Insights into human neutral ceramidase transcription and the role of ceramide metabolism in the development of an inhibitor of restenosis

Atherosclerosis is the predominant underlying cause of cardiovascular disease (CVD). CVD is the leading cause of death in the United States and much of Europe, thus the prevention and successful treatment of atherosclerosis is of utmost importance in combating CVD. Atherosclerosis is an inflammatory disease of the arteries where inflammation induces a build up of fat- and cholesterol-laden immune cells and hyperproliferative vascular smooth muscle cells (VSMC). Sphingolipids are naturally occurring lipids with important structural and signaling roles and have identified roles in multiple diseases including atherosclerosis, often due to dysfunctional regulation of their metabolism. The focus of our studies is to better understand the regulation of sphingolipid metabolism and how this may apply to the therapeutic treatment of atherosclerosis.;An important enzyme in sphingolipid metabolism is neutral ceramidase (nCDase). NCDase catalyzes the first step in the conversion of apoptotic ceramide into anti-apoptotic/pro-mitogenic sphingosine-1-phosphate (S-1-P). S-1-P has been reported to induce pro-atherogenic effects on VSMCs. Moreover, inflammatory cytokines involved in the atherogenic process, such as tumor necrosis factor-alpha and interleukin-1beta can induce nCDase transcription and protein expression, leading to increased S-1-P levels. As nCDase could have important ramifications on the progression of atherosclerosis, we chose to study the regulation of nCDase at the level of transcription. Our studies determined the proximal promoter of the nCDase gene and identified multiple functional transcription response elements within the proximal promoter. Furthermore, we determined that the growth factor- and cytokine-activated transcription factor, AP-1, regulated nCDase transcription. These studies have given us an insight into the transcription regulation of nCDase that may be exploited to control its expression.;Our studies of sphingolipid metabolism also focused on the catabolism of ceramide in a vascular system and its applicability to restenosis therapy in animal models of atherosclerosis. A common treatment for atherosclerosis is angioplasty with, or without, stent implantation, but angioplasty often leads to restenosis, or reclogging of the artery due to hyperproliferation of VSMCs in response to the arterial injury induced by the angioplasty. This body of work extends previous work and shows that cell-permeable C6-ceramide can limit stenosis in multiple large arterial beds in response to stretch injury. Furthermore, while the ceramide inhibited VSMC growth in vivo and in vitro, vascular endothelial cells (EC) were not as susceptible to the growth inhibitory effects of ceramide and the endothelium was able to wound heal after angioplasty with C6-ceramide-coated balloon catheters. The role of ceramide metabolism was explored in the differential response of VSMCs and vascular ECs to ceramide treatment. Interestingly, vascular ECs, compared to VSMCs, were found to contain higher levels of ceramide kinase, but not nCDase, and had an enhanced capacity to metabolize the administered ceramide in into ceramide-1-phosphate (C-1-P). In addition, we also demonstrated that C-1-P protected cell apoptosis induced by serum-starvation. These studies indicate that the differential metabolism of ceramide by arterial cells makes exogenous ceramide a promising therapeutic for the prevention of restenosis.;The observations described in this dissertation increase our understanding of the transcriptional regulation of an important enzyme, nCDase, in sphingolipid metabolism, which may eventually be used in the design of drugs to regulate nCDase in multiple diseases including atherosclerosis. Other studies in sphingolipid metabolism reveal that C6-ceramide has strong potential to be an effective therapeutic in the prevention of restenosis. Collectively, the results of this dissertation emphasize the prospects of exploiting sphingolipids and sphingolipid metabolism in drug design.