ATP-binding Cassette Subfamily A Member 1 Research Articles

Macrophage miR-34a Is a Key Regulator of Cholesterol Efflux and Atherosclerosis.

Macrophages play a crucial role in the pathogenesis of atherosclerosis, but the molecular mechanisms remain poorly understood.Here we show that microRNA-34a (miR-34a) is a key regulator of macrophage cholesterol efflux and reverse cholesterol transport by modulating ATP-binding cassette transporters ATP-binding cassette subfamily A member 1 (ABCA1) and ATP-binding cassette subfamily G member 1 (ABCG1). miR-34a also regulates M1 and M2 macrophage polarizationvia liver X receptor α. Furthermore, global loss of miR-34a reduces intestinal cholesterol or fat absorption by inhibiting cytochrome P450 enzymes CYP7A1 and sterol 12α-hydroxylase (CYP8B1). Consistent with these findings, macrophage-selective or global ablation of miR-34a markedly inhibits the development of atherosclerosis. Finally, therapeutic inhibition of miR-34a promotes atherosclerosis regression and reverses diet-induced metabolic disorders. Our studies outline a central role of miR-34a in regulating macrophage cholesterol efflux, inflammation, and atherosclerosis, suggesting that miR-34a is a promising target for treatment of cardiometabolic diseases.

Prenatal ethanol exposure-induced a low level of foetal blood cholesterol and its mechanism of IGF1-related placental cholesterol transport dysfunction

Clinical researches showed that intrauterine growth retardation (IUGR) foetus had decreased blood cholesterol levels. The present study aimed to confirm that prenatal ethanol exposure (PEE) caused decreased blood cholesterol levels in IUGR foetal rats and elucidate its placental mechanism. Pregnant Wistar rats were intragastrically administrated with ethanol (4 g/kg.d) on gestational days 9–20 (GD9–20). in vivo, PEE increased the levels of total cholesterol (TCH), high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) in maternal serum, whereas decreased them in both female and male foetal serum. Moreover, the expression of cholesterol transport genes, scavenger receptor class B type 1 (SCARB1), low-density lipoprotein receptor (LDLR), ATP binding cassette subfamily A member 1 (ABCA1) and ATP binding cassette subfamily G member 1 (ABCG1) was reduced in female and male placentas in the PEE group. Meanwhile, the proliferation decreased and the apoptosis increased in female and male placentas, and the insulin like growth factor 1 (IGF1) signal pathway was inhibited. in vitro, after being treated with ethanol (15, 30, 60, 120 mM) for 72 h, the expression of cholesterol transport genes was decreased, the apoptosis was increased, the proliferation was decreased and the IGF1 signal pathway was inhibited in BeWo cells, whereas exogenous IGF1 reversed these changes. In conclusion, by inhibiting the IGF1 signal pathway in placentas, PEE induced apoptosis and inhibited proliferation, thus decreased the cholesterol transport in placentas, and eventually leading to low blood cholesterol levels in foetal rats.

ATP-binding cassette sub-family a member1 gene mutation improves lipid metabolic abnormalities in diabetes mellitus

BackgroundPatients with diabetes mellitus were often accompanied with hyperlipidemia. ATP-binding cassette sub-family A member1 (ABCA1) promotes the efflux of lipids and thereby mediates the metabolism of cholesterol. The aim of our study was to determine the associations of ABCA1 gene polymorphisms with the risks of diabetes mellitus and dyslipidemia in diabetic patients.MethodsWe retrieved literature about the relationship between ABCA1 gene polymorphisms (C69T and R230C) and the risk of diabetes through PubMed, Web of Science, EMBASE, Wanfang Database, China National Knowledge Infrastructure (CNKI) and Cochrane database. Weighted mean difference (WMD) and odds ratio (OR) were used to compare continuous and dichotomous variables, respectively, accompanied by their 95% confidence interval (CI).ResultsA total of 1746 diabetic patients and 1292 non-diabetic controls were enrolled. All subjects were Caucasians. ABCA1 R230C T allele was significantly associated with reduced the risk of diabetes (OR = 0.75, 95% CI = 0.57–0.98, P = 0.04). There was no association of ABCA1 C69T gene polymorphisms with the risk of diabetes. However, subgroup analyses showed that the ABCA1 C69T gene mutation significantly reduced the risk of hypertriglyceridemia in diabetic patients as compared with that in non-diabetic subjects (dominant model: WMD =0.66, 95% CI = 0.52–0.8, P < 0.0001; recessive model: WMD = 0.47, 95%CI = 0.11–0.83, P = 0.01).ConclusionsABCA1 R230C T allele gene mutation is a protective in decreasing the risk of diabetes in Caucasians and ABCA1 C69T gene mutation markedly influences the level of lipid metabolism in diabetic patients.

