Date of Award
Doctor of Philosophy (PhD)
The enzyme thrombin has been demonstrated through experimental and clinical studies to play a crucial role in mediating both inflammation and thrombosis in atherosclerosis. The cellular effects of thrombin in promoting atherosclerosis involve the activation of signaling pathways that result in the secretion of a host of various chemokines, cytokines, cell adhesion molecules, etc. that promote vascular inflammation. Due in part to thrombin and other pro-atherogenic molecules, this prolonged inflammatory state in atherosclerosis results in the deterioration of the endothelium, increasing the risk of focal thrombosis. Current treatment strategies to address the role of thrombin in atherosclerosis, despite efficacy of anticoagulant activity, suffer from significant bleeding side effects. Thus, the goal of this dissertation was to address the central role that thrombin plays in atherosclerosis and thrombosis through the application of perfluorocarbon nanoparticles carrying the direct thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone, to impart focal anti-inflammatory and anticoagulant effects at sites of high thrombin activity. In this work, we demonstrate the ability of thrombin-inhibiting nanoparticles and liposomes to form localized "anticlotting" surfaces that inhibit the growth of clots both in vivo and on ex vivo applications on bare metal vascular stents. We also investigate the use of quantitative fluorine magnetic resonance spectroscopy (19F-MRS) to demonstrate an inverse relationship between endothelial barrier integrity and propensity for thrombosis. We show that increased exposure of an atherosclerotic animal to an inflammatory stimulus (e.g. cholesterol) over time decreases endothelial barrier integrity and increases hypercoagulability, however resolution of these symptoms after removal of the inflammatory stimulus can be tracked using 19F-MRS. Furthermore, we demonstrate through proof-of-concept imaging studies, the possibility for non-invasive imaging of endothelial barrier disrupted atherosclerotic plaques. These concepts in mind, our final objective was to utilize the ability to deliver localized thrombin inhibition with thrombin-inhibiting nanoparticles and demonstrate their ability to limit the effect of vascular inflammation on loss of endothelial barrier integrity, as measured by 19F-MRS. Our results indicate that within one month of treatment with thrombin-inhibiting nanoparticles, ApoE-null atherosclerotic mice presented with diminished endothelial barrier loss, reduced hypercoagulability, and an overall 22.5% decrease in aortic arch plaque deposition.
Future work on this platform would involve the improvement of dosing regimens that may address earlier time points of atherosclerosis development that may have a greater effect on induction of vascular inflammation. Nevertheless, these results demonstrate for the first time, the utility of focal thrombin inhibition as a means for a limiting vascular inflammation and hypercoagulability in atherosclerosis.
Shelly Sakiyama-Elbert, Donald Elbert, Dana Abendschein, J. Evan Sadler