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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.
Assuntos
Proteínas de Ligação a DNA , Células Endoteliais , Endotélio Vascular , RNA Circular , Fatores de Transcrição , Animais , Humanos , Camundongos , Circulação Sanguínea , Proteínas de Ligação a DNA/genética , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Ligação Proteica , RNA Circular/genética , RNA Circular/metabolismo , Sindecana-4/metabolismo , Fatores de Transcrição/genéticaRESUMO
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.
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RNA Longo não Codificante , Animais , Humanos , Camundongos , Cromatina , DNA Helicases/genética , DNA Helicases/metabolismo , Células Endoteliais/metabolismo , Camundongos SCID , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/genética , Neovascularização FisiológicaRESUMO
The process of angiogenesis is involved in several pathological conditions, such as tumor growth or age-related macular degeneration. Although the available anti-angiogenic drugs have improved the therapy of these diseases, major drawbacks, such as unwanted side effects and resistances, still exist. Consequently, the search for new anti-angiogenic substances is still ongoing. Narciclasine, a plant alkaloid from different members of the Amaryllidaceae family, has extensively been characterized as anti-tumor compound. Beyond the field of cancer, the compound has recently been shown to possess anti-inflammatory properties. Surprisingly, potential actions of narciclasine on endothelial cells in the context of angiogenesis have been neglected so far. Thus, we aimed to analyze the effects of narciclasine on angiogenic processes in vitro and in vivo and to elucidate the underlying mechanism. Narciclasine (100-300â¯nM) effectively inhibited the proliferation, undirected and directed migration, network formation and angiogenic sprouting of human primary endothelial cells. Moreover, narciclasine (1â¯mg/kg/day) strongly reduced the VEGF-triggered angiogenesis in vivo (Matrigel plug assay in mice). Narciclasine mediated its anti-angiogenic effects in part by a RhoA-independent activation of the Rho kinase ROCK. Most importantly, however, the compound reduced the de novo protein synthesis in endothelial cells by approx. 50% without exhibiting considerable cytotoxic effects. As a consequence, narciclasine diminished the presence of proteins with a short half-life, such as the VEGF receptor 2, which is the basis for its anti-angiogenic effects. Taken together, our study highlights narciclasine as an interesting anti-angiogenic compound that is worth to be further evaluated in preclinical studies.
Assuntos
Alcaloides de Amaryllidaceae/farmacologia , Neovascularização Patológica/tratamento farmacológico , Fenantridinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Quinases Associadas a rho/genética , Amaryllidaceae/química , Alcaloides de Amaryllidaceae/química , Inibidores da Angiogênese/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno/química , Combinação de Medicamentos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Laminina/química , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Fenantridinas/química , Proteoglicanas/química , Transdução de Sinais/efeitos dos fármacos , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
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.
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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.
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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.
Assuntos
Envelhecimento , Senescência Celular , Coração , MicroRNAs , Densidade Microvascular , Miocárdio , Semaforina-3A , Coração/inervação , Microcirculação , MicroRNAs/genética , MicroRNAs/metabolismo , Semaforina-3A/genética , Animais , Camundongos , Envelhecimento/genética , Envelhecimento/patologia , Masculino , Camundongos Endogâmicos C57BL , Senescência Celular/genética , Miocárdio/patologia , AxôniosRESUMO
OBJECTIVE: The NADPH oxidase Nox4 is an important source of H2O2. Nox4-derived H2O2 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.
