Cholesteryl Ester Hydrolysis Research Articles

Cholesteryl Ester Storage Disease: an Easily Missed Diagnosis in Oligosymptomatic Children

Cholesteryl ester storage disease (CESD) is a rare, autosomal recessively inherited disorder resulting from deficient activity of lysosomal acid lipase (LAL). LAL is the key enzyme hydrolyzing cholesteryl esters and triglycerides stored in lysosomes after LDL receptor-mediated endocytosis. Mutations within the LIPA gene locus on chromosome 10q23.2-q23.3 may result either in the always fatal Wolman disease, where no LAL activity is found, or in the more benign disorder CESD with a reduced enzymatic activity, leading to massive accumulation of cholesteryl esters and triglycerides in many body tissues. CESD affects mostly the liver, the spectrum is ranging from isolated hepatomegaly to liver cirrhosis. Chronic diarrhea has been reported in some pediatric cases, while calcifications of the adrenal glands, the hallmark of Wolman disease, are rarely observed. Hypercholesterolemia and premature atherosclerosis are other typical disease manifestations. Hepatomegaly as a key finding has been reported in all 71pediatric patients and in 134 of 135 adult cases in the literature. We present a 13-year-old boy with mildly elevated liver enzymes in the absence of hepatomegaly, finally diagnosed with CESD. Under pravastatine treatment, the patient has normal laboratory findings and is clinically unremarkable since 5years of follow-up. To our knowledge, this is the first pediatric case of genetically and biopsy confirmed CESD without hepatomegaly, suggesting that this diagnosis can be easily missed. It further raises the question about the natural course and the therapy required for this oligosymptomatic form.

Many ABCs make light work with cholesterol

Cholesterol-centric model of the Universe is gaining recognition. The key role of cholesterol metabolism in cardiovascular diseases has been accepted long ago; emerging evidence now supports the central role of cholesterol metabolism in neurodegenerative, metabolic, skin and infectious diseases. Cellular cholesterol content has to be maintained within tight limits, or else; not surprisingly, therefore, a number of regulatory mechanisms provide multi-layer protection of cholesterol homeostasis. Deficiency of cholesterol is rarely an issue: all human nucleated cells have efficient cholesterol biosynthesis machinery capable of satisfying the need in cholesterol under almost any circumstance. Excess of cholesterol is a different matter, it almost inevitably causes severe pathology with clinical manifestation depending on the tissue affected. Difficulty is withstanding excessive cholesterol is because the only cell type capable of degrading cholesterol is hepatocyte; other cells and tissues have to find a way around it. Reducing production of cholesterol and its uptake from lipoproteins provide some relief, but can only go so far. Esterification of cholesterol, although reduces toxicity, is nevertheless a trap as reversing it (hydrolysis of cholesteryl esters) is a very slow process. The most important regulatory pathway in maintaining cholesterol homeostasis is therefore reverse cholesterol transport (RCT). The essence of RCT is in taking excessive cholesterol from any cell of the body and transporting it through the blood to the liver and intestine where it can be degraded and/or secreted. Cholesterol has limited aqueous solubility and in the blood it has to be carried by lipoproteins. A lipoprotein that takes cholesterol from cells and carries it to the liver/intestine is high density lipoprotein (HDL)/apolipoprotein A-I (apoA-I). Although limited amount of cholesterol can passively diffuse from cells to lipoproteins, most of excessive cholesterol is released in energy-dependent and controlled manner, via a process termed cholesterol efflux. Several transporters control cholesterol efflux, but the most important is ATP binding cassette transporter A1 (ABCA1). ABCA1 interacts with extracellular apoA-I loading it with cellular phospholipids and cholesterol; by doing so it transforms apoA-I into nascent HDL and relieves cell of excessive cholesterol. The rate of the efflux depends on the abundance of ABCA1 and its functionality, both are regulated on several levels. On transcriptional level ABCA1 is regulated by the Liver X Receptor (LXR), a nuclear receptor capable of stimulation transcription of ABCA1 gene when bound to an agonist. On post-transcriptional level abundance of ABCA1 is regulated through its degradation in both lysosomes and proteasomes as well as through action of calpain. Functionality of ABCA1 is regulated through its phosphorylation and trafficking to and from plasma membrane. Different levels of regulation are interconnected; for example removal of ABCA1 from plasma membrane reduces its functionality, but also leads to degradation. Hozoji-Inada et al. have recently proposed that LXR regulates ABCA1 on both transcriptional and post-translational levels [1]. They suggested that one isoform of LXR, LXRβ, binds to ABCA1 preventing ATP hydrolysis and shutting down its function. LXR agonist disrupts this complex, on the one hand, restoring ABCA1 functionality and on the other, allowing LXRβ to travel to the nucleus and to initiate transcription of the ABCA1 gene. We have recently found another player in this game [2]. ABCA12 is known for its role in maintaining skin barrier function. Deficiency in ABCA12 is the cause of Harlequin ichthyosis, an often fatal skin disease. We however noticed that fibroblasts from ABCA12−/− mouse are extremely susceptible to challenge with excessive cholesterol [3]. Mechanistic studies on macrophages demonstrated that ABCA12 deficient cells fail to respond to activation with LXR agonist. Interestingly, expression of the ABCA1 gene was properly stimulated, but increases in ABCA1 protein abundance were blunted and cholesterol efflux was not stimulated at all. Another unexpected effect of ABCA12 deficiency was a fall in abundance of LXRβ; overexpression of LXRβ reversed the effects of ABCA12 deficiency. Like Hozoji-Inada we found that LXRβ binds to ABCA1, ABCA12 binds to both LXRβ and ABCA1, and while in ABCA12+/+ cells LXR dissociates when agonist is added, this didn't happen in ABCA12−/− cells. In vivo, when apoe−/− mice were transplanted with apoe−/−/ Abca12−/− bone marrow, this lead to impairment of reverse cholesterol transport and significant acceleration of development of atherosclerosis. Our conclusion is that ABCA12, along with LXRβ, is a part of a regulatory complex controlling ABCA1 functionality. Reverse cholesterol transport and specifically ABCA1 are involved in many diseases that are common in middle to late ages, most importantly in type 2 diabetes [4] and Alzheimer disease [5]. And yet, epidemiological data connecting ABCA1 polymorphism and susceptibility to these diseases are not very convincing [6]. It is conceivable that mutations in proteins within pathways regulating ABCA1 functionality (such as LXRβ or ABCA12) are more important for the outcomes than polymorphism of ABCA1 itself. On the other hand, the elements of the regulatory pathways may present many potential targets for therapeutic interventions.

