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.
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•Inducible deletion of endothelial ERG in mice causes defective angiogenesis•ERG controls vascular stability through Wnt/β-catenin signaling•β-catenin activation rescues the angiogenic defect in vivo in ERG-deficient mice•Overexpression of ERG in vivo stabilizes VEGF-induced angiogenesis
Birdsey, Shah et al. show that the endothelial ETS factor ERG controls Wnt/β-catenin signaling by promoting β-catenin stability, through pathways mediated by VE-cadherin and the Wnt receptor Frizzled-4. In vivo, ERG overexpression stabilizes VEGF-dependent angiogenesis. Thus, ERG is an essential regulator of angiogenesis and vascular stability through Wnt signaling.
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 (Erg
) and inducible homozygous deficient mice (Erg
), in a SMAD3-dependent manner. Acute administration of the TNF-α inhibitor etanercept inhibits carbon tetrachloride (CCL
)-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.The transcription factor ERG is key to endothelial lineage specification and vascular homeostasis. Here the authors show that ERG balances TGFβ signalling through the SMAD1 and SMAD3 pathways, protecting the endothelium from endothelial-to-mesenchymal transition and consequent liver fibrosis in mice via a SMAD3-dependent mechanism.
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-associated single nucleotide polymorphisms from genome-wide association studies lie within noncoding regions and perturb transcription factor recognition sequences in relevant cell types. Analysis of genome-wide association studies data shows significant enrichment of risk variants for cardiovascular disease and other diseases, at ERG endothelial enhancers and super-enhancers.
CONCLUSIONS:The transcription factor ERG promotes endothelial homeostasis via regulation of lineage-specific enhancers and super-enhancers. Enrichment of cardiovascular disease-associated single nucleotide polymorphisms at ERG super-enhancers suggests that ERG-dependent transcription modulates disease risk.
Intercellular junctions are crucial for mechanotransduction, but whether tight junctions contribute to the regulation of cell-cell tension and adherens junctions is unknown. Here, we demonstrate that ...the tight junction protein ZO-1 regulates tension acting on VE-cadherin-based adherens junctions, cell migration, and barrier formation of primary endothelial cells, as well as angiogenesis in vitro and in vivo. ZO-1 depletion led to tight junction disruption, redistribution of active myosin II from junctions to stress fibers, reduced tension on VE-cadherin and loss of junctional mechanotransducers such as vinculin and PAK2, and induced vinculin dissociation from the α-catenin-VE-cadherin complex. Claudin-5 depletion only mimicked ZO-1 effects on barrier formation, whereas the effects on mechanotransducers were rescued by inhibition of ROCK and phenocopied by JAM-A, JACOP, or p114RhoGEF down-regulation. ZO-1 was required for junctional recruitment of JACOP, which, in turn, recruited p114RhoGEF. ZO-1 is thus a central regulator of VE-cadherin-dependent endothelial junctions that orchestrates the spatial actomyosin organization, tuning cell-cell tension, migration, angiogenesis, and barrier formation.
Endothelial junctions control functions such as permeability, angiogenesis and contact inhibition. VE-Cadherin (VECad) is essential for the maintenance of intercellular contacts. In confluent ...endothelial monolayers, N-Cadherin (NCad) is mostly expressed on the apical and basal membrane, but in the absence of VECad it localizes at junctions. Both cadherins are required for vascular development. The intercellular adhesion molecule (ICAM)-2, also localized at endothelial junctions, is involved in leukocyte recruitment and angiogenesis.
In human umbilical vein endothelial cells (HUVEC), both VECad and NCad were found at nascent cell contacts of sub-confluent monolayers, but only VECad localized at the mature junctions of confluent monolayers. Inhibition of ICAM-2 expression by siRNA caused the appearance of small gaps at the junctions and a decrease in NCad junctional staining in sub-confluent monolayers. Endothelioma lines derived from WT or ICAM-2-deficient mice (IC2neg) lacked VECad and failed to form junctions, with loss of contact inhibition. Re-expression of full-length ICAM-2 (IC2 FL) in IC2neg cells restored contact inhibition through recruitment of NCad at the junctions. Mutant ICAM-2 lacking the binding site for ERM proteins (IC2 ΔERM) or the cytoplasmic tail (IC2 ΔTAIL) failed to restore junctions. ICAM-2-dependent Rac-1 activation was also decreased in these mutant cell lines. Barrier function, measured in vitro via transendothelial electrical resistance, was decreased in IC2neg cells, both in resting conditions and after thrombin stimulation. This was dependent on ICAM-2 signalling to the small GTPase Rac-1, since transendothelial electrical resistance of IC2neg cells was restored by constitutively active Rac-1. In vivo, thrombin-induced extravasation of FITC-labeled albumin measured by intravital fluorescence microscopy in the mouse cremaster muscle showed that permeability was increased in ICAM-2-deficient mice compared to controls.
