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Cystathionine γ lyase (CSE) is the major source of hydrogen sulfide-derived species (H2Sn) 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. H2Sn 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.
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 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.
DGK and DZHK position paper on genome editing: basic science applications and future perspective
(2021)
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.
More than 97 percent of the transcribed RNA in mammalian cells is not coding for proteins. Among these are micro RNAs (miRs), transfer RNAs (tRNA) as well as ribosomal RNAs (rRNA) but also long non-coding RNAs (lncRNAs). This RNA class is only defined by its sequence length of more than 200 nucleotides and its lack of protein coding potential. The human genome encodes for more than 18.000 lncRNAs which contribute to gene expression control. Here, we discuss the function of these lncRNAs and how they modulate the angiogenic process of vessel growth.
Ribosome biogenesis is fundamental for cellular life, but surprisingly little is known about the underlying pathway. In eukaryotes a comprehensive collection of experimentally verified ribosome biogenesis factors (RBFs) exists only for Saccharomyces cerevisiae. Far less is known for other fungi, animals or plants, and insights are even more limited for archaea. Starting from 255 yeast RBFs, we integrated ortholog searches, domain architecture comparisons and, in part, manual curation to investigate the inventories of RBF candidates in 261 eukaryotes, 26 archaea and 57 bacteria. The resulting phylogenetic profiles reveal the evolutionary ancestry of the yeast pathway. The oldest core comprising 20 RBF lineages dates back to the last universal common ancestor, while the youngest 20 factors are confined to the Saccharomycotina. On this basis, we outline similarities and differences of ribosome biogenesis across contemporary species. Archaea, so far a rather uncharted domain, possess 38 well-supported RBF candidates of which some are known to form functional sub-complexes in yeast. This provides initial evidence that ribosome biogenesis in eukaryotes and archaea follows similar principles. Within eukaryotes, RBF repertoires vary considerably. A comparison of yeast and human reveals that lineage-specific adaptation via RBF exclusion and addition characterizes the evolution of this ancient pathway.
Epigenetic control of microsomal prostaglandin E synthase-1 by HDAC-mediated recruitment of p300
(2017)
Nonsteroidal anti-inflammatory drugs are the most widely used medicine to treat pain and inflammation, and to inhibit platelet function. Understanding the expression regulation of enzymes of the prostanoid pathway is of great medical relevance. Histone acetylation crucially controls gene expression. We set out to identify the impact of histone deacetylases (HDACs) on the generation of prostanoids and examine the consequences on vascular function. HDAC inhibition (HDACi) with the pan-HDAC inhibitor, vorinostat, attenuated prostaglandin (PG)E2 generation in the murine vasculature and in human vascular smooth muscle cells. In line with this, the expression of the key enzyme for PGE2 synthesis, microsomal PGE synthase-1 (PTGES1), was reduced by HDACi. Accordingly, the relaxation to arachidonic acid was decreased after ex vivo incubation of murine vessels with HDACi. To identify the underlying mechanism, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis were performed. These results suggest that HDACs are involved in the recruitment of the transcriptional activator p300 to the PTGES1 gene and that HDACi prevented this effect. In line with the acetyltransferase activity of p300, H3K27 acetylation was reduced after HDACi and resulted in the formation of heterochromatin in the PTGES1 gene. In conclusion, HDAC activity maintains PTGES1 expression by recruiting p300 to its gene.
Epigenetic marks critically control gene expression and thus the cellular activity state. The functions of many epigenetic modifiers in the vascular system have not yet been studied. We screened for histone modifiers in endothelial cells and observed a fairly high expression of the histone plant homeodomain finger protein 8 (PHF8). Given its high expression, we hypothesize that this histone demethylase is important for endothelial cell function. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. As a functional readout endothelial migration and tube formation was studied. PHF8 siRNA attenuated the capacity for migration and developing of capillary-like structures. Given the impact of PHF8 on cell cycle genes, endothelial E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4 but not E2F1 expression in endothelial cells. Consistently, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Conclusion: PHF8 by controlling E2F4 expression maintains endothelial function.