Liver-Specific Cholesteryl Ester Hydrolase Deficiency Attenuates Sterol Elimination in the Feces and Increases Atherosclerosis in Ldlr −/− Mice

Liver is the major organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or as bile acids. Intracellular hydrolysis of lipoprotein-derived cholesteryl esters (CEs) is essential to generate the free cholesterol required for this process. Earlier, we demonstrated that overexpression of human CE hydrolase (Gene symbol CES1) increased bile acid synthesis in human hepatocytes and enhanced reverse cholesterol transport in mice. The objective of the present study was to demonstrate that liver-specific deletion of its murine ortholog, Ces3, would decrease cholesterol elimination from the body and increase atherosclerosis. Liver-specific Ces3 knockout mice (Ces3-LKO) were generated, and Ces3 deficiency did not affect the expression of genes involved in cholesterol homeostasis and free cholesterol or bile acid transport. The effects of Ces3 deficiency on the development of Western diet-induced atherosclerosis were examined in low density lipoprotein receptor knock out(-/-) mice. Despite similar plasma lipoprotein profiles, there was increased lesion development in low density lipoprotein receptor knock out(-/-)Ces3-LKO mice along with a significant decrease in the bile acid content of bile. Ces3 deficiency significantly reduced the flux of cholesterol from [(3)H]-CE-labeled high-density lipoproteins to feces (as free cholesterol and bile acids) and decreased total fecal sterol elimination. Our results demonstrate that hepatic Ces3 modulates the hydrolysis of lipoprotein-delivered CEs and thereby regulates free cholesterol and bile acid secretion into the feces. Therefore, its deficiency results in reduced cholesterol elimination from the body, leading to significant increase in atherosclerosis. Collectively, these data establish the antiatherogenic role of hepatic CE hydrolysis.

Plasmin Promotes Foam Cell Formation by Increasing Macrophage Catabolism of Aggregated Low-Density Lipoprotein