Potentiation of mitotane action by rosuvastatin: New insights for adrenocortical carcinoma management

Mitotane (also termed o,p'‑DDD) is the most effective therapy for advanced adrenocortical carcinoma(ACC). Mitotane‑induced dyslipidemia is treated with statins. Mitotane and statins are known to exert anti‑proliferative effects invitro; however, the effects of statins have never been directly evaluated in patients with ACC and ACC cells, at least to the best of our knowledge. Thus, in this study, we aimed to examine the effects of the rosuvastatin on ACC cells. It has been shown that the combined use of mitotane and statins significantly increases the tumor control rate in patients with ACC; however, it would be of interest to elucidate the molecular mechanisms involved in this potentiation. In this study, we examined the effects of mitotane, rosuvastatin and their combination in NCI‑H295R human ACC cells using proliferation assays, gene expression analyses and free intracellular cholesterol measurements. The results revealed that mitotane dose‑dependently reduced cell viability, induced apoptosis and increased intracellular free cholesterol levels, considered as one of the key features of mitotane action, while rosuvastatin alone reduced cell viability and increased apoptosis at high concentrations. We also demonstrated that rosuvastatin potentiated the effects of mitotane by reducing cell viability, inducing apoptosis, increasing intracellular free cholesterol levels, and by decreasing the expression of 3‑hydroxy‑3‑methylglutaryl‑CoA reductase (HMGCR) and ATP binding cassette subfamily a member1 (ABCA1), genes involved in cholesterol metabolism, and inhibiting steroidogenesis. Collectively, potentiating the effects of mitotane with the use of rosuvastatin may provide novel therapeutic strategies for ACC, given that the combination of these drugs, pending clinical validation, may lead to the better management of ACC.

MicroRNA-106b Regulates Milk Fat Metabolism via ATP Binding Cassette Subfamily A Member 1 ( ABCA1) in Bovine Mammary Epithelial Cells.

Research on the mechanisms that regulate milk fat synthesis in dairy cows is essential to identify potential molecular targets that in the long term can help develop appropriate molecular breeding programs. Although some studies have revealed that microRNA (miRNA) affect lipid metabolism by targeting specific genes, joint analysis of miRNA and target mRNA data from bovine mammary tissue has revealed few clues regarding the underlying mechanisms controlling milk fat synthesis. The objective of the present study was to use high-throughput sequencing and bioinformatics analysis to identify miRNA and mRNA pairs and explore further their potential roles in regulating milk fat synthesis. A total of 233 pairs of negatively associated miRNA and mRNA pairs were detected. Among those, there were 162 pairs in which the miRNAs were down-regulated and the target mRNAs were up-regulated. Among the identified miRNA, miR-106b can bind the 3'-UTR of the ATP binding cassette subfamily A member 1 ( ABCA1), a gene previously identified as having a positive association with bovine milk fat synthesis. The overexpression of miR-106b in bovine mammary epithelial cells caused a decrease in triglyceride and cholesterol content while the inhibition of miR-106b increased triglyceride and cholesterol content, confirming its role in lipid metabolism. The present study allowed for the construction of a miR-106b- ABCA1 regulatory network map, thus providing a theoretical basis to target this gene in the molecular breeding of dairy cows.

Associations of Genetic Variations in ABCA1 and Lifestyle Factors with Coronary Artery Disease in a Southern Chinese Population with Dyslipidemia: A Nested Case-Control Study.

Coronary artery disease has become a major health concern over the past several decades. We aimed to explore the association of single nucleotide polymorphisms (SNPs) in the ATP-binding cassette subfamily A member 1 (ABCA1) and lifestyle factors with coronary artery disease (CAD) in dyslipidemia. This nested case-control study included 173 patients with CAD and 500 matched control individuals (1:3, case: control) from a district in southern China. We collected medical reports, lifestyle details, and blood samples of individuals with dyslipidemia and used the polymerase chain reaction-ligase detection reaction method to genotype the SNPs. The CC genotype of the additive and recessive models of rs4149339, together with regular intake of fried foods or dessert, increased the risk of CAD (adjusted odd ratio (OR) = 1.91, p = 0.030; adjusted OR = 1.97, p = 0.017; adjusted OR = 1.80, p = 0.002; adjusted OR = 1.98, p = 0.001). The AT + AA genotype of the dominant model of rs4743763 and moderate/heavy physical activity reduced the risk of CAD (adjusted OR = 0.66, p = 0.030; adjusted OR = 0.44, p = 0.001). The CT + CC genotype of the dominant model of rs2472386 reduced the risk of CAD only in males (adjusted OR = 0.36, p = 0.001). The interaction between rs4149339 and rs4743763 of ABCA1 and haplotype CTT (comprising rs4149339, rs4743763, and rs2472386) appeared to increase the risk of CAD (relative excess risk due to interaction (RERI) = 3.19, p = 0.045; OR = 1.49, p = 0.019). Polymorphisms of rs4149339, rs4743763 and rs2472386 in ABCA1 and three lifestyle factors (physical activity, fried food intake, and dessert intake) were associated with CAD in people with dyslipidemia in southern China. These results provide the theoretical basis for gene screening and the prevention of chronic cardiovascular diseases.

