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Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNALINC00607to be highly enriched in human endothelial cells.LINC00607was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout ofLINC00607attenuated VEGF-A-induced angiogenic sprouting.LINC00607knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression ofLINC00607in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq afterLINC00607knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically,LINC00607interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout ofBRG1in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion,LINC00607is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis.

Microvascular dysfunction is a pathological hallmark of diabetes, and is central to the ethology of diabetes-associated cardiac events. Herein, previous studies have highlighted the role of the vasoactive micro-RNA 92a (miR-92a) in small, as well as large, animal models. In this study, we explore the effects of miR-92a on mouse and human cardiac microvascular endothelial cells (MCMEC, HCMEC), and its underlying molecular mechanisms. Diabetic HCMEC displayed impaired angiogenesis and a pronounced inflammatory phenotype. Quantitative PCR (qPCR) showed an upregulation of miR-92a in primary diabetic HCMEC. Downregulation of miR-92a by antagomir transfection in diabetic HCMEC rescued angiogenesis and ameliorated diabetic endothelial bed inflammation. Furthermore, additional analysis of potential in silico-identified miR-92a targets in diabetic HCMEC revealed the miR-92a dependent downregulation of an essential metalloprotease, ADAM10. Accordingly, downregulation of ADAM10 impaired angiogenesis and wound healing in MCMEC. In myocardial tissue slices from diabetic pigs, ADAM10 dysregulation in micro- and macro-vasculature could be shown. Altogether, our data demonstrate the role of miR-92a in cardiac microvascular dysfunction and inflammation in diabetes. Moreover, we describe for the first time the metalloprotease ADAM10 as a novel miR-92a target, mediating its anti-angiogenic effect.

To develop a free-breathing myocardialFree-running (free-breathing, retrospective cardiac gating) IR radial FLASH is used for data acquisition at 3T. First, to reduce the waiting time between inversions, an analytical formula is derived that takes the incompleteIn comparison to an IR spin-echo reference, phantom results confirm goodMotion-resolved myocardial

Coronary heart disease is one of the leading causes of death in industrialized nations. Even though revascularization strategies improved the outcome of patients after acute myocardial infarction, about 30% of patients develop chronic heart failure. Ischemic heart disease and heart failure are characterized by an adverse remodeling of the heart, featuring cardiomyocyte hypertrophy, increased fibrosis, and capillary rarefaction. Therefore, novel therapeutic approaches for the treatment of heart failure, such as reducing ischemia/reperfusion injury, fibrosis, or hypertrophy, are needed. Here microRNAs (miRNAs) come into play. For heart failure, cardiac stress and several cardiovascular diseases, individual miRNAs, and whole miRNA clusters have been implicated as disease relevant and dysregulated. miRNAs are short non-coding RNA molecules of about 22 nucleotides, and their inhibitors are 8-15 nucleotides long plus a sugar-ring (LNA, locked nucleid acid) or cholesterol ending (AntagomiR). Modulation of miRNAs might serve as therapeutic targets through miRNA knockdown or overexpression via miRNA mimics. Due to their pleiotropic mode of action and the presence of individual miRNAs in a variety of tissues and cells, a local, target region-oriented application is important to achieve therapeutic effects and at the same time reducing adverse effects in other off-target organs and tissues. Due to their small size, the miRNA inhibitors are able to pass endothelial barrier at both arterial and venous sides of the bloodstream vessels. For these reasons, the gold standard administration route of miRNA modulators for therapeutic approaches of the left ventricle is the anterograde application into one or both coronary arteries via an over-the-wire (OTW) balloon. In this chapter we provide a comprehensive description of the anterograde application procedure in a large animal model such as pig. Of a particular note, this methodology is a standard procedure in catheter laboratories, a key characteristic that allows therapeutic translation from large animals to patients.

