Oxidative modification of LDL to an atherogenic particle is an early event that inappropriately supplies cholesterol to infiltrating monocytes, and attracts and stimulates monocytes and smooth muscle cells into the intema. We find oxidants attack the phosphatidylcholine (PC) that comprises 25 percent of the mass of LDL to create potent inflammatory agents that activate monocytes through at least two mechanisms. First, oxidation fragments PC to several compounds that activate the receptor for the potent inflammatory phospholipid PAF (platelet-activating factor). We find these potent PAF mimetics in animal, and now human, blood after exposure to cigarette smoke, showing that these compounds occur in vivo. These cause demonstrable systemic inflammation that is prevented by dietary antioxidants. Second, we find that monocytes respond to other oxidatively-fragmented PC from oxidized LDL by synthesizing the CxC chemokine MCP-1 and the CC chemokine IL-8. This additionally depends on outside-in signaling: freshly isolated human monocytes in suspension do not do this; PAF and PAF mimetics stimulate cytokine secretion when monocytes to certain surfaces, but respond only to oxidized PC on others. We find adhesion differentially induces PPAR (peroxisome proliferator-activated receptor) expression, and that oxidized PC will drive PPAR promoter-reporter constructs. We found a functional PPAR responsive element in the MCP-1 promoter and that prototypical PPAR agonists will induce MCP-1 production and expression from an MCP-1 promoter-reporter construct. We have identified a new route to stimulate the production of inflammatory cytokines by adherent monocytes that through a mechanism that is not present in circulating monocytes. Here we will: determine the molecular mechanism by which certain oxidized PC activate inflammatory cytokines; determine which oxidized PC activate PPAR, which PPAR isotypes are responsible; identify other cytokines activated in this way; determine how oxidized PC enter adherent monocytes and how they are metabolized; and, determine how and when PAF mimetics and PPAR ligands accumulate in vivo. This application will define new mechanisms by which inflammatory cytokines are made by monocytes after entry into vessel walls such that oxidant stresses convert short lived lipid mediators into long term chronic inflammatory stimuli.
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