Cholesteryl Ester Hydrolase Research Articles

Sterol O-acyltransferase (SOAT/ACAT) activity is required to form cholesterol crystals in hepatocyte lipid droplets

ObjectiveExcess cholesterol storage can induce the formation of cholesterol crystals in hepatocyte lipid droplets. Such crystals distinguish metabolic dysfunction associated steatohepatitis (MASH) from simple steatosis and may underlie its pathogenesis by causing cell damage that triggers liver inflammation. The mechanism linking cholesterol excess to its crystallization in lipid droplets is unclear. As cholesteryl esters localize to and accumulate in lipid droplets more readily than unesterified free cholesterol, we investigated whether cholesterol esterification by sterol O-acyltransferase (SOAT), also known as acyl co-A cholesterol acyltransferase (ACAT), is required for hepatocyte lipid droplet crystal formation. MethodCholesterol crystals were measured in cholesterol loaded Hep3B hepatocytes, RAW264.7 macrophages, and mouse liver using polarizing light microscopy. We examined the effect of blocking SOAT activity on crystal formation and compared these results to features of cholesterol metabolism and the progression to intracellular crystal deposits. ResultsCholesterol loading of Hep3B cells caused robust levels of lipid droplet localized crystal formation in a dose- and time-dependent manner. Co-treatment with SOAT inhibitors and genetic ablation of SOAT1 blocked crystal formation. SOAT inhibitor also blocked crystal formation in low density lipoprotein (LDL) treated Hep3B cells, acetylated LDL treated RAW 264.7 macrophages, and in the liver of mice genetically predisposed to hepatic cholesterol overload and in mice with cholesterol enriched diet-induced MASH. ConclusionSOAT1-mediated esterification may underlie cholesterol crystals associated with MASH by concentrating it in lipid droplets. These findings imply that inhibiting hepatocyte SOAT1 may be able to alleviate cholesterol associated MASH. Moreover, that either a lipid droplet localized cholesteryl ester hydrolase is required for cholesterol crystal formation, or the crystals are composed of cholesteryl ester.

Lipid Metabolic Reprogramming Extends beyond Histologic Tumor Demarcations in Operable Human Pancreatic Cancer.

In clinically operable pancreatic cancer, regions distant from malignant cells already display proteomic changes related to lipid transport and metabolism that affect prognosis and may be pharmacologically targeted.

Off-target effects of the lysosomal acid lipase inhibitors Lalistat-1 and Lalistat-2 on neutral lipid hydrolases

ObjectivesLysosomal acid lipase (LAL) is the key enzyme, which degrades neutral lipids at an acidic pH in lysosomes. The role of LAL in various cellular processes has mostly been studied in LAL-knockout mice, which share phenotypical characteristics with humans suffering from LAL deficiency. In vitro, the cell-specific functions of LAL have been commonly investigated by using the LAL inhibitors Lalistat-1 and Lalistat-2. MethodsWe performed lipid hydrolase activity assays and serine hydrolase-specific activity-based labeling combined with quantitative proteomics to investigate potential off-target effects of Lalistat-1 and -2. ResultsPharmacological LAL inhibition but not genetic loss of LAL impairs isoproterenol-stimulated lipolysis as well as neutral triglyceride and cholesteryl ester hydrolase activities. Apart from LAL, Lalistat-1 and -2 also inhibit major cytosolic lipid hydrolases responsible for lipid degradation in primary cells at neutral pH through off-target effects. Their binding to the active center of the enzymes leads to a decrease in neutral lipid hydrolase activities in cells overexpressing the respective enzymes. ConclusionsOur findings are critically important since they demonstrate that commonly used concentrations of these inhibitors are not suitable to investigate the role of LAL-specific lipolysis in lysosomal function, signaling pathways, and autophagy. The interpretation of their effects on lipid metabolism should be taken with caution and the applied inhibitor concentrations in cell culture studies should not exceed 1 μM.

