The RNA editor ADAR2 promotes immune cell trafficking by enhancing endothelial responses to interleukin-6 during sterile inflammation.

Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.

Age-Dependent RGS5 Loss in Pericytes Induces Cardiac Dysfunction and Fibrosis.

BACKGROUND: Pericytes are capillary-associated mural cells involved in the maintenance and stability of the vascular network. Although aging is one of the main risk factors for cardiovascular disease, the consequences of aging on cardiac pericytes are unknown. METHODS: In this study, we have combined single-nucleus RNA sequencing and histological analysis to determine the effects of aging on cardiac pericytes. Furthermore, we have conducted in vivo and in vitro analysis of RGS5 (regulator of G-protein signaling 5) loss of function and finally have performed pericytes-fibroblasts coculture studies to understand the effect of RGS5 deletion in pericytes on the neighboring fibroblasts. RESULTS: Aging reduced the pericyte area and capillary coverage in the murine heart. Single-nucleus RNA sequencing analysis further revealed that the expression of Rgs5 was reduced in cardiac pericytes from aged mice. In vivo and in vitro studies showed that the deletion of RGS5 impaired cardiac function, induced fibrosis, and morphological changes in pericytes characterized by a profibrotic gene expression signature and the expression of different ECM (extracellular matrix) components and growth factors, for example, TGFB2 and PDGFB. Indeed, culturing fibroblasts with the supernatant of RGS5-deficient pericytes induced their activation as evidenced by the increased expression of αSMA (alpha smooth muscle actin) in a TGFß (transforming growth factor beta)2-dependent mechanism. CONCLUSIONS: Our results have identified RGS5 as a crucial regulator of pericyte function during cardiac aging. The deletion of RGS5 causes cardiac dysfunction and induces myocardial fibrosis, one of the hallmarks of cardiac aging.

Loss of Y Chromosome and Cardiovascular Events in Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease represents one of the strongest risk factors for cardiovascular diseases, and particularly for heart failure. Despite improved pharmaceutical treatments, mortality remains high. Recently, experimental studies demonstrated that mosaic loss of Y chromosome (LOY) associates with cardiac fibrosis in male mice. Since diffuse cardiac fibrosis is the common denominator for progression of all forms of heart failure, we determined the association of LOY on mortality and cardiovascular disease outcomes in patients with chronic kidney disease. METHODS: LOY was quantified in men with stable chronic kidney disease (CARE for HOMe study [XXX], n=279) and dialysis patients (4D study, n=544). The association between LOY and mortality, combined cardiovascular and heart failure-specific end points, and echocardiographic measures was assessed. RESULTS: In CARE for HOMe, the frequency of LOY increased with age. LOY >17% was associated with increased mortality (heart rate, 2.58 [95% CI, 1.33-5.03]) and risk for cardiac decompensation or death (heart rate, 2.30 [95% CI, 1.23-4.27]). Patients with LOY >17% showed a significant decline of ejection fraction and an increase of E/E' within 5 years. Consistently, in the 4D study, LOY >17% was significantly associated with increased mortality (heart rate, 2.76 [95% CI, 1.83-4.16]), higher risk of death due to heart failure and sudden cardiac death (heart rate, 4.11 [95% CI, 2.09-8.08]), but not atherosclerotic events. Patients with LOY >17% showed significantly higher plasma levels of soluble interleukin 1 receptor-like 1, a biomarker for myocardial fibrosis. Mechanistically, intermediate monocytes from patients with LOY >17% showed significantly higher C-C chemokine receptor type 2 expression and higher plasma levels of the C-C chemokine receptor type 2 chemokine (C-C motif) ligand 2, which may have contributed to increased heart failure events. CONCLUSIONS: LOY identifies male patients with chronic kidney disease at high risk for mortality and heart failure events.

Endothelial Heterogeneity in the Response to Autophagy Drives Small Vessel Muscularization in Pulmonary Hypertension.

