Abstract Over the last few years, the ETS transcription factor ERG has emerged as a major regulator of endothelial function. Multiple studies have shown that ERG plays a crucial role in promoting ...angiogenesis and vascular stability during development and after birth. In the mature vasculature ERG also functions to maintain endothelial homeostasis, by transactivating genes involved in key endothelial functions, while repressing expression of pro-inflammatory genes. Its homeostatic role is lineage-specific, since ectopic expression of ERG in 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 vascular endothelium, its transcriptional targets and its functional partners and the emerging evidence on the pathways regulating ERG's activity and expression.
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.
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.
A decline in the function of the macro- and micro-vasculature occurs with ageing. DNA damage also accumulates with ageing, and thus DNA damage and repair have important roles in physiological ageing. ...Considerable evidence also supports a crucial role for DNA damage in the development and progression of macrovascular disease such as atherosclerosis. These findings support the concept that prolonged exposure to risk factors is a major stimulus for DNA damage within the vasculature, in part via the generation of reactive oxygen species. Genomic instability can directly affect vascular cellular function, leading to cell cycle arrest, apoptosis and premature vascular cell senescence. In contrast, the study of age-related impaired function and DNA damage mechanisms in the microvasculature is limited, although ageing is associated with microvessel endothelial dysfunction. This review examines current knowledge on the role of DNA damage and DNA repair systems in macrovascular disease such as atherosclerosis and microvascular disease. We also discuss the cellular responses to DNA damage to identify possible strategies for prevention and treatment.
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 endothelial ETS transcription factor Erg plays an important role in homeostasis and angiogenesis by regulating many endothelial functions including survival and junction stability. Here we show ...that Erg regulates endothelial cell (EC) migration. Transcriptome profiling of Erg-deficient ECs identified ∼ 80 genes involved in cell migration as candidate Erg targets, including many regulators of Rho- GTPases. Inhibition of Erg expression in HUVECs resulted in decreased migration in vitro, while Erg overexpression using adenovirus caused increased migra-tion. Live-cell imaging of Erg-deficient HUVECs showed a reduction in lamellipodia, in line with decreased motility. Both actin and tubulin cytoskeletons were disrupted in Erg-deficient ECs, with a dramatic increase in tubulin acetylation. Among the most significant microarray hits was the cytosolic histone deacetylase 6 (HDAC6), a regulator of cell migration. Chromatin immunoprecipitation (ChIP) and transactivation studies demonstrated that Erg regulates HDAC6 expression. Rescue experiments confirmed that HDAC6 mediates the Erg-dependent regulation of tubulin acetylation and actin localization. In vivo, inhibition of Erg expression in angiogenic ECs resulted in decreased HDAC6 expression with increased tubulin acetylation. Thus, we have identified a novel function for the transcription factor Erg in regulating HDAC6 and multiple pathways essential for EC migration and angiogenesis.
Inflammation drives atherosclerotic plaque rupture. Although inflammation can be measured using fluorine-18-labeled fluorodeoxyglucose positron emission tomography (
FFDG PET),
FFDG lacks cell ...specificity, and coronary imaging is unreliable because of myocardial spillover.
This study tested the efficacy of gallium-68-labeled DOTATATE (
Ga-DOTATATE), a somatostatin receptor subtype-2 (SST
)-binding PET tracer, for imaging atherosclerotic inflammation.
We confirmed
Ga-DOTATATE binding in macrophages and excised carotid plaques.
Ga-DOTATATE PET imaging was compared to
FFDG PET imaging in 42 patients with atherosclerosis.
Target SSTR2 gene expression occurred exclusively in "proinflammatory" M1 macrophages, specific
Ga-DOTATATE ligand binding to SST
receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and carotid SSTR2 mRNA was highly correlated with in vivo
Ga-DOTATATE PET signals (r = 0.89; 95% confidence interval CI: 0.28 to 0.99; p = 0.02).
Ga-DOTATATE mean of maximum tissue-to-blood ratios (mTBR
) correctly identified culprit versus nonculprit arteries in patients with acute coronary syndrome (median difference: 0.69; interquartile range IQR: 0.22 to 1.15; p = 0.008) and transient ischemic attack/stroke (median difference: 0.13; IQR: 0.07 to 0.32; p = 0.003).
