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Endothelial cells (ECs) are highly heterogenous across different organs and within the vascular bed, in both health and disease. However, EC heterogeneity has not been extensively dissected in the context of pulmonary arterial hypertension (PAH). It is a rare and progressive disease characterised by remodelling of the distal pulmonary vasculature. Using an EC lineage-tracing mouse model and single-cell RNA-sequencing (scRNA-seq), I first investigated how enriched mouse ECs respond to Sugen 5416/Hypoxia-induced (SuHx) PAH. Globally, ECs adopt an immunophenotype in PAH, with upregulation of genes relating to the major histocompatibility class II (MHC-II) complex. The strongest upregulation is observed in Artery and one of the two capillary ECs (Capillary A/gCap). The other capillary EC cluster (Capillary B/aerocyte) had a distinct response to PAH, with upregulation of genes regulating apoptosis, migration, and angiogenesis. Comparison with whole-lung scRNA-seq in rat and human PAH identified several candidate genes conserved across all species. Following this, I further examined heterogeneity within Artery and Capillary A ECs, the pulmonary vasculature predominantly affected in PAH. Artery ECs can be subdivided into Proximal and Distal Artery ECs, with both groups having distinct biomarkers. In PAH, a loss of Proximal Artery EC identity and a gain in Distal Artery EC identity is observed. As for Capillary A ECs, three subpopulations were identified. In Control, two of these subclusters (CapillaryA_0, CapillaryA_1) were marked by high expression of Distal Artery markers, and low expression of Proximal Artery markers, whilst the reverse was true for the third CapillaryA_2 subcluster. There was also significantly more CapillaryA_2 ECs in PAH. Differential gene expression analysis found some genes uniquely upregulated in each CapillaryA subcluster: senescence-associated genes in CapillaryA_0 (Cd9, H3f3b, Neat1), signalling genes in CapillaryA_1 (Rab5a, Slc6a6, Tmem252), and both inflammatory (H2-Q4, Irgm1, Tgtp2) and angiogenesis regulatory genes (Cldn5, Cxcl12, Esam, Rhoj) in CapillaryA_2. Overall, this work has revealed in high-resolution, how ECs in different vascular beds distinctly respond to PAH.

Breast cancer survival rates have increased over the years due to early detection and therapeutic efficacy. However, after many years of what appears to be disease-free health, cancer can return in the form of a secondary metastatic tumour. Formation of new blood vessels is a crucial stage in the progression of primary tumours to metastatic tumours. In many cases primary breast tumours that metastasise to the brain occur. Brain tumours are heterogenous malignancies with a low survival rate. Tumours often development therapy resistance by secreting different pro-angiogenic growth factors that allow them to overcome the effect of anti-angiogenic drugs. Thus, characterising the mechanisms that promote tumour angiogenesis may help to stop the development of tumour metastasis. Here it was determined that ATF2 is a downstream molecule activated (phosphorylated) by major pro-angiogenic factors and that its suppression in HUVECs resulted in increased upregulation of Notch signalling pathway (major regulator of angiogenesis) ligand DLL1 and DLL4. Additionally, we investigated genomic changes that may be involved in the development and progression of breast to brain metastasis (BBM). Whole exome sequencing (WES) of 26 breast to brain metastases was carried out. Bioinformatic analysis of WES data identified recurrent genomic alterations in several genes that may be associated with BBM development including an ARFEFG protein family member gene, BIG3. Functional analysis of BIG3 showed that this gene is involved in the regulation of neurotransmitter receptors subunits in the BIG3-knockout MCF-7 breast cancer cell line. Neurotransmitter regulation has been shown as one mechanism through which brain tumours integrate into the neuronal signalling network to promote colonisation of the brain. Results presented here show that investigating the possible mechanisms behind tumour angiogenesis, and the molecular changes that may be involved in metastatic tumour development and brain colonisation offers a better understanding of tumour mechanisms for growth and survival which can offer an opportunity for the development of new therapeutic targets.

Angiogenesis is a physiological process involved in the formation of blood vessels from pre-existing ones and is tightly regulated by a balance between pro- and anti- angiogenic signals. Disturbance to this balance is associated to human diseases characterised by excessive or insufficient angiogenesis. MicroRNA (miRNA) are small non-coding RNA molecules, which inhibit gene expression by inducing mRNA degradation or suppressing protein translation. Emerging evidence highlights a novel role for miRNAs as regulators of angiogenesis. In endothelial cells miR-133a is expressed at very low levels in physiological conditions however, increased expression of this microRNA in the endothelium has been strongly associated with cardiovascular disease. Previous studies have reported conflicting results regarding the effect of miR-133a expression in endothelial cells during blood vessel formation. The study involved assessing the specific effect of mature miR-133a strands in angiogenesis and the expression of endothelial angiogenic genes. The study evaluated the consequences of aberrant expression of miR-133a in endothelial cells via transfection of miR-133a-3p, -5p, or negative control mimics in primary endothelial cells. This significantly inhibited endothelial cell proliferation, migration, and tubular morphogenesis. The screened gene arrays were performed to identify genes involved in the regulation of signalling pathways, which play a key role in angiogenesis. The results have been further validated by qPCR, which revealed that aberrant expression of miR-133a-3p led to a decrease in the expression of genes encoding pro-angiogenic molecules, whilst increasing those with anti-angiogenic functions. Ingenuity Pathway Analysis of a network of genes differentially expressed in cells harbouring miR-133a-3p, predicted decreased cellular functions related to vasculature branching and cell cycle progression, underlining the inhibitory role of miR-133a-3p in angiogenic cellular processes. The results indicate that enhanced expression of miR-133a-3p in endothelial cells during cardiovascular disease impairs pro-angiogenic cellular processes by altering the expression of specific target genes. Therefore, the results suggest that controlled delivery of miR-133a-3p mimics in diseased endothelial cells may open new therapeutic interventions to treat patients suffering from cardiovascular pathologies associated with excessive or insufficient blood vessel formation.

The endothelial-to-haematopoietic transition (EHT) gives rise to the first murine definitive haematopoietic stem cells (HSCs) in the aorta-gonads-mesonephros (AGM) region around embryonic stage E10.5. While our understanding of the transcriptional networks regulating HSC emergence is ever-increasing, the mechanisms governing the EHT process are still being investigated. The transcription factor Gata3, whose function has previously been characterized in the sympathetic nervous system AGM niche, is expressed directly in the haemogenic endothelium(HE), the tissue giving rise to haematopoietic stem and progenitor cells (HSPCs), prior to EHT. Previous studies have shown that Gata3-expressing endothelial cells (ECs) have a greater capacity to produce haematopoietic colonies, over Gata3-negative EC, while EC-specific Gata3 knockout leads to greatly decreased HSC activity. The cyclin-dependent kinase inhibitorp57Kip2 is notably upregulated in Gata3+ cells of the AGM, indicating a potential role for Gata3 as a cell cycle regulator. This thesis outlines the investigation of Gata3, p57Kip2 and the cell cycle during the EHT, both through functional experiments, as well as via single-cell RNA sequencing (scRNA-seq) of Gata3+ ECs and haematopoietic cells (HCs). ScRNA-seq data of Gata3 knockout (KO) and p57Kip2 KO cells is further used to explore any non-cell cycle related role of Gata3 and p57Kip2 in the HE. This thesis goes on to describe the identification of the potential Gata3 downstream-target CD200R1 and its ligand CD200 as new functional markers for the enrichment of HSPCs, initially discovered within the scRNA-seq dataset via trajectory analysis of Gata3+ ECs differentiating into HSPCs. Finally, this thesis includes the scRNA-seq-level characterization of mesenchymal/stromal cells, a cell type whose AGM niche supportive function is important, but insufficiently defined. Taken together, the data detailed in this thesis will deliver new insights in the generation of the first HSCs and the environment in which they emerge.

