Cholesterol Efflux Assay Research Articles

Human glia‐specific functional dysregulations affected by APOE ε4 risk of Alzheimer's disease

AbstractBackgroundApolipoprotein E (APOE) is the strongest genetic risk factor for late‐onset Alzheimer's disease (AD). APOE ɛ4/ɛ4 homozygosity increases AD risk by >15‐fold. Although its association with AD is well‐established, the mechanisms underlying this genetic risk on particular brain cell types remains elusive. We hypothesized that the APOE ε4/ε4 genotype contributes to disease risk through cell autonomous mechanisms in glia.MethodWe used three models: human induced pluripotent stem cells (hiPSCs), post‐mortem brains and human APOE‐targeted replacement (TR) mice. We have differentiated microglia, astrocytes, cortical neurons and brain microvascular endothelial cells from hiPSC derived from non‐isogenic and isogenic cohort of cells selected based on APOE genotype. We also deconvoluted AD vs control whole brain transcriptome by cell types to confirm the findings from iPSCs in human brain tissue. Primary mouse microglia and astrocytes were purified from APOE‐TR mice. Global transcriptomic analyses were performed to identify APOE ɛ4 impact on particular cell type and to compare species differences. We validated predicted functional pathways in vitro using isogenic hiPSC lines using gas chromatograph‐mass spectrometry, fluorescently tagged cholesterol efflux assay, protein biochemistry and Luminex multiplex immunoassay.ResultGene set enrichment and pathway analyses of whole transcriptome profiles showed that APOE ε4 is associated with dysregulation of cholesterol homeostasis in human glia but not mouse astrocytes and microglia. Elevated matrisome signaling associated with chemotaxis, glial activation and lipid biosynthesis in APOE ε4 mixed neuron/astrocyte cultures parallels altered pathways uncovered in cell‐type deconvoluted transcriptomic data from APOE ε4 glia and AD post‐mortem brains. Experimental validation of the transcriptomic findings showed that isogenic APOE ε4 is associated with increased lysosomal cholesterol levels and decreased cholesterol efflux, demonstrating decoupled lipid metabolism. APOE ε4 glia also secrete higher levels of proinflammatory chemokines, cytokines and growth factors, indicative of glial activation.ConclusionThus, CNS cell type based models allowed us to elucidate APOE ε4‐driven cell autonomous effects; APOE ε4 induces human glia‐specific dysregulation in reactivity of the cells and associated lipid metabolism that may initiate AD risk.

Punicalagin Regulates Key Processes Associated with Atherosclerosis in THP-1 Cellular Model.

Atherosclerosis may lead to cardiovascular diseases (CVD), which are the primary cause of death globally. In addition to conventional therapeutics for CVD, use of nutraceuticals that prevents cholesterol deposition, reduce existing plaques and hence anti-atherosclerotic effects of nutraceuticals appeared to be promising. As such, in the present study we evaluated the beneficial effects of punicalagin, a phytochemical against an atherosclerotic cell model in vitro. Cytotoxicity assays were examined for 10 µM concentration of punicalagin on THP-1 macrophages. Real-time-polymerase chain reaction (RT-PCR) was used to analyze monocyte chemoattractant protein-1 (MCP-1) and Intercellular adhesion molecule (ICAM-1) expressions. Monocyte migration and cholesterol efflux assays were performed to investigate punicalagin’s further impact on the key steps of atherosclerosis. Cytotoxicity assays demonstrated no significant toxicity for punicalagin (10 µM) on THP-1 macrophages. Punicalagin inhibited the IFN-γ-induced overexpression of MCP-1 and ICAM-1 in macrophages by 10 fold and 3.49 fold, respectively, compared to the control. Punicalagin also reduced the MCP-1- mediated migration of monocytes by 28% compared to the control. Percentages of cellular cholesterol efflux were enhanced in presence or absence of IFN-γ by 88% and 84% compared to control with 58% and 62%, respectively. Punicalagin possesses anti-inflammatory and anti-atherosclerotic effects. Punicalagin also did not exhibit any cytotoxicity and therefore can be considered a safe and potential candidate for the treatment and prevention of atherosclerosis.

Facile Cholesterol Loading with a New Probe ezFlux Allows for Streamlined Cholesterol Efflux Assays.

