Lysosomal cholesterol accumulation inhibits subsequent hydrolysis of lipoprotein cholesteryl ester

Abstract

Human macrophages incubated for prolonged periods with mildly oxidized LDL (oxLDL) or cholesteryl ester-rich lipid dispersions (DISP) accumulate free and esterified cholesterol within large, swollen lysosomes similar to those in foam cells of atherosclerosis. The cholesteryl ester (CE) accumulation is, in part, the result of inhibition of lysosomal hydrolysis due to increased lysosomal pH mediated by excessive lysosomal free cholesterol (FC). To determine if the inhibition of hydrolysis was long lived and further define the extent of the lysosomal defect, we incubated THP-1 macrophages with oxLDL or DISP to produce lysosome sterol engorgement and then chased with acetylated LDL (acLDL). Unlike oxLDL or DISP, CE from acLDL normally is hydrolyzed rapidly. Three days of incubation with oxLDL or DISP produced an excess of CE in lipid-engorged lysosomes, indicative of inhibition. After prolonged oxLDL or DISP pretreatment, subsequent hydrolysis of acLDL CE was inhibited. Coincident with the inhibition, the lipid-engorged lysosomes failed to maintain an acidic pH during both the initial pretreatment and subsequent acLDL incubation. This indicates that the alterations in lysosomes were general, long lived, and affected subsequent lipoprotein metabolism. This same phenomenon, occurring within atherosclerotic foam cells, could significantly affect lesion progression.

Figure 1

Electron micrograph of THP-1 incubated for 3 days with oxLDL conjugated to 15 nm colloidal gold followed by 3 day incubation with acLDL conjugated to 30 nm colloidal gold. Lipid engorged lysosomes contain both 15 nm and 30 nm gold particles. Bar= 200 nm.

Figure 2

THP-1 macrophages incubated for 3 days with acLDL (Ac) or oxLDL (Ox), for 6 days (media refreshed after 3 days) with acLDL (Ac/Ac) or oxLDL (Ox/Ox), for 3 days with acLDL followed by a 3 day incubation with oxLDL (Ac/Ox), or for 3 days with oxLDL followed by a 3 day incubation with acLDL (Ox/Ac). Data represents the mean and standard error for 3 experiments. A) Total cholesterol and its distribution between free (FC) and esterified (CE) form in cells after incubation. B) Volume of cell occupied by lipid stores and the distribution between lysosomes (Lys) and cytoplasm droplets (Drop). Bars with the same letter indicate means which are not statistically different (p < 0.05).

Figure 3

Effect of pretreatment of cells with oxLDL for 6 hours prior to incubation for 3 days with acLDL (Ox/Ac) compared to cells incubated with acLDL alone (Ac). Data represents the mean and standard error for 3 experiments. A) Total cholesterol and its distribution between free (FC) and esterified (CE) cholesterol. B) Volume of cell occupied by lipid stores and the distribution between lysosomes (Lys) and cytoplasm droplets (Drop).

Figure 4

Filipin staining of THP-1 macrophages incubated for 6 days (media refreshed after 3 days) with acLDL (A) or oxLDL (B), for 3 days with acLDL followed by 3 day incubation with oxLDL (C), or for 3 days with oxLDL followed by a 3 day incubation with acLDL (D). Bar= 5 μm.

Figure 5

Distribution of free (FC) and esterified (CE) cholesterol determined for THP-1 macrophages incubated without lipoprotein (No) or for 6 days (media refreshed after 3 days) with acLDL (Ac/Ac) or oxLDL (Ox/Ox), for 3 days with acLDL followed by a 3 day incubation with oxLDL (Ac/Ox), or for 3 days with oxLDL followed by a 3 day incubation with acLDL (Ox/Ac). Incubations with lipoprotein were done either in the absence (−) or presence (+) of the ACAT inhibitor, CP113,818.

Figure 6

Time course of hydrolysis of acLDL-derived [³H]CE. THP-1 macrophages were incubated for 6 days with acLDL (A) or 3 days with oxLDL followed by 3 days with acLDL. Following sterol loading, cells were incubated with [³H]CE-labeled acLDL for up to 24 hours in the presence of the ACAT inhibitor CP113,818. At the indicated times, cells and media were harvested and the [³H]CE and [³H]FC were measured. Results are expressed as mean +/− SD for triplicate dishes.

Figure 7

THP-1 macrophages incubated for 6 days (media refreshed after 3 days) with acLDL (Ac/Ac) or CE-rich dispersions (DISP/DISP), for 3 days with acLDL followed by a 3 day incubation with dispersions (Ac/DISP), or for 3 days with dispersions followed by a 3 day incubation with acLDL (DISP/Ac). Data represents the mean and standard error for 3 experiments. A) Total cholesterol and its distribution between free (FC) and esterified (CE) form in cells after incubation. B) Volume of cell occupied by lipid stores and the distribution between lysosomes (Lys) and cytoplasm droplets (Drop).

Figure 8

HMM incubated for 6 days (media refreshed after 3 days) with acLDL (Ac/Ac) or oxLDL (Ox/Ox), for 3 days with AcLDL followed by 3 day incubation with oxLDL (Ac/Ox), or for 3 days with oxLDL followed by a 3 day incubation with acLDL (Ox/Ac). Data represents the mean and standard error for 3 experiments. A) Total cholesterol and its distribution between free (FC) and esterified (CE) form in cells after incubation. B) Volume of cell occupied by lipid stores and the distribution between lysosomes (Lys) and cytoplasm droplets (Drop). Bars with the same letter indicate means which are not statistically different (p < 0.05).

Figure 9

Percentage of LysoSensor Yellow/Blue staining vesicles which show an active pH (pH<4.8) in THP-1 macrophages in media alone (No LP), treated for 3 days with oxLDL (Ox), 3 days with oxLDL followed by 3 days in media alone (Ox/Med), 3 days with acLDL (Ac), 6 days with AcLDL (Ac/Ac), or 3 days with oxLDL followed by 3 days with acLDL. (Ox/Ac). Bars with the same letter indicate means which are not statistically different (p < 0.05).