Excess Free Cholesterol Research Articles

Quantifying ABCA1/apoA-1 Signaling Pathways with AFM Imaging and Lipidomic Analysis.

The role of lipid metabolic pathways in the pathophysiology of primary open-angle glaucoma (POAG) has been thoroughly elucidated, with pathways involved in lipid-related disorders such as hypercholesterolemia and hyperlipoprotein accumulation being of particular interest. The ABCA1/apoA-1 transduction pathway moderates reverse cholesterol transport (RCT), facilitating the transport of free cholesterol (FC) and phospholipids (PL) and preventing intracellular lipid aggregates in retinal ganglion cells (RGCs) due to excess FCs and PLs. A deficiency of ABCA1 transporters, and thus, dysregulation of the ABCA1/apoA-1 transduction pathway, may potentiate cellular lipid accumulation, which affects the structural and mechanical features of the cholesterol-rich RGC membranes. Atomic force microscopy (AFM) is a cutting-edge imaging technique suitable for imaging topographical surfaces of a biological specimen and determining its mechanical properties and structural features. The versatility and precision of this technique may prove beneficial in understanding the effects of ABCA1/apoA-1 pathway downregulation and decreased cholesterol efflux in RGCs and their membranes. In this protocol, ABCA1-/- RGC mouse models are prepared over the course of 3days and are then compared with non-knockout ABCA1 RGC mouse models through AFM imaging of topographical surfaces to examine the difference in membrane dynamics of knockout vs. non-knockout models. Intracellular and extracellular levels of lipids are quantified through high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS).

Identifying the Transcriptional Drivers of Metastasis Embedded within Localized Melanoma.

We analyzed human melanoma transcriptomics data to identify a gene signature predictive of metastasis. To rapidly test clinical signatures, we built a genetic metastasis platform in adult zebrafish and identified GRAMD1B as a suppressor of melanoma metastasis. GRAMD1B-associated cholesterol overload activates an AP-1 program to promote melanoma invasion. This article is highlighted in the In This Issue feature, p. 1.

MicroRNA-379-5p regulates free cholesterol accumulation and relieves diet induced-liver damage in db/db mice via STAT1/HMGCS1 axis

Lipotoxicity induced by the overload of lipid in the liver, especially excess free cholesterol (FC), has been recognized as one of driving factors in the transition from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). MicroRNA (miR)-379-5p has been reported to play regulatory roles in hepatic triglyceride homeostasis, but the relationship of miR-379-5p and hepatic cholesterol homeostasis has never been touched. In the current study, we found that hepatic miR-379-5p levels were decreased obviously in NAFLD patients and model mice compared with their controls. Moreover, miR-379-5p was discovered to be able to inhibit intracellular FC accumulation and alleviate mitochondrial damage induced by palmitic acid (PA) in vitro. Furthermore, overexpression of miR-379-5p in HFHC-fed db/db mice could reduce the level of hepatic total cholesterol (TC) and FC, and ameliorate hepatic injury reflected by the lower serum alanine aminotransferase (ALT) and aspartate transaminase (AST). Subsequently, by combining spectrometry (MS) and luciferase assay, we identified miR-379-5p suppressed STAT1 through transcriptional and translational regulation. Finally, we confirmed that STAT1 was a transcriptional factor of HMGCS1. In conclusion, miR-379-5p inhibits STAT1 expression and regulates cholesterol metabolism through the STAT1/HMGCS1 axis, suggesting miR-379-5p might be applied to improve lipotoxicity in the future.

Kupffer cells are protective in alcoholic steatosis

Massive accumulation of lipids is a characteristic of alcoholic liver disease. Excess of hepatic fat activates Kupffer cells (KCs), which affect disease progression. Yet, KCs contribute to the resolution and advancement of liver injury. Aim of the present study was to evaluate the effect of KC depletion on markers of liver injury and the hepatic lipidome in liver steatosis (Lieber-DeCarli diet, LDC, female mice, mixed C57BL/6J and DBA/2J background). LDC increased the number of dead hepatocytes without changing the mRNA levels of inflammatory cytokines in the liver. Animals fed LDC accumulated elevated levels of almost all lipid classes. KC ablation normalized phosphatidylcholine and phosphatidylinositol levels in LDC livers, but had no effect in the controls. A modest decline of trigylceride and diglyceride levels upon KC loss was observed in both groups. Serum aminotransferases and hepatic ceramide were elevated in all animals upon KC depletion, and in particular, cytotoxic very long-chain ceramides increased in the LDC livers. Meta-biclustering revealed that eight lipid species occurred in more than 40% of the biclusters, and four of them were very long-chain ceramides. KC loss was further associated with excess free cholesterol levels in LDC livers. Expression of inflammatory cytokines did, however, not increase in parallel. In summary, the current study described a function of KCs in hepatic ceramide and cholesterol metabolism in an animal model of LDC liver steatosis. High abundance of cytotoxic ceramides and free cholesterol predispose the liver to disease progression suggesting a protective role of KCs in alcoholic liver diseases.