BACKGROUND: Endothelial cell (EC) apoptosis and proliferation of apoptosis-resistant cells is a hallmark of pulmonary hypertension (PH). Yet, why some ECs die and others proliferate and how this contributes to vascular remodeling is unclear. We hypothesized that this differential response may: (1) relate to different EC subsets, namely pulmonary artery (PAECs) versus microvascular ECs (MVECs); (2) be attributable to autophagic activation in both EC subtypes; and (3) cause replacement of MVECs by PAECs with subsequent distal vessel muscularization. METHODS: EC subset responses to chronic hypoxia were assessed by single-cell RNA sequencing of murine lungs. Proliferative versus apoptotic responses, activation, and role of autophagy were assessed in human and rat PAECs and MVECs, and in precision-cut lung slices of wild-type mice or mice with endothelial deficiency in the autophagy gene Atg7 (Atg7EN-KO). Abundance of PAECs versus MVECs in precapillary microvessels was assessed in lung tissue from patients with PH and animal models on the basis of structural or surface markers. RESULTS: In vitro and in vivo, PAECs proliferated in response to hypoxia, whereas MVECs underwent apoptosis. Single-cell RNA sequencing analyses support these findings in that hypoxia induced an antiapoptotic, proliferative phenotype in arterial ECs, whereas capillary ECs showed a propensity for cell death. These distinct responses were prevented in hypoxic Atg7EN-KO mice or after ATG7 silencing, yet replicated by autophagy stimulation. In lung tissue from mice, rats, or patients with PH, the abundance of PAECs in precapillary arterioles was increased, and that of MVECs reduced relative to controls, indicating replacement of microvascular by macrovascular ECs. EC replacement was prevented by genetic or pharmacological inhibition of autophagy in vivo. Conditioned medium from hypoxic PAECs yet not MVECs promoted pulmonary artery smooth muscle cell proliferation and migration in a platelet-derived growth factor-dependent manner. Autophagy inhibition attenuated PH development and distal vessel muscularization in preclinical models. CONCLUSIONS: Autophagic activation by hypoxia induces in parallel PAEC proliferation and MVEC apoptosis. These differential responses cause a progressive replacement of MVECs by PAECs in precapillary pulmonary arterioles, thus providing a macrovascular context that in turn promotes pulmonary artery smooth muscle cell proliferation and migration, ultimately driving distal vessel muscularization and the development of PH.

Poison cassette exon splicing of SRSF6 regulates nuclear speckle dispersal and the response to hypoxia.

Hypoxia induces massive changes in alternative splicing (AS) to adapt cells to the lack of oxygen. Here, we identify the splicing factor SRSF6 as a key factor in the AS response to hypoxia. The SRSF6 level is strongly reduced in acute hypoxia, which serves a dual purpose: it allows for exon skipping and triggers the dispersal of nuclear speckles. Our data suggest that cells use dispersal of nuclear speckles to reprogram their gene expression during hypoxic adaptation and that SRSF6 plays an important role in cohesion of nuclear speckles. Down-regulation of SRSF6 is achieved through inclusion of a poison cassette exon (PCE) promoted by SRSF4. Removing the PCE 3' splice site using CRISPR/Cas9 abolishes SRSF6 reduction in hypoxia. Aberrantly high SRSF6 levels in hypoxia attenuate hypoxia-mediated AS and impair dispersal of nuclear speckles. As a consequence, proliferation and genomic instability are increased, while the stress response is suppressed. The SRSF4-PCE-SRSF6 hypoxia axis is active in different cancer types, and high SRSF6 expression in hypoxic tumors correlates with a poor prognosis. We propose that the ultra-conserved PCE of SRSF6 acts as a tumor suppressor and that its inclusion in hypoxia is crucial to reduce SRSF6 levels. This may prevent tumor cells from entering the metastatic route of hypoxia adaptation.

CVD-associated SNPs with regulatory potential reveal novel non-coding disease genes.

