Development and Application of Engineered Vascular Tissues for Vascular Disease Modelling

Abstract

In healthy arteries smooth muscle cells (SMCs) exist in a quiescent contractile state. SMC dedifferentiation characterised by loss of contractile SMC markers and altered extracellular matrix protein (ECM) secretion is associated with the onset and exacerbation of detrimental arterial pathologies including arterial stiffening and atherosclerosis. Currently, most studies rely on traditional two-dimensional (2D) cell culture techniques to investigate SMC behaviour. However, 2D cultures are limited by not representing the in vivo cell environment. This is in part due to the non-physiological stiffness of tissue culture polystyrene (TCP) and absence of 3D cell-ECM interactions. Conceptually, 3D cell culture models can alleviate some of the limitations associated with 2D cell culture.

Within this thesis, 3D fibrin-based engineered vascular tissues (EVTs) have been developed and characterised. The aim of this work was to produce a novel pathophysiological model to investigate ECM production and turnover within the context of SMC disease. EVTs were formed from primary murine or human aortic SMCs by adapting the protocol for the formation of Engineered Heart Tissues (EHTs), an established model for cardiovascular disease (CVD). Employment of light microscopy and molecular biology techniques revealed that SMCs within murine and human EVTs were aligned, viable and secretory. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis detected 135 core ECM proteins which were retained within the 3D EVT environment over prolonged culture. In contrast to 2D SMCs, the evolution of the ECM within EVTs was characterised by an initial deposition of proteoglycans, including the hyalectan versican, at day seven followed by basement membrane proteins and collagens by day 14 of culture.

The efficacy of murine EVTs to model disease was determined in three scenarios:

1) Proteoglycan turnover within EVTs: Versican was one of the most upregulated proteins in EVTs under baseline conditions. Our laboratory has previously described a role for ADAM Metallopeptidase with Thrombospondin Type 1 Motif 5 (ADAMTS-5) in vascular pathologies including aortic aneurysm formation, stent-induced neointima formation and atherosclerosis.
Therefore, to investigate ADAMTS-5-mediated turnover of versican, EVTs were seeded with SMCs derived from wild-type mice and mutant mice that lack the catalytically active domain in the ADAMTS-5 enzyme (Adamts5ΔCat). Altered ECM secretion in EVTs was compared to
an ex vivo aorta culture model.

2) Transforming growth factor beta 1 (TGFβ-1) signalling: Murine EVTs responded to TGFβ- 1 treatment by a dose-dependent increase in construct contractility and ECM secretion, including a significant induction in elastin synthesis. This contrasted with treatment with the commercial TGFβ receptor inhibitor, SB 431542, and the endogenous inhibitor of TGFβ signalling, basic fibroblast growth factor (FGF2). As well as ECM ligands, the discovery LC- MS/MS approach facilitated the identification of ECM receptors providing insight into how SMCs within EVTs sense and interact with their extracellular environment.

3) Calcification assays: The ECM acts as a nidus for calcium phosphate deposition in the arterial wall. The onset and extent of calcification in EVTs and 2D SMCs cultured under high calcium and phosphate conditions was determined qualitatively by Alizarin Red S staining and quantitively by an o-Cresolphthalein assay. Calcified EVTs appeared opaque and bone- like and displayed increased tissue stiffness by up to 30% compared to non-calcified controls. Compared to the rapid calcification of SMCs in 2D cultures, EVTs sustained expression of the calcification inhibitor, matrix Gla protein, and allowed for better discrimination of the calcification propensity between biological replicates.

In summary, EVTs have been comprehensively characterised under baseline and disease conditions. Unlike in traditional 2D cultures, retention of the nascent SMC ECM within EVTs provides a more pathophysiological relevant model.

Date of Award	1 Dec 2022
Original language	English
Awarding Institution	King's College London
Supervisor	Manuel Mayr (Supervisor) & Philip Chowienczyk (Supervisor)