18 results found
Peghaire C, Dufton N, Lang M, et al., 2019, The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature, Nature Communications, Vol: 10, Pages: 1-17, ISSN: 2041-1723
Endothelial cells actively maintain an anti-thrombotic environment; loss of this protective function may lead to thrombosis and systemic coagulopathy. The transcription factor ERG is essential to maintain endothelial homeostasis. Here we show that inducible endothelial ERG deletion (ErgiEC-KO) in mice is associated with spontaneous thrombosis, hemorrhages and systemic coagulopathy. We find that ERG drives transcription of the anti-coagulant thrombomodulin (TM), as shown by reporter assays and chromatin immunoprecipitation. TM expression is regulated by shear stress (SS) via Krüppel-like factor 2 (KLF2). In vitro, ERG regulates TM expression under low SS conditions, by facilitating KLF2 binding to the TM promoter. However, ERG is dispensable for TM expression in high SS conditions. In ErgiEC-KO mice, TM expression is decreased in liver and lung microvasculature exposed to low SS but not in blood vessels exposed to high SS. Our study identifies an endogenous, vascular bed- specific anti-coagulant pathway in microvasculature exposed to low SS.
Issitt T, Bosseboeuf E, De Winter N, et al., 2019, Neuropilin-1 controls endothelial homeostasis by regulating mitochondrial function and iron-dependent oxidative stress via ABCB8, iScience, Vol: 11, Pages: 205-223, ISSN: 2589-0042
The transmembrane protein Neuropilin-1 (NRP1) promotes vascular endothelial growth factor (VEGF) and extracellular matrix signalling in endothelial cells (ECs). Although it is established that NRP1 is essential for angiogenesis, little is known about its role in EC homeostasis. Here, we report that NRP1 promotes mitochondrial function in ECs by preventing iron accumulation and iron-induced oxidative stress through a VEGF-independent mechanism in non-angiogenic ECs. Furthermore, NRP1-deficient ECs have reduced growth and show the hallmarks of cellular senescence. We show that a subcellular pool of NRP1 localises in mitochondria and interacts with the mitochondrial transporter ATP-binding-cassette-B8 (ABCB8). NRP1 loss reduces ABCB8 levels, resulting in iron accumulation, iron-induced mitochondrial superoxide production and iron-dependent EC senescence. Treatment of NRP1-deficient ECs with the mitochondria-targeted antioxidant compound mitoTEMPO or with the iron chelator deferoxamine restores mitochondrial activity, inhibits superoxide production and protects from cellular senescence. This finding identifies an unexpected role of NRP1 in EC homeostasis.
Nowak-Sliwinska P, Alitalo K, Allen E, et al., 2018, Consensus guidelines for the use and interpretation of angiogenesis assays, Angiogenesis, Vol: 21, Pages: 425-532, ISSN: 0969-6970
The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.
Dufton NP, peghaire CR, Osuna-Almagro L, et al., 2017, Dynamic regulation of canonical TGFβ signaling by endothelial transcription factor ERG protects from liver fibrogenesis, Nature Communications, Vol: 8, Pages: 1-14, ISSN: 2041-1723
The role of the endothelium in protecting from chronic liver disease and TGFβ-mediated fibrosis remains unclear. Here we describe how the endothelial transcription factor ETS-related gene (ERG) promotes liver homoeostasis by controlling canonical TGFβ-SMAD signalling, driving the SMAD1 pathway while repressing SMAD3 activity. Molecular analysis shows that ERG binds to SMAD3, restricting its access to DNA. Ablation of ERG expression results in endothelial-to-mesenchymal transition (EndMT) and spontaneous liver fibrogenesis in EC-specific constitutive hemi-deficient (ErgcEC-Het) and inducible homozygous deficient mice (ErgiEC-KO), in a SMAD3-dependent manner. Acute administration of the TNF-α inhibitor etanercept inhibits carbon tetrachloride (CCL4)-induced fibrogenesis in an ERG-dependent manner in mice. Decreased ERG expression also correlates with EndMT in tissues from patients with end-stage liver fibrosis. These studies identify a pathogenic mechanism where loss of ERG causes endothelial-dependent liver fibrogenesis via regulation of SMAD2/3. Moreover, ERG represents a promising candidate biomarker for assessing EndMT in liver disease.