Prostaglandin A1 Inhibits the Cognitive Decline of APP/PS1 Transgenic Mice via PPARγ/ABCA1-dependent Cholesterol Efflux Mechanisms.

Prostaglandins (PGs) are early and key contributors to chronic neurodegenerative diseases. As one important member of classical PGs, PGA1 has been reported to exert potential neuroprotective effects. However, the mechanisms remain unknown. To this end, we are prompted to investigate whether PGA1 is a useful neurological treatment for Alzheimer's disease (AD) or not. Using high-throughput sequencing, we found that PGA1 potentially regulates cholesterol metabolism and lipid transport. Interestingly, we further found that short-term administration of PGA1 decreased the levels of the monomeric and oligomeric β-amyloid protein (oAβ) in a cholesterol-dependent manner. In detail, PGA1 activated the peroxisome proliferator-activated receptor-gamma (PPARγ) and ATP-binding cassette subfamily A member 1 (ABCA1) signalling pathways, promoting the efflux of cholesterol and decreasing the intracellular cholesterol levels. Through PPARγ/ABCA1/cholesterol-dependent pathway, PGA1 decreased the expression of presenilin enhancer protein 2 (PEN-2), which is responsible for the production of Aβ. More importantly, long-term administration of PGA1 remarkably decreased the formation of Aβ monomers, oligomers, and fibrils. The actions of PGA1 on the production and deposition of Aβ ultimately improved the cognitive decline of the amyloid precursor protein/presenilin1 (APP/PS1) transgenic mice.

Persistent Neuroadaptations in the Expression of Genes Involved in Cholesterol Homeostasis Induced by Chronic, Voluntary Alcohol Intake in Rats.

Alcohol use disorder (AUD) is associated with persistent adaptations in the brain that are believed to participate in the long-lasting vulnerability to relapse after abstinence. Cholesterol, the major sterol compound found in the central nervous system (CNS), plays a major role in maintenance of neuronal morphology, synaptogenesis and synaptic communication and may be involved in alcohol-induced neuroadaptations. In this study, we investigated whether alcohol consumption in a two-bottle choice paradigm followed by 3 weeks of abstinence could alter the expression of genes encoding proteins involved in cholesterol homeostasis in brain regions involved in addiction and relapse, namely the prefrontal cortex (PFC), the nucleus accumbens (NAc), the mesencephalon and the amygdala. We found that voluntary alcohol intake followed by 3 weeks of forced abstinence produces changes in the transcription of several genes encoding proteins directly involved in cholesterol synthesis such as 3-hydroxyl-3-methylglutaryl-coenzyme A (HMGCoA) reductase, farnesyl-diphosphate farnesyltransferase 1 (FDFT1) and farnesyl diphosphate synthase (FDPS) and in its regulation such as sterol regulatory element-binding factor-2 (SREBF2), in cholesterol transport such as ATP-binding cassette subfamily A member 1 (ABCA1) and in cholesterol degradation such as CYP46A1. Interestingly, these changes appeared to be region-specific and suggest that previous chronic exposure to alcohol might durably increase cholesterol metabolism in the PFC, the NAc and the mesencephalon and decrease cholesterol metabolism in the amygdala. Altogether, these results suggest that alcohol consumption leads to durable deregulations in cholesterol metabolism in key areas involved in loss of control over drug use and addiction. These long-term neuroadaptations may participate in the changes in brain structure and functioning that are responsible for the long-lasting risks of relapse to alcohol.

ABCA1 Overexpression in Endothelial Cells In Vitro Enhances ApoAI-Mediated Cholesterol Efflux and Decreases Inflammation.