Assuntos
NADPH Oxidase 4 , Neointima , Lesões do Sistema Vascular , Animais , Células Cultivadas , Peróxido de Hidrogênio , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso , NADPH Oxidase 4/genéticaRESUMO
OBJECTIVE: Oxidative stress is a risk factor for atherosclerosis. NADPH oxidases of the Nox family produce ROS but their contribution to atherosclerosis development is less clear. Nox2 promotes and Nox4 rather limits atherosclerosis. Although Nox1 with its cytosolic co-factors are largely expressed in epithelial cells, a role for Nox1 for atherosclerosis development was suggested. To further define the role of this homologue, the role of its essential cytosolic cofactor, NoxO1, was determined for atherosclerosis development with the aid of knockout mice. METHODS AND RESULTS: Wildtype (WT) and NoxO1 knockout mice were treated with high fat diet and adeno-associated virus (AAV) overexpressing pro-protein convertase subtilisin/kexin type 9 (PCSK9) to induce hepatic low-density lipoprotein (LDL) receptor loss. As a result, massive hypercholesterolemia was induced and spontaneous atherosclerosis developed within three month. Deletion of NoxO1 reduced atherosclerosis formation in brachiocephalic artery and aortic arch in female but not male NoxO1-/- mice as compared to WT littermates. This was associated with a reduced pro-inflammatory cytokine signature in the plasma of female but not male NoxO1-/- mice. MACE-RNAseq of the vessel did not reveal this signature and the expression of the Nox1/NoxO1 system was low to not detectable. CONCLUSIONS: The scaffolding protein NoxO1 plays some role in atherosclerosis development in female mice probably by attenuating the global inflammatory burden.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Aterosclerose , Pró-Proteína Convertase 9 , Animais , Aterosclerose/genética , Feminino , Camundongos , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismoRESUMO
Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and death worldwide. Peroxynitrite, formed from nitric oxide, which is derived from inducible nitric oxide synthase, and superoxide, has been implicated in the development of emphysema, but the source of the superoxide was hitherto not characterized. Here, we identify the non-phagocytic NADPH oxidase organizer 1 (NOXO1) as the superoxide source and an essential driver of smoke-induced emphysema and pulmonary hypertension development in mice. NOXO1 is consistently upregulated in two models of lung emphysema, Cybb (also known as NADPH oxidase 2, Nox2)-knockout mice and wild-type mice with tobacco-smoke-induced emphysema, and in human COPD. Noxo1-knockout mice are protected against tobacco-smoke-induced pulmonary hypertension and emphysema. Quantification of superoxide, nitrotyrosine and multiple NOXO1-dependent signalling pathways confirm that peroxynitrite formation from nitric oxide and superoxide is a driver of lung emphysema. Our results suggest that NOXO1 may have potential as a therapeutic target in emphysema.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/efeitos dos fármacos , Enfisema/tratamento farmacológico , Enfisema/genética , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/efeitos dos fármacos , Enfisema/etiologia , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Doença Pulmonar Obstrutiva Crônica/complicações , Transdução de Sinais/genética , Superóxidos/metabolismo , Poluição por Fumaça de Tabaco/efeitos adversos , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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AIMS: The protein Scrib (Scribble 1) is known to control apico-basal polarity in epithelial cells. The role of polarity proteins in the vascular system remains poorly characterized; however, we previously reported that Scrib maintains the endothelial phenotype and directed migration. On this basis, we hypothesized that Scrib has anti-atherosclerotic functions. METHODS AND RESULTS: Tamoxifen-induced Scrib-knockout mice were crossed with ApoE-/- knockout mice and spontaneous atherosclerosis under high-fat diet (HFD), as well as accelerated atherosclerosis in response to partial carotid artery ligation and HFD, was induced. Deletion of Scrib resulted in increased atherosclerosis development in both models. Mechanistically, flow- as well as acetylcholine-induced endothelium-dependent relaxation and AKT phosphorylation was reduced by deletion of Scrib, whereas vascular permeability and leucocyte extravasation were increased after Scrib knockout. Scrib immune pull down in primary carotid endothelial cells and mass spectrometry identified Arhgef7 (Rho Guanine Nucleotide Exchange Factor 7, ßPix) as interaction partner. Scrib or Arhgef7 down-regulation by siRNA reduced the endothelial barrier function in human umbilical vein endothelial cells. Gene expression analysis from murine samples and from human biobank material of carotid endarterectomies indicated that loss of Scrib resulted in endothelial dedifferentiation with a decreased expression of endothelial signature genes. CONCLUSIONS: By maintaining a quiescent endothelial phenotype, the polarity protein Scrib elicits anti-atherosclerotic functions.