The plasmin/plasminogen system is involved in atherosclerosis. However, the mechanisms by which it stimulates disease are not fully defined. A key event in atherogenesis is the deposition of low-density lipoprotein (LDL) on arterial walls where it is modified, aggregated, and retained. Macrophages are recruited to clear the lipoproteins, and they become foam cells. The goal of this study was to assess the role of plasmin in macrophage uptake of aggregated LDL and foam cell formation. Plasminogen treatment of macrophages catabolizing aggregated LDL significantly accelerated foam cell formation. Macrophage interaction with aggregated LDL increased the surface expression of urokinase-type plasminogen activator receptor and plasminogen activator activity, resulting in increased ability to generate plasmin at the cell surface. The high local level of plasmin cleaves cell-associated aggregated LDL, allowing a portion of the aggregate to become sequestered in a nearly sealed, yet extracellular, acidic compartment. The low pH in the plasmin-induced compartment allows lysosomal enzymes, delivered via lysosome exocytosis, greater activity, resulting in more efficient cholesteryl ester hydrolysis and delivery of a large cholesterol load to the macrophage, thereby promoting foam cell formation. These findings highlight a critical role for plasmin in the catabolism of aggregated LDL by macrophages and provide a new context for considering the atherogenic role of plasmin.

Abstract 408: Macrophage-mediated Low Density Lipoprotein Modification Reduces Foam Cell Formation

Macrophage metabolism of LDL-derived cholesterol is thought to be a key process regulating the composition and progression of atherosclerotic lesions, including the generation of cholesterol-loaded macrophage foam cells and the lipid-rich necrotic core. Many mechanisms contribute to macrophage endocytosis of LDL, and we recently reported that selective cholesteryl ester (CE) uptake contributes to LDL-induced macrophage cholesterol accumulation. Selective uptake is expected to result in the net removal of cholesteryl ester from LDL; however, the effect of this and other LDL modifications that may occur during incubation with macrophages is not understood. In the current investigation, we pre-incubated LDL for 1-2 days with macrophages in order to promote macrophage-mediated LDL modification. Surprisingly, when macrophage-conditioned LDL was transferred to fresh macrophages, much less cholesterol accumulation resulted compared to cells incubated with unconditioned LDL, as indicated by cholesterol quantification and by neutral lipid staining with Oil Red-O. This difference was not accounted for by changes in uptake of the modified 125I-LDLs, but instead was associated with a large decrease in selective [3H]CE uptake. In contrast, uptake of the non-hydrolyzable CE analog [3H]cholesteryl ether was not reduced, suggesting that reduced uptake in macrophage-conditioned LDL may be due to changes in the hydrolysis of LDL-derived CE or its subsequent metabolism. Cell-mediated LDL oxidation was considered as a possible mechanism for changes in selective CE uptake. However, agarose gel electrophoretic mobility of LDL and analysis of LDL cholesteryl esters indicated that reduced selective uptake and foam cell formation did not depend on extensive oxidative modifications of LDL. These results indicate the importance of selective CE and suggest that macrophage-mediated LDL modification may reduce foam cell formation in some circumstances.

Abstract 66: SR-BI Mediates Cholesteryl Ester (CE) Sorting in Tetraspanin Microdomains with CD81 Facilitating CE Nano-endocytosis and CE Transport to Lipid Droplets

SR-BI functions as a neutral lipid transporter facilitating cholesteryl ester (CE) sorting, internalization and accumulation in LDs. The mechanism of CE transport remains obscure. We hypothesized that HDL lipid transport requires CD81 as a co-receptor and tetraspanin enriched domains (TED) for CE sorting, similar to hepatitis C uptake. Utilizing BODIPY-CE labeled recombinant HDL (rHDL/BP-CE) based on either human apoA-I or an apoA-I mimetic peptide, L-37pA, and confocal and TIRF microscopy, we found that after initially binding to SR-BI at the cellular filopodia and lamellipodia, HDL CE was laterally transported to the bases of filopodia, accumulating in 1-2 μm plasma membrane microdomains. While CE was rapidly internalized, neither apoA-I, L37pA nor SR-BI was, suggesting that these microdomains are an important part of the CE sorting mechanism. Internalized CE was transported in 50-100 nm CE-containing intracellular nano-vesicles. In a process of multiple collisions potentially involving transient nano-vesicle/LD fusion, BODIPY-CE was transferred to LDs. To identify CE-sorting microdomains, SR-BI expressing HeLa cells were transfected with GFP-CD81 or GFP-CD82. After a 1 h incubation with rHDL/BP-CE, CE was co-localized with both tetraspanin proteins demonstrating that these are TED (the observation was confirmed by anti-CD81 and -CD-82 antibody immunostaining). Following a 2 h chase, only GFP-CD81, not CD82, was found internalized and co-localized with CE transporting nano-vesicles. CE transport was not affected by inhibitors of de novo neutral lipid synthesis such as the ACSL-3 inhibitor, triacsin c, ACAT inhibitor, Sandoz 58-035 or ATGL inhibitor, A926500, indicating that CE was directly transported through a novel nano-endocytosis mechanism involving CD81 and not requiring CE hydrolysis and de novo synthesis. We conclude that TED function as CE sorting sites where CD81 helps to form nano-vesicles delivering CE to LDs. Further understanding of their function will help develop novel anti-atherogenic compounds accelerating reverse cholesterol transport.

