62 results found
Wiggins BG, Wang Y-F, Burke A, et al., 2023, Endothelial sensing of AHR ligands regulates intestinal homeostasis, Nature, Pages: 1-32, ISSN: 0028-0836
Endothelial cells (ECs) line the blood and lymphatic vasculature, and act as an essential physical barrier, control nutrient transport, facilitate tissue immunosurveillance, and coordinate angiogenesis/ lymphangiogenesis1,2. In the intestine, dietary and microbial cues are particularly important in the regulation of organ homeostasis. However, whether enteric ECs actively sense and integrate such signals is currently unknown. Here, we show that the aryl hydrocarbon receptor (AHR) acts as a critical node for EC-sensing of dietary metabolites in adult mice and human primary ECs. We first established a comprehensive single-cell endothelial atlas of the mouse small intestine, uncovering the cellular complexity and functional heterogeneity of blood and lymphatic ECs. Analyses of AHR mediated responses at single-cell resolution identified tissue-protective transcriptional signatures and regulatory networks promoting cellular quiescence and vascular normalcy at steady state. Endothelial AHR-deficiency in adult mice resulted in dysregulated inflammatory responses, and the initiation of proliferative pathways. Furthermore, endothelial sensing of dietary AHR ligands was required for optimal protection against enteric infection. In human ECs, AHR signalling promoted quiescence and restrained activation by inflammatory mediators. Together, our data provide a comprehensive dissection of the impact of environmental sensing across the spectrum of enteric endothelia, demonstrating that endothelial AHR signalling integrates dietary cues to maintain tissue homeostasis by promoting EC quiescence and vascular normalcy.
Schafer CM, Martin-Almedina S, Kurylowicz K, et al., 2023, Cytokine-mediated degradation of the transcription factor ERG impacts the pulmonary vascular response to systemic inflammatory challenge, Arteriosclerosis, Thrombosis and Vascular Biology, Vol: 43, Pages: 1412-1428, ISSN: 1079-5642
BACKGROUND: During infectious diseases, proinflammatory cytokines transiently destabilize interactions between adjacent vascular endothelial cells (ECs) to facilitate the passage of immune molecules and cells into tissues. However, in the lung, the resulting vascular hyperpermeability can lead to organ dysfunction. Previous work identified the transcription factor ERG (erythroblast transformation-specific-related gene) as a master regulator of endothelial homeostasis. Here we investigate whether the sensitivity of pulmonary blood vessels to cytokine-induced destabilization is due to organotypic mechanisms affecting the ability of endothelial ERG to protect lung ECs from inflammatory injury. METHODS: Cytokine-dependent ubiquitination and proteasomal degradation of ERG were analyzed in cultured HUVECs (human umbilical vein ECs). Systemic administration of TNFα (tumor necrosis factor alpha) or the bacterial cell wall component lipopolysaccharide was used to cause a widespread inflammatory challenge in mice; ERG protein levels were assessed by immunoprecipitation, immunoblot, and immunofluorescence. Murine Erg deletion was genetically induced in ECs (Ergfl/fl;Cdh5[PAC]-CreERT2), and multiple organs were analyzed by histology, immunostaining, and electron microscopy. RESULTS: In vitro, TNFα promoted the ubiquitination and degradation of ERG in HUVECs, which was blocked by the proteasomal inhibitor MG132. In vivo, systemic administration of TNFα or lipopolysaccharide resulted in a rapid and substantial degradation of ERG within lung ECs but not ECs of the retina, heart, liver, or kidney. Pulmonary ERG was also downregulated in a murine model of influenza infection. Ergfl/fl;Cdh5(PAC)-CreERT2 mice spontaneously recapitulated aspects of inflammatory challenges, including lung-predominant vascular hyperpermeability, immune cell recruitment, and fibrosis. These phenotypes were associated with a lung-specific decrease in the expression of Tek-a gene target of
Nagy D, Maude H, Birdsey GM, et al., 2023, Liver sinusoidal endothelial transcription factors in metabolic homeostasis and disease, Journal of Molecular Endocrinology, Vol: 71, Pages: 1-20, ISSN: 0952-5041
Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells that form the liver microvasculature. LSECs maintain liver homeostasis, scavenging bloodborne molecules, regulating immune response, and actively promoting hepatic stellate cell quiescence. These diverse functions are underpinned by a suite of unique phenotypical attributes distinct from other blood vessels. In recent years, studies have begun to reveal the specific contributions of LSECs to liver metabolic homeostasis and how LSEC dysfunction associates with disease aetiology. This has been particularly evident in the context of non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome, which is associated with loss of key LSEC phenotypical characteristics and molecular identity. Comparative transcriptome studies of LSECs and other endothelial cells, together with rodent knockout models, have revealed that loss of LSEC identity through disruption of core transcription factor activity leads to impaired metabolic homeostasis and to hallmarks of liver disease. This review explores the current knowledge of LSEC transcription factors, covering their roles in LSEC development and maintenance of key phenotypic features, which, when disturbed, lead to loss of liver metabolic homeostasis and promote features of chronic liver diseases, such as non-alcoholic liver disease.