BACKGROUND: Cardiovascular diseases (CVDs) are the leading cause of death worldwide. Genome-wide association studies (GWAS) have identified many single nucleotide polymorphisms (SNPs) appearing in non-coding genomic regions in CVDs. The SNPs may alter gene expression by modifying transcription factor (TF) binding sites and lead to functional consequences in cardiovascular traits or diseases. To understand the underlying molecular mechanisms, it is crucial to identify which variations are involved and how they affect TF binding. METHODS: The SNEEP (SNP exploration and analysis using epigenomics data) pipeline was used to identify regulatory SNPs, which alter the binding behavior of TFs and link GWAS SNPs to their potential target genes for six CVDs. The human-induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs), monoculture cardiac organoids (MCOs) and self-organized cardiac organoids (SCOs) were used in the study. Gene expression, cardiomyocyte size and cardiac contractility were assessed. RESULTS: By using our integrative computational pipeline, we identified 1905 regulatory SNPs in CVD GWAS data. These were associated with hundreds of genes, half of them non-coding RNAs (ncRNAs), suggesting novel CVD genes. We experimentally tested 40 CVD-associated non-coding RNAs, among them RP11-98F14.11, RPL23AP92, IGBP1P1, and CTD-2383I20.1, which were upregulated in hiPSC-CMs, MCOs and SCOs under hypoxic conditions. Further experiments showed that IGBP1P1 depletion rescued expression of hypertrophic marker genes, reduced hypoxia-induced cardiomyocyte size and improved hypoxia-reduced cardiac contractility in hiPSC-CMs and MCOs. CONCLUSIONS: IGBP1P1 is a novel ncRNA with key regulatory functions in modulating cardiomyocyte size and cardiac function in our disease models. Our data suggest ncRNA IGBP1P1 as a potential therapeutic target to improve cardiac function in CVDs.

Locus-Conserved Circular RNA cZNF292 Controls Endothelial Cell Flow Responses.

BACKGROUND: Circular RNAs (circRNAs) are generated by back splicing of mostly mRNAs and are gaining increasing attention as a novel class of regulatory RNAs that control various cellular functions. However, their physiological roles and functional conservation in vivo are rarely addressed, given the inherent challenges of their genetic inactivation. Here, we aimed to identify locus conserved circRNAs in mice and humans, which can be genetically deleted due to retained intronic elements not contained in the mRNA host gene to eventually address functional conservation. METHODS AND RESULTS: Combining published endothelial RNA-sequencing data sets with circRNAs of the circATLAS databank, we identified locus-conserved circRNA retaining intronic elements between mice and humans. CRISPR/Cas9 mediated genetic depletion of the top expressed circRNA cZfp292 resulted in an altered endothelial morphology and aberrant flow alignment in the aorta in vivo. Consistently, depletion of cZNF292 in endothelial cells in vitro abolished laminar flow-induced alterations in cell orientation, paxillin localization and focal adhesion organization. Mechanistically, we identified the protein SDOS (syndesmos) to specifically interact with cZNF292 in endothelial cells by RNA-affinity purification and subsequent mass spectrometry analysis. Silencing of SDOS or its protein binding partner Syndecan-4, or mutation of the SDOS-cZNF292 binding site, prevented laminar flow-induced cytoskeletal reorganization thereby recapitulating cZfp292 knockout phenotypes. CONCLUSIONS: Together, our data reveal a hitherto unknown role of cZNF292/cZfp292 in endothelial flow responses, which influences endothelial shape.

The splicing-regulatory lncRNA NTRAS sustains vascular integrity.

Vascular integrity is essential for organ homeostasis to prevent edema formation and infiltration of inflammatory cells. Long non-coding RNAs (lncRNAs) are important regulators of gene expression and often expressed in a cell type-specific manner. By screening for endothelial-enriched lncRNAs, we identified the undescribed lncRNA NTRAS to control endothelial cell functions. Silencing of NTRAS induces endothelial cell dysfunction in vitro and increases vascular permeability and lethality in mice. Biochemical analysis revealed that NTRAS, through its CA-dinucleotide repeat motif, sequesters the splicing regulator hnRNPL to control alternative splicing of tight junction protein 1 (TJP1; also named zona occludens 1, ZO-1) pre-mRNA. Deletion of the hnRNPL binding motif in mice (Ntras∆CA/∆CA ) significantly repressed TJP1 exon 20 usage, favoring expression of the TJP1α- isoform, which augments permeability of the endothelial monolayer. Ntras∆CA/∆CA mice further showed reduced retinal vessel growth and increased vascular permeability and myocarditis. In summary, this study demonstrates that NTRAS is an essential gatekeeper of vascular integrity.