Bioactive components to inhibit foam cell formation in atherosclerosis.

The production of lipid-laden cells in macrophages after significant ingestion of oxidized low-density lipoprotein is considered the most critical phase in the creation of atherosclerotic lesions, which is known as foam cell formation. Targeting foam cell development to find a potential therapeutic strategy for the management of atherosclerosis has yielded numerous promising outcomes. Multiple variables influence foam cell growth, including scavenger receptor expression, cholesterol transporter expression acyl CoA: cholesterol acyltransferase activity, and neutral cholesteryl ester hydrolase activity. Plants used during herbal therapy have been shown to assist with a variety of ailments. In this study, we found medicinal plants and their bioactive components suppress foam cell formation in a variety of ways; some inhibit cholesterol transporter and lectin-like oxidized low-density lipoprotein receptor-1 upregulation, while others inhibit the function of acyl CoA: cholesterol acyltransferase activity, and neutral cholesteryl ester hydrolase activity. Recent study findings related to the synthesis of the new active component from plant sources by focusing on the typical process involved in the generation of foam cells. We're also looking at using a cellular target-based therapeutic approach to generate novel plant-based medications for the cure of atherosclerosis.

Abnormal histidine metabolism promotes macrophage lipid accumulation under Ox-LDL condition

Distinct macrophage populations exert highly heterogeneity and perform various functions, among which, a crucial function of lipid metabolism is highlighted. However, the role of histidine metabolism disorder in macrophage lipid metabolism remains elusive. Addressed this question, we sorted and cultured the bone marrow-derived macrophages (BMDMs) of histidine decarboxylase (Hdc) knockout (Hdc−/-) mice with an in vitro oxidized low-density lipoprotein (ox-LDL) model, and detected the intracellular lipids by Oil Red O staining as well as lipid probe staining. Astemizole, a canonical and long-acting histamine H1 receptor (H1R) antagonist, was applied to elucidate the impact of antagonizing the H1R-dependent signaling pathway on macrophage lipid metabolism. Subsequently, the differential expressed genes were screened and analyzed in the bone marrow-derived CD11b+ immature myeloid cells of Hdc−/- and Hdc+/+ mice with a high fat diet by the microarray study. The expression levels of cholesterol metabolism-related genes were examined by qRT-PCR to explore underlying mechanisms. Lastly, we used a high-sensitivity histidine probe to detect the intracellular histidine in the BMDMs after oxidative stress. The results revealed that histidine metabolism disorder and histamine deficiency aggravated lipid accumulation in the ox-LDL-treated BMDMs. The expression level of H1R gene in the BMDMs was down-regulated after ox-LDL stimulation. The disruption of the H1R-dependent signaling pathway by astemizole further exacerbated ox-LDL-induced lipid deposition in the BMDMs partly by up-regulating scavenger receptor class A (SR-A) for lipid intake, down-regulating neutral cholesteryl ester hydrolase (nCEH) for cholesterol esterification and down-regulating ATP-binding cassette transporters A1 (ABCA1) and ABCG1 for reverse cholesterol transport. The intracellular histidine increased under ox-LDL condition, which was further increased by Hdc knockout. Collectively, these results partially reveal the relationship between histidine metabolism and lipid metabolism in the BMDMs and offer a novel strategy for lipid metabolism disorder-associated diseases.

