Publications by authors named "Ralf P Brandes"

212 Publications

Long non-coding RNAs: novel regulators of cellular physiology and function.

Pflugers Arch 2021 Nov 18. Epub 2021 Nov 18.

Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60590, Frankfurt, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00424-021-02641-zDOI Listing
November 2021

Integrative analysis of epigenetics data identifies gene-specific regulatory elements.

Nucleic Acids Res 2021 Oct;49(18):10397-10418

Cluster of Excellence for Multimodal Computing and Interaction, Saarland University, Saarland Informatics Campus, 66123 Saarbrücken, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkab798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8501997PMC
October 2021

Myeloid cell-specific deletion of inducible nitric oxide synthase protects against smoke-induced pulmonary hypertension in mice.

Eur Respir J 2021 Sep 2. Epub 2021 Sep 2.

Cardio-Pulmonary Institute (CPI), Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Justus-Liebig-University, Giessen, Germany

Pulmonary hypertension (PH) is a common complication of chronic obstructive pulmonary disease (COPD), associated with increased mortality and morbidity. Intriguingly, pulmonary vascular alterations have been suggested to drive emphysema development. We previously 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.To address this question we used 1) myeloid cell-specific iNOS knockout mice in chronic smoke exposure, 2) co-cultures of macrophages and pulmonary artery smooth muscle cells (PASMC) to decipher underlying signalling pathways.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. , smoke-induced PASMC proliferation in co-cultures with M2-polarised macrophages could be abolished by iNOS deletion in phagocytic cells, as well as by ERK inhibition in PASMC. Crucially, CD206-positive and iNOS-positive macrophages accumulated in proximity of remodelled vessels in the lungs of COPD patients, as shown by immunohistochemistry.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 PASMC in underlying pulmonary vascular remodelling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1183/13993003.01153-2021DOI Listing
September 2021

Mitofusin 2 Deficiency Causes Pro-Inflammatory Effects in Human Primary Macrophages.

Front Immunol 2021 12;12:723683. Epub 2021 Aug 12.

Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, Frankfurt, Germany.

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 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2021.723683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8397414PMC
August 2021

Is It in the EPIgenome?: Epigenetics Marks at Birth Are Associated With Arterial Stiffness in Children.

Hypertension 2021 Sep 11;78(3):801-803. Epub 2021 Aug 11.

Institute for Cardiovascular Physiology, Faculty of Medicine, Goethe-University Frankfurt, Germany. DZHK - German Center for Cardiovascular Research, Partner site Rhine-Main.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/HYPERTENSIONAHA.121.17777DOI Listing
September 2021

Nox4 Maintains Blood Pressure during Low Sodium Diet.

Antioxidants (Basel) 2021 Jul 10;10(7). Epub 2021 Jul 10.

Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern Kai 7, 60590 Frankfurt, Germany.

The NADPH oxidase Nox4 is a hydrogen peroxide (HO)-producing enzyme, with the highest expression in the kidney. As the kidney is involved in volume and blood pressure control through sodium handling, we set out to determine the impact of a low sodium diet on these parameters in WT and Nox4-/- mice. Nox4 expression in the murine kidney was restricted to the proximal tubule. Nevertheless, low-sodium-induced weight loss and sodium sparing function was similar in WT and Nox4-/- mice, disputing an important function of renal Nox4 in sodium handling. In contrast, a low sodium diet resulted in a reduction in systolic blood pressure in Nox4-/- as compared to WT mice. This was associated with a selectively lower pressure to heart-rate ratio, as well as heart to body weight ratio. In general, a low sodium diet leads to activation of sympathetic tone and the renin angiotensin system, which subsequently increases peripheral resistance. Our observations suggest that the control by this system is attenuated in Nox4-/- mice, resulting in lower blood pressure in response to low sodium.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/antiox10071103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8301203PMC
July 2021

Understanding Arteriosclerosis 2.0: Making Sense of Genetic Variants with scATAC.