Due to severely limited donor heart availability, durable mechanical circulatory support remains the only treatment option for many patients with end-stage heart failure. However, treatment complexity persists due to its univentricular support modality and continuous contact with blood. We investigated the function and safety of reBEAT (AdjuCor GmbH), a novel, minimal invasive mechanical circulatory support device that completely avoids blood contact and provides pulsatile, biventricular support.For each animal tested, an accurately sized cardiac implant was manufactured from computed tomography scan analyses. The implant consists of a cardiac sleeve with three inflatable cushions, 6 epicardial electrodes and driveline connecting to an electro-pneumatic, extracorporeal portable driver. Continuous epicardial electrocardiogram signal analysis allows for systolic and diastolic synchronization of biventricular mechanical support. In 7 pigs (weight, 50-80 kg), data were analyzed acutely (under beta-blockade, n = 5) and in a 30-day long-term survival model (n = 2). Acquisition of intracardiac pressures and aortic and pulmonary flow data were used to determine left ventricle and right ventricle stroke work and stroke volume, respectively.Each implant was successfully positioned around the ventricles. Automatic algorithm electrocardiogram signal annotations resulted in precise, real-time mechanical support synchronization with each cardiac cycle. Consequently, progressive improvements in cardiac hemodynamic parameters in acute animals were achieved. Long-term survival demonstrated safe device integration, and clear and stable electrocardiogram signal detection over time.The present study demonstrates biventricular cardiac support with reBEAT. Various demonstrated features are essential for realistic translation into the clinical setting, including safe implantation, anatomical fit, safe device-tissue integration, and real-time electrocardiogram synchronized mechanical support, result in effective device function and long-term safety.

The high mortality seen in sepsis is caused by a systemic hypotension in part owing to a drastic increase in vascular permeability accompanied by a loss of pericytes. As has been shown previously, pericyte retention in the perivascular niche during sepsis can enhance the integrity of the vasculature and promote survival via recruitment of adhesion proteins such as VE-cadherin and N-cadherin. Sphingosine-1-phosphate (S1P) represents a lipid mediator regulating the deposition of the crucial adhesion molecule VE-cadherin at sites of interendothelial adherens junctions and of N-cadherin at endothelial–pericyte adherens junctions. Furthermore, in septic patients, S1P plasma levels are decreased and correlate with mortality in an indirectly proportional way. In the present study, we investigated the potential of S1P to ameliorate a lipopolysaccharide-induced septic hypercirculation in mice. Here we establish S1P as an antagonist of pericyte loss, vascular hyperpermeability, and systemic hypotension, resulting in an increased survival in mice. During sepsis S1P preserved VE-cadherin and N-cadherin deposition, mediated by a reduction of Src and cadherin phosphorylation. At least in part, this effect is mediated by a reduction of globular actin and a subsequent increase in nuclear translocation of MRTF-A (myocardin-related transcription factor A). These findings indicate that S1P may counteract pericyte loss and microvessel disassembly during sepsis and additionally emphasize the importance of pericyte–endothelial interactions to stabilize the vasculature.

BACKGROUND miR-21 is a central regulator of cardiac fibrosis, and its inhibition in small-animal models has been shown to be an effective antifibrotic strategy in various organs, including the heart. Effective delivery of therapeutic antisense micro-ribonucleic acid (antimiR) molecules to the myocardium in larger organisms is challenging, though, and remains to be established for models of chronic heart failure.OBJECTIVES The aims of this study were to test the applicability and therapeutic efficacy of local, catheter-based delivery of antimiR-21 in a pig model of heart failure and determine its effect on the cardiac transcriptomic signature and cellular composition.METHODS Pigs underwent transient percutaneous occlusion of the left coronary artery and were followed up for 33 days. AntimiR-21 (10 mg) was applied by intracoronary infusion at days 5 and 19 after the injury. Cardiac function was assessed in vivo, followed by histological analyses and deep ribonucleic acid sequencing (RNA-seq) of the myocardium and genetic deconvolution analysis.RESULTS AntimiR-21 effectively suppressed the remodeling-associated increase of miR-21. At 33 days after ischemia/reperfusion injury, LNA-21-treated hearts exhibited reduced cardiac fibrosis and hypertrophy and improved cardiac function. Deep RNA-seq revealed a significant derepression of the miR-21 targetome in antimiR-21-treated myocardium and a suppression of the inflammatory response and mitogen-activated protein kinase signaling. A genetic deconvolution approach built on deep RNA-seq and single-cell RNA-seq data identified reductions in macrophage and fibroblast numbers as the key cell types affected by antimiR-21 treatment.CONCLUSIONS This study provides the first evidence for the feasibility and therapeutic efficacy of miR-21 inhibition in a large animal model of heart failure. (C) 2020 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation.

