Atherosclerosis is a chronic inflammatory disease, characterized by specific infiltration of monocytic cells, such as monocytes and T-cells, while neutrophils are essentially absent in the fatty streak lesion. One of the earliest steps in the development of the atherosclerotic lesion is the adhesion of monocytes to endothelial cells of the vessel wall. Previously it was demonstrated that, if stimulated with minimally modified LDL (MM-LDL), endothelial cells are activated to specifically bind monocytes, but not neutrophils. This specificity towards mononuclear cells was subsequently observed with the activation of endothelial cells by oxidized 1-palmitoyl-2-arachidonoyl-s/7-glycero-3-phosphocholine (OxPAPC), implying lipid oxidation products as culprits in chronic inflammation. We hypothesize that specific phospholipid oxidation products (OxPL) trigger vascular inflammation and determine monocyte specificity characteristic of atherosclerosis and other chronic inflammatory diseases. The outcome of the proposed studies will add to our understanding of how atherosclerosis is initiated and propagated and should lead to new strategies in the treatment of chronic inflammatory disorders.
The specific aims addressed in this proposal are: ? ? Specific Aim #1: To structurally identify individual OxPL that determine monocyte specific inflammation and to investigate the hypothesis that OxPL-induced signaling involves TLR4. We will investigate intracellular signaling pathways induced by OxPL that lead to specific monocyte adhesion in vitro, using HUVEC as well as isolated murine aortic EC. Furthermore, we will test the hypothesis that POVPC is one active component in OxPAPC that determines monocyte specificity and determine structure-function relationships. We will examine the role of TLR-4 and the involvement of the TLR-4 adaptor proteins MyD88 and TRIP, as well as respective downstream elements in OxPL-mediated signalling events. ? ? Specific Aim #2: To explore the hypothesis that OxPL are triggers of vascular inflammation in vivo. To mimic accumulation of OxPL in the vascular wall, we will topically apply OxPL to carotid arteries in mice and measure chemokine and adhesion molecule expression. Furthermore, we will use ex vivo perfused carotid arteries to study OxPL-induced monocyte rolling and adhesion. ? ? Specific Aim #3: To examine the hypothesis that OxPL-induced specific mononuclear cell accumulation in vivo requires a specific chemokine expression pattern and involves 12 lipoxygenase. Using the mouse air pouch model, we will examine OxPL-induced leukocyte accumulation in vivo and characterize leukocyte subsets. We will investigate the chemokine expression pattern as well as time course of expression in OxPL-induced compared to LPS-induced inflammation. Furthermore, we will investigate the role of 12-LO in OxPL-induced inflammation in vivo. ? ? ?
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