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The laboratory rat emerges as a useful tool for studying the interaction between the host and its microbiome. To advance principles relevant to the human microbiome, we systematically investigated and defined a multi-tissue full lifespan microbial biogeography for healthy Fischer 344 rats. Microbial community profiling data was extracted and integrated with host transcriptomic data from the Sequencing Quality Control (SEQC) consortium. Unsupervised machine learning, Spearman’s correlation, taxonomic diversity, and abundance analyses were performed to determine and characterize the rat microbial biogeography and the identification of four inter-tissue microbial heterogeneity patterns (P1-P4). The 11 body habitats harbor a greater diversity of microbes than previously suspected. Lactic acid bacteria (LAB) abundances progressively declined in lungs from breastfeed newborn to adolescence/adult and was below detectable levels in elderly rats. LAB’s presence and levels in lungs were further evaluated by PCR in the two validation datasets. The lung, testes, thymus, kidney, adrenal, and muscle niches were found to have age-dependent alterations in microbial abundance. P1 is dominated by lung samples. P2 contains the largest sample size and is enriched for environmental species. Liver and muscle samples were mostly classified into P3. Archaea species were exclusively enriched in P4. The 357 pattern-specific microbial signatures were positively correlated with host genes in cell migration and proliferation (P1), DNA damage repair and synaptic transmissions (P2), as well as DNA transcription and cell cycle in P3. Our study established a link between metabolic properties of LAB with lung microbiota maturation and development. Breastfeeding and environmental exposure influence microbiome composition and host health and longevity. The inferred rat microbial biogeography and pattern-specific microbial signatures would be useful for microbiome therapeutic approaches to human health and good quality of life.Graphical Abstract

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disease that causes arteriovenous vascular malformations (AVMs) in different organs, including the lung. Three genes, ENG (endoglin), ACVRL1 (ALK1) and SMAD4, all members of the TGF-β/BMPR2 signaling pathway, are responsible for over 85% of all HHT cases. However, how these loss-of-function gene mutations lead to AVMs formation and what common downstream signaling they target is unknown. Here, using a combination of siRNA-mediated gene silencing, whole transcriptomic RNA sequencing, bioinformatic analysis, transcriptomic-based drug discovery, endothelial cells functional assays and VEGF signaling analysis, andex vivoprecision cut lung slice (PCLS) cultures approach, we uncovered common downstream transcriptomic gene signatures of HHT-casing genes and identified promising drug for HHT. We found the commonly used BMPR2-signaling downstream target ID1 is not a common downstream target of all the three HHT genes knockdown in human pulmonary microvascular endothelial cells (PMVECs). We identified novel common downstream targets of all the three HHT-causing genes that were enriched for HHT-related biological process and signaling pathways. Among those downstream genes, LYVE1, GPNMB, and MC5R were strong downstream targets that could serve as a better common downstream target than ID1. Furthermore, using the common downstream upregulated genes (HHT disease signature) following HHT gene knockdown, we identified a small molecule drug, Brivanib, that reversed the HHT disease signature, and inhibited VEGF-induced ERK1/2 phosphorylation, proliferation, and angiogenesis in PMVECs and inhibited some of the upregulated HHT disease genes in PCLS. Our findings suggest that Brivanib could be an emerging new drug for HHT.

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its embryonic transparency, genetic tractability, and amenity to rapid, high-throughput studies. However, the loss of transparency limits heart function analysis at the adult stage, which complicates modeling of age-related heart conditions. To overcome such limitations, high-frequency ultrasound echocardiography in zebrafish is emerging as a viable option. Here, we present a detailed protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using high-frequency ultrasound. The method allows visualization and analysis of zebrafish heart dimension and quantification of important functional parameters, including heart rate, stroke volume, cardiac output, and ejection fraction. In this method, the fish are anesthetized and kept underwater and can be recovered after the procedure. Although high-frequency ultrasound is an expensive technology, the same imaging platform can be used for different species (e.g., murine and zebrafish) by adapting different transducers. Zebrafish echocardiography is a robust method for cardiac phenotyping, useful in the validation and characterization of disease models, particularly late-onset diseases; drug screens; and studies of heart injury, recovery, and regenerative capacity.

Supplemental Digital Content is available in the text., Background: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. Methods: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II–mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. Results: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. Conclusions: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.

Arteriovenous malformations are a vascular anomaly typically present at birth, characterized by an abnormal connection between an artery and a vein (bypassing the capillaries). These high flow lesions can vary in size and location. Therapeutic approaches are limited, and AVMs can cause significant morbidity and mortality. Here, we describe our current understanding of the pathogenesis of arteriovenous malformations based on preclinical and clinical findings. We discuss past and present accomplishments and challenges in the field and identify research gaps that need to be filled for the successful development of therapeutic strategies in the future.