In recent years, the interest in extracellular vesicles (EV) functions has been growing. However, methods for isolating these small vesicles from tissue are still not trivial. Few protocols are available that allows EV isolation from whole tissue samples, including the heart. Those protocols are based on organ perfusion using the Langendorff method. In this work, aiming to analysing in vivo biology of small EVs, we implemented a simple method to obtain enrichment of these vesicles from murine heart tissue. We tested a titration of Liberase for tissue digestion, which was subjected to differential ultracentrifugation combined with iodixanol cushion and presented the step-by-step procedure of this protocol. Validation was done with Nanoparticle Tracking Analysis, transmission Electron Microscope and Western Blot analysis of EV markers and organelles contaminants. Furthermore, we tested the suitability of the protocol for isolating EVs from heart tissue obtained from a pre-clinical translational non-human primate animal model. Therefore, this protocol may be suitable for isolating vesicle from human heart tissue. Additionally, this method can potentially be applied beyond heart tissue.

Heart failure is one of the most common causes of morbidity and mortality. Both maturational abnormalities and age-associated cardiac pathologies contribute to heart failure. Imaging-based assessment to discern detailed cardiac structure at various maturational stages is imperative for understanding mechanisms behind cardiac growth and aging. Using multiphoton nonlinear optical microscopy (NLOM) based label-free imaging, we investigated cardiac structural composition in a human-relevant aging model, the common marmoset monkey (Callithrix jacchus). Animals were divided into three different age groups including neonatal, young adult and old. By devising a unique strategy for segregating collagen and myosin emitted second harmonic generation (SHG) signals, we performed a volumetric assessment of collagen and total scattering tissue (collagen + myosin). Aged marmoset hearts exhibited an increase in collagen and total scattering tissue volume at the sites of severe tissue remodelling indicating age-related cardiac fibrosis. Significantly low scattering tissue volume in neonatal marmoset hearts was attributed to a lack of binding between the myofibrils in maturing cardiac tissue. Comprehensive quantitative assessment of structural composition during maturation and aging of marmoset hearts revealed significant differences in myofibril length, alignment, curvature and angular distribution. In conclusion, label-free high-resolution NLOM facilitates visualization and quantification of subcellular structural features for understanding vital age-related morphological alterations in the marmoset heart.

Cardiovascular diseases are the leading cause of mortality worldwide. Understanding the mechanisms at the basis of these diseases is necessary in order to generate therapeutic approaches. Recently, cardiac tissue engineering and induced pluripotent stem cell (iPSC) reprogramming has led to a skyrocketing number of publications describing cardiovascular regeneration as a promising option for cardiovascular disease treatment. Generation of artificial tissue and organoids derived from induced pluripotent stem cells is in the pipeline for regenerative medicine. The present review summarizes the multiple approaches of heart regeneration with a special focus on iPSC application. In particular, we describe the strength of iPSCs as a tool to study the molecular mechanisms driving cardiovascular pathologies, as well as their potential in drug discovery. Moreover, we will describe some insights into novel discoveries of how stem-cell-secreted biomolecules, such as exosomes, could affect cardiac regeneration, and how the fine tuning of the immune system could be a revolutionary tool in the modulation of heart regeneration.

