Year: 2016
Type: dissertation
Abstract: Abdominal aortic aneurysm (AAA) is an asymptomatic dilatation of the abdominal aorta which ultimately leads to rupture without intervention. Rupture is typically fatal. Due to the silent nature of the disease, the National Abdominal Aortic Aneurysm Screening Programme (NAAASP) has recently been implemented in the UK. It is an unprecedented opportunity to explore the nature of early AAA development and uncover new targets for therapeutics. \nSmooth muscle cells (SMCs) are the principle component of the arterial wall and have the ability to stabilise the pathological remodelling which is characteristic of AAA. In AAA tissue, SMCs have been shown to be dysfunctional. Phenotypic switching of SMCs has been reported as an early event in AAA development. \nThe aim of this study was therefore to further characterise an ex vivo porcine model of AAA and temporally characterise SMC phenotype. The approach was \nto harvest porcine carotid arteries and subject them to a protease pre-treatment. \nThey were then cultured under flow and pressure in a vascular bioreactor for specified periods of time to model the early and end stage of AAA. The model \nwas then temporally characterised in terms of the structure and function of arterial tissue and SMCs. This study directly followed a previous study in our \nlaboratory which validated SMCs from the end-stage model with human AAA SMCs. \nHistology and immunohistochemistry for SMCs and components of the extracellular matrix were used to qualitatively analyse tissue structure in the early and end-stage models. Uniaxial tensile testing was used to \nbiomechanically characterise the function of the tissue. Whole vessel pressuredilation analysis was also evaluated as a method of biomechanical characterisation for this model. \nThe structure of the SMCs in the early and end-stage models were characterised using image analysis and fluorescence microscopy of the actin cytoskeleton. \nSMC circularity was an indication of a change in phenotype. The function of the SMCs was also assessed using proliferation assays, scratch wound migration \nassays, senescence associated beta-galactosidase assays and gelatin zymography for matrix metalloproteinase-2 and 9 (MMP-2 and -9). \nIt was determined that a combination of the protease pre-treatment and bioreactor culture was required to induce the phenotypic switch seen previously in the end-stage model. This study managed to reproduce the results seen in the previous study; in the end-stage model, protease pre-treatment followed by bioreactor culture for twelve days induced an increase in SMC circularity, impaired proliferation, elevated levels of senescence and decreased levels of MMP secretion. These characteristics are also observed in human AAA SMCs. \nA period of SMC hyperproliferation was also observed in the early-stage model. The SMCs had not yet undergone the phenotypic switch characteristic of the end-stage model. The SMCs were also more active in terms of MMP secretion \nand had much lower levels of senescence compared to the end-stage model. \nIn terms of tissue structure, Sirius Red histological staining revealed a periluminal deposition of collagen in the end-stage model which was not present in the early-stage model. It was hypothesised that this was an attempt at an ECM stabilisation mechanism. The early-stage model also reached aneurysmal dimensions but inwards remodelling of the artery had occurred by the end stage \nof the model. \nUniaxial tensile testing revealed that culture in the bioreactor caused arterial remodelling regardless of pre-treatment. Generally, vessels which received protease pre-treatment tended to be thinner, weaker and less compliant then those without pre-treatment. \nThis study shows that it may not be appropriate to characterise AAA SMCs according to classical SMC phenotype. The early-stage model may be used to \nilluminate potential targets which is particularly pertinent given the recent advent of AAA screening, providing a window of opportunity for early \ntherapeutic intervention.
Author: Emily R. Clark
Language: English
Subfield: Surgery
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2578239678