Investigating the role of N-WASP in the development of colorectal cancer

Colorectal cancer (CRC) is the second most common cancer in Europe, with the second highest mortality rate. Although prognosis is improving, survival rates remain poor for those presenting with the most advanced stages of the disease. There is therefore a need for improved early diagnosis and thus a greater understanding of the early stages of the development of colorectal tumours is desirable. Additionally, as most deaths in colorectal cancer are due to advanced metastatic disease, it is of great interest to explore any potential mechanisms by which metastatic disease can be inhibited. N-WASP is a ubiquitously expressed protein with multiple intracellular roles including actin regulation and maintaining stability of epithelial cell-cell junctions. Through its role as an actin regulator, it has been implicated in the processes of invasion and metastasis of multiple cancer types. Its role in the development and progression of colorectal cancer however has not been fully explored. This thesis will present a series of in vitro and in vivo studies that were carried out with the aim of answering the following questions: • Does N-Wasp have a role in normal intestinal homeostasis? • Does N-Wasp knockout affect the development of tumours in a mouse model of intestinal tumourigenesis? • Does N-Wasp knockout affect the invasive properties of intestinal cancer in vitro? • Does N-WASP correlate with prognosis or other indicators in human colorectal cancer TMAs? Findings from the in vivo experiments, using an inducible, gut-specific knockout model, have uncovered potential roles for N-Wasp in regulating differentiation and migration of intestinal epithelial cells. Although it had no effect in short term models of intestinal hyperproliferation, N-Wasp knockout increased tumour burden and decreased survival in an established in vivo model of intestinal tumourigenesis, in which there is heterozygous loss of Apc (Apcfl/+). No effect was seen on tumour development or survival when additional N-WASP knockout was introduced into a more rapid model, with heterozygous loss of Apc and mutation of Kras (Apcfl/+ KrasG12D/+). N-WASP expression in human colorectal cancer was assessed using immunohistochemical staining of two tissue microarrays. Low levels of N-WASP expression were found to be associated with presence of MMR deficiency. There was no statistically significant difference in overall or cancer specific survival based on N-WASP expression. Collectively, the data presented here suggest a previously unreported role for N-WASP in regulation of intestinal epithelial differentiation and indicate that it may act as a tumour suppressor against development of benign precursor lesions of colorectal cancer. Further research is warranted to delineate the mechanisms underlying these processes.

TRIB2 in normal and malignant hematopoiesis - a transcription factor network

Hematopoiesis is the tightly controlled and complex process in which the entire blood system is formed and maintained by a rare pool of hematopoietic stem cells (HSCs), and its dysregulation results in the formation of leukaemia. TRIB2, a member of the Tribbles family of serine/threonine pseudokinases, has been implicated in a variety of cancers and is a potent murine oncogene that induces acute myeloid leukaemia (AML) in vivo via modulation of the essential myeloid transcription factor CCAAT-enhancer binding protein α (C/EBPα). C/EBPα, which is crucial for myeloid cell differentiation, is commonly dysregulated in a variety of cancers, including AML. Two isoforms of C/EBPα exist - the full-length p42 isoform, and the truncated oncogenic p30 isoform. TRIB2 has been shown to selectively degrade the p42 isoform of C/EBPα and induce p30 expression in AML. In this study, overexpression of the p30 isoform in a bone marrow transplant (BMT) leads to perturbation of myelopoiesis, and in the presence of physiological levels of p42, this oncogene exhibited weak transformative ability. It was also shown by BMT that despite their degradative relationship, expression of C/EBPα was essential for TRIB2 mediated leukaemia. A conditional mouse model was used to demonstrate that oncogenic p30 cooperates with TRIB2 to reduce disease latency, only in the presence of p42. At the molecular level, a ubiquitination assay was used to show that TRIB2 degrades p42 by K48-mediated proteasomal ubiquitination and was unable to ubiquitinate p30. Mutation of a critical lysine residue in the C-terminus of C/EBPα abrogated TRIB2 mediated