Rebuttal to: Liver Steatosis is a Driving Factor of Inflammation.

Hepatosteatosis, a hallmark of alcoholic and nonalcoholic fatty liver disease (NAFLD), is characterized by accumulation of lipid storage organelles called lipid droplets (LDs) within hepatocytes. LDs primarily function to maintain cellular metabolism and protect cells from lipotoxicity by storing excess free fatty acids and cholesterol esters during nutrient excess. Conversely, LDs serve as energy sources during periods of nutrient deprivation or tissue growth.1 During chronic or excess caloric intake when lipid availability exceeds the capacity of LDs to sequester excess free fatty acids, LDs accumulate in hepatocytes.

Current models of apolipoprotein A-I lipidation by adenosine triphosphate binding cassette transporter A1.

The primary cardioprotective function of high-density lipoprotein (HDL) is to remove excess cellular free cholesterol (FC) from peripheral tissues and deliver it to the liver. Here, we summarize recent research that examines apolipoprotein A-I (apoA-I) lipidation models by adenosine triphosphate binding cassette transporter A1 (ABCA1) and discuss its relevance in atherosclerotic cardiovascular disease (ASCVD). The first step in HDL formation involves the interaction between apoA-I and ABCA1, where ABCA1 mediates the removal of FC and phospholipids from lipid-laden macrophages to form discoidal nascent HDL (nHDL). However, there are currently no clear-cut systematic models that characterize HDL formation. A number of recent studies have investigated the importance of apoA-I C- and N-terminal domains required for optimal cholesterol efflux and nHDL production. Furthermore, functional ABCA1 is required for direct or indirect binding to apoA-I where ABCA1 dimer-monomer interconversion facilitates apoA-I lipidation from plasma membrane microdomains. Microparticles are also another lipid source for apoA-I solubilization into nHDL. ApoA-I and ABCA1 are key factors in macrophage-mediated cholesterol efflux and nHDL production. Understanding of the key steps in HDL formation may unlock the therapeutic potential of HDL and improve clinical management of ASCVD.

Excess HDL free cholesterol bioavailability drives free cholesterol accretion into macrophages and erythrocytes in SCRAB1-/- mice.

Excess HDL free cholesterol bioavailability drives free cholesterol accretion into macrophages and erythrocytes in SCRAB1-/- mice.

Serum opacity factor normalizes dysfunctional high density lipoproteins in SRBI -/- female mice and rescues fertility

Background and Aims: Previous views about the role of HDL are shifting from, “HDL is good,” to “Some HDL is dysfunctional.” HDL from SR-B1-/- mice is dysfunctional; these mice are atherosusceptible, have dysfunctional erythrocytes and platelets, and females are infertile. Notably, SR-B1-/- mice have high plasma levels of HDL that is free cholesterol (FC)-rich relative to WT mice producing a state of excess FC bioavailability. Dietary probucol, an HDL-lowering drug, partially reverses infertility in SR-B1-/- mice. The mechanisms underlying ovaritoxicity is not known and interventions that fully reverse this state have not been identified. We hypothesized that a bacterial protein, serum opacity factor (SOF), which acts on HDL and lowers cholesterol in WT mice by ∼43% in 3 hours, would normalize HDL and rescue infertility in SRBI -/- female mice.