Mechanisms of Action of Hormone-sensitive Lipase in Mouse Leydig Cells: ITS ROLE IN THE REGULATION OF THE STEROIDOGENIC ACUTE REGULATORY PROTEIN

Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of cholesteryl esters in steroidogenic tissues and, thus, facilitates cholesterol availability for steroidogenesis. The steroidogenic acute regulatory protein (StAR) controls the rate-limiting step in steroid biosynthesis. However, the modes of action of HSL in the regulation of StAR expression remain obscure. We demonstrate in MA-10 mouse Leydig cells that activation of the protein kinase A (PKA) pathway, by a cAMP analog Bt2cAMP, enhanced expression of HSL and its phosphorylation (P) at Ser-660 and Ser-563, but not at Ser-565, concomitant with increased HSL activity. Phosphorylation and activation of HSL coincided with increases in StAR, P-StAR (Ser-194), and progesterone levels. Inhibition of HSL activity by CAY10499 effectively suppressed Bt2cAMP-induced StAR expression and progesterone synthesis. Targeted silencing of endogenous HSL, with siRNAs, resulted in increased cholesteryl ester levels and decreased cholesterol content in MA-10 cells. Depletion of HSL affected lipoprotein-derived cellular cholesterol influx, diminished the supply of cholesterol to the mitochondria, and resulted in the repression of StAR and P-StAR levels. Cells overexpressing HSL increased the efficacy of liver X receptor (LXR) ligands on StAR expression and steroid synthesis, suggesting HSL-mediated steroidogenesis entails enhanced oxysterol production. Conversely, cells deficient in LXRs exhibited decreased HSL responsiveness. Furthermore, an increase in HSL was correlated with the LXR target genes, steroid receptor element-binding protein 1c and ATP binding cassette transporter A1, demonstrating HSL-dependent regulation of steroidogenesis predominantly involves LXR signaling. LXRs interact/cooperate with RXRs and result in the activation of StAR gene transcription. These findings provide novel insight and demonstrate the molecular events by which HSL acts to drive cAMP/PKA-mediated regulation of StAR expression and steroidogenesis in mouse Leydig cells.

Porcine Pancreas: A Superior Source of Cholesterol Esterase for Total Serum Cholesterol Assay by the Enzymatic Kinetic Method

Accurate determination of cholesterol requires complete hydrolysis of cholesteryl esters and must be very fast for the kinetic cholesterol assay. We investigated the properties of cholesterol esterase derived from Pseudomonas fluorescens, Candida cylindracea, bovine pancreas, and porcine pancreas for cholesterol determination in human serum. Optimization of four enzymes and effect of sodium cholate concentration were performed. We evaluated and compared their performances in enzymatic kinetic cholesterol determination. The optimal sodium cholate concentration was 3, 5, 15, and 12 mmol/l with the enzyme activities at 200, 100, 100, and 100 U/l for P. fluorescens, C. cylindracea, bovine pancreas, and porcine pancreas, respectively. Linearity obtained from all enzymes was up to 16.3 mmol/l. All assays were compared favorably with standardized endpoint method. Only the cholesterol esterase derived from porcine pancreas demonstrated acceptable precision within the acceptable criteria (%CV < 3.0). Also, this esterase was least affected by interfering substances and showed longer stability than that of C. cylindracea and bovine pancreas. Porcine pancreas cholesterol esterase is superior to that obtained from P. fluorescens, C. cylindracea, and bovine pancreas for total serum cholesterol determination by the kinetic method because of its lower cost, better accuracy and precision, less interference, and longer stability.

High-capacity selective uptake of cholesteryl ester from native LDL during macrophage foam cell formation

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Recent studies have demonstrated that at high concentrations associated with hypercholesterolemia, native LDL induces macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol from native LDL during foam cell formation has not been clearly defined. Previous reports have suggested that selective cholesteryl ester (CE) uptake might contribute to cholesterol uptake from LDL independently of particle endocytosis. In this study we demonstrate that the majority of macrophage LDL-derived cholesterol is acquired by selective CE uptake in excess of LDL pinocytosis and degradation. Macrophage selective CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. In contrast to CE uptake, macrophages exhibit little selective uptake of free cholesterol (FC) from LDL. Following selective uptake from LDL, CE is rapidly hydrolyzed by a novel chloroquine-sensitive pathway. FC released from LDL-derived CE hydrolysis is largely effluxed from cells but also is subject to ACAT-mediated reesterification. These results indicate that selective CE uptake plays a major role in macrophage metabolism of LDL.

