The Dutch National TissueArchive Portal enables efficient, consistent, and transparent procurement of diagnostic tissue samples for scientific use.

Biobanks play a crucial role in enabling biomedical research by facilitating scientific use of valuable human biomaterials. The PALGA foundation-a nationwide network and registry of histo- and cytopathology in the Netherlands-was established to promote the provision of data within and between pathology departments, and to make the resulting knowledge available for healthcare. Apart from the pathology data, we aimed to utilize PALGA's nationwide network to find and access the rich wealth of Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples for scientific use.  We implemented the Dutch National TissueArchive Portal (DNTP) to utilize PALGA's nationwide network for requesting FFPE tissue samples. The DNTP consists of (1) a centrally organized internet portal to improve the assessing, processing, harmonization, and monitoring of the procurement process, while (2) dedicated HUB-employees provide practical support at peripheral pathology departments. Since incorporation of the DNTP, both the number of filed requests for FFPE tissue samples and the amount of HUB-mediated support increased 55 and 29% respectively. In line, the sample procurement duration time decreased significantly (- 47%). These findings indicate that implementation of the DNTP improved the frequency, efficiency, and transparency of FFPE tissue sample procurement for research in the Netherlands. To conclude, the need for biological resources is growing persistently to enable precision medicine. Here, we access PALGA's national, pathology network by implementation of the DNTP to allow for efficient, consistent, and transparent exchange of FFPE tissue samples for research across the Netherlands.

Reversal of hypoxia in murine atherosclerosis prevents necrotic core expansion by enhancing efferocytosis.

OBJECTIVE: Advanced murine and human plaques are hypoxic, but it remains unclear whether plaque hypoxia is causally related to atherogenesis. Here, we test the hypothesis that reversal of hypoxia in atherosclerotic plaques by breathing hyperoxic carbogen gas will prevent atherosclerosis. APPROACH AND RESULTS: Low-density lipoprotein receptor-deficient mice (LDLR(-/-)) were fed a Western-type diet, exposed to carbogen (95% O2, 5% CO2) or air, and the effect on plaque hypoxia, size, and phenotype was studied. First, the hypoxic marker pimonidazole was detected in murine LDLR(-/-) plaque macrophages from plaque initiation onwards. Second, the efficacy of breathing carbogen (90 minutes, single exposure) was studied. Compared with air, carbogen increased arterial blood pO2 5-fold in LDLR(-/-) mice and reduced plaque hypoxia in advanced plaques of the aortic root (-32%) and arch (-84%). Finally, the effect of repeated carbogen exposure on progression of atherosclerosis was studied in LDLR(-/-) mice fed a Western-type diet for an initial 4 weeks, followed by 4 weeks of diet and carbogen or air (both 90 min/d). Carbogen reduced plaque hypoxia (-40%), necrotic core size (-37%), and TUNEL(+) (terminal uridine nick-end labeling positive) apoptotic cell content (-50%) and increased efferocytosis of apoptotic cells by cluster of differentiation 107b(+) (CD107b, MAC3) macrophages (+36%) in advanced plaques of the aortic root. Plaque size, plasma cholesterol, hematopoiesis, and systemic inflammation were unchanged. In vitro, hypoxia hampered efferocytosis by bone marrow-derived macrophages, which was dependent on the receptor Mer tyrosine kinase. CONCLUSIONS: Carbogen restored murine plaque oxygenation and prevented necrotic core expansion by enhancing efferocytosis, likely via Mer tyrosine kinase. Thus, plaque hypoxia is causally related to necrotic core expansion.

Extension of Indication for Authorised Oncology Products in the European Union: A Joint Effort of Multiple Stakeholders.

After marketing authorisation, the development of a medicinal product often continues with studies investigating new therapeutic indications. Positive results can potentially lead to changes to the terms of the marketing authorisation, such as an extension of therapeutic indication(s). These studies can be initiated and sponsored by the marketing authorisation holder (MAH) or by others. When results from an investigator-initiated trial suggest that an authorised medicinal product is safe and effective for a new therapeutic indication, physicians may want to treat their patients with this medicinal product. In such a situation, it is desirable to extend the therapeutic indication(s) via the regulatory approval process, as this can facilitate patient access within the European Union. There may however be challenges when the MAH did not conduct the study and might not have access to the data. In this perspective, we focus on the possibilities to extend the therapeutic indication(s) of an already authorised medicinal product based on results from investigator-initiated trials. We address: (1) the advantages of an extension of indication; (2) the regulatory requirements for a variation application; (3) investigator-initiated trials as a basis for regulatory approval; (4) the role of the MAH in extending the indication. With this article, we want to emphasize the importance of a collaborative approach and dialogue between stakeholders with the aim to facilitate access to effective medicinal products.

Reviewing the Limitations of Adult Mammalian Cardiac Regeneration: Noncoding RNAs as Regulators of Cardiomyogenesis.