Adrenal cortex hypoxia modulates aldosterone production in heart failure

Plasma aldosterone concentration increases in proportion to the severity of heart failure, even during treatment with renin-angiotensin system inhibitors. This study investigated alternative regulatory mechanisms of aldosterone production that are significant in heart failure. Dahl salt-sensitive rats on a high-salt diet, a rat model of heart failure with cardio-renal syndrome, had high plasma aldosterone levels and elevated β3-adrenergic receptor expression in hypoxic zona glomerulosa cells. In H295R cells (a human adrenocortical cell line), hypoxia-induced β3-adrenergic receptor expression. Hypoxia-mediated β3-adrenergic receptor expression augmented aldosterone production by facilitating hydrolysis of lipid droplets though ERK-mediated phosphorylation of hormone-sensitive lipase, also known as cholesteryl ester hydrolase. Hypoxia also accelerated the synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase, thereby increasing the cholesterol ester content in lipid droplets. Thus, hypoxia enhanced aldosterone production by zona glomerulosa cells via promotion of the accumulation and hydrolysis of cholesterol ester in lipid droplets. In conclusion, hypoxic zona glomerulosa cells with heart failure show enhanced aldosterone production via increased catecholamine responsiveness and activation of cholesterol trafficking, irrespective of the renin-angiotensin system.

Cholesterol accumulation, lipid droplet formation, and steroid production in Leydig cells: Role of translocator protein (18-kDa).

Cholesterol import into the mitochondria of steroid-producing cells is the rate-determining step in steroidogenesis. Numerous studies have provided evidence that the cholesterol-binding translocator protein (18kDa TSPO) plays an important role in cholesterol translocation into mitochondria and that it also might act on cholesterol homeostasis. Several TSPO-specific ligands have been shown to increase steroid production in vitro and in vivo. The present study assessed the effects of the TSPO drug ligand FGIN-1-27 on cholesterol accumulation and lipid droplet formation in relationship to steroid formation. Using MA-10 and primary Leydig cells, immunocytochemical and molecular methods were used to examine cholesterol accumulation, the formation of lipid droplets, and steroid formation in response to LH and FGIN-1-27. Additionally, we determined the effects of Tspo knockout by CRISPR/Cas9, and of siRNA knockdowns of Tspo and Plin2 (Perilipin 2; also known as adipose differentiation-related protein, ADFP) on LH- and FGIN-1-27-induced steroidogenesis. In response to LH and FGIN-1-27, cultured MA-10 cells and primary Leydig cells increased steroid formation, cholesterol accumulation, and lipid droplet formation. Cholesterol accumulation in the lipid droplets also was increased in Tspo knockout cells. Knockout of Tspo or its knockdown in MA-10 cells resulted in reduced progesterone formation in response to both LH and FGIN-1-27, as did knockdown of Plin2. Steroid production also was inhibited by the cholesteryl ester hydrolase inhibitor diethylumbelliferyl phosphate. These results support the conclusion that FGIN-1-27 stimulates steroid formation by increasing TSPO-mediated cholesterol translocation into the inner mitochondria for steroidogenesis, as well as into the cytosol for lipid droplet formation. FGIN-1-27 also increased steroid formation at least in part by inducing the conversion of cholesteryl ester located in lipid droplets to cholesterol, thus making available more substrate for steroid formation.

Redox regulation of hormone sensitive lipase: Potential role in the mechanism of MEHP-induced stimulation of basal steroid synthesis in MA-10 Leydig cells

Mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate (DEHP), is a plasticizer with endocrine disruptor activity that has been shown to stimulate basal steroid biosynthesis in Leydig cells. The mechanism by which it does so is unknown. Using MA-10 mouse tumor Leydig cells, we assessed the effects of MEHP on reactive oxygen species (ROS) levels, and on the signal transduction pathways that mobilize cholesterol. Exposure to 0–300 μM MEHP stimulated basal progesterone production in a dose-dependent manner. Progesterone stimulation was correlated with increases in the phosphorylation of hormone-sensitive lipase (HSL; aka cholesteryl ester hydrolase), which is involved in the production of free cholesterol, and of steroidogenic acute regulatory (STAR) protein expression. Co-treating MA-10 cells with MEHP and the ROS scavenger N-acetyl cysteine (NAC) blocked the activation of HSL, blunted MEHP-induced STAR, and reduced basal progesterone formation. These observations suggest that ROS generation by MEHP leads to activation of HSL and increase in STAR which, together, result in increased free-cholesterol bioavailability and progesterone formation.