Ga-DOTATATE mTBR
predicted high-risk coronary computed tomography features (receiver operating characteristics area under the curve ROC AUC: 0.86; 95% CI: 0.80 to 0.92; p < 0.0001), and correlated with Framingham risk score (r = 0.53; 95% CI: 0.32 to 0.69; p <0.0001) and
FFDG uptake (r = 0.73; 95% CI: 0.64 to 0.81; p < 0.0001).
FFDG mTBR
differentiated culprit from nonculprit carotid lesions (median difference: 0.12; IQR: 0.0 to 0.23; p = 0.008) and high-risk from lower-risk coronary arteries (ROC AUC: 0.76; 95% CI: 0.62 to 0.91; p = 0.002); however, myocardial
FFDG spillover rendered coronary
FFDG scans uninterpretable in 27 patients (64%). Coronary
Ga-DOTATATE PET scans were readable in all patients.
We validated
Ga-DOTATATE PET as a novel marker of atherosclerotic inflammation and confirmed that
Ga-DOTATATE offers superior coronary imaging, excellent macrophage specificity, and better power to discriminate high-risk versus low-risk coronary lesions than
FFDG. (Vascular Inflammation Imaging Using Somatostatin Receptor Positron Emission Tomography VISION; NCT02021188).
Inflammation drives atherosclerotic plaque rupture. Although inflammation can be measured using fluorine-18-labeled fluorodeoxyglucose positron emission tomography (18FFDG PET), 18FFDG lacks cell ...specificity, and coronary imaging is unreliable because of myocardial spillover.
This study tested the efficacy of gallium-68-labeled DOTATATE (68Ga-DOTATATE), a somatostatin receptor subtype-2 (SST2)-binding PET tracer, for imaging atherosclerotic inflammation.
We confirmed 68Ga-DOTATATE binding in macrophages and excised carotid plaques. 68Ga-DOTATATE PET imaging was compared to 18FFDG PET imaging in 42 patients with atherosclerosis.
Target SSTR2 gene expression occurred exclusively in “proinflammatory” M1 macrophages, specific 68Ga-DOTATATE ligand binding to SST2 receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and carotid SSTR2 mRNA was highly correlated with in vivo 68Ga-DOTATATE PET signals (r = 0.89; 95% confidence interval CI: 0.28 to 0.99; p = 0.02). 68Ga-DOTATATE mean of maximum tissue-to-blood ratios (mTBRmax) correctly identified culprit versus nonculprit arteries in patients with acute coronary syndrome (median difference: 0.69; interquartile range IQR: 0.22 to 1.15; p = 0.008) and transient ischemic attack/stroke (median difference: 0.13; IQR: 0.07 to 0.32; p = 0.003). 68Ga-DOTATATE mTBRmax predicted high-risk coronary computed tomography features (receiver operating characteristics area under the curve ROC AUC: 0.86; 95% CI: 0.80 to 0.92; p < 0.0001), and correlated with Framingham risk score (r = 0.53; 95% CI: 0.32 to 0.69; p <0.0001) and 18FFDG uptake (r = 0.73; 95% CI: 0.64 to 0.81; p < 0.0001). 18FFDG mTBRmax differentiated culprit from nonculprit carotid lesions (median difference: 0.12; IQR: 0.0 to 0.23; p = 0.008) and high-risk from lower-risk coronary arteries (ROC AUC: 0.76; 95% CI: 0.62 to 0.91; p = 0.002); however, myocardial 18FFDG spillover rendered coronary 18FFDG scans uninterpretable in 27 patients (64%). Coronary 68Ga-DOTATATE PET scans were readable in all patients.
We validated 68Ga-DOTATATE PET as a novel marker of atherosclerotic inflammation and confirmed that 68Ga-DOTATATE offers superior coronary imaging, excellent macrophage specificity, and better power to discriminate high-risk versus low-risk coronary lesions than 18FFDG. (Vascular Inflammation Imaging Using Somatostatin Receptor Positron Emission Tomography VISION; NCT02021188)
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