Abstract
Background: Aortic aneurysms are estimated to account for 150 000 to 200 000 deaths per year world wide. As the vessel wall becomes enlarged its mechanical integrity is reduced exposing patients to risk of highly fatal aortic dissections or ruptures. This aortic dilatation is caused by breakdown of essential extra-cellular matrix proteins in the tissue mediated by aberrant vascular cell remodelling. Aneurysms are challenging to diagnose with symptoms presenting late in the disease progression. Furthermore, the rate of aneurysms in the population is rapidly increasing and there is therefore a pressing need to develop better metrics to stratify patient risk and new therapeutic strategies. Aneurysms are associated with a whole host of cardio-vascular diseases. In this work we are predominately focused on Bicuspid Aortic Valve disease (BAV), a congenital condition characterised by the malformation of the aortic valve resulting in only 2 leaflets. Indeed, BAV presents a high risk of thoracic aortic aneurysms (30-70%) but the mechanism driving their development is not well understood. Improvements in imaging techniques have now allowed pathological blood flow patterns to be correlated to aneurysm morphology and indeed there is increasing evidence that mechanotransduction from flow may be a driver of aneurysm development. Blood flow imparts mechanical forces to cells through stretching and frictional shear forces and these are known to regulate vascular cell behaviour. In particular, we are focused on interstitial flows through the vascular wall and their impact on vascular smooth muscle cell (VSMC) ECM interactions. We hypothesise that resulting interstitial fluid stresses may play a role in aberrant ECM remodelling and that this may be used to correlate disrupted flow patterns seen in patients to the morphology changes in their aortas.Aim: To test this hypothesis we aimed to create a reductionist in-vitro model culturing VSMCs in 3D under interstitial flow. By creating a model that allows us to observe cell mediated matrix remodelling in these conditions, we may be able to infer the role, if any, that interstitial flows play in VSMC-ECM interaction.
Methods: To this end, we combined a synthetic PEG based ECM mimicking hydrogel with primary cultures of both bovine and human VSCMs with a microfludics setup. We used both experimental and modelling techniques to characterise the hydrogel’s properties looking at degradability, permeability to flow and mass transport within the material. With these data in hand, we then sought to establish the optimum encapsulation conditions for our cultures of primary VSMCs using imaging techniques to assess cell viability and morphology in different hydrogel conditions. We performed long term cultures of our cells and used immunostaining techniques to test cells’ ability to synthesise nascent proteins within the hydrogels. Finally, we optimised a microfluidics setup to expose our cultures to interstitial flow and explored the impact these had on cell viability and morphology.
Results: We showed hydrogels doctored with degradable bioactive peptides could be broken down by exogenous MMPs as well our ability to control the rate of degradation by changing the number of these cross-links. We measured hydrogel permeability using 2 separate techniques which confirmed the material to have a low permeability compared to other standard hydrogels, on the order of 10−16 m2. Furthermore, we characterised mass transport of solutes within hydrogels and were able to demonstrate a negligible impact of changing polymer solid content on diffusivity in hydrogels of up to 5% weight/volume for molecules up to 40 kDa in size. We identified that encapsulated cells preferred softer hydrogels of up to 3 kPa in stiffness, including 1 mM of degradable and adhesive bioactive peptides with cells encapsulated up to a density of 4 × 106 cells/ml. Via production of MMPs, VSMCs are able to break down the hydrogel and importantly by day 7 of culture, we detected newly synthesised fibronectin, collagen 1 and 4 proteins. Finally we showed we are able to stimulate encapsulated cells with interstitial flows demonstrating that cells align to the flow direction and remain rounder in morphology compared to in static controls. As an extension of this work, we also present a novel bioreactor design to impart a fuller range of mechanical forces to cell cultures in the future.
Conclusion: We have developed a novel in-vitro platform to study vascular cell-ECM interactions in response to flow, addressing the gap in the literature of studies in 3D focused on interstitial flows. Our experiments are underpinned by predictive mathematical models and can be extended to study multiple different vascular pathologies other than aneurysms. This type of reductionist model can be a powerful tool to elucidate the individual contributions of multiple factors involved in disease progression. Finally, these types of platforms combined with in-vivo studies may serve to identify new therapeutic targets and importantly identify novel markers to determine patient risk of disease.
| Date of Award | 1 Dec 2023 |
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| Original language | English |
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| Supervisor | Eileen Gentleman (Supervisor) & Pablo Lamata de la Orden (Supervisor) |