PO-305 An 8-week, low carbohydrate, high fat, ketogenic diet enhanced exercise capacity through improved ketolysis and lipolysis in mice

PO-305 An 8-week, low carbohydrate, high fat, ketogenic diet enhanced exercise capacity through improved ketolysis and lipolysis in mice

Abstract 169: A Non-Viral Functionalized Nanoparticle Based Hepatocyte-Targeting Cholesteryl Ester Hydrolase Gene Delivery Strategy for Potential Alleviation of Atherosclerosis

Current atherosclerosis treatment strategies primarily focus on limiting further cholesteryl ester (CE) accumulation within the plaques by reducing plasma LDL-cholesterol levels (the primary source of plaque CE) using multiple strategies. No therapy is currently available to enhance the removal of CE, a crucial step to reduce the burden of existing disease. Increasing hepatic CE hydrolysis by adenovirus or transgene mediated over-expression of CE hydrolase (CEH) enhances final elimination of cholesterol from the body and is anti-atherosclerotic. In an effort to advance towards application of this anti-atherosclerotic strategy, in the present study we developed a non-viral hepatocyte-specific platform, namely, galactose-functionalized polyamidoamine (PAMAM) dendrimer G5.0 (Gal-G5) for delivery of CEH expression vector via the hepatocyte-specific asialoglycoprotein receptor (ASGPR). ASGPR is triggered by binding of galactose residues and facilitates receptor-mediated endocytosis of large molecules across the hepatocyte plasma membrane. The data presented herein show increased specific uptake of Gal-G5 by hepatocytes in vitro (See Figure, Panel A) and liver in vivo (Panel B). Gal-G5 mediated delivery of CEH expression vector upregulated CEH expression in hepatocytes (Panel C), led to increased intracellular hydrolysis of HDL-CE and subsequent conversion/secretion of released FC into bile acids (Panel D). Gal-G5 mediated increased CEH expression in the liver significantly increased the flux of FC from HDL-CE into bile (Panel E) as well as feces (Panel F). These data are consistent with adenovirus or transgene-mediated over-expression of CEH and are likely to be anti-atherogenic. In conclusion, the development of this relatively non-toxic and efficient liver-specific gene delivery platform is an encouraging step towards clinical translation of strategies to enhance hepatic processes involved in final elimination of cholesterol from the body.

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.

Bolstering cholesteryl ester hydrolysis in liver: A hepatocyte-targeting gene delivery strategy for potential alleviation of atherosclerosis.

Current atherosclerosis treatment strategies primarily focus on limiting further cholesteryl esters (CE) accumulation by reducing endogenous synthesis of cholesterol in the liver. No therapy is currently available to enhance the removal of CE, a crucial step to reduce the burden of the existing disease. Given the central role of hepatic cholesteryl ester hydrolase (CEH) in the intrahepatic hydrolysis of CE and subsequent removal of the resulting free cholesterol (FC), in this work, we applied galactose-functionalized polyamidoamine (PAMAM) dendrimer generation 5 (Gal-G5) for hepatocyte-specific delivery of CEH expression vector. The data presented herein show the increased specific uptake of Gal-G5/CEH expression vector complexes (simply Gal-G5/CEH) by hepatocytes invitro and invivo. Furthermore, the upregulated CEH expression in the hepatocytes significantly enhanced the intracellular hydrolysis of high density lipoprotein-associated CE (HDL-CE) and subsequent conversion/secretion of hydrolyzed FC as bile acids (BA). The increased CEH expression in the liver significantly increased the flux of HDL-CE to biliary as well as fecal FC and BA. Meanwhile, Gal-G5 did not induce hepatic or renal toxicity. It was also not immunotoxic. Because of these encouraging pre-clinical testing results, using this safe and highly efficient hepatocyte-specific gene delivery platform to enhance the hepatic processes involved in cholesterol elimination is a promising strategy for the alleviation of atherosclerosis.

