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Background: KCNQ1-T587M is a trafficking-deficient long QT syndrome (LQTS) missense mutation. Affected patients exhibit severe clinical phenotypes that are not explained by the mutant's effects on I(Ks). Previous work showed a KCNH2 and KCNQ1 alpha-subunit interaction that increases KCNH2 membrane localization and function.Objective: We hypothesized that failure of trafficking-deficient KCNQ1-T587M to enhance KCNH2 membrane expression could reduce KCNH2 current versus wild-type KCNQ1 (KCNQ1-WT), contributing to the LQTS phenotype of KCNQ1-T587M carriers.Methods: Patch-clamp, protein biochemical studies, confocal imaging, and in vivo transfection of guinea pig cardiomyocytes were performed.Results: KCNQ1-T587M failed to generate functional current when coexpressed with KCNE1 and caused haploinsufficiency when coexpressed with KCNQ1-WT/KCNE1. Coexpression of KCNQ1-WT with KCNH2 increased I(KCNH2) versus KCNH2 alone (P <.05). Immunoblots and confocal microscopy indicated increased plasma membrane localization of KCNH2 alpha-subunits in cells cotransfected with KCNQ1-WT plasmid, while total KCNH2 protein synthesis and KCNH2 glycosylation remained unaffected, which suggests a chaperone effect of KCNQ1-WT to enhance the membrane localization of KCNH2. KCNH2 also coimmunoprecipitated with KCNQ1-WT. Although KCNQ1-T587M coprecipitated with KCNH2, the mutant was retained intracellularly and failed to increase KCNH2 membrane localization, abolishing the KCNQ1-WT chaperone function and reducing I(KCNH2) upon coexpression substantially compared with coexpression with KCNQ1-WT (P <.05). In vivo transfection of KCNQ1-T587M in guinea pigs suppressed I(Kr) in isolated cardiomyocytes.Conclusion: The trafficking-deficient LQTS mutation KCNQ1-T587M fails to show the chaperoning function that enhances KCNH2 membrane localization with KCNQ1-WT. This novel mechanism results in reduced I(KCNH2), which would be expected to decrease repolarization reserve and synergize with reduced I(KCNQ1) caused directly by the mutation, potentially explaining the malignant clinical phenotype in affected patients. [ABSTRACT FROM AUTHOR]

RNA splicing enables the functional adaptation of cells to changing contexts. Impaired splicing has been associated with diseases, including retinitis pigmentosa, but the underlying molecular mechanisms and cellular responses remain poorly understood. In this work, we report that deficiency of ubiquitin-specific protease 39 (USP39) in human cell lines, zebrafish larvae, and mice led to impaired spliceosome assembly and a cytotoxic splicing profile characterized by the use of cryptic 5' splice sites. Disruptive cryptic variants evaded messenger RNA (mRNA) surveillance pathways and were translated into misfolded proteins, which caused proteotoxic aggregates, endoplasmic reticulum (ER) stress, and, ultimately, cell death. The detrimental consequence of splicing-induced proteotoxicity could be mitigated by up-regulating the ubiquitin-proteasome system and selective autophagy. Our findings provide insight into the molecular pathogenesis of spliceosome-associated diseases.

Human induced pluripotent stem cells (hiPSCs) are an invaluable tool to study molecular mechanisms on a human background. Culturing stem cells at an oxygen level different from their microenvironmental niche impacts their viability. To understand this mechanistically, dermal skin fibroblasts of 52 probands were reprogrammed into hiPSCs, followed by either hyperoxic (20 % O 2 ) or physioxic (5 % O 2 ) culture and proteomic profiling. Analysis of chromosomal stability by Giemsa-banding revealed that physioxic -cultured hiPSC clones exhibited less pathological karyotypes than hyperoxic (e.g. 6 % vs. 32 % mosaicism), higher pluripotency as evidenced by higher Stage-Specific Embryonic Antigen 3 positivity, higher glucose consumption and lactate production. Global proteomic analysis demonstrated lower abundance of several subunits of NADH:ubiquinone oxidoreductase (complex I) and an underrepresentation of pathways linked to oxidative phosphorylation and cellular senescence. Accordingly, release of the pro-senescent factor IGFBP3 and β-galactosidase staining were lower in physioxic hiPSCs. RNA- and ATAC-seq profiling revealed a distinct hypoxic transcription factor-binding footprint, amongst others higher expression of the HIF1α-regulated target NDUFA4L2 along with increased chromatin accessibility of the NDUFA4L2 gene locus. While mitochondrial DNA content did not differ between groups, physioxic hiPSCs revealed lower polarized mitochondrial membrane potential, altered mitochondrial network appearance and reduced basal respiration and electron transfer capacity. Blue-native polyacrylamide gel electrophoresis coupled to mass spectrometry of the mitochondrial complexes detected higher abundance of NDUFA4L2 and ATP5IF1 and loss of incorporation into complex IV or V, respectively. Taken together, physioxic culture of hiPSCs improved chromosomal stability, which was associated with downregulation of oxidative phosphorylation and senescence and extensive re-wiring of mitochondrial complex composition., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: TE is a shareholder and a member of the scientific advisory board of DiNAQOR, which is not relating to this manuscript. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Monocytes, the circulating macrophage precursors, contribute to diseases like atherosclerosis and asthma. Long non-coding RNAs (lncRNAs) have been shown to modulate the phenotype and inflammatory capacity of monocytes. We previously discovered the lncRNA SMANTIS, which contributes to cellular phenotype expression by controlling BRG1 in mesenchymal cells. Here, we report that SMANTIS is particularly highly expressed in monocytes and lost during differentiation into macrophages. Moreover, different types of myeloid leukemia presented specific SMANTIS expression patterns. Interaction studies revealed that SMANTIS binds RUNX1, a transcription factor frequently mutated in AML, primarily through its Alu-element on the RUNT domain. RNA-seq after CRISPR/Cas9-mediated deletion of SMANTIS or RUNX1 revealed an association with cell adhesion and both limited the monocyte adhesion to endothelial cells. Mechanistically, SMANTIS KO reduced RUNX1 genomic binding and altered the interaction of RUNX1 with EP300 and CBFB. Collectively, SMANTIS interacts with RUNX1 and attenuates monocyte adhesion, which might limit monocyte vascular egress., (© 2024. The Author(s).)

