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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.

Arteriovenous fistula creation is the preferred vascular access for haemodialysis therapy, but has a large failure rate in the maturation period. This period generally lasts six to eight weeks after surgical creation, in which the vein and artery undergo extensive vascular remodelling, largely due to the haemodynamics. The primary aim of this thesis was to develop a fundamental understanding of the arteriovenous fistula maturation and the associated vascular remodelling. Previous research, considering the remodelling mechanisms and risks associated with failure-to-mature patients through the use of computational fluid dynamics, has been limited by obtaining the patient-specific boundary conditions at only a few points in the patient history. Here, a non-invasive imaging system was specifically developed to reconstruct the three-dimensional vasculature and use computational fluid dynamics to analyse the haemodynamics. An initial investigation with six patients demonstrated that each patient had unique vasculature which altered the maturation process. Wall shear stress metrics used to describe disturbed flow were calculated for both successful and unsuccessful maturations. While no clear correlation could determine the cause of failure, high multi-directional wall shear stress was present in the patient before failure and could be an indicator for disease development. The results from this study also showed that vascular remodelling was not complete by six weeks, and therefore another patient was recruited for long-term analysis with high temporal data. The extended analysis revealed evidence of a control mechanism, which adjusted the lumen diameter to keep the wall shear stress near constant in the proximal regions of the vein and artery. Additionally, the vein and artery were shown to remodel at different growth rates, but the growth rates were also dependent on proximity to the anastomosis. Finally, the key factors that influenced the maturation were identified from the patient data set. High blood flow rates immediately after creation were found to have positive contribution on successful outward remodelling. Current clinical surveillance was shown to be inadequate for identifying a failing arteriovenous fistula, and highlighted the need for early and regular surveillance of the haemodynamics.

Hypertension represents a major risk factor for cardiovascular diseases. Increased mechanical stress/hypertension in the vessel wall triggers the hypertrophic signaling pathway, resulting in structural remodeling of vasculature. We have developed a novel transgenic mouse model by overexpressing the cDNA of human profilin1 in the blood vessels of transgenic mice. We showed that increased expression of profilin1 protein in the medial layer of the aorta induces stress fiber formation, triggering the hypertrophic signaling resulting in vascular hypertrophy and, ultimately, hypertension in older mice. Our results showed significant increases in the expression of alpha1integrin (280%), beta1 integrin (325%) and the activation of Rho/ROCKII pathway (260% and 350%, respectively) in profilin1 mesenteric arteries. The activation of Rho/ROCK led to the inhibition of eNOS expression (39%) and phosphorylation (35%), while an increase in myosin light chain phosphorylation (p-MLC20) (372%). There were also increases in hypertrophic signaling pathways i.e. phospho-ERK1/2 (p-ERK1/2) (312.15%) and phospho-JNK (p-JNK) (232.5%). Functional analyses of profilin1 mesenteric arteries showed significant increases in the vascular responses towards phenylephrine, while significant decreases in response towards ROCK inhibitor Y27632, acetylcholine (Ach), sodium nitrite NaNO2 and cytochalasin D. In addition, we assessed the effects of losartan, amlodipine or atenolol on vascular hypertrophy-associated hypertension, by treating the profilin1 transgenic mice for 4 weeks. Our myograph results showed improvement in the contraction response towards phenylephrine and in the relaxation response towards Ach and NaNO2 in losartan- and amlodipine-treated profilin1 mice. Western blot analyses using mesenteric arteries of lasortan and amlodipine-treated profilin1 mice showed significant decreases in their signaling respectively as follows: the expression of alpha1 integrin (104% and 93%) and beta1 integrin (116% and 109%); p-ERK1/2 (149% and 130% ) and p-JNK (171% and 137%); p-MLC20 (117% and 150%) and the ROCKII expression (125% and 180). Conversely, there were significant increases in the eNOS expression (82% and 80%) and activation (p-eNOS) (78 and 76%). On the other hand, atenolol-treated profilin1 mice showed no significant change in all measured parameters. Furthermore, we assessed the effects of elevated blood pressure on the cardiac structure and function of old hypertensive transgenic mice (OHP1). Our results showed significant increases in profilin1 expression (179%) and STAT3 activation (161.2%) in the hearts of OHP1. In addition, there was an increase in alpha1 integrin (215%), beta1 (305%) integrin and ROCKII (176%) in the hearts of OHP1 as well as p-ERK1/2 (185%) and p-JNK (265%). Echocardiography and magnetic resonance imaging demonstrated a significant decrease in left ventricle (LV) ejection fraction, fractional shortening while a significant increase in LV mass in OHP1. There was also an increase in heart/body weight ratio in OHP1. In conclusion, the changes in vascular responses in the mesenteric arteries of profilin1 mice are due to vascular hypertrophy and hypertension. In addition, the cardiac remodeling in OHP1 mice may enable us to understand the signaling mechanisms involved in the development of cardiac hypertrophy-associated hypertension in human and may reveal a new therapeutic target to inhibit the cardiac remodeling in chronic hypertensive patients.