Neutral Cholesterol Ester Hydrolase Research Articles

Deficiency of neutral cholesterol ester hydrolase 1 (NCEH1) impairs endothelial function in diet-induced diabetic mice

BackgroundNeutral cholesterol ester hydrolase 1 (NCEH1) plays a critical role in the regulation of cholesterol ester metabolism. Deficiency of NCHE1 accelerated atherosclerotic lesion formation in mice. Nonetheless, the role of NCEH1 in endothelial dysfunction associated with diabetes has not been explored. The present study sought to investigate whether NCEH1 improved endothelial function in diabetes, and the underlying mechanisms were explored.MethodsThe expression and activity of NCEH1 were determined in obese mice with high-fat diet (HFD) feeding, high glucose (HG)-induced mouse aortae or primary endothelial cells (ECs). Endothelium-dependent relaxation (EDR) in aortae response to acetylcholine (Ach) was measured.ResultsResults showed that the expression and activity of NCEH1 were lower in HFD-induced mouse aortae, HG-exposed mouse aortae ex vivo, and HG-incubated primary ECs. HG exposure reduced EDR in mouse aortae, which was exaggerated by endothelial-specific deficiency of NCEH1, whereas NCEH1 overexpression restored the impaired EDR. Similar results were observed in HFD mice. Mechanically, NCEH1 ameliorated the disrupted EDR by dissociating endothelial nitric oxide synthase (eNOS) from caveolin-1 (Cav-1), leading to eNOS activation and nitric oxide (NO) release. Moreover, interaction of NCEH1 with the E3 ubiquitin-protein ligase ZNRF1 led to the degradation of Cav-1 through the ubiquitination pathway. Silencing Cav-1 and upregulating ZNRF1 were sufficient to improve EDR of diabetic aortas, while overexpression of Cav-1 and downregulation of ZNRF1 abolished the effects of NCEH1 on endothelial function in diabetes. Thus, NCEH1 preserves endothelial function through increasing NO bioavailability secondary to the disruption of the Cav-1/eNOS complex in the endothelium of diabetic mice, depending on ZNRF1-induced ubiquitination of Cav-1.ConclusionsNCEH1 may be a promising candidate for the prevention and treatment of vascular complications of diabetes.

KIAA1363-A Multifunctional Enzyme in Xenobiotic Detoxification and Lipid Ester Hydrolysis.

KIAA1363, annotated as neutral cholesterol ester hydrolase 1 (NCEH1), is a member of the arylacetamide deacetylase (AADAC) protein family. The name-giving enzyme, AADAC, is known to hydrolyze amide and ester bonds of a number of xenobiotic substances, as well as clinical drugs and of endogenous lipid substrates such as diglycerides, respectively. Similarly, KIAA1363, annotated as the first AADAC-like protein, exhibits enzymatic activities for a diverse substrate range including the xenobiotic insecticide chlorpyrifos oxon and endogenous substrates, acetyl monoalkylglycerol ether, cholesterol ester, and retinyl ester. Two independent knockout mouse models have been generated and characterized. However, apart from reduced acetyl monoalkylglycerol ether and cholesterol ester hydrolase activity in specific tissues and cell types, no gross-phenotype has been reported. This raises the question of its physiological role and whether it functions as drug detoxifying enzyme and/or as hydrolase/lipase of endogenous substrates. This review delineates the current knowledge about the structure, function and of the physiological role of KIAA1363, as evident from the phenotypical changes inflicted by pharmacological inhibition or by silencing as well as knockout of KIAA1363 gene expression in cells, as well as mouse models, respectively.

Correlation of cholesteryl ester metabolism to pathogenesis, progression and disparities in colorectal Cancer