Background Pathological cardiac hypertrophy is a result of afterload-increasing pathologies including untreated hypertension and aortic stenosis. It features progressive adverse cardiac remodeling, myocardial dysfunction, capillary rarefaction, and interstitial fibrosis often leading to heart failure. Objectives This study aimed to establish a novel porcine model of pressure-overload–induced heart failure and to determine the effect of inhibition of microribonucleic acid 132 (miR-132) on heart failure development in this model. Methods This study developed a novel porcine model of percutaneous aortic constriction by implantation of a percutaneous reduction stent in the thoracic aorta, inducing progressive remodeling at day 56 (d56) after pressure-overload induction. In this study, an antisense oligonucleotide specifically inhibiting miR-132 (antimiR-132), was regionally applied via intracoronary injection at d0 (percutaneous transverse aortic constriction induction) and d28. Results At d56, antimiR-132 treatment diminished cardiomyocyte cross-sectional area (188.9 ± 2.8 vs. 258.4 ± 9.0 μm2 in untreated hypertrophic hearts) and improved global cardiac function (ejection fraction 48.9 ± 1.0% vs. 36.1 ± 1.7% in control hearts). Moreover, at d56 antimiR-132-treated hearts displayed less increase of interstitial fibrosis compared with sham-operated hearts (Δsham 1.8 ± 0.5%) than control hearts (Δsham 10.8 ± 0.6%). Of note, cardiac platelet and endothelial cell adhesion molecule 1+ capillary density was higher in the antimiR-132–treated hearts (647 ± 20 cells/mm2) compared with in the control group (485 ± 23 cells/mm2). Conclusions The inhibition of miR-132 is a valid strategy in prevention of heart failure progression in hypertrophic heart disease and may be developed as a treatment for heart failure of nonischemic origin.

Aims Increased shedding of extracellular vesicles (EVs)—small, lipid bilayer-delimited particles with a role in paracrine signalling—has been associated with human pathologies, e.g. atherosclerosis, but whether this is true for cardiac diseases is unknown. Methods and results Here, we used the surface antigen CD172a as a specific marker of cardiomyocyte (CM)-derived EVs; the CM origin of CD172a+ EVs was supported by their content of cardiac-specific proteins and heart-enriched microRNAs. We found that patients with aortic stenosis, ischaemic heart disease, or cardiomyopathy had higher circulating CD172a+ cardiac EV counts than did healthy subjects. Cellular stress was a major determinant of EV release from CMs, with hypoxia increasing shedding in in vitro and in vivo experiments. At the functional level, EVs isolated from the supernatant of CMs derived from human-induced pluripotent stem cells and cultured in a hypoxic atmosphere elicited a positive inotropic response in unstressed CMs, an effect we found to be dependent on an increase in the number of EVs expressing ceramide on their surface. Of potential clinical relevance, aortic stenosis patients with the highest counts of circulating cardiac CD172a+ EVs had a more favourable prognosis for transcatheter aortic valve replacement than those with lower counts. Conclusion We identified circulating CD172a+ EVs as cardiac derived, showing their release and function and providing evidence for their prognostic potential in aortic stenosis patients.

Purpose: To develop a free-breathing myocardial T1 mapping technique using inversion-recovery (IR) radial fast low-angle shot (FLASH) and calibrationless motion-resolved model-based reconstruction. Methods: Free-running (free-breathing, retrospective cardiac gating) IR radial FLASH is used for data acquisition at 3T. First, to reduce the waiting time between inversions, an analytical formula is derived that takes the incomplete T1 recovery into account for an accurate T1 calculation. Second, the respiratory motion signal is estimated from the k-space center of the contrast varying acquisition using an adapted singular spectrum analysis (SSA-FARY) technique. Third, a motion-resolved model-based reconstruction is used to estimate both parameter and coil sensitivity maps directly from the sorted k-space data. Thus, spatiotemporal total variation, in addition to the spatial sparsity constraints, can be directly applied to the parameter maps. Validations are performed on an experimental phantom, eleven human subjects, and a young landrace pig with myocardial infarction. Results: In comparison to an IR spin-echo reference, phantom results confirm good T1 accuracy when reducing the waiting time from five seconds to one second using the new correction. The motion-resolved model-based reconstruction further improves precision compared to the spatial regularization-only reconstruction. Aside from showing that a reliable respiratory motion signal can be estimated using modified SSA-FARY, in vivo studies demonstrate that dynamic myocardial T1 maps can be obtained within two minutes with good precision and repeatability. Conclusion: Motion-resolved myocardial T1 mapping during free-breathing with good accuracy, precision and repeatability can be achieved by combining inversion-recovery radial FLASH, self-gating and a calibrationless motion-resolved model-based reconstruction., Comment: Part of this work has been presented at the ISMRM Annual Conference 2021 (Virtual), accepted by Magnetic Resonance in Medicine

