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Cardiac homeostasis relies on the appropriate provision of nutrients and functional specialization of local endothelial cells. Previously we reported in this journal that the endothelial Eph-ephrin signalling, in particular the ligand EphB4, is required for the maintenance of vascular integrity and correct fatty acid transport uptake in the heart via regulating the caveolar trafficking of the fatty acid receptor CD36. In the mouse, endothelial specific loss-of-function of the receptor EphB4, or its ligand ephrin-B2, induces Dilated Cardiomyopathy (DCM) like defects (Luxán et al., 2019).Here, we have identified new rareEPHB4variants in a cohort of 573 DCM patients. Similar to what we had observed in the EphB4 mutant mice,EPHB4variants carrying patients show an altered expression pattern of CD36 and CAV1 in the myocardium.Our study confirms a crucial role of the Eph-ephrin signalling pathway, and in particular the receptor EPHB4, in the development of DCM in humans.

Endothelial cells can acquire a mesenchymal phenotype through a process called Endothelial-to-Mesenchymal transition (EndMT). This event is found in embryonic development, but also in pathological conditions. Blood vessels lose their ability to maintain vascular homeostasis and ultimately develop atherosclerosis, pulmonary hypertension, or fibrosis. An increase in inflammatory signals causes an upregulation of EndMT transcription factors, mesenchymal markers, and a decrease in endothelial markers. In our study, we show that the induction of EndMT results in an increase in long non-coding RNA AERRIE expression. JMJD2B, a known EndMT regulator, induces AERRIE and subsequently SULF1. Silencing of AERRIE shows a partial regulation of SULF1 but showed no effect on the endothelial and mesenchymal markers. Additionally, the overexpression of AERRIE results in no significant changes in EndMT markers, suggesting that AERRIE is marginally regulating mesenchymal markers and transcription factors. This study identifies AERRIE as a novel factor in EndMT, but its mechanism of action still needs to be elucidated.

Significance Here we show that the histone demethylase JMJD2B is induced in endothelial cells by EndMT provoking stimuli and thereby contributes to the acquirement of a mesenchymal/smooth muscle phenotype. Silencing of JMJD2B inhibited EndMT in vitro and reduced the induction of EndMT after myocardial infarction in vivo. Inhibition of JMJD2B prevents the demethylation of repressive trimethylated histone H3 at lysine 9 (H3K9me3) at promoters of mesenchymal and EndMT-controlling genes, thereby reducing EndMT. Together, our study reports a crucial role for JMJD2B in controlling histone modifications during the transition of endothelial cells toward a mesenchymal phenotype., 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-β2-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-β 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.

Rationale: Long noncoding RNAs (lncRNAs) are emerging as important regulators of diverse biological functions. Their role in pulmonary arterial hypertension (PAH) remains to be explored. Objectives: To elucidate the role of TYKRIL (tyrosine kinase receptor–inducing lncRNA) as a regulator of p53/ PDGFRβ (platelet-derived growth factor receptor β) signaling pathway and to investigate its role in PAH. Methods: Pericytes and pulmonary arterial smooth muscle cells exposed to hypoxia and derived from patients with idiopathic PAH were analyzed with RNA sequencing. TYKRIL knockdown was performed in above-mentioned human primary cells and in precision-cut lung slices derived from patients with PAH. Measurements and Main Results: Using RNA sequencing data, TYKRIL was identified to be consistently upregulated in pericytes and pulmonary arterial smooth muscles cells exposed to hypoxia and derived from patients with idiopathic PAH. TYKRIL knockdown reversed the proproliferative (n = 3) and antiapoptotic (n = 3) phenotype induced under hypoxic and idiopathic PAH conditions. Owing to the poor species conservation of TYKRIL, ex vivo studies were performed in precision-cut lung slices from patients with PAH. Knockdown of TYKRIL in precision-cut lung slices decreased the vascular remodeling (n = 5). The number of proliferating cell nuclear antigen–positive cells in the vessels was decreased and the number of terminal deoxynucleotide transferase–mediated dUTP nick end label–positive cells in the vessels was increased in the LNA (locked nucleic acid)-treated group compared with control. Expression of PDGFRβ, a key player in PAH, was found to strongly correlate with TYKRIL expression in the patient samples (n = 12), and TYKRIL knockdown decreased PDGFRβ expression (n = 3). From the transcription factor–screening array, it was observed that TYKRIL knockdown increased the p53 activity, a known repressor of PDGFRβ. RNA immunoprecipitation using various p53 mutants demonstrated that TYKRIL binds to the N-terminal of p53 (an important region for p300 interaction with p53). The proximity ligation assay revealed that TYKRIL interferes with the p53–p300 interaction (n = 3) and regulates p53 nuclear translocation. Conclusions: TYKRIL plays an important role in PAH by regulating the p53/PDGFRβ axis.