These results indicate that ICAM-2 regulates endothelial barrier function and permeability through a pathway involving N-Cadherin, ERMs and Rac-1.
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.
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
deletion was genetically induced in ECs (
), and multiple organs were analyzed by histology, immunostaining, and electron microscopy.
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.
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
-a gene target of ERG previously implicated in maintaining pulmonary vascular stability during inflammation.
Collectively, our data highlight a unique role for ERG in pulmonary vascular function. We propose that cytokine-induced ERG degradation and subsequent transcriptional changes in lung ECs play critical roles in the destabilization of pulmonary blood vessels during infectious diseases.
The ETS transcription factor ERG is constitutively expressed in endothelial cells (EC) and acts as a master-regulator of endothelial function. ERG drives expression of genes which determine ...endothelial lineage and control homeostasis, such as VE-Cadherin, ICAM-2 and HDAC-6. ERG also represses expression of pro-inflammatory genes such as ICAM-1 and IL-8, through inhibition of NF-kB activity (1), thus acting as a gatekeeper for endothelial activation. Interestingly, ERG expression is down-regulated in EC by pro-inflammatory agents such as TNF-α, suggesting that down-regulation of ERG during inflammation is necessary to allow full NF-kB activation. This is in line with the decrease of ERG expression in the endothelium overlaying the “inflamed” regions of human atherosclerotic plaques (1). In vitro, ERG deletion in EC results in enhanced leukocyte adhesion, whilst over-expression of ERG by adenovirus inhibits TNF-induced leukocyte adhesion in vitro and acute TNF-induced inflammation in mouse (1). To confirm the role of endothelial ERG in controlling vascular inflammation in vivo, we used Tie2-Cre/Ergfl/+ mice (Birdsey et al, under review) in a zymosan-induced peritonitis model. Zymosan injection resulted in enhanced leukocyte infiltration in Tie2-Cre/Ergfl/+ mice compared to littermate controls (Figure 2), confirming that endothelial ERG can negatively control acute inflammation. Thus targetting ERG to promote its anti-inflammatory effects could be beneficial against vascular inflammation. A novel small molecule ETS inhibitor, YK-4–279, has recently been shown to bind to ERG and disrupt protein-protein interactions (2). By in silico molecular dynamic modelling, we confirmed YK-4–279 binds the putative protein-binding site within the pointed domain of ERG. In vitro, analysis of protein and gene expression following YK-4–279 treatment of HUVEC resulted in upregulation of ICAM-1 and IL-8 expression in an NF-kB dependent manner, in line with ERG siRNA (Figure 1). However, YK-4–279 did not affect the expression of genes transactivated by ERG, ICAM-2 and HCAD-6, suggesting that ERG’s transactivation and repression functions can be separated. In vivo , i.p. injection of YK-4–279 caused exacerbated leukocyte infiltration into the peritoneum under basal conditions and after Zymosan challenge. Abstract 210 Figure 1 qPCR of HUVEC treated for 24 hours with either YK-4-279, 3μM, (Black) or Erg siRNA (White). Both treatment significantly elevated ICAM-1 and IL-8 expression compared to vehicle (DMSO) or control siRNA, respectively, by one way ANOVA (n = 3) Abstract 210 Figure 2 Total cell infiltration after 24 hours Zymosaninduced peritonitis in Littermate controls (Black) or Tie2-Cre/ERGfl/+ mice (White). Tie2-Cre/ERGfl/+ had significantly elevated leukocyte infiltration compared to littermate controls by T-test (n = 8) Together, these findings confirm that ERG acts to prevent vascular inflammation in vitro and in vivo, and that compounds can be developed which specifically target ERG’s anti-inflammatory activity. Therefore, the development of ERG mimetic molecules to restore ERG’s anti-inflammatory activity may be a novel therapeutic approach to reducing vascular inflammation. References Sperone, et al, ATVB 2011 Rahim, et al. Plos One 2011
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