The human endothelial colony-forming cells (ECFCs), a subpopulation of endothelial progenitor cells (EPCs), offer a potential platform for cellular assessment due to their remarkable angiogenic expansion, regenerative, and self-renewable potentials in vitro. This thesis investigated how ECFC function and proteome profiles are altered in young adults with elevated cardiovascular disease (CVD) risk factors, including increased blood pressure (BP). It also demonstrated the effect of an exercise intervention on ECFC function and the potential to use ECFC to predict response to exercise intervention. The main investigations of this thesis were based on a cohort of young adult participants aged 18 to 35 years, recruited through the Trial of Exercise to Prevent HypeRtension in young Adults (TEPHRA). Detailed clinical measurements, including BP measurements, retinal micro-vascular imaging, pulse wave velocity, blood biochemistry and lipid profiles, as well as ECFC culture and cellular assessments, were performed at baseline pre-exercise intervention. Measurements were repeated following a 16-week exercise intervention training period in a subgroup of participants. Our data presented a distinct proteomic signature with altered angiogenic mechanisms in dysfunctional ECFCs. An increase in CVD risks, including higher BP, body mass index and impaired lipid profile, were associated with a reduction in expansion and vasculogenic capacity in dysfunctional ECFCs. Further analysis revealed that the young adults with elevated BP had significantly reduced ECFC functionality and presented a potentially adverse retinal micro-vascular structure and greater macro-vascular resistance. Although no significant changes were observed in the ECFCs after exercise intervention, we demonstrated that baseline ECFC functional characteristics might positively affect aerobic training response. In addition, baseline ECFC functional characteristics may predict response to exercise, suggesting that mechanisms or substances modulating ECFC colony formation and function may be essential factors that affect an individual's response to exercise.

Von Willebrand disease (VWD) is the most common inherited bleeding disorder. It is defined by a deficiency or dysfunction of plasma von Willebrand factor (VWF), a glycoprotein with a multifaceted role in haemostasis. The majority of circulating VWF is synthesised and released by endothelial cells (ECs). VWD is caused by rare DNA sequence variants in the VWF gene. However, coupling genotype with phenotype is complicated by factors including incomplete penetrance and the trans-acting effect of the ABO histo-group. High throughput sequencing (HTS) is becoming the standard of care for the diagnosis of inherited bleeding disorders, including VWD. This raises several challenges. First, how should candidate VWF variants be searched for and their pathogenicity assessed? Second, if a pathogenic variant (PV) for VWD is identified how does this influence bleeding risk? Third, if the mechanism of the identified PV is unknown, how can its effect be elucidated? These questions are sequentially addressed in this thesis. I curated 1,455 unique VWF variants into a single repository called VWDbase. Variants were only included if they had been previously linked to VWD. Two thirds of VWDbase variants had previously been deemed causal of VWD and were termed Putatively Aetiological VWD Variants (PAVVs). Of these, 194 PAVVs were identified in the whole exome sequencing data of 140,327 participants in UK Biobank (UKB). These data were used to accurately determine the minor allele frequency (MAF) of these PAVVs. The pathogenicity of each PAVV was then scrutinised using published data. Seventy three of 194 PAVVs were rejected as being pathogenic for VWD. In over half of cases this was because the PAVV occurred too frequently to be compatible with VWD prevalence. The PAVVs that were accepted as being pathogenic for VWD were identified in 401 UKB participants (the 'genetically accepted VWD' [ga] group). Hospital inpatient data were analysed for UKB participants from 1997 to 2020. These were used to create the ICD-bleeding assessment tool (ICD-BAT) to assess the presence or absence of bleeding episodes across 16 different domains and the time over which UKB participants lived without experiencing an episode (bleeding free survival). There was no difference in the ICD-BAT score or bleeding-free survival when the gaVWD group was compared to the rest of the UKB population. However, blood group O predicted for both an increased ICD-BAT score and a reduced risk of bleeding-free survival over the observation period. VWDbase was then utilised to analyse 10 patients with VWD in whom no molecular diagnosis had previously been identified. The patient with the most severe (type 3) VWD phenotype was homozygous for a rare PAVV, c.8155+6T > A, situated in the donor splice site of the penultimate exon-intron junction. Analysis of platelet mRNA demonstrated that c.8155+6T > A results in a transcript with a frameshift and premature termination codon (PTC). Evaluation of patient-derived endothelial colony forming cells (ECFCs) revealed that c.8155+6T > A resulted in VWF that was mostly retained in a perinuclear position as opposed to being packed into Weibel-Palade bodies (WPBs). In order to overcome the finite supply of ECFCs and assess the effect of c.8155+6T > A in a different genetic context, a new cellular model of VWD was created. Human induced pluripotent stem cells (hiPSCs) were edited using CRISPR/Cas9 to contain a PTC in exon 50, positioned 10 nucleotides 5' of c.8155+6T > A. They were then differentiated to ECs and the findings in the patient ECFCs were replicated. The effect of c.8155+6T > A is likely to be due to the truncation of VWF prior to the C-terminal cysteine knot (CK), the domain which is crucial for VWF dimerisation and exit from the endoplasmic reticulum. In summary, this thesis highlights the utility of large reference populations and hiPSC-derived ECs (iECs) in the critical appraisal of PAVVs.

Diabetic macular oedema (DMO) is an important cause of vision loss worldwide. It is characterized by hyperpermeability of retinal vessels due to breakdown of the inner blood-retinal barrier. The goal of this thesis was to better understand the mechanisms through which bradykinin (BK) and thrombin (THR) trigger vasopermeability responses in retinal microvascular endothelial cells (RMECs), with particular emphasis on the role of intracellular Ca2+ and the Ca2+/calmodulin-dependent protein kinase, CaMKII. Ca2+ imaging studies revealed that BK and THR elicit biphasic intracellular Ca2+ responses in cultured bovine RMECs. The initial peak phase resulted from endoplasmic reticulum Ca2+ release via the Gαq-PLC-IP3 signalling pathway, while the sustained phase derived from Ca2+ influx via store-operated Ca2+ channels. Receptor pharmacology studies demonstrated that BK acts primarily through the bradykinin B2 receptor, whereas THR signals through protease-activated receptor 1 (PAR1). Experiments to determine CAMKII phosphorylation upon BK and THR exposure were undertaken in bovine and human RMECs. Western blotting studies identified a time-dependent increase in CaMKII activity upon agonist treatment persisting for up to 24 hours. Hyperglycaemic conditions for 21 days did not have a significant effect on basal or agonist induced CaMKII phosphorylation. Increased FITC-dextran leakage was observed in bovine RMEC monolayers challenged with BK and THR and these effects could be largely abolished in the presence of the CaMKII inhibitor, KN-93, but not its inactive analogue, KN-92. To investigate the relevance of these findings to DMO, the effects of systemic administration of KN-93 and KN-92 on retinal vascular leakage in streptozotocin-induced diabetic mice were investigated. These experiments confirmed an important role for CaMKII in mediating retinal vasopermeability responses in the diabetic retina. Taken together, the work in this thesis suggests that CaMKII could represent a novel therapeutic target for DMO treatment, although further studies will be required to confirm this.