The adult mammalian heart is incapable of regeneration following cardiac injury, leading to a decline in function and eventually heart failure. One of the most evident barriers limiting cardiac regeneration is the inability of cardiomyocytes to divide. It has recently become clear that the mammalian heart undergoes limited cardiomyocyte self-renewal throughout life and is even capable of modest regeneration early after birth. These exciting findings have awakened the goal to promote cardiomyogenesis of the human heart to repair cardiac injury or treat heart failure. We are still far from understanding why adult mammalian cardiomyocytes possess only a limited capacity to proliferate. Identifying the key regulators may help to progress towards such revolutionary therapy. Specific noncoding RNAs control cardiomyocyte division, including well explored microRNAs and more recently emerged long noncoding RNAs. Elucidating their function and molecular mechanisms during cardiomyogenesis is a prerequisite to advance towards therapeutic options for cardiac regeneration. In this review, we present an overview of the molecular basis of cardiac regeneration and describe current evidence implicating microRNAs and long noncoding RNAs in this process. Current limitations and future opportunities regarding how these regulatory mechanisms can be harnessed to study myocardial regeneration will be addressed.

tRNAs and tRNA fragments as modulators of cardiac and skeletal muscle function.

Transfer RNAs (tRNAs) and their processing enzymes have long-recognized roles in cardiac and skeletal muscle pathophysiology. Recently, tRNA fragments have emerged as a new class of non-coding RNAs involved in the regulation of cell function. In this review, we provide a synopsis of the molecular processes that regulate the biogenesis, post-transcriptional regulation and functional roles of tRNAs in cardiac and skeletal muscle. In addition, we list the (dys)regulated expression profiles and putative functional roles of tRNA-derived small RNAs in the heart and skeletal muscle. Finally, the technical challenges surrounding tRNA research are discussed alongside suggestions to advance research in this field.

Functional Screening Identifies MicroRNAs as Multi-Cellular Regulators of Heart Failure.

Heart failure (HF) is the leading cause of death in the Western world. Pathophysiological processes underlying HF development, including cardiac hypertrophy, fibrosis and inflammation, are controlled by specific microRNAs (miRNAs). Whereas most studies investigate miRNA function in one particular cardiac cell type, their multicellular function is poorly investigated. The present study probed 194 miRNAs -differentially expressed in cardiac inflammatory disease - for regulating cardiomyocyte size, cardiac fibroblasts collagen content, and macrophage polarization. Of the tested miRNAs, 13%, 26%, and 41% modulated cardiomyocyte size, fibroblast collagen production, and macrophage polarization, respectively. Seventeen miRNAs affected all three cellular processes, including miRNAs with established (miR-210) and unknown roles in cardiac pathophysiology (miR-145-3p). These miRNAs with a multi-cellular function commonly target various genes. In-depth analysis in vitro of previously unstudied miRNAs revealed that the observed phenotypical alterations concurred with changes in transcript and protein levels of hypertrophy-, fibrosis- and inflammation-related genes. MiR-145-3p and miR-891a-3p were identified to regulate the fibrotic response, whereas miR-223-3p, miR-486-3p, and miR-488-5p modulated macrophage activation and polarisation. In conclusion, miRNAs are multi-cellular regulators of different cellular processes underlying cardiac disease. We identified previously undescribed roles of miRNAs in hypertrophy, fibrosis, and inflammation, and attribute new cellular effects to various well-known miRNAs.

AntagomiR-103 and -107 Treatment Affects Cardiac Function and Metabolism.

MicroRNA-103/107 regulate systemic glucose metabolism and insulin sensitivity. For this reason, inhibitory strategies for these microRNAs are currently being tested in clinical trials. Given the high metabolic demands of the heart and the abundant cardiac expression of miR-103/107, we questioned whether antagomiR-mediated inhibition of miR-103/107 in C57BL/6J mice impacts on cardiac function. Notably, fractional shortening decreased after 6 weeks of antagomiR-103 and -107 treatment. This was paralleled by a prolonged systolic radial and circumferential time to peak and by a decreased global strain rate. Histology and electron microscopy showed reduced cardiomyocyte area and decreased mitochondrial volume and mitochondrial cristae density following antagomiR-103 and -107. In line, antagomiR-103 and -107 treatment decreased mitochondrial OXPHOS complexes' protein levels compared to scrambled, as assessed by mass spectrometry-based label-free quantitative proteomics. MiR-103/107 inhibition in primary cardiomyocytes did not affect glycolysis rates, but it decreased mitochondrial reserve capacity, reduced mitochondrial membrane potential, and altered mitochondrial network morphology, as assessed by live-cell imaging. Our data indicate that antagomiR-103 and -107 decrease cardiac function, cardiomyocyte size, and mitochondrial oxidative capacity in the absence of pathological stimuli. These data raise concern about the possible cardiac implications of the systemic use of antagomiR-103 and -107 in the clinical setting, and careful cardiac phenotyping within ongoing trials is highly recommended.