[Heart and blood: clonal hematopoiesis]. / Herz und Blut: klonale Hämatopoese.

Cardiovascular diseases are among the leading causes of death worldwide, with well-known modifiable risk factors, such as smoking, overweight, lipid metabolism disorders, lack of physical activity and high blood pressure playing a significant role. Recent studies have now identified "clonal hematopoiesis" as a novel blood-based risk factor. Clonal hematopoiesis arises from mutations in hematopoietic stem cells, which lead to the expansion of mutated blood cells. Mutated cell clones can be detected in over 40% of individuals over 50 years old, with more than 15% of those over 90 years old harboring large clones. Surprisingly, mutated cells predispose to the development of leukemia only to a minor extent, leading to the term clonal hematopoiesis of indeterminate potential (CHIP); however, it has been shown that CHIP is associated with an increased risk of cardiovascular diseases. Individuals with CHIP-associated gene mutations have an elevated risk of atherosclerotic vascular diseases, stroke and thrombosis. Patients with heart failure with reduced ejection fraction (HFrEF), whether of ischemic or non-ischemic origin and patients with heart failure with preserved ejection fraction (HFpEF) exhibit an increased number of mutated cells in the blood. The presence of CHIP mutations is linked to a poorer prognosis in patients with existing cardiovascular diseases. Future research should aim at a better understanding of the specific effects of different mutations, clone sizes and combinations to develop personalized therapeutic approaches. Various anti-inflammatory therapeutic drugs are available, which can be tested in controlled studies.

Mosaic loss of Y chromosome in monocytes is associated with lower survival after transcatheter aortic valve replacement.

AIMS: Mosaic loss of Y chromosome (LOY) in blood cells is the most common acquired mutation, increases with age, and is related to cardiovascular disease. Loss of Y chromosome induces cardiac fibrosis in murine experiments mimicking the consequences of aortic valve stenosis, the prototypical age-related disease. Cardiac fibrosis is the major determinant of mortality even after transcatheter aortic valve replacement (TAVR). It was hypothesized that LOY affects long-term outcome in men undergoing TAVR. METHODS AND RESULTS: Using digital PCR in DNA of peripheral blood cells, LOY (Y/X ratio) was assessed by targeting a 6 bp sequence difference between AMELX and AMELY genes using TaqMan. The genetic signature of monocytes lacking the Y chromosome was deciphered by scRNAseq. In 362 men with advanced aortic valve stenosis undergoing successful TAVR, LOY ranged from -4% to 83.4%, and was >10% in 48% of patients. Three-year mortality increased with LOY. Receiver operating characteristic (ROC) curve analysis revealed an optimal cut-off of LOY >17% to predict mortality. In multivariate analysis, LOY remained a significant (P < 0.001) independent predictor of death during follow-up. scRNAseq disclosed a pro-fibrotic gene signature with LOY monocytes displaying increased expression of transforming growth factor (TGF) ß-associated signaling, while expression of TGFß-inhibiting pathways was down-regulated. CONCLUSION: This is the first study to demonstrate that LOY in blood cells is associated with profoundly impaired long-term survival even after successful TAVR. Mechanistically, the pro-fibrotic gene signature sensitizing the patient-derived circulating LOY monocytes for the TGFß signaling pathways supports a prominent role of cardiac fibrosis in contributing to the effects of LOY observed in men undergoing TAVR.

Cardiomyocyte hyperplasia and immaturity but not hypertrophy are characteristic features of patients with RASopathies.