Circ Res 2021 07 8;129(2):259-261. Epub 2021 Jul 8.

Institute for Cardiovascular Physiology, Faculty of Medicine, Goethe-University Frankfurt, Germany (R.P.B., R.G.).

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.121.319585DOI Listing
July 2021

The hydrogen-peroxide producing NADPH oxidase 4 does not limit neointima development after vascular injury in mice.

Redox Biol 2021 09 18;45:102050. Epub 2021 Jun 18.

Institute for Cardiovascular Physiology, Goethe-University, Frankfurt Am Main, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Rhein Main, Frankfurt Am Main, Germany. Electronic address:

Objective: The NADPH oxidase Nox4 is an important source of HO. Nox4-derived HO 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2021.102050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8256285PMC
September 2021

Glycolysis and Inflammation: Partners in Crime!

Circ Res 2021 06 24;129(1):30-32. Epub 2021 Jun 24.

Institute for Cardiovascular Physiology, Faculty of Medicine, Goethe-University Frankfurt, Germany (R.P.B., F.R.).

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.121.319447DOI Listing
June 2021

Genetic deletion of Nox4 enhances cancerogen-induced formation of solid tumors.

Proc Natl Acad Sci U S A 2021 03;118(11)

Institute for Cardiovascular Physiology, Goethe University, 60590 Frankfurt am Main, Germany;

Reactive oxygen species (ROS) can cause cellular damage and promote cancer development. Besides such harmful consequences of overproduction of ROS, all cells utilize ROS for signaling purposes and stabilization of cell homeostasis. In particular, the latter is supported by the NADPH oxidase 4 (Nox4) that constitutively produces low amounts of HO By that mechanism, Nox4 forces differentiation of cells and prevents inflammation. We hypothesize a constitutive low level of HO maintains basal activity of cellular surveillance systems and is unlikely to be cancerogenic. Utilizing two different murine models of cancerogen-induced solid tumors, we found that deletion of Nox4 promotes tumor formation and lowers recognition of DNA damage. Nox4 supports phosphorylation of H2AX (γH2AX), a prerequisite of DNA damage recognition, by retaining a sufficiently low abundance of the phosphatase PP2A in the nucleus. The underlying mechanism is continuous oxidation of AKT by Nox4. Interaction of oxidized AKT and PP2A captures the phosphatase in the cytosol. Absence of Nox4 facilitates nuclear PP2A translocation and dephosphorylation of γH2AX. Simultaneously AKT is left phosphorylated. Thus, in the absence of Nox4, DNA damage is not recognized and the increased activity of AKT supports proliferation. The combination of both events results in genomic instability and promotes tumor formation. By identifying Nox4 as a protective source of ROS in cancerogen-induced cancer, we provide a piece of knowledge for understanding the role of moderate production of ROS in preventing the initiation of malignancies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2020152118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980388PMC
March 2021

Nox4-dependent upregulation of S100A4 after peripheral nerve injury modulates neuropathic pain processing.

Free Radic Biol Med 2021 05 28;168:155-167. Epub 2021 Mar 28.

Institute of Pharmacology and Clinical Pharmacy, Goethe University, 60438 Frankfurt am Main, Germany. Electronic address:

Previous studies suggested that reactive oxygen species (ROS) produced by NADPH oxidase 4 (Nox4) affect the processing of neuropathic pain. However, mechanisms underlying Nox4-dependent pain signaling are incompletely understood. In this study, we aimed to identify novel Nox4 downstream interactors in the nociceptive system. Mice lacking Nox4 specifically in sensory neurons were generated by crossing Advillin-Cre mice with Nox4 mice. Tissue-specific deletion of Nox4 in sensory neurons considerably reduced mechanical hypersensitivity and neuronal action potential firing after peripheral nerve injury. Using a proteomic approach, we detected various proteins that are regulated in a Nox4-dependent manner after injury, including the small calcium-binding protein S100A4. Immunofluorescence staining and Western blot experiments confirmed that S100A4 expression is massively up-regulated in peripheral nerves and dorsal root ganglia after injury. Furthermore, mice lacking S100A4 showed increased mechanical hypersensitivity after peripheral nerve injury and after delivery of a ROS donor. Our findings suggest that S100A4 expression is up-regulated after peripheral nerve injury in a Nox4-dependent manner and that deletion of S100A4 leads to an increased neuropathic pain hypersensitivity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2021.03.021DOI Listing
May 2021