BackgroundColorectal cancer (CRC) is one of the most common cancers worldwide characterized by disparities in age, gender, race and anatomic sites. The mechanism underlying pathogenesis, progression and disparities of CRC remains unclear. This study aims to reveal the association of expression levels of enzymes related to cholesteryl ester (CE) metabolism with pathogenesis, progression and disparities of CRC.MethodsThe differences in gene expression levels were analyzed for enzymes in CE synthesis (acyl CoA: cholesterol acyltransferase 1 and 2, ACAT1, and ACAT2), and in CE hydrolysis (neutral cholesterol ester hydrolase, NCEH1 and lysosomal acid lipase, LAL) on TNMplot platform between CRC and normal colorectal tissues (NCT) in a large cohort. The differences in protein expression levels for these enzymes were determined by Immunochemistry (IHC) performed on tissue microarray containing 96 pairs of CRC and benign colorectal tissues (BCT) from different patient populations. The expression level represented as IHC score of each enzyme was compared between CRC and BCT in entire population and populations stratified by race, gender and anatomic sites. Student’s t-test, Fisher exact test and ANOVA were used for data analysis. Significant p value was set at P<0.05.ResultsThe gene expression level of ACAT1 was significantly lower in CRC than in NCT (P = 2.15e-119). The gene expression level of ACAT2 was not statistically different between CRC and NCT. The gene expression level of LIPA (encoding LAL) was significantly higher in CRC than in NCT (P = 2.01e-14). No data was found for the gene expression level of NCEH1. The IHC score of ACAT1was significantly lower in CRC than in BCT in all studied populations and in sub site of colon, but not in that of rectum. The IHC score of ACAT2 was not statistically different between CRC and BCT. IHC score of NCEH1 was significantly higher in CRC than in BCT only in African American (AA) population. The IHC score of LAL was significantly higher in CRC than in BCT in all studied populations and in all sub sites. In addition, decreased ACAT1 in CRC significantly correlated to progression of CRC: the lower IHC score of ACAT1, the more advanced clinical stage of CRC will be.ConclusionsThis study revealed that altered expression levels in enzymes related to CE metabolism highly correlate to the pathogenesis, clinical progression and disparities of CRC. The results will add revenue in elucidating mechanisms underlying progression of CRC, and shed light on seeking biomarkers and exploring therapeutic targets for CRC in a new direction.

KIAA1363 affects retinyl ester turnover in cultured murine and human hepatic stellate cells

Large quantities of vitamin A are stored as retinyl esters (REs) in specialized liver cells, the hepatic stellate cells (HSCs). To date, the enzymes controlling RE degradation in HSCs are poorly understood. In this study, we identified KIAA1363 (also annotated as arylacetamide deacetylase 1 or neutral cholesterol ester hydrolase 1) as a novel RE hydrolase. We show that KIAA1363 is expressed in the liver, mainly in HSCs, and exhibits RE hydrolase activity at neutral pH. Accordingly, addition of the KIAA1363-specific inhibitor JW480 largely reduced RE hydrolase activity in lysates of cultured murine and human HSCs. Furthermore, cell fractionation experiments and confocal microscopy studies showed that KIAA1363 localizes to the endoplasmic reticulum. We demonstrate that overexpression of KIAA1363 in cells led to lower cellular RE content after a retinol loading period. Conversely, pharmacological inhibition or shRNA-mediated silencing of KIAA1363 expression in cultured murine and human HSCs attenuated RE degradation. Together, our data suggest that KIAA1363 affects vitamin A metabolism of HSCs by hydrolyzing REs at the endoplasmic reticulum, thereby counteracting retinol esterification and RE storage in lipid droplets.

Expression and biological role of the neutral cholesterol ester hydrolase 1 gene in liver cancer tissue and cell lines

Objective To investigate the expression of the neutral cholesterol ester hydrolase 1 (NCEH1) gene in liver cancer tissue and human hepatoma cell lines and the effect of NCEH1 gene knockdown on the proliferation, apoptosis, invasion, and metastasis abilities of human hepatoma SMMC-7721 cells. Methods Liver cancer tissue samples and adjacent tissue samples were collected from 32 patients with liver cancer who underwent surgical treatment in Guangzhou Red Cross Hospital Affiliated to Jinan University from January 2013 to June 2019, and quantitative real-time PCR was used to measure the relative expression level of the NCEH1 gene. Gene expression data of liver cancer samples up to September 2020 were downloaded from the ICGC database, and R software was used to analyze the data and obtain the expression level of the NCEH1 gene in each sample. The paired Wilcoxon signed-rank test and the Wilcoxon rank-sum test were used to investigate the differences between liver cancer tissue and adjacent tissue. Quantitative real-time PCR was used to measure the expression level of the NCEH1 gene in human hepatoma SMMC-7721, Bel-7402, HepG2, and Hep3B cells and normal human HL7702 liver cells. The lentivirus-mediated small interfering RNA (siRNA) technique was used to establish a human hepatoma SMMC-7721 cell line with NCEH1 gene knockdown, and the cells were divided into NCEH1 knockdown group (KD group) and negative control group (NC group); quantitative real-time PCR was used to measure the knockdown efficiency of the NCEH1 gene, and then MTT assay, flow cytometry with Annexin V-APC single staining, wound healing assay, Transwell assay, and Transwell chamber invasion assay were used to measure the proliferation, apoptosis, metastasis, and invasion abilities of SMMC-7721 cells in both groups. The t-test was used for statistical analysis of data between the two groups. Results The mean expression level of the NCEH1 gene in liver cancer tissue was significantly higher than that in adjacent tissue (specimens from our hospital: Z=2.263, P=0.024; ICGC database: U=18 768, P Conclusion There is a significant increase in the expression of the NCEH1 gene in liver cancer tissue and cell lines, and the NCEH1 gene can promote the growth, proliferation, invasion, and metastasis of hepatoma cells and inhibit their apoptosis, suggesting that it may be a potential therapeutic target for liver cancer.