Association of LIPA Gene Polymorphisms With Obesity‐Related Metabolic Complications Among Severely Obese Patients

The lipase A, lysosomal acid, cholesterol esterase enzyme (LIPA) is involved in the hydrolysis of triglycerides (TGs) and cholesteryl esters (CEs) delivered to lysosomes. LIPA deficiency in human causes two distinct phenotypes characterized by intracellular storage of CE and derangements in the control of cholesterol production, namely the Wolman disease (WD) and the CE storage disease (CESD). To test the potential association of LIPA gene polymorphisms with obesity-related metabolic complications, promoter, exons, and intronic flanking regions of the LIPA gene were first sequenced in 25 individuals. From the 14 common polymorphisms identified, 12 tagging single-nucleotide polymorphisms (tSNPs) were genotyped in a cohort of 1,751 obese individuals. After adjustments for the effect of age, sex, diabetes, and medication, the C allele of SNP rs1051338 was associated with lower blood pressure (BP; systolic (SBP) P = 0.004; diastolic (DBP) P = 0.006). Three of the tested SNPs were associated with modifications of the plasma lipid profile. The G/G genotype of rs2071509 was associated with higher high-density lipoprotein cholesterol (HDL-C) levels (P = 0.009) and minor allele of rs1131706 was also associated with higher HDL-C (P = 0.004) and an association between rs3802656 and total cholesterol (total-C)/HDL-C ratio was identified (P = 0.04). These results thus suggest that LIPA polymorphisms contribute to the interindividual variability observed in obesity-related metabolic complications.

The Chlamydia trachomatis CT149 protein exhibits esterase activity in vitro and catalyzes cholesteryl ester hydrolysis when expressed in HeLa cells

Chlamydia, like other intracellular bacteria, are auxotrophic for a variety of essential metabolites and obtain cholesterol and fatty acids from their eukaryotic host cell, however not many Chlamydia-specific enzymes have been identified that are involved in lipid metabolism. In silico analysis of one candidate Chlamydia trachomatis enzyme, annotated as a conserved putative hydrolase (CT149), identified two lipase/esterase GXSXG motifs, and a potential cholesterol recognition/interaction amino acid consensus (CRAC) sequence. His-tag purified recombinant CT149 exhibited ester hydrolysis activity in a nitrophenyl acetate-based cell-free assay system. When cholesteryl linoleate was used as substrate, ester hydrolysis occurred and production of cholesterol was detected by high performance liquid chromatography. Exogenous expression of transfected CT149 in HeLa cells resulted in a significant decrease of cytoplasmic cholesteryl esters within 48 h. These results demonstrate that CT149 has cholesterol esterase activity and is likely to contribute to the hydrolysis of eukaryotic cholesteryl esters during intracellular chlamydial growth.

Cholesteryl ester accumulation and accelerated cholesterol absorption in intestine-specific hormone sensitive lipase-null mice

Hormone sensitive lipase (HSL) regulates the hydrolysis of acylglycerols and cholesteryl esters (CE) in various cells and organs, including enterocytes of the small intestine. The physiological role of this enzyme in enterocytes, however, stayed elusive. In the present study we generated mice lacking HSL exclusively in the small intestine (HSLiKO) to investigate the impact of HSL deficiency on intestinal lipid metabolism and the consequences on whole body lipid homeostasis. Chow diet-fed HSLiKO mice showed unchanged plasma lipid concentrations. In addition, feeding with high fat/high cholesterol (HF/HC) diet led to unaltered triglyceride but increased plasma cholesterol concentrations and CE accumulation in the small intestine. The same effect was observed after an acute cholesterol load. Gavaging of radioactively labeled cholesterol resulted in increased abundance of radioactivity in plasma, liver and small intestine of HSLiKO mice 4h post-gavaging. However, cholesterol absorption determined by the fecal dual-isotope ratio method revealed no significant difference, suggesting that HSLiKO mice take up the same amount of cholesterol but in an accelerated manner. mRNA expression levels of genes involved in intestinal cholesterol transport and esterification were unchanged but we observed downregulation of HMG-CoA reductase and synthase and consequently less intestinal cholesterol biosynthesis. Taken together our study demonstrates that the lack of intestinal HSL leads to CE accumulation in the small intestine, accelerated cholesterol absorption and decreased cholesterol biosynthesis, indicating that HSL plays an important role in intestinal cholesterol homeostasis.