A hierarchical regulatory network analysis of the vitamin D induced transcriptome reveals novel regulators and complete VDR dependency in monocytes.

Sci Rep 2021 03 22;11(1):6518. Epub 2021 Mar 22.

Institute for Cardiovascular Physiology, Medical Faculty, Goethe University, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany.

The transcription factor vitamin D receptor (VDR) is the high affinity nuclear target of the biologically active form of vitamin D (1,25(OH)D). In order to identify pure genomic transcriptional effects of 1,25(OH)D, we used VDR cistrome, transcriptome and open chromatin data, obtained from the human monocytic cell line THP-1, for a novel hierarchical analysis applying three bioinformatics approaches. We predicted 75.6% of all early 1,25(OH)D-responding (2.5 or 4 h) and 57.4% of the late differentially expressed genes (24 h) to be primary VDR target genes. VDR knockout led to a complete loss of 1,25(OH)D-induced genome-wide gene regulation. Thus, there was no indication of any VDR-independent non-genomic actions of 1,25(OH)D modulating its transcriptional response. Among the predicted primary VDR target genes, 47 were coding for transcription factors and thus may mediate secondary 1,25(OH)D responses. CEBPA and ETS1 ChIP-seq data and RNA-seq following CEBPA knockdown were used to validate the predicted regulation of secondary vitamin D target genes by both transcription factors. In conclusion, a directional network containing 47 partly novel primary VDR target transcription factors describes secondary responses in a highly complex vitamin D signaling cascade. The central transcription factor VDR is indispensable for all transcriptome-wide effects of the nuclear hormone.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-86032-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985518PMC
March 2021

Vascular biotransformation of organic nitrates is independent of cytochrome P450 monooxygenases.

Br J Pharmacol 2021 04 18;178(7):1495-1506. Epub 2021 Feb 18.

Institute for Cardiovascular Physiology, Faculty of Medicine, Goethe University, Frankfurt am Main, Germany.

Background And Purpose: Organic nitrates such as nitroglycerin (NTG) or pentaerythritol tetranitrate (PETN) have been used for over a century in the treatment of angina or ischaemic heart disease. These compounds are prodrugs which release their nitrovasodilators upon enzymic bioactivation by aldehyde dehydrogenase (ALDH2) or cytochromes P450 (CYP). Whereas ALDH2 is known to directly activate organic nitrates in vessels, the contribution of vascular CYPs is unknown and was studied here.

Experimental Approach: As all CYPs depend on cytochrome P450 reductase (POR) as electron donor, we generated a smooth muscle cell-specific, inducible knockout mouse of POR (smcPOR ) to investigate the contribution of POR/CYP to vascular biotransformation of organic nitrates.

Key Results: Microsomes containing recombinant CYPs expressed in human vascular tissues released nitrite from NTG and PETN with CYP2C9 and CYP2C8 being most efficient. SFK525, a CYP suicide inhibitor, blocked this effect. smcPOR mice exhibited no obvious cardiovascular phenotype (normal cardiac weight and endothelium-dependent relaxation) and plasma and vascular nitrite production was similar to control (CTL) animals. NTG- and PETN-induced relaxation of isolated endothelium-intact or endothelium-denuded vessels were identical between CTL and smcPOR . Likewise, nitrite release from organic nitrates in aortic rings was not affected by deletion of POR in smooth muscle cells (SMCs). In contrast, inhibition of ALDH2 by benomyl (10 μM) inhibited NTG-induced nitrite production and relaxation. Deletion of POR did not modulate this response.