Novel insights: Dynamic foam cells derivedfrom the macrophage in atherosclerosis.

Atherosclerosis can be regarded as a chronic disease derivedfrom the interaction between disordered lipoproteins and an unsuitable immune response. The evolution of foam cells is not only a significant pathological change in the early stage of atherosclerosisbut also a key stagein the occurrence and development of atherosclerosis. The formation of foam cells is mainly caused by the imbalance among lipids uptake, lipids treatment, and reverse cholesterol transport. Although a large number of studies have summarized the source of foam cells and the mechanism of foam cells formation, we propose anew ideaabout foam cells in atherosclerosis. Rather than an isolated microenvironment,the macrophage multiple lipiduptake pathways, lipidinternalization, lysosome, mitochondria, endoplasmic reticulum, neutral cholesterol ester hydrolase (NCEH), acyl-coenzyme A-cholesterol acyltransferase (ACAT), and reverse cholesterol transport are mutually influential, andforma dynamic process under multi-factor regulation. The macrophage takes on different uptake lipidstatuses depending on multiple uptake pathways and intracellular lipids, lipid metabolites versus pro-inflammatory factors. Except for NCEH and ACAT, the lipid internalization of macrophages also depends on multicellular organelles including the lysosome, mitochondria, and endoplasmic reticulum, which are associated with each other. A dynamic balance between esterification and hydrolysis of cholesterol for macrophages isessential for physiology and pathology. Therefore, we propose that the foam cell in the process of atherosclerosis may be dynamicunder multi-factor regulation, and collate thisstudy to provide a holistic and dynamic idea ofthe foam cell.

NAD(P)-dependent steroid dehydrogenase-like protein and neutral cholesterol ester hydrolase 1 serve as novel markers for early detection of gastric cancer identified using quantitative proteomics.

BackgroundGastric cancer (GC) is the third most common cause of cancer deaths worldwide. In the present study, we aimed to identify novel GC biomarkers by integrating isobaric tags of relative and absolute quantitation (iTRAQ) for aberrantly expressed proteins in GC patients.MethodsUsing stable isotope tags, we labeled an initial discovery group comprising four paired gastric cancer and adjacent gastric tissue samples, and subjected them to LC‐ESI‐MS/MS. We used a validation set comprising 129 paired gastric cancer and adjacent gastric tissues from patients and benign healthy controls to validate the candidate targets.ResultsWe identified two proteins, NAD(P)‐dependent steroid dehydrogenase‐like (NSDHL) and neutral cholesterol ester hydrolase 1 (NCEH1), that were significantly overexpressed in GC tissues. The sensitivity and specificity of NSDHL were 80.6% and 74.4%, respectively, in GC compared with a sensitivity of 25.6% in adjacent tissues and 24% in benign healthy controls. The area under the ROC curve (AUC) for NSDHL was 0.810 for GC detection. Overexpression of NSDHL in GC was significantly correlated with local tumor invasion. The sensitivity and specificity of NCEH1 were 77.5% and 73.6%, respectively, in GC compared with a sensitivity of 26.4% in adjacent tissues and 20% in benign controls. The AUC for NSDHL was 0.792. Overexpression of NCEH1 was significantly associated with tumor histological classification and local invasion. Moreover, a combined analysis of NSDHL and NCEH1 achieved a sensitivity and specificity of 85.7% and 83%, respectively, and the AUC was 0.872. The combined analysis of NSDHL and NCEH1 was significantly correlated with histological grade and TNM Ⅱ‐Ⅳ staging.ConclusionsiTRAQ‐labeled quantitative proteomics represents a powerful method to identify novel cancer biomarkers. The present study identified NSDHL and NCEH1 as useful biomarkers for screening, diagnosis, and prognosis of patients with gastric cancer.

2-Hydroxypropyl-β-cyclodextrin reduces retinal cholesterol in wild-type and Cyp27a1-/- Cyp46a1-/- mice with deficiency in the oxysterol production.