Oxidized phospholipids (oxPAPC) induce endothelial dysfunction and atherosclerosis. Here we show that oxPAPC induce a gene network regulating serine-glycine metabolism with the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) as a causal regulator using integrative network modeling and Bayesian network analysis in human aortic endothelial cells. The cluster is activated in human plaque material and by atherogenic lipoproteins isolated from plasma of patients with coronary artery disease (CAD). Single nucleotide polymorphisms (SNPs) within the MTHFD2-controlled cluster associate with CAD. The MTHFD2-controlled cluster redirects metabolism to glycine synthesis to replenish purine nucleotides. Since endothelial cells secrete purines in response to oxPAPC, the MTHFD2-controlled response maintains endothelial ATP. Accordingly, MTHFD2-dependent glycine synthesis is a prerequisite for angiogenesis. Thus, we propose that endothelial cells undergo MTHFD2-mediated reprogramming toward serine-glycine and mitochondrial one-carbon metabolism to compensate for the loss of ATP in response to oxPAPC during atherosclerosis.
Background: Unruptured intracranial aneurysm (UIA) poses a therapeutic dilemma in which the risk-benefit analysis of invasive intervention has to be balanced against the natural history of the disease. To date, there is no medical treatment to prevent aneurysm development and subsequent progression to rupture. We explored the vitamin D system because of its known anti-inflammatory and anti-tissue-remodeling effect as a potential treatment for UIA.
Methods: 25-vitaminD3 levels tested between 2008-2016 and data of SAH patients admitted during the months with a peak versus nadir of VitD3-values were analyzed, retrospectively. We prospectively correlated VitD3 with size and number of aneurysms at the rupture time in patients admitted between 2017-2019. An experimental mice shear stress model and cell culture model were used to investigate the effect of 1,25-dihydroxy-vitaminD3 (1,25-VitD3) and acting mediators in this mechanism.
Results: Based on the retrospective analysis demonstrating an increased frequency of aneurysm rupture rate in patients during the low vitamin D period in winter, we started the prospective study evaluating plasma vitamin D levels at admission. VitD levels were inversely correlated with aneurysm size as well as number of aneurysms. Low number of aneurysms was significantly associated with sufficient plasma Vitamin D level as an independent factor in a multivariate analysis.
From bedside back to bench, active 1,25-VitD3 hormone attenuated the natural history of remodeling in mice basilar artery. Deletion of the vitamin-D-receptor in myeloid cells decreased the protective 1,25-VitD3 effect. Cell-culture of vascular fibroblasts confirmed the anti-tissue remodeling effect of 1,25-VitD3.
Conclusion: 1,25-VitD3 attenuates aneurysm development and subsequent progression to rupture. However, VitD-administration should be tested as optional treatment in management of patients with UIA.
Propranolol as a potentially novel treatment of arteriovenous malformations: from bench to bedside
(2022)
Background: Propranolol is a non-selective blocker of the β-adrenergic receptor and has been used for treatment of proliferative infantile hemangiomas. The vasoconstrictive and antiangiogenic effects of propranolol led us to explore its potential application for the treatment of AVMs.
Methods: AVM tissue was cultured after surgical resection in the presence of 100μM propranolol or solvent DMSO. After incubation for 72 hours, tissue was harvested for testing. The expression levels of SDF1α, CXCR4, VEGF and HIF-1 was measured by rt-PCR. Furthermore, data of patients in 2 vascular centres harboring AVM was retrospectively interrogated for a time period of 20 years. The database included information about hemorrhage, AVM size and antihypertensive medication. Descriptive analyses were performed, focusing on the risk of hemorrhage, size of the lesion at presentation and clinical follow-up in patients on β-blocker medication versus those who were not.
Results: Among 483 patients, 73 (15%) were under β-blocker-treatment. 48% AVMs presented with hemorrhage at diagnosis. Patients under β-blocker-treatment had a lower risk of hemorrhage at the time of diagnosis in a univariate analysis (p<0,0001;OR13). Patients under β-blocker-treatment showed a significant higher chance for a lower Spetzler-Martin-grade ≤III (p<0,0001;OR6,5) and a lower risk for the presence of an associated aneurysm (p<0,0001;OR3,6).
Multivariate analysis including Spetzler-Martin-Grading, young age ≤50, presence of associated aneurysm and β-blocker-treatment showed reduced risk for hemorrhage under β-blocker-treatment (p<0,01,OR0,2).
The expression of CXCR4 was suppressed by propranolol most likely through the HIF-1-pathways. The gene-expression of vasculogenesis factors was decreased in with propranolol incubated AVMs.
Conclusion: β-Blocker medication seems to be associated with a decreased risk of AVM-related hemorrhage and AVM-size at presentation or during follow-up. Propranolol inhibits SDF1α-induced vasculogenesis by suppressing the expression of CXCR4 most likely through the HIF-1-pathways. Therefore, SDF1α/CXCR4 axis plays an important role in the vasculogenesis and migration of inflammatory cells in AVM lesions.