Amyloid light chain (AL) amyloidosis results from tissue deposition of clonal light chains, most commonly produced by clonal plasma cells. AL amyloidosis is closely associated with multiple myeloma (MM), another disease which arises from clonal plasma cell proliferation. Here we aim to identify gene signatures to distinguish AL cardiomyopathy (AL-CM) risk in MM patients. We utilized publicly available data sets and applied Graph Cluster Perturbation approach to cluster the co-expression networks based on pathways in MM and AL-CM utilizing 225 samples from four datasets: GSE42955 study (12 dilated CM, 12 ischemic CM and 5 control LV human heart tissues), GSE95077 study (16 amiloride drug treated/untreated myeloma cell line samples), GSE24128 study (16 newly diagnosed AL with monoclonal plasma cell samples over- or under-expressing cyclin D1), and GSE6477 study (76 primary bone marrow samples from hyper-diploid myeloma patients and 80 non-hyperdiploid MM patients). From these data sets, the networks for extracellular matrix organization, immune system, innate immune system, metabolism, and neutrophil degranulation pathways for MM and amyloid CM were extracted. Summary: Ranking of the perturbed genes based on pathways similarity index in MM and AL resulted in a panel of genes: CD44, NRAS, GRAP2, CTLA4, GSN, CCND1, NFKB1, and IRF1 (p= Figure , the high hazard ratio of this gene cluster in MM with AL-CM (3.76; p=0.021) compared to MM without CM (0.96; p=0.032) suggests the potential of this gene panel to distinguish high or low risk groups in MM based on survival.

Background: Neonatal mammalian hearts have the unique characteristic to fully repair and regenerate following injury. This discovery has led to visionary endeavors to understand and subsequently reawaken regeneration processes in the human adult heart that are lost after birth, contributing to heart failure and death after ischemic insults. In utero exposure to tobacco smoke has detrimental effects on fetal development and growth. However, the consequences of prenatal exposure to cigarette smoke on murine neonate’s cardiac regeneration have never been explored. Methods: To study the impact of cigarette smoke during the entire pregnancy on cardiac regeneration in the offspring, plugged female wild type C57LB/6J mice were exposed either to cigarette smoke (n=6) or filtered air (control, n=6). Two days after birth, 5 pups from each litter were assigned to the two experimental groups: Myocardial infarction (MI) and sham. Detrimental effects of in utero exposure to tobacco smoke on the recovery of cardiac function following MI surgery were followed using two-dimensional speckle tracking echocardiography and strain imaging. To investigate the underlying mechanism, cardiac tissue of the infarct and remote zone was collected for non-coding RNA analysis. Results: In utero exposure to cigarette smoke significantly compromised cardiac regeneration in neonates. At both early and late time points in the phase of cardiac repair, hearts of neonates exposed to cigarette smoke during pregnancy showed a marked impairment of cardiac regenerative potential. In particular, ejection fraction, fractional area change and shortening, as well as left ventricular internal diameter during systole remained pathologically changed following MI insult in the smoked group until the very endpoint. Conclusions: Collectively, we here provide evidence that in utero exposure to cigarette smoke strongly compromises cardiac regeneration in the newborn offspring. This result reinforces smoking cessation during pregnancy. Additionally, understanding the changes in non-coding RNA expression in a setting of preserved versus disrupted repair of the heart might be an important first step towards the identification of key cellular processes in cardiac regeneration after injury.

Long non-coding RNAs (lncRNAs) have potential as novel therapeutic targets in cardiovascular diseases, but detailed information about the intercellular lncRNA shuttling mechanisms in the heart is lacking. Here, we report an important novel crosstalk between cardiomyocytes and fibroblasts mediated by the transfer of lncRNA-enriched extracellular vesicles (EVs) in the context of cardiac ischemia. lncRNA profiling identified two hypoxia-sensitive lncRNAs: ENSMUST00000122745 was predominantly found in small EVs, whereas lncRNA Neat1 was enriched in large EVs in vitro and in vivo. Vesicles were taken up by fibroblasts, triggering expression of profibrotic genes. In addition, lncRNA Neat1 was transcriptionally regulated by P53 under basal conditions and by HIF2A during hypoxia. The function of Neat1 was further elucidated in vitro and in vivo. Silencing of Neat1 in vitro revealed that Neat1 was indispensable for fibroblast and cardiomyocyte survival and affected fibroblast functions (reduced migration capacity, stalled cell cycle, and decreased expression of fibrotic genes). Of translational importance, genetic loss of Neat1 in vivo resulted in an impaired heart function after myocardial infarction highlighting its translational relevance., Graphical Abstract