Atherosclerosis, a disease of blood vessels, is driven by cholesterol accumulation and inflammation. Gene therapy that removes cholesterol from blood vessels and decreases inflammation is a promising approach for prevention and treatment of atherosclerosis. In previous work, we reported that helper-dependent adenoviral (HDAd) overexpression of apolipoprotein A-I (apoAI) in endothelial cells (ECs) increases cholesterol efflux in vitro and reduces atherosclerosis in vivo. However, the effect of HDAdApoAI on atherosclerosis is partial. To improve this therapy, we considered concurrent overexpression of ATP-binding cassette subfamily A, member 1 (ABCA1), a protein that is required for apoAI-mediated cholesterol efflux. Before attempting combined apoAI/ABCA1 gene therapy, we tested whether an HDAd that expresses ABCA1 (HDAdABCA1) increases EC cholesterol efflux, whether increased cholesterol efflux alters normal EC physiology, and whether ABCA1 overexpression in ECs has anti-inflammatory effects. HDAdABCA1 increased EC ABCA1 protein (∼3-fold; p < 0.001) and apoAI-mediated cholesterol efflux (2.3-fold; p = 0.007). Under basal culture conditions, ABCA1 overexpression did not alter EC proliferation, metabolism, migration, apoptosis, nitric oxide production, or inflammatory gene expression. However, in serum-starved, apoAI-treated EC, ABCA1 overexpression had anti-inflammatory effects: decreased inflammatory gene expression (∼50%; p ≤ 0.02 for interleukin [IL]-6, tumor necrosis factor [TNF]-α, and vascular cell adhesion protein-1); reduced lipid-raft Toll-like receptor 4 (80%; p = 0.001); and a trend towards increased nitric oxide production (∼55%; p = 0.1). In ECs stimulated with lipopolysaccharide, ABCA1 overexpression markedly decreased inflammatory gene expression (∼90% for IL-6 and TNF-α; p < 0.001). Therefore, EC ABCA1 overexpression has no toxic effects and counteracts the two key drivers of atherosclerosis: cholesterol accumulation and inflammation. In vivo testing of HDAdABCA1 is warranted.

MicroRNA-217 attenuates intima\u2013media complex thickness of ascending aorta measured by ultrasound bio-microscopy and inhibits inflammation and lipid metabolism in atherosclerotic models of ApoE\u2212/\u2212 mice

BackgroundLittle investigation was done to test the efficiency of microRNA-217 (miR-217) on atherosclerosis in vivo.MethodsApoE−/− mice were used to construct atherosclerotic models and ultrasound bio-microscopy (UBM) was applied to detect the intima–media thickness (IMT) of the ascending aorta. The serum level of miR-217 and correlation with IMT was investigated. After miR-217 mimic administration, the IMT, inflammation, and lipid-associated molecules were assayed.ResultsThe serum level of miR-217 was reduced in ApoE−/− mice and showed a negative correlation with the IMT of the ascending aorta (r2 = 0.5899, p < 0.0001). miR-217 mimic administration attenuated IMT and down-regulated the level of serum triglyceride (TG), total cholesterol (TC), and low-density-lipoprotein cholesterol (LDL-C), while it could up-regulate high-density lipoprotein cholesterol (HDL-C). Inflammation relevant genes, such as F4/80, tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, and monocyte chemoattractant protein (MCP)-1, and lipid metabolism associated gene, such as LDL receptor, class A scavenger receptors (SR-A), scavenger receptor class B type I (SR-BI), CD36, ATP binding cassette subfamily A member 1 (ABCA1), and ATP binding cassette subfamily G member 1 (ABCG1) in the aorta were significantly down-regulated in miR-217 group when compared with atherosclerosis group.ConclusionmiR-217 could down-regulate IMT and modulate the inflammation and lipid metabolism process, which indicates that miR-217 could be a potential treatment option.

Association Between Four ABCA1 gene Polymorphisms and Risk of Non-Alcoholic Fatty Liver Disease in a Chinese Han Population

Background: Non-alcoholic fatty liver disease (NAFLD) as a severe health problem is the leading cause of morbidity and mortality from the chronic liver disease worldwide. NAFLD is tightly associated with dyslipidemia although the etiology is still unclear. ATP binding cassette subfamily A member 1 (ABCA1) is involved in cholesterol efflux, fatty acid oxidation, and inflammation. Although some reports show that the ABCA1 polymorphisms affect the lipids metabolism and severity of clinical liver diseases, the effects of ABCA1 polymorphisms on the development of NAFLD are unknown. Objectives: The current study was performed to investigate the association between the ABCA1 polymorphisms and the development of NAFLD and the effect of the four ABCA1 SNPs on the serum lipid levels. Methods: The ABCA1 polymorphisms (rs1800977, rs2066714, rs2066715, and rs2230808) were determined in 265 NAFLD patients and 126 healthy controls using the sequencing and polymerase chain reaction analysis. Serum lipid profiles and liver enzymes were examined using standard clinical laboratory methods. Results: There was a significant difference (P < 0.05) in the genotype of the ABCA1 rs1800977 G/A polymorphisms between NAFLD patients and healthy controls. No significant differences were found in genotypes and allele frequencies of the ABCA1 rs2066714T/C, rs2066715T/C, and rs2230808C/T between NAFLD patients and healthy controls. The ABCA1 rs1800977 A was independently associated with NAFLD after adjusting for the effects of age, gender, and BMI. Compared to the noncarriers in NAFLD patients, the carriers of ABCA1 rs2066714 C showed a significantly higher level of LDL (P = 0.045) and the carriers of ABCA1 rs2230808 T showed a significantly lower level of HDL (P = 0.039). Conclusions: We first demonstrated the association between the ABCA1 polymorphisms and the risk of NAFLD in a Chinese Han population. The ABCA1 rs1800977 may be a protective factor against the development of NAFLD. The ABCA1 rs2066714 C allele could increase the serum LDL cholesterol level, and the ABCA1 rs2230808 T allele could decrease the serum HDL cholesterol level in NAFLD patients.