2-Hydroxypropyl-β-cyclodextrin (HPCD) is an FDA approved vehicle for drug delivery and an efficient cholesterol-lowering agent. HPCD was proposed to lower tissue cholesterol via multiple mechanisms including those mediated by oxysterols. CYP27A1 and CYP46A1 are the major oxysterol-producing enzymes in the retina that convert cholesterol to 27- and 24-hydroxycholesterol, respectively. We investigated whether HPCD treatments affected the retina of wild-type and Cyp27a1-/- Cyp46a1-/- mice that do not produce the major retinal oxysterols. HPCD administration was either by i.p., p.o. or s.c. Delivery to the retina was confirmed by angiography using the fluorescently labelled HPCD. Effects on the levels of retinal sterols, mRNA and proteins were evaluated by GC-MS, qRT-PCR and label-free approach, respectively. In both wild-type and Cyp27a1-/- Cyp46a1-/- mice, HPCD crossed the blood-retinal barrier when delivered i.p. and lowered the retinal cholesterol content when administered p.o. and s.c. In both genotypes, oral HPCD treatment affected the expression of cholesterol-related genes as well as the proteins involved in endocytosis, lysosomal function and lipid homeostasis. Mechanistically, liver X receptors and the altered expression of Lipe (hormone-sensitive lipase), Nceh1 (neutral cholesterol ester hydrolase 1) and NLTP (non-specific lipid-transfer protein) could mediate some of the HPCD effects. HPCD treatment altered retinal cholesterol homeostasis and is a potential therapeutic approach for the reduction of drusen and subretinal drusenoid deposits, cholesterol-rich lesions and hallmarks of age-related macular degeneration. This article is part of a themed issue on Oxysterols, Lifelong Health and Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.16/issuetoc.

Retinoic acid receptor-related orphan receptor \u03b1 reduces lipid droplets by upregulating neutral cholesterol ester hydrolase 1 in macrophages

BackgroundNeutral cholesterol ester hydrolase 1 (NCEH1) catalyzes the hydrolysis of cholesterol ester (CE) in macrophages. Genetic ablation of NCEH1 promotes CE-laden macrophages and the development of atherosclerosis in mice. Dysregulation of NCEH1 levels is involved in the pathogenesis of multiple disorders including metabolic diseases and atherosclerosis; however, relatively little is known regarding the mechanisms regulating NCEH1. Retinoic acid receptor-related orphan receptor α (RORα)-deficient mice exhibit several phenotypes indicative of aberrant lipid metabolism, including dyslipidemia and increased susceptibility to atherosclerosis.ResultsIn this study, inhibition of lipid droplet formation by RORα positively regulated NCEH1 expression in macrophages. In mammals, the NCEH1 promoter region was found to harbor putative RORα response elements (ROREs). Electrophoretic mobility shift, chromatin immunoprecipitation, and luciferase reporter assays showed that RORα binds and responds to ROREs in human NCEH1. Moreover, NCEH1 was upregulated through RORα via a phorbol myristate acetate-dependent mechanism during macrophage differentiation from THP1 cells. siRNA-mediated knockdown of RORα significantly downregulated NCEH1 expression and accumulated lipid droplets in human hepatoma cells. In contrast, NCEH1 expression and removal of lipid droplets were induced by RORα agonist treatments and RORα overexpression in macrophages.ConclusionThese data strongly suggested that NCEH1 is a direct RORα target, defining potential new roles for RORα in the inhibition of lipid droplet formation through NCEH1.

GPR120 facilitates cholesterol efflux in macrophages through activation of AMPK signaling pathway.

Cholesterol efflux from macrophages is the initial step of reverse cholesterol transport, an important process for high-density lipoprotein-mediated atheroprotection. G protein-coupled receptor (GPR) 120, which functions as long-chain fatty acid receptor, is well known for its anti-inflammatory and insulin-sensitizing function in macrophages. However, the role of GPR120 on macrophage foam cell formation, the hallmark of atherosclerotic plaques, has not been verified. In this study, we found for the first time that stimulation of GPR120 by its agonist GW9508 elevated the expression of ATP-binding cassette transporters (ABC) A1 and ABCG1 in THP-1 macrophage-derived foam cells and Raw264.7 macrophages, and promoted ABCA1- and ABCG1-mediated cholesterol efflux and reduced cellular cholesteryl ester (CE) content as well. In addition, GPR120 activation was accompanied with the stimulation of AMPK pathway in macrophages; however, the effect of GPR120 on macrophage cholesterol efflux was largely abolished by AMPK inhibition. Moreover, the AMPK activity and the expression of ABCA1 and ABCG1 were markedly abrogated by knockdown of GPR120, or application of phospholipase C (PLC) inhibitor, calcium chelator, or CaMKK inhibitor. Because only free cholesterol can be effluxed from macrophages, we found that activation of AMPK could lead to increase both neutral CEs hydrolysis by upregulation of neutral cholesterol ester hydrolase expression and acid CEs hydrolysis by activation of ULK1. In conclusion, these results demonstrated that GPR120 facilitated ABCA1- and ABCG1-mediated cholesterol efflux through activation of PLC/Ca2+ /CaMKK/AMPK signaling pathway, which induced CE hydrolysis and elevated the expression of ABCA1 and ABCG1 in macrophages.