Telomere length is a marker of biological aging. Short leukocyte telomere length has been associated with various conditions including cardiovascular disorders. Here, we evaluated if patients with hypertrophic cardiomyopathy have altered leukocyte telomere length and whether this is associated with disease severity. A quantitative polymerase chain reaction-based method was used to measure peripheral blood leukocyte telomere length in 59 healthy control subjects and a well-characterized cohort of 88 patients diagnosed with hypertrophic cardiomyopathy: 32 patients with non-obstructive cardiomyopathy (HNCM) and 56 patients with obstructive cardiomyopathy (HOCM). We observed shorter leukocyte telomeres in both HNCM and HOCM patients compared to healthy controls. Furthermore, leukocyte telomere length was inversely associated with HCM even after adjusting for age and sex. Telomere length of HOCM patients was also inversely correlated with left ventricular outflow tract obstruction. Therefore, HOCM patients were categorized by tertiles of telomere length. Patients in the first tertile (shortest telomeres) had a significantly increased left ventricular posterior wall thickness at end-diastole and higher left ventricular outflow tract gradients, whereas the left ventricular end-diastolic diameter was lower compared with patients in the second and third tertile. In summary, telomere length is associated with the severity of the disease in the HOCM subtype., This work was supported by Deutsche Forschungsgemeinschaft (grant number BA5631/2-1 to CB), the Hannover Medical School REBIRTH Excellence cluster (to TT and JB) and the KFO311 (to TT and JB). DdG-C was recipient of a Juan de la Cierva-Incorporación grant from the Ministerio de Economía y Competitividad (IJCI-2016-29393). CIBER Cardiovascular (CB16/11/00403 to DdG-C) is a project of the Instituto de Salud Carlos III.

Introduction Amyloidosis is caused by dysregulation of protein folding resulting in systemic or organ specific amyloid aggregation. When affecting the heart, amyloidosis can cause severe heart failure, which is associated with a high morbidity and mortality. Different subtypes of cardiac amyloidosis exist e.g. transthyretin cardiac amyloidosis and senile cardiac amyloidosis. Today, diagnostics is primarily based on cardiac biopsies and no clinically used circulating blood-based biomarkers existing. Therefore, our aim was to identify circulating microRNAs in patients with different forms of amyloidosis. Methods Blood was collected from healthy subjects (n = 10), patients with reduced ejection fraction (EF < 35%; n = 10), patients affected by transthyretin cardiac amyloidosis (n = 13) as well as senile cardiac amyloidosis (n = 11). After performing TaqMan array profiling, promising candidates, in particular miR-99a-5p, miR-122-5p, miR-27a-3p, miR-221-3p, miR-1180-3p, miR-155-5p, miR-339-3p, miR-574-3p, miR-342-3p and miR-329-3p were validated via quantitative real time PCR. Results The validation experiments revealed a significant upregulation of miR-339-3p in patients affected with senile cardiac amyloidosis compared to controls. This corresponded to the array profiling results. In contrast, there was no deregulation in the other patient groups. Conclusion MiR-339-3p was increased in blood of patients with senile cardiac amyloidosis. Therefore, miR-339-3p is a potential candidate as biomarker for senile cardiac amyloidosis in future studies. Larger patient cohorts should be investigated.

The mammalian cell cycle is a complex and tightly controlled event. Myriads of different control mechanisms are involved in its regulation. Long non-coding RNAs (lncRNA) have emerged as important regulators of many cellular processes including cellular proliferation. However, a more global and unbiased approach to identify lncRNAs with importance for cell proliferation is missing. Here, we present a lentiviral shRNA library-based approach for functional lncRNA profiling. We validated our library approach in NIH3T3 (3T3) fibroblasts by identifying lncRNAs critically involved in cell proliferation. Using stringent selection criteria we identified lncRNA NR_015491.1 out of 3842 different RNA targets represented in our library. We termed this transcript Ntep (non-coding transcript essential for proliferation), as a bona fide lncRNA essential for cell cycle progression. Inhibition of Ntep in 3T3 and primary fibroblasts prevented normal cell growth and expression of key fibroblast markers. Mechanistically, we discovered that Ntep is important to activate P53 concomitant with increased apoptosis and cell cycle blockade in late G2/M. Our findings suggest Ntep to serve as an important regulator of fibroblast proliferation and function. In summary, our study demonstrates the applicability of an innovative shRNA library approach to identify long non-coding RNA functions in a massive parallel approach.