Tangshen Formula Attenuates Diabetic Nephropathy by Promoting ABCA1-Mediated Renal Cholesterol Efflux in db/db Mice.

The commonly prescribed Tangshen Formula (TSF) is a traditional Chinese formulation that has been shown to reduce plasma lipid metabolism and proteinuria and improve the estimated glomerular filtration rate (eGFR) in patients with diabetic kidney disease. This study investigated the underlying mechanism whereby TSF regulates renal lipid accumulation and ameliorates diabetic renal injuries in spontaneous diabetic db/db mice and in vitro in sodium palmitate (PA)-stimulated and Abca1-SiRNA-transfected mouse tubular epithelial cells (mTECs). The results revealed that TSF treatment significantly ameliorated the renal injuries by lowering urinary albumin excretion and improving renal tissue injuries in diabetic (db/db) mice. Interestingly, the treatment with TSF also resulted in decreased cholesterol levels in the renal tissues of db/db mice, which was associated with increased expression of the peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), the Liver X receptors (LXR), and ATP-binding cassette subfamily A member 1 (ABCA1), suggesting that TSF might attenuate diabetic kidney injury via a mechanism associated with improving cholesterol efflux in the diabetic kidney. This was investigated in vitro in mTECs, and the results showed that TSF reduced the PA-stimulated cholesterol accumulation in mTECs. Mechanistically, the addition of TSF was capable of reversing PA-induced downregulation of PGC-1α, LXR, and ABCA1 expression and cholesterol accumulation in mTECs, suggesting that TSF might act the protection via the PGC-1α-LXR-ABCA1 pathway to improve the cholesterol efflux in the renal tissues of db/db mice. This was further confirmed by silencing ABCA1 to block the promotive effect of TSF on cholesterol efflux in vitro. In conclusion, TSF might ameliorate diabetic kidney injuries by promoting ABCA1-mediated renal cholesterol efflux.

Efatutazone and T0901317 exert synergistically therapeutic effects in acquired gefitinib-resistant lung adenocarcinoma cells.

The development of acquired EGFR‐TKI therapeutic resistance is still a serious clinical problem in the management of lung adenocarcinoma. Peroxisome proliferator activated receptor gamma (PPAR γ) agonists may exhibit anti‐tumor activity by transactivating genes which are closely associated with cell proliferation, apoptosis, and differentiation. However, it remains not clear whether efatutazone has similar roles in lung adenocarcinoma cells of gefitinib resistant such as HCC827‐GR and PC9‐GR. It has been demonstrated by us that efatutazone prominently increased the mRNA and protein expression of PPAR γ, liver X receptor alpha (LXR α),as well as ATP binding cassette subfamily A member 1 (ABCA1). In the presence of GW9662 (a specific antagonist of PPAR γ) or GGPP (a specific antagonist of LXR α), efatutazone (40 μmol/L) restored the proliferation of both HCC827‐GR and PC9‐GR cells and obviously inhibited the increased protein and mRNA expression of PPAR‐gamma, LXR‐alpha, and ABCA1 induced by efatutazone. LXR α knockdown by siRNA (si‐LXR α) significantly promoted the HCC827‐GR and PC9‐GR cells proliferation, whereas incubation efatutazone with si‐LXR α restored the proliferation ability compared with the control group. In addition, combination of efatutazone and LXR α agonist T0901317 showed a synergistic therapeutic effect on lung adenocarcinoma cell proliferation and PPAR gamma, LXR A and ABCA1 protein expression. These results indicate that efatutazone could inhibit the cells proliferation of HCC827‐GR and PC9‐GR through PPAR γ/LXR α/ABCA1 pathway, and synergistic therapeutic effect is achieved when combined with T0901317.