C/EBPα ubiquitination suggesting that this site, which is frequently mutated in AML, is the site at which TRIB2 mediates its degradative effects. The TRIB2-C/EBPα axis was effectively targeted by proteasome inhibition. AML is a very difficult disease to target therapeutically due to the extensive array of chromosomal translocations and genetic aberrations that contribute to the disease. The cell from which a specific leukaemia arises, or leukaemia initiating cell (LIC), can affect the phenotype and chemotherapeutic response of the resultant disease. The LIC has been elucidated for some common oncogenes but it is unknown for TRIB2. The data presented in this thesis investigate the ability of the oncogene TRIB2 to transform hematopoietic stem and progenitor cells in vitro and in vivo. TRIB2 overexpression conferred in vitro serially replating ability to all stem and progenitor cells studied. Upon transplantation, only TRIB2 overexpressing HSCs and granulocyte/macrophage progenitors (GMPs) resulted in the generation of leukaemia in vivo. TRIB2 induced a mature myeloid leukaemia from the GMP, and a mixed lineage leukaemia from the HSC. As such the role of TRIB2 in steady state hematopoiesis was also explored using a Trib2-/- mouse and it was determined that loss of Trib2 had no effect on lineage distribution in the hematopoietic compartment under steady-state conditions. The process of hematopoiesis is controlled by a host of lineage restricted transcription factors. Recently members of the Nuclear Factor 1 family of transcription factors (NFIA, NFIB, NFIC and NFIX) have been implicated in hematopoiesis. Little is known about the role of NFIX in lineage determination. Here we describe a novel role for NFIX in lineage fate determination. In human and murine datasets the expression of Nfix was shown to decrease as cells differentiated along the lymphoid pathway. NFIX overexpression resulted in enhanced myelopoiesis in vivo and in vitro and a block in B cell development at the pre-pro-B cell stage. Loss of NFIX resulted in disruption of myeloid and lymphoid differentiation in vivo. These effects on stem and progenitor cell fate correlated with changes in the expression levels of key transcription factors involved in hematopoietic differentiation including a 15-fold increase in Cebpa expression in Nfix overexpressing cells. The data presented support a role for NFIX as an important transcription factor influencing hematopoietic lineage specification. The identification of NFIX as a novel transcription factor influencing lineage determination will lead to further study of its role in hematopoiesis, and contribute to a better understanding of the process of differentiation. Elucidating the relationship between TRIB2 and C/EBPα not only impacts on our understanding of the pathophysiology of AML but is also relevant in other cancer types including lung and liver cancer. Thus in summary, the data presented in this thesis provide important insights into key areas which will facilitate the development of future therapeutic approaches in cancer treatment.

Oxidative stress and its haemodynamic consequences in chronic kidney disease

Chronic kidney disease (CKD) is associated with increased cardiovascular risk in comparison with the general population. This can be observed even in the early stages of CKD, and rises in proportion to the degree of renal impairment. Not only is cardiovascular disease (CVD) more prevalent in CKD, but its nature differs too, with an excess of morbidity and mortality associated with congestive cardiac failure, arrhythmia and sudden death, as well as the accelerated atherosclerosis which is also observed. Conventional cardiovascular risk factors such as hypertension, dyslipidaemia, obesity, glycaemia and smoking, are highly prevalent amongst patients with CKD, although in many of these examples the interaction between risk factor and disease differs from that which exists in normal renal function. Nevertheless, the extent of CVD cannot be fully explained by these conventional risk factors, and non-conventional factors specific to CKD are now recognised to contribute to the burden of CVD. Oxidative stress is a state characterised by excessive production of reactive oxygen species (ROS) and other radical species, a reduction in the capacity of antioxidant systems, and disturbance in normal redox homeostasis with depletion of protective vascular signalling molecules such as nitric oxide (NO). This results in oxidative damage to macromolecules such as lipids, proteins and DNA which can alter their