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The effect of haemodynamic stresses on endothelial permeability to macromolecules is important to normal physiology and in the pathogenesis of atherosclerosis. I developed and applied novel methods to evaluate effects on such transport of acute or chronic exposure to flow along and across cultured endothelium. Porcine aortic endothelial cells were isolated and cultured at passage 1-3 within the porous capillaries of a FiberCell bioreactor. At confluence they were exposed to acute (4 h) or chronic (3-10 day) steady or pulsatile luminal flow (mean shear 3.75 dyne/cm2), with or without transendothelial flow (4 x 10-7 cm/s). Permeability to rhodamine-labelled albumin was assessed by fluorimetry. Confluence of monolayers was confirmed by confocal and scanning electron microscopy and by demonstrating established effects of vasoactive agents on permeability: 10 U/ml thrombin increased permeability, as did 500 μM Nω-nitro-Larginine methyl ester, compared to controls. Permeability was increased by acute pulsatile shear and decreased by chronic pulsatile shear compared to static controls. A decrease in PECAM-1 expression under chronic pulsatile flow was demonstrated by flow cytometry. Steady flow gave higher permeability than pulsatile flow. The introduction of transendothelial flow increased apparent permeability more than could be explained by the addition of the convective transport itself. Preliminary studies suggested that albumin transport may partially be an active process and demonstrated the potential for engineered fibre walls that would allow effects of cyclic strain to be investigated. In conclusion, the hollow fibre bioreactor allowed endothelial permeability to be measured with or without exposure to luminal flow and transendothelial flow over 30 days, permitting the investigation of effects of mechanical stresses. Effects of shear stress varied with duration, pulsatility and direction relative to the endothelial surface.

Transcriptional programs establish and maintain cell identities. Regulation of gene expression is mediated by sequence-specific transcription factors (TFs) and cis-regulatory elements present in the genome. More recently, cell identity has been associated with lineage-defining super-enhancers: comprising dense TF platforms. Endothelial cells are key players of vascular integrity. The ETS TF ERG is constitutively expressed in endothelial cells and essential for endothelial lineage specification, vascular homeostasis and angiogenesis. However, the genomic programs that are regulated by ERG in endothelial cells are poorly understood. In this thesis, I show that ERG densely occupies super-enhancers in human umbilical vein endothelial cells (HUVEC), and its high occupancy can identify super-enhancers. I find that variants associated with cardiovascular disease are enriched in ERG-defined super-enhancers providing insight to the endothelial contribution to complex disease. Depletion of ERG causes profound modulation of the active enhancer mark H3K27ac genome-wide and in recruitment of the transcriptional co-activator Mediator complex. Loss of ERG leads to a decrease in 107 endothelial super-enhancers that have reduced co-occupancy of TFs GATA2 and AP-1. This indicates that ERG plays an essential role as a positive regulator of a core set of endothelial super-enhancers. Interestingly, aberrant ERG overexpression in prostate cancer via androgen-responsive TMPRSS2:ERG fusion proteins is oncogenic. Comparison between HUVEC and prostate cancer TMPRSS2:ERG fusion-positive VCaP cells revealed distinct lineage-specific transcriptome and super-enhancer profiles. In endothelial cells, I show that ERG is required at promoters and enhancers yet assembles distinct TF complexes at these two regions. ERG also colocalises with structural chromatin regulator CTCF in HUVEC, implying a role for ERG in coordinating chromatin structural organisation. Finally, I adopt CRISPR-Cas9 gene editing technology to genetically dissect the super-enhancer of adhesion molecular VE-cadherin. The mechanistic exploration of ERG and endothelial super-enhancers provide insight into the regulation of the endothelial-specific gene expression program.