Greene D, Genomics England Research Consortium, Pirri D, et al., 2023, Genetic association analysis of 77,539 genomes reveals rare disease etiologies, Nature Medicine, Vol: 29, Pages: 679-688, ISSN: 1078-8956
The genetic etiologies of more than half of rare diseases remain unknown. Standardized genome sequencing and phenotyping of large patient cohorts provide an opportunity for discovering the unknown etiologies, but this depends on efficient and powerful analytical methods. We built a compact database, the ‘Rareservoir’, containing the rare variant genotypes and phenotypes of 77,539 participants sequenced by the 100,000 Genomes Project. We then used the Bayesian genetic association method BeviMed to infer associations between genes and each of 269 rare disease classes assigned by clinicians to the participants. We identified 241 known and 19 previously unidentified associations. We validated associations with ERG, PMEPA1 and GPR156 by searching for pedigrees in other cohorts and using bioinformatic and experimental approaches. We provide evidence that (1) loss-of-function variants in the Erythroblast Transformation Specific (ETS)-family transcription factor encoding gene ERG lead to primary lymphoedema, (2) truncating variants in the last exon of transforming growth factor-β regulator PMEPA1 result in Loeys–Dietz syndrome and (3) loss-of-function variants in GPR156 give rise to recessive congenital hearing impairment. The Rareservoir provides a lightweight, flexible and portable system for synthesizing the genetic and phenotypic data required to study rare disease cohorts with tens of thousands of participants.
Schafer CM, Martin-Almedina S, Kurylowicz K, et al., 2023, Cytokine-Mediated Degradation of the Transcription Factor ERG Impacts the Pulmonary Vascular Response to Systemic Inflammatory Challenge., bioRxiv
BACKGROUND: During infectious diseases, pro-inflammatory cytokines transiently destabilize interactions between adjacent vascular endothelial cells (ECs) to facilitate the passage of immune molecules and cells into tissues. However, in the lung the resulting vascular hyperpermeability can lead to organ dysfunction. Previous work identified the transcription factor ERG as a master regulator of endothelial homeostasis. Here we investigate whether the sensitivity of pulmonary blood vessels to cytokine-induced destabilization is due to organotypic mechanisms affecting the ability of endothelial ERG to protect lung ECs from inflammatory injury. METHODS: Cytokine-dependent ubiquitination and proteasomal degradation of ERG was analyzed in cultured Human Umbilical Vein ECs (HUVECs). Systemic administration of TNFα or the bacterial cell wall component lipopolysaccharide (LPS) was used to cause a widespread inflammatory challenge in mice; ERG protein levels were assessed by immunoprecipitation, immunoblot, and immunofluorescence. Murine Erg deletion was genetically induced in ECs ( Erg fl/fl ;Cdh5(PAC)Cre ERT2 ), and multiple organs were analyzed by histology, immunostaining, and electron microscopy. RESULTS: In vitro, TNFα promoted the ubiquitination and degradation of ERG in HUVECs, which was blocked by the proteasomal inhibitor MG132. In vivo, systemic administration of TNFα or LPS resulted in a rapid and substantial degradation of ERG within lung ECs, but not ECs of the retina, heart, liver, or kidney. Pulmonary ERG was also downregulated in a murine model of influenza infection. Erg fl/fl ;Cdh5(PAC)-Cre ERT2 mice spontaneously recapitulated aspects of inflammatory challenges, including lung-predominant vascular hyperpermeability, immune cell recruitment, and fibrosis. These phenotypes were associated with a lung-specific decrease in the expression of Tek , a gene target of ERG previously implicated in maintaining pulmonary vascular stability during infl