functionality. Moreover, many enzymes are sensitive to redox regulation such that oxidative modification to cysteine thiol groups results in activation of signalling cascades which result in adverse cardiovascular effects such as vascular and endothelial dysfunction. Endothelial dysfunction and oxidative stress are present in association with many conventional cardiovascular risk factors, and can be observed even prior to the development of overt, clinical, vascular pathology, suggesting that these phenomena represent the earliest stages of CVD. In the presence of CKD, there is increased ROS production due to upregulated NADPH oxidase (NOX), increase in a circulating asymmetric dimethylarginine (ADMA), uncoupling of endothelial nitric oxide synthase (eNOS) as well as other mechanisms. There is also depletion in exogenous antioxidants such as ascorbic acid and tocopherol, and a reduction in activity of endogenous antioxidant systems regulated by the master gene regulator Nrf-2. In previous studies, circulating markers of oxidative stress have been shown to be increased in CKD, together with a reduction in endothelial function in a stepwise fashion relating to the severity of renal impairment. Not only is CVD linked to oxidative stress, but the progression of CKD itself is also in part dependent on redox sensitive mechanisms. For example, administration of the ROS scavenger tempol attenuates renal injury and reduces renal fibrosis seen on biopsy in a mouse model of CKD, whilst conversely, supplementation with the NOS inhibitor L-NAME causes proteinuria and renal impairment. Previous human studies examining the effect of antioxidant administration on vascular and renal function have been conflicting however. The work contained in this thesis therefore examines the effect of antioxidant administration on vascular and endothelial function in CKD. Firstly, 30 patients with CKD stages 3 – 5, and 20 matched hypertensive controls were recruited. Participants with CKD had lower ascorbic acid, higher TAP and ADMA, together with higher augmentation index and pulse wave velocity. There was no difference in baseline flow mediated dilatation (FMD) between groups. Intravenous ascorbic acid increased TAP and O2-, and reduced central BP and augmentation index in both groups, and lowered ADMA in the CKD group only. No effect on FMD was observed. The effects of ascorbic acid on kidney function was then investigated, however this was hindered by the inherent drawbacks of existing methods of non-invasively measuring kidney function. Arterial spin labelling MRI is an emerging imaging technique which allows measurement of renal perfusion without administration of an exogenous contrast agent. The technique relies upon application of an inversion pulse to blood within the vasculature proximal to the kidneys, which magnetically labels protons allowing measurement upon transit to the kidney. At the outset of this project local experience using ASL MRI was limited and there ensued a prolonged pre-clinical phase of testing with the aim of optimising imaging strategy. A study was then designed to investigate the repeatability of ASL MRI in a group of 12 healthy volunteers with normal renal function. The measured T1 longitudinal relaxation times and ASL MRI perfusion values were in keeping with those found in the literature; T1 time was 1376 ms in the cortex and 1491 ms in the whole kidney ROI, whilst perfusion was 321 mL/min/100g in the cortex, and 228 mL/min/100g in the whole kidney ROI. There was good reproducibility demonstrated on Bland Altman analysis, with a CVws was 9.2% for cortical perfusion and 7.1% for whole kidney perfusion. Subsequently, in a study of 17 patients with CKD and 24 healthy volunteers, the effects of ascorbic acid on renal perfusion was investigated. Although no change in renal perfusion was found following ascorbic acid, it was found that ASL MRI demonstrated significant differences between those with normal renal function and participants with CKD stages 3 – 5, with increased cortical and whole kidney T1, and reduced cortical and whole kidney perfusion. Interestingly, absolute perfusion showed a weak but significant correlation with progression of kidney disease over the preceding year. Ascorbic acid was therefore shown to have a significant effect on vascular biology both in CKD and in those with normal renal function, and to reduce ADMA only in patients with CKD. ASL MRI has shown promise as a non-invasive investigation of renal function and as a biomarker to identify individuals at high risk of progressive renal impairment.