Abstract P1-10-09: EPHA2-targeting enhances eicosapentaenoic acid cytotoxicity against triple-negative inflammatory breast cancer via ABCA1 inhibition–mediated membrane rigidity

Abstract Background: Effective treatment options for triple-negative inflammatory breast cancer (TN-IBC), the most aggressive form of breast cancer, are currently lacking. We previously reported that mediators of inflammation promote the growth of TN-IBC xenografts. Eicosapentaenoic acid (EPA), an omega-3 fatty acid (fish oil) with anti-inflammatory properties, is an emerging FDA-approved therapeutic with a favorable toxicology profile. Here we aimed to develop a novel approach to enhance EPA efficacy against TN-IBC by identifying a kinase inhibitor that synergizes with EPA's antitumor activity. Methods and Results: Using a high-throughput siRNA screen in the TN-IBC cell line SUM149PT, we identified inhibition of ephrin type-A receptor 2 (EPHA2), an oncogenic receptor tyrosine kinase, as a target that sensitizes TN-IBC cells to EPA therapy. To determine the clinical relevance of EPHA2, we investigated a meta-analysis of breast cancer mRNA expression data sets and found that high EPHA2 tumor expression, compared with low expressing, correlated significantly with poor overall survival in TN-IBC patients (P = 0.01), while not with other subtypes. Similar findings were observed in vitro, were EPHA2 protein and mRNA overexpression occurred predominantly in the TN subtypes among 49 and 51 breast cancer cell lines (63% and 47%, respectively), highlighting EPHA2 translational potential. Functional expression studies using proliferation and apoptosis assays in vitro, and xenografts in vivo, were performed in two EPHA2-expressing TN-IBC cell lines, SUM149PT and BCX010, to validate EPHA2 as a synergistic combinational target with EPA. EPHA2 gene silencing in combination with EPA significantly reduced cell growth, and enhanced apoptosis, compared with untreated and monotherapy in vitro (P &amp;lt; 0.05), and in vivo (P &amp;lt; 0.001). To translate our findings to the clinic, we validated dasatinib, an FDA-approved small molecule inhibitor of EPHA2, in combination to EPA to significantly enhance apoptosis of TN-IBC cells in vitro (P &amp;lt; 0.05) and in vivo (P &amp;lt; 0.05), compared with untreated and monotherapies. Using membrane fluidity assessment and cholesterol quantification we determined that apoptosis induction after combination therapy was due to increased membrane rigidity and cholesterol concentrations in the plasma membrane of TN-IBC cells (P &amp;lt; 0.05, compared with monotherapies). Finally, we discovered by western blot and gain/loss-of-expression studies that combination therapy inhibited the cholesterol efflux protein ATP-binding cassette sub-family A member 1 (ABCA1), which plays a significant role mediating increased cellular cholesterol (P &amp;lt; 0.05), cell membrane rigidity (P &amp;lt; 0.05), and induction of apoptosis (P &amp;lt; 0.05) in TN-IBC after EPA and EPHA2-targeting combination therapy. Conclusions: This is the first study demonstrating that EPA can enhance conventional targeted therapy against breast cancer. Our study provides molecular and preclinical evidence to support the development of an EPA/EPHA2-inhibition–based phase I clinical trial for patients with EPHA2-positive TN-IBC; our study further suggests the use of EPHA2 and ABCA1 protein expression as biomarkers for patient selection and therapeutic response. Citation Format: Torres-Adorno AM, Vitrac H, Qi Y, Tan L, Levental KR, Fan Y-Y, Yang P, Chapkin RS, Eckhardt BL, Ueno NT. EPHA2-targeting enhances eicosapentaenoic acid cytotoxicity against triple-negative inflammatory breast cancer via ABCA1 inhibition–mediated membrane rigidity [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-09.

Differences between the Molecular Mechanisms Underlying Ruptured and Non-Ruptured Carotid Plaques, and the Significance of ABCA1.

Background and Purpose Carotid plaques are a strong risk factor for ischemic stroke, and plaque rupture poses an even higher risk. Although many studies have investigated the pathogenic mechanisms of carotid plaque formation, few have studied the differences in molecular mechanisms underlying the rupture and non-rupture of carotid plaques. In addition, since early diagnosis and treatment of carotid plaque rupture are critical for the prevention of ischemic stroke, many studies have sought to identify the important target molecules involved in the rupture. However, a target molecule critical in symptomatic ruptured plaques is yet to be identified.Methods A total of 79 carotid plaques were consecutively collected, and microscopically divided into ruptured and non-ruptured groups. Quantitative polymerase chain reaction array, proteomics, and immunohistochemistry were performed to compare the differences in molecular mechanisms between ruptured and non-ruptured plaques. Enzyme-linked immunosorbent assay was used to measure the differences in ATP-binding cassette subfamily A member 1 (ABCA1) levels in the serum.Results The expression of several mRNAs and proteins, including ABCA1, was higher in ruptured plaques than non-ruptured plaques. In contrast, the expression of other proteins, including β-actin, was lower in ruptured plaques than non-ruptured plaques. The increased expression of ABCA1 was consistent across several experiments, ABCA1 was positive only in the serum of patients with symptomatic ruptured plaques.Conclusions This study introduces a plausible molecular mechanism underlying carotid plaque rupture, suggesting that ABCA1 plays a role in symptomatic rupture. Further study of ABCA1 is needed to confirm this hypothesis.