Background: Ischemic heart diseases are leading causes of death and reduced life quality worldwide. Although revascularization strategies significantly reduce mortality after acute myocardial infarction (MI), a large number of patients with MI develop chronic heart failure over time. We previously reported that a fragment of the extracellular matrix protein agrin promotes cardiac regeneration after MI in adult mice. Methods: To test the therapeutic potential of agrin in a preclinical porcine model, we performed ischemia–reperfusion injuries using balloon occlusion for 60 minutes followed by a 3-, 7-, or 28-day reperfusion period. Results: We demonstrated that local (antegrade) delivery of recombinant human agrin to the infarcted pig heart can target the affected regions in an efficient and clinically relevant manner. A single dose of recombinant human agrin improved heart function, infarct size, fibrosis, and adverse remodeling parameters 28 days after MI. Short-term MI experiments along with complementary murine studies revealed myocardial protection, improved angiogenesis, inflammatory suppression, and cell cycle reentry as agrin’s mechanisms of action. Conclusions: A single dose of agrin is capable of reducing ischemia–reperfusion injury and improving heart function, demonstrating that agrin could serve as a therapy for patients with acute MI and potentially heart failure.

Cardiovascular diseases remain the leading cause of death in the developed world, accounting for more than 30% of all deaths. In a large proportion of these patients, acute myocardial infarction is usually the first manifestation, which might further progress to heart failure. In addition, the human heart displays a low regenerative capacity, leading to a loss of cardiomyocytes and persistent tissue scaring, which entails a morbid pathologic sequela. Novel therapeutic approaches are urgently needed. Stem cells, such as induced pluripotent stem cells or embryonic stem cells, exhibit great potential for cell-replacement therapy and an excellent tool for disease modeling, as well as pharmaceutical screening of novel drugs and their cardiac side effects. This review article covers not only the origin of stem cells but tries to summarize their translational potential, as well as potential risks and clinical translation.

For treatment of advanced heart failure, current strategies include cardiac transplantation or blood-contacting pump technology associated with complications, including stroke and bleeding. This study investigated an individualized biventricular epicardial augmentation technology in a drug-induced porcine failing heart model. A total of 11 pigs were used, for the assessment of hemodynamics and cardiac function under various conditions of support pressures and support durations (n = 4), to assess device positioning and function by in vivo computer tomographic imaging (n = 3) and to investigate a minimally invasive implantation on the beating heart (n = 4). Support pressures of 20-80 mm Hg gradually augmented cardiac function parameters in this animal model as indicated by increased left ventricular stroke volume, end-systolic pressures, and decreased end-diastolic pressures. Strong evidence was found regarding the necessity of mechanical synchronization of support end with the isovolumetric relaxation phase of the heart. In addition, the customized, self-expandable implant enabled a marker-guided minimally invasive implantation through a 4 cm skin incision using fluoroscopy. Correct positioning was confirmed in computer tomographic images. Continued long-term survival investigations will deliver preclinical evidence for further development of this concept.