Conclusions And Implications: Our data suggest that metabolism by vascular CYPs does not contribute to the pharmacological function of organic nitrates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/bph.15362DOI Listing
April 2021

DGK and DZHK position paper on genome editing: basic science applications and future perspective.

Basic Res Cardiol 2021 01 15;116(1). Epub 2021 Jan 15.

Institute of Developmental Genetics, Helmholtz Zentrum München Germany, Technische Universität München-Weihenstephan, Neuherberg, Munich, Germany.

For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00395-020-00839-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810637PMC
January 2021

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

Circulation 2021 Mar 14;143(9):935-948. Epub 2020 Dec 14.

Institute for Vascular Signalling (S-I.B., J.H., J.W., M.K.D., V.R., F.D.L., B.F., S.Z., A.K., A.F.O.J., I.F.), Goethe University, Frankfurt am Main, Germany.

Background: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (HS), that exert their biological actions via cysteine -sulfhydration of target proteins. This study set out to map the "-sulfhydrome" (ie, the spectrum of proteins targeted by HS) in human endothelial cells.

Methods: Liquid chromatography with tandem mass spectrometry was used to identify -sulfhydrated cysteines in endothelial cell proteins and β3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction.

Results: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing HS donor, SG1002. The endothelial cell "-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on β3 integrin in detail we found that -sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the β leg. β3 integrin -sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of -sulfhydration impaired interactions between β3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low HS generation, impaired flow-induced dilatation, and failure to detect β3 integrin -sulfhydration, all of which were rescued after the administration of an HS supplement.

Conclusions: Vascular disease is associated with marked changes in the -sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term HS supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.120.051877DOI Listing
March 2021

Vitamin D-A New Perspective in Treatment of Cerebral Vasospasm.

Neurosurgery 2021 02;88(3):674-685

Institute for Cardiovascular Physiology, Goethe-University, Frankfurt, Germany.

Background: Cerebral vasospasm (CVS) is a frequent complication after subarachnoid hemorrhage (SAH), with no sufficient therapy and a complex pathophysiology.

Objective: To explore the vitamin D system as a potential treatment for CVS.

Methods: 25-vitamin D3 levels tested between 2007 and 2015 and data of SAH patients admitted during the months with a peak vs nadir of VitD3 values were analyzed, retrospectively. We prospectively correlated VitD3 and vasospasm/outcome data in SAH patients admitted in 2017. An experimental mice SAH model and cell culture model were used to investigate the effect of 1,25-dihydroxyvitamin D3 (1,25-VitD3). Additionally, the mediators acting in the VitD mechanism were researched and detected.

Results: Based on the retrospective analysis demonstrating an increased frequency of vasospasm in SAH patients during the low vitamin D period in winter, we started basic research experiments. Active 1,25-VitD3 hormone attenuated CVS, neurological deficit, and inflammation after intrathecal blood injection in mice. Deletion of the vitamin D receptor in the endothelium or in myeloid cells decreased the protective 1,25-VitD3 effect. Co-culture experiments of myeloid and endothelial cells with blood confirmed the anti-inflammatory 1,25-VitD3 effect but also revealed an induction of stroma-cell-derived factor 1α (SDF1α), vascular endothelial growth factor, and endothelial nitric oxide synthase by 1,25-VitD3. In mice, SDF1α mimicked the protective effect of 1,25-VitD3 against CVS. From bench to bedside, CVS severity was inversely correlated with vitamin D plasma level, prospectively. Patients with more severe CVS exhibited attenuated expression of SDF1α and 1,25-VitD3-responsive genes on circulating myeloid cells.

Conclusion: 1,25-VitD3 attenuates CVS after SAH by inducing SDF1α. However, VitD administration should be tested as optional treatment to prevent CVS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/neuros/nyaa484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884149PMC
February 2021

ZNF354C is a transcriptional repressor that inhibits endothelial angiogenic sprouting.