Identification of a novel intracellular cholesteryl ester hydrolase (carboxylesterase 3) in human macrophages: compensatory increase in its expression after carboxylesterase 1 silencing

Cholesteryl ester (CE) hydrolysis is the rate-limiting step in the removal of free cholesterol (FC) from macrophage foam cells, and several enzymes have been identified as intracellular CE hydrolases in human macrophages. We have previously reported the antiatherogenic role of a carboxylesterase [carboxylesterase 1 (CES1)], and the objective of the present study was to determine the contribution of CES1 to total CE hydrolytic activity in human macrophages. Two approaches, namely, immune depletion and short hairpin (sh)RNA-mediated knockdown, were used. Immuneprecipitation by a CES1-specific antibody resulted in a 70-80% decrease in enzyme activity, indicating that CES1 is responsible for >70% of the total CE hydrolytic activity. THP1-shRNA cells were generated by stably transfecting human THP1 cells with four different CES1-specific shRNA vectors. Despite a significant (>90%) reduction in CES1 expression both at the mRNA and protein levels, CES1 knockdown neither decreased intracellular CE hydrolysis nor decreased FC efflux. Examination of the underlying mechanisms for the observed lack of effects of CES1 knockdown revealed a compensatory increase in the expression of a novel CES, CES3, which is only expressed at <30% of the level of CES1 in human macrophages. Transient overexpression of CES3 led to an increase in CE hydrolytic activity, mobilization of intracellular lipid droplets, and a reduction in cellular CE content, establishing CES3 as a bona fide CE hydrolase. This study provides the first evidence of functional compensation whereby increased expression of CES3 restores intracellular CE hydrolytic activity and FC efflux in CES1-deficient cells. Furthermore, these data support the concept that intracellular CE hydrolysis is a multienzyme process.

Abstract 23: Identification of a Novel Intracellular Cholesteryl Ester Hydrolase (CES3) in Human Macrophages: Compensatory Increase Following CES1 Silencing

Removal of free cholesterol (FC) from lesion-associated macrophage foam cells represents the major mechanism for reducing the lipid burden of atherosclerotic plaques and intracellular cholesteryl ester (CE) hydrolysis is recognized as the rate-limiting step in this process. We have earlier reported cloning of human macrophage CE hydrolase (gene symbol CES1) that associated with cytoplasmic lipid droplets and enhanced cellular CE mobilization. Macrophage-specific transgenic expression of CES1 attenuated diet-induced atherosclerosis and lesion necrosis in ldlr -/- mice. Several enzymes have since been identified as intracellular CE hydrolases in human macrophages and the objective of the present study was to determine the relative contribution of CES1 to the total CE hydrolytic activity in human macrophages. Immune-precipitation by CES1-specific antibody resulted in 70-80% decrease in enzyme activity indicating that CES1 is responsible for &gt;70% of the total CE hydrolytic activity (Panel A). To examine the effects of CES1 depletion/knockdown on FC efflux, human monocyte/macrophages (THP1) were stably transfected with 4 different CES1-specific shRNA vectors (p81-p84) to generate THP1-shRNA cells; cells transfected with pRSL vector were used as controls. Despite significant reduction in CES1 expression both at mRNA and protein levels, CES1 knockdown neither decreased intracellular CE hydrolysis nor decreased FC efflux. Examination of the underlying mechanisms for the observed lack of effects of CES1 knockdown revealed a compensatory increase in the expression of a novel carboxylesterase, CES3 (Panel B). Human macrophage CES3 was identical to the reported CES3 except it lacked exon 8 and, in wild type THP1 cells it was only expressed at 29.2±6.1 percent of CES1. Transient over-expression of this novel variant of CES3 led to an increase in CE hydrolytic activity (&gt;4 fold), mobilization of intracellular lipid droplets and reduction in cellular CE content (108.1±7.65 vs 160.9±7.00, P=0.0005), establishing CES3 as a bona fide CE hydrolase. Reciprocal backup circuits, where deficiency of a gene leads to an increase in another gene with similar function, have been described and this study provides first evidence of functional compensation in intracellular CE hydrolysis whereby increased expression of CES3 restores intracellular CE hydrolytic activity and FC efflux in CES1-deficient cells. To gain insight into the mechanisms underlying the observed increase in CES3 following CES1 deficiency in silico analyses of CES3 promoter was performed and several NF-kB binding sites were identified. Based on our earlier demonstration of a decrease in NF-kB activation by CES1 over-expression, it is speculated that conversely, deficiency of CES1 increases NF-kB activation leading to compensatory increase in CES3 expression.