The establishment of induced pluripotent stem cells (iPSCs) and cardiomyocytes differentiated from them generated a new platform to study pathophysiological processes and to generate drug screening platforms and iPSC-derived tissues as therapeutic agents. Although major advances have been made in iPSC-reprogramming, cardiac differentiation and EHT production, reprogramming efficiency and the maturity of iPSC-CMs need to be further improved.In this review, the authors summarize the current state of the field of iPSC research, the methodology of cardiac differentiation of iPSCs, the use of iPSC-CMs as disease models and toxicity screening platforms, and the potential of EHTs as therapeutic agents. The authors furthermore highlight the mechanisms by which Thymosin β4 might enhance the production of iPSC-CMs and EHTs to improve their maturity and performance.iPSCs derived cardiomyocytes and EHTs represent a still young research field with many problems and pitfalls that need to be resolved to realize the full potential of iPSC-CMs and EHTs. Given that Thymosin β4 directly enhances cardiac differentiation while also promoting angiogenic sprouting and vessel maturation, Tβ4 might be of particular interest as a novel agent in tackling the difficulty of iPSC-CMs and engineered heart tissue grafts.

BACKGROUND Heart failure following myocardial infarction (MI) remains one of the major causes of death worldwide, and its treatment is a crucial challenge of cardiovascular medicine. An attractive therapeutic strategy is to stimulate endogenous mechanisms of myocardial regeneration.OBJECTIVES This study evaluates the potential therapeutic treatment with annexin A1 (AnxA1) to induce cardiac repair after MI.METHODS AnxA1 knockout (AnxA1(-/-)) and wild-type mice underwent MI induced by ligation of the left anterior descending coronary artery. Cardiac functionality was assessed by longitudinal echocardiographic measurements. Histological, fluorescence-activated cell sorting, dot blot analysis, and in vitro/ex vivo studies were used to assess the myocardial neovascularization, macrophage content, and activity in response to AnxA1.RESULTS AnxA1(-/-) mice showed a reduced cardiac functionality and an expansion of proinflammatory macrophages in the ischemic area. Cardiac macrophages from AnxA1(-/-) mice exhibited a dramatically reduced ability to release the proangiogenic mediator vascular endothelial growth factor (VEGF)-A. However, AnxA1 treatment enhanced VEGF-A release from cardiac macrophages, and its delivery in vivo markedly improved cardiac performance. The positive effect of AnxA1 treatment on cardiac performance was abolished in wild-type mice transplanted with bone marrow derived from Cx(3)cr1cre(ERT2)Vegf(flox/flox) or in mice depleted of macrophages. Similarly, cardioprotective effects of AnxA1 were obtained in pigs in which full-length AnxA1 was overexpressed by use of a cardiotropic adeno-associated virus.CONCLUSIONS AnxA1 has a direct action on cardiac macrophage polarization toward a pro-angiogenic, reparative phenotype. AnxA1 stimulated cardiac macrophages to release high amounts of VEGF-A, thus inducing neovascularization and cardiac repair. (C) 2019 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation.

SummaryAdvanced glycation end-products (AGEs) interact with their receptor RAGE, leading to an inflammatory state. We investigated the role of RAGE in postischaemic leukocyte adhesion after myocardial infarction and its effect on postischaemic myocardial function. Wildtype (WT), ICAM-1-/-, RAGE-/- or ICAM-1/RAGE-/- mice underwent 20 minutes (min) of LAD-occlusion followed by 15 min of reperfusion. We applied in vivo fluorescence microscopy visualising Rhodamine-6G labelled leukocytes. To differentiate between endothelial and leukocyte RAGE, we generated bone marrow chimeric mice. Invasive hemodynamic measurements were performed in mice undergoing 45 min of myocardial ischaemia (via LAD-occlusion) followed by 24 hours of reperfusion. Left-ventricular developed pressure (LVDP) was assessed by insertion of a millar-tip catheter into the left ventricle. In the acute model of myocardial ischaemia, leukocyte retention (WT 68 ± 4 cells/ hpf) was significantly reduced in ICAM-1-/- (40 ± 3 cells/hpf) and RAGE-/- mice (38 ± 4 cells/hpf). ICAM-1/RAGE-/- mice displayed an additive reduction of leukocyte retention (ICAM-1/RAGE-/- 15 ± 3 cells/ hpf). Ly-6G+ neutrophil were predominantly reduced in ICAM-1/RAGE-/- hearts (28%), whereas Ly-6C+ proinflammatory monocytes decreased to a lesser extent (55%). Interestingly, PMN recruitment was not affected in chimeric mice with RAGE deficiency in BM cells (WT mice reconstituted with ICAM-1/RAGE-/- BM: 55 ± 4 cells/hpf) while in mice with global RAGE deficiency (ICAM-1/RAGE-/- mice reconstituted with ICAM-1/RAGE-/- BM) leucocyte retention was significantly reduced (13 ± 1 cells/hpf), similar to non-transplanted ICAM/ RAGE-/- mice. Furthermore, postischaemic LVDP increased in ICAM-1/RAGE-/- animals (98 ± 4 mmHg vs 86 ± 4 mmHg in WT mice). In conclusion, combined deficiency of ICAM-1 and RAGE reduces leukocyte influx into infarcted myocardium and improves LV function during the acute phase after myocardial ischaemia and reperfusion. RAGE represents an additional pro-inflammatory endothelial mediator of ischaemia-reperfusion injury.