Kharwadkar R, Ulrich BJ, Chu M, et al., 2023, ERG functionally overlaps with other Ets proteins in promoting TH9 cell expression of Il9 during allergic lung inflammation., Journal of Immunology, ISSN: 0022-1767
CD4+ TH cells develop into subsets that are specialized in the secretion of particular cytokines to mediate restricted types of inflammation and immune responses. Among the subsets that promote development of allergic inflammatory responses, IL-9-producing TH9 cells are regulated by a number of transcription factors. We have previously shown that the E26 transformation-specific (Ets) family members PU.1 and Ets translocation variant 5 (ETV5) function in parallel to regulate IL-9. In this study we identified a third member of the Ets family of transcription factors, Ets-related gene (ERG), that mediates IL-9 production in TH9 cells in the absence of PU.1 and ETV5. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during murine TH9 polarization. Knockdown or knockout of ERG during murine or human TH9 polarization in vitro led to a decrease in IL-9 production in TH9 cells. Deletion of ERG in vivo had modest effects on IL-9 production in vitro or in vivo. However, in the absence of PU.1 and ETV5, ERG was required for residual IL-9 production in vitro and for IL-9 production by lung-derived CD4 T cells in a mouse model of chronic allergic airway disease. Thus, ERG contributes to IL-9 regulation in TH9 cells.
Gomez-Salinero J, Itkin T, Houghton S, et al., 2022, Cooperative ETS transcription factors enforce adult endothelial cell fate and cardiovascular homeostasis., Nature Cardiovascular Research, Vol: 1, Pages: 882-899, ISSN: 2731-0590
Current dogma dictates that, during adulthood, endothelial cells (ECs) are locked in an immutable stable homeostatic state. By contrast, herein we show that maintenance of EC fate and function are linked and active processes, which depend on the constitutive cooperativity of only two ETS transcription factors (TFs), ERG and Fli1. Although deletion of either ERG or Fli1 manifests subtle vascular dysfunction, their combined genetic deletion in adult ECs results in acute vasculopathy and multi-organ failure, due to loss of EC fate and integrity, hyperinflammation and spontaneous thrombosis, leading to death. ERG and Fli1 co-deficiency causes rapid transcriptional silencing of pan and organotypic vascular core genes, with dysregulation of inflammation and coagulation pathways. Vascular hyperinflammation leads to impaired hematopoiesis with myeloid skewing. Accordingly, enforced ERG and FLI1 expression in adult human mesenchymal stromal cells activates vascular programs and functionality, enabling in vivo engraftment of a perfusable vascular network. Genome-wide association study analysis identified vascular diseases that are associated with FLI1/ERG mutations. Constitutive expression of ERG and Fli1 upholds EC fate, physiological function and resilience in adult vasculature, whereas their functional loss can contribute to systemic human diseases.