Abstract 283: Novel Mechanism of Low-Density Lipoprotein--Derived Cholesteryl Ester Uptake and Hydrolysis During Macrophage Foam Cell Formation

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Classically, scavenger receptor-mediated uptake of oxidized or enzyme-modified LDL is cited as a mechanism for foam cell formation. Recent studies have demonstrated that at high concentrations, native LDL can also induce macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol derived from native LDL during macrophage foam cell formation has not been clearly defined. Previous studies suggested that selective CE uptake might contribute to cholesterol uptake independently of LDL endocytosis. In this study we demonstrate that the majority of cholesterol taken up from LDL is acquired by selective CE uptake and not by pinocytosis and LDL degradation. CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. LDL modification and scavenger receptors do not appear to play a major role in macrophage selective CE uptake. Following uptake from LDL, CE is rapidly hydrolyzed in macrophages by a novel chloroquine- and temperature-sensitive pathway. Our findings suggest that selective CE uptake plays a major role in macrophage cholesterol homeostasis during foam cell formation.

Abstract 497: Cholesterol and Toll-like Receptor Signaling Are Important Regulators of Macrophage Interactions with Atherosclerotic Lipoprotein Aggregates

Macrophages encounter deposits of aggregated low-density lipoproteins (agLDL) in the subendothelial space of blood vessels during the first stages of atherosclerotic plaque formation. Notably, current models for the mechanism of macrophage internalization of cholesterol in early atherosclerotic plaques are incomplete due to the lack of attention paid to the unique cellular mechanisms that are required for macrophages to degrade aggregates of LDL in particular, which can comprise &gt;90% of the LDL in atherosclerotic plaques. In fact, internalization of cholesterol from cholesteryl esters in agLDL involves the development of intriguing cellular processes in which extracellular acidic compartments, lysosomal synapses (LSs), are formed whereby agLDL is partially degraded prior to internalization. This process requires extensive cytoskeletal rearrangements and secretion of lysosomal enzymes responsible for hydrolysis of cholesteryl esters from the agLDL. Subsequent delivery of free cholesterol from agLDL to the macrophage plasma membrane is central for development of the LS. Nonetheless, the molecular mechanism underlying initiation and propagation of the LS are currently largely unknown. This research proposal aims to elucidate the molecular mechanisms of LS formation and the role that cholesterol plays in eliciting these morphological responses to agLDL. Fluorescence microscopy assays were used to identify activation of TLR4 and downstream signaling involving PI3K and Akt as important events leading to LS formation. Furthermore, morphological responses of macrophages to cholesterol overloading require overlapping signaling pathways, indicating the role of interplay of cholesterol and TLR4 signaling in development of this novel macrophage interaction with aggregated LDL found in plaques. Identification of specific molecular pathways involved in this process will not only contribute to the basic understanding of one of the primary cellular processes contributing to atherosclerosis, one of the primary causes of heart disease, but also provide tangible molecular targets for the ultimate development of therapies.

Early steps in reverse cholesterol transport

Several controversies exist related to the molecular identity and subcellular localization of the enzyme catalyzing macrophage cholesteryl ester hydrolysis. Some of these issues have been reviewed earlier and this review summarizes new developments that describe effects of overexpression or gene ablation. The main objective is to highlight the disagreement between lack of gene expression and incomplete abolition of macrophage cholesteryl ester hydrolytic activity and to emphasize the importance of redundancy. New information resulting from the continuing characterization of the various cholesteryl ester hydrolases (hormone-sensitive lipase, HSL; cholesteryl ester hydrolase, CEH; and KIAA1363/NCEH1) is reviewed. Whereas CEH overexpression leads to beneficial effects such as decreased inflammation, improved glucose tolerance/insulin sensitivity, and attenuation of atherosclerotic lesion progression, deficiency/ablation of HSL or KIAA1363/NCEH1 results in incomplete loss of macrophage cholesteryl ester hydrolysis/turnover. New paradigms challenging the classical view of cytoplasmic cholesteryl ester hydrolysis and reverse cholesterol transport are also presented. The observed beneficial effects of CEH overexpression identify macrophage cholesteryl ester hydrolysis as an important therapeutic target and future studies will determine whether similar effects are obtained with overexpression of HSL or KIAA1363/NCEH1. It is imperative that, for clinical benefit, mechanisms to enhance endogenous cholesteryl ester hydrolase(s) are established.