Sci Rep 2020 11 5;10(1):19079. Epub 2020 Nov 5.

Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany.

Zinc finger proteins (ZNF) are a large group of transcription factors with diverse functions. We recently discovered that endothelial cells harbour a specific mechanism to limit the action of ZNF354C, whose function in endothelial cells is unknown. Given that ZNF354C has so far only been studied in bone and tumour, its function was determined in endothelial cells. ZNF354C is expressed in vascular cells and localises to the nucleus and cytoplasm. Overexpression of ZNF354C in human endothelial cells results in a marked inhibition of endothelial sprouting. RNA-sequencing of human microvascular endothelial cells with and without overexpression of ZNF354C revealed that the protein is a potent transcriptional repressor. ZNF354C contains an active KRAB domain which mediates this suppression as shown by mutagenesis analysis. ZNF354C interacts with dsDNA, TRIM28 and histones, as observed by proximity ligation and immunoprecipitation. Moreover, chromatin immunoprecipitation revealed that the ZNF binds to specific endothelial-relevant target-gene promoters. ZNF354C suppresses these genes as shown by CRISPR/Cas knockout and RNAi. Inhibition of endothelial sprouting by ZNF354C is dependent on the amino acids DV and MLE of the KRAB domain. These results demonstrate that ZNF354C is a repressive transcription factor which acts through a KRAB domain to inhibit endothelial angiogenic sprouting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-76193-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645770PMC
November 2020

Deletion of NoxO1 limits atherosclerosis development in female mice.

Redox Biol 2020 10 4;37:101713. Epub 2020 Sep 4.

Institute for Cardiovascular Physiology, Goethe-University, Theodor-Stern Kai 7, 60590, Frankfurt Am Main, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Rhein Main, Theodor-Stern Kai 7, 60590, Frankfurt Am Main, Germany. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2020.101713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502371PMC
October 2020

NADPH oxidase subunit NOXO1 is a target for emphysema treatment in COPD.

Nat Metab 2020 06 8;2(6):532-546. Epub 2020 Jun 8.

Department of Surgery, Justus-Liebig University, Giessen, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42255-020-0215-8DOI Listing
June 2020

Aging-regulated anti-apoptotic long non-coding RNA Sarrah augments recovery from acute myocardial infarction.

Nat Commun 2020 04 27;11(1):2039. Epub 2020 Apr 27.

Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam, the Netherlands.

Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-15995-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184724PMC
April 2020

The endocannabinoid anandamide has an anti-inflammatory effect on CCL2 expression in vascular smooth muscle cells.

Basic Res Cardiol 2020 04 22;115(3):34. Epub 2020 Apr 22.

Fachbereich Medizin, Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, 60590, Frankfurt, Germany.

Endocannabinoids are important lipid-signaling mediators. Both protective and deleterious effects of endocannabinoids in the cardiovascular system have been reported but the mechanistic basis for these contradicting observations is unclear. We set out to identify anti-inflammatory mechanisms of endocannabinoids in the murine aorta and in human vascular smooth muscle cells (hVSMC). In response to combined stimulation with cytokines, IL-1β and TNFα, the murine aorta released several endocannabinoids, with anandamide (AEA) levels being the most significantly increased. AEA pretreatment had profound effects on cytokine-induced gene expression in hVSMC and murine aorta. As revealed by RNA-Seq analysis, the induction of a subset of 21 inflammatory target genes, including the important cytokine CCL2 was blocked by AEA. This effect was not mediated through AEA-dependent interference of the AP-1 or NF-κB pathways but rather through an epigenetic mechanism. In the presence of AEA, ATAC-Seq analysis and chromatin-immunoprecipitations revealed that CCL2 induction was blocked due to increased levels of H3K27me3 and a decrease of H3K27ac leading to compacted chromatin structure in the CCL2 promoter. These effects were mediated by recruitment of HDAC4 and the nuclear corepressor NCoR1 to the CCL2 promoter. This study therefore establishes a novel anti-inflammatory mechanism for the endogenous endocannabinoid AEA in vascular smooth muscle cells. Furthermore, this work provides a link between endogenous endocannabinoid signaling and epigenetic regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00395-020-0793-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176595PMC
April 2020

Thymosin beta-4x LINCs SPAAR to its non-coding function.