Autoinflammation in addition to combined immunodeficiency: SLC29A3 gene defect

IntroductionH Syndrome is an autosomal recessive (AR) disease caused by defects in SLCA29A3 gene. This gene encodes the equilibrative nucleoside transporter, the protein which is highly expressed in spleen, lymph node and bone marrow. Autoinflammation and autoimmunity accompanies H Syndrome (HS). AimThe aim was to further elucidate the mechanisms of disease by molecular studies in a patient with SLC29A3 gene defect. Patient and MethodsMitochondrial dysfunction, lysosomal integrity, cytokine response in response to stimulation with different pattern recognition receptor ligands, and circulating cell-free mitochondrial-DNA(ccf-mtDNA) level in plasma were analyzed compared to controls to understand the cellular triggers of autoinflammation. RNA sequencing (RS) analyses were also performed in monocytes before/after culture with lipopolysaccharide. ResultsPatient had progressive destructive arthropathy in addition to clinical findings due to combined immunodeficiency. Pure red cell aplasia (PRCA), vitiligo, diabetes, multiple autoantibody positivity, lymphopenia, increased acute phase reactants were present. Recent thymic emigrants (RTE), naïve T cells were decreased, effector memory CD4 + T cells, nonclassical inflammatory monocytes were increased. Patient’s peripheral blood mononuclear cells secreted more IL-1β and IL-6, showed lysosomal disruption and significant mitochondrial dysfunction compared to healthy controls. Plasma ccf-mtDNA level was significantly elevated compared to age-matched controls (p < 0.05). RNA sequencing studies revealed decreased expression of NLR Family Caspase Recrument-Domain Containing 4(NLRC4), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4(PFKFB4), serine dehydratase(SDS), heparan sulfate(Glucosamine) 3-O-sulfotransferase 1(HS3ST1), neutral cholesterol ester hydrolase 1 (NCEH1), and interleukin-8 (IL-8) in patient’s monocytes compared to controls. Longstanding PRCA, which is possibly autoimmune, resolved after initiating monthly intravenous immunoglobulins (IVIG) and low dose steroids to the patient. ConclusionAlthough autoinflammation and autoimmunity are reported in HS, by functional analyses we here show in the present patient that over-active inflammasome pathway in HS might be related with mitochondrial and lysosomal dysfunction. Increased plasma ccf-mtDNA may be used as a biomarker of inflammasomopathy in HS. HS should be included in the classification of primary immunodeficiency diseases.

Abstract 4378: The association of enzymes related to cholesterol esters metabolism with hepatocellular carcinoma

Abstract Objectives: The roles of cholesteryl esters (CE) in the pathogenesis of hepatocellular carcinoma (HCC) have not been completely elucidated. This study is aimed to investigate differences in the expression level of enzymes involving in synthesis and degradation of CEs between benign hapatic tissues (BHT) and HCC, and between male and female patients from African American (AA) and Caucasian American (CA). Methods: TMAs were constructed with 97 BHT and 91 HCC samples from patients enrolled in the University of Mississippi Medical Center from 2010 to 2017. Immunochemistry (IHC) was performed on the TMAs for enzymes involved in CE synthesis (acyl CoA:cholesterol acyltransferase 1, ACAT1 and acyl CoA:cholesterol acyltransferase 2, ACAT2), and in CE degradation (lysosomal acid lipase, LAL and neutral cholesterol ester hydrolase 1, NCEH1) following the manufacturer’s instructions. The expression level for each enzyme was expressed as IHC score calculated with a scoring system. Two-tailed Student’s t-test was used to compare the difference in the expression level of each enzyme between two groups. Significant p value was set at p&amp;lt;0.05. Results: The IHC score of ACAT2 was significantly higher in HCC than in BHT in all population (6.21±2.42 vs (4.89.4±2.40, P=0.0006) and in stratified male population (6.15±2.33 vs 5.08±2.32, p=0.046), but not in stratified women, AA and CA populations. There was no significant difference in IHC score for ACAT1 between HCC and BHT in all population and populations stratified by race and gender. The IHC score of LAL was significantly higher in HCC as compared to BHT in all population (3.57±2.31 vs 2.40±1.30, P =0.0001) and in male population (3.33±2.01 vs 2.39±1.16, p=0.015), but not in stratified women, AA or CA population. The IHC score of NCEH1 was not significantly different between HCC and BHT in all studied population and population stratified by gender. However, it was disparate between AA and CA populations: The IHC score of NCEH1 was significantly lower in HCC than in BHT in AA population (5.59±2.04 vs 6.66±1.74, p=0.031) but it was higher in HCC than in BHT in CA population (6.84±1.96 vs 7.12±1.70, p&amp;gt;0.05). Conclusions: These results suggest that alterations in the expression levels of enzymes involving CE metabolism are associated with pathogenesis, progression and disparities in race and gender of HCC. This study also suggest that ACAT2 predominates CE synthesis as compared to ACAT1, and that LAL plays major role in CE degradation as compared to NCEH1 in association with HCC. Citation Format: Zhirong Liu, Akram Shalaby, Charu Subramony, Jinghe Mao, Xinchun Zhou. The association of enzymes related to cholesterol esters metabolism with hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4378.