McCracken I, Saginc G, He L, et al., 2022, Lack of evidence of ACE2 expression and replicative infection by SARS-CoV-2 in human endothelial cells, Publisher: Cold Spring Harbor Laboratory
Abstract: A striking feature of severe COVID-19 is thrombosis in large as well as small vessels of multiple organs. This has led to the assumption that SARS-CoV-2 virus directly infects and damages the vascular endothelium. However, endothelial expression of ACE2, the cellular receptor for SARS-CoV-2, has not been convincingly demonstrated. Interrogating human bulk and single-cell transcriptomic data, we found <jats:italic>ACE2</jats:italic> expression in endothelial cells to be extremely low or absent <jats:italic>in vivo</jats:italic> and not upregulated by exposure to inflammatory agents <jats:italic>in vitro</jats:italic>. Also, the endothelial chromatin landscape at the <jats:italic>ACE2</jats:italic> locus showed presence of repressive and absence of activation marks, suggesting that the gene is inactive in endothelial cells. Finally, we failed to achieve infection and replication of SARS-CoV-2 in cultured human endothelial cells, which were permissive to productive infection by coronavirus 229E that uses CD13 as the receptor. Our data suggest that SARS-Cov-2 is unlikely to infect endothelial cells directly; these findings are consistent with a scenario where endothelial injury is indirectly caused by the infection of neighbouring epithelial cells and/or due to systemic effects mediated by immune cells, platelets, complement activation, and/or proinflammatory cytokines
D'Amico G, Fernandez I, Gómez-Escudero J, et al., 2022, ERG activity is regulated by endothelial FAK coupling with TRIM25/USP9x in vascular patterning, Development, Vol: 149, ISSN: 0950-1991
Precise vascular patterning is critical for normal growth and development. The ERG transcription factor drives Delta like ligand 4 (DLL4)/Notch signalling and is thought to act as pivotal regulators of endothelial cell (EC) dynamics and developmental angiogenesis. However, molecular regulation of ERG activity remains obscure. Using a series of EC specific Focal Adhesion Kinase (FAK)-knockout (KO) and point-mutant FAK-knockin mice, we show that loss of ECFAK, its kinase activity or phosphorylation at FAK-Y397, but not FAK-Y861, reduces ERG and DLL4 expression levels together with concomitant aberrations in vascular patterning. Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins identified that endothelial nuclear-FAK interacts with the de-ubiquitinase USP9x and the ubiquitin ligase TRIM25 enzymes. Further in silico analysis corroborates that ERG interacts with USP9x and TRIM25. Moreover, ERG levels are reduced in FAKKO ECs via a ubiquitin-mediated post-translational modification programme involving USP9x and TRIM25. Re-expression of ERG in vivo and in vitro rescues the aberrant vessel sprouting defects observed in the absence of ECFAK. Our findings identify ECFAK as a regulator of retinal vascular patterning by controlling ERG protein degradation via TRIM25/USP9x.
McCracken IR, Saginc G, He L, et al., 2021, Lack of evidence of ACE2 expression and replicative infection by SARSCoV-2 in human endothelial cells, Circulation, Vol: 143, Pages: 865-868, ISSN: 0009-7322
Dufton NP, Peghaire CR, Osuna-Almagro L, et al., 2020, Dynamic regulation of canonical TGF beta signalling by endothelial transcription factor ERG protects from liver fibrogenesis (vol 31, pg 450, 2017), Nature Communications, Vol: 11, Pages: 1-1, ISSN: 2041-1723
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.
Garonna E, Botham KM, Birdsey GM, et al., 2019, Vascular Endothelial Growth Factor Receptor-2 Couples Cyclo-Oxygenase-2 with Pro-Angiogenic Actions of Leptin on Human Endothelial Cells (vol 6, e18823, 2011), PLOS ONE, Vol: 14, ISSN: 1932-6203
Kalna V, Yang Y, Peghaire C, et al., 2019, The transcription factor ERG regulates super-enhancers associated with an endothelial-specific gene expression program, Circulation Research, Vol: 124, Pages: 1337-1349, ISSN: 0009-7330
Rationale:The ETS (E-26 transformation-specific) transcription factor ERG (ETS-related gene) is essential for endothelial homeostasis, driving expression of lineage genes and repressing proinflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG’s homeostatic function is lineage-specific, because aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers).Objective:To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers.Methods and Results:Chromatin immunoprecipitation with high-throughput sequencing in human umbilical vein endothelial cells showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4 (Delta-like protein 4), CLDN5 (claudin-5), VWF (von Willebrand factor), and CDH5 (VE-cadherin). Comparison between human umbilical vein endothelial cell and prostate cancer TMPRSS2 (transmembrane protease, serine-2):ERG fusion-positive human prostate epithelial cancer cell line (VCaP) cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and MED (Mediator complex subunit)-1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 (GATA-binding protein 2) and AP-1 (activator protein 1) is significantly lower compared with super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in endothelial cells and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-assoc
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.
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.