Global inactivation of carboxylesterase 1 (Ces1/Ces1g) protects against atherosclerosis in Ldlr\u2212/\u2212 mice

Atherosclerotic cardiovascular disease is a leading cause of death in the western world. Increased plasma triglyceride and cholesterol levels are major risk factors for this disease. Carboxylesterase 1 (Ces1/Ces1g) has been shown to play a role in metabolic control. So far, the role of mouse Ces1/Ces1g deficiency in atherosclerosis is not elucidated. We generated Ces1/Ces1g−/− mice. Compared to wild-type mice, Ces1/Ces1g−/− mice had reduced plasma cholesterol levels. We then generated Ces1g−/−Ldlr−/− double knockout (DKO) mice, which were fed a Western diet for 16 weeks. Compared to Ldlr−/− mice, DKO mice displayed decreased plasma cholesterol and TG levels and reduced atherosclerotic lesions. Interestingly, knockdown of hepatic Ces1/Ces1g in Apoe−/− mice resulted in hyperlipidemia and exacerbated Western diet-induced atherogenesis. Mechanistically, global inactivation of Ces1/Ces1g inhibited intestinal cholesterol and fat absorption and Niemann-Pick C1 like 1 expression, and increased macrophage cholesterol efflux by inducing ATP-binding cassette subfamily A member 1 (ABCA1) and ABCG1. Ces1/Ces1g ablation also promoted M2 macrophage polarization and induced hepatic cholesterol 7α-hydroxylase and sterol 12α-hydroxylase expression. In conclusion, global loss of Ces1/Ces1g protects against the development of atherosclerosis by inhibiting intestinal cholesterol and triglyceride absorption and promoting macrophage cholesterol efflux.

Understanding Chylomicron Retention Disease Through Sar1b Gtpase Gene Disruption: Insight From Cell Culture.

Understanding the specific mechanisms of rare autosomal disorders has greatly expanded insights into the complex processes regulating intestinal fat transport. Sar1B GTPase is one of the critical proteins governing chylomicron secretion by the small intestine, and its mutations lead to chylomicron retention disease, despite the presence of Sar1A paralog. The central aim of this work is to examine the cause-effect relationship between Sar1B expression and chylomicron output and to determine whether Sar1B is obligatory for normal high-density lipoprotein biogenesis. The SAR1B gene was totally silenced in Caco-2/15 cells using the zinc finger nuclease technique. SAR1B deletion resulted in significantly decreased secretion of triglycerides (≈40%), apolipoprotein B-48 (≈57%), and chylomicron (≈34.5%). The absence of expected chylomicron production collapse may be because of the compensatory SAR1A elevation observed in our experiments. Therefore, a double knockout of SAR1A and SAR1B was engineered in Caco-2/15 cells, which led to almost complete inhibition of triglycerides, apolipoprotein B-48, and chylomicron output. Further experiments with labeled cholesterol revealed the downregulation of high-density lipoprotein biogenesis in cells deficient in SAR1B or with the double knockout of the 2 SAR1 paralogs. Similarly, there was a fall in the movement of labeled cholesterol from cells to basolateral medium containing apolipoprotein A-I, thereby limiting newly synthesized high-density lipoprotein in genetically modified cells. The decreased cholesterol efflux was associated with impaired expression of ABCA1 (ATP-binding cassette subfamily A member 1). These findings demonstrate that the deletion of the 2 SAR1 isoforms is required to fully eliminate the secretion of chylomicron in vitro. They also underscore the limited high-density lipoprotein production by the intestinal cells in response to SAR1 knockout.

Effects of sepsis on the metabolism of sphingomyelin and cholesterol in mice with liver dysfunction.