Loss of ACAT1 Attenuates Atherosclerosis Aggravated by Loss of NCEH1 in Bone Marrow-Derived Cells.

Aim: Acyl-CoA cholesterol acyltransferase 1 (ACAT1) esterifies free cholesterol to cholesteryl esters (CE), which are subsequently hydrolyzed by neutral cholesterol ester hydrolase 1 (NCEH1). The elimination of ACAT1 in vitro reduces the amounts of CE accumulated in Nceh1-deficient macrophages. The present study aimed at examining whether the loss of ACAT1 attenuates atherosclerosis which is aggravated by the loss of NCEH1 in vivo.Methods: Low density lipoprotein receptor (Ldlr)-deficient mice were transplanted with bone marrow from wild-type mice and mice lacking ACAT1, NCEH1, or both. The four types of mice were fed a high-cholesterol diet and, then, were examined for atherosclerosis.Results: The cross-sectional lesion size of the recipients of Nceh1-deficient bone marrow was 1.6-fold larger than that of the wild-type bone marrow. The lesions of the recipients of Nceh1-deficient bone marrow were enriched with MOMA2-positive macrophages compared with the lesions of the recipients of the wild-type bone marrow. The size and the macrophage content of the lesions of the recipients of bone marrow lacking both ACAT1 and NCEH1 were significantly smaller than the recipients of the Nceh1-deficient bone marrow, indicating that the loss of ACAT1 decreases the excess CE in the Nceh1-deficient lesions. The collagen-rich and/or mucin-rich areas and en face lesion size were enlarged in the recipients of the Acat1−/− bone marrow compared with those of the recipients of the WT bone marrow.Conclusion: The loss of ACAT1 in bone marrow-derived cells attenuates atherosclerosis, which is aggravated by the loss of NCEH1, corroborating the in vitro functions of ACAT1 (formation of CE) and NCEH1 (hydrolysis of CE).

Inhibition of the NLRP3 inflammasome attenuates foam cell formation of THP-1 macrophages by suppressing ox-LDL uptake and promoting cholesterol efflux

The NOD-like receptor family, pyrin domain–containing protein 3 (NLRP3) inflammasome plays an important role in the development of atherosclerosis. The activated NLRP3 inflammasome has been reported to promote macrophage foam cell formation, but not all studies have obtained the same result, and how NLRP3 inflammasome is involved in the formation of foam cells remains elusive. We used selective NLRP3 inflammasome inhibitors and NLRP3-deficient THP-1 cells to assess the effect of NLRP3 inflammasome inhibition on macrophage foam cell formation, oxidized low-density lipoprotein (ox-LDL) uptake, esterification, and cholesterol efflux, as well as the expression of associated proteins. Inhibition of the NLRP3 inflammasome attenuated foam cell formation, diminished ox-LDL uptake, and promoted cholesterol efflux from THP-1 macrophages. Moreover, it downregulated CD36, acyl coenzyme A: cholesterol acyltransferase-1 and neutral cholesterol ester hydrolase expression; upregulated ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression; but had no effect on the expression of scavenger receptor class A and ATP-binding cassette transporter G1. Collectively, our findings show that inhibition of the NLRP3 inflammasome decreases foam cell formation of THP-1 macrophages via suppression of ox-LDL uptake and enhancement of cholesterol efflux, which may be due to downregulation of CD36 expression and upregulation of ABCA1 and SR-BI expression, respectively.

Mechanisms of foam cell formation in atherosclerosis.