Cardiovasc Res 2020 10;116(12):1927-1928

Institute für Kardiovaskuläre Physiologie, Fachbereich Medizin der Goethe-Universität, Theodor-Stern Kai 7, 60590 Frankfurt am Main, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cvr/cvaa098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8599718PMC
October 2020

Glucose-Stimulated Insulin Secretion Fundamentally Requires HO Signaling by NADPH Oxidase 4.

Diabetes 2020 07 3;69(7):1341-1354. Epub 2020 Apr 3.

Department of Mitochondrial Physiology, No. 75, Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic

NADPH facilitates glucose-stimulated insulin secretion (GSIS) in pancreatic islets (PIs) of β-cells through an as yet unknown mechanism. We found NADPH oxidase isoform 4 (NOX4) to be the main producer of cytosolic HO, which is essential for GSIS; an increase in ATP alone was insufficient for GSIS. The fast GSIS phase was absent from PIs from NOX4-null, β-cell-specific knockout mice (NOX4βKO) (though not from NOX2 knockout mice) and from NOX4-silenced or catalase-overexpressing INS-1E cells. Lentiviral NOX4 overexpression or HO rescued GSIS in PIs from NOX4βKO mice. NOX4 silencing suppressed Ca oscillations, and the patch-clamped K channel opened more frequently when glucose was high. Mitochondrial HO, decreasing upon GSIS, provided alternative redox signaling when 2-oxo-isocaproate or fatty acid oxidation formed superoxides through electron-transfer flavoprotein:Q-oxidoreductase. Unlike GSIS, such insulin secretion was blocked with mitochondrial antioxidant SkQ1. Both NOX4 knockout and NOX4βKO mice exhibited impaired glucose tolerance and peripheral insulin resistance. Thus, the redox signaling previously suggested to cause β-cells to self-check hypothetically induces insulin resistance when it is absent. In conclusion, increases in ATP and HO constitute an essential signal that switches on insulin exocytosis for glucose and branched-chain oxoacids as secretagogues (it does so partially for fatty acids). Redox signaling could be impaired by cytosolic antioxidants; hence, those targeting mitochondria should be preferred for clinical applications to treat (pre)diabetes at any stage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db19-1130DOI Listing
July 2020

Shear stress regulates cystathionine γ lyase expression to preserve endothelial redox balance and reduce membrane lipid peroxidation.

Redox Biol 2020 01 13;28:101379. Epub 2019 Nov 13.

Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt am Main, Germany; German Center of Cardiovascular Research (DZHK), Partner site RheinMain, Frankfurt am Main, Germany. Electronic address:

Cystathionine γ lyase (CSE) is the major source of hydrogen sulfide-derived species (HS) in endothelial cells and plays an important role in protecting against atherosclerosis. Here we investigated the molecular mechanisms underlying the regulation of CSE expression in endothelial cells by fluid shear stress/flow. Fluid shear stress decreased CSE expression in human and murine endothelial cells and was negatively correlated with the transcription factor Krüppel-like factor (KLF) 2. CSE was identified as a direct target of the KLF2-regulated microRNA, miR-27b and high expression of CSE in native human plaque-derived endothelial cells, was also inversely correlated with KLF2 and miR-27b levels. One consequence of decreased CSE expression was the loss of Prx6 sulfhydration (on Cys47), which resulted in Prx6 hyperoxidation, decamerization and inhibition, as well as a concomitant increase in endothelial cell reactive oxygen species and lipid membrane peroxidation. HS supplementation in vitro was able to reverse the redox state of Prx6. Statin therapy, which is known to activate KLF2, also decreased CSE expression but increased CSE activity by preventing its phosphorylation on Ser377. As a result, the sulfhydration of Prx6 was partially restored in samples from plaque containing arteries from statin-treated donors. Taken together, the regulation of CSE expression by shear stress/disturbed flow is dependent on KLF2 and miR-27b. Moreover, in murine and human arteries CSE acts to maintain endothelial redox balance at least partly by targeting Prx6 to prevent its decamerization and inhibition of its peroxidase activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.redox.2019.101379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880097PMC
January 2020