Heart failure is a major contributor to the healthcare burden and mortality worldwide. Current treatment strategies are able to slow down the transition of healthy heart into the failing one; nevertheless better understanding of the complex genetic regulation of maladaptive remodeling in the failing heart is essential for new drug discovery. Noncoding RNAs are key epigenetic regulators of cardiac gene expression and thus significantly influence cardiac homeostasis and functions.In this chapter we will discuss characteristics of noncoding RNAs, especially miRNAs, long noncoding RNAs, and circular RNAs, and review recent evidences proving their profound involvement during different stages of heart failure progression. Several open questions still prevent the extensive use of noncoding RNA-modulating therapies in clinics; yet they are becoming an attractive target to define novel regulatory mechanisms in the heart. In-depth study of their interaction with gene networks will refine our current view of heart failure and revolutionize the drug development in coming years.

Heart failure is a major contributor to the healthcare burden and mortality worldwide. Current treatment strategies are able to slow down the transition of healthy heart into the failing one; nevertheless better understanding of the complex genetic regulation of maladaptive remodeling in the failing heart is essential for new drug discovery. Noncoding RNAs are key epigenetic regulators of cardiac gene expression and thus significantly influence cardiac homeostasis and functions.

Background: The transcription factor GATA2 orchestrates the expression of many endothelial-specific genes, illustrating its crucial importance for endothelial cell function. The capacity of this transcription factor in orchestrating endothelial-important microRNAs (miRNAs/miR) is unknown. Methods: Endothelial GATA2 was functionally analyzed in human endothelial cells in vitro. Endogenous short interfering RNA–mediated knockdown and lentiviral-based overexpression were applied to decipher the capacity of GATA2 in regulating cell viability and capillary formation. Next, the GATA2-dependent miR transcriptome was identified by using a profiling approach on the basis of quantitative real-time polymerase chain reaction. Transcriptional control of miR promoters was assessed via chromatin immunoprecipitation, luciferase promoter assays, and bisulfite sequencing analysis of sites in proximity. Selected miRs were modulated in combination with GATA2 to identify signaling pathways at the angiogenic cytokine level via proteome profiler and enzyme-linked immunosorbent assays. Downstream miR targets were identified via bioinformatic target prediction and luciferase reporter gene assays. In vitro findings were translated to a mouse model of carotid injury in an endothelial GATA2 knockout background. Nanoparticle-mediated delivery of proangiogenic miR-126 was tested in the reendothelialization model. Results: GATA2 gain- and loss-of-function experiments in human umbilical vein endothelial cells identified a key role of GATA2 as master regulator of multiple endothelial functions via miRNA-dependent mechanisms. Global miRNAnome-screening identified several GATA2-regulated miRNAs including miR-126 and miR-221. Specifically, proangiogenic miR-126 was regulated by GATA2 transcriptionally and targeted antiangiogenic SPRED1 and FOXO3a contributing to GATA2-mediated formation of normal vascular structures, whereas GATA2 deficiency led to vascular abnormalities. In contrast to GATA2 deficiency, supplementation with miR-126 normalized vascular function and expression profiles of cytokines contributing to proangiogenic paracrine effects. GATA2 silencing resulted in endothelial DNA hypomethylation leading to induced expression of antiangiogenic miR-221 by GATA2-dependent demethylation of a putative CpG island in the miR-221 promoter. Mechanistically, a reverted GATA2 phenotype by endogenous suppression of miR-221 was mediated through direct proangiogenic miR-221 target genes ICAM1 and ETS1. In a mouse model of carotid injury, GATA2 was reduced, and systemic supplementation of miR-126–coupled nanoparticles enhanced miR-126 availability in the carotid artery and improved reendothelialization of injured carotid arteries in vivo. Conclusions: GATA2-mediated regulation of miR-126 and miR-221 has an important impact on endothelial biology. Hence, modulation of GATA2 and its targets miR-126 and miR-221 is a promising therapeutic strategy for treatment of many vascular diseases.

Background: Myocardial fibrosis is a hallmark of the failing heart, contributing to the most common causes of deaths worldwide. Several microRNAs (miRNAs, miRs) controlling cardiac fibrosis were identified in recent years; however, a more global approach to identify miRNAs involved in fibrosis is missing. Methods and results: Functional miRNA mimic library screens were applied in human cardiac fibroblasts (HCFs) to identify annotated miRNAs inducing proliferation. In parallel, miRNA deep sequencing was performed after subjecting HCFs to proliferating and resting stimuli, additionally enabling discovery of novel miRNAs. In-depth in vitro analysis confirmed the pro-fibrotic nature of selected, highly conserved miRNAs miR-20a-5p and miR-132-3p. To determine downstream cellular pathways and their role in the fibrotic response, targets of the annotated miRNA candidates were modulated by synthetic siRNA. We here provide evidence that repression of autophagy and detoxification of reactive oxygen species by miR-20a-5p and miR-132-3p explain some of their pro-fibrotic nature on a mechanistic level. Conclusion: We here identified both miR-20a-5p and miR-132-3p as crucial regulators of fibrotic pathways in an in vitro model of human cardiac fibroblast biology. European Research Council