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-β2-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-β 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.

Aims Circulating immune cells have a significant impact on progression and outcome of heart failure. Long non-coding RNAs (lncRNAs) comprise novel epigenetic regulators which control cardiovascular diseases and inflammatory disorders. We aimed to identify lncRNAs regulated in circulating immune cells of the blood of heart failure patients. Methods and results Next-generation sequencing revealed 110 potentially non-coding RNA transcripts differentially expressed in peripheral blood mononuclear cells of heart failure patients with reduced ejection fraction. The up-regulated lncRNA Heat2 was further functionally characterized. Heat2 expression was detected in whole blood, PBMNCs, eosinophil and basophil granulocytes. Heat2 regulates cell division, invasion, transmigration and immune cell adhesion on endothelial cells. Conclusion Heat2 is an immune cell enriched lncRNA that is elevated in the blood of heart failure patients and controls cellular functions.

Objective— Endothelial cells play important roles in tissue homeostasis and vascularization, a function that is impaired by aging. Here, we aim to decipher the role of the microenvironment underlying the impairment of endothelial cell functions by aging. Approach and Results— RNA sequencing of isolated cardiac endothelial cells derived from young and 18-month-old mouse hearts revealed that aging affects the endothelial expression of genes encoding extracellular matrix proteins, specifically the laminin β1 ( Lamb1 ) and laminin β2 ( Lamb2 ) chains. Whereas Lamb1 was upregulated, Lamb2 was decreased in endothelial cells in old mice compared with young controls. A similar change in expression patterns was observed after induction of acute myocardial infarction. Mimicking aging and injury conditions by plating endothelial cells on laminin β1–containing laminin 411 matrix impaired endothelial cell adhesion, migration, and tube formation and augmented endothelial-to-mesenchymal transition and endothelial detachment compared with laminin 421, which contains the laminin β2 chain. Because laminins can signal via integrin receptors, we determined the activation of ITGB1 (integrin β1). Laminin 421 coating induced a higher activation of ITGB1 compared with laminin 411. siRNA-mediated silencing of ITGB1 reduced laminin β2–dependent adhesion, suggesting that laminin β2 more efficiently activates ITGB1. Conclusions— Mimicking age-related modulation of laminin β1 versus β2 chain expression changes the functional properties and phenotype of endothelial cells. The dysregulation of the extracellular matrix during vascular aging may contribute to age-associated impairment of organ function and fibrosis.

Rationale: Vascularization is critical to maintain organ function. Although many molecular pathways were shown to control vessel growth, the genuine process of capillary formation under different conditions is unclear. Objective: Here, we elucidated whether clonal expansion contributes to vessel growth by using Confetti mice for genetic tracing of clonally expanding endothelial cells (ECs). Methods and Results: In postnatal retina angiogenesis, we predominantly observed random distribution of fluorescence labeled ECs indicative of random integration or cell mixing. However, in models of pathophysiological angiogenesis (retinopathy of prematurity), as well as ischemia-induced angiogenesis in limbs and hearts, clonally expanded ECs were significantly more abundant (≤69%). Inhibition of VEGFR2 (vascular endothelial growth factor receptor 2) reduced clonal expansion after ischemia. To determine the mechanism underlying clonal expansion in vivo, we assessed gene expression specifically in clonally expanded ECs selected by laser capture microscopy. Clonally expanded ECs showed an enrichment of genes involved in endothelial-to-mesenchymal transition. Moreover, hypoxia-induced clonal expansion and endothelial-to-mesenchymal transition in ECs in vitro suggesting that hypoxia-enhanced endothelial-to-mesenchymal transition might contribute to vessel growth under ischemia. Conclusions: Our data suggest that neovascularization after ischemia is partially mediated by clonal expansion of ECs. Identification of the pathways that control clonal expansion may provide novel tools to augment therapeutic neovascularization or treat pathological angiogenesis.

