Developing a 3D tissue-engineered model to study the biology and treatment of atherosclerosis

Abstract

Coronary heart disease is the primary global cause of morbidity and mortality, accounting for about 33% of global deaths in 2013. Atherosclerosis is the principal cause of coronary heart disease and is caused by inflammation of the arterial wall. This begins with the accumulation of foam cells in the subendothelial space of an inflamed segment of the endothelium to create the fatty streak. The accumulation of these cells, and their apoptosis creates a proinflammatory necrotic core. This triggers smooth muscle cells migration into the subendothelial space, where these cells form a fibrous cap that mechanically strengthens the plaque. The ongoing inflammatory condition infit smooth muscle cell apoptosis which leads to cap thinning and eventual rupture of the plaque, triggering thrombus formation. Recreating this complex multi-step pathogenesis has principally relied on animal studies. However, key differences have been observed between the human and animal plaques. This has triggered attempts to develop a humanised in vitro models, however none of these have been demonstrated to reach later stages of plaque development, where plaque rupture trigger atherothrombosis. Previouslyour lab has used a layer-by-layer fabrication method to create a healthy tissue-engineered arterial construct. In this project, the aim was to develop and validate a simple 3D cell cultured neointimal model that can be inserted into this arterial construct to provide a novel tissue-engineered atherosclerotic plaque model. A protocol was developed to generate a 3D neointimal culture model in which the THP-1 monocytic cell line can be differentiated into THP-1-derived foam cells within a compressed collagen hydrogel. The cells were demonstrated to remain viable and secrete greater quantities of proinflammatory cytokines (such as TNF-a and IL-6) than macrophages.
The neointimal construct was found to possess significant tissue factor activity and could be observed to induce a slow platelet aggregation. These prothrombotic effects were reduced when the 3D neointimal model was treated with atorvastatin during the ox LDL loading period of the culture. These results provided the first demonstration that a tissue-engineered atherosclerotic plaque model could replicate the prothrombotic properties of the native neointima. A co-culturing method was successfully developed that allowed reversible attachment of the neointimal model to the previously developed tissue-engineered medial layer using a fibrin hydrogel. Through treatment with plasmin containing solutions, the different layers of the co-culture could be shown to detach from one another, providing a basis for creating the first plaque rupture model in an in vitro atherosclerosis model. Additionally, it was possible to observe the migration of human coronary artery smooth muscle cells from the medial layer into the neointima. This provides the first evidence that tissue-engineered atherosclerosis models can elicit this key event in the development of the advanced stage of fibroatheroma. Overall, this thesis demonstrates the power of using a layer-by-layer fabrication method to develop a 3D human neointimal model that can replicate the early events in fibroatheroma. This ability to replicate both early and more advanced stage events highlight the potential for this construct to be further developed into an effective model of atherosclerotic plaque rupture to allow us to study human atherothrombosis more effectively in an ex vivo environment, and as a replacement to current in vivo animal models.