Role of Free Apolipoprotein A-I in Cholesterol Efflux

Abstract

Abstract This article characterizes products formed by the interaction of purified apolipoprotein (apo) A-I and human fibroblasts. Fibroblasts were incubated with different concentrations of purified apoA-I (1 to 30 μg/mL) in tissue culture medium for different periods of time (0 to 24 hours). The medium was then characterized by one- (agarose) and two-dimensional (agarose : polyacrylamide nondenaturing gradient gel) electrophoresis. At any given concentration of apoA-I, the rate of cellular cholesterol efflux appeared linear over 24 hours. Incubating purified apoA-I with fibroblasts for 4 hours, we detected five pre-α lipoproteins with particle sizes between 114 and 684 kDa. Formation of pre-α lipoproteins was concentration-dependent. At low concentrations (below 5 μg/mL apoA-I), all purified apoA-I (with pre-β mobility) was converted to pre-α lipoproteins. At higher concentrations (greater than 5 μg/mL apoA-I), more apoA-I remained with pre-β mobility. The pre-α lipoproteins were characterized by colocalization of apoA-I particles with 14 C-cholesterol and 32 P-phospholipids. Results showed that the pre-α particle of lowest molecular weight contained phospholipid and apoA-I but no cholesterol. The remaining pre-α particles contained all three substances. When pre-α particles were subjected to ultracentrifugation, all particles floated at d<1.21 g/mL with some of the smallest phospholipid apoA-I only particles being present in the d>1.21 g/mL fraction. Based on these results, we postulated that in the first stages of reverse cholesterol transport, pre-α lipoproteins are formed by the interaction of lipid free apoA-I and peripheral cells.