Perbellini F, Watson SA, Scigliano M, et al., 2017, Investigation of cardiac fibroblasts using myocardial slices, Cardiovascular Research, Vol: 114, Pages: 77-89, ISSN: 1755-3245
AimsCardiac fibroblasts (CFs) are considered the principal regulators of cardiac fibrosis. Factors that influence CF activity are difficult to determine. When isolated and cultured in vitro, CFs undergo rapid phenotypic changes including increased expression of α-SMA. Here we describe a new model to study CFs and their response to pharmacological and mechanical stimuli using in vitro cultured mouse, dog and human myocardial slices.Methods and resultsUnloading of myocardial slices induced CF proliferation without α-SMA expression up to 7 days in culture. CFs migrating onto the culture plastic support or cultured on glass expressed αSMA within 3 days. The cells on the slice remained αSMA(−) despite transforming growth factor-β (20 ng/ml) or angiotensin II (200 µM) stimulation. When diastolic load was applied to myocardial slices using A-shaped stretchers, CF proliferation was significantly prevented at Days 3 and 7 (P < 0.001).ConclusionsMyocardial slices allow the study of CFs in a multicellular environment and may be used to effectively study mechanisms of cardiac fibrosis and potential targets.
Shah AV, Birdsey GM, Peghaire C, et al., 2017, The endothelial transcription factor ERG mediates Angiopoietin-1-dependent control of Notch signalling and vascular stability, Nature Communications, Vol: 8, Pages: 1-16, ISSN: 2041-1723
Notch and Angiopoietin-1 (Ang1)/Tie2 pathways are crucial for vascular maturation and stability. Here we identify the transcription factor ERG as a key regulator of endothelial Notch signalling. We show that ERG controls the balance between Notch ligands by driving Delta-like ligand 4 (Dll4) while repressing Jagged1 (Jag1) expression. In vivo, this regulation occurs selectively in the maturing plexus of the mouse developing retina, where Ang1/Tie2 signalling is active. We find that ERG mediates Ang1-dependent regulation of Notch ligands and is required for the stabilizing effects of Ang1 in vivo. We show that Ang1 induces ERG phosphorylation in a phosphoinositide 3-kinase (PI3K)/Akt-dependent manner, resulting in ERG enrichment at Dll4 promoter and multiple enhancers. Finally, we demonstrate that ERG directly interacts with Notch intracellular domain (NICD) and β-catenin and is required for Ang1-dependent β-catenin recruitment at the Dll4 locus. We propose that ERG coordinates Ang1, β-catenin and Notch signalling to promote vascular stability.
Beuerle MG, Dufton NP, Randi AM, et al., 2016, Molecular dynamics studies on the DNA-binding process of ERG, Molecular BioSystems, Vol: 12, Pages: 3600-3610, ISSN: 1742-206X
The ETS family of transcription factors regulate gene targets by binding to a core GGAA DNA-sequence. The ETS factor ERG is required for homeostasis and lineage-specific functions in endothelial cells, some subset of haemopoietic cells and chondrocytes; its ectopic expression is linked to oncogenesis in multiple tissues. To date details of the DNA-binding process of ERG including DNA-sequence recognition outside the core GGAA-sequence are largely unknown. We combined available structural and experimental data to perform molecular dynamics simulations to study the DNA-binding process of ERG. In particular we were able to reproduce the ERG DNA-complex with a DNA-binding simulation starting in an unbound configuration with a final root-mean-square-deviation (RMSD) of 2.1 Å to the core ETS domain DNA-complex crystal structure. This allowed us to elucidate the relevance of amino acids involved in the formation of the ERG DNA-complex and to identify Arg385 as a novel key residue in the DNA-binding process. Moreover we were able to show that water-mediated hydrogen bonds are present between ERG and DNA in our simulations and that those interactions have the potential to achieve sequence recognition outside the GGAA core DNA-sequence. The methodology employed in this study shows the promising capabilities of modern molecular dynamics simulations in the field of protein DNA-interactions.