MicroRNA-221/222 Family Counteracts Myocardial Fibrosis in Pressure Overload-Induced Heart Failure.

Pressure overload causes cardiac fibroblast activation and transdifferentiation, leading to increased interstitial fibrosis formation and subsequently myocardial stiffness, diastolic and systolic dysfunction, and eventually heart failure. A better understanding of the molecular mechanisms underlying pressure overload-induced cardiac remodeling and fibrosis will have implications for heart failure treatment strategies. The microRNA (miRNA)-221/222 family, consisting of miR-221-3p and miR-222-3p, is differentially regulated in mouse and human cardiac pathology and inversely associated with kidney and liver fibrosis. We investigated the role of this miRNA family during pressure overload-induced cardiac remodeling. In myocardial biopsies of patients with severe fibrosis and dilated cardiomyopathy or aortic stenosis, we found significantly lower miRNA-221/222 levels as compared to matched patients with nonsevere fibrosis. In addition, miRNA-221/222 levels in aortic stenosis patients correlated negatively with the extent of myocardial fibrosis and with left ventricular stiffness. Inhibition of both miRNAs during AngII (angiotensin II)-mediated pressure overload in mice led to increased fibrosis and aggravated left ventricular dilation and dysfunction. In rat cardiac fibroblasts, inhibition of miRNA-221/222 derepressed TGF-ß (transforming growth factor-ß)-mediated profibrotic SMAD2 (mothers against decapentaplegic homolog 2) signaling and downstream gene expression, whereas overexpression of both miRNAs blunted TGF-ß-induced profibrotic signaling. We found that the miRNA-221/222 family may target several genes involved in TGF-ß signaling, including JNK1 (c-Jun N-terminal kinase 1), TGF-ß receptor 1 and TGF-ß receptor 2, and ETS-1 (ETS proto-oncogene 1). Our findings show that heart failure-associated downregulation of the miRNA-221/222 family enables profibrotic signaling in the pressure-overloaded heart.

Multiple common comorbidities produce left ventricular diastolic dysfunction associated with coronary microvascular dysfunction, oxidative stress, and myocardial stiffening.

Aims: More than 50% of patients with heart failure have preserved ejection fraction characterized by diastolic dysfunction. The prevalance of diastolic dysfunction is higher in females and associates with multiple comorbidities such as hypertension (HT), obesity, hypercholesterolemia (HC), and diabetes mellitus (DM). Although its pathophysiology remains incompletely understood, it has been proposed that these comorbidities induce systemic inflammation, coronary microvascular dysfunction, and oxidative stress, leading to myocardial fibrosis, myocyte stiffening and, ultimately, diastolic dysfunction. Here, we tested this hypothesis in a swine model chronically exposed to three common comorbidities. Methods and results: DM (induced by streptozotocin), HC (produced by high fat diet), and HT (resulting from renal artery embolization), were produced in 10 female swine, which were followed for 6 months. Eight female healthy swine on normal pig-chow served as controls. The DM + HC + HT group showed hyperglycemia, HC, hypertriglyceridemia, renal dysfunction and HT, which were associated with systemic inflammation. Myocardial superoxide production was markedly increased, due to increased NOX activity and eNOS uncoupling, and associated with reduced NO production, and impaired coronary small artery endothelium-dependent vasodilation. These abnormalities were accompanied by increased myocardial collagen content, reduced capillary/fiber ratio, and elevated passive cardiomyocyte stiffness, resulting in an increased left ventricular end-diastolic stiffness (measured by pressure-volume catheter) and a trend towards a reduced E/A ratio (measured by cardiac MRI), while ejection fraction was maintained. Conclusions: The combination of three common comorbidities leads to systemic inflammation, myocardial oxidative stress, and coronary microvascular dysfunction, which associate with myocardial stiffening and LV diastolic dysfunction with preserved ejection fraction.

MiRNA Deregulation in Cardiac Aging and Associated Disorders.

The prevalence of age-related diseases is increasing dramatically, among which cardiac disease represents the leading cause of death. Aging of the heart is characterized by various molecular and cellular hallmarks impairing both cardiomyocytes and noncardiomyocytes, and resulting in functional deteriorations of the cardiac system. The aging process includes desensitization of ß-adrenergic receptor (ßAR)-signaling and decreased calcium handling, altered growth signaling and cardiac hypertrophy, mitochondrial dysfunction and impaired autophagy, increased programmed cell death, low-grade inflammation of noncanonical inflammatory cells, and increased ECM deposition. MiRNAs play a fundamental role in regulating the processes underlying these detrimental changes in the cardiac system, indicating that MiRNAs are crucially involved in aging. Among others, MiR-34, MiR-146a, and members of the MiR-17-92 cluster, are deregulated during senescence and drive cardiac aging processes. It is therefore suggested that MiRNAs form possible therapeutic targets to stabilize the aged failing myocardium.