Here, we report a nanoparticle-based probe that affords facile cell labeling with cholesterol in cholesterol efflux (CE) assays. This probe, called ezFlux, was optimized through a screening of multiple nanoformulations engineered with a Förster resonance energy transfer (FRET) reporter. The physicochemical- and bio-similarity of ezFlux to standard semi-synthetic acetylated low-density lipoprotein (acLDL) was confirmed by testing uptake in macrophages, the intracellular route of degradation, and performance in CE assays. A single-step fast self-assembly fabrication makes ezFlux an attractive alternative to acLDL. We also show that CE testing using ezFlux is significantly cheaper than that performed using commercial kits or acLDL. Additionally, we analyze clinical trials that measure CE and show that ezFlux has a place in many research and clinical laboratories worldwide that use CE to assess cellular and lipoprotein function.

The anti-inflammatory properties of HDLs are impaired in gout.

We sought to determine whether high-density lipoprotein (HDL) function was altered in gout patients. The study included 95 gout patients and 68 healthy controls. The concentrations of interleukin (IL)-1β and IL-9 were measured by ELISA, and indicators such as blood uric acid, liver and kidney function, blood glucose, and blood lipids were detected. To test for the anti-inflammatory and reverse cholesterol transport (RCT) function of HDL, 11 gout patients and 11 healthy controls were randomly selected for the BioVision cholesterol efflux test, which detects the RCT activity of HDL. To assess the anti-inflammatory function of HDL, cells in co-culture with HDL were treated with inflammatory stimuli such as tumor necrosis factor-α (TNF-α), and then, the cells were assayed for the expression of intercellular adhesion molecules (ICAMs) and vascular cell adhesion molecule-1 (VCAM-1). In total, this study enrolled 163 participants, including 95 non-hyperlipidemic gout patients and 68 healthy controls. IL-1β and IL-9 levels were significantly higher in the gout group than in the control group (85.26 ± 23.16 vs. 41.47 ± 6.48 and 33.77 ± 12.68 vs. 23.66 ± 4.53, respectively, P < 0.001). Additionally, plasma IL-1β and IL-9 levels were increased along with those of blood uric acid (R2 = 0.4116 and R2 = 0.4150, respectively, P < 0.001). Compared with the healthy controls, gout patients showed no differences in plasma apoA-1 levels or in the cholesterol efflux assay. Gout patients had increased ICAM-1 expression compared with the healthy controls (88.79 ± 3.68 vs. 86.27 ± 4.64, P < 0.05), but no difference in VCAM-1 expression was found (0.87 ± 0.43 vs. 0.98 ± 0.96, P > 0.05). In this assay, higher values indicate less suppression of ICAM-1 induction, which correlates with a reduced anti-inflammatory capacity. The anti-inflammatory activities of HDLs are impaired in gout patients. Key Points • Gout patients show chronic inflammation. • The anti-inflammatory activity of high-density lipoprotein is impaired in gout patients.

Di-n-butyl phthalate promotes lipid accumulation via the miR200c-5p-ABCA1 pathway in THP-1 macrophages

Di-n-butyl phthalate (DBP) is ubiquitously in the environment and has been detected in almost all of human bodies. Few data could be found about the effects of DBP on cardiovascular system, though its reproductive toxicities have been studied extensively. This study aimed to explore the effects of DBP on lipid metabolism, a key step during the formation of atherosclerosis, since DBP was recently reported to be associated with atherosclerosis. THP-1 macrophages were employed and exposed to various levels of DBP (10−8, 10−7, 10−6, 10−5 and 10−4 mol/L) or DMSO as control. Lipid accumulation was determined by detection of cellular total cholesterol, free cholesterol, cholesterol ester and content of lipid drops. Expressions of mRNA/miRNAs and proteins were measured by qRT-PCR and western blotting, respectively. Bioinformatic analysis and dual luciferase reporter assay were used to analyze the combination between miR200c-5p and ATP-binding cassette transporter A1 (ABCA1). Cholesterol efflux assay was executed to study the inhibitory effects of DBP on cholesterol efflux capability. Results revealed that DBP at 10−7 mol/L prompted THP-1 macrophages lipid accumulation by inhibiting cholesterol efflux via suppressing ABCA1 expression. In addition, a non-linear inverted U-shaped relationship between DBP and lipid accumulation could be observed. Moreover, miR200c-5p could directly targets to ABCA1 3′UTR and modulate ABCA1 expression. Besides, downregulation of ABCA1 expression and reduction of lipid efflux induced by DBP were due to the miR200c-5p upregulation. Collectively, these data suggested that DBP at levels relative to human exposure could increase lipid accumulation in THP-1 macrophages by decreasing cholesterol efflux through miR200c-5p-ABCA1, then potentiate the formation of atherosclerosis.