Bauer A, Mylroie H, Thornton C, et al., 2016, Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis, Scientific Reports, Vol: 6, ISSN: 2045-2322
Angiogenesis is an essential physiological process and an important factor in diseasepathogenesis. However, its exploitation as a clinical target has achieved limited success and novelmolecular targets are required. Although heme oxygenase-1 (HO-1) acts downstream of vascularendothelial growth factor (VEGF) to modulate angiogenesis, knowledge of the mechanismsinvolved remains limited. We set out identify novel HO-1 targets involved in angiogenesis. HO-1depletion attenuated VEGF-induced human endothelial cell (EC) proliferation and tube formation.The latter response suggested a role for HO-1 in EC migration, and indeed HO-1 siRNA negativelyaffected directional migration of EC towards VEGF; a phenotype reversed by HO-1 overexpression.EC from Hmox1-/- mice behaved similarly. Microarray analysis of HO-1-depleted andcontrol EC exposed to VEGF identified cyclins A1 and E1 as HO-1 targets. Migrating HO-1-deficient EC showed increased p27, reduced cyclin A1 and attenuated cyclin-dependent kinase 2activity. In vivo, cyclin A1 siRNA inhibited VEGF-driven angiogenesis, a response reversed by AdHO-1.Proteomics identified structural protein vimentin as an additional VEGF-HO-1 target. HO-1depletion inhibited VEGF-induced calpain activity and vimentin cleavage, while vimentin silencingattenuated HO-1-driven proliferation. Thus, vimentin and cyclins A1 and E1 represent VEGFactivatedHO-1-dependent targets important for VEGF-driven angiogenesis.
Shah AV, Birdsey GM, Randi AM, 2016, Regulation of endothelial homeostasis, vascular development and angiogenesis by the transcription factor ERG, Vascular Pharmacology, Vol: 86, Pages: 3-13, ISSN: 1879-3649
Over the last few years, the ETS transcription factor ERG has emerged as a major regulator ofendothelial function. Multiple studies have shown that ERG plays a crucial role in promotingangiogenesis and vascular stability during development and after birth. In the maturevasculature ERG also functions to maintain endothelial homeostasis, by transactivatinggenes involved in key endothelial functions, whilst repressing expression of pro‐inflammatory genes. Its homeostatic role is lineage-specific, since ectopic expression of ERGin non-endothelial tissues such as prostate is detrimental and contributes to oncogenesis.This review summarises the main roles and pathways controlled by ERG in the vascularendothelium, its transcriptional targets and its functional partners and the emergingevidence on the pathways regulating ERG’s activity and expression.
Thornton CC, Al-Rashed F, Calay D, et al., 2016, Methotrexate-mediated activation of an AMPK-CREB-dependent pathway: a novel mechanism for vascular protection in chronic systemic inflammation, Annals of the Rheumatic Diseases, Vol: 75, Pages: 439-448, ISSN: 0003-4967
Aims Premature cardiovascular events complicate chronic inflammatory conditions. Low-dose weekly methotrexate (MTX), the most widely used disease-modifying drug for rheumatoid arthritis (RA), reduces disease-associated cardiovascular mortality. MTX increases intracellular accumulation of adenosine monophosphate (AMP) and 5-aminoimidazole-4-carboxamide ribonucleotide which activates AMP-activated protein kinase (AMPK). We hypothesised that MTX specifically protects the vascular endothelium against inflammatory injury via induction of AMPK-regulated protective genes.Methods/results In the (NZW×BXSB)F1 murine model of inflammatory vasculopathy, MTX 1 mg/kg/week significantly reduced intramyocardial vasculopathy and attenuated end-organ damage. Studies of human umbilical vein endothelial cells (HUVEC) and arterial endothelial cells (HAEC) showed that therapeutically relevant concentrations of MTX phosphorylate AMPKαThr172, and induce cytoprotective genes including manganese superoxide dismutase (MnSOD) and haem oxygenase-1 (HO-1). These responses were preserved when HUVECs were pretreated with tumour necrosis factor-α to mimic dysfunctional endothelium. Furthermore, MTX protected against glucose deprivation-induced endothelial apoptosis. Mechanistically, MTX treatment led to cyclic AMP response element-binding protein (CREB)Ser133 phosphorylation, while AMPK depletion attenuated this response and the induction of MnSOD and HO-1. CREB siRNA inhibited upregulation of both cytoprotective genes by MTX, while chromatin immunoprecipitation demonstrated CREB binding to the MnSOD promoter in MTX-treated EC. Likewise, treatment of (NZW×BXSB)F1 mice with MTX enhanced AMPKαThr172 phosphorylation and MnSOD, and reduced aortic intercellular adhesion molecule-1 expression.Conclusions These data suggest that MTX therapeutically conditions vascular endothelium via activation of AMPK-CREB. We propose that this mechanism contributes to the protection against
Birdsey GM, Shah AV, Randi AM, 2015, Regulation of vascular development and angiogenesis by the ETS transcription factor ERG, ACTA PHYSIOLOGICA, Vol: 215, Pages: 19-19, ISSN: 1748-1708
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.
