Non-Invasive Monitoring of Inflammation in a Genetically-Modified, Tissue Engineered Vascular Model Abstract Across the world, the leading cause of death is cardiovascular disease. An underlying cause of many cardiovascular diseases is atherosclerosis, a process in which plaque builds up in the vascular wall and prevents the steady flow of blood to-and-from the heart. Currently, medicines targeting atherosclerosis undergo the same laborious process as all drugs. This research aims to improve the efficiency of this process by creating a tissue-engineered model of an atherosclerotic artery to serve as a preclinical tool for screening anti- atherosclerosis drugs. Cyclooxygenase-2 (COX-2) regulates cellular inflammatory processes and mediates atherogenic events. The goal of this work was to develop a method for non-invasively monitoring COX- 2 expression within the tissue-engineered vascular endothelium as a real-time measure of inflammation. To measure COX-2 expression, rat brain endothelial cells (RBECs) were transfected with Ad- COX2 -Luc an adenoviral vector with a COX-2 controlled luciferase reporter. Transfected RBECs were then seeded into three poly-dimethylsiloxane tubes and treated with varying amounts of interleukin 1-beta (IL-1), a pro-inflammatory mediator that induces COX-2 expression. Luciferin was added to the cells to initiate a bioluminescent-response. The Xenogen-IVIS Imaging System was used to detect and quantify luciferase activity within each tube. The results obtained indicated that luciferase expression correlated with the level of IL-1. The tissue- engineered vascular model created in this research demonstrated a COX-2 inflammatory response following exposure to a pro- inflammatory cytokine. This response was monitored through a luciferase reporter and bioluminescent-imaging. Conclusively, this research project resulted in an accurate, non-invasive method of monitoring inflammation in a tissue-engineered artery. Introduction Atherosclerosis is a progressive disease that prevents proper blood flow. An artery becomes atherosclerotic when high blood pressure, smoking, and/or high cholesterol causes damage to its endothelial layer. This damage leads to the formation of plaque, a substance composed of cholesterol, fat, calcium, and other substances, that builds up in the vascular walls of arteries. Over time plaque hardens and narrows the infected arteries, limiting the flow of oxygen-rich blood throughout the human body. Infected arteries demonstrate an inflammatory response, increasing the blockage caused by the plaque. Purpose Atherosclerosis can affect any artery in the human body, including those in the heart, brain, limbs, and kidney. Due to this versatility, atherosclerosis is a condition present in a variety of illnesses Coronary Heart Disease, Carotid Artery Disease, Peripheral Arterial Disease, Chronic Kidney Disease, etc. The purpose of the research is to create a model on which anti-atherosclerosis drugs can be tested. Goal This research aims to tissue engineer a three-dimensional vascular endothelial layer to non-invasively measure COX-2 expression. Hypothesis Transfection of endothelial cells with a COX-2 -dependent luciferase gene will enable non-invasive monitoring of COX-2 activity in an engineered vascular endothelium. Sources 1.Boer, O., Wal, A., & Becker, A. (2000). Atherosclerosis, inflammation, and infection. The Journal of Pathology, 190 (3), Crofford, L. J., Wilder, R. L., Ristimki, A. P., Sano, H., Remmers, E. F., Epps, H. R., & Hla, T. (1994). Cyclooxygenase-1 and -2 expression in rheumatoid synovial tissues. Effects of interleukin-1 beta, phorbol ester, and corticosteroids. Journal of Clinical Investigation, 93 (3), 1095 Quint, C., Kondo, Y., Manson, R., Lawson, J., Dardik, A., & Niklason, L. (2011). Decellularized Tissue-engineered Blood Vessel As An Arterial Conduit. Proceedings of the National Academy of Sciences, 108 (22), Williams, C., Mann, M., & Dubois, R. (1999). The role of cyclooxygenases in inflammation, cancer, and development. Oncogene, 18 (55), Yazdani, S., Watts, B., Machingal, M., Jarajapu, Y., Dyke, M., & Christ, G. (2008). Smooth Muscle Cell Seeding of Decellularized Scaffolds: The Importance of Bioreactor Preconditioning to Development of a More Native Architecture for Tissue-Engineered Blood Vessels. Tissue Engineering Part A, Future Research Future research will validate COX-2 gene expression with luciferase expression using PCR. This research will also redesign the bioreactor system to introduce a larger number of vessels, resulting in higher throughput. The new design will also introduce dynamic culture conditions including pulsatile flow and pressure to mimic physiologic blood flow conditions. These improvements will increase the efficiency of the vascular model as a tool for screening anti-atherosclerotic drugs. Conclusion The data obtained throughout this research project supports the method of non-invasive monitoring of inflammation. Levels of IL-1 directly correlated with levels of luciferase expression, which is an indirect measure of COX-2 activity. Based on this, we conclude that transfection of endothelial cells with a COX-2 -dependent luciferase gene does enable non-invasive monitoring of COX-2 activity in an engineered vascular endothelium Materials Trypsin DMEM cell media with 10% FBS Rat Brain Endothelial Cells Ad- COX2 -Luc Interleukin 1-beta (IL-1) Luciferin Sylgard 184 Silicone Elastomer Base Sylgard 184 Silicon Elastomer Curing Agent PDMS tube mold PDMS tubes 24-well cell plates 3-channel bioreactor system Figure A Varying Levels of Ad- COX2 -Luc in a 2D Model Figure A. RBECs were seeded into a 24-well black cell culture plate. After 24 hours, the cells in each column were transfected with Ad- COX2 -Luc at increasing multiplicities of infection (MOI). After 24 hours, the cells were stimulated with a constant dose of Tumor Necrosis Factor-alpha (TNF-), a pro- inflammatory cytokine. After a 4 hour treatment period, the cells were washed with PBS to remove the treatment. Luciferin was added to each well and luciferase expression measured with bioluminescence imaging. (A-1) Representative bioluminescence image captured by the Xenogen-IVIS Imaging System. In this image, the MOI of 100 shows a high total count of luciferase activity and it appears that no other MOI successfully transfected the RBECs. However, using the Living Image software associated with the Xenogen System, the total counts of luciferase expressed by cells transfected with other MOIs were quantified. (A-2) The total luciferase counts for each MOI were quantified using matching region of interest (ROI) analysis. The MOIs of 1, 10, and 100 successfully transfected the RBECs and were significantly different than uninfected cells ( *p