Sepsis is characterized by a severe inflammatory response to infection. With the spread of sepsis, various tissues, including the lungs, liver and kidney, may be damaged. This may finally develop into multiple organ dysfunction syndrome. Sphingomyelin and cholesterol are two main lipids involved in sepsis. The metabolism of sphingomyelin and cholesterol in the livers of mice with sepsis needs to be clarified. To achieve this, the present study intraperitoneally injected mice with PBS, lipopolysaccharide (LPS; 10 mg/kg) and LPS + pyrrolidine dithiocarbamate (PDTC; 30 mg/kg). Subsequently, sphingomyelin and cholesterol content were measured using kits, the sphingomyelin synthase (SMS) activity was measured using thin layer chromatography, and the expression levels of SMS1 and 2, hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), ATP binding cassette subfamily A member 1 (ABCA1), scavenger receptor class B member 1 (SR-B1) and apolipoprotein A1 (Apo A1) were determined by western blotting in the livers of mice. Results demonstrated that, in the LPS group, sphingomyelin and cholesterol content was significantly increased (P<0.001; n=6), the SMS activity significantly enhanced (P<0.001; n=6), the expression levels of SMS2, HMGCR, ABCA1 and SR-B1 were augmented (P<0.05; n=6), and the expression of Apo A1 was decreased (P<0.05; n=6), whereas SMS1 level only slightly increased with no statistical significance (P>0.05; n=6), compared to the levels in the control group. However, PDTC was able to attenuate these alterations. These results indicated that sphingomyelin and cholesterol content may increase in the liver dysfunction of sepsis by increasing the expression of SMS2, HMGCR, SR-B1 and ABCA1, and downregulating Apo A1.

CRISPR/Cas9-Mediated Gene Editing in Human iPSC-Derived Macrophage Reveals Lysosomal Acid Lipase Function in Human Macrophages-Brief Report.

To gain mechanistic insights into the role of LIPA (lipase A), the gene encoding LAL (lysosomal acid lipase) protein, in human macrophages. We used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) technology to knock out LIPA in human induced pluripotent stem cells and then differentiate to macrophage (human-induced pluripotent stem cells-derived macrophage [IPSDM]) to explore the human macrophage LIPA loss-of-function phenotypes. LIPA was abundantly expressed in monocyte-derived macrophages and was markedly induced on IPSDM differentiation to comparable levels as in human monocyte-derived macrophage. IPSDM with knockout of LIPA (LIPA-/-) had barely detectable LAL enzymatic activity. Control and LIPA-/- IPSDM were loaded with [3H]-cholesteryl oleate-labeled AcLDL (acetylated low-density lipoprotein) followed by efflux to apolipoprotein A-I. Efflux of liberated [3H]-cholesterol to apolipoprotein A-I was abolished in LIPA-/- IPSDM, indicating deficiency in LAL-mediated lysosomal cholesteryl ester hydrolysis. In cells loaded with [3H]-cholesterol-labeled AcLDL, [3H]-cholesterol efflux was, however, not different between control and LIPA-/- IPSDM. ABCA1 (ATP-binding cassette, subfamily A, member 1) expression was upregulated by AcLDL loading but to a similar extent between control and LIPA-/- IPSDM. In nonlipid loaded state, LIPA-/- IPSDM had high levels of cholesteryl ester mass compared with minute amounts in control IPSDM. Yet, with AcLDL loading, overall cholesteryl ester mass was increased to similar levels in both control and LIPA-/- IPSDM. LIPA-/- did not impact lysosomal apolipoprotein-B degradation or expression of IL1B, IL6, and CCL5. CONCLUSIONS: LIPA-/- IPSDM reveals macrophage-specific hallmarks of LIPA deficiency. CRISPR/Cas9 and IPSDM provide important tools to study human macrophage biology and more broadly for future studies of disease-associated LIPA genetic variation in human macrophages.

CCAAT/enhancer-binding protein delta promotes intracellular lipid accumulation in M1 macrophages of vascular lesions.

Lipid homeostasis is reprogrammed in the presence of inflammation, which results in excessive lipid accumulation in macrophages, and leads to the formation of lipid-laden foam cells. We aimed to link an inflammation-responsive transcription factor CCAAT/enhancer-binding protein delta (CEBPD) with polarized macrophages and dissect its contribution to lipid accumulation. We found that CEBPD protein colocalized with macrophages in human and mouse (C57BL/6, Apoe-/-) atherosclerotic plaques and that Cebpd deficiency in bone marrow cells suppressed atherosclerotic lesions in hyperlipidemic Apoe-/- mice. CEBPD was responsive to modified low-density lipoprotein (LDL) via the p38MAPK/CREB pathway, and it promoted lipid accumulation in M1 macrophages but not in M2 macrophages. CEBPD up-regulated pentraxin 3 (PTX3), which promoted the macropinocytosis of LDL, and down-regulated ATP-binding cassette subfamily A member 1 (ABCA1), which impaired the intracellular cholesterol efflux in M1 macrophages. We further found that simvastatin (a HMG-CoA reductase inhibitor) could target CEBPD to block lipid accumulation in a manner not directly related to its cholesterol-lowering effect in M1 macrophages. This study underscores how CEBPD functions at the junction of inflammation and lipid accumulation in M1 macrophages. Therefore, CEBPD-mediated lipid accumulation in M1 macrophages could represent a new therapeutic target for the treatment of cardiovascular diseases.