AIMS: RASopathies are caused by mutations in genes that alter the MAP kinase pathway and are marked by several malformations with cardiovascular disorders as the predominant cause of mortality. Mechanistic insights in the underlying pathogenesis in affected cardiac tissue are rare. The aim of the study was to assess the impact of RASopathy causing mutations on the human heart. METHODS AND RESULTS: Using single cell approaches and histopathology we analyzed cardiac tissue from children with different RASopathy-associated mutations compared to age-matched dilated cardiomyopathy (DCM) and control hearts. The volume of cardiomyocytes was reduced in RASopathy conditions compared to controls and DCM patients, and the estimated number of cardiomyocytes per heart was ∼4-10 times higher. Single nuclei RNA sequencing of a 13-year-old RASopathy patient (carrying a PTPN11 c.1528C > G mutation) revealed that myocardial cell composition and transcriptional patterns were similar to <1 year old DCM hearts. Additionally, immaturity of cardiomyocytes is shown by an increased MYH6/MYH7 expression ratio and reduced expression of genes associated with fatty acid metabolism. In the patient with the PTPN11 mutation activation of the MAP kinase pathway was not evident in cardiomyocytes, whereas increased phosphorylation of PDK1 and its downstream kinase Akt was detected. CONCLUSION: In conclusion, an immature cardiomyocyte differentiation status appears to be preserved in juvenile RASopathy patients. The increased mass of the heart in such patients is due to an increase in cardiomyocyte number (hyperplasia) but not an enlargement of individual cardiomyocytes (hypertrophy).

Pathogenetic mechanisms and therapeutic approaches of acute-to-chronic liver failure.

Liver cirrhosis is the end stage of all chronic liver diseases and contributes significantly to overall mortality of 2% globally. The age-standardized mortality from liver cirrhosis in Europe is between 10 and 20% and can be explained by not only the development of liver cancer but also the acute deterioration in the patient's overall condition. The development of complications including accumulation of fluid in the abdomen (ascites), bleeding in the gastrointestinal tract (variceal bleeding), bacterial infections, or a decrease in brain function (hepatic encephalopathy) define an acute decompensation that requires therapy and often leads to acute-on-chronic liver failure (ACLF) by different precipitating events. However, due to its complexity and organ-spanning nature, the pathogenesis of ACLF is poorly understood, and the common underlying mechanisms leading to the development of organ dysfunction or failure in ACLF are still elusive. Apart from general intensive care interventions, there are no specific therapy options for ACLF. Liver transplantation is often not possible in these patients due to contraindications and a lack of prioritization. In this review, we describe the framework of the ACLF-I project consortium funded by the Hessian Ministry of Higher Education, Research and the Arts (HMWK) based on existing findings and will provide answers to these open questions.

Clonal Hematopoiesis-Driver DNMT3A Mutations Alter Immune Cells in Heart Failure.

RATIONALE: Clonal hematopoiesis driven by mutations of DNMT3A (DNA methyltransferase 3a) is associated with increased incidence of cardiovascular disease and poor prognosis of patients with chronic heart failure (HF) and aortic stenosis. Although experimental studies suggest that DNMT3A clonal hematopoiesis-driver mutations may enhance inflammation, specific signatures of inflammatory cells in humans are missing. OBJECTIVE: To define subsets of immune cells mediating inflammation in humans using single-cell RNA sequencing. METHODS AND RESULTS: Transcriptomic profiles of peripheral blood mononuclear cells were analyzed in n=6 patients with HF harboring DNMT3A clonal hematopoiesis-driver mutations and n=4 patients with HF and no DNMT3A mutations by single-cell RNA sequencing. Monocytes of patients with HF carrying DNMT3A mutations demonstrated a significantly increased expression of inflammatory genes compared with monocytes derived from patients with HF without DNMT3A mutations. Among the specific upregulated genes were the prototypic inflammatory IL (interleukin) IL1B (interleukin 1B), IL6, IL8, the inflammasome NLRP3, and the macrophage inflammatory proteins CCL3 and CCL4 as well as resistin, which augments monocyte-endothelial adhesion. Silencing of DNMT3A in monocytes induced a paracrine proinflammatory activation and increased adhesion to endothelial cells. Furthermore, the classical monocyte subset of DNMT3A mutation carriers showed increased expression of T-cell stimulating immunoglobulin superfamily members CD300LB, CD83, SIGLEC12, as well as the CD2 ligand and cell adhesion molecule CD58, all of which may be involved in monocyte-T-cell interactions. DNMT3A mutation carriers were further characterized by increased expression of the T-cell alpha receptor constant chain and changes in T helper cell 1, T helper cell 2, T helper cell 17, CD8+ effector, CD4+ memory, and regulatory T-cell-specific signatures. CONCLUSIONS: This study demonstrates that circulating monocytes and T cells of patients with HF harboring clonal hematopoiesis-driver mutations in DNMT3A exhibit a highly inflamed transcriptome, which may contribute to the aggravation of chronic HF.