Tornavaca O, Chia M, Dufton N, et al., 2015, ZO-1 CONTROLS ENDOTHELIAL ADHERENS JUNCTIONS, CELL-CELL TENSION, ANGIOGENESIS AND BARRIER FORMATION, ANTICANCER RESEARCH, Vol: 35, Pages: 4304-4305, ISSN: 0250-7005
Dowsett L, Piper S, Slaviero A, et al., 2015, Endothelial dimethylarginine dimethylaminohydrolase 1 Is an important regulator of angiogenesis but does not regulate vascular reactivity or hemodynamic homeostasis, Circulation, Vol: 131, Pages: 2217-2225, ISSN: 0009-7322
Background—Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthesis and a risk factor for cardiovascular disease. Dimethylarginine dimethylaminohydrolase (DDAH) enzymes are responsible for ADMA breakdown. It has been reported that endothelial DDAH1 accounts for the majority of ADMA metabolism. However, we and others have shown strong DDAH1 expression in a range of nonendothelial cell types, suggesting that the endothelium is not the only site of metabolism. We have developed a new endothelium-specific DDAH1 knockout mouse (DDAH1En−/−) to investigate the significance of endothelial ADMA in cardiovascular homeostasis.Methods and Results—DDAH1 deletion in the DDAH1En−/− mouse was mediated by Tie-2 driven Cre expression. DDAH1 deletion was confirmed through immunocytochemistry, whereas Western blotting showed that DDAH1 remained in the kidney and liver, confirming expression in nonendothelial cells. Plasma ADMA was unchanged in DDAH1En−/− mice, and cultured aortas released amounts of ADMA to similar to controls. Consistent with these observations, vasoreactivity ex vivo and hemodynamics in vivo were unaltered in DDAH1En−/− mice. In contrast, we observed significantly impaired angiogenic responses both ex vivo and in vivo.Conclusions—We demonstrate that endothelial DDAH1 is not a critical determinant of plasma ADMA, vascular reactivity, or hemodynamic homeostasis. DDAH1 is widely expressed in a range of vascular and nonvascular cell types; therefore, the additive effect of DDAH1 expression in multiple organ systems determines plasma ADMA concentrations. Endothelial deletion of DDAH1 profoundly impairs the angiogenic capacity of endothelial cells, indicating that intracellular ADMA is a critical determinant of endothelial cell response.
Tornavaca O, Chia M, Dufton N, et al., 2015, ZO-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation, JOURNAL OF CELL BIOLOGY, Vol: 208, Pages: 821-838, ISSN: 0021-9525
Birdsey GM, Shah AV, Dufton N, et al., 2015, The endothelial transcription factor ERG promotes vascular stability and growth through Wnt/beta-catenin signaling, Developmental Cell, Vol: 32, Pages: 82-96, ISSN: 1534-5807
Blood vessel stability is essential for embryonic development; in the adult, many diseases are associated with loss of vascular integrity. The ETS transcription factor ERG drives expression of VE-cadherin and controls junctional integrity. We show that constitutive endothelial deletion of ERG (ErgcEC-KO) in mice causes embryonic lethality with vascular defects. Inducible endothelial deletion of ERG (ErgiEC-KO) results in defective physiological and pathological angiogenesis in the postnatal retina and tumors, with decreased vascular stability. ERG controls the Wnt/β-catenin pathway by promoting β-catenin stability, through signals mediated by VE-cadherin and the Wnt receptor Frizzled-4. Wnt signaling is decreased in ERG-deficient endothelial cells; activation of Wnt signaling with lithium chloride, which stabilizes β-catenin levels, corrects vascular defects in ErgcEC-KO embryos. Finally, overexpression of ERG in vivo reduces permeability and increases stability of VEGF-induced blood vessels. These data demonstrate that ERG is an essential regulator of angiogenesis and vascular stability through Wnt signaling.
Dufton N, Natividad J, Verdu EF, et al., 2012, Hydrogen sulfide and resolution of acute inflammation: A comparative study utilizing a novel fluorescent probe, Scientific Reports, Vol: 2
Vong L, Ferraz JGP, Dufton N, et al., 2012, Up-Regulation of Annexin-A1 and Lipoxin A4 in Individuals with Ulcerative Colitis May Promote Mucosal Homeostasis, PLoS ONE, Vol: 7, Pages: e39244-e39244
Maderna P, Cottell DC, Toivonen T, et al., 2010, FPR2/ALX receptor expression and internalization are critical for lipoxin A<sub>4</sub>and annexin‐derived peptide‐stimulated phagocytosis, The FASEB Journal, Vol: 24, Pages: 4240-4249, ISSN: 0892-6638
Dufton N, Perretti M, 2010, Therapeutic anti-inflammatory potential of formyl-peptide receptor agonists, Pharmacology & Therapeutics, Vol: 127, Pages: 175-188, ISSN: 0163-7258
Dufton N, Hannon R, Brancaleone V, et al., 2010, Anti-Inflammatory Role of the Murine Formyl-Peptide Receptor 2: Ligand-Specific Effects on Leukocyte Responses and Experimental Inflammation, JOURNAL OF IMMUNOLOGY, Vol: 184, Pages: 2611-2619, ISSN: 0022-1767
Maione F, Paschalidis N, Mascolo N, et al., 2009, Interleukin 17 sustains rather than induces inflammation, Biochemical Pharmacology, Vol: 77, Pages: 878-887, ISSN: 0006-2952
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