Caco-2 Cells for Measuring Intestinal Cholesterol Transport - Possibilities and Limitations

BackgroundThe human Caco-2 cell line is a common in vitro model of the intestinal epithelial barrier. As the intestine is a major interface in cholesterol turnover and represents a non-biliary pathway for cholesterol excretion, Caco-2 cells are also a valuable model for studying cholesterol homeostasis, including cholesterol uptake and efflux. Currently available protocols are, however, either sketchy or not consistent among different laboratories. Our aim was therefore to generate a collection of optimized protocols, considering the different approaches of the different laboratories and to highlight possibilities and limitations of measuring cholesterol transport with this cell line.ResultsWe developed comprehensive and quality-controlled protocols for the cultivation of Caco-2 cells on filter inserts in a single tight monolayer. A cholesterol uptake as well as a cholesterol efflux assay is described in detail, including suitable positive controls. We further show that Caco-2 cells can be efficiently transfected for luciferase reporter gene assays in order to determine nuclear receptor activation, main transcriptional regulators of cholesterol transporters (ABCA1, ABCB1, ABCG5/8, NPC1L1). Detection of protein and mRNA levels of cholesterol transporters in cells grown on filter inserts can pose challenges for which we highlight essential steps and alternative approaches for consideration. A protocol for viability assays with cells differentiated on filter inserts is provided for the first time.ConclusionsThe Caco-2 cell line is widely used in the scientific community as model for the intestinal epithelium, although with highly divergent protocols. The herein provided information and protocols can be a common basis for researchers intending to use Caco-2 cells in the context of cellular cholesterol homeostasis.

First eight residues of apolipoprotein A-I mediate the C-terminus control of helical bundle unfolding and its lipidation.

The crystal structure of a C-terminal deletion of apolipoprotein A-I (apoA1) shows a large helical bundle structure in the amino half of the protein, from residues 8 to 115. Using site directed mutagenesis, guanidine or thermal denaturation, cell free liposome clearance, and cellular ABCA1-mediated cholesterol efflux assays, we demonstrate that apoA1 lipidation can occur when the thermodynamic barrier to this bundle unfolding is lowered. The absence of the C-terminus renders the bundle harder to unfold resulting in loss of apoA1 lipidation that can be reversed by point mutations, such as Trp8Ala, and by truncations as short as 8 residues in the amino terminus, both of which facilitate helical bundle unfolding. Locking the bundle via a disulfide bond leads to loss of apoA1 lipidation. We propose a model in which the C-terminus acts on the N-terminus to destabilize this helical bundle. Upon lipid binding to the C-terminus, Trp8 is displaced from its interaction with Phe57, Arg61, Leu64, Val67, Phe71, and Trp72 to destabilize the bundle. However, when the C-terminus is deleted, Trp8 cannot be displaced, the bundle cannot unfold, and apoA1 cannot be lipidated.

The ABCA1 blocking agent probucol decreases capacitation in ejaculated dog spermatozoa

BackgroundThe ATP binding cassette (ABC) transporters participate in the cholesterol and phospholipid transport within and through cell membranes of many cells including spermatozoa. Cholesterol efflux is important for capacitation of spermatozoa. ABCA1 expression has been assessed in canine spermatozoa previously but its role in capacitation still has to be determined. The aim of the study was to test whether inhibition of ABCA1 (1) decreases capacitation in ejaculated and epididymal canine sperm samples and (2) decreases cholesterol efflux in the same samples. Twenty-one ejaculates and sperm from 22 epididymal tails were collected from healthy dogs. Motility was measured by CASA and viability assessed after staining with SYBR-14/PI. Samples from ejaculated sperm and sperm from epididymal tails were aliquoted. One part was incubated with the ABCA1 inhibitor probucol, the other served as a negative control. In all samples, capacitation was evaluated by chlortetracyclin (CTC) assay and cholesterol was measured by cholesterol efflux assay and colorimetric enzymatic assay.ResultsIn ejaculated sperm, blockade of ABCA1 with 100 µM of probucol/mL of sample resulted in a significantly higher percentage of uncapacitated and acrosome reacted spermatozoa (P < 0.001 and P = 0.031), capacitation was significantly decreased (35% in probucol samples vs 54.2% in controls, P < 0.001). In probucol inhibited sperm samples from epididymal tails, the percentage of capacitated spermatozoa did not differ between groups but the percentage of acrosome reacted spermatozoa increased significantly (P = 0.014). The cholesterol measurement revealed significantly lower cholesterol concentration in the probucol group when compared to the controls (P = 0.035), however only in ejaculated sperm samples.ConclusionsCTC assay and cholesterol measurement revealed significant differences between groups; we conclude that inhibition of ABCA1 significantly decreased capacitation and cholesterol efflux in ejaculated canine spermatozoa. The inhibition was not complete but ABCA1 is supposed to contribute to capacitation in canine ejaculated spermatozoa. ABCA1 is probably not important for capacitation of epididymal spermatozoa but might exert other functions during spermatozoa ripening.