The functional role of long noncoding RNAs in the endothelium is highly diverse. Among their many functions, regulation of transcription factor activity and abundance is one of the most relevant. This review summarizes the recent progress in the research on the lncRNA-transcription factor axes and their implications for the vascular endothelium under physiological and pathological conditions. The focus is on transcription factors critical for the endothelial response to external stressors, such as hypoxia, inflammation, and shear stress, and their lncRNA interactors. These regulatory interactions will be exemplified by a selected number of lncRNAs that have been identified in the endothelium under physiological and pathological conditions that are influencing the activity or protein stability of important transcription factors. Thus, lncRNAs can add a layer of cell type-specific function to transcription factors. Understanding the interaction of lncRNAs with transcription factors will contribute to elucidating cardiovascular disease pathologies and the development of novel therapeutic approaches.

Interactions of RNA with DNA are principles of gene expression control that have recently gained considerable attention. Among RNA-DNA interactions are R-loops and RNA-DNA hybrid G-quadruplexes, as well as RNA-DNA triplexes. It is proposed that RNA-DNA triplexes guide RNA-associated regulatory proteins to specific genomic locations, influencing transcription and epigenetic decision making. Although triplex formation initially was considered solely an in vitro event, recent progress in computational, biochemical, and biophysical methods support in vivo functionality with relevance for gene expression control. Here, we review the central methodology and biology of triplexes, outline paradigms required for triplex function, and provide examples of physiologically important triplex-forming long non-coding RNAs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Long non-coding RNAs (lncRNAs) are important regulators of gene expression and can associate with DNA as RNA : DNA heteroduplexes or RNA ⋅ DNA : DNA triple helix structures. Here, we review in vitro biochemical and biophysical experiments including electromobility shift assays (EMSA), circular dichroism (CD) spectroscopy, thermal melting analysis, microscale thermophoresis (MST), single-molecule Förster resonance energy transfer (smFRET) and nuclear magnetic resonance (NMR) spectroscopy to investigate RNA ⋅ DNA : DNA triple helix and RNA : DNA heteroduplex formation. We present the investigations of the antiparallel triplex-forming lncRNA MEG3 targeting the gene TGFB2 and the parallel triplex-forming lncRNA Fendrr with its target gene Emp2. The thermodynamic properties of these oligonucleotides lead to concentration-dependent heterogeneous mixtures, where a DNA duplex, an RNA : DNA heteroduplex and an RNA ⋅ DNA : DNA triplex coexist and their relative populations are modulated in a temperature-dependent manner. The in vitro data provide a reliable readout of triplex structures, as RNA ⋅ DNA : DNA triplexes show distinct features compared to DNA duplexes and RNA : DNA heteroduplexes. Our experimental results can be used to validate computationally predicted triple helix formation between novel disease-relevant lncRNAs and their DNA target genes., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Corepressors negatively regulate gene expression by chromatin compaction. Targeted regulation of gene expression could provide a means to control endothelial cell phenotype. We hypothesize that by targeting corepressor proteins, endothelial angiogenic function can be improved. To study this, the expression and function of nuclear corepressors in human umbilical vein endothelial cells (HUVEC) and in murine organ culture was studied. RNA-seq revealed that nuclear receptor corepressor 1 (NCoR1), silencing mediator of retinoid and thyroid hormone receptors (SMRT) and repressor element-1 silencing transcription factor (REST) are the highest expressed corepressors in HUVECs. Knockout and knockdown strategies demonstrated that the depletion of NCoR1 increased the angiogenic capacity of endothelial cells, whereas depletion of SMRT or REST did not. Interestingly, the effect was VEGF signaling independent. NCoR1 depletion significantly upregulated angiogenesis-associated genes, especially tip cell genes, including ESM1, DLL4 and NOTCH4, as observed by RNA- and ATAC-seq. Confrontation assays comparing cells with and without NCoR1-deficiency revealed that loss of NCoR1 promotes a tip-cell position during spheroid sprouting. Moreover, a proximity ligation assay identified NCoR1 as a direct binding partner of the Notch-signaling-related transcription factor RBPJk. Luciferase assays showed that siRNA-mediated knockdown of NCOR1 promotes RBPJk activity. Furthermore, NCoR1 depletion prompts upregulation of several elements in the Notch signaling cascade. Downregulation of NOTCH4, but not NOTCH1, prevented the positive effect of NCOR1 knockdown on spheroid outgrowth. Collectively, these data indicate that decreasing NCOR1 expression is an attractive approach to promote angiogenic function., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: The development of cognitive dysfunction is not necessarily associated with diet-induced obesity. We hypothesized that cognitive dysfunction might require additional vascular damage, for example, in atherosclerotic mice., Methods: We induced atherosclerosis in male C57BL/6N mice by injecting AAV-PCSK9 DY (2x10 11 VG) and feeding them a cholesterol-rich Western diet. After 3 months, mice were examined for cognition using Barnes maze procedure and for cerebral blood flow. Cerebral vascular morphology was examined by immunehistology., Results: In AAV-PCSK9 DY -treated mice, plaque burden, plasma cholesterol, and triglycerides are elevated. RNAseq analyses followed by KEGG annotation show increased expression of genes linked to inflammatory processes in the aortas of these mice. In AAV-PCSK9 DY -treated mice learning was delayed and long-term memory impaired. Blood flow was reduced in the cingulate cortex (-17%), caudate putamen (-15%), and hippocampus (-10%). Immunohistological studies also show an increased incidence of string vessels and pericytes (CD31/Col IV staining) in the hippocampus accompanied by patchy blood-brain barrier leaks (IgG staining) and increased macrophage infiltrations (CD68 staining)., Discussion: We conclude that the hyperlipidemic PCSK9 DY mouse model can serve as an appropriate approach to induce microvascular dysfunction that leads to reduced blood flow in the hippocampus, which could explain the cognitive dysfunction in these mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hernandez Torres, Rezende, Peschke, Will, Hövener, Spiecker, Özorhan, Lampe, Stölting, Aherrahrou, Künne, Kusche-Vihrog, Matschl, Hille, Brandes, Schwaninger, Müller and Raasch.)