Epithelial ovarian cancer (EOC) frequently metastasises to the omentum. Metastatic growth requires angiogenesis (new blood vessel formation), to provide nutrients and oxygen to the growing secondary tumour. This process requires the activation of local microvascular endothelial cells (ECs), by tumour cell secreted pro-angiogenic factors, such as vascular endothelial growth factor (VEGF). The prognosis for patients with advanced metastatic EOC remains poor and the search for effective treatments is on-going. However, therapeutic targeting of angiogenesis and, in particular, the VEGF pathway has been relatively unsuccessful, suggesting the involvement of other pro-angiogenic factors that may offer alternative therapeutic targets. Cathepsin-L (CL), a lysosomal protease, has been shown to induce galectin-1 (gal-1) secretion, a small glycoprotein, from disease-relevant human omental microvascular ECs (HOMECs). Gal-1 is upregulated in advanced EOC, and has been implicated in metastatic processes, including angiogenesis. The mechanisms by which gal-1 may contribute to HOMEC pro-metastatic and angiogenic activity are unknown, and therefore the aims of this thesis were to a) improve a method to isolate HOMECs from omental samples, b) to examine pro-metastatic and angiogenic effects of gal-1 in EOC cells and HOMECs, and c) to identify pro-proliferative gal-1 activated receptors and downstream signalling pathways in HOMECs. HOMEC cultures were obtained by enzymatic digestions and immunoselection, and characterised with immunocytochemistry (ICC). A2780 and SKOV3 EOC cell lines secreted gal-1, and HOMECs secreted significantly more gal-1 in response to CL. Gal-1 pre-treatment of either A2780/SKOV3 cells or HOMEC monolayers, significantly increased A2780/SKOV3 cell adhesiveness to HOMEC monolayers. CL significantly increased extracellular surface gal-1 on HOMECs, and HOMECs were shown to be able to bind exogenous gal-1 to their cell surface. Exogenous gal-1 significantly induced HOMEC proliferation in WST-1 and BrdU assays, as well as HOMEC migration in chamber but not scratch assays. Receptor tyrosine kinase and intracellular phosphokinase arrays identified gal-1 induced phosphorylation of VEGF receptor 2 (VEGFR2) and phospholipase Cγ1 (PLCγ1) independently of VEGF, findings which were confirmed by ELISA and flow cytometry. It was hypothesised that gal-1 was inducing pro-proliferative effects by binding to complex N-glycans on the cell surface (possibly on VEGFR2). Inhibition of complex N-glycan synthesis with swainsonine (SW) significantly inhibited gal-1 induced HOMEC proliferation, phosphorylation of VEGFR2, and preliminarily suggested inhibition of PLCγ1 phosphorylation. Gal-1 also significantly increased retention of VEGF activated VEGFR2 complexes at the cell surface. In conclusion, these data suggest that gal-1 is a potential pro-metastatic and pro-angiogenic molecule that may contribute to metastasis of EOC to, and within the omentum. In disease-relevant microvascular ECs (HOMECs) gal-1 activates pro-angiogenic responses via the VEGF receptor independently of VEGF, potentially by binding to complex N-glycans on VEGFR2 and initiating pro-proliferative intracellular pathways. Thus gal-1 may represent a new therapeutic target for the treatment of advanced EOC.

Over 17 million yearly deaths are caused by cardiovascular diseases worldwide, and up to 80% are due to heart attacks and strokes caused by atherosclerosis: fatty plaque build-up within artery walls restricting blood flow. Atherosclerotic plaque can also build up in arteries supplying the extremities such as the arms and legs; this is called peripheral arterial disease and is the 3rd most common atherosclerotic disease following that of the coronary arteries and cerebral arteries. With no cure for chronic ischemic diseases, clinical management includes reducing risk factors and utilising drug therapies to help with ailments that exacerbate disease such as diabetes and hypertension. Surgical intervention is a last resort with a high number of peripheral arterial disease patients requiring limb amputation. To avoid this, many clinical trials have attempted to increase patients' blood flow by targeting endothelial cells and stimulating angiogenesis, the development of new blood vessels from pre-existing vessels. However, none of these attempts have led to a curative therapy yet. To prevent ischemic disease from escalating to amputation, heart attacks, or strokes, it is vital we find a way to combat them at early stages. Endothelial dysfunction, the aberrant or extended activation of adaptive endothelial behaviours, is an early event in atherosclerotic development, hence further understanding of endothelial molecular mechanisms is required. Long non-coding RNAs (lncRNAs) regulate many cell functions but are not well characterised and are poorly understood due to their previous categorisation as 'junk DNA'. Several lncRNAs have been identified in aspects of cardiovascular pathophysiology, however, the human genome is estimated to possess ~ 270,000 lncRNAs, ergo many remain undiscovered. High-throughput RNA-sequencing in a human embryonic stem cell to endothelial cell differentiation protocol identified the lncRNA LINC00961 as endothelial enriched. This locus houses a micropeptide, small peptide of amino acid regulation (SPAAR), and has a mouse homologue; unique factors suggesting an important and evolutionary conserved function. Therefore, this project sought to investigate the role of the LINC00961 locus in the endothelium. Knock down of LINC00961 expression by ~90% was achieved in human umbilical vein endothelial cells which significantly reduced several endothelial functions; tubule formation, proliferation, adhesion, migration, and barrier integrity. The LINC00961 locus knock out mouse line showed no lethality; however, a foetal growth restriction like phenotype was identified in male LINC00961-/- animals; these offspring were significantly smaller and lighter with an increased brain weight to body ratio at 9 weeks of age. Cardiac ultrasound at 8 weeks of age found no differences in cardiac output between female LINC00961-/- and wildtype controls. However, reduced left ventricular wall diameter, slower mitral valve deceleration, and isovolumetric contraction time were observed in these mice. This restricted heart filling and compromised myocardial relaxation indicates the early stages of diastolic dysfunction. Adult male LINC00961-/- and wild type control mice underwent surgically induced hind limb ischemia. Comparable to in vitro data, LINC00961 deletion caused transient changes to capillary number during early hypoxic injury, and a lack of mature α- smooth muscle actin vessels at baseline, indicating underlying issues with vessel physiology. Crucially, lentiviral overexpression cassettes showed LINC00961 acted independently of SPAAR in human umbilical vein endothelial cells, and LINC00961 and SPAAR were linked to the actin binding proteins thymosin β-4 and SYNE1, respectively. LINC00961 and SPAAR are encoded by the same locus but have opposing effects on angiogenesis. Reduction of locus expression also affected other endothelial behaviours; thus, this locus contributes to maintaining proper endothelial function. This refinement of angiogenic control may be in part due to actin cytoskeletal regulation via thymosin β-4 and SYNE1 interactions. Murine LINC00961 contributes to blood vessel physiology and may also have a role in heart physiology given the altered parameters in LINC00961-/- hearts. Therefore, this locus has important roles in several aspects of cardiovascular biology and is a potential novel target for therapeutic regulation of angiogenesis in patients with compromised blood flow.

Diabetic endotheliopathy is the main cause for impaired angiogenesis and reduced neovascularization that lead to microvascular injury and vascular complications. The pathogenesis for vascular complications arising from diabetes is complex. Elucidation of key underlying mechanisms will help the development of novel therapies and the discovery of potential biomarkers. The ability to generate functional endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) is a novel and powerful tool for cell-based therapies. Human iPSC-derived ECs (iPS-ECs) have a broad range of clinical applications including cell-based therapy, disease modelling and drug screening; they can be used in mechanistic studies towards the development of novel therapies and in the discovery of new biomarkers to be applied in regenerative medicine and treatment of diabetic vasculopathy. Here we utilize transcriptomic and proteomic technologies to assess patient-specific iPS-ECs from diabetic (DiPS-ECs) and non-diabetic (NiPS-ECS) donors in order to investigate the mechanisms driving endotheliopathy in diabetes. Our in vitro and in vivo models recapitulate the effects of hyperglycaemia on the vasculature in the clinical setting. RNA-seq data showed differential expression of genes and proteins involved in EC function and angiogenesis were significantly downregulated in DiPS-ECs in comparison to NDiPS-ECs. Moreover, factors involved endothelial-to-mesenchymal transition were increased in DiPS-ECs. DiPS-ECs had significantly reduced impaired tube formation and barrier stability in vitro. DiPS-ECs displayed impaired angiogenic function demonstrated by poor post-transplant engraftment and decreased blood flow recovery (BFR) when injected to the hindlimb of mice following femoral artery ligation. Proteomic and transcriptomic analysis confirmed imbalances in several pathways involved in endothelial function. Pathways involved in metabolism, homeostasis and stress response were found to be underrepresented in DiPS-ECs. RNA-seq revealed differences in the transcriptome of DiPS-ECs. EndMT related gene ontology was enriched in DiPS-ECs. Endothelial specific Roundabout protein 4 (ROBO4) is highly involved in pathways related to angiogenesis, barrier stability and endothelial health. Expression of ROBO4 was found to be impaired in DiPS-ECs and transcriptomic analysis along with in vitro and in vivo studies revealed its importance in vascular specification and angiogenesis. ROBO4 improved ECs function of DiPS-ECs and recovered their lost angiogenic function. Our data suggest that DiPS-ECs carry an imprint of the diabetic milieu which is reflected in their dysfunction. An unregulated EndMT process is proposed to cause a phenotype drift and poor angiogenic functionality observed in DiPS-ECs. ROBO4 seems to act as a fine tuning factor of this process. To the best of our knowledge, we aim to identify and report a novel disease-specific signature in diabetic iPS-ECs. Therefore, our human iPS-ECs model may serve as a valuable tool to study biological pathways and identify new treatments for diabetes-induced endotheliopathy.