New prophylactic and therapeutic treatments to combat pathogenic Enterohaemorrhagic Escherichia coli

Bacterial diarrhoeal diseases have significant influence on global human health, and are a leading cause of preventable death in the developing world. Enterohaemorrhagic Escherichia coli (EHEC), pathogenic strains of E. coli that carry potent toxins, have been associated with a high number of large-scale outbreaks caused by contaminated food and water sources. This pathotype produces diarrhoea and haemorrhagic colitis in infected humans, and in some patients leads to the development of haemolytic uremic syndrome (HUS), which can result in mortality and chronic kidney disease. A major obstacle to the treatment of EHEC infections is the increased risk of HUS development that is associated with antibiotic treatment, and rehydration and renal support are often the only options available. New treatments designed to prevent or clear E. coli infections and reduce symptoms of illness would therefore have large public health and economic impacts. The three main aims of this thesis were: to explore mouse models for pre-clinical evaluation in vivo of small compounds that inhibit a major EHEC colonisation factor, to assess the production and role of two proteins considered promising candidates for a broad-spectrum vaccine against pathogenic E. coli, and to investigate a novel compound that has recently been identified as a potential inhibitor of EHEC toxin production. As EHEC cannot be safely tested in humans due to the risk of HUS development, appropriate small animal models are required for in vivo testing of new drugs. A number of different mouse models have been developed to replicate different features of EHEC pathogenesis, several of which we investigated with a focus on colonisation mediated by the Type III Secretion System (T3SS), a needle-like structure that translocates bacterial proteins into host cells, resulting in a tight, intimate attachment between pathogen and host, aiding colonisation of the gastrointestinal tract. As E. coli models were found not to depend significantly on the T3SS for colonisation, the Citrobacter rodentium model, a natural mouse pathogen closely related to E. coli, was deemed the most suitable mouse model currently available for in vivo testing of T3SS-targeting compounds. Two bacterial proteins, EaeH (an outer membrane adhesin) and YghJ (a putative secreted lipoprotein), highly conserved surface-associated proteins recently identified as III protective antigens against E. coli infection of mice, were explored in order to determine their suitability as candidates for a human vaccine against pathogenic E. coli. We focused on the expression and function of these proteins in the EHEC O157:H7 EDL933 strain and the adherent-invasive E. coli (AIEC) LF82 strain. Although expression of EaeH by other E. coli pathotypes has recently been shown to be upregulated upon contact with host intestinal cells, no evidence of this upregulation could be demonstrated in our strains. Additionally, while YghJ was produced by the AIEC strain, it was not secreted by bacteria under conditions that other YghJ-expressing E. coli pathotypes do, despite the AIEC strain carrying all the genes required to encode the secretion system it is associated with. While our findings indicate that a vaccine that raises antibodies against EaeH and YghJ may have limited effect on the EHEC and AIEC strains we used, recent studies into these proteins in different E. coli pathogens have suggested they are still excellent candidates for a broadly effective vaccine against E. coli. Finally, we characterised a small lead compound, identified by high-throughput screening as a possible inhibitor of Shiga toxin expression. Shiga toxin production causes both the symptoms of illness and development of HUS, and thus reduction of toxin production, release, or binding to host receptors could therefore be an effective way to treat infections and decrease the risk of HUS. Inhibition of Shiga toxin production by this compound was confirmed, and was shown to be caused by an inhibitory effect on activation of the bacterial SOS response rather than on the Shiga toxin genes themselves. The bacterial target of this compound was identified as RecA, a major regulator of the SOS response, and we hypothesise that the compound binds covalently to its target, preventing oligomerisation of RecA into an activated filament. Altogether, the results presented here provide an improved understanding of these different approaches to combating EHEC infection, which will aid the development of safe and effective vaccines and anti-virulence treatments against EHEC.