Pseudoprotodioscin inhibits SREBPs and microRNA 33a/b levels and reduces the gene expression regarding the synthesis of cholesterol and triglycerides

The extract of Dioscorea zingiberensis C.H. Wright rhizomes is found to be effective in the therapy of cardiovascular disease. Steroidal saponins make substantial contribution. Previous study has proposed that methylprotodioscin (MP) may promote cholesterol efflux by increasing ABCA1 expression. But the other main saponins ingredients are not referred to. The aim of the present work was to reveal the effect and mechanism of protodioscin (PD), MP and pseudoprotodioscin (PPD) on the synthesis-related gene expression of cholesterol and triglycerides. MTT assay apoptosis assay with annexin AV-APC and 7-AAD double staining were performed. MicroRNA assay and qRT-PCR were used to analyze the gene expression which regulates synthesis of cholesterol and triglycerides. Western blot was to demonstrate the levels of target proteins. Cholesterol efflux assay was executed to study the stimulative effect of saponins on cholesterol efflux. In Hep G2 cells, PPD increased ABCA1 protein and mRNA levels, and promoted the effluxion of ApoA-1-mediated cholesterol. The underlying mechanisms involved that PPD inhibited SREBP1c and SREBP2 transcription by decreasing microRNA 33a/b levels. This procedure reciprocally led to the increase of ABCA1 levels. In THP-1 macrophages, PPD showed the similar effect, which reduced HMGCR, FAS and ACC mRNA levels and promoted low density lipoprotein receptor by decreasing the PCSK9 levels. These studies demonstrated that PPD is a potential agent for cholesterol efflux, SREBPs and microRNA 33a/b inhibition, which related to the gene expression for the synthesis of cholesterol and triglycerides.

Downregulation of ABCA1 and ABCG1 transporters by simvastatin in cholangiocarcinoma cells.

Disturbances in cholesterol homeostasis of the bile duct epithelium, including transport interruption and the hyperaccumulation of intracellular cholesterol can lead to the initiation and progression of cholangiocarcinoma (CCA). Statins, which are lipid-lowering drugs, have been previously documented to exhibit anti-cancer properties. The role of statins in CCA cell cholesterol transport through the expression and function of ATP-binding cassette (ABC) A1 and ABCG1 was investigated in the current study. In four CCA cell lines, ABCA1 and ABCG1 expression was identified. However, neither ABCG5 nor ABCG8 expression was observed. Immunocytochemistry revealed that the expression of ABCA1 was localized in the proximity of the nucleus, while ABCG1 was more dispersed throughout the cytoplasm of KKU-100 cells. A cholesterol efflux assay was performed using bodipy cholesterol, and the translocation of cholesterol via ABCA1 and ABCG1 to Apo-A1 and high density lipoprotein was confirmed, respectively. Simvastatin and atorvastatin demonstrated the inhibitory effects on CCA cell viability. A reduction in intracellular lipid level and a lower expression of ABCA1 and ABCG1 were observed in KKU-100 cells under simvastatin treatment. The pre-exposure of KKU-100 cells to cholesterol diminished the statin effect. Furthermore, when KKU-100 cells were pre-loaded with cholesterol, ABCA1 and ABCG1-mediated exports were unaffected even though they were treated with simvastatin. The results of the current study indicated the limitations of the use of statin in CCA therapy, particularly under hypercholesterolemia conditions.

A reliable and reproducible assay for determining the effect of natural product on macrophages lipid uptake and cholesterol efflux: A case study of maslinic acid

