PMA activation of macrophages alters macrophage metabolism of aggregated LDL

Wei Huang,Itsuko Ishii,Wei-Yang Zhang,Miyahiko Sonobe,Howard S Kruth

doi:10.1194/jlr.m100436-jlr200

Abstract

Aggregation of LDL may contribute to its retention in atherosclerotic lesions. Previously, we showed that aggregated LDL induces and enters surface-connected compartments (SCCs) in human monocyte-derived macrophages by a process we have named patocytosis. Aggregated LDL was disaggregated and released from SCCs of macrophages when exposed to human lipoprotein-deficient serum. The serum factor that mediated aggregated LDL release and disaggregation was plasmin generated from plasminogen by macrophage urokinase plasminogen activator. We now show that activation of macrophages with PMA inhibits plasmin-mediated release of aggregated LDL from macrophages. With macrophage activation, plasminogen released about 60% less cholesterol and 63% less TCA-insoluble (125)I-aggregated LDL than when macrophages were not activated. Electron microscopy showed that PMA did not cause SCCs to close, which could have trapped aggregated LDL within the SCCs and limited protease access to aggregated LDL. Rather, PMA decreased macrophage generation of plasmin by 61%, and stimulated lysosomal degradation of aggregated LDL by more than 2-fold. Degradation was mediated by protein kinase C, shown by the finding that degradation was inhibited by the protein kinase C inhibitor Gö6976. PMA-stimulated degradation of aggregated LDL was associated with a 3-fold increase in cholesterol esterification, consistent with hydrolysis and re-esterification of aggregated LDL-derived cholesteryl ester. In conclusion, macrophage activation with PMA causes more of the aggregated LDL that enters macrophage SCCs to be metabolized by lysosomes. This results in more cholesterol to be stored in macrophages and less aggregated LDL to be available for plasmin-mediated release from macrophage SCCs.

Highlights

Aggregation of LDL may contribute to its retention in atherosclerotic lesions
We recently showed that macrophage conversion of serum plasminogen to the active serine-protease, plasmin, causes release of aggregated LDL (AgLDL) from macrophage surface-connected compartment (SCC) [10]
We examined what would happen if macrophages with SCCs containing AgLDL were exposed to serum plasminogen and at the same time were activated with PMA

Summary

EXPERIMENTAL PROCEDURES

Human LDL and human lipoprotein-deficient serum (LPDS) were obtained from Intracel; human 125I-LDL from Biomedical Technologies; [9,10-3H]oleic acid in ethanol from American Radiolabeled Chemicals; bovine lung aprotinin from ICN; human plasminogen and PMA from Calbiochem; human plasmin from American Diagnostica; D-Val-Leu-Lys-AFC plasmin substrate from Enzyme Systems Products; chloroquine, ␧-amino-N-caproic acid, sodium oleate, and fatty acid-free BSA from Sigma; RPMI-1640 medium from Cellgro; pooled human AB, heat-inactivated serum from Pel-Freez; mouse monoclonal anti-cathepsin D (clone C5) from Biogenesis; mouse monoclonal anti-Aspergillus major glucose oxidase (clone DAK-G09) from Dako; Gö6976 and wortmannin from Biomol; polysilica acid gel-impregnated glass fiber sheets from Pall; Plastek C culture plates from MatTek; Lab-Tek chamber slides from Nalge-Nunc; and 20-nm BSA-gold from British Biocell. Cell-associated 125I-AgLDL was determined by rinsing macrophages five times with Dulbecco’s phosphate-buffered saline (DPBS) containing Ca2ϩ, Mg2ϩ, and 0.2% BSA (three quick rinses and two 10-min incubations, all on ice). Values were subtracted for 125I radioactivity determined for wells incubated with 125IAgLDL but without macrophages These values were Ͻ1% of the cell-associated 125I-AgLDL. Macrophages were rinsed three times with DPBS plus Ca2ϩ and Mg2ϩ, fixed at room temperature in glutaraldehyde, and labeled with ruthenium red as described previously [17]. Macrophages were rinsed again, and incubated 24 h in RPMI-1640 medium plus 0.35% BSA without or with 0.1 ␮g/ml PMA Following this incubation, the macrophage cultures were processed for electron microscopy as described above.

RESULTS

99 Ϯ 3 315 Ϯ 17 134 Ϯ 6 192 Ϯ 9 190 Ϯ 18 343 Ϯ 36

DISCUSSION