Investigating Angiotensin II in cardiac remodelling and the counter-regulatory actions of Angiotensin-(1-9)

Cardiovascular diseases (CVDs) including, hypertension, coronary heart disease and heart failure are the leading cause of death worldwide. Hypertension, a chronic increase in blood pressure above 140/90 mmHg, is the single main contributor to deaths due to heart disease and stroke. In the heart, hypertension results in adaptive cardiac remodelling, including LV hypertrophy to normalize wall stress and maintain cardiac contractile function. However, chronic increases in BP results in the development of hypertensive heart disease (HHD). HHD describes the maladaptive changes during cardiac remodelling which result in reduced systolic and diastolic function and eventually heart failure. This includes ventricular dilation due to eccentric hypertrophy, cardiac fibrosis which stiffens the ventricular wall and microvascular rarefaction resulting in a decrease in coronary blood flow albeit an increase in energy demand. Chronic activation of the renin-angiotensin-system (RAS) with its effector peptide angiotensin (Ang)II plays a key role in the development of hypertension and the maladaptive changes in HHD. Ang II acts via the angiotensin type 1 receptor (AT1R) to mediate most of its pathological actions during HHD, including stimulation of cardiomyocyte hypertrophy, activation of cardiac fibroblasts and increased collagen deposition. The counter-regulatory axis of the RAS which is centred on the ACE2/Ang-(1-7)/Mas axis has been demonstrated to counteract the pathological actions of Ang II in the heart and vasculature. Ang-(1-7) via the Mas receptor prevents Ang II-induced cardiac hypertrophy and fibrosis and improves cardiac contractile function in animal models of HHD. In contrast, less is known about Ang-(1-9) although evidence has demonstrated that Ang-(1-9) also antagonises Ang II and is anti-hypertrophic and anti-fibrotic in animal models of acute cardiac remodelling. However, so far it is not well documented whether Ang-(1-9) can reverse established cardiac dysfunction and remodelling and whether it is beneficial when administered chronically. Therefore, the main aim of this thesis was to assess the effects of chronic Ang-(1-9) administration on cardiac structure and function in a model of Ang II-induced cardiac remodelling. Furthermore, this thesis aimed to investigate novel pathways contributing to the pathological remodelling in response to Ang II. First, a mouse model of chronic Ang II infusion was established and characterised by comparing the structural and functional effects of the infusion of a low and high dose of Ang II after 6 weeks. Echocardiographic measurements demonstrated that low dose Ang II infusion resulted in a gradual decline in cardiac function while a high dose of Ang II induced acute cardiac contractile dysfunction. Both doses equally induced the development of cardiac hypertrophy and cardiac fibrosis characterised by an increase in the deposition of collagen I and collagen III. Moreover, increases in gene expression of fibrotic and hypertrophic markers could be detected following high dose Ang II infusion over 6 weeks. Following this characterisation, the high dose infusion model was used to assess the effects of Ang-(1-9) on cardiac structural and functional remodelling in established disease. Initially, it was evaluated whether Ang-(1-9) can reverse Ang II-induced cardiac disease by administering Ang-(1-9) for 2-4 weeks following an initial 2 week infusion of a high dose of Ang II to induce cardiac contractile dysfunction. The infusion of Ang-(1-9) for 2 weeks was associated with a significant improvement of LV fractional shortening compared to Ang II infusion. However, after 4 weeks fractional shortening declined to Ang II levels. Despite the transient improvement in cardiac contractile function, Ang-(1-9) did not modulate blood pressure, LV hypertrophy or cardiac fibrosis. To further investigate the direct cardiac effects of Ang-(1-9), cardiac contractile performance in response to Ang-(1-9) was evaluated in the isolated Langendorff-perfused rat heart. Perfusion of Ang-(1-9) in the paced and spontaneously beating rat heart mediated a positive inotropic effect characterised by an increase in LV developed pressure, cardiac contractility and relaxation. This was in contrast to Ang II and Ang-(1-7). Furthermore, the positive inotropic effect to Ang-(1-9) was blocked by the AT1R antagonist losartan and the protein kinase A inhibitor H89. Next, endothelial-to-mesenchymal transition (EndMT) as a novel pathway that may contribute to Ang II-induced cardiac remodelling was assessed in Ang II-infused mice in vivo and in human