperfusion, and enhanced cardiac function after myocardial infarction. These findings also confirm that dilative remodeling can be attenuated by topical transplantation of CTS but not skeletal muscle tissue grafts.

Although cellular therapies hold great promise for the treatment of human disease, results from several initial clinical trials have not shown a level of efficacy required for their use as a first line therapy. Here we discuss how in vivo molecular imaging has helped identify barriers to clinical translation and potential strategies that may contribute to successful transplantation and improved outcomes, with a focus on cardiovascular and neurological diseases. We conclude with a perspective on the future role of molecular imaging in defining safety and efficacy for clinical implementation of stem cell therapies.

Rationale: Human embryonic stem cell (hESC) derivatives are attractive candidates for therapeutic use. The engraftment and survival of hESC derivatives as xenografts or allografts require effective immunosuppression to prevent immune cell infiltration and graft destruction. Objective: To test the hypothesis that a short-course, dual-agent regimen of two costimulation-adhesion blockade agents can induce better engraftment of hESC derivatives compared to current immunosuppressive agents. Methods and Results: We transduced hESCs with a double fusion reporter gene construct expressing firefly luciferase (Fluc) and enhanced green fluorescent protein, and differentiated these cells to endothelial cells (hESC-ECs). Reporter gene expression enabled longitudinal assessment of cell engraftment by bioluminescence imaging. Costimulation-adhesion therapy resulted in superior hESC-EC and mouse EC engraftment compared to cyclosporine therapy in a hind limb model. Costimulation-adhesion therapy also promoted robust hESC-EC and hESC-derived cardiomyocyte survival in an ischemic myocardial injury model. Improved hESC-EC engraftment had a cardioprotective effect after myocardial injury, as assessed by magnetic resonance imaging. Mechanistically, costimulation-adhesion therapy is associated with systemic and intragraft upregulation of T-cell immunoglobulin and mucin domain 3 (TIM3) and a reduced proinflammatory cytokine profile. Conclusions: Costimulation-adhesion therapy is a superior alternative to current clinical immunosuppressive strategies for preventing the post-transplant rejection of hESC derivatives. By extending the window for cellular engraftment, costimulation-adhesion therapy enhances functional preservation following ischemic injury. This regimen may function through a TIM3-dependent mechanism.

The downstream consequences of inflammation in the adult mammalian heart are formation of a non-functional scar, pathological remodelling and heart failure. In zebrafish, hydrogen peroxide (H2O2) released from a wound is the initial instructive chemotactic cue for the infiltration of inflammatory cells, however, the identity of a subsequent resolution signal(s), to attenuate chronic inflammation, remains unknown. Here we reveal that Thymosin β4-Sulfoxide inhibits interferon-γ, and increases monocyte dispersal and cell death, lies downstream of H2O2 in the wounded fish and triggers depletion of inflammatory macrophages at the injury site. This function is conserved in the mouse and observed after cardiac injury, where it promotes wound healing and reduced scarring. In human T cell/CD14+ monocyte co-cultures, Tβ4-SO inhibits IFN-γ and increases monocyte dispersal and cell death, likely by stimulating superoxide production. Thus, Tβ4-SO is a putative target for therapeutic modulation of the immune response, resolution of fibrosis and cardiac repair.

The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7(Mtu) and Rps7(Zma)) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes.

Efficient cardiac regeneration post-infarction (MI) requires the replacement of lost cardiomyocytes, formation of new coronary vessels and appropriate modulation of the inflammatory response. However, insight into how to stimulate repair of the human heart is currently limited. Utilising the embryonic paradigm of regeneration, we demonstrated that the actin-binding peptide Thymosin β4 (Tβ4), required for epicardium-derived coronary vasculogenesis, can recapitulate its embryonic role and activate quiescent adult epicardial cells (EPDCs). Once stimulated, EPDCs facilitate neovascularisation of the ischaemic adult heart and, moreover, contribute bona fide cardiomyocytes. EPDC-derived cardiomyocytes structurally and functionally integrate with resident muscle, to regenerate functional myocardium, limiting pathological remodelling and effecting an improvement in cardiac function. Alongside pro-survival and anti-inflammatory properties, these regenerative roles, via EPDCs, markedly expand the range of therapeutic benefits of Tβ4 to sustain and repair the myocardium after ischaemic damage.

Summary Early inflammation following status epilepticus has been implicated in the development of epilepsy and the evolution of brain injury, yet its precise role remains unclear. The development of non-invasive imaging markers of inflammation would enable researchers to test this hypothesis in vivo and study its temporal progression in relation to epileptogenic insults. In this study we have investigated the potential of a targeted magnetic resonance imaging contrast agent – vascular cell adhesion molecule 1 antibody labelled iron oxide – to image the inflammatory process following status epilepticus in the rat lithium–pilocarpine model. Intravascular administration of the targeted contrast agent was performed at approximately 1 day following status epilepticus. The control group received diazepam prior to pilocarpine to prevent status epilepticus. Magnetic resonance imaging of rats was performed before and after contrast administration. Comparison with quantitative T2 measurements was also performed. At the end of the study, brains were removed for ex vivo magnetic resonance imaging and histology. Marked focal hypointensities caused by contrast agent binding were observed on in vivo magnetic resonance images in the post status epilepticus group. In particular these occurred in the periventricular organs, the hippocampus and the cerebral cortex. Relatively little contrast agent binding was observed in the control group. T2 relaxation times were not significantly increased for the hippocampus or the cerebral cortex in post status epilepticus animals. These results demonstrate the feasibility of in vivo imaging of seizure-induced inflammation in an animal model of epilepsy. The antibody targeted MRI contrast agent identified regions of acute inflammation following status epilepticus and may provide an early marker of brain injury. This technique could be used to determine the role of inflammation in models of epileptogenesis and to study the potential for anti-inflammatory therapeutic interventions.