Low-density lipoprotein (LDL) and cholesterol homeostasis in the peripheral blood is maintained by specialized cells, such as macrophages. Macrophages express a variety of scavenger receptors (SR) that interact with lipoproteins, including SR-A1, CD36, and lectin-like oxLDL receptor-1 (LOX-1). These cells also have several cholesterol transporters, including ATP-binding cassette transporter ABCA1, ABCG1, and SR-BI, that are involved in reverse cholesterol transport. Lipids internalized by phagocytosis are transported to late endosomes/lysosomes, where lysosomal acid lipase (LAL) digests cholesteryl esters releasing free cholesterol. Free cholesterol in turn is processed by acetyl-CoA acetyltransferase (ACAT1), an enzyme that transforms cholesterol to cholesteryl esters. The endoplasmic reticulum serves as a depot for maintaining newly synthesized cholesteryl esters that can be processed by neutral cholesterol ester hydrolase (NCEH), which generates free cholesterol that can exit via cholesterol transporters. In atherosclerosis, pro-inflammatory stimuli upregulate expression of scavenger receptors, especially LOX-1, and downregulate expression of cholesterol transporters. ACAT1 is also increased, while NCEH expression is reduced. This results in deposition of free and esterified cholesterol in macrophages and generation of foam cells. Moreover, other cell types, such as endothelial (ECs) and vascular smooth muscle cells (VSMCs), can also become foam cells. In this review, we discuss known pathways of foam cell formation in atherosclerosis.

NCEH-1 modulates cholesterol metabolism and protects against α-synuclein toxicity in a C. elegans model of Parkinson's disease.

Parkinson's disease (PD) is an aging-associated neurodegenerative disease affecting millions worldwide. Misfolding, oligomerization and accumulation of the human α-synuclein protein is a key pathological hallmark of PD and is associated with the progressive loss of dopaminergic neurons over the course of aging. Lifespan extension via the suppression of IGF-1/insulin-like signaling (IIS) offers a possibility to retard disease onset through induction of metabolic changes that provide neuroprotection. The nceh-1 gene of Caenorhabditis elegans encodes an ortholog of neutral cholesterol ester hydrolase 1 (NCEH-1), an IIS downstream protein that was identified in a screen as a modulator of α-synuclein accumulation in vivo. The mechanism whereby cholesterol metabolism functionally impacts neurodegeneration induced by α-synuclein is undefined. Here we report that NCEH-1 protects dopaminergic neurons from α-synuclein-dependent neurotoxicity in C. elegans via a mechanism that is independent of lifespan extension. We discovered that the presence of cholesterol, LDLR-mediated cholesterol endocytosis, and cholesterol efflux are all essential to NCEH-1-mediated neuroprotection. In protecting from α-synuclein neurotoxicity, NCEH-1 also stimulates cholesterol-derived neurosteroid formation and lowers cellular reactive oxygen species in mitochondria. Collectively, this study augments our understanding of how cholesterol metabolism can modulate a neuroprotective mechanism that attenuates α-synuclein neurotoxicity, thereby pointing toward regulation of neuronal cholesterol turnover as a potential therapeutic avenue for PD.

Convergent Signaling Pathways Controlled by LRP1 (Receptor-related Protein 1) Cytoplasmic and Extracellular Domains Limit Cellular Cholesterol Accumulation

The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (α) chain of LRP1 mediates TGFβ-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (β) chain of LRP1 suffices to limit cholesterol accumulation in LRP1(-/-) cells. Through binding of Erk2 to the second of its carboxyl-terminal NPXY motifs, LRP1 β-chain positively regulates the expression of ATP binding cassette transporter A1 (ABCA1) and of neutral cholesterol ester hydrolase (NCEH1). These results highlight the unexpected functions of LRP1 and the canonical Wnt5a pathway and new therapeutic potential in cholesterol-associated disorders including cardiovascular diseases.

Counteractive effects of omentin-1 against atherogenesis†.