coronary artery endothelial cells (HCAEC) in vitro. Infusion of Ang II to mice for 2-6 weeks resulted in a significant decrease in myocardial capillary density and this was associated with the occurrence of dual labelling of endothelial cells for endothelial and mesenchymal markers. In vitro stimulation of HCAEC with TGFβ and Ang II revealed that Ang II exacerbated TGF-induced gene expression of mesenchymal markers. This was not correlated with any changes in SMAD2 or ERK1/2 phosphorylation with co-stimulation of TGFβ and Ang II. However, superoxide production was significantly increased in HCAEC stimulated with Ang II but not TGFβ. Finally, the role of Ang II in microvesicle (MV)-mediated cardiomyocyte hypertrophy was investigated. MVs purified from neonatal rat cardiac fibroblasts were found to contain detectable Ang II and this was increased by stimulation of fibroblasts with Ang II. Treatment of cardiomyocytes with MVs derived from Ang II-stimulated fibroblasts induced cardiomyocyte hypertrophy which could be blocked by the AT1R antagonist losartan and an inhibitor of MV synthesis and release brefeldin A. Furthermore, Ang II was found to be present in MVs isolated from serum and plasma of Ang II-infused mice and SHRSP and WKY rats. Overall, the findings of this thesis demonstrate for the first time that the actions of Ang-(1-9) in cardiac pathology are dependent on its time of administration and that Ang-(1-9) can reverse Ang II-induced cardiac contractile dysfunction by acting as a positive inotrope. Furthermore, this thesis demonstrates evidence for an involvement of EndMT and MV signalling as novel pathways contributing to Ang II-induced cardiac fibrosis and hypertrophy, respectively. These findings provide incentive to further investigate the therapeutic potential of Ang-(1-9) in the treatment of cardiac contractile dysfunction in heart disease, establish the importance of novel pathways in Ang II-mediated cardiac remodelling and evaluate the significance of the presence of Ang II in plasma-derived MVs.

The role of non-coding RNA in the development of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease of the small pulmonary arteries, characterised by pulmonary vascular remodelling due to excessive proliferation and resistance to apoptosis of pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs). The increased pulmonary vascular resistance and elevated pulmonary artery pressures result in right heart failure and premature death. Germline mutations of the bone morphogenetic protein receptor-2 (bmpr2) gene, a receptor of the transforming growth factor beta (TGF-β) superfamily, account for approximately 75%-80% of the cases of heritable form of PAH (HPAH) and 20% of sporadic cases or idiopathic PAH (IPAH). IPAH patients without known bmpr2 mutations show reduced expression of BMPR2. However only ~ 20% of bmpr2-mutation carriers will develop the disease, due to an incomplete penetrance, thus the need for a ‘second hit’ including other genetic and/or environmental factors is accepted. Diagnosis of PAH occurs most frequently when patients have reached an advanced stage of disease. Although modern PAH therapies can markedly improve a patient’s symptoms and slow the rate of clinical deterioration, the mortality rate from PAH remains unacceptably high. Therefore, the development of novel therapeutic approaches is required for the treatment of this multifaceted disease. Noncoding RNAs (ncRNAs) include microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). MiRNAs are ~ 22 nucleotide long and act as negative regulators of gene ex-pression via degradation or translational inhibition of their target mRNAs. Previous studies showed extensive evidence for the role of miRNAs in the development of PAH. LncRNAs are transcribed RNA molecules greater than 200 nucleotides in length. Similar to classical mRNA, lncRNAs are translated by RNA polymerase II and are generally alternatively spliced and polyadenylated. LncRNAs are highly versatile and function to regulate gene expression by diverse mechanisms. Unlike miRNAs, which exhibit well-defined actions in negatively regulating gene expression via the 3’-UTR of mRNAs, lncRNAs play more diverse and unpredictable regulatory roles. Although a number of lncRNAs have been intensively investigated in the cancer field, studies of the role of lncRNAs in vascular diseases such as PAH are still at a very early stage. The aim of this study was to investigate the involvement of specific ncRNAs in the development of PAH using experimental animal models and cell culture. The first ncRNA we focused on was miR-143, which is up-regulated in the lung and right ventricle tissues of various animal models of PH, as well