Differential lipid metabolism in monocytes and macrophages: influence of cholesterol loading

The influence of the hypercholesterolemia associated with atherosclerosis on monocytes is poorly understood. Monocytes are exposed to high concentrations of lipids, particularly cholesterol and lysophosphatidylcholine (lyso-PC). Indeed, in line with recent reports, we found that monocytes accumulate cholesteryl esters (CEs) in hypercholesterolemic mice, demonstrating the need for studies that analyze the effects of lipid accumulation on monocytes. Here we analyze the effects of cholesterol and lyso-PC loading in human monocytes and macrophages. We found that cholesterol acyltransferase and CE hydrolase activities are lower in monocytes. Monocytes also showed a different expression profile of cholesterol influx and efflux genes in response to lipid loading and a different pattern of lyso-PC metabolism. In monocytes, increased levels of CE slowed the conversion of lyso-PC into PC. Interestingly, although macrophages accumulated glycerophosphocholine, phosphocholine was the main water-soluble choline metabolite being generated in monocytes, suggesting a role for mono- and diacylglycerol in the chemoattractability of these cells. In summary, monocytes and macrophages show significant differences in lipid metabolism and gene expression profiles in response to lipid loading. These findings provide new insights into the mechanisms of atherosclerosis and suggest potentials for targeting monocyte chemotactic properties not only in atherosclerosis but also in other diseases.

Cholesterol crystal induced arterial inflammation and destabilization of atherosclerotic plaque.

Evolution of plaque that is prone to rupture is characterized by inflammation and physical changes. Accumulation of low-density lipoprotein in the sub-intima provides esterified cholesterol (ESC) to macrophages and smooth muscle cells that convert it into free cholesterol (FRC) by cholesteryl ester hydrolases (CEHs). Membrane-bound cholesterol carriers transport FRC to high-density lipoprotein (HDL). Impaired HDL transport function and altered composition can lead to extracellular accumulation of FRC, whereas impaired membrane carrier activity can lead to intracellular FRC accumulation. Saturation of FRC can result in cholesterol crystallization with cell death and intimal injury. Disequilibrium between ESC and FRC can impact foam cell and cholesterol crystal (CC) formation. Cholesterol crystals initiate inflammation via NLRP3 inflammasome leading to interleukin-1β (IL-1β) production inducing C-reactive protein. Eventually, crystals growing from within the plaque and associated inflammation destabilize the plaque. Thus, inhibition of inflammation by antagonists to IL-1β or agents that dissolve or prevent CC formation may stabilize vulnerable plaques.

Abstract 13493: Intracellular Cholesterol Transport Proteins Enhance Hydrolysis of HDL-delivered Cholesteryl Esters and Facilitate Preferential Elimination of Resulting Cholesterol Into Bile as Bile Acids

HDL-associated cholesterol, >80% esterified (HDL-CE) entering the hepatocyte via SR-BI can have multiple fates: 1) re-secretion as nascent HDL; 2) re-esterification and either storage of resulting CE or secretion of CE as VLDL; 3) secretion of free cholesterol into bile; and 4) conversion to and secretion of bile acids into bile. While fates 1 and 2 will lead to recirculation of HDL-CE returning to the liver from the periphery including plaque associated macrophage foam cells, fates 3 and 4 will lead to final elimination of cholesterol from the body and would be anti-atherogenic. Although we earlier identified cholesteryl ester hydrolase (CEH) as the hepatic enzyme responsible for the hydrolysis of SR-BI delivered HDL-CE, intracellular factors affecting the directional movement of HDL-derived FC remain undefined. In the present study, we examined the hypothesis that intracellular cholesterol transport proteins (SCP-2 and FABP1) not only facilitate CEH mediated hydrolysis of HDL-CE but also enhance elimination of cholesterol into bile. Primary C57BL/6 mouse hepatocytes were used and expression of CEH, FABP1 or SCP2 was modulated by using respective adenoviruses; AdLacZ was used as control. Over-expression of SCP2 or FABP1 significantly increased hydrolysis of HDL-[ 3 H]-CE (A) and reduced the re-secretion of HDL-derived cholesterol as nascent HDL (B). However, over-expression of SCP2 or FABP1 increased the secretion of cholesterol from HDL-[ 3 H]-CE as bile acids (C). Flux of HDL-[ 3 H]-CE into bile was monitored in vivo using C57BL/6 mice. Similar to the effects seen in vitro, adenovirus-mediated over-expression of FABP1 and SCP2 significantly increased the flux of cholesterol from HDL-[ 3 H]-CE to bile acids (D). Taken together, these studies demonstrate that SCP2 and FABP1 facilitate the preferential movement of HDL-associated cholesterol to bile for final elimination from the body. Studies are in progress to establish the anti-atherogenic role of these proteins