Impaired or excessive growth of endothelial cells contributes to several diseases. However, the functional involvement of regulatory long non-coding RNAs in these processes is not well defined. Here, we show that the long non-coding antisense transcript of GATA6 (GATA6-AS) interacts with the epigenetic regulator LOXL2 to regulate endothelial gene expression via changes in histone methylation. Using RNA deep sequencing, we find that GATA6-AS is upregulated in endothelial cells during hypoxia. Silencing of GATA6-AS diminishes TGF-β2-induced endothelial–mesenchymal transition in vitro and promotes formation of blood vessels in mice. We identify LOXL2, known to remove activating H3K4me3 chromatin marks, as a GATA6-AS-associated protein, and reveal a set of angiogenesis-related genes that are inversely regulated by LOXL2 and GATA6-AS silencing. As GATA6-AS silencing reduces H3K4me3 methylation of two of these genes, periostin and cyclooxygenase-2, we conclude that GATA6-AS acts as negative regulator of nuclear LOXL2 function., LncRNAs influence endothelial cell function via a number of mechanisms. Here the authors show that the lncRNA GATA6-AS regulates endothelial gene expression through interaction with the nuclear deaminase LOXL2, with functional consequences on endothelial-mesenchymal transition and angiogenesis.

Objective— Jumonji C (JmjC) domain–containing proteins modify histone and nonhistone proteins thereby controlling cellular functions. However, the role of JmjC proteins in angiogenesis is largely unknown. Here, we characterize the expression of JmjC domain–containing proteins after inducing endothelial differentiation of murine embryonic stem cells and study the function of JmjC domain–only proteins in endothelial cell (EC) functions. Approach and Results— We identified a large number of JmjC domain–containing proteins regulated by endothelial differentiation of murine embryonic stem cells. Among the family of JmjC domain–only proteins, Jmjd8 was significantly upregulated on endothelial differentiation. Knockdown of Jmjd8 in ECs significantly decreased in vitro network formation and sprouting in the spheroid assay. JMJD8 is exclusively detectable in the cytoplasm, excluding a function as a histone-modifying enzyme. Mass spectrometry analysis revealed JMJD8-interacting proteins with known functions in cellular metabolism like pyruvate kinase M2. Accordingly, knockdown of pyruvate kinase M2 in human umbilical vein ECs decreased endothelial sprouting in the spheroid assay. Knockdown of JMJD8 caused a reduction of EC metabolism as measured by Seahorse Bioscience extracellular flux analysis. Conversely, overexpression of JMJD8 enhanced cellular oxygen consumption rate of ECs, reflecting an increased mitochondrial respiration. Conclusions— Jmjd8 is upregulated during endothelial differentiation and regulates endothelial sprouting and metabolism by interacting with pyruvate kinase M2.

Rationale: Pericytes are essential for vessel maturation and endothelial barrier function. Long noncoding RNAs regulate many cellular functions, but their role in pericyte biology remains unexplored. Objective: Here, we investigate the effect of hypoxia-induced endoplasmic reticulum stress regulating long noncoding RNAs (HypERlnc, also known as ENSG00000262454) on pericyte function in vitro and its regulation in human heart failure and idiopathic pulmonary arterial hypertension. Methods and Results: RNA sequencing in human primary pericytes identified hypoxia-regulated long noncoding RNAs, including HypERlnc. Silencing of HypERlnc decreased cell viability and proliferation and resulted in pericyte dedifferentiation, which went along with increased endothelial permeability in cocultures consisting of human primary pericyte and human coronary microvascular endothelial cells. Consistently, Cas9-based transcriptional activation of HypERlnc was associated with increased expression of pericyte marker genes. Moreover, HypERlnc knockdown reduced endothelial-pericyte recruitment in Matrigel assays ( P P P P P Conclusions: Here, we show that HypERlnc regulates human pericyte function and the endoplasmic reticulum stress response. In addition, RNA sequencing analyses in conjunction with reduced expression of HypERlnc in heart failure and correlation with pericyte markers in idiopathic pulmonary arterial hypertension indicate a role of HypERlnc in human cardiopulmonary disease.