as in the lungs and PASMCs of PAH patients. We show that genetic ablation of miR-143 is protective against the development of chronic hypoxia induced PH in mice, assessed via measurement of right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling. We further report that knockdown of miR-143-3p in WT mice via anti-miR-143-3p administration prior to exposure of mice to chronic hypoxia significantly decreases certain indices of PH (RVSP) although no significant changes in RVH and pulmo-nary vascular remodelling were observed. However, a reversal study using antimiR-143-3p treatment to modulate miR-143-3p demonstrated a protective effect on RVSP, RVH, and muscularisation of pulmonary arteries in the mouse chronic hypoxia induced PH model. In vitro experiments showed that miR-143-3p overexpression promotes PASMC migration and inhibits PASMC apoptosis, while knockdown miR-143-3p elicits the opposite effect, with no effects observed on cellular proliferation. Interestingly, miR-143-3p-enriched exosomes derived from PASMCs mediated cell-to-cell communication between PASMCs and PAECs, contributing to the pro-migratory and pro-angiogenic phenotype of PAECs that underlies the pathogenesis of PAH. Previous work has shown that miR-145-5p expression is upregulated in the chronic hypoxia induced mouse model of PH, as well as in PAH patients. Genetic ablation and pharmacological inhibition (subcutaneous injection) of miR-145-5p exert a protective against the de-velopment of PAH. In order to explore the potential for alternative, more lung targeted delivery strategies, miR-145-5p expression was inhibited in WT mice using intranasal-delivered antimiR-145-5p both prior to and post exposure to chronic hypoxia. The decreased expression of miR-145-5p in lung showed no beneficial effect on the development of PH compared with control antimiRNA treated mice exposed to chronic hypoxia. Thus, miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while the inhibition of miR-143-3p prevented the development of experimental pulmonary hypertension. We focused on two lncRNAs in this project: Myocardin-induced Smooth Muscle Long noncoding RNA, Inducer of Differentiation (MYOSLID) and non-annotated Myolnc16, which were identified from RNA sequencing studies in human coronary artery smooth muscle cells (HCASMCs) that overexpress myocardin. MYOSLID was significantly in-creased in PASMCs from patients with IPAH compared to healthy controls and increased in circulating endothelial progenitor cells (EPCs) from bmpr2 mutant PAH patients. Exposure of PASMCs to hypoxia in vitro led to a significant upregulation in MYOSLID expres-sion. MYOSLID expression was also induced by treatment of PASMC with BMP4, TGF-β and PDGF, which are known to be triggers of PAH in vitro. Small interfering RNA (siR-NA)-mediated knockdown MYOSLID inhibited migration and induced cell apoptosis without affecting cell proliferation and upregulated several genes in the BMP pathway in-cluding bmpr1α, bmpr2, id1, and id3. Modulation of MYOSLID also affected expression of BMPR2 at the protein level. In addition, MYOSLID knockdown affected the BMP-Smad and BMP-non-Smad signalling pathways in PASMCs assessed by phosphorylation of Smad1/5/9 and ERK1/2, respectively. In PAECs, MYOSLID expression was also induced by hypoxia exposure, VEGF and FGF2 treatment. In addition, MYOSLID knockdown sig-nificantly decreased the proliferation of PAECs. Thus, MYOSLID may be a novel modulator in pulmonary vascular cell functions, likely through the BMP-Smad and –non-Smad pathways. Treatment of PASMCs with inflammatory cytokines (IL-1 and TNF-α) significantly in-duced the expression of Myolnc16 at a very early time point. Knockdown of Myolnc16 in vitro decreased the expression of il-6, and upregulated the expression of il-1 and il-8 in PASMCs. Moreover, the expression levels of chemokines (cxcl1, cxcl6 and cxcl8) were sig-nificantly decreased with Myolnc16 knockdown. In addition, Myolnc16 knockdown decreased the MAP kinase signalling pathway assessed by phosphorylation of ERK1/2 and p38 MAPK and inhibited cell migration and proliferation in PASMCs. Thus, Myolnc16 may a novel modulator of PASMCs functions through anti-inflammatory signalling pathways. In summary, in this thesis we have demonstrated how miR-143-3p plays a protective role in the development of PH both in vivo animal models and patients, as well as in vitro cell cul-ture. Moreover, we have showed the role of two novel lncRNAs in pulmonary vascular cells. These ncRNAs represent potential novel therapeutic targets for the treatment of PAH with further work addressing to investigate the target genes, and the pathways modulated by these ncRNAs during the development of PAH.