sPLA2 GV and GX Huydrolysis of LDL Results in Free Fatty Acid and Lyso-Phospholipid Generation and Affects Macrophage Functions

Several lines of evidence show that secretory phospholipase A2 (sPLA2) modification of LDL is proatherogenic. For example hydrolysis of LDL by Group IIA sPLA2 results in formation of smaller and denser particles with increased affinity to proteoglycans and enhanced retention in the vessel wall. Recent studies have shown that Group V (GV) and Group (GX) sPLA2 have higher activity towards LDL-phospholipids (PL) compared to Group IIA. To date, it has been shown that GV and GX sPLA2 are present in atherosclerotic lesions and GV or GX sPLA2 modified LDL promote foam cell formation in vitro. In addition to the structural changes, LDL-PL hydrolysis by sPLA2 produces free fatty acids (FFA) and Iyso-PL. Possible effects of the FFA and Iyso-PL generated after LDL-PL hydrolysis on macrophage functions have not been defined. The goal of this project is to determine the effects of LDL modified by GV or GX sPLA2 on macrophage activation and to delineate the role of FFA/lyso-PL in the foam cell formation mediated by GV or GX modified LDL. To test our hypothesis we have designed two specific aims. The objective of Specific Aim I is to test whether GV or GX sPLA2 modified LDL alter TNF-alpha, IL-G and IL-10 secretion by macrophages in vitro, and to determine whether FFA/lyso-PL contribute to this effect. In Specific Aim II we will determine how FFA and Iyso-PL affect GV or GX modified LDL uptake by macrophages. To do this, we will use mouse peritoneal macrophages to compare the cellular binding, association and degradation of 1251-labeled control LDL,1251-GV or 1251-GX modified LDL and 1251-GV or GX modified LDL depleted of FFA and Iyso-PL. As an alternative approach, we will quantify the amount of cholesteryl esters accumulated in macrophages after incubation with control LDL, GV/GX-LDL, GV or GX-LDL depleted of FFA/lyso-PL. If performed, this study will define the effects of GV/GXmodified LDL on macrophage activation and macrophage ability to take up lipoproteins, which is important to our understanding of atherosclerotic inflammation and lipid accumulation in the arterial wall.