Background: Upregulation of microRNA-21 (miR-21) has been associated with myocardial disease and its systemic inhibition was effective in reducing cardiac fibrosis and dysfunction in rodent models. However, the translation to human disease is challenging, as systemic application of miR inhibitors does not achieve adequate delivery in most organs including the heart. Here, we sought to test the applicability and therapeutic efficacy of local, catheter-based delivery of antimiR-21 in a large animal model of heart failure. Methods and results: Pigs underwent percutaneous occlusion of the left coronary artery for 60 minutes and were followed up for 33 days. A synthetic inhibitor against miR-21 (LNA-antimiR-21, 10 mg per application) was applied locally by intracoronary infusion at days 5 and 19 after the injury. AntimiR-21 effectively suppressed the I/R-induced increase of miR-21. At 33 days after I/R, LNA-21-treated hearts exhibited reduced cardiac fibrosis and hypertrophy, and improved cardiac function (ejection fraction: I/R control 22±1% vs. I/R LNA-21 33±2%). RNA sequencing of ischemic myocardium revealed a significant enrichment for miR-21 targets in anti-miR-21 treated hearts. Mechanistically, antimiR-21 interfered with a number of signalling pathways involved in myocardial remodelling including the MAPK-ERK signalling cascade. Conclusion: Taken together, our study provides the first evidence for the feasibility and therapeutic efficacy of miR-21 inhibition in a large animal model of myocardial ischemia. AntimiR-21 prevented the structural and functional consequences of ischemic myocardial damage.

Translations of new therapeutic options for cardiovascular disease from animal studies into a clinical setting have been hampered, in part by an improper reflection of a relevant patient population in animal models. In this study, we investigated the impact of thymosin β4 (Tβ4), which promotes collateralization and capillarization, during hypercholesterolemia, a known risk factor of coronary artery disease. Initial in vitro results highlighted an improved endothelial cell function upon Tβ4 treatment under control conditions and during hypercholesterolemic stress (scratch area [pixels]: oxidized low-density lipoprotein [oxLDL], 191,924 ± 7,717; and oxLDL + Tβ4, 105,621 ± 11,245). To mimic the common risk factor of hypercholesterolemia in vivo, pigs on regular (NC) or high-fat (HC) diet underwent chronic myocardial ischemia followed by recombinant adeno-associated virus (rAAV)-mediated transduction of Tβ4 or LacZ as a control. We show that Tβ4 overexpression improves capillarization and collateralization (collaterals: NC + rAAV.LacZ, 2.1 ± 0.5; NC + rAAV.Tβ4, 6.7 ± 0.5; HC + rAAV.LacZ, 3.0 ± 0.3; and HC + rAAV.Tβ4, 6.0 ± 0.4), ultimately leading to an improved myocardial function in both diet groups (ejection fraction [EF] at day 56 [%]: NC + rAAV.LacZ, 26 ± 1.1; NC + rAAV.Tβ4, 45 ± 1.5; HC + rAAV.LacZ, 26 ± 2.5; and HC + rAAV.Tβ4, 41 ± 2.6). These results demonstrate the potency of Tβ4 in a patient-relevant large animal model of chronic myocardial ischemia.