Diabetic retinopathy (DR) is the most frequently occurring microvascular complication of diabetes mellitus, where the later stages of the disease are often is chaemia driven which can result in severe vision loss. A key component cell of the retinal neurovascular unit is the vascular endothelium, which is significantly impacted by the diabetic milieu. It has now been established that the endothelium relies on glycolytic metabolism predominantly for ATP generation, however the degree of flux through this pathway in some non-retinal endothelial cell types has been shown to vary in disease conditions. This PhD project has assessed the nature of glycolysis and related plasticity in human retinal microvascular endothelial cells (HRMECs) exposed to in vitro conditions relevant to DR.HRMECs were obtained from human donors and were exposed to either normal glucose conditions of 5 m M D-glucose (NG), 20 m ML-Glucose + 5 m M D-Glucose (HLG), or 25 m M D-Glucose (HDG) for four weeks, along with a hypoxic exposure (1% O2, 5% CO2 )for 24 hours before experiments were performed, mimicking diabetic-like conditions. Assessments of in vitro metabolism were assessed using the Seahorse XF system and qPCR to highlight potential changes in metabolic pathways. Functional assays including 3D-Matrigelexperiments, Clonogenics, proliferation, migration, and barrier integrity were also implemented to ascertain what changes to functionality occur after HDG exposure. Furthermore, the use of pharmacological compounds such as the glycolytic inhibitor 3PO ((2E)-3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one),and Proxison (7-decyl-3-hydroxy-2-(3, 4, 5-trihdroxyphenyl)-4-chromenone) a synthetic flavonoid were also implemented in this study to determine if pharmacological interventions/ modifications to metabolism could be effective in reducing HDG exposure affects. In an attempt to understand angiogenic pathways in more detail, single cell sequencing was also performed on cells recovered from 3D environment. This project together helps to understand the nature of this complex disease and identify potential new targets of therapeutic interventions.

Induced pluripotent stem cell-derived endothelial cells (iPS-ECs) hold clear potential for therapeutic angiogenesis in ischaemic diseases, although many challenges remain such as enrichment and expansion of homogenous, mature, highly proliferative and functional ECs. It is well established that reactive oxygen species (ROS), which are centrally involved in ischaemic disease, are important regulators of EC and stem cell biology, with recent evidence specifically highlighting NADPH oxidases as a key source. In this thesis, we investigated the role of NADPH oxidase-dependent redox signalling in differentiation of mouse and human iPS-ECs, and provide evidence implicating ROS derived from the NOX4 isoform during the differentiation process, which is likely to be important when considering their therapeutic potential. Our findings show that miPS-EC differentiation is associated with induction of NOX2 and NOX4 isoforms together with increased ROS generation and general antioxidant suppression. We found that NOX4 promotes iPS-EC differentiation in a ROS-dependent manner, whilst opposite effects are evident with NOX4 knockdown. Mechanistically, we demonstrated that NOX4 specifically regulates eNOS signalling in miPS-ECs and hiPS-ECs and antioxidant expression via activation of NRF2 and downstream pro-angiogenic intermediates including STAT3, JUN, NFⱩB, ERK, Akt and Jnk. Nevertheless, we highlighted the differentiation process as a highly oxidative event mediated by superoxide generation and inhibition of NRF2 and downstream antioxidant genes. Our data also reveal that oxidative stress may be damaging and underlie reduced differentiation efficiency, which was improved by the pan-NADPH oxidase inhibitor, VAS2870, whilst increased ROS levels may be important in directing early differentiation, before subsiding as derived ECs mature. Taken together, the data in this thesis indicate that NOX4 NADPH oxidase plays a significant role in regulating iPS-EC differentiation and function, whilst highlighting complexity of ROS signalling and importance of defining key aspects of iPS-EC signalling towards their clinical application for treatment of ischaemic cardiovascular disease.

Endothelial cells, which form the inner lining of all blood vessels, maintain various critical functions to enable the efficient transport of oxygen and nutrients to all metabolising cells of the body. Finely tuned regulation of endothelial function is important for the establishment and maintenance of an efficient and functional vascular transport system. As such, endothelial dysfunction can result in the development of tissue ischaemia and has been highlighted as a major step in the development of various degenerative diseases that affect the micro and macro vasculature including a variety of retinopathies such as Retinopathy of Prematurity (ROP). A vast repertoire of signalling pathways have been identified in regulating the functional maintenance of endothelial cells. Two such pathways known to have crucial regulatory roles governing endothelial function are the Notch and endothelial nitric oxide synthase (eNOS) signalling pathways: currently, the interaction between these pathways is poorly described. In part 1 we developed a heterologous system to investigate the effect of eNOS on Notch activation by overexpressing eNOS in HEK 293 cells; cells which are free of endogenous eNOS. eNOS overexpression resulted in increased cell-cell aggregation as well as increased N-Cadherin and Notch1 expression, the latter of which correlated with increased availability of the Notch intracellular domain (NICD). The results from chapter1 implicate eNOS in promoting an epithelial-like phenotype with increased cell-cell adhesion which was associated with enhanced Notch1-Dll4 signalling. In the presence of induced Dll4 expression the epithelial-like phenotype was further enhanced culminating in excessive cell-cell aggregation and the lack of any observable effect with γ-secretase inhibition indicated that augmented Dll4/Notch1 binding enhances cell aggregation. This provides evidence of the presence of Dll4 and Notch1 expression within adjacent cells leading to heterotypic cell-cell binding/adhesion. In part 2 of the study we investigated the functional role of constitutively expressed Dll4 on Notch1 activation in mature primary retinal microvascular endothelial cells (RMECs). We demonstrated using lentiviral-mediated shRNA knockdown that Dll4 has important roles in mediating cell-cell junction and that removal of Dll4 induces an enlarged senescent-like phenotype. These findings were linked to decreased Notch1 activation evidenced by reduced NICD levels indicating Notch 1 as the receptor mediating these effects. In retinal pericytes, the Notch1 receptor also showed evidence of regulating smooth muscle properties through increased Notch 3 signalling. Overall, these findings suggest a previously unknown role for the Notch1 isoform in retinal pericytes (RP) and endothelial cells (EC) in facilitating EC-P interaction. In part 3 using hyperoxia to model the early pathology of ROP we investigated the effect of hyperoxia on EC and on Notch signalling in particular. We demonstrated hyperoxia to have detrimental effects upon endothelial functions such as decreased proliferation with increased senescence and apoptosis while inducing a phenotypic switch to an elongated, spindle-shaped morphology with evidential decreased cell-cell adhesion as shown from epifluorescent and phase contrast images. We also presented evidence of increased Notch signalling under hyperoxia which included increased Notch1 cleavage, upregulation of target genes and activation of a fluorescent Notch1 reporter. Such target genes included the arterial markers Dll4 and Hey2, thus suggesting an arterial phenotype switch under hyperoxia. Collectively, the results generated in this thesis provide new insights into the complexity of the Dll4 and Notch1 signalling pathway in particular the differential signalling dynamics observed in the presence of eNOS and identified functional roles of Dll4 in mature retinal EC. The current study also identified detrimental effects of hyperoxia on endothelial function including decreased proliferation coinciding with increased apoptosis and senescence, all of which correlated with increased Notch signalling. The results provide insight into the pathology of ROP on a molecular and cellular level in particular the role of Notch1.

