Abstract 259: Generation of Model HDL Complexes to Dissect the Role of Apolipoprotein A-II on Antioxidative and Anti-inflammatory Properties of HDL

Abstract

Human high density lipoproteins (HDL) are divided into two major subpopulations based on lipoprotein composition. The predominant subpopulation contains both major apolipoproteins apoA-I and apoA-II (termed LpA-I/A-II), while the minor subpopulation contains only apoA-I (termed LpA-I). It is speculated that LpA-I/A-II could demonstrate different anti-oxidative and anti-inflammatory properties compared to LpA-I. The goal of the current work was to dissect out any contributions from apoA-II on such beneficial functions of HDL. We generated a series of reconstituted (r)LpA-I/A-II in which major apolipoprotein ratios mimic native LpA-I/A-II (apoA-I: apoA-II; 2:1, 2:2 and 2:3) but lack the anti-oxidative enzymes in native HDL. First, we generated the precursor rLpA-I by reacting discoidal HDL with recombinant lecithin cholesterol acyl transferase (LCAT) in the presence of low density lipoproteins. The rLpA-I consist of a single population of ∼93 Å particles. Cross-linking followed by MALDI-MS indicated exactly three apoA-I per rLpA-I. Different rLpA-I/A-II complexes were generated by adding the required amount of apoA-II into rLpA-I followed by isolation of rLpA-I:A-II using size exclusion chromatography. All rLpA-I/A-II showed comparable particle diameter to rLpA-I. Electron microscopy clearly captured the spherical nature of these particles and agarose gel electrophoresis demonstrated the presence of a neutral lipid core in rLpA-I and rLpA-I/A-II compared to discoidal HDL. All particles contain comparable amounts of total protein 36-40%, phospholipids 40-45%, cholesteryl esters 17-22%, free cholesterol <1% and triglycerides <1%. In vitro anti-oxidative functional assays utilizing copper-mediated oxidation demonstrated ∼20% better anti-oxidative capacity for rLpA-I/A-II (apoA-I: apoA-II, 2:1) compared to rLpA-I. We are currently in the process of testing anti-oxidative properties of other rLpA-I/A-II complexes. Experiments are currently underway to assess differences of anti-inflammatory properties of these complexes by quantitative measurements of adhesion molecule VCAM, ICAM and E-selection using flow cytometry in cell culture.