Macrophage foam cell formation represents a key feature that contributes to the development of atherosclerotic lesions. Assessment of cardioprotective natural compounds targeting macrophage foam cell formation processes including lipid uptake and cholesterol efflux could lead to the identification of potential lead compounds for development into novel anti-atherosclerotic drugs. In this case study, maslinic acid, a natural product was used to study the effect on lipid uptake and cholesterol efflux in THP-1-derived macrophages. Oil red O (ORO) staining and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled oxidized low-density lipoprotein (Dil-labeled oxLDL) uptake assays were performed to determine lipid uptake by macrophages while cholesterol efflux was assessed using 3-hexanoyl-NBD labeled cholesterol. ORO-stained images were further analyzed using ImageJ analysis software to determine intracellular lipid droplets accumulation and flow cytometric analysis of mean fluorescence intensity were obtained to quantify Dil-labeled oxLDL uptake by macrophages. Meanwhile, 3-hexanoyl-NBD labeled cholesterol uptake and efflux from THP-1-derived macrophages were characterized. The fluorescence intensity values obtained from the medium and cell lysates were then converted into percentage of cholesterol efflux. The results have shown that incubation with maslinic acid suppressed oxLDL-induced macrophage foam cell formation which may be contributed from its effect in reducing lipid uptake and enhancing cholesterol efflux. In conclusion, the optimized ORO staining, Dil-labeled oxLDL uptake, and fluorescent-labeled cholesterol efflux assays provide reproducible and reliable results for assessment of foam cells formation.

AIBP and APOA-I synergistically inhibit intestinal tumor growth and metastasis by promoting cholesterol efflux

BackgroundThe roles played by cholesterol in cancer development and progression represent a popular field in the cancer community. High cholesterol levels are positively correlated with the risk of various types of cancer. APOA-I binding protein (AIBP) promotes the reverse cholesterol transport pathway (RCT) in cooperation with Apolipoprotein A-I (APOA-I) or high-density lipoprotein cholesterol. However, the combined effect of AIBP and APOA-I on intestinal tumor cells is still unclear.MethodsImmunohistochemistry, western blot and qPCR were performed to investigate the expression of AIBP and APOA-I in intestinal tumor tissues and cell lines. The anti-tumor activity of AIBP and APOA-I was evaluated by overexpression or recombinant protein treatment. Cholesterol efflux and localization of lipid raft-related proteins were analyzed by a cholesterol efflux assay and lipid raft fraction assay, respectively.ResultsHere, we reported that both AIBP expression and APOA-I expression were associated with the degree of malignancy in intestinal tumors. Co-overexpression of AIBP and APOA-I more potently inhibited colon cancer cell-mediated tumor growth and metastasis compared to overexpression of each protein individually. Additionally, the recombinant fusion proteins of AIBP and APOA-I exhibited a significant therapeutic effect on tumor growth in Apcmin/+ mice as an inherited intestinal tumor model. The synergistic effect of the two proteins inhibited colon cancer cell migration, invasion and tumor-induced angiogenesis by promoting cholesterol efflux, reducing the membrane raft content, and eventually disrupting the proper localization of migration- and invasion-related proteins on the membrane raft. Moreover, cyclosporine A, a cholesterol efflux inhibitor, rescued the inhibitory effect induced by the combination of AIBP and APOA-I.ConclusionsThese results indicate that the combination of APOA-I and AIBP has an obvious anticancer effect on colorectal cancer by promoting cholesterol efflux.

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay.

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

TISSUE-SELECTIVE ABCA1 AGONISTS AS POTENTIAL ALZHEIMER'S DISEASE THERAPEUTICS: HIT-TO-LEAD OPTIMIZATION

The apolipoprotein E (APOE) ε4 allele is the strongest known risk factor for sporadic Alzheimer's disease (AD), which accounts for over 95% of all AD cases. ApoE4-containing lipoproteins contain a lower lipid content, which decreases stability and contributes to loss of lipoprotein function. To correct these deficits and restore proper lipoprotein function, we have developed tissue-selective ABCA1 agonists (TSAAgs) that induce central nervous system expression of the cholesterol transport protein ABCA1, thereby increasing lipid content of apoE4-containing lipoproteins, with minimal impact on peripheral lipogenesis. TSAAgs also ameliorate additional aspects of AD, including neuroinflammation and insulin resistance. High-throughput screening (HTS) utilized CCF-STTG1 astrocytoma (primary screen) and HepG2 hepatocellular carcinoma cells (counterscreen) expressing luciferase response elements linked to ABCA1 and SREBP1c promoters, respectively. Novel structural analogs of HTS hits were generated via iterative chemical synthesis. In vitro analysis of analog activity included PCR/immunoblot for both lipid- and insulin-related genes, ELISA for inflammatory markers, and a fluorescent cholesterol efflux assay. Prioritized HTS hits – those demonstrating anti-inflammatory and insulin-sensitizing properties in addition to TSAAg activity – served as scaffolds to generate a library of structural analogs. Evaluation of this analog library in vitro established structure-activity relationships that identified compounds with improved TSAAg activity and guided further structural modification. These results demonstrated a proof-of-concept to develop TSAAgs possessing multifunctional therapeutic potential against Alzheimer's disease. Our study represents a novel strategy to develop small molecule drug candidates that target multiple aspects of AD pathology. Future in vivo experiments are proposed to establish pharmacokinetic profiles, determine magnitude and mechanisms of tissue-selective ABCA1 induction, and monitor alterations in peripheral lipogenesis. Finally, treatments in the EFAD mouse model will assess TSAAg effects on cognitive and pathological deficits. Successful completion of this project could result in establishment of TSAAg compounds as leads for further pharmaceutical development and human clinical testing.