A major challenge in regenerative medicine is the development of grafts that can be vascularised successfully. Capillary networks facilitate processes that are essential for tissue survival including the supply of nutrients to cells and waste exchange. Therefore, in order to prevent core degradation of tissue-engineered grafts, it is important to provide appropriate physico-chemical conditions to encourage angiogenesis in the period immediately following implantation. Ice-templated collagen scaffolds have been used in tissue engineering due to their bioactivity, tunable biodegradability and the ability to produce architectures with tailored porosity, but until now very little work has focused on vascularisation of these structures. The aim of the work described in this thesis was to develop in vitro cell culture models, to study the effects of structural features and surface chemistry properties on the formation of microvessels. Firstly, the influence of mould design on architectural features of collagen scaffolds fabricated using lyophilisation was assessed using micro-computed tomography (Micro-CT) and scanning electron microscopy (SEM). A range of mould designs was investigated and those made of tissue culture polystyrene, and perspex with a stainless steel base mould were found to produce the most suitable isotropic and anisotropic structures, respectively. Both moulds allowed control and optimisation of the scaffold porosity through freezing protocol modification, enabling analysis of cell behaviour in response to scaffold structure. Secondly, the mechanical and biological functionality of the substrates were investigated with the aim of establishing and improving vascularisation in collagen scaffolds. A co-culture of primary human osteoblasts (hOBs) and human dermal microvascular endothelial cells (HDMECs) was chosen to achieve microvessel formation in vitro. Initially, mono-culture studies were carried out to gain an in-depth understanding of the behaviour of the individual cell types involved in co-culture on substrates with varying mechanical properties and architectural features. Mechanical stability of 2D collagen films and 3D scaffolds was adjusted using 1-ethyl- 3-(3-dimethylaminopropyl)-carbodiimide hydrochloride cross-linking in the presence of N-hydroxysuccinimide (EDC/NHS) at concentrations ranging from 0 to 100% of standard conditions (SC) reported widely in the literature. Cell behaviour was quantified using cell metabolic activity, cytotoxicity, proliferation, adhesion and migration assays. A cross-linking treatment at 30% SC provided the most appropriate combination of mechanical stiffness (Young's modulus: 6 kPa) and specific cell attachment for both cell types. Analysis of cell behaviour in response to structural properties revealed that, at early time points, an increase in percolation diameter had the strongest influence on cell invasion, while the effect of pore size became more prominent at later stages (18 days of incubation). Both hOBs and HDMECs exhibited the highest invasion efficiency on scaffolds with the lowest degree of anisotropy and the highest mean pore size and percolation diameter (approximately 175 and 130 μm, respectively). Films and scaffolds cross-linked at 30% SC were taken forward for structural analysis in co-culture. For in vitro vessel formation, an equilibrium is required between the cell types involved in co-culture. To obtain the optimal co-culture conditions on both collagen films and scaffolds, a range of seeding densities and cell ratios were investigated in relation to their influence on cell- metabolic activity and proliferation behaviour. A systematic study using various hOB:HDMEC cell ratios showed that 70:30 and 50:50 provided the optimal conditions for microvessel formation on 2D films and 3D scaffolds, respectively. Nevertheless, vascularisation of these collagen substrates remained limited. Further assessment in 2D revealed that osteoblast detachment was the main factor restricting vessel formation. Pre-coating of the substrates with a 5 μg/mL fibronectin solution provided the appropriate conditions for osteoblast stabilisation, resulting in abundant vessel formation on collagen substrates. Furthermore, the protein coating was found to counteract the negative influence of high cross-linking levels on specific cell attachment. Overall, fibronectin-coated collagen scaffolds seeded with a ratio of 50:50 hOB:HDMEC resulted in abundant microvascular network formation with multiple branching points and lumen formation. Lastly, the influence of scaffold architecture on vessel formation and invasion was examined using the established co-culture system. On all samples, long term survival of microvessels was achieved up to approximately 31 days of incubation. Structural alignment of the scaffold architecture was found to have the strongest effect on vessel density and ingrowth, with a higher mean pore size and percolation diameter as secondary factors. Anisotropic scaffolds with a mean pore size and percolation diameter of approximately 118 and 108 μm, respectively, exhibited the highest vessel density and invasion depth. The work described in this thesis offers new insights into the factors that underpin microvessel formation and demonstrates that by careful control of scaffold architecture and surface chemistry it has been possible to develop a successful in vitro co-culture model.

Endothelial-to-mesenchymal transition (EndMT) is a dynamic biological process present during development and involved in a variety of pathological vascular remodelling scenarios. However, despite our growing understanding of the key cellular alterations required, the precise molecular determinants governing this phenotypical transition remain elusive. With long non-coding RNAs (lncRNA) now emerging as powerful regulators of gene expression we sought to understand their role in the process of EndMT. To replicate EndMT in vitro and characterise its molecular signature, human umbilical vein endothelial cells (HUVEC) and human pulmonary artery endothelial cells (HPAEC) were exposed to a continuous co-treatment of transforming growth factor-beta 2 (TGF-β2) and interleukin 1 beta (IL-1β) for a total of 7 days. Using high-throughput RNA-sequencing analysis of these cells, a total of 103 differential expressed lncRNAs were identified. Of these, the downregulation of the lncRNA MIR503HG was found to be a prevalent feature present in multiple human primary EC types undergoing EndMT in vitro. Further analysis revealed that depletion of MIR503HG was sufficient to elicit a robust EndMT phenotype, with a significant increase in the expression of SNAI2, ACTA2 and COL1A1, accompanied by repression of CD31. Conversely, ectopic expression of a single MIR503HG transcript suppressed these hallmark EndMT-associated changes despite TGF-β2 and IL-1β co-treatment. Accompanying RNA-sequencing of these cells showed that the overexpression of MIR503HG alone was able to inhibit over 25% of the EndMT transcriptional profile. Crucially, these changes were found to be independent of the functional regulation of miR-503 and miR-424, found within the MIR503HG locus. Our findings were then confirmed in vivo using a sugen/hypoxia-induced model of pulmonary hypertension (PH) established in endothelial lineage-tracing mice. Here, the expression of the MIR503HG mouse homolog (Gm28730) was significantly downregulated in association with an EndMT profile in the lung. Conversely, targeted up-regulation of MIR503HG in the mouse lung significantly suppressed the appearance of mesenchymal markers in CD31+ cells during PH. Notably, MIR503HG availability was also found to be decreased in lung tissue sections from patients with idiopathic pulmonary arterial hypertension (IPAH) and cultured blood outgrowth ECs isolated from patients with heritable pulmonary arterial hypertension (HPAH). Collectively, our studies identify MIR503HG as essential in maintaining EC phenotypical commitment and preventing EndMT both in vitro and during disease.

Pulmonary arterial hypertension (PAH) is a rare, devastating disease with no cure. Current treatment consists of a cocktail of vasodilators which relieve symptoms of PAH but do not treat the cause. Thus, there is a need for novel drugs that target the underlying pathological causes of PAH. PAH is a multi-factorial, but one key contributor is the pro-inflammatory cytokine IL-6 which stimulates pro-inflammatory and pro-angiogenic signalling mediated by the JAK/STAT pathway. One way in which IL-6 signalling via JAK/STAT is inhibited is via SOCS3 in a type of negative feedback loop whereby IL-6 induces transcription of SOCS3, which then attenuates further JAK/STAT signalling. SOCS3 can also be induced by cAMP. This is interesting as prostanoids, a type of drug used in the treatment of PAH due to its vasodilator effects and the only type to show any efficacy improving the life expectancy of PAH patients, acts by mobilising cAMP. Thus, prostanoid stimulation of cAMP could potentially limit IL-6 signalling via the induction of SOCS3. This is a novel mechanism of prostanoids which has not previously been considered. This study investigated the capability of prostanoids to limit the pro-inflammatory/pro-angiogenic effects of IL-6 that enable PAH to develop. Initial experiments confirmed that vascular endothelial cells responded to prostanoids which increased SOCS3 and limited IL-6 signalling activity. Further experiments utilising SOCS3 KO endothelial cell models demonstrated prostanoid inhibition of IL-6 signalling was due in part to SOCS3. In conclusion, this project has confirmed that prostanoids do limit the pro-inflammatory effects induced by IL-6 and that this is in part due to SOCS3. Although the exact mechanism is yet to be discovered, it will be beneficial in the treatment of PAH as it provides currently unexploited drug targets which can be considered for future PAH therapies.