The ETS transcription factor ERG is highly expressed in endothelial cells (EC) and has a crucial role in controlling homeostasis and angiogenesis. ERG drives expression of genes which promote EC identity, monolayer integrity, survival and angiogenesis. Studies in non-EC have identified multiple ERG isoforms that display functional differences. Notably, some ERG isoforms are phosphorylated through the MAPK pathway, which is downstream of pro-angiogenic growth factors, such as VEGF. The objective of this study was to identify the expression pattern and activity of ERG isoforms in EC and to define their functional role during angiogenesis and in the maintenance of vascular quiescence. Using a PCR screen of human umbilical vein EC (HUVEC) we identify multiple ERG isoforms which differ in the use of alternative transcription and translation initiation sites, and in alternative splicing of a highly evolutionary conserved 24 amino acid domain encoded by exon 7b (ex7b). Isoforms containing ex7b were found to be predominantly expressed in EC. Molecular tools such as ERG isoform specific siRNA and over expression plasmids were designed and validated to investigate the functional role of endothelial ERG isoforms in HUVEC. Selective siRNA targeting of ERG isoforms in HUVEC followed by in vitro Matrigel tube formation assays and propidium iodide staining/flow cytometry demon strated an enhanced role for ex7b-containing isoforms in the regulation of EC tube formation and cell survival. PCR screening of HUVEC treated with ERG isoform-specific siRNAs identified genes regulating cell survival and the cell cycle as differentially regulated. Furthermore, we demonstrate that VEGF stimulation increased endothelial ERG phosphorylation at S215 in an ERK2 and ex7b-dependent manner; leading to enhanced EC proliferation, tube formation and increased expression of multiple ERG target genes. Thus, we show that the responses to VEGF are dependent on the presence of ex7b-containing ERG isoforms, suggesting that alternative splicing of ERG ex7b may provide a mechanism to regulate ERG's transcriptional activity in response to growth factors.

Von Willebrand Factor (VWF) is a large glycoprotein synthesised by endothelial cells (EC) and megakaryocytes. Defects in VWF cause the most common inherited bleeding disorder in humans, Von Willebrand disease (VWD). Some patients with VWD present with vascular malformations in the gastrointestinal (GI) tract (angiodysplasia), which can lead to severe intractable bleeding. Angiodysplasia may be linked to the ability of VWF to regulate blood vessel formation. VWF drives the formation of Weibel Palade Bodies (WPB) which store a key regulator of angiogenesis and permeability, Angiopoietin 2 (Ang-2). In this study, I investigate how VWF controls vascular formation and function through Ang-2. Inhibition of VWF expression in ECs caused enhanced synthesis and release of Ang-2. In addition, ECs isolated from a patient with a severe quantitative defect in VWF (type 3 VWD) showed enhanced synthesis and release of Ang-2, confirming that VWF regulates Ang-2 storage and synthesis in ECs. I investigate whether VWF regulates Ang-2 in vivo, and find that Ang-2 expression is increased in the hearts but not lungs of VWF-deficient mice, indicating a tissue specific regulation. I then investigate the pathway though which VWF may regulate Ang-2 expression. I show that VWF controls Ang-2 synthesis through a pathway involving Akt phosphorylation and the transcription factor FOXO1. I demonstrate a role for atorvastatin in pharmacological manipulation of this pathway, which may be relevant for patients with angiodysplasia and therapeutic manipulation of Ang-2 levels. Inhibition of Ang-2 was able to normalise the VWF-dependent increase in in vitro vascular sprouting. In vivo, increased expression of Ang-2 in the heart of VWF-deficient mice correlates with enhanced vascularisation. Furthermore, cardiac vessels from VWF-deficient mice showed enhanced permeability compared to controls assessed by perfusion of low (4.4 x 103) molecular weight dextran. The results of this thesis provides evidence for the role of VWF in controlling angiogenesis via an Ang-2 dependent pathway. Thus may suggest a novel molecular mechanism which may contribute to VWD associated vascular malformations.