SIRT6 inhibits cholesterol crystal-induced vascular endothelial dysfunction via Nrf2 activation

Sirtuin 6 (SIRT6), a nicotinamide adenine dinucleotide-dependent deacetylase, participates in various age-related disorders, such as dyslipidemia and cardiovascular diseases. Recent studies have revealed that minute cholesterol crystals (CCs), which are generated after excess free cholesterol accumulation, form not only in mature atherosclerotic plaques but also extremely early in atherosclerosis. Since endothelial dysfunction is an early feature of atherogenesis, this study was designed to investigate the role of SIRT6 in minute CC-induced endothelial dysfunction and the related mechanism. We found that minute CCs could be endocytosed by endothelial cells (ECs), which then decreased nitric oxide (NO) levels and endothelial nitric oxide synthase (eNOS) activity and expression, upregulated the expression of adhesion molecules and enhanced monocyte adhesion to ECs. In addition, minute CCs significantly suppressed SIRT6 expression in ECs. Moreover, the overexpression of SIRT6 could mitigate minute CC-induced endothelial dysfunction. In addition, the expression of Nuclear factor erythroid2-related factor2 (Nrf2) was suppressed after minute CC treatment, whereas SIRT6 overexpression reversed this decrease in Nrf2 expression. More importantly, Nrf2 activation also notably attenuated minute CC-induced endothelial dysfunction. In vivo experiments further indicated that endothelium-specific SIRT6 depletion impaired vascular endothelial function and suppressed Nrf2 expression in hyperlipidemic mice. Taken together, these results indicate that SIRT6 rescues minute CC-induced endothelial dysfunction partly via Nrf2 activation.

Schnyder corneal dystrophy-associated UBIAD1 mutations cause corneal cholesterol accumulation by stabilizing HMG-CoA reductase.

Schnyder corneal dystrophy (SCD) is a rare genetic eye disease characterized by corneal opacification resulted from deposition of excess free cholesterol. UbiA prenyltransferase domain-containing protein-1 (UBIAD1) is an enzyme catalyzing biosynthesis of coenzyme Q10 and vitamin K2. More than 20 UBIAD1 mutations have been found to associate with human SCD. How these mutants contribute to SCD development is not fully understood. Here, we identified HMGCR as a binding partner of UBIAD1 using mass spectrometry. In contrast to the Golgi localization of wild-type UBIAD1, SCD-associated mutants mainly resided in the endoplasmic reticulum (ER) and competed with Insig-1 for HMGCR binding, thereby preventing HMGCR from degradation and increasing cholesterol biosynthesis. The heterozygous Ubiad1 G184R knock-in (Ubiad1G184R/+) mice expressed elevated levels of HMGCR protein in various tissues. The aged Ubiad1G184R/+ mice exhibited corneal opacification and free cholesterol accumulation, phenocopying clinical manifestations of SCD patients. In summary, these results demonstrate that SCD-associated mutations of UBIAD1 impair its ER-to-Golgi transportation and enhance its interaction with HMGCR. The stabilization of HMGCR by UBIAD1 increases cholesterol biosynthesis and eventually causes cholesterol accumulation in the cornea.

Revisiting Reverse Cholesterol Transport in the Context of High-Density Lipoprotein Free Cholesterol Bioavailability.

Dysregulated free cholesterol (FC) metabolism has been implicated in nearly all stages of atherosclerosis, the underlying cause of most cardiovascular disease. According to a widely cited model, the burden of macrophage FC in the arterial wall is relieved by transhepatic reverse cholesterol transport (RCT), which comprises three successive steps: (1) macrophage FC efflux to high-density lipoprotein (HDL) and/or its major protein, apolipoprotein AI; (2) FC esterification by lecithin:cholesterol acyltransferase (LCAT); and (3) HDL-cholesteryl ester (CE) uptake via the hepatic HDL-receptor, scavenger receptor class B type 1 (SR-B1). Recent studies have challenged the validity of this model, most notably the role of LCAT, which appears to be of minor importance. In mice, most macrophage-derived FC is rapidly cleared from plasma (t1/2 < 5 min) without esterification by hepatic uptake; the remainder is taken up by multiple tissue and cell types, especially erythrocytes. Further, some FC is cleared by the nonhepatic transintestinal pathway. Lastly, FC movement among lipid surfaces is reversible, so that a higher-than-normal level of HDL-FC bioavailability-defined by high plasma HDL levels concurrent with a high mol% HDL-FC-leads to the transfer of excess FC to cells in vivo. SR-B1-/- mice provide an animal model to study the mechanistic consequences of high HDL-FC bioavailability that provokes atherosclerosis and other metabolic abnormalities. Future efforts should aim to reduce HDL-FC bioavailability, thereby reducing FC accretion by tissues and the attendant atherosclerosis.

