Since HDL has been demonstrated to facilitate lipid and cholesterol efflux in macrophages, we sought to determine whether HDL has similar effect on lipid efflux from RPE cells. RPE cells were cultured in Transwell plates and fed 14
C‐DHA labelled bovine POS in the apical chambers in the presence or absence of purified lipoproteins added to the bottom media. Lipoprotein acceptors included LDL (100 µg/ml), HDL (100 µg/ml), and LDL+HDL (50 µg/ml each). After 36 hours 14
C in basal media was determined by liquid scintillation counting. As shown in figure 1, total 14
C in basal media was significantly increased by HDL (p
0.0027, two tailed t
test). HDL stimulated basal 14
C labelled lipid efflux 1.9‐fold compared to no lipoprotein acceptor. LDL did not significantly increase basal efflux of 14
C labelled lipids (p
0.4293, two tailed t
test). When LDL and HDL were present together, stimulation of 14
C labelled lipid efflux was about half that of HDL alone (1.4‐fold), although this was not significantly different from the control (p
0.0719, two tailed t
In order to determine whether basally effuxed 14C labelled lipids associated with lipoproteins, like samples were combined and lipoproteins were purified from basal media by ultracentrifugation at a density of 1.21 g/ml. The amount of 14C in the d<1.21 g/ml density fraction for each sample was determined by liquid scintillation and is given in table 1.
Table 114C labelled lipid associated with lipoprotein
HDL bound about 14‐fold more 14C labelled lipids than did LDL. When both LDL and HDL were present, 14C in the d<1.21 g/ml fraction was intermediate to the amount when either HDL or LDL were present alone. In the absence of added lipoproteins, control media had low, but measurable, levels of radioactivity in the d<1.21 g/ml fraction. The ultracentrifuged media lipoprotein fractions were resolved by non‐denaturing PAGE (fig 2). The Coomassie stained components observed in HDL (fig 2, lane 3) and LDL (fig 2, lane 4) are typical lipoprotein profiles expected of pure LDL and HDL. For purposes of comparison control basal medium (fig 2, lane 1) and purified plasma lipoprotein (fig 2, lane 2) profiles are also shown.
Figure 2Repurification of HDL and LDL following incubation with RPE cells fed 14C labelled POS. Shown are Coomassie stained gel lanes containing samples: control medium (lane 1), purified plasma lipoproteins (lane 2), repurified HDL (lane 3), (more ...)
The distribution of 14C labelled lipids among the lipoproteins in HDL and LDL samples was determined. Gel lanes (fig 2, lanes 3 and 4) were fractionated and counted. As shown in figure 3, radioactivity was confined to the lipoproteins present in each sample: HDL (1783 cpm), LDL (266 cpm). HDL+LDL was separated on another gel (not shown) and yielded 966 cpm in the HDL band and 380 cpm in the LDL band. Again, HDL was a better acceptor (sixfold to sevenfold) than LDL when tested as a pure lipoprotein and in plasma. When purified LDL and HDL were combined, HDL exhibited a twofold to threefold higher affinity for basally effluxed 14C labelled lipids.
Figure 3Distribution of radioactivity in repurified lipoproteins. Polyacrylamide gel lanes were fractionated from the top (fraction 1) to the bottom (fraction 28). 14C was quantified by liquid scintillation counting. Coomassie blue stained fractions (more ...)
Lipids were extracted from the HDL fraction, purified as above, and partially purified by one dimensional TLC. As shown in figure 4 several lipid spots could be identified. Most of the 14C label was in phosphatidyl choline (PC) and cholesterol (C), with lesser amounts in phosphatidyl inosotol (PI), phosphatidyl ethanolamine (PE), triglycerides (TG) and cholesterol esters (CE) (table 2). The remaining 14C label was in a dozen other, as yet unidentified, spots.
Figure 4TLC separation and identification of some lipids bound to HDL. Following incubation with RPE cells fed 14C labelled POS, HDL bound lipids were extracted and separated by TLC (bottom of plate at the left). Standards for pure phosphatidyl (more ...)
Table 214C labelled lipid bound to HDC
As a first step in determining which HDL fraction was the most potent stimulator of 14C labelled lipid efflux, we fractionated plasma HDL (1.063<d<1.210) by ultracentrifugation in a continuous KBr density gradient. Ten HDL fractions ranging in density (1.07–1.18 g/ml) and particle size (6–11 nm, Stoke's diameter) (fig 5) were tested in equivalent protein concentrations (100 µg/ml). All HDL fractions stimulated basal efflux of 14C labelled lipids more than twofold (p<0.0005, t test) (fig 6). In addition, all HDL fractions bound effluxed 14C labelled lipids (not shown).
As a first step in identifying the components of HDL necessary and sufficient for stimulating basal efflux of 14
C labelled lipids, an artificial HDL, consisting of purified apoA‐I, cholesterol, and DMPC, was synthesised as described in Methods. Purified artificial HDL (apoA‐I vesicles), average Stoke's diameter of 10 nm, is shown in figure 7, fractions 44–49. The ability of purified apoA‐I and apoA‐I vesicles (fractions 44–49), to stimulate basal 14
C labelled lipid efflux was tested. Both purified apoA‐I and apoA‐I vesicles stimulated 14
C labelled lipid efflux by about 1.5‐fold to 2‐fold (p
0.0079, Mann‐Whitney test) (fig 8).
Figure 7Purification of synthetic HDL (apoA‐I vesicles). Discrete lipid particles from sodium cholate dispersions of DMPC, cholesterol, and apo A‐I were purified by FPLC and fractions were analysed by non‐denaturing PAGE (more ...)