Birdsey GM, Shah AV, Dufton N, et al., 2015, REGULATION OF VASCULAR DEVELOPMENT AND ANGIOGENESIS BY THE TRANSCRIPTION FACTOR ERG, Joint Meeting of the European-Society-for-Microcirculation (ESM) and European-Vascular-Biology-Organisation (EVBO), Publisher: KARGER, Pages: 17-18, ISSN: 1018-1172
Birdsey GM, Shah AV, Dufton N, et al., 2015, The endothelial transcription factor ERG promotes vascular stability and growth through Wnt/β-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 (Erg(cEC-KO)) in mice causes embryonic lethality with vascular defects. Inducible endothelial deletion of ERG (Erg(iEC-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 Erg(cEC-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.
Shah AV, Birdsey GM, Reynolds LE, et al., 2014, The endothelial transcription factor ERG promotes vascular stability and growth through Wnt/beta-catenin signaling, ANGIOGENESIS, Vol: 17, Pages: 715-715, ISSN: 0969-6970
Amsellem V, Dryden NH, Martinelli R, et al., 2014, ICAM-2 regulates vascular permeability and N-cadherin localization through ezrin-radixin-moesin (ERM) proteins and Rac-1 signalling, CELL COMMUNICATION AND SIGNALING, Vol: 12, ISSN: 1478-811X
Shapiro SE, Nowak AA, Wooding C, et al., 2014, The von Willebrand factor predicted unpaired cysteines are essential for secretion, JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Vol: 12, Pages: 246-254, ISSN: 1538-7933
Birdsey GM, Shah A, Reynolds L, et al., 2014, Regulation of vascular development and angiogenesis by the ETS transcription factor Erg through canonical Wnt signalling, ANGIOGENESIS, Vol: 17, Pages: 276-277, ISSN: 0969-6970
Kostourou V, Lechertier T, Reynolds LE, et al., 2013, FAK-heterozygous mice display enhanced tumour angiogenesis, Nature Communications, Vol: 4, ISSN: 2041-1723
Genetic ablation of endothelial focal adhesion kinase (FAK) can inhibit pathological angiogenesis, suggesting that loss of endothelial FAK is sufficient to reduce neovascularization. Here we show that reduced stromal FAK expression in FAK-heterozygous mice unexpectedly enhances both B16F0 and CMT19T tumour growth and angiogenesis. We further demonstrate that cell proliferation and microvessel sprouting, but not migration, are increased in serum-stimulated FAK-heterozygous endothelial cells. FAK-heterozygous endothelial cells display an imbalance in FAK phosphorylation at pY397 and pY861 without changes in Pyk2 or Erk1/2 activity. By contrast, serum-stimulated phosphorylation of Akt is enhanced in FAK-heterozygous endothelial cells and these cells are more sensitive to Akt inhibition. Additionally, low doses of a pharmacological FAK inhibitor, although too low to affect FAK autophosphorylation in vitro, can enhance angiogenesis ex vivo and tumour growth in vivo. Our results highlight a potential novel role for FAK as a nonlinear, dose-dependent regulator of angiogenesis where heterozygous levels of FAK enhance angiogenesis.
Birdsey GM, Dryden NH, Shah AV, et al., 2012, The transcription factor Erg regulates expression of HDAC6 and multiple pathways involved in endothelial cell migration and angiogenesis, 6th European Meeting on Vascular Biology and Medicine (EMVBM), Publisher: ELSEVIER SCIENCE INC, Pages: 348-349, ISSN: 1537-1891
Bauer A, Thornton CC, Mylroie H, et al., 2012, Investigation of the regulatory role of heme oxygenase-1 and its products in VEGF-mediated angiogenesis, 6th European Meeting on Vascular Biology and Medicine (EMVBM), Publisher: ELSEVIER SCIENCE INC, Pages: 345-345, ISSN: 1537-1891
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