Cell–cell and cell–matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell–cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA‐ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis‐inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages.

Recombinant adeno-associated virus vectors (rAAVs) represent a reliable tool for basic and translational research, while rAAVs are also making strides in early clinical trials as vehicles for gene transfer. Their low immunogenicity, tissue tropism, and relative safety due to their low rate of genomic integration represent key features, making rAAVs promising instruments as vectors for future gene therapy approaches. Specifically, for cardiovascular gene therapy, rAAVs appear superior to other vector systems such as lenti- and adenoviral vectors due to the ease of accomplishing long-term cardiac expression of target genes and the reduced risk of provoking immune responses or triggering malignancies through genomic integration. However, major obstacles remain to be resolved if rAAVs are to achieve their full potential as gene therapy vectors in clinical trials. The main hurdles prohibiting their sustained success are their limited capacity to carry transgenes of larger sizes, the prevalence of neutralizing antibodies in the general population, and their tissue specificity, which leaves room for improvement. This review discusses the properties of rAAV that make them useful tools in experimental studies and the treatment of cardiovascular disease in patients.

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need1–3. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative4. Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons5. This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once6. Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation7.

Background: Heme oxygenase-1 (HO-1) is an inducible stress-responsive enzyme converting heme to bilirubin, carbon monoxide, and free iron, which exerts anti-inflammatory and antiapoptotic e...

The actin polymerization regulator Thymosin β4 (Tβ4) has been shown to be involved in angiogenesis, wound healing, cell survival and anti-inflammatory responses. We have previously shown that Tβ4 is capable of recruiting pericytes, thus stabilizing the endothelial barrier function. Here, we analyzed whether treatment with Tβ4 is able to reduce the pericytes loss in lipopolysaccharides (LPS)-induced sepsis and to improve the hemodynamic function and survival in C57BL/6 mice.Fourteen days before LPS injection, the mice were injected with an adeno-associated virus carrying the Tβ4 (rAAV.Tβ4) or LacZ gene (rAAV.LacZ). A sepsis-severity score was assessed, and non-invasive hemodynamic and permeability measurements were performed. Heart and muscle samples were analyzed for PECAM-1(+) capillaries and NG2(+)pericytes.At 36 h, there was a decrease of sepsis severity score in rAAV.Tβ4-treated animals as compared to rAAV.LacZ-treated control. rAAV.Tβ4-treated animals displayed lower perivascular leakage and higher blood pressure compared to control. Of note, the rAAV.Tβ4 group showed a higher pericyte count in heart and peripheral muscle samples. Finally, Tβ4-treatment reduced mortality compared to control.The data indicate a preventive role of Tβ4 in septic hypercirculation and highlight Tβ4 as a potential therapeutic target in severe sepsis.

Purpose of review To discuss the possible therapeutic options for miRNA as targets in coronary heart disease. Noncoding RNAs regulate gene expression at a posttranscriptional state. Modulation of miRNAs might be a new therapeutic option in coronary heart disease. Recent findings Noncoding RNAs (long and short noncoding RNAs) might be used as biomarkers in cardiovascular disease, as they are differentially regulated and released in the pathophysiological situation of coronary heart disease. In acute myocardial infarction already a lot of miRNAs are investigated as biomarkers, still not superior to high-sensitive Troponin T. In rare cardiovascular diseases such as Tako-Tsubo cardiomyopathy or the different stages of heart failure, development of new biomarkers is even more important. In addition, miRNA inhibition via antimiRs is capable of attenuating cardiovascular disease in small and large animal models. Over-expression of 'protective' miRNAs (miR mimics) improved the outcome of cardiovascular disease in vitro and in first small animal models. Summary Noncoding RNAs are promising new biomarkers for cardiovascular disease. Directly targeting miRNA for disease modulation is possible for the specific inhibition, as well as for overexpression of 'protective' miRNAs. However, additional preclinical and clinical testing has to be performed before this therapy will enter clinical routine.