Background: Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury., Methods: Native endothelial cells were isolated from young (20±3.4 years) and aged (80±2.3 years) individuals and subjected to molecular analyses to assess global transcriptional and metabolic changes. In vitro studies were conducted using primary human and murine endothelial cells. A murine aortic re-endothelialization model was used to examine endothelial cell regenerative capacity in vivo., Results: RNA sequencing of native endothelial cells revealed that aging resulted in p53-mediated reprogramming to express senescence-associated genes and suppress glycolysis. Reduced glucose uptake and ATP contributed to attenuated assembly of the telomerase complex, which was required for endothelial cell proliferation. Enhanced p53 activity in aging was linked to its acetylation on K120 due to enhanced activity of the acetyltransferase MOZ (monocytic leukemic zinc finger). Mechanistically, p53 acetylation and translocation were, at least partially, attributed to the loss of the vasoprotective enzyme, CSE (cystathionine γ-lyase). CSE physically anchored p53 in the cytosol to prevent its nuclear translocation and CSE absence inhibited AKT (Protein kinase B)-mediated MOZ phosphorylation, which in turn increased MOZ activity and subsequently p53 acetylation. In mice, the endothelial cell-specific deletion of CSE activated p53, induced premature endothelial senescence, and arrested vascular repair after injury. In contrast, the adeno-associated virus 9-mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in native endothelial cells from aged individuals maintained low p53 activity and reactivated telomerase to revert endothelial cell senescence., Conclusions: Aging-associated impairment of vascular repair is partly determined by the vasoprotective enzyme CSE., Competing Interests: Disclosures None.

Background: Pain in early life may impact on development and risk of chronic pain. We developed an optogenetic Cre/loxP mouse model of "early-life-pain" (ELP) using mice with transgenic expression of channelrhodopsin-2 (ChR2) under control of the Advillin (Avil) promoter, which drives expression of transgenes predominantly in isolectin B4 positive non-peptidergic nociceptors in postnatal mice. Avil-ChR2 (Cre +) and ChR2-flfl control mice were exposed to blue light in a chamber once daily from P1-P5 together with their Cre-negative mother., Results: ELP caused cortical hyperexcitability at P8-9 as assessed via multi-electrode array recordings that coincided with reduced expression of synaptic genes (RNAseq) including Grin2b, neurexins, piccolo and voltage gated calcium and sodium channels. Young adult (8-16 wks) Avil-ChR2 mice presented with nociceptive hypersensitivity upon heat or mechanical stimulation, which did not resolve up until one year of age. The persistent hypersensitivy to nociceptive stimuli was reflected by increased calcium fluxes in primary sensory neurons of aged mice (1 year) upon capsaicin stimulation. Avil-ChR2 mice behaved like controls in maze tests of anxiety, social interaction, and spatial memory but IntelliCage behavioral studies revealed repetitive nosepokes and corner visits and compulsive lickings. Compulsiveness at the behavioral level was associated with a reduction of sphingomyelin species in brain and plasma lipidomic studies. Behavioral studies were done with female mice., Conclusion: The results suggest that ELP may predispose to chronic "pain" and compulsive psychopathology in part mediated by alterations of sphingolipid metabolism, which have been previously described in the context of addiction and psychiatric diseases., (© 2023. Society of Chinese Bioscientists in America (SCBA).)

Aging is a major risk factor for impaired cardiovascular health. Because the aging myocardium is characterized by microcirculatory dysfunction, and because nerves align with vessels, we assessed the impact of aging on the cardiac neurovascular interface. We report that aging reduces nerve density in the ventricle and dysregulates vascular-derived neuroregulatory genes. Aging down-regulates microRNA 145 (miR-145) and derepresses the neurorepulsive factor semaphorin-3A. miR-145 deletion, which increased Sema3a expression or endothelial Sema3a overexpression, reduced axon density, mimicking the aged-heart phenotype. Removal of senescent cells, which accumulated with chronological age in parallel to the decline in nerve density, rescued age-induced denervation, reversed Sema3a expression, preserved heart rate patterns, and reduced electrical instability. These data suggest that senescence-mediated regulation of nerve density contributes to age-associated cardiac dysfunction.

MicroRNAs (miRNAs) are post-transcriptional regulators that finetune gene expression via translational repression or degradation of their target mRNAs. Despite their functional relevance, frameworks for the scalable and accurate detection of miRNA orthologs are missing. Consequently, there is still no comprehensive picture of how miRNAs and their associated regulatory networks have evolved. Here we present ncOrtho, a synteny informed pipeline for the targeted search of miRNA orthologs in unannotated genome sequences. ncOrtho matches miRNA annotations from multi-tissue transcriptomes in precision, while scaling to the analysis of hundreds of custom-selected species. The presence-absence pattern of orthologs to 266 human miRNA families across 402 vertebrate species reveals four bursts of miRNA acquisition, of which the most recent event occurred in the last common ancestor of higher primates. miRNA families are rarely modified or lost, but notable exceptions for both events exist. miRNA co-ortholog numbers faithfully indicate lineage-specific whole genome duplications, and miRNAs are powerful markers for phylogenomic analyses. Their exceptionally low genetic diversity makes them suitable to resolve clades where the phylogenetic signal is blurred by incomplete lineage sorting of ancestral alleles. In summary, ncOrtho allows to routinely consider miRNAs in evolutionary analyses that were thus far reserved to protein-coding genes., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Electronic cigarette (e-cigarette) vapour is gaining popularity as an alternative to tobacco smoking and can induce acute lung injury. However, the specific role of nicotine in e-cigarette vapour and its long-term effects on the airways, lung parenchyma and vasculature remain unclear., Results: In vitro exposure to nicotine-containing e-cigarette vapour extract (ECVE) or to nicotine-free e-cigarette vapour extract (NF ECVE) induced changes in gene expression of epithelial cells and pulmonary arterial smooth muscle cells (PASMCs), but ECVE in particular caused functional alterations ( e.g. a decrease in human and mouse PASMC proliferation by 29.3±5.3% and 44.3±8.4%, respectively). Additionally, acute inhalation of nicotine-containing e-cigarette vapour (ECV) but not nicotine-free e-cigarette vapour (NF ECV) increased pulmonary endothelial permeability in isolated lungs. Long-term in vivo exposure of mice to ECV for 8 months significantly increased the number of inflammatory cells, in particular lymphocytes, compared to control and NF ECV in the bronchoalveolar fluid (BALF) (ECV: 853.4±150.8 cells·mL -1 ; control: 37.0±21.1 cells·mL -1 ; NF ECV: 198.6±94.9 cells·mL -1 ) and in lung tissue (ECV: 25.7±3.3 cells·mm -3 ; control: 4.8±1.1 cells·mm -3 ; NF ECV: 14.1±2.2 cells·mm -3 ). BALF cytokines were predominantly increased by ECV. Moreover, ECV caused significant changes in lung structure and function ( e.g. increase in airspace by 17.5±1.4% compared to control), similar to mild tobacco smoke-induced alterations, which also could be detected in the NF ECV group, albeit to a lesser degree. In contrast, the pulmonary vasculature was not significantly affected by ECV or NF ECV., Conclusions: NF ECV components induce cell type-specific effects and mild pulmonary alterations, while inclusion of nicotine induces significant endothelial damage, inflammation and parenchymal alterations., Competing Interests: Conflict of interest: All authors have nothing to disclose., (Copyright ©The authors 2023.)

