During the current grant period normal HDL was shown to inhibit three steps in the formation of mildly oxidized LDL (MM-LDL). The function of HDL (its ability to prevent LDL oxidation and inactivate oxidized phospholipids (Ox-PAPC) in MM-LDL) was found to better predict atherosclerosis in some patients than HDL-cholesterol. MKP-1 was required for Ox-PAPC to induce endothelial cells to produce MCP-1. Paraoxonase (PON)-2 was found to be an intracellular enzyme capable of inactivating Ox-PAPC while PON-3 was shown to be an HDL associated enzyme that like PON-1 inactivates Ox-PAPC, but unlike PON-1 is not regulated by Ox-PAPC. Ox-PAPC regulated hepatic PON-1 and apoJ, but not MCP-1, via IL-6. Following influenza A infection in mice, HDL lost PON activity and lost the ability to protect LDL against oxidation. When an apoA-I mimetic peptide synthesized from all D-amino acids (D-4F) was given orally to LDL receptor null mice on a Western diet or apoE null mice on a chow diet, there was a dramatic improvement in HDL's ability to inhibit LDL oxidation accompanied by a dramatic decrease in atherosclerotic lesions independent of total plasma or HDL-cholesterol. When D-4F was given to LDL receptor null mice after a Western diet and influenza A infection there was a dramatic reduction in macrophage traffic into the aortic arch and innominate arteries. In the next grant period the mechanisms by which MKP-1 mediates the inflammatory response induced by Ox-PAPC will be determined in genetically engineered mice. A link between reverse cholesterol transport and LDL oxidation will be explored in mice. The mechanisms of action of D-4F will be determined in mouse models of atherosclerosis. The ability of D-4F to promote the formation and cycling of pre-beta HDL-like particles through the reverse cholesterol transport pathway will also be studied. The mechanism by which D-4F inhibits macrophage traffic into arteries after influenza infection will be determined. The mechanisms by which oral administration of a synthetic phospholipid raises HDL and PON levels, and decreases atherosclerosis in mouse models will be determined. Finally we will determine if HDL function is a sensitive indicator of the presence or absence of atherosclerosis in mice and humans. This proposal will identify potential diagnostic and therapeutic targets by elucidating the molecular and genetic mechanisms that enhance or inhibit the inflammatory response to oxidized phospholipids.
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