Omentin-1, a novel adipocytokine expressed in visceral fat tissue, is negatively correlated with obesity, insulin resistance, and stable coronary artery disease (CAD). However, there have been no previous reports regarding the effects of omentin-1 on atherogenesis. This study was performed to evaluate the atheroprotective effects of omentin-1 on human monocyte-derived macrophages, human aortic smooth muscle cells (HASMCs) in vitro, and aortic lesions in Apoe(-/-) mice in vivo. The histological expression of omentin-1 in coronary artery lesions and epicardial adipose tissues and its plasma levels were compared between CAD and non-CAD patients. Omentin-1 was abundantly expressed in human umbilical vein endothelial cells, macrophages, HASMCs, and human coronary artery SMCs in vitro. Omentin-1 promoted anti-inflammatory M2 phenotype during differentiation of human monocytes into macrophages. Omentin-1 suppressed oxidized low-density lipoprotein-induced foam cell formation associated with down-regulation of CD36, scavenger receptor class A, and acyl-CoA:cholesterol acyltransferase-1 and up-regulation of neutral cholesterol ester hydrolase in human macrophages. Omentin-1 suppressed angiotensin II-induced migration and platelet-derived growth factor-BB-induced proliferation, and collagen-1 and -3 expression in HASMCs. Four-week infusion of omentin-1 into Apoe(-/-) mice retarded the development of aortic atherosclerotic lesions with reduced contents of monocytes/macrophages, SMCs, and collagen fibres along with peritoneal M2-activated macrophages with inflammasome down-regulation and lowered plasma total cholesterol levels. Omentin-1 levels were markedly reduced in coronary endothelium and epicardial fat but increased in plasma and atheromatous plaques (macrophages/SMCs) in CAD patients compared with non-CAD patients. This study provided the first evidence that omentin-1 may serve as a novel therapeutic target for atherosclerosis and CAD.

Psoriasis Skin Inflammation-Induced microRNA-26b Targets NCEH1 in Underlying Subcutaneous Adipose Tissue

Psoriasis is an immune-mediated inflammatory disease, which is associated with a high risk of developing systemic comorbidities, such as obesity, cardiovascular disease, and diabetes mellitus. However, the mechanistic links between psoriatic skin inflammation and systemic comorbidities remain largely unknown. MicroRNAs (miRNAs) are recently discovered gene regulators that play important roles in psoriasis skin inflammation. In this study we aimed to explore whether the skin inflammation in psoriasis affects miRNA expression of the underlying subcutaneous adipose tissue and whether this may be a link between psoriasis and comorbidities. To this end, we compared the miRNA expression profile of subcutaneous adipose tissue underneath lesional and nonlesional psoriatic skin. We further validated the differential expression of several miRNAs and characterized their expression patterns in different cell types present in subcutaneous adipose tissue. We focused on miR-26b-5p, which was highly up-regulated in subcutaneous adipose tissue underneath lesional psoriasis skin. We showed that it targets and down-regulates neutral cholesterol ester hydrolase 1, an enzyme essential for cholesterol efflux, in monocytes/macrophages, adipocytes, vascular endothelial cells, and fibroblasts. We conclude that this miRNA may serve as a mechanistic link between psoriatic skin inflammation and its systemic comorbidities.

Abstract 9742: Counteractive Effects of Omentin-1 Against Atherogenesis

Introduction: Omentin-1, a novel adipocytokine expressed in visceral fat tissue, is negatively correlated with obesity, insulin resistance, and stable coronary artery disease (CAD). However, there have been no previous reports regarding the effects of omentin-1 on atherogenesis. Objective and Methods: We aimed to evaluate the atheroprotective effects of omentin-1 on human monocyte-derived macrophages, human aortic smooth muscle cells (HASMCs) in vitro , and aortic lesions in apolipoprotein E-deficient (ApoE -/- ) mice in vivo . The histological expression of omentin-1 in coronary artery lesions and epicardial adipose tissues and its plasma levels were compared between CAD and non-CAD patients. Results: Omentin-1 was abundantly expressed in human umbilical vein endothelial cells, macrophages, HASMCs, and human coronary artery SMCs in vitro . Omentin-1 promoted antiinflammatory M2 phenotype via PPAR-γ upregulation and NF-κB downregulation during differentiation of human monocytes into macrophages. Omentin-1 suppressed oxidized LDL-induced foam cell formation associated with downregulation of CD36, scavenger receptor class A, and acyl-CoA:cholesterol acyltransferase-1 and upregulation of neutral cholesterol ester hydrolase in human macrophages. In HASMCs, omentin-1 suppressed angiotensin II-induced migration and PDGF-BB-induced proliferation, and collagen-1 and collagen-3 expressions via ERK1/2 and NF-κB downregulation. Four-week infusion of omentin-1 into ApoE -/- mice retarded the development of aortic atherosclerotic lesions with reduced contents of monocytes/macrophages, SMCs, and collagen fibers. In addition, it increased peritoneal M2-activated macrophages, downregulated inflammasomes such as CRP, NF-κB, COX-2, and apoptosis-associated speck-like protein containing a caspase recruitment domain, and lowered plasma total cholesterol levels in ApoE -/- mice. Omentin-1 levels were markedly reduced in coronary endothelium and epicardial fat but increased in plasma and atheromatous plaques (macrophages/SMCs) in CAD patients compared with non-CAD patients. Conclusions: Our translational research provided the first evidence that omentin-1 may serve as a novel therapeutic target for atherosclerosis and CAD.