ERG is a crucial regulator of endothelial gene expression and controls endothelial functions including cell survival and monolayer permeability. Previous studies indicate a role for ERG in angiogenesis and vascular development, however the pathways through which ERG controls angiogenesis are unclear. Transcriptome profiling comparing ERG-positive and ERG-deficient endothelial cells has previously shown that ERG controls a network of genes that are essential to angiogenesis. This analysis identified genes involved in the Wnt, Notch and Angiopoietin1/Tie2 signalling pathways as candidate ERG targets. ERG has been shown to drive expression of the junction molecule vascular endothelial (VE)-cadherin, which binds β-catenin, a crucial mediator of Wnt signalling, at the cell membrane. Here, I show that ERG controls β-catenin stability, by driving expression of both VE-cadherin and the Wnt receptor Frizzled-4- the balance of which regulates β-catenin localisation and activity. ERG promotes angiogenesis via Wnt/β-catenin signalling, since activation of Wnt signalling with lithium chloride, which stabilises β-catenin, corrects the angiogenic defect in ERG-deficient endothelial cells. The Notch signalling pathway is critical for promoting vascular quiescence and I demonstrate that ERG controls Notch signalling by regulating the levels of two Notch ligands, Delta like ligand (Dll)-4 and Jagged-1, with reported opposing roles in the vasculature. ERG simultaneously drives expression of pro-quiescent Dll4 and represses expression of pro-angiogenic Jagged-1, which has been shown to antagonize Dll4-mediated signalling. The Angiopoietin1/Tie2 pathway, also connected to the Wnt and Notch pathways, is a regulatory growth factor system essential for vessel maturation and quiescence. The results from this thesis suggest that ERG mediates growth factor Angiopoietin-1-dependent signals and ERG is required for Angiopoietin-1-induced Notch and Wnt signalling. Thus, ERG is able to integrate with three signalling pathways controlling vascular growth and stability - Wnt, Notch, Angiopoietin1/Tie2- which may function downstream of ERG to regulate blood vessel patterning during angiogenesis.

Angiogenesis is fundamental to many physiological processes, and associated with various pathologies, including atherosclerosis and malignant disease. Increasing evidence suggests a role for the cytoprotective enzyme heme oxygenase-1 (HO-1) and its products in angiogenesis. However, the mechanisms through which HO-1 exerts its effects remain elusive. This study aims to identify signalling pathways and novel HO-1 downstream targets regulating angiogenesis. I show that inhibition of HO-1 with synthetic antagonist (ZnPP) or specific siRNA alters the angiogenic process at various levels. HO-1 inhibition significantly reduced vascular endothelial growth factor A (VEGF)-mediated human endothelial cell (EC) proliferation and inhibited capillary-like formation on 2D-Matrigel. Further, I demonstrate that VEGF-induced EC cell cycle progression is inhibited by HO-1 siRNA; an observation associated with decreased expression of cell cycle regulators cyclin A1 and cyclin E1. In contrast, HO-1-deficient cells were still protected from apoptosis by VEGF, most likely through induction of anti-apoptotic genes Bcl-2 and A1. Interestingly, HO-1 depletion negatively affected directional migration of EC towards a VEGF gradient; a phenotype reversed by HO-1 over-expression using an adenoviral vector. Moreover, migrating HO-1-deficient cells showed decreased cyclin A1 protein accompanied by decreased cyclin-dependent kinase 2 activity. Importantly, a combined proteomics and microarray approach has identified downstream targets of HO-1 and their potential roles in HO-1-driven angiogenesis have been investigated. For instance, HO-1 depletion results in impaired assembly of the intermediate filament vimentin. HO-1-deficient cells show reduced activity of the calcium-dependent protease calpain in response to VEGF; this observation was accompanied by a decrease in vimentin cleavage. The differences in vimentin cleavage and filament assembly may in turn account for the impaired angiogenic phenotype of HO-1-deficient cells. Identification of HO-1 downstream target genes may reveal potential therapeutic approaches for enhancing angiogenesis at sites of ischaemia or wound healing, or alternatively inhibiting angiogenesis associated with atherosclerosis or tumourogenesis.