The histone demethylase JMJD2B regulates endothelial-to-mesenchymal transition.

Endothelial cells play an important role in maintenance of the vascular system and the repair after injury. Under proinflammatory conditions, endothelial cells can acquire a mesenchymal phenotype by a process named endothelial-to-mesenchymal transition (EndMT), which affects the functional properties of endothelial cells. Here, we investigated the epigenetic control of EndMT. We show that the histone demethylase JMJD2B is induced by EndMT-promoting, proinflammatory, and hypoxic conditions. Silencing of JMJD2B reduced TGF-ß2-induced expression of mesenchymal genes, prevented the alterations in endothelial morphology and impaired endothelial barrier function. Endothelial-specific deletion of JMJD2B in vivo confirmed a reduction of EndMT after myocardial infarction. EndMT did not affect global H3K9me3 levels but induced a site-specific reduction of repressive H3K9me3 marks at promoters of mesenchymal genes, such as Calponin (CNN1), and genes involved in TGF-ß signaling, such as AKT Serine/Threonine Kinase 3 (AKT3) and Sulfatase 1 (SULF1). Silencing of JMJD2B prevented the EndMT-induced reduction of H3K9me3 marks at these promotors and further repressed these EndMT-related genes. Our study reveals that endothelial identity and function is critically controlled by the histone demethylase JMJD2B, which is induced by EndMT-promoting, proinflammatory, and hypoxic conditions, and supports the acquirement of a mesenchymal phenotype.

Single-cell technologies to decipher cardiovascular diseases.

Cardiovascular disease remains the leading cause of death worldwide. A deeper understanding of the multicellular composition and molecular processes may help to identify novel therapeutic strategies. Single-cell technologies such as single-cell or single-nuclei RNA sequencing provide expression profiles of individual cells and allow for dissection of heterogeneity in tissue during health and disease. This review will summarize (i) how these novel technologies have become critical for delineating mechanistic drivers of cardiovascular disease, particularly, in humans and (ii) how they might serve as diagnostic tools for risk stratification or individualized therapy. The review will further discuss technical pitfalls and provide an overview of publicly available human and mouse data sets that can be used as a resource for research.

Single Nuclei Sequencing Reveals Novel Insights Into the Regulation of Cellular Signatures in Children With Dilated Cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a leading cause of death in children with heart failure. The outcome of pediatric heart failure treatment is inconsistent, and large cohort studies are lacking. Progress may be achieved through personalized therapy that takes age- and disease-related pathophysiology, pathology, and molecular fingerprints into account. We present single nuclei RNA sequencing from pediatric patients with DCM as the next step in identifying cellular signatures. METHODS: We performed single nuclei RNA sequencing with heart tissues from 6 children with DCM with an age of 0.5, 0.75, 5, 6, 12, and 13 years. Unsupervised clustering of 18 211 nuclei led to the identification of 14 distinct clusters with 6 major cell types. RESULTS: The number of nuclei in fibroblast clusters increased with age in patients with DCM, a finding that was confirmed by histological analysis and was consistent with an age-related increase in cardiac fibrosis quantified by cardiac magnetic resonance imaging. Fibroblasts of patients with DCM >6 years of age showed a profoundly altered gene expression pattern with enrichment of genes encoding fibrillary collagens, modulation of proteoglycans, switch in thrombospondin isoforms, and signatures of fibroblast activation. In addition, a population of cardiomyocytes with a high proregenerative profile was identified in infant patients with DCM but was absent in children >6 years of age. This cluster showed high expression of cell cycle activators such as cyclin D family members, increased glycolytic metabolism and antioxidative genes, and alterations in ß-adrenergic signaling genes. CONCLUSIONS: Novel insights into the cellular transcriptomes of hearts from pediatric patients with DCM provide remarkable age-dependent changes in the expression patterns of fibroblast and cardiomyocyte genes with less fibrotic but enriched proregenerative signatures in infants.