Endosomal Actin Remodeling by Coronin-1A Controls Lipoprotein Uptake and Degradation in Macrophages

The actin cytoskeleton has been implicated in the processing of atherogenic lipoproteins in macrophages. However, the functional role of actin and the regulatory proteins involved are unknown. Coronin-1A (Coro1A) was identified as a differentially expressed transcript in wild-type versus Niemann-Pick type C1 deficient macrophages exposed to acetylated low-density lipoproteins (AcLDL). We investigated whether Coro1A plays a role in the uptake or processing of modified lipoproteins in macrophages and if this is related to its actin regulatory functions. In wild-type primary macrophages, filamentous actin transiently decorated AcLDL containing endosomes that also recruited Coro1A. This dynamic association of F-actin with endosomes was disturbed in Coro1A deficient macrophages. In Coro1A knockout macrophages the uptake of AcLDL was increased, rate of AcLDL delivery to lysosomes enhanced, and lipoprotein-derived cholesteryl ester hydrolysis accelerated. Overexpression of wild-type Coro1A normalized AcLDL uptake in Coro1A knockout macrophages while a Coro1A actin binding mutant did not. Furthermore, the effects of macrophage Coro1A silencing on endosomal actin association and AcLDL delivery to lysosomes resembled those of cofilin silencing. Coro1A controls actin association with endocytic organelles, thereby negatively regulating endo-lysosomal delivery, degradation of modified lipoproteins and cholesterol deposition in macrophages.

Anti-Atherosclerotic Potential of Dihydropyridine Calcium Channel Blockers

Dihydropyridine calcium channel blockers (CCBs) are widely used agents for patients with hypertension. Dihydropyridine CCBs lower blood pressure mainly through vasodilation and reduction of peripheral resistance, and several clinical studies have demonstrated that they have clinical benefits in patients with cardiovascular diseases. In addition, some studies have indicated that dihydropyridine CCBs have anti-atherogenic effects beyond their blood pressure-lowering effects. In fact, several studies using atherosclerotic model animals have revealed that dihydropyridine CCBs suppress atherosclerotic lesion formation. It is well known that the production of reactive oxygen species (ROS) is involved in the progression of atherosclerosis by stimulating the production of inflammatory factors such as chemokines, cytokines and adhesion molecules. Dihydropyridine CCBs can suppress ROS generation and subsequent inflammatory actions in vascular cells and arterial walls. Furthermore, several reports have revealed that dihydropyridine CCBs suppress the expression of adhesion molecules, thereby inhibiting monocyte adhesion to endothelial cells, which is thought to be an early step in the pathogenesis of atherosclerosis. In smooth muscle cells, dihydropyridine CCBs suppress cell proliferation and migration in vitro and in vivo. In macrophages, dihydropyridine CCBs decrease cholesterol accumulation and intracellular cholesterol esterification, and increase cholesteryl ester hydrolysis. Moreover, dihydropyridine CCBs suppress the expression of matrix metalloproteinases, which affects the stability of atheromatous plaques. Interestingly, recent studies have revealed that the anti-atherosclerotic effects of dihydropyridine CCBs are mediated, at least in part, via the activation of peroxisome proliferator-activated receptor-γ. In this review, we focus on the anti-atherosclerotic effects of dihydropyridine CCBs beyond their blood pressure-lowering effects.

Regulation of Lipid Droplet Cholesterol Efflux From Macrophage Foam Cells

Cholesterol efflux from macrophages is the first and potentially most important step in reverse cholesterol transport, a process especially relevant to atherosclerosis and to the regression of atherosclerotic plaques. Increasingly, lipid droplet (LD) cholesteryl ester (CE) hydrolysis is being recognized as a rate-limiting step in cholesterol efflux. The traditional view on macrophage CE hydrolysis is that this pathway is entirely dependent on the action of neutral hydrolases, and numerous candidate CE hydrolases have been proposed to play a role in lipid hydrolysis in macrophages and atherogenesis. Although the exact identity of macrophage-specific CE hydrolases remains to be clarified, a common point to all of these studies is that enhancing LD-associated CE hydrolysis increases cholesterol efflux and is antiatherogenic. Understanding how cholesterol is mobilized from LDs offers new steps for modulating cholesterol efflux, and recently a role for autophagy and lysosomal acid lipase in macrophage lipolysis has emerged. Autophagy and lysosomal acid lipase thus represent novel therapeutic targets to enhance macrophage reverse cholesterol transport. This review discusses our current understanding of the relationship between macrophage LDs and atherosclerosis and presents recent insights into the mechanisms for LD CE hydrolysis in macrophage foam cells.