Die Finanzmarktkrise der Jahre 2008 und 2009 hat deutlich gemacht, dass die bislang praktizierten Methoden zur Risikoanalyse um Systemkomponenten zur Krisenfrühaufklärung ergänzt werden müssen. Verhaltensrisiken, das Auftreten „finanzwirtschaftlicher Blasen“ und volkswirtschaftliche Abhängigkeiten führten bei Banken und Unternehmen zu erheblichen Bedrohungen. Dieses Buch stellt das komplexe Thema Krisenaufklärung aus wissenschaftlicher, aufsichtsrechtlicher und unternehmenspraktischer Perspektive dar. Besonders hilfreich sind die anhand authentischer Praxisfälle vorgestellten Instrumente zur Krisenerkennung und -vermeidung. Dazu werden interessante Einblicke in konkretes Projektmanagement und konkrete Bilanzpolitik geboten.

Management of intestinal failure remains a clinical challenge and total parenteral nutrition, intestinal elongation and/or transplantation are partial solutions. In this study, using a detergent-enzymatic treatment (DET), we optimize in rats a new protocol that creates a natural intestinal scaffold, as a base for developing functional intestinal tissue. After 1 cycle of DET, histological examination and SEM and TEM analyses showed removal of cellular elements with preservation of the native architecture and connective tissue components. Maintenance of biomechanical, adhesion and angiogenic properties were also demonstrated strengthen the idea that matrices obtained using DET may represent a valid support for intestinal regeneration.

Understanding the molecular and cellular mechanisms that mediate magnetosensation in vertebrates is a formidable scientific problem. One hypothesis is that magnetic information is transduced into neuronal impulses by using a magnetite-based magnetoreceptor. Previous studies claim to have identified a magnetic sense system in the pigeon, common to avian species, which consists of magnetite-containing trigeminal afferents located at six specific loci in the rostral subepidermis of the beak. These studies have been widely accepted in the field and heavily relied upon by both behavioural biologists and physicists. Here we show that clusters of iron-rich cells in the rostro-medial upper beak of the pigeon Columbia livia are macrophages, not magnetosensitive neurons. Our systematic characterization of the pigeon upper beak identified iron-rich cells in the stratum laxum of the subepidermis, the basal region of the respiratory epithelium and the apex of feather follicles. Using a three-dimensional blueprint of the pigeon beak created by magnetic resonance imaging and computed tomography, we mapped the location of iron-rich cells, revealing unexpected variation in their distribution and number--an observation that is inconsistent with a role in magnetic sensation. Ultrastructure analysis of these cells, which are not unique to the beak, showed that their subcellular architecture includes ferritin-like granules, siderosomes, haemosiderin and filopodia, characteristics of iron-rich macrophages. Our conclusion that these cells are macrophages and not magnetosensitive neurons is supported by immunohistological studies showing co-localization with the antigen-presenting molecule major histocompatibility complex class II. Our work necessitates a renewed search for the true magnetite-dependent magnetoreceptor in birds.

Our aim was to compare different magnet arrangements for magnetic cell delivery to human lower leg arteries and investigate the theoretical targeting efficiency under realistic flow conditions as a possible treatment after angioplasty. Additionally the potential of scaling down or translating the magnetic actuation device for preclinical studies was explored.Using finite element methods, the magnetic field distribution was calculated in 3D for the optimization of magnet arrangements. Computational fluid dynamics simulations were performed for the human posterior tibial artery with the geometry and boundary condition data derived from magnetic resonance imaging (MRI) studies. These simulations were used to trace the trajectories of cells for an optimized magnet arrangement. Additionally the behavior of cells close to the vessel wall was investigated using a fluid-structure interaction model.The optimal magnet for the lower leg arteries was a Halbach cylinder k3 variety (12 elements with 900 rotation steps for the magnetization orientation). With this magnet, numerical simulations predict a targeting efficiency of 6.25% could be achieved in the posterior tibial artery for cells containing 150 pg iron. Similar simulations, which were scaled down to rabbit dimensions while keeping the forces acting on a cell constant, lead to similar predicted targeting efficiencies. Fluid dynamic and fluid-structure interaction simulations predict that magnetically labeled cells within a 0.5% radii distance to the vessel wall would be attracted and remain at the wall under physiological flow conditions.First pass capture of magnetically labeled cells under pulsatile flow conditions in human lower leg arteries leads to low targeting efficiencies. However, this can be increased to almost 100% by stopping the blood flow for 5 min. A magnetic actuation device can be designed for animal models that generate magnetic forces achievable for cells in human leg arteries.

International audience; A simple analytical model is presented which enables rapid interactive prediction and control of magnetically labelled cells in an arterial bifurcation using magnetic field gradients produced by a magnetic resonance imaging system (MRI). This model is compared against experimental results for human mononuclear cells labelled with micron sized superparamagnetic iron oxide particles. Experimental and theoretical results highlight the importance of cell aggregation for magnetic targeting in a strong magnetic field. These predicted aggregates are confirmed via confocal endoscopy which allows the visualisation of cell aggregates and their movement inside a vascular flow model in a 9.4T preclinical MRI scanner.