Mapping the Endothelial Cell S-Sulfhydrome Highlights the Crucial Role of Integrin Sulfhydration in Vascular Function.

BACKGROUND: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the "S-sulfhydrome" (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. METHODS: Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and ß3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. RESULTS: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell "S-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on ß3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the ß leg. ß3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between ß3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect ß3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. CONCLUSIONS: Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

Why is endothelial resilience key to maintain cardiac health?

Myocardial injury as induced by myocardial infarction results in tissue ischemia, which critically incepts cardiomyocyte death. Endothelial cells play a crucial role in restoring oxygen and nutrient supply to the heart. Latest advances in single-cell multi-omics, together with genetic lineage tracing, reveal a transcriptional and phenotypical adaptation to the injured microenvironment, which includes alterations in metabolic, mesenchymal, hematopoietic and pro-inflammatory signatures. The extent of transition in mesenchymal or hematopoietic cell lineages is still debated, but it is clear that several of the adaptive phenotypical changes are transient and endothelial cells revert back to a naïve cell state after resolution of injury responses. This resilience of endothelial cells to acute stress responses is important for preventing chronic dysfunction. Here, we summarize how endothelial cells adjust to injury and how this dynamic response contributes to repair and regeneration. We will highlight intrinsic and microenvironmental factors that contribute to endothelial cell resilience and may be targetable to maintain a functionally active, healthy microcirculation.

Reduction of A-to-I RNA editing in the failing human heart regulates formation of circular RNAs.

Alterations of RNA editing that affect the secondary structure of RNAs can cause human diseases. We therefore studied RNA editing in failing human hearts. Transcriptome sequencing showed that adenosine-to-inosine (A-to-I) RNA editing was responsible for 80% of the editing events in the myocardium. Failing human hearts were characterized by reduced RNA editing. This was primarily attributable to Alu elements in introns of protein-coding genes. In the failing left ventricle, 166 circRNAs were upregulated and 7 circRNAs were downregulated compared to non-failing controls. Most of the upregulated circRNAs were associated with reduced RNA editing in the host gene. ADAR2, which binds to RNA regions that are edited from A-to-I, was decreased in failing human hearts. In vitro, reduction of ADAR2 increased circRNA levels suggesting a causal effect of reduced ADAR2 levels on increased circRNAs in the failing human heart. To gain mechanistic insight, one of the identified upregulated circRNAs with a high reduction of editing in heart failure, AKAP13, was further characterized. ADAR2 reduced the formation of double-stranded structures in AKAP13 pre-mRNA, thereby reducing the stability of Alu elements and the circularization of the resulting circRNA. Overexpression of circAKAP13 impaired the sarcomere regularity of human induced pluripotent stem cell-derived cardiomyocytes. These data show that ADAR2 mediates A-to-I RNA editing in the human heart. A-to-I RNA editing represses the formation of dsRNA structures of Alu elements favoring canonical linear mRNA splicing and inhibiting the formation of circRNAs. The findings are relevant to diseases with reduced RNA editing and increased circRNA levels and provide insights into the human-specific regulation of circRNA formation.

Noncoding RNAs in Vascular Diseases.

The advent of deep sequencing technologies led to the identification of a considerable amount of noncoding RNA transcripts, which are increasingly recognized for their functions in controlling cardiovascular diseases. MicroRNAs have already been studied for a decade, leading to the identification of several vasculoprotective and detrimental species, which might be considered for therapeutic targeting. Other noncoding RNAs such as circular RNAs, YRNAs, or long noncoding RNAs are currently gaining increasing attention, and first studies provide insights into their functions as mediators or antagonists of vascular diseases in vivo. The present review article will provide an overview of the different types of noncoding RNAs controlling the vasculature and focus on the developing field of long noncoding RNAs.