Abstract 364: An NPC1 and NPC2 Independent Pathway Mediates Trafficking of Cholesterol From Lysosomes to the Extracellular Matrix

We previously reported that cholesterol-enriched macrophages deposit cholesterol into the extracellular matrix and this process depends on ABCA1 and ABCG1. The extracellular cholesterol deposits can be mobilized by HDL, or ApoA-I, the latter dependent on ABCA1-mediated lipidation of ApoA-I. The objective of the current study was to determine the effects of other genes and inhibitors that affect cellular cholesterol trafficking on the deposition of extracellular cholesterol. We used a monoclonal antibody that labels cholesterol microdomains to detect the extracellular cholesterol deposits. Progesterone and U18666A previously were shown to induce accumulation of cholesterol within lysosomes, and we found that these agents also blocked macrophage deposition of extracellular cholesterol. Brefeldin A, an inhibitor of golgi and vesicular trafficking, inhibited extracellular cholesterol deposition. On the other hand, vacuolin, an agent that inhibits the fusion between autophagosomes and lysosomes and thereby induces cellular accumulation of autophagosomes, did not inhibit extracellular cholesterol deposition. To examine the effect of mutant genes known to affect cholesterol trafficking, we examined mutant cultured fibroblasts that were cholesterol-enriched by incubation with LDL plus the LXR agonist, T0901317. Like macrophages, normal cholesterol-enriched fibroblasts deposited extracellular cholesterol that was dependent on ABCA1, because ABCA1-deficient fibroblasts did not deposit extracellular cholesterol. While progesterone and U18666A inhibited fibroblast extracellular cholesterol deposition, surprisingly, fibroblasts deficient in NPC1 or NPC2, which also abnormally accumulate cholesterol within lysosomes during cholesterol enrichment, nevertheless, deposited extracellular cholesterol similar to normal fibroblasts. That lysosomal hydrolysis of LDL cholesteryl ester is involved in extracellular cholesterol deposition was shown by substantially reduced extracellular cholesterol deposition by fibroblasts lacking acid cholesteryl ester hydrolase. Our findings indicate that a pathway not dependent on NPC1 or NPC2 mediates trafficking of cholesterol from lysosomes to the extracellular space.

Macrophage-specific transgenic expression of cholesteryl ester hydrolase attenuates hepatic lipid accumulation and also improves glucose tolerance in ob/ob mice

Cellular cholesterol homeostasis is increasingly being recognized as an important determinant of the inflammatory status of macrophages, and a decrease in cellular cholesterol levels polarizes macrophages toward an anti-inflammatory or M2 phenotype. Cholesteryl ester hydrolase (CEH) catalyzes the hydrolysis of stored intracellular cholesteryl esters (CE) and thereby enhances free cholesterol efflux and reduces cellular CE content. We have reported earlier reduced atherosclerosis as well as lesion necrosis and improved insulin sensitivity (due to decreased adipose tissue inflammation) in macrophage-specific CEH transgenic (CEHTg) mice in the LDLR−/− background. In the present study, we examined the effects of reduced intracellular accumulation of CE in CEHTg macrophages in an established diabetic mouse model, namely the leptin-deficient ob/ob mouse. Macrophage-specific transgenic expression of CEH improved glucose tolerance in ob/ob-CEHTg mice significantly compared with ob/ob nontransgenic littermates, but with no apparent change in macrophage infiltration into the adipose tissue. However, there was a significant decrease in hepatic lipid accumulation in ob/ob-CEHTg mice. Consistently, decreased [14C]acetate incorporation into total lipids and triglycerides was noted in precision-cut liver slices from ob/ob-CEHTg mice. In the primary hepatocyte-macrophage coculture system, macrophages from CEHTg mice significantly reduced the incorporation of [14C]acetate into triglycerides in hepatocytes, indicating a direct effect of macrophages on hepatocyte triglyceride biosynthesis. Kupffer cells isolated from ob/ob-CEHTg mice were polarized toward an anti-inflammatory M2 (Ly6Clo) phenotype. Taken together, these studies demonstrate that transgenic overexpression of CEH in macrophages polarizes hepatic macrophages (Kupffer cells) to an anti-inflammatory M2 phenotype that attenuates hepatic lipid synthesis and accumulation.

Phospholipase A2 Mediates Apolipoprotein-Independent Uptake of Chylomicron Remnant-Like Particles by Human Macrophages

Apolipoprotein E-receptor-mediated pathways are the main routes by which macrophages take up chylomicron remnants, but uptake may also be mediated by receptor-independent routes. To investigate these mechanisms, triacylglycerol (TG) accumulation induced by apolipoprotein-free chylomicron remnant-like particles (CRLPw/o) in human monocyte-derived macrophages was evaluated. Macrophage TG content increased about 5-fold after incubation with CRLPw/o, and this effect was not reduced by the inhibition of phagocytosis, macropinocytosis, apolipoprotein E function, or proteoglycan bridging. The role of lipases, including lipoprotein lipase, cholesteryl ester hydrolase, and secretory (sPLA2) and cytosolic phospholipase A2, was studied using [3H]TG-labelled CRLPw/o. Total cell radioactivity after incubation with [3H]TG CRLPw/o was reduced by 15–30% by inhibitors of lipoprotein lipase and cholesteryl ester hydrolase and by about 45% by inhibitors of sPLA2 and cytosolic PLA2. These results suggest that macrophage lipolytic enzymes mediate the internalization of postprandial TG-rich lipoproteins and that sPLA2and cytosolic PLA2, play a more important role than extracellular lipoprotein lipase-mediated TG hydrolysis.