Acute-Phase-HDL Remodeling by Heparan Sulfate Generates a Novel Lipoprotein with Exceptional Cholesterol Efflux Activity from Macrophages

During episodes of acute-inflammation high-density lipoproteins (HDL), the carrier of so-called good cholesterol, experiences a major change in apolipoprotein composition and becomes acute-phase HDL (AP-HDL). This altered, but physiologically important, HDL has an increased binding affinity for macrophages that is dependent on cell surface heparan sulfate (HS). While exploring the properties of AP-HDL∶HS interactions we discovered that HS caused significant remodeling of AP-HDL. The physical nature of this change in structure and its potential importance for cholesterol efflux from cholesterol-loaded macrophages was therefore investigated. In the presence of heparin, or HS, AP-HDL solutions at pH 5.2 became turbid within minutes. Analysis by centrifugation and gel electrophoresis indicated that AP-HDL was remodeled generating novel lipid poor particles composed only of apolipoprotein AI, which we designate β2. This remodeling is dependent on pH, glycosaminoglycan type, is promoted by Ca2+ and is independent of protease or lipase activity. Compared to HDL and AP-HDL, remodeled AP-HDL (S-HDL-SAA), containing β2 particles, demonstrated a 3-fold greater cholesterol efflux activity from cholesterol-loaded macrophage. Because the identified conditions causing this change in AP-HDL structure and function can exist physiologically at the surface of the macrophage, or in its endosomes, we postulate that AP-HDL contains latent functionalities that become apparent and active when it associates with macrophage cell surface/endosomal HS. In this way initial steps in the reverse cholesterol transport pathway are focused at sites of injury to mobilize cholesterol from macrophages that are actively participating in the phagocytosis of damaged membranes rich in cholesterol. The mechanism may also be of relevance to aspects of atherogenesis

Acute-phase-HDL remodeling by heparan sulfate generates a novel lipoprotein with exceptional cholesterol efflux activity from macrophages.

During episodes of acute-inflammation high-density lipoproteins (HDL), the carrier of so-called good cholesterol, experiences a major change in apolipoprotein composition and becomes acute-phase HDL (AP-HDL). This altered, but physiologically important, HDL has an increased binding affinity for macrophages that is dependent on cell surface heparan sulfate (HS). While exploring the properties of AP-HDLratioHS interactions we discovered that HS caused significant remodeling of AP-HDL. The physical nature of this change in structure and its potential importance for cholesterol efflux from cholesterol-loaded macrophages was therefore investigated. In the presence of heparin, or HS, AP-HDL solutions at pH 5.2 became turbid within minutes. Analysis by centrifugation and gel electrophoresis indicated that AP-HDL was remodeled generating novel lipid poor particles composed only of apolipoprotein AI, which we designate beta2. This remodeling is dependent on pH, glycosaminoglycan type, is promoted by Ca(2+) and is independent of protease or lipase activity. Compared to HDL and AP-HDL, remodeled AP-HDL (S-HDL-SAA), containing beta2 particles, demonstrated a 3-fold greater cholesterol efflux activity from cholesterol-loaded macrophage. Because the identified conditions causing this change in AP-HDL structure and function can exist physiologically at the surface of the macrophage, or in its endosomes, we postulate that AP-HDL contains latent functionalities that become apparent and active when it associates with macrophage cell surface/endosomal HS. In this way initial steps in the reverse cholesterol transport pathway are focused at sites of injury to mobilize cholesterol from macrophages that are actively participating in the phagocytosis of damaged membranes rich in cholesterol. The mechanism may also be of relevance to aspects of atherogenesis

Non-denaturing polyacylamide gel electrophoresis (N-PAGE) of normal and heparin treated HDLs.

<p>AP-HDL, S-HDL-SAA and HDL were resolved by N-PAGE using 4–20% gradient gels with a continuous buffer system consisting of 0.1 M glycine-Tris-HCl, pH 8.6. Samples (30 µg) were loaded onto the gel and electrophoresis carried out at 4°C for 6 h at 150 volts. Bands were detected by staining with Coomassie Blue R250. A), lane 1, AP-HDL, lane 2, S-HDL-SAA, lane 3, float sample after re-centrifugation of S-HDL-SAA at d = 1.25 g/ml. Molecular weight markers are indicated on the left; B), lane 1, HDL, lane 2, S-HDL, lane 3, delipidated apoA-I.</p

The aggregation of AP-HDL at mildly acid pH when incubated with heparin or HS.

<p>A) HDL or AP-HDL at pH 5.2 or 7.2 were incubated for 30 min with increasing concentrations of heparin, HS+1 mM Ca²⁺, CS (chondroitin sufate) or DS (dermatan sulfate). The absorbance at 400 nm was then plotted against glycosaminoglycan concentration. B) AP-HDL (1 mg/ml) at pH 5.2 in 125 mM NaCl, 25 mM Na acetate, was incubated with 0.2 mg/ml heparin and absorbance at 400 nm read at intervals to 60 min. C) AP-HDL (1 mg/ml) incubated for 20 min at 37°C with heparin, or different chemically modified heparins, at 0.1 mg/ml as described in Panel B; CR-hep. = carboxy-reduced heparin; de-NS-hep. = N-desulfated heparin; de-2-O-hep. = de-2-O-sulfated heparin; de-6-O-hep. = de-6-O-sulfated heparin; de-OS-hep. = de-2,6-O-sulfated heparin. D) Incubations, supplemented with 1 mM Ca²⁺, greatly increased the AP-HDL aggregation activity of HS. AP-HDL was incubated at pH 5.2 with heparin, HS, CS, DS with or without Ca²⁺, as in C). The results in C) and D) are the mean±SEM of 3 experiments. The results with CS and DS were virtually identical.</p

Cholesterol efflux from J774 monocytes incubated with different lipoproteins.

<p>Monolayer cultures of J774 monocytes were loaded with cholesterol (22.7 µg/well) and [³H]-cholesterol (0.45 µCi) overnight, washed twice with DMEM in 0.2% BSA (1 ml) and once with DMEM alone. Cholesterol-loaded cells were then incubated with different lipoprotein preparations (50 µg/ml) in 1 ml DMEM, 0.2% BSA; A) S-HDL-SAA prepared with heparin; S-HDL-SAA prepared with HS+1 mM Ca2+; A-HDL-SAA; AP-HDL; HDL; S-HDL prepared with heparin. B) Lipid was extracted from cell and analyzed by thin-layer chromatography as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003867#pone.0003867-Kisilevsky2" target="_blank">[35]</a> to determine the amount of [3H]-cholestryl ester remaining in the cells relative to cholesterol-loaded cells. C) S-HDL-SAA; S-HDL-SAA preincubated with DEAE-Sepharose to remove any heparin; S-HDL-SAA recentrifuged at 1.25 g/ml to remove lipid poor species, A-HDL-SAA and delipidated apoA-I. Aliquots of medium were taken at the time intervals shown and the quantity of [³H]-cholesterol exported from the cells determined and expressed as a percentage of total [³H]-cholesterol in loaded cells before incubations.</p