6-Dihydroparadol, a Ginger Constituent, Enhances Cholesterol Efflux from THP-1-Derived Macrophages.

ScopeGinger is reported to be used for the prevention and treatment of cardiovascular diseases (CVD). Cholesterol efflux from macrophage foam cells is an important process in reverse cholesterol transport, whose increase may help to prevent or treat CVD. In this study, we investigated the effects of 6‐dihydroparadol from ginger on macrophage cholesterol efflux.Methods and resultsWe show that 6‐dihydroparadol concentration‐dependently enhances both apolipoprotein A1‐ and human plasma–mediated cholesterol efflux from cholesterol‐loaded THP‐1‐derived macrophages using macrophage cholesterol efflux assay. 6‐Dihydroparadol increases protein levels of both ATP‐binding cassette transporters A1 and G1 (ATP‐binding cassette transporter A1 [ABCA1] and ATP‐binding cassette transporter G1 [ABCG1]) according to Western blot analysis. The ABCA1 inhibitor probucol completely abolishes 6‐dihydroparadol‐enhanced cholesterol efflux. Furthermore, increased ABCA1 protein levels in the presence of 6‐dihydroparadol were associated with both increased ABCA1 mRNA levels and increased ABCA1 protein stability. Enhanced ABCG1 protein levels were only associated with increased protein stability. Increased ABCA1 protein stability appeared to be the result of a reduced proteasomal degradation of the transporter in the presence of 6‐dihydroparadol.ConclusionWe identified 6‐dihydroparadol from ginger as a novel promoter of cholesterol efflux from macrophages that increases both ABCA1 and ABCG1 protein abundance. This newly identified bioactivity might contribute to the antiatherogenic effects of ginger.

Validation and Application of a Novel Cholesterol Efflux Assay Using Immobilized Liposomes as a Substitute for Cultured Cells

Estimation of the function as well as the amount of high-density lipoprotein (HDL) is required to predict the risk of cardiovascular disease development. Cholesterol efflux capacity (CEC) is the key metric for determining the antiatherosclerotic function of HDL. However, the assay methods currently used to calculate CEC are not ideal for clinical use as they require the culture of cells. In the present study, we developed a novel CEC assay using immobilized liposome-bound gel beads (ILGs), containing fluorescently labeled cholesterol, as a substitute for cultured cells. When apolipoprotein B-100 depleted serum, obtained by polyethylene glycol precipitation, was used as the cholesterol acceptors, the basic properties of this method, such as the available range of HDL-cholesterol, efflux temperature and time, and normalization parameters, indicate that this method is sufficient to estimate CEC. Furthermore, the CEC values obtained with this ILG method were also correlated with those obtained with a conventional method using THP-1 macrophages derived foam cells and 3H-cholesterol as a tracer (r = 0.932). Overall, this novel cholesterol efflux assay method is a realistic and effective alternative to current methods in the field while also being easier to use in clinical laboratories as neither cell culture, radioisotope nor ultracentrifugation is required.

Abstract 164: Synthetic High-Density Lipoprotein Cholesterol Scavengers for the Treatment of Niemann-Pick C Disease