Macrophage-mediated cholesterol handling in atherosclerosis.

Formation of foam cells is a hallmark at the initial stages of atherosclerosis. Monocytes attracted by pro-inflammatory stimuli attach to the inflamed vascular endothelium and penetrate to the arterial intima where they differentiate to macrophages. Intimal macrophages phagocytize oxidized low-density lipoproteins (oxLDL). Several scavenger receptors (SR), including CD36, SR-A1 and lectin-like oxLDL receptor-1 (LOX-1), mediate oxLDL uptake. In late endosomes/lysosomes of macrophages, oxLDL are catabolysed. Lysosomal acid lipase (LAL) hydrolyses cholesterol esters that are enriched in LDL to free cholesterol and free fatty acids. In the endoplasmic reticulum (ER), acyl coenzyme A: cholesterol acyltransferase-1 (ACAT1) in turn catalyses esterification of cholesterol to store cholesterol esters as lipid droplets in the ER of macrophages. Neutral cholesteryl ester hydrolases nCEH and NCEH1 are involved in a secondary hydrolysis of cholesterol esters to liberate free cholesterol that could be then out-flowed from macrophages by cholesterol ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 and SR-BI. In atherosclerosis, disruption of lipid homoeostasis in macrophages leads to cholesterol accumulation and formation of foam cells.

Cholesterol mobilization from hepatic lipid droplets during endotoxemia is altered in obese ob/ob mice

The innate immune response to pathogens during the acute phase response includes lipid metabolism adaptations. Hepatic triacylglycerol (TG) and cholesteryl ester (CE) storage in and mobilization from lipid droplets (LDs) respond to metabolic changes under the control of liver X receptor (LXR) transactivation and cytokine transduction. To evaluate whether alterations of these mechanisms have an impact in the adaptive response to endotoxemia, we analysed liver metabolism changes in lipopolysaccharide (LPS)-treated ob/ob mice, which show altered metabolic and innate responses and a higher sensitivity to sepsis. Lipid composition of serum lipoproteins and hepatic LDs was determined in wild type and ob/ob mice 24 h after LPS treatment. Liver metabolic profiling was done by measuring enzyme activities and mRNA levels. Increased CE hydrolase activity in LDs from endotoxemic mice was accompanied by a lower content of CE and low or no induction of LXR-mediated expression of genes involved in HDL secretion. The attenuated response in liver lipid mobilization accompanied by the strain-specific cholesterol enrichment of secreted VLDL might lead to accumulation of LDL cholesterol. According to our findings, obese leptin-deficient mice present an altered control of hepatic lipid metabolism responses to LPS, which might be, in part at least, a consequence of impaired LXR.