The blood-brain barrier (BBB) is a unique physical and enzymatic barrier, formed by the endothelial cells of the cerebral capillaries. The BBB regulates movement of endogenous and exogenous compounds into, and out of, the central nervous system (CNS) and protects the brain from exposure to xenobiotics. A number of ATP-binding cassette (ABC) transporters are expressed in brain endothelial cells and actively efflux an array of substances from the cell into the systemic circulation, thereby limiting CNS penetration. Alzheimer's disease is a progressive, fatal neurodegenerative disorder and the most common form of dementia. One of the key pathological characteristics of Alzheimer's disease is aggregation of amyloid-ß (Aß(1-42)) peptide within the brain, leading to production of cytotoxic oligomers, plaque formation, inflammation and subsequent neurological deficit. Aß(1-42) is transported across the BBB, with influx into the brain mediated by the receptor for advanced glycation end products (RAGE) and clearance via the low density lipoprotein-related protein (LRP) receptor 1 (LRP1) and it has been postulated that reduced Aß(1-42) clearance from the brain may be associated with accumulation of Aß(1-42) in Alzheimer's disease. ABC transportermediated transport of amyloid-ß has also been reported. The overall aims of this thesis were (i) to investigate the roles of ABCB1 (MDR1, P-glycoprotein), ABCC5 (multidrug resistance-associated protein 5, MRP5) and ABCG2 (breast cancer resistance protein, BCRP) in Aß(1-42) transport, (ii) determine the effects of Aß(1-42) exposure on ABC transporter expression and function and (iii) determine the effects of Aß(1-42) exposure on expression of the constitutive androstane receptor (CAR) and pregnane X receptor (PXR), key nuclear receptors governing ABC transporter expression (iv) determine the ability of the ABCB1, ABCC5 and ABCG2 inhibitors and inducers to modify transcellular Aß(1–42) transport, (v) determine the effect of inflammatory conditions on ABCB1 and ABCG2 activity and expression. In this thesis, primary porcine brain endothelial cells (PBECs) were used as an in vitro BBB model since this model retains phenotypic features of the in vivo BBB, including expression of marker enzymes, ABC transporters, PXR and CAR. Aß(1-42) significantly inhibited ABCB1, ABCG2 and ABCC5 activity, as determined by increased intracellular accumulation of fluorescent probe substrates, and expression in PBECs, as measured densitometrically by Western blotting, by up to 55%. The inhibitory effect of Aß(1-42) was both concentration- and time-dependent. Aß(1-42) treatment also significantly reduced expression of both PXR and CAR in PBECs up to 18 % and 30 %, respectively. Further studies investigated the effects of therapeutic drugs classed as ABC transporter inducers (dexamethasone, doxorubicin, ritonavir and rifampicin) or inhibitors (lovastatin, indomethacin, olanzapine, glibenclamide, chlorpromazine, telmisartan, pantoprazole, quinidine, sulfasalazine and nefazodone) on Aß(1-42) transport. When PBECs were co-treated with inhibitors and Aß(1-42) there was a significant increase in intracellular accumulation of Aß(1-42). Furthermore, when PBECs were pre-loaded with Aß(1-42), and treated with inhibitors, Aß(1-42) efflux was significantly reduced. In a Transwell® model of the BBB, treatment of PBECs with inducer therapeutic drugs significantly reduced Aß(1-42) apical to basal penetration (i.e. uptake) up to 1.75-fold, but significantly increased basal to apical penetration (i.e. efflux clearance) up to 2-fold. Treatment of PBECs with the pro-inflammatory cytokine interleukin-1ß significantly increased ABCB1 expression and activity by 32% and 48% respectively, but no significant effect on ABCG2 expression and activity was observed. Under conditions of oxidative stress (i.e. treatment of PBECs with hydrogen peroxide) no significant change in ABCB1 and ABCG2 expression and activity were observed. In summary, Aß(1-42) significantly inhibits the activities and the expression of BBB ABCB1, ABCC5 and ABCG2, and modulates expression of CAR and PXR, key components of the transporter regulatory pathway. Aß(1-42) therefore has the potential to impact on penetration of ABC transporter substrates, including therapeutic drugs, across the BBB. This thesis demonstrates that therapeutic drugs can induce expression of ABC transporters in BBB endothelial cells which may aid clearance of Aß(1-42) from within the brain. Furthermore, therapeutic drugs that inhibit ABC transporters may reduce Aß(1-42) clearance from brain via a drug-Aß(1-42) interaction.

Drug safety is a major cause of attrition in contemporary drug development. Many promising candidate drugs are terminated in the developmental process or withdrawn in the post-approval stage because of adverse drug reactions (ADRs). Among these, cardiovascular (CV) ADRs occur with a high incidence. Affecting the heart, blood vessels and blood components, CV ADRs can lead to hypertension, heart failure, cardiac arrhythmias and thrombosis. The prompt identification of CV toxicity would reduce both the delay and cost of drug development and increase patient safety. Drug-induced vascular injury (DIVI) in pre-clinical toxicology studies involves damage to endothelial (EC) and smooth muscle cells (SMC) in small and medium sized vessels, but little is known about the mechanisms of DIVI. Damage includes microhaemorrhage, endothelial junction disruption, SMC necrosis and inflammation. DIVI is often observed preclinically, driving a need to study this in human tissue. The aim of this thesis was to develop and characterise an in vitro human model that would allow the study of DIVI in human tissue during drug development. The first stage was to effectively reproduce DIVI in an animal model. To reduce the utilisation of live animals, a vessel explant was used. To determine whether DIVI was recapitulated ex vivo von Willebrand factor (VWF) release, one of the hallmarks of DIVI, was analysed using four drugs reported in the literature to produce DIVI. The same four drugs were then tested in human EC and SMC for the following features of DIVI: (1) VWF release; (2) EC monolayer disruption; (3) inflammation; (4) EC and SMC death. Both ex vivo in rats and in vitro in humans, the drugs did not exhibit common characteristics and each expressed DIVI features in different ways. The human in vitro model was also assessed in a co-culture of EC and SMC, and in the presence of a flow-mimetic that reproduces in vivo-derived hemodynamics. It was concluded that DIVI cannot be fully recapitulated in vitro in such a reductionist cellular model, suggesting that exposure to the complex microenvironment of the vasculature may be required for the lesions to develop, or that DIVI may, in fact, not be relevant to humans at all.

One of the greatest obstacles to clinical translation of bone tissue engineering is the inability to effectively and efficiently vascularise scaffolds. This limits the size of defects that can be repaired, as blood perfusion is necessary to provide nutrient and waste exchange to tissue at the core of scaffolds. The goal of this work was to systematically explore whether architecture, at a scale of hundreds of microns, can be used to direct the growth of microvessels into the core of scaffolds. A pipeline was developed for the production of hydroxyapatite surfaces with controlled architecture. Three batches of hydroxyapatite were used with two different particle morphologies and size distributions. On sintering, one batch remained phase pure and the other two batches were biphasic mixtures of α-tricalcium phosphate (α-TCP) and hydroxyapatite. Sample production methods based on slip casting of a hydroxyapatite-gelatin slurry were explored. The most successful of these involved the use of curable silicone to produce moulds of high-resolution, three dimensional (3D) printed parts with the desired design. Parts were dried and sintered to produce patterned surfaces with higher resolution than obtainable through conventional 3D printing techniques. Given the difficulties associated with the structural reproducibility of concave pores architectures in 3D reported in the literature, in this work, a 2.5D model has been developed that varies architectural parameters in a controlled manner. Six contrasting architectures consisting of semi-circular ridges and grooves were produced. Grooves and ridges were designed to have widths of 330 μm and 660 μm, with periodicities, respectively, of 1240 μm and 630 μm. Groove depth was varied between 150 μm and 585 μm. Co-cultures of endothelial cells and osteoblasts were optimised and used to grow microcapillary-like structures (referred to as "microvessels") on substrates. Literature shows that these precursors to microcapillaries contain lumina and can produce functional vasculature, demonstrating their clinical promise. The effects of the composition and surface texture of grooved samples on microvessel formation were studied. It was found that surface microtopography and phase purity (α-TCP content) did not affect microvessel formation. However, hydroxyapatite architecture was found to significantly affect microvessel location and orientation. Microvessels were found to form predominantly in grooves or between convexities. Two metrics - the degree of alignment (DOA) and the degree of containment (DOC) - were developed to measure the alignment of endothelial cell structures and their localisation in grooves. For all patterned samples, the CD31 (an endothelial cell marker) signal was at least 2.5 times higher along grooves versus perpendicular to grooves. In addition, the average signal was at least two times higher within grooves than outside grooves for all samples. Small deep grooves had the highest DOA and DOC (6.13 and 4.05 respectively), and individual, highly aligned microvessels were formed. An image analysis method that compares sample X-ray microtomography sections to original designs to quantify architectural distortion was developed. This method will serve as a useful tool for improvements to architectural control for future studies. This body of work shows the crucial influence of architecture on microvessel self-assembly at the hundreds of micron scale. It also highlights that microvessel formation has a relatively low sensitivity to phase composition and microtopography. These findings have important implications for the design of porous scaffolds and the refinement of fabrication technologies. While important results were shown for six preliminary architectures, this work represents a toolkit that can be applied to screen any 2.5D architecture for its angiogenic potential. This work has laid the foundations that will allow elucidating the precise correspondence between architecture and microvessel organisation, ultimately enabling the "engineering" of microvasculature by tuning local scaffold design to achieve desirable microvessel properties.

