Neil Dufton

ZO-1 controls endothelial adherens junctions, cell–cell tension, angiogenesis, and barrier formation

ZO-1 regulates VE-cadherin–dependent endothelial junctions and actomyosin organization, thereby influencing cell–cell tension, migration, angiogenesis, and barrier formation

The Endothelial Transcription Factor ERG Promotes Vascular Stability and Growth through Wnt/β-Catenin Signaling

Summary 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.

Endothelial DDAH1 is an Important Regulator of Angiogenesis but Does Not Regulate Vascular Reactivity or Hemodynamic Homeostasis

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.

Endothelial Dimethylarginine Dimethylaminohydrolase 1 Is an Important Regulator of Angiogenesis but Does Not Regulate Vascular Reactivity or Hemodynamic Homeostasis

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.

Consensus guidelines for the use and interpretation of angiogenesis assays

AbstractThe 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.

The endothelial transcription factor ERG mediates Angiopoietin-1-dependent control of Notch signalling and vascular stability

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.

Investigation of cardiac fibroblasts using myocardial slices

Abstract Aims Cardiac 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 results Unloading 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). Conclusions Myocardial slices allow the study of CFs in a multicellular environment and may be used to effectively study mechanisms of cardiac fibrosis and potential targets.

Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis

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.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.

210 The Endothelial Transcription Factor ERG Inhibits Vascular Inflammation

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