Background: Niemann-Pick C disease (NPC) is genetic disorder caused by an accumulation of unesterified cholesterol in late endosomes and lysosomes due to defects in NPC genes. The levels of ABCA1 gene expression and concentration of high-density lipoproteins (HDL) are significantly decreased in NPC patients. Infusion of synthetic HDL (sHDL) in patients with atherosclerosis has been previously found safe (up to 100 mg/kg) and effective at reducing cholesterol in atheroma. The objective of this study is to identify sHDL composition capable of rescuing cholesterol storage in NPC. Materials and Methods: A panel of sHDL formulations was prepared by lyophilization method using commercially available Apolipoprotein A-I mimetic peptide, 5A, complexed in various ratios with phospholipids such as sphingomyelin (SM), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylcholine (DMPC). The efficacy of 5A alone and sHDLs was determined in primary patient NPC and wild type fibroblast cells using fluorescent filipin staining and cholesterol efflux assay. Trafficking of sHDL in NPC fibroblasts was assessed by confocal microscopy using fluorescently-labeled sHDL. Finally, in vivo study was executed to examine effects of our best sHDL formulation 5A-SM in NPC1 I1061T homozygotes and littermate controls treated with vehicle or sHDL (100 mg/kg, i.p., 3x/wk) for 4 weeks, starting at 7 wks of age. Neurological correction was accessed by the balance beam tests and body weight changes were tracked. Results: Treatment of NPC fibroblasts with sHDL resulted in a dose- and time-dependent rescue of lipid storage (5A-POPC&lt;5A-SM&lt;5A-DMPC). Cellular toxicity was observed only for 5A-DMPC (~30%). HDL trafficking studies revealed that sHDLs got endocytosed into cells and co-localized with LAMP1. Additionally, administration of 100 mg/kg 5A-SM resulted in a significant rescue of body weight (p&lt;0.01) in the NPC mice with no toxicity to animals. However, neuro-correction after sHDL treatment in adult mice was not detected. The absence of neuro-correction observed in adult NPC mice suggest that the alternative delivery routes, treatment durations, or sHDL compositions are still needed.

Diets Low in Saturated Fat with Different Unsaturated Fatty Acid Profiles Similarly Increase Serum-Mediated Cholesterol Efflux from THP-1 Macrophages in a Population with or at Risk for Metabolic Syndrome: The Canola Oil Multicenter Intervention Trial

Cholesterol efflux plays an important role in preventing atherosclerosis progression. Vegetable oils with varying unsaturated fatty acid profiles favorably affect multiple cardiovascular disease risk factors; however, their effects on cholesterol efflux remain unclear. The objectives of this study were to examine the effects of diets low in saturated fatty acids (SFAs) with varying unsaturated fatty acid profiles on serum-mediated cholesterol efflux and its association with the plasma lipophilic index and central obesity. The present study is a randomized, crossover, controlled-feeding study. Participants [men: n=50; women: n=51; mean ± SE age: 49.5±1.2 y; body mass index (in kg/m2): 29.4±0.4] at risk for or with metabolic syndrome (MetS) were randomly assigned to 5 isocaloric diets containing the treatment oils: canola oil, high oleic acid-canola oil, DHA-enriched high oleic acid-canola oil, corn oil and safflower oil blend, and flax oil and safflower oil blend. These treatment oils were incorporated into smoothies that participants consumed 2 times/d. For a 3000-kcal diet, 60 g of treatment oil was required to provide 18% of total energy per day. Each diet period was 4 wk followed by a 2- to 4-wk washout period. We quantified cholesterol efflux capacity with a validated ex vivo high-throughput cholesterol efflux assay. Statistical analyses were performed with the use of the SAS mixed-model procedure. The 5 diets increased serum-mediated cholesterol efflux capacity from THP-1 macrophages similarly by 39%, 34%, 55%, 49% and 51%, respectively, compared with baseline (P<0.05 for all). Waist circumference and abdominal adiposity were negatively correlated with serum-mediated cholesterol efflux capacity (r=-0.25, P=0.01, r=-0.33, P=0.02, respectively). Diets low in SFAs with different monounsaturated fatty acid and polyunsaturated fatty acid profiles improved serum-mediated cholesterol efflux capacity in individuals with or at risk for MetS. This mechanism may account, in part, for the cardiovascular disease benefits of diets low in SFAs and high in unsaturated fatty acids. Importantly, central obesity is inversely associated with cholesterol efflux capacity. This trial was registered at www.clinicaltrials.gov as NCT01351012.

Validation and application of a novel cholesterol efflux assay using immobilized liposomes as a substitute for cultured cells.

Estimation of the function as well as the amount of high-density lipoprotein (HDL) is required to predict the risk of cardiovascular disease development. Cholesterol efflux capacity (CEC) is the key metric for determining the antiatherosclerotic function of HDL. However, the assay methods currently used to calculate CEC are not ideal for clinical use as they require the culture of cells. In the present study, we developed a novel CEC assay using immobilized liposome-bound gel beads (ILGs), containing fluorescently labeled cholesterol, as a substitute for cultured cells. When apolipoprotein B-100 depleted serum, obtained by polyethylene glycol precipitation, was used as the cholesterol acceptors, the basic properties of this method, such as the available range of HDL-cholesterol, efflux temperature and time, and normalization parameters, indicate that this method is sufficient to estimate CEC. Furthermore, the CEC values obtained with this ILG method were also correlated with those obtained with a conventional method using THP-1 macrophages derived foam cells and 3H-cholesterol as a tracer (r = 0.932). Overall, this novel cholesterol efflux assay method is a realistic and effective alternative to current methods in the field while also being easier to use in clinical laboratories as neither cell culture, radioisotope nor ultracentrifugation is required.