Cardiovascular disease is a major cause of morbidity and mortality around the globe, which is linked to athero-thrombosis. The risk factors for atherothrombosis, thus cardiovascular disease is impaired anti-thrombotic and antiinflammatory functions of the endothelium. Thrombosis is a hallmark of cardiovascular disease/complications characterised by increased platelet activation and increased secretion of platelet micro-particles that induce angiogenesis. This study determined the role of platelet micro-particles derived microRNA in the regulation of angiogenesis and migration, with a focus on the regulation of thrombospondin-1 release by platelet micro-particles delivered Let- 7a. The role of thrombospondin-1 receptors (integrin beta-1 and integrin associated protein) and downstream caspase-3 activation were explored by Let-7a inhibition prior to PMP treatment. MicroRNA dependent modulation of proangiogenic proteins including monocyte chemoattractant protein-1 and placental growth factor, and recruitment of activating transcription factor-4 protein to their promoter regions were explored. Main findings are: 1. Platelet micro-particles induce angiogenesis, migration, and release of novel cytokine subsets specific to platelet micro-particle’s RNA content. 2. The targeting of thrombospondin-1 mRNA by platelet micro-particles’ transferred Let-7a chiefly modulate the angiogenic effect on endothelial cells. 3. The inhibition of thrombospondin-1 translation enable platelet micro-particles to increase angiogenesis and migration in the presence of functional integrin beta-1 and integrin associated protein, and reduced cleaving of caspase-3. 4. Platelet micro-particle modulate the transcription of monocyte chemoattractant protein-1 and placental growth factor in a Let-7a dependent manner. 5. Let-7a induce angiogenesis ii independent of other platelet micro-particle’s microRNAs. Platelet micro-particle derived Let-7a is a master regulator of endothelial cell function in this model, which presents an opportunity for the development of new biomarkers and therapeutic approaches in the management of cardiovascular disease. Future studies should aim to confirm these findings in-vivo.

Epithelial ovarian cancer frequently metastasizes to the omentum, a process that requires pro-angiogenic activation of local microvascular endothelial cells (ECs) by tumour-secreted factors. We have previously shown that ovarian cancer cells secrete factors, other than vascular endothelial growth factor (VEGF), with possible roles in metastatic angiogenesis including the lysosomal proteases cathepsin L (CathL) and cathepsin D (CathD), and insulin-like growth factor binding protein 7 (IGFBP7). However, the mechanisms by which these factors may contribute to omental endothelial angiogenic changes are unknown. Therefore the aims of this thesis were a) to examine disease relevant human omental microvascular endothelial cell (HOMEC) proliferation, migration and angiogenesis tube-formation induced by CathL, CathD and IGFBP7; b) to investigate whether CathL and CathD act via a proteolytic or non-proteolytic mechanism; c) to identify activated downstream intracellular signalling cascades in HOMECs and their activation in proliferation and migration; and finally d) to identify activated cell surface receptors by these factors. CathL, CathD and IGFBP7 significantly induced proliferation and migration in HOMECs, with CathL and CathD acting in a non-proteolytic manner. Proteome-profiler and ELISA data identified increased phosphorylation of the ERK1/2 and AKT (protein kinase B) pathways in HOMECs in response to these factors. CathL induced HOMEC proliferation and migration via the ERK1/2 pathway, whereas, although CathD-induced proliferation was mediated by activation of ERK1/2, its migratory effect was dependent on both ERK1/2 and AKT pathways. Interestingly, CathL induced secretion of galectin-1 (Gal1) from HOMECs which in turn significantly induced HOMEC proliferation via ERK1/2. However, none of the ERK1/2 or AKT pathways was observed to be active in Gal1-induced HOMEC migration. Interestingly, Gal1-induced proliferation and migration were significantly inhibited by L-glucose, suggesting a role for a receptor with extracellular sugar moieties. IGFBP7-induced migration was shown to be mediated via activation of the ERK1/2 pathway only. CathL, Gal1 and IGFBP7 significantly induced angiogenesis tube-formation in HOMECs which was not observed in CathD-treated cells. Receptor tyrosine kinase array revealed activation of Tie-1 and VEGF receptor type 2 (VEGFR2) in CathL and IGFBP7-treated HOMECs respectively. In conclusion, all CathL, CathD, Gal1 and IGFBP7 have the potential to act as proangiogenic factors in the metastasis of ovarian cancer to the omentum. These in vitro data suggest all four factors activate intracellular pathways which are involved in well-known angiogenesis models.