Narciclasine inhibits angiogenic processes by activation of Rho kinase and by downregulation of the VEGF receptor 2.

J Mol Cell Cardiol 2019 10 2;135:97-108. Epub 2019 Aug 2.

Institute of Pharmaceutical Biology, Faculty of Biochemistry, Chemistry and Pharmacy, Goethe University, Frankfurt, Germany. Electronic address:

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 (100-300 nM) effectively inhibited the proliferation, undirected and directed migration, network formation and angiogenic sprouting of human primary endothelial cells. Moreover, narciclasine (1 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yjmcc.2019.08.001DOI Listing
October 2019

Pleiotropic effects of laminar flow and statins depend on the Krüppel-like factor-induced lncRNA MANTIS.

Eur Heart J 2019 08;40(30):2523-2533

Institute for Cardiovascular Physiology, Goethe University, Theodor-Stern-Kai 7, Frankfurt, Germany.

Aims: To assess the functional relevance and therapeutic potential of the pro-angiogenic long non-coding RNA MANTIS in vascular disease development.

Methods And Results: RNA sequencing, CRISPR activation, overexpression, and RNAi demonstrated that MANTIS, especially its Alu-element, limits endothelial ICAM-1 expression in different types of endothelial cells. Loss of MANTIS increased endothelial monocyte adhesion in an ICAM-1-dependent manner. MANTIS reduced the binding of the SWI/SNF chromatin remodelling factor BRG1 at the ICAM-1 promoter. The expression of MANTIS was induced by laminar flow and HMG-CoA-reductase inhibitors (statins) through mechanisms involving epigenetic rearrangements and the transcription factors KLF2 and KLF4. Mutation of the KLF binding motifs in the MANTIS promoter blocked the flow-induced MANTIS expression. Importantly, the expression of MANTIS in human carotid artery endarterectomy material was lower compared with healthy vessels and this effect was prevented by statin therapy. Interestingly, the protective effects of statins were mediated in part through MANTIS, which was required to facilitate the atorvastatin-induced changes in endothelial gene expression. Moreover, the beneficial endothelial effects of statins in culture models (spheroid outgrowth, proliferation, telomerase activity, and vascular organ culture) were lost upon knockdown of MANTIS.

Conclusion: MANTIS is tightly regulated by the transcription factors KLF2 and KLF4 and limits the ICAM-1 mediated monocyte adhesion to endothelial cells and thus potentially atherosclerosis development in humans. The beneficial effects of statin treatment and laminar flow are dependent on MANTIS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/eurheartj/ehz393DOI Listing
August 2019

Tolerizing CTL by Sustained Hepatic PD-L1 Expression Provides a New Therapy Approach in Mouse Sepsis.

Theranostics 2019 16;9(7):2003-2016. Epub 2019 Mar 16.

Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, Frankfurt, Germany.

Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option. To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line and the mouse model of polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved by adenoviral infection and transposon-based gene transfer using hydrodynamic injection. We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers. These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7150/thno.28057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485280PMC
April 2020

The polarity protein Scrib limits atherosclerosis development in mice.

Cardiovasc Res 2019 Dec;115(14):1963-1974

Institute for Cardiovascular Physiology, Goethe-University, Theodor-Stern Kai 7, Frankfurt, Frankfurt am Main, Germany.

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, βPix) 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cvr/cvz093DOI Listing
December 2019
-->