Increased 27-hydroxycholesterol production during luteolysis may mediate the progressive decline in progesterone secretion.

Does 27-hydroxycholesterol (27OH) actively facilitate the progression of luteolysis? There is increased mRNA expression of the enzyme that produces 27OH during luteolysis in vivo in rhesus macaques and sheep, and 27OH reduces progesterone secretion from human luteinized granulosa cells. There is an increase in mRNA expression of liver x receptor (LXR) and a decrease in sterol regulatory element binding protein 2 (SREBP2) target genes during spontaneous luteolysis in primates, which could result in reduced cholesterol availability for steroidogenesis. Concentrations of 27OH are also increased in primate corpora lutea (CL) during luteolysis, and 27OH is a dual LXR agonist and SREBP2 inhibitor. This was an in vitro study using primary human luteinized granulosa cells in a control versus treatment(s) design. Analyses of CL from sheep undergoing induced or spontaneous luteolysis were also performed, along with database mining of microarray data from rhesus macaque CL. Primary luteinizing granulosa cells were obtained from 37 women aged 24-44 who were undergoing oocyte donation or IVF for male factor or idiopathic infertility, and cells were further luteinized in vitro using human chorionic gonadotropin. Three approaches to test the effect of 27OH produced via CYP27A1 (cytochrome p450, family 27, subfamily A, polypeptide 1) on luteinized granulosa cells were used: (i) direct 27OH supplementation, (ii) induction of endogenous CYP27A1 activity via pharmacologic inhibition of steroidogenesis, and (iii) siRNA-mediated knockdown to directly inhibit CYP27A1 as well as cholesterol transport into the mitochondria via the steroidogenic acute regulatory protein (STAR). Endpoints included: progesterone (P4) secretion into culture media determined by enzyme immunoassay, cholesterol efflux and uptake assays using fluorescent lipid analogs, and mRNA expression determined via semi-quantitative real-time PCR (QPCR). An additional experiment involved QPCR analysis of 40 CL collected from ewes undergoing induced or spontaneous luteolysis, as well as database mining of microarray data generated from 16 rhesus macaque CL collected during spontaneous luteolysis and 13 macaque CL collected during a luteinizing hormone ablation and replacement protocol. The mRNA expression of CYP27A1 was significantly increased during luteolysis in rhesus macaques and sheep in vivo, and CYP27A1 transcription was suppressed by luteinizing hormone and hCG. There was a significant decrease in hCG-stimulated P4 secretion from human luteinized granulosa cells caused by 27OH treatment, and a significant increase in basal and hCG-stimulated P4 synthesis when endogenous 27OH production was inhibited via CYP27A1 knockdown, indicating that 27OH inhibits steroidogenesis. Pharmacologic inhibition of steroidogenesis by aminoglutethimide significantly induced LXR and inhibited SREBP2 target gene mRNA expression, indicating that increased oxysterol production occurs when steroidogenesis is suppressed. Inhibiting cholesterol delivery into the mitochondria via knockdown of STAR resulted in reduced SREBP2 target gene mRNA expression, indicating that STAR function is necessary to maintain SREBP2-mediated transcription. The effects of 27OH treatment on markers of LXR and SREBP2 activity were moderate, and knockdown of CYP27A1 did not prevent aminoglutethimide-induced changes in LXR and SREBP2 target gene mRNA expression. These observations indicate that 27OH inhibits P4 secretion partially via mechanisms separate from its role as an LXR agonist and SREBP2 inhibitor, and also demonstrate that other oxysterols are involved in modulating LXR and SREBP2-mediated transcription when steroidogenesis is suppressed. None. Luteinized granulosa cells may differ from luteal cells, and the effect on luteal function in vivo was not directly tested. The mechanisms that cause the initial rise in CYP27A1 mRNA expression during luteolysis are also not clear. The factors causing luteolysis in primates have not yet been determined. This study provides functional evidence of a novel mechanism via increased 27OH synthesis during luteolysis, which subsequently represses progesterone secretion. Increased 27OH may also facilitate the progression of luteolysis in domestic animal species. The authors have nothing to disclose. Support was provided by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH), award number R00HD067678 to R.L.B.