Background: Our laboratory identified ELTD1, an orphan GPCR belonging to the adhesion GPCR family (aGPCR), as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. ELTD1 is normally expressed in both endothelial cells and vascular smooth muscle cells and expression is significantly increased in the tumour vasculature. The aim of this project was to analyse ELTD1's function in endothelial cells and its role in breast cancer. Method: 62 sequenced vertebrate genomes were interrogated for ELTD1 conservation and domain alterations. A phylogenetic timetree was assembled to establish time estimates for ELTD1's evolution. After ELTD1 silencing, mRNA array profiling was performed on primary human umbilical vein endothelial cells (HUVECs) and validated with qPCR and confocal microscopy. ELTD1's signalling was investigated by applying the aGPCR ‘Stinger/tethered-agonist Hypothesis'. For this, truncated forms of ELTD1 and peptides analogous to the proposed tethered agonist region were designed. FRET-based 2nd messenger (Cisbio IP-1;cAMP) and luciferase-reporter assays (NFAT; NFÎoB; SRE; SRF-RE; CREB) were performed to establish canonical GPCR activation. To further investigate ELTD1's role in endothelial cells, ELTD1 was stably overexpressed in HUVECS. Functional angiogenesis assays and mRNA array profiling were then performed. To investigate ELTD1 in breast cancer, a panel of cell lines representative of all molecular subtypes were screened using qPCR. Furthermore, an exploratory pilot study was performed on matched primary and regional nodal secondary breast cancers (n=43) which were stained for ELTD1 expression. Staining intensity was then scored and compared with relapse free survival and overall survival. Results: ELTD1 arose 435 million years ago (mya) in bony fish and is present in all subsequent vertebrates. ELTD1 has 3 evolutionary variants of which 2 are most common: one variant with 3 EGFs and a variant with 2 EGFs. Additionally, ELTD1 may be ancestral to members of aGPCR family 2. HUVEC mRNA expression profiling after ELTD1 silencing showed upregulation of the mitochondrial citrate transporter SLC25A1, and ACLY which converts cytoplasmic citrate to Acetyl CoA, feeding fatty acid and cholesterol synthesis, and acetylation. A review of lipid droplet (fatty acid and cholesterol) accumulation by confocal microscopy and flow cytometry (FACS) revealed no changes with ELTD1 silencing. Silencing was also shown to affect the Notch pathway (downregulating the Notch ligand JAG1 and target gene HES2; upregulating the Notch ligand DLL4) and inducing KIT, a mediator of haematopoietic (HSC) and endothelial stem cell (ESC) maintenance. Signalling experiments revealed that unlike other aGPCRs, ELTD1 does not couple to any canonical GPCR pathways (Gαi, Gαs, Gαq, Gα12/13). ELTD1 overexpression in HUVECS revealed that ELTD1 induces an endothelial tip cell phenotype by promoting sprouting and capillary formation, inhibiting lumen anastomoses in mature vessels and lowering proliferation rate. There was no effect on wound healing or adhesion to angiogenesis associated matrix components. Gene expression changes following ELTD1 overexpression included upregulation of angiogenesis associated ANTRX1 as well as JAG1 and downregulation of migration associated CCL15 as well as KIT and DLL4. In breast cancer, none of the representative breast cancer cell lines screened expressed ELTD1. ELTD1 breast cancer immunohistochemistry revealed higher levels of vascular ELTD1 staining intensity within the tumour stroma contrasted to normal stroma and expression within tumour epithelial cells. Additionally, ELTD1 expression in tumour vessels was differentially expressed between the primary breast cancer microenvironment and that of the matched regional node. Due to the small size of the pilot study population, survival comparisons between the various subgroups did not yield significant results. Conclusion: ELTD1 is a novel regulator of endothelial metabolism through its suppression of ACLY and the related citrate transporter SLC25A1. ELTD1 also represses KIT, which is known to mediate haematopoietic and endothelial progenitors stem cell maintenance, a possible mechanism through which endothelial cells maintain terminal endothelial differentiation. ELTD1 does not signal like other adhesion GPCRS with CTF and FL forms of ELTD1 not signalling canonically. Additionally, ELTD1 regulates various functions of endothelial cell behaviour and function, inducing an endothelial tip cell phenotype and is highly evolutionarily conserved. Lastly, ELTD1 is differentially expressed in tumour vessels between primary breast cancer and regional nodal metastases and is also expressed in a small subset of breast cancer cells in vivo despite no cancer cell lines expressing ELTD1. The pilot study investigating ELTD1 in the primary breast cancer and regional involved nodes will be followed up with a larger study including the investigation of ELTD1 in distant metastases.

Tumour angiogenesis is a hallmark of cancer. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in endothelial cells (ECs) survival, proliferation and migration. FAK has several tyrosine phosphorylation sites thought to be involved in FAK function but the requirement of phosphorylation of these residues in vivo is unknown. We have generated mice where endogenous FAK is deleted simultaneously with the expression of nonphosphorylatable FAK-Y397F or FAK-Y861F mutated or wild type forms of FAK in adult endothelium in order to test this. My data show that EC-FAK-Y397FKI mice present with decreased tumour angiogenesis (in sygeneic B16F0, CMT19T and LLC) but impaired B16F0 and CMT19T tumour growth only, with increased tumour hypoxia. FAK-Y397F tumour endothelium is not perfusion, leakage or vascular maturation defective. This mutation affects VEGF-, PlGF- and bFGF-driven angiogenesis in vivo and VEGF+Ang2 administration is able to partially rescue this phenotype ex vivo. In contrast, endothelial FAK-Y861F mutation leads to an initial delay in B16F0 tumour angiogenesis, that subsequently resolves, and does not affect B16F0 tumour growth. LLC and CMT19T tumour growth and angiogenesis are not affected by the endothelial FAK-Y861F mutation; neither are tumour blood vessel perfusion, leakage, vascular maturation or tumour hypoxia. VEGF-, PlGFand bFGF-driven angiogenesis in vivo and ex vivo was not affected by the endothelial FAK-Y861F mutation, whereas increased in vivo angiogenesis was triggered by Ang2 administration. Lastly, to understand whether cytokine profiles that might affect angiocrine signalling are affected differentially in FAK-Y397F vs FAK-Y861F endothelial cells, I show that CCL1 and CCL2 are increased in FAK-Y397F but IL- 13, IL-1F3, CCL4, IL-1F1, CCL2 and others are increased in FAK-Y861F endothelial cells. Overall my data indicates that endothelial-specific FAK mutations on two phosphorylation sites has different effects on tumour angiogenesis, tumour growth, growth factor stimulated angiogenesis in vivo and ex-vivo and cytokine production.

Introduction and aims: The branched-chain aminotransferase (BCAT) enzymes are important in the regulation of brain L-glutamate. A unique function of these aminotransferases is their regulation by the redox environment, where our group have shown their function as oxidoreductases, and their ability to refold misfolded proteins in particular when S-glutathionylated. Our group recently showed a significant increase in the level of these proteins in Alzheimer’s disease brain. An increase in BCAT metabolism could generate excess glutamate, contributing to the excitotoxic environment observed under pathogenic conditions. Alternatively, we hypothesize that if this protein is modified in response to cellular stress, it may play a more prominent role in regulating redox status or protein folding. To address these questions this project focussed on the design of chemical inhibitors and knock-down models together with a co-culture model of the human cerebrovasculature and specifically targeted key metabolic and redox pathways. Methods: Several chemical inhibitors based on 4-Benzyloxyphenylacetic acid were identified using the DockBlaster and Schrödinger software suites, synthesized and structurally verified using proton nuclear magnetic resonance (NMR) spectroscopy. The functional impact and specificity of these inhibitors was assessed using the BCAT radiolabelled assay and a coupled-enzyme assay. In tandem, siRNA was used to knock-down both isoforms in SH-SY5Y cells, and validated using Western blot analysis and RT-PCR. The impact of BCAT inhibition on the expression of redox proteins, in addition to selected metabolic proteins, was assessed by Western blot analysis. Functional redox assays, including glutathione concentration and metabolic activity, were also used to investigate the impact of human BCAT (hBCAT) expression in neuronal cells. Finally, a model of the blood-brain barrier (BBB) was developed and validated for studies into the role of mitochondrial hBCAT (hBCATm) in brain microvasculature. Results: For the first time we have identified a family of chemical inhibitors of hBCATm. In particular, benzofenac has a two-fold greater enzyme affinity for hBCATm (Ki=43 μM) than cytosolic hBCAT (hBCATc) (Ki=93 μM) and a four-fold greater inhibition relative to alanine transaminase (ALT; Ki=167 μM). These inhibitors will require further optimisation but have potential as tools to assess the cellular function of hBCAT. In separate studies, knock-down of hBCATm in SH-SY5Y neuronal cells demonstrated that hBCATm expression has an impact on the metabolic and redox status of the cell. In particular, knock-down caused a >70% decrease in glutaredoxin (GRx), thioredoxin (TRx), branched-chain α-keto acid dehydrogenase α-subunit (BCKDHA), and AU-rich binding homolog of enoyl-CoA hydratase (AUH) expression. Interestingly, this effect was attenuated when cells were treated with L-leucine, indicating that these mechanisms may be regulated by a metabolic signal. L-glutamate treatment was also found to significantly increase hBCATm expression, but decreased glutamate dehydrogenase (GDH) expression, except in cells overexpressing hBCATm, suggesting a metabolic synergy between the two enzymes. Finally, total glutathione concentration was significantly decreased on hBCATm knock-down, while sensitivity to L-glutamate toxicity was significantly increased. Discussion: Results from this work has significantly contributed to the design of cellular models, which can be used to further investigate the role of BCAT in metabolic and redox metabolism. A model of the human blood-brain barrier, developed in this thesis, will also contribute to evaluating the endothelial role of hBCATm, particular to humans. The initial impact of limiting BCAT expression is both a reduction in the expression of key metabolic proteins and also the cellular reductants of the cell. Together this had an impact on cell viability and survival. Knowledge of these pathways and their regulation will be important to our understanding, of not only the regulation of brain glutamate, but also the role of BCAT in protein folding and as a contributor to cellular redox status. These factors are fundamentally important to the development of neurodegenerative conditions but also to tumour development such as gliomas.