Endothelial cells (EC) maintain homeostasis through the tightly controlled balance between expression of protective genes and repression of pro-inflammatory genes, and loss of this balance can cause endothelial dysfunction, leading to inflammatory diseases including atherosclerosis. We have previously shown that the ETS transcription factor Erg is involved in maintaining EC homeostasis through transactivation of genes involved in key functions including angiogenesis, migration and survival. In addition to the role for Erg as a transcriptional activator, recent genome wide gene expression analysis has also highlighted a role for Erg in the repression of multiple pro-inflammatory genes. In this Thesis I describe a novel mechanism for Erg-mediated repression of these pro-inflammatory genes using ICAM-1 as a model. We identify two ETS binding sites (EBS) within the ICAM-1 promoter (EBS-118 and -181) which are required for Erg mediated repression. One of these EBS is within a functional NF-κB binding site. We show that the increase in ICAM-1 expression upon Erg inhibition is NF-κB dependent, and that Erg prevents NF-κB p65 from binding to the ICAM-1 promoter, suggesting a direct mechanism of interference. Gene Set Enrichment Analysis (GSEA) of transcriptome profiles of Erg and NF-κB dependent genes, together with chromatin immunoprecipitation (ChIP) studies, reveals that this mechanism is common to other pro-inflammatory genes, including cIAP2 and IL8. We investigate the role of chromatin modifying enzymes and histone modifications in Erg-mediated repression and show that in quiescent EC the ICAM-1 promoter is also bound by the histone methyltransferase ESET, and by HDAC1, both indicators of a repressed chromatin structure. Moreover, in silico data on histone modifications suggest that in quiescent EC the ICAM-1 promoter is in a repressed conformation. The results from this Thesis suggest that Erg acts as a gatekeeper to inhibit transactivation of pro-inflammatory genes in quiescent EC, providing an important barrier to protect against inappropriate endothelial activation.

Pro-inflammatory and atherogenic stimuli up-regulate the expression of endothelial adhesion molecules which support the extravasation of leukocytes into the sub-endothelial space. This process drives plaque progression and contributes to its complications. Erg is an ETS transcription factor constitutively expressed in EC which regulates angiogenesis and endothelial homeostasis. Recently, Erg was shown to inhibit endothelial expression of the chemokine IL-8. In EC, Erg expression is down-regulated by the pro-inflammatory cytokine TNF-alpha, through a mechanism which appears to involve Erg protein degradation. These data suggest that Erg may be involved in inflammation. To test this hypothesis, I modulated Erg expression in HUVEC by inhibition with antisense or over-expression using adenovirus encoding for Erg (AdErg). Basal ICAM-1 expression was up-regulated following Erg inhibition, and down-regulated by Erg over-expression. Both of these effects were transcriptional, as shown by a luciferase reporter assay. Over-expression of Erg could inhibit TNF-dependent up-regulation of ICAM-1 and ICAM-1 promoter activity. Thus Erg acts as a transcriptional repressor of ICAM-1 expression in resting and activated EC. This pathway involves NF-kB, since both basal and TNF-α-induced NF-kB activity was repressed by AdErg, and Erg over-expression inhibited the TNF-α induction of NF-kB p65 phosphorylation. In vitro, Erg over-expression led to decreased adhesion of leukocytes to TNF-stimulated HUVEC. In vivo, local injection of AdErg in the mouse paw resulted in reduction of TNF-α-induced inflammation. Interestingly, staining of human coronary artery plaques showed that Erg is absent from activated endothelium over the vulnerable region of the plaque. In conclusion, I propose that Erg exerts a protective role in the endothelium by repressing pro- inflammatory signaling and gene expression. These results suggest a novel approach to anti-inflammatory therapies.