The establishment and use of tissue engineered vascular models to investigate the pleiotropic effects of statins

Abstract

Cardiovascular disease (CVD) has been identified as the leading cause of mortality in westernised society. The triggering factor for the majority of cardiovascular diseases is atherosclerosis, defined as an accumulation of fatty materials in the vascular sub-endothelial space. This results in the initiation and propagation of inflammatory responses that result in the narrowing of the vascular lumen, as well as thickening and hardening of arteries. The initiating stimulus in atherosclerosis is elevated levels of low-density lipoprotein (LDL) in circulation, and the cells involved in this process are primarily endothelial cells-which interact with blood-, smooth muscle cells-which facilitate the contraction and relaxation of large diameter blood vessels-, and macrophages, which are immune cells that are able to take up and store lipids. As inflammation progresses, the endothelium becomes dysfunctional and expresses adhesion molecules that facilitate the entry of monocytes into the sub-endothelial space, where they differentiate into macrophages, take up LDL and become foam cells. These lipid rich foam cells are a key component of atherosclerotic plaques, together with dead cells that make up the necrotic core. Statins have been established as the gold standard for the treatment of atherosclerosis, and have been useful in decreasing morbidity and mortality in CVD patients. They function by preventing cholesterol synthesis through inhibition of HMG-CoA, thus lowering amounts of circulating cholesterol. In addition to this function, a number of pleiotropic effects have been associated with statin treatment including, increasing numbers of circulating endothelial progenitor cells (EPCs), reduce inflammation, improve atherosclerotic plaque stability and improve engraftment of MSCs into sites of vascular injury. To investigate these pleiotropic effects of this ubiquitous drug used in the treatment of the most prevalent disease, we developed tissue engineered blood vessel models that incorporated endothelial cells (Human umbilical vein endothelial cells (HUVECs)) and smooth muscle cells (Human cardiac artery smooth muscle cells (HCASMCs)) to represent the intimal and medial layers of the vasculature and could be used individually (Tissue engineered intimal layer-TEIL and tissue engineered medial layer-TEML) or in concert as a full blood vessel (tissue engineered blood vessel-TEBV). These vessel models/constructs were subjected to shear stress and used to evaluate the effect atorvastatin has on the homing of endothelial progenitor cells, the production of SDF-1 and expression of its receptor CXCR4. Further to this, the effect of atorvastatin on initiating cholesterol efflux was also investigated with considerations made to examine the role of HUVECs and smooth muscle cells in this process. The experiments conducted for this thesis were able to determine that atorvastatin increases the density of cells attached onto the surface of a lesioned construct. This was observed for the partial blood vessel models (TEIL and TEML) as well as the TEBV. This effect was noted for human mesenchymal stem cells (hMSCs) as well as EPCs. Observations in EPCs were consistent under both high (22.16 dyne/cm2) and low (2.2 dyne/cm2) shear stress. We were also able to determine that atorvastatin is more functional when used in conditions of oxidative stress through examination of different lesioning techniques. FeCl3 induced oxidative damage resulted in the recruitment of more cells to the surfaces of the lesioned constructs as well as higher levels of SDF-1, compared to the mechanical lesion which generated a mild surface abrasion. It was also possible to demonstrate that atorvastatin increases secretion of SDF-1 and expression of CXCR4, which are the main cytokine and receptor associated with cell homing and migration. This effect was determined to be both time and dose dependent. Through the use of different blood vessel models, it was determined that the cells in each layer have differing responses to the composite tissue model i.e., observations of cell attachment and SDF-1 production on TEBV were an amalgam of TEIL and TEML responses. Through the use of nanofiber inserts to create a novel HUVEC RAW264 co-culture system, we were able to demonstrate that atorvastatin triggers consistent cholesterol efflux from cultured foam cells compared to drug free controls, resulting in up to a 13% reduction in amounts off internalised cholesterol, a phenomenon that is affected by HUVEC integrity i.e., lesioned HUVECs promoted cholesterol efflux, especially in the presence of atorvastatin and IFN-?. Atorvastatin was also able to restrict nitric oxide (NO) production in macrophages and may reverse the effects of the inflammatory cytokine IFN ?. The models used here proved a useful tool for investigating the effects of atorvastatin, and could prove useful in evaluating cellular responses to a wider array of pharmacologic, or other stimuli.