Rationale: Tobacco smoking and air pollution are primary causes of chronic obstructive pulmonary disease (COPD). However, only a minority of smokers develop COPD. The mechanisms underlying the defense against nitrosative/oxidative stress in nonsusceptible smokers to COPD remain largely unresolved. Objectives: To investigate the defense mechanisms against nitrosative/oxidative stress that possibly prevent COPD development or progression. Methods: Four cohorts were investigated: 1 ) sputum samples (healthy, n  = 4; COPD, n  = 37), 2 ) lung tissue samples (healthy, n  = 13; smokers without COPD, n  = 10; smoker+COPD, n  = 17), 3 ) pulmonary lobectomy tissue samples (no/mild emphysema, n  = 6), and 4 ) blood samples (healthy, n  = 6; COPD, n  = 18). We screened 3-nitrotyrosine (3-NT) levels, as indication of nitrosative/oxidative stress, in human samples. We established a novel in vitro model of a cigarette smoke extract (CSE)-resistant cell line and studied 3-NT formation, antioxidant capacity, and transcriptomic profiles. Results were validated in lung tissue, isolated primary cells, and an ex vivo model using adeno-associated virus-mediated gene transduction and human precision-cut lung slices. Measurements and Main Results: 3-NT levels correlate with COPD severity of patients. In CSE-resistant cells, nitrosative/oxidative stress upon CSE treatment was attenuated, paralleled by profound upregulation of heme oxygenase-1 (HO-1). We identified carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) as a negative regulator of HO-1-mediated nitrosative/oxidative stress defense in human alveolar type 2 epithelial cells (hAEC2s). Consistently, inhibition of HO-1 activity in hAEC2s increased the susceptibility toward CSE-induced damage. Epithelium-specific CEACAM6 overexpression increased nitrosative/oxidative stress and cell death in human precision-cut lung slices on CSE treatment. Conclusions: CEACAM6 expression determines the hAEC2 sensitivity to nitrosative/oxidative stress triggering emphysema development/progression in susceptible smokers.

Treatment of vascular stenosis with angioplasty results in acute vascular damage, which may lead to restenosis. Owing to the highly complex cellularity of blood vessels, the healing response following this damage is incompletely understood. To gain further insight into this process, scRNA-seq of mouse carotid tissue after wire injury was performed. Stages of acute inflammation, resolution and remodeling were recapitulated in these data. To identify cell types which give rise to neointima, analyses focused on smooth muscle cell and fibroblast populations, and included data integration with scRNA-seq data from myocardial infarction and atherosclerosis datasets. Following carotid injury, a subpopulation of smooth muscle cells which also arises during atherosclerosis and myocardial infarction was identified. So-called stem cell/endothelial cell/monocyte (SEM) cells are candidates for repopulating injured vessels, and were amongst the most proliferative cell clusters following wire-injury of the carotid artery. Importantly, SEM cells exhibit specific transcriptional profiles which could be therapeutically targeted. SEM cell gene expression patterns could also be detected in bulk RNA-sequencing of neointimal tissue isolated from injured carotid vessels by laser capture microdissection. These data indicate that phenotypic plasticity of smooth muscle cells is highly important to the progression of lumen loss following acute carotid injury. Interference with SEM cell formation could be an innovative approach to combat development of restenosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Warwick, Buchmann, Pflüger-Müller, Spaeth, Schürmann, Abplanalp, Tombor, John, Weigert, Leo-Hansmann, Dimmeler and Brandes.)

Long non-coding RNAs (lncRNAs) can act as regulatory RNAs which, by altering the expression of target genes, impact on the cellular phenotype and cardiovascular disease development. Endothelial lncRNAs and their vascular functions are largely undefined. Deep RNA-Seq and FANTOM5 CAGE analysis revealed the lncRNA LINC00607 to be highly enriched in human endothelial cells. LINC00607 was induced in response to hypoxia, arteriosclerosis regression in non-human primates, post-atherosclerotic cultured endothelial cells from patients and also in response to propranolol used to induce regression of human arteriovenous malformations. siRNA knockdown or CRISPR/Cas9 knockout of LINC00607 attenuated VEGF-A-induced angiogenic sprouting. LINC00607 knockout in endothelial cells also integrated less into newly formed vascular networks in an in vivo assay in SCID mice. Overexpression of LINC00607 in CRISPR knockout cells restored normal endothelial function. RNA- and ATAC-Seq after LINC00607 knockout revealed changes in the transcription of endothelial gene sets linked to the endothelial phenotype and in chromatin accessibility around ERG-binding sites. Mechanistically, LINC00607 interacted with the SWI/SNF chromatin remodeling protein BRG1. CRISPR/Cas9-mediated knockout of BRG1 in HUVEC followed by CUT&RUN revealed that BRG1 is required to secure a stable chromatin state, mainly on ERG-binding sites. In conclusion, LINC00607 is an endothelial-enriched lncRNA that maintains ERG target gene transcription by interacting with the chromatin remodeler BRG1 to ultimately mediate angiogenesis., (© 2023. The Author(s).)

Long non-coding RNAs (lncRNAs) impact cell function via numerous mechanisms. In the nucleus, interactions between lncRNAs and DNA and the consequent formation of non-canonical nucleic acid structures seems to be particularly relevant. Along with interactions between single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA), such as R-loops, ssRNA can also interact with double-stranded DNA (dsDNA) to form DNA:DNA:RNA triplexes. A major challenge in the study of DNA:DNA:RNA triplexes is the identification of the precise RNA component interacting with specific regions of the dsDNA. As this is a crucial step towards understanding lncRNA function, there exist several computational methods designed to predict these sequences. This review summarises the recent progress in the prediction of triplex formation and highlights important DNA:DNA:RNA triplexes. In particular, different prediction tools ( Triplexator , LongTarget , TRIPLEXES , Triplex Domain Finder , TriplexFFP , TriplexAligner and Fasim-LongTarget ) will be discussed and their use exemplified by selected lncRNAs, whose DNA:DNA:RNA triplex forming potential was validated experimentally. Collectively, these tools revealed that DNA:DNA:RNA triplexes are likely to be numerous and make important contributions to gene expression regulation.