Silencing the ACAT1 Gene in Human SH-SY5Y Neuroblastoma Cells Inhibits the Expression of Cyclo-Oxygenase 2 (COX2) and Reduces β-Amyloid-Induced Toxicity Due to Activation of Protein Kinase C (PKC) and ERK

BackgroundAcyl-coenzymeA: cholesterol acyltransferase (ACAT) 1, a key enzyme converting excess free cholesterol to cholesterol esters, has been demonstrated to be associated with the pathogenesis of Alzheimer disease (AD). However, the mechanism underlying the protective role of ACAT1 blockage in AD progression remains elusive.Material/MethodsHuman neuroblastoma SH-SY5Y cells were treated for 24 h with increasing concentrations of aggregated Aβ25–35 (5, 15, 25, and 45 μmol) with or without the ACAT1 siRNA pretreatment. Cell viability analysis was measured by CCK-8 assay. The genome-wide correlation between ACAT1 and all other probe sets was measured by the Pearson correlation coefficient (r). Western blotting was used to detect the ACAT1 protein expression in the hippocampus of APP/PSN transgenic AD mice. The mRNA level for each target was analyzed by qPCR. Western blotting was used to detect the ACAT1, cyclo-oxygenase-2 (Cox2), Calcium voltage-gated channel subunits (CACNAs), and ERK/PKC proteins in SH-SY5Y cells with or without the ACAT1 siRNA pretreatment in the presence of Aβ25–35.ResultsThe expression of ACAT1 was significantly increased in the hippocampus of APP/PSN mice, and also showed an increasing trend when SH-SY5Y cells were exposed to Aβ25–35. Silencing ACAT1 significantly attenuated Aβ-induced cytotoxicity and cell apoptosis in SH-SY5Y cells. The genome-wide correlation analysis showed that Ptgs2 had the most significant correlation with Acat1 in the hippocampus of BXD RI mice. We further determined the regulatory effect of ACAT1 on COX2 expression by silencing or over-expressing ACAT1 in SH-SY5Y cells and found that silencing ACAT1 played a protective role in AD progression by regulating CACNAs and PKC/ERK signaling cascades.ConclusionsSilencing ACAT1 attenuates Aβ25–35-induced cytotoxicity and cell apoptosis in SH-SY5Y cells, which may due to the synergistic effect of ACAT1 and COX2 through PKC/ERK pathways.

Abstract 4416: Studies on role of acyl-CoA cholesterol acyltransferase (ACAT) expression in ovarian/endometrial cancer progression - An invitro study

Abstract INTRODUCTION: Cancer progression and tumorigenesis have been linked to alterations in cellular metabolic pathways of lipid and cholesterol synthesis in many cancers. Increased levels of cholesterol esters (CE) have been reported in breast cancer, leukemia, glioma and prostate cancer, but not exploited in detail in ovarian and endometrial cancers. Inside cells, excess free cholesterol is esterified by acyl-CoA cholesterol acyltransferase (ACAT) and stored as cholesteryl ester (CE) in lipid droplets (LDs). Cholesteryl ester accumulation may be a consequence of loss of tumor suppressor PTEN and subsequent activation of PI3K/AKT pathway as observed in pancreatic cancer. In the present study, we focused on the role of ACAT and cholesterol esters in altered cholesterol metabolism in ovarian/endometrial cancer cell lines. METHODS: We studied the effect of abrogation of cholesterol esterification either by an ACAT-1 inhibitor/shRNA knockdown or via inhibition of PI3K/AKT/mTOR pathways and subsequent effect on ovarian/endometrial cancer cells proliferation, invasion, cell migration and tumorigenesis. The expression of ACAT was studied at the protein and mRNA levels in a panel of ovarian/ endometrial cancer cell lines and also in normal and ovarian cancer clinical samples. ACAT expression was assessed by western blotting, ELISA, quantitative RT-PCR and immunostaining techniques. RESULTS: Our results reveal an elevated expression of ACAT-1 at both protein and mRNA levels in ovarian and endometrial cancer cell lines as compared to primary cells. Our results also provide preliminary data on involvement of ACAT and cholesterol esters in the progression and metastasis of ovarian cancer. CONCLUSION: These studies may aid in the development of new biomarkers or treatment targets for ovarian/endometrial cancer. Citation Format: Vijayalakshmi N. Ayyagari, Xinjia Wang, Laurent Brard. Studies on role of acyl-CoA cholesterol acyltransferase (ACAT) expression