Abstract 13: Development of Functionalized Nanoparticle Platform for Reducing Atherosclerosis

No therapy is currently available to enhance the removal of cholesteryl esters (CE) from existing atherosclerotic plaques to facilitate plaque regression. Such a strategy is crucial to reduce the burden of existing disease in addition to preventing the progression targeted by the current therapeutics. Earlier studies from our laboratory have established the anti-atherogenic role of CE hydrolase (CEH)-mediated CE mobilization from macrophage foam cells and final elimination of cholesterol by the liver. While transgenic expression of CEH was used in pre-clinical animal studies, increase in human CEH by activation of Liver-X-receptor (LXR) was also established. Increased lipogenesis induced by LXR ligands precludes their use. The current studies focused on the development of mannose-functionalized dendrimer nanoparticles (DNPs) for the delivery of LXR ligand (TO901317) or CEH expression vector to plaque associated macrophage foam cells. As shown in the Figure, mannose functionalization restricts the uptake of DNPs to macrophages and minimal uptake was seen with primary hepatocytes ( A ). Western diet fed LDLR-/- mice were injected (iv) with DNPs and tissues harvested 48 later to monitor gene expression by QPCR. DNP-mediated delivery of LXR ligand (DNP-LXR) increased the target gene expression (ABCA1, ABCG1) in plaque associated macrophage foam cells in the aortic arch with no effects on target gene expression in the liver ( B ) demonstrating the specific delivery of LXR ligand. Comparable increase in CEH activity was seen following exposure of macrophages to free LXR ligand and DNP-delivered LXR ligand ( C ) and DNP-mediated delivery of CEH expression vectors driven either by CMV or SR-A promoter induced dramatic increase in CEH expression ( D ). These data establish functionalized DNP as a suitable platform for specific and functional delivery of drugs or DNA to plaque associated macrophages to enhance processes involved in cholesterol removal and plaque regression.

Role of Cholesteryl Ester (CE) Hydrolase Mediated Mobilization of Intracellular CE in Regulating Inflammasome Activation

Cellular cholesterol content and inflammatory pathways are thought to be linked but the underlying mechanisms are not completely defined. Dramatic (>20 fold) reduction in circulating IL-1b in macrophage-specific CE hydrolase (CEH) transgenic (Tg) mice indicates a role of macrophage cholesterol homeostasis in regulating IL-1b production. Cellular priming for inducing transcription via NF-kB (Signal 1) and activation of NLRP3 inflammasome for proteolytic cleavage of pro-IL-1b (Signal 2) are required for IL-1b secretion. While activation of surface TLR4 represents Signal 1, several disparate signals can activate inflammasome and decrease in cellular K+ by increased efflux is an important convergence point. Since membrane cholesterol/lipid-raft levels regulate the function of cell surface receptors and K+ channels, we hypothesized that CEH-mediated reduction in cellular cholesterol attenuates IL-1b secretion by reducing inflammasome activation. Mouse peritoneal macrophages (MPM) from C57BL/6 (WT) or CEHTg mice were used for all the studies. Membrane cholesterol content was reduced by CEH over-expression with significant reduction in lipid-raft cholesterol content (A). Delayed reappearance of TLR4 (MFI, B) following LPS stimulation in CEHTg MPMs indicates attenuation of Signal 1. Efflux of K+ (Signal 2) required for inflammasome activation occurs via K+ channels and single cell patch clamp studies were performed to monitor channel activity. Significantly lower outward K+ currents in CEHTg MPMs were observed in CEHTg MPMs (C). Pharmacological inhibition of CEH increased IL-1b secretion from MPMs (D) confirming the role of CEH-mediated cellular cholesterol homeostasis in regulating inflammasome activation.

Enolase is regulated by Liver X Receptors

Enolase is a glycolytic enzyme known to inhibit cholesteryl ester hydrolases (CEHs). Cholesteryl ester loading of macrophages, as occurs during atherosclerosis, is accompanied by increased Enolase protein and activity. Here, we describe that J774 macrophages treated with LXR agonists exhibit reduced Enolase transcript and protein abundance. Moreover, we show that this reduction is further potentiated by activation of the LXR/RXR heterodimer with the RXR ligand 9-cis retinoic acid. Enolase levels are also reduced in vivo following activation of LXRs in the intestine, but not in the liver. This effect is lost in Lxrαβ−/− mice. In aggregate, our study identified Enolase as a new target of LXRs in vivo, which may promote cholesterol mobilization for subsequent efflux.