The cytochrome P450 reductase (POR) transfers electrons to all microsomal cytochrome P450 enzymes (CYP450) thereby driving their activity. In the vascular system, the POR/CYP450 system has been linked to the production of epoxyeicosatrienoic acids (EETs) but also to the generation of reactive oxygen species. In cardiac myocytes (CMs), EETs have been shown to modulate the cardiac function and have cardioprotective effects. The functional importance of the endothelial POR/CYP450 system in the heart is unclear and was studied here using endothelial cell-specific, inducible knockout mice of POR (ecPOR -/- ). RNA sequencing of murine cardiac cells revealed a cell type-specific expression of different CYP450 homologues. Cardiac endothelial cells mainly expressed members of the CYP2 family which produces EETs, and of the CYP4 family that generates omega fatty acids. Tamoxifen-induced endothelial deletion of POR in mice led to cardiac remodelling under basal conditions, as shown by an increase in heart weight to body weight ratio and an increased CM area as compared to control animals. Endothelial deletion of POR was associated with a significant increase in endothelial genes linked to protein synthesis with no changes in genes of the oxidative stress response. CM of ecPOR -/- mice exhibited attenuated expression of genes linked to mitochondrial function and an increase in genes related to cardiac myocyte contractility. In a model of pressure overload (transverse aortic constriction, TAC with O-rings), ecPOR -/- mice exhibited an accelerated reduction in cardiac output (CO) and stroke volume (SV) as compared to control mice. These results suggest that loss of endothelial POR along with a reduction in EETs leads to an increase in vascular stiffness and loss in cardioprotection, resulting in cardiac remodelling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lopez, Malacarne, Ramanujam, Warwick, Müller, Hu, Dewenter, Weigert, Günther, Gilsbach, Engelhardt, Brandes and Rezende.)

RNA.DNA:DNA triple helix (triplex) formation is a form of RNA-DNA interaction which regulates gene expression but is difficult to study experimentally in vivo. This makes accurate computational prediction of such interactions highly important in the field of RNA research. Current predictive methods use canonical Hoogsteen base pairing rules, which whilst biophysically valid, may not reflect the plastic nature of cell biology. Here, we present the first optimization approach to learn a probabilistic model describing RNA-DNA interactions directly from motifs derived from triplex sequencing data. We find that there are several stable interaction codes, including Hoogsteen base pairing and novel RNA-DNA base pairings, which agree with in vitro measurements. We implemented these findings in TriplexAligner, a program that uses the determined interaction codes to predict triplex binding. TriplexAligner predicts RNA-DNA interactions identified in all-to-all sequencing data more accurately than all previously published tools in human and mouse and also predicts previously studied triplex interactions with known regulatory functions. We further validated a novel triplex interaction using biophysical experiments. Our work is an important step towards better understanding of triplex formation and allows genome-wide analyses of RNA-DNA interactions., (© The Author(s) 2022. Published by Oxford University Press.)

In healthy vessels, endothelial cells maintain a stable, differentiated, and growth-arrested phenotype for years. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. Here we report the identification of the endothelial cell-enriched long non-coding RNA (lncRNA) PCAT19, which contributes to the proliferative switch and acts as a safeguard for the endothelial genome. PCAT19 is enriched in confluent, quiescent endothelial cells and binds to the full replication protein A (RPA) complex in a DNA damage- and cell-cycle-related manner. Our results suggest that PCAT19 limits the phosphorylation of RPA2, primarily on the serine 33 (S33) residue, and thereby facilitates an appropriate DNA damage response while slowing cell cycle progression. Reduction in PCAT19 levels in response to either loss of cell contacts or knockdown promotes endothelial proliferation and angiogenesis. Collectively, PCAT19 acts as a dynamic guardian of the endothelial genome and facilitates rapid switching from quiescence to proliferation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing of the RNA in the major groove of the DNA duplex have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-AS1 reduces the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1α-AS1 is down-regulated in pulmonary hypertension and loss-of-function approaches not only result in gene de-repression but also enhance angiogenic capacity. As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control., (© 2022. The Author(s).)

Rationale: Nox4 is a constitutively active NADPH oxidase that constantly produces low levels of H 2 O 2 . Thereby, Nox4 contributes to cell homeostasis and long-term processes, such as differentiation. The high expression of Nox4 seen in endothelial cells contrasts with the low abundance of Nox4 in stem cells, which are accordingly characterized by low levels of H 2 O 2 . We hypothesize that Nox4 is a major contributor to endothelial differentiation, is induced during the process of differentiation, and facilitates homeostasis of the resulting endothelial cells., Objective: To determine the role of No×4 in differentiation of murine inducible pluripotent stem cells (miPSC) into endothelial cells (ECs)., Methods and Results: miPSC, generated from mouse embryonic wildtype (WT) and Nox4 -/- fibroblasts, were differentiated into endothelial cells (miPSC-EC) by stimulation with BMP4 and VEGF. During this process, Nox4 expression increased and knockout of Nox4 prolonged the abundance of pluripotency markers, while expression of endothelial markers was delayed in differentiating Nox4-depleted iPSCs. Eventually, angiogenic capacity of iPSC-ECs is reduced in Nox4 deficient cells, indicating that an absence of Nox4 diminishes stability of the reached phenotype. As an underlying mechanism, we identified JmjD3 as a redox target of Nox4. iPSC-ECs lacking Nox4 display a lower nuclear abundance of the histone demethylase JmjD3, resulting in an increased triple methylation of histone 3 (H3K27me3), which serves as a repressive mark for several genes involved in differentiation., Conclusions: Nox4 promotes differentiation of miPSCs into ECs by oxidation of JmjD3 and subsequent demethylation of H3K27me3, which forced endothelial differentiation and stability., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Human 5-lipoxygenase (5-LO) is the key enzyme of leukotriene biosynthesis, mostly expressed in leukocytes and thus a crucial component of the innate immune system. In this study, we show that 5-LO, besides its canonical function as an arachidonic acid metabolizing enzyme, is a regulator of gene expression associated with euchromatin. By Crispr-Cas9-mediated 5-LO knockout (KO) in MonoMac6 (MM6) cells and subsequent RNA-Seq analysis, we identified 5-LO regulated genes which could be clustered to immune/defense response, cell adhesion, transcription and growth/developmental processes. Analysis of differentially expressed genes identified cyclooxygenase-2 (COX2, PTGS2) and kynureninase (KYNU) as strongly regulated 5-LO target genes. 5-LO knockout affected MM6 cell adhesion and tryptophan metabolism via inhibition of the degradation of the immunoregulator kynurenine. By subsequent FAIRE-Seq and 5-LO ChIP-Seq analyses, we found an association of 5-LO with euchromatin, with prominent 5-LO binding to promoter regions in actively transcribed genes. By enrichment analysis of the ChIP-Seq results, we identified potential 5-LO interaction partners. Furthermore, 5-LO ChIP-Seq peaks resemble patterns of H3K27ac histone marks, suggesting that 5-LO recruitment mainly takes place at acetylated histones. In summary, we demonstrate a noncanonical function of 5-LO as transcriptional regulator in monocytic cells., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: POR (cytochrome P450 reductase) provides electrons for the catalytic activity of the CYP (cytochrome P450) monooxygenases. CYPs are dual-function enzymes as they generate protective vasoactive mediators derived from polyunsaturated fatty acids but also reactive oxygen species. It is not known in which conditions the endothelial POR/CYP system is beneficial versus deleterious. Here, the activity of all CYP enzymes was eliminated in the vascular endothelium to examine its impact on vascular function., Methods: An endothelial-specific, tamoxifen-inducible POR knockout mouse (ecPOR - /- ) was generated. Vascular function was studied by organ chamber experiments. eNOS (endothelial nitric oxide synthase) activity was accessed by heavy arginine/citrulline LC-MS/MS detection and phosphorylation of serine1177 in aortic rings. CYP-derived epoxyeicosatrienoic acids and prostanoids were measured by LC-MS/MS. Gene expression of aorta and endothelial cells was profiled by RNA sequencing. Blood pressure was measured by telemetry., Results: Acetylcholine-induced endothelium-dependent relaxation was attenuated in isolated vessels of ecPOR -/- as compared with control mice. Additionally, ecPOR -/- mice had attenuated eNOS activity and eNOS/AKT phosphorylation. POR deletion reduced endothelial stores of CYP-derived epoxyeicosatrienoic acids but increased vascular prostanoids. This phenomenon was paralleled by the induction of genes implicated in eicosanoid generation. In response to Ang II (angiotensin II) infusion, blood pressure increased significantly more in ecPOR - /- mice. Importantly, the cyclooxygenase inhibitor Naproxen selectively lowered the Ang II-induced hypertension in ecPOR - /- mice., Conclusions: POR expression in endothelial cells maintains eNOS activity and its loss results in an overactivation of the vasoconstrictor prostanoid system. Through these mechanisms, loss of endothelial POR induces vascular dysfunction and hypertension.

