Lipolysis and inflammatory response in endothelial cells

Endothelial lipase (EL) is a member of the triglycerid lipase gene family, expressed by various cell types including endothelial cells. The action of EL on its major substrate HDL-phosphatidylcholine (HDL-PC), yields a complex mixture of bioactive molecules (EL/HDL-mixture), which gas the ability to up-regulate the expression of inflammatory genes, including vascular cell adhesion molecule-1 (VCAM-1), E selectin, interleukin-6 (IL-6) and RANTES (regulated on activation normal T cell expressed) in human placental artery endothelial cells (HPAEC) and human aortic endothelial cells (HAEC). Because excessive activation of inflammatory genes in endothelial cells promotes enhanced adhesion of monocytes to the vascular endothelium, which is one of the first events in the development of atherosclerosis the following aims will be addressed: 1. To determine the relative impact of the most prominent bioactive lipolytic products found in the EL/HDL mixture on the up-regulation of inflammatory genes in endothelial cells. 2. To identify underlying intracellular signaling pathways and transcription factors accounting for the up-regulation of inflammatory genes by EL-generated lipolytic products. 3. To examine the functional implications of the altered inflammatory gene expression in endothelial cells exposed to EL-generated lipolytic products. To address these goals the expression of the four target genes will be monitored by real-time RT-PCR, Western blot and ELISA assays in HPAEC and HAEC exposed to the most prominent free fatty acids (FFAs) and lyso-PCs found in the EL/HDL-mixture as well as to EL-modified HDL and FFA- and lyso-PC-enriched HDL. The impact of lyso-PCs on inflammatory gene expression in vivo will be examined in lyso-PC-injected mice by real-time RT- PCR, ELISA-assays and fluorescent immunohistochemistry. Monitoring the expression levels of the target genes in the presence of pharmacological inhibitors and antioxidants as well as Western blot analysis of phosphorylated kinases will help to identify signaling pathways activated by the selected FFAs and lyso-PCs. By using protein/DNA microarray technology and the transcription factor decoy oligodeoxynucleotide approach, we aim at identifying EL-lipolytic products-inducible transcription factors in endothelial cells. By a functional approach, we will investigate the adhesion of monocytes to HPAEC and HAEC exposed to EL-specific lipolytic products as well as to aortic strips from EL-deficient mice and their wild type littermates. Identification of the key signaling pathways and subset of transcription factors responsible for lyso-PC and/or FFAs-induced pro-inflammatory gene expression in human endothelial cells may lead to a specific drug development approach for the prevention and therapy of atherosclerosis.

The aim of the project was to examine how bioactive lipids, generated on the surface of vascular endothelial cells, modulate function of those cells. Vascular endothelial cells constitute a very active and important barrier between circulating blood and deeper layers of the blood vessel wall. Healthy endothelial cells produce vasoprotective compounds, which maintain normal vascular tone and blood pressure, attenuate inflammation and excessive blood coagulation, thereby preventing the development of atherosclerosis. Various factors including increased blood glucose, oxidized lipids, fatty acids or bioactive lipids have been shown to disturb normal endothelial function. Our studies focused on four structurally related bioactive lipids known as lysophosphatidylcholine (LPC), whose, rather slight structural differences (fatty acid component) and high bioavailability, prompted us to study their capacity to modulate endothelial function. Experiments were performed in cultured human aortic endothelial cells. Our experiments have show that tested bioactive lipids which are natural constituents of the circulating blood and whose abundance increases during inflammation exert dual effect on vascular endothelial cells: On one hand these lipids promote production of the vasoprotective compounds in endothelial cells and on the other, they stimulate endothelial cells to produce inflammatory molecules. The magnitude and duration of the effects elicited by the tested bioactive lipids was related to their structural differences. Interestingly, analysis of molecular mechanisms revealed that the tested bioactive lipids use the same mechanisms for the induction of both the vasoprotective and inflammatory compounds. Thus, these bioactive lipids are both benefitial and detrimental for the vessel wall. This might have potential implications on pharmacologic interventions. A therapeutic agent, designed to modulate molecular mechanisms activated by the tested bioactive lipids, aiming to improve production of vasoprotective compounds, would simultaneously stimulate endothelial production of inflammatory compounds. Oppositely, a therapeutic intervention to attenuate inflammatory actions of bioactive lipids would abolish their induction of vasoprotective compounds and their benefitial effect on the vessel wall. Importantly, our findings on underlying mechanisms of bioactive lipids emerged from cell culture experiments, where the complexity as found in vivo in terms of interactions with different chemical compounds in blood as well as with circulating cells and cells of the vessel wall, is not given. It would be of utmost interest to find out under which patho(physiological) conditions the vasoprotective and inflammatory mechanisms induced by bioactive lipids are balanced and under which conditions one of those prevails. Therefore, the functional implications of the tested bioactive lipids, regarding induction of vasoprotective as well as inflammatory compounds will be studied under more physiological conditions, in vivo and ex vivo in a follow-up project.