Epoxyeicosatrienoic acids (EETs) are signaling lipids produced by the cytochrome P450-(CYP450)-mediated epoxygenation of arachidonic acid. EETs have numerous biological effects on the vascular system, but aspects including their species specificity make their effects on vascular tone controversial. CYP450 enzymes require the 450-reductase (POR) for their activity. We set out to determine the contribution of endothelial CYP450 to murine vascular function using isolated aortic ring preparations from tamoxifen-inducible endothelial cell-specific POR knockout mice (ecPOR -/- ). Constrictor responses to phenylephrine were similar between control (CTR) and ecPOR -/- mice. Contrastingly, sensitivity to the thromboxane receptor agonist U46619 and prostaglandin E2 (PGE2) was increased following the deletion of POR. Ex vivo incubation with a non-hydrolyzable EET (14,15-EE-8(Z)-E, EEZE) reversed the increased sensitivity to U46619 to the levels of CTR. EETs had no effect on vascular tone in phenylephrine-preconstricted vessels, but dilated vessels contracted with U46619 or PGE2. As U46619 acts through RhoA-dependent kinase, this system was analyzed. The deletion of POR affected the expression of genes in this pathway and the inhibition of Rho-GTPase with SAR407899 decreased sensitivity to U46619. These data suggest that EET and prostanoid crosstalk at the receptor level and that lack of EET production sensitizes vessels to vasoconstriction via the induction of the Rho kinase system.

Spatial genome organization is tightly controlled by several regulatory mechanisms and is essential for gene expression control. Nuclear receptors are ligand-activated transcription factors that modulate physiological and pathophysiological processes and are primary pharmacological targets. DNA binding of the important loop-forming insulator protein CCCTC-binding factor (CTCF) was modulated by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). We performed CTCF HiChIP assays to produce the first genome-wide dataset of CTCF long-range interactions in 1,25(OH)2D3-treated cells, and to determine whether dynamic changes of spatial chromatin interactions are essential for fine-tuning of nuclear receptor signaling. We detected changes in 3D chromatin organization upon vitamin D receptor (VDR) activation at 3.1% of all observed CTCF interactions. VDR binding was enriched at both differential loop anchors and within differential loops. Differential loops were observed in several putative functional roles including TAD border formation, promoter-enhancer looping, and establishment of VDR-responsive insulated neighborhoods. Vitamin D target genes were enriched in differential loops and at their anchors. Secondary vitamin D effects related to dynamic chromatin domain changes were linked to location of downstream transcription factors in differential loops. CRISPR interference and loop anchor deletion experiments confirmed the functional relevance of nuclear receptor ligand-induced adjustments of the chromatin 3D structure for gene expression regulation., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Pulmonary hypertension (PH) is a common complication of COPD, associated with increased mortality and morbidity. Intriguingly, pulmonary vascular alterations have been suggested to drive emphysema development. Previously, we identified inducible nitric oxide synthase (iNOS) as an essential enzyme for development and reversal of smoke-induced PH and emphysema, and showed that iNOS expression in bone-marrow-derived cells drives pulmonary vascular remodelling, but not parenchymal destruction. In this study, we aimed to identify the iNOS-expressing cell type driving smoke-induced PH and to decipher pro-proliferative pathways involved., Methods: To address this question we used 1) myeloid-cell-specific iNOS knockout mice in chronic smoke exposure and 2) co-cultures of macrophages and pulmonary artery smooth muscle cells (PASMCs) to decipher underlying signalling pathways., Results: Myeloid-cell-specific iNOS knockout prevented smoke-induced PH but not emphysema in mice. Moreover, iNOS deletion in myeloid cells ameliorated the increase in expression of CD206, a marker of M2 polarisation, on interstitial macrophages. Importantly, the observed effects on lung macrophages were hypoxia-independent, as these mice developed hypoxia-induced PH. In vitro , smoke-induced PASMC proliferation in co-cultures with M2-polarised macrophages could be abolished by iNOS deletion in phagocytic cells, as well as by extracellular signal-regulated kinase inhibition in PASMCs. Crucially, CD206-positive and iNOS-positive macrophages accumulated in proximity of remodelled vessels in the lungs of COPD patients, as shown by immunohistochemistry., Conclusion: In summary, our results demonstrate that iNOS deletion in myeloid cells confers protection against PH in smoke-exposed mice and provide evidence for an iNOS-dependent communication between M2-like macrophages and PASMCs in underlying pulmonary vascular remodelling., Competing Interests: Conflict of interest: M. Gredic has nothing to disclose. Conflict of interest: C-Y. Wu has nothing to disclose. Conflict of interest: S. Hadzic has nothing to disclose. Conflict of interest: O. Pak has nothing to disclose. Conflict of interest: R. Savai has nothing to disclose. Conflict of interest: B. Kojonazarov has nothing to disclose. Conflict of interest: S. Doswada has nothing to disclose. Conflict of interest: A. Weiss has nothing to disclose. Conflict of interest: A. Weigert has nothing to disclose. Conflict of interest: A. Guenther has nothing to disclose. Conflict of interest: R.P. Brandes has nothing to disclose. Conflict of interest: R.T. Schermuly has nothing to disclose. Conflict of interest: F. Grimminger has nothing to disclose. Conflict of interest: W. Seeger reports personal fees from Actelion, Bayer AG, Novartis, Vectura, Medspray and United Therapeutics, outside the submitted work. Conflict of interest: N. Sommer reports personal fees from Actelion, outside the submitted work. Conflict of interest: S. Kraut has nothing to disclose. Conflict of interest: N. Weissmann reports grants from the German Research Foundation, during the conduct of the study; and has a patent L-NIL as inhibitor for regenerating the lung of a patient suffering from COPD (EP2591777A2) issued., (Copyright ©The authors 2022.)

Background: Pulmonary hypertension (PH) is a life-threatening disease, characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary arterial pressure and right heart hypertrophy. PH can be caused by chronic hypoxia, leading to hyper-proliferation of pulmonary arterial smooth muscle cells (PASMCs) and apoptosis-resistant pulmonary microvascular endothelial cells (PMVECs). On reexposure to normoxia, chronic hypoxia-induced PH in mice is reversible. In this study, the authors aim to identify novel candidate genes involved in pulmonary vascular remodeling specifically in the pulmonary vasculature., Methods: After microarray analysis, the authors assessed the role of SPARC (secreted protein acidic and rich in cysteine) in PH using lung tissue from idiopathic pulmonary arterial hypertension (IPAH) patients, as well as from chronically hypoxic mice. In vitro studies were conducted in primary human PASMCs and PMVECs. In vivo function of SPARC was proven in chronic hypoxia-induced PH in mice by using an adeno-associated virus-mediated Sparc knockdown approach., Results: C57BL/6J mice were exposed to normoxia, chronic hypoxia, or chronic hypoxia with subsequent reexposure to normoxia for different time points. Microarray analysis of the pulmonary vascular compartment after laser microdissection identified Sparc as one of the genes downregulated at all reoxygenation time points investigated. Intriguingly, SPARC was vice versa upregulated in lungs during development of hypoxia-induced PH in mice as well as in IPAH, although SPARC plasma levels were not elevated in PH. TGF-β1 (transforming growth factor β1) or HIF2A (hypoxia-inducible factor 2A) signaling pathways induced SPARC expression in human PASMCs. In loss of function studies, SPARC silencing enhanced apoptosis and reduced proliferation. In gain of function studies, elevated SPARC levels induced PASMCs, but not PMVECs, proliferation. Coculture and conditioned medium experiments revealed that PMVECs-secreted SPARC acts as a paracrine factor triggering PASMCs proliferation. Contrary to the authors' expectations, in vivo congenital Sparc knockout mice were not protected from hypoxia-induced PH, most probably because of counter-regulatory proproliferative signaling. However, adeno-associated virus-mediated Sparc knockdown in adult mice significantly improved hemodynamic and cardiac function in PH mice., Conclusions: This study provides evidence for the involvement of SPARC in the pathogenesis of human PH and chronic hypoxia-induced PH in mice, most likely by affecting vascular cell function.

Background: Blood pressure is known to be increased in kidney donors following living-donor kidney transplantation. However, the physiological underpinnings of the blood-pressure increase following uninephrectomy remain unclear. We hypothesized that changes in sympathetic tone or in parasympathetic modulation of sinus node function are involved in the blood-pressure increase following experimental kidney-mass reduction., Methods: C57BL6N mice (6 to 11 per group) subjected to sham surgery (controls) or uninephrectomy with or without a one-week course of sodium chloride-enriched, taurine-deficient diet were studied. Uninephrectomized mice treated with a subcutaneous infusion of angiotensin-II over a period of one week were positive controls. A transfemoral aortic catheter with telemetry unit was implanted, readings of heart-rate and blood-pressure were recorded. Powerspectral analysis of heart rate and systolic blood pressure was performed to gain surrogate parameters of sympathetictone and parasympathetic modulation of sinus node function. Baroreflex sensitivity of heart rate was determined from awake, unrestrained mice using spontaneous baroreflex gain technique., Results: Systolic arterial blood pressure, heart rate and baroreflex sensitivity were not different in uninephrectomized mice when compared to controls. Parasympathetic modulation of sinus node function was less in uninephrectomized mice in comparison to controls. Uninephrectomized mice of the high-angiotensin-II model or of the high-salt and taurine-deficiency model had an increased systolic arterial blood pressure., Conclusions: Uninephrectomy associated with less parasympathetic modulation of sinus node function. The combination of uninephrectomy, taurine-deficiency and high-salt intake led to arterial hypertension., Competing Interests: The authors have declared that no competing interests exist.

Reactive oxygen species (ROS) are important mediators of both physiological and pathophysiological signal transduction in the cardiovascular system. The effects of ROS on cellular processes depend on the concentration, localization, and duration of exposure. Cellular stress response mechanisms have evolved to mitigate the negative effects of acute oxidative stress. In this study, we investigate the short-term and long-term metabolic and transcriptomic response of human umbilical vein endothelial cells (HUVEC) to different types and concentrations of ROS. To generate intracellular H 2 O 2 , we utilized a lentiviral chemogenetic approach for overexpression of human D-amino acid oxidase (DAO). DAO converts D-amino acids into their corresponding imino acids and H 2 O 2 . HUVEC stably overexpressing DAO (DAO-HUVEC) were exposed to D-alanine (3 mM), exogenous H 2 O 2 (10 µM or 300 µM), or menadione (5 µM) for various timepoints and subjected to global untargeted metabolomics (LC-MS/MS) and RNAseq by MACE (Massive analysis of cDNA ends). A total of 300 µM H 2 O 2 led to pronounced changes on both the metabolic and transcriptomic level. In particular, metabolites linked to redox homeostasis, energy-generating pathways, and nucleotide metabolism were significantly altered. Furthermore, 300 µM H 2 O 2 affected genes related to the p53 pathway and cell cycle. In comparison, the effects of menadione and DAO-derived H 2 O 2 mainly occurred at gene expression level. Collectively, all types of ROS led to subtle changes in the expression of ribosomal genes. Our results show that different types and concentration of ROS lead to a different metabolic and transcriptomic response in endothelial cells.

Long non-coding RNAs were once considered as "junk" RNA produced by aberrant DNA transcription. They are now understood to play central roles in diverse cellular processes from proliferation and migration to differentiation, senescence and DNA damage control. LncRNAs are classed as transcripts longer than 200 nucleotides that do not encode a peptide. They are relevant to many physiological and pathophysiological processes through their control of fundamental molecular functions. This review summarises the recent progress in lncRNA research and highlights the far-reaching physiological relevance of lncRNAs. The main areas of lncRNA research encompassing their characterisation, classification and mechanisms of action will be discussed. In particular, the regulation of gene expression and chromatin landscape through lncRNA control of proteins, DNA and other RNAs will be introduced. This will be exemplified with a selected number of lncRNAs that have been described in numerous physiological contexts and that should be largely representative of the tens-of-thousands of mammalian lncRNAs. To some extent, these lncRNAs have inspired the current thinking on the central dogmas of epigenetics, RNA and DNA mechanisms., (© 2021. The Author(s).)

The transcription factor hypoxia-inducible factor 1 (HIF1) is an important driver of cancer and is therefore an attractive drug target. Acriflavine (ACF) has been suggested to inhibit HIF1, but its mechanism of action is unknown. Here we investigated the interaction of ACF with DNA and long non-coding RNAs (lncRNAs) and its function in human endothelial cells. ACF promoted apoptosis and reduced proliferation, network formation, and angiogenic capacity. It also induced changes in gene expression, as determined by RNA sequencing (RNA-seq), which could not be attributed to specific inhibition of HIF1. A similar response was observed in murine lung endothelial cells. Although ACF increased and decreased a similar number of protein-coding genes, lncRNAs were preferentially upregulated under normoxic and hypoxic conditions. An assay for transposase accessibility with subsequent DNA sequencing (ATAC-seq) demonstrated that ACF induced strong changes in chromatin accessibility at lncRNA promoters. Immunofluorescence showed displacement of DNA:RNA hybrids. Such effects might be due to ACF-mediated topoisomerase inhibition, which was indeed the case, as reflected by DNA unwinding assays. Comparison with other acridine derivatives and topoisomerase inhibitors suggested that the specific function of ACF is an effect of acridinium-class compounds. This study demonstrates that ACF inhibits topoisomerases rather than HIF specifically and that it elicits a unique expression response of lncRNAs., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Understanding how epigenetic variation in non-coding regions is involved in distal gene-expression regulation is an important problem. Regulatory regions can be associated to genes using large-scale datasets of epigenetic and expression data. However, for regions of complex epigenomic signals and enhancers that regulate many genes, it is difficult to understand these associations. We present StitchIt, an approach to dissect epigenetic variation in a gene-specific manner for the detection of regulatory elements (REMs) without relying on peak calls in individual samples. StitchIt segments epigenetic signal tracks over many samples to generate the location and the target genes of a REM simultaneously. We show that this approach leads to a more accurate and refined REM detection compared to standard methods even on heterogeneous datasets, which are challenging to model. Also, StitchIt REMs are highly enriched in experimentally determined chromatin interactions and expression quantitative trait loci. We validated several newly predicted REMs using CRISPR-Cas9 experiments, thereby demonstrating the reliability of StitchIt. StitchIt is able to dissect regulation in superenhancers and predicts thousands of putative REMs that go unnoticed using peak-based approaches suggesting that a large part of the regulome might be uncharted water., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: The NADPH oxidase Nox4 is an important source of H 2 O 2 . Nox4-derived H 2 O 2 limits vascular inflammation and promotes smooth muscle differentiation. On this basis, the role of Nox4 for restenosis development was determined in the mouse carotid artery injury model., Methods and Results: Genetic deletion of Nox4 by a tamoxifen-activated Cre-Lox-system did not impact on neointima formation in the carotid artery wire injury model. To understand this unexpected finding, time-resolved single-cell RNA-sequencing (scRNAseq) from injured carotid arteries of control mice and massive-analysis-of-cDNA-ends (MACE)-RNAseq from the neointima harvested by laser capture microdissection of control and Nox4 knockout mice was performed. This revealed that resting smooth muscle cells (SMCs) and fibroblasts exhibit high Nox4 expression, but that the proliferating de-differentiated SMCs, which give rise to the neointima, have low Nox4 expression. In line with this, the first weeks after injury, gene expression was unchanged between the carotid artery neointimas of control and Nox4 knockout mice., Conclusion: Upon vascular injury, Nox4 expression is transiently lost in the cells which comprise the neointima. NADPH oxidase 4 therefore does not interfere with restenosis development after wire-induced vascular injury., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Mitofusin 2 (MFN2) is a mitochondrial outer membrane GTPase, which modulates mitochondrial fusion and affects the interaction between endoplasmic reticulum and mitochondria. Here, we explored how MFN2 influences mitochondrial functions and inflammatory responses towards zymosan in primary human macrophages. A knockdown of MFN2 by small interfering RNA decreased mitochondrial respiration without attenuating mitochondrial membrane potential and reduced interactions between endoplasmic reticulum and mitochondria. A MFN2 deficiency potentiated zymosan-elicited inflammatory responses of human primary macrophages, such as expression and secretion of pro-inflammatory cytokines interleukin-1β, -6, -8 and tumor necrosis factor α, as well as induction of cyclooxygenase 2 and prostaglandin E 2 synthesis. MFN2 silencing also increased zymosan-induced nuclear factor kappa-light-chain-enhancer of activated B cells and mitogen-activated protein kinases inflammatory signal transduction, without affecting mitochondrial reactive oxygen species production. Mechanistic studies revealed that MFN2 deficiency enhanced the toll-like receptor 2-dependent branch of zymosan-triggered responses upstream of inhibitor of κB kinase. This was associated with elevated, cytosolic expression of interleukin-1 receptor-associated kinase 4 in MFN2-deficient cells. Our data suggest pro-inflammatory effects of MFN2 deficiency in human macrophages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Khodzhaeva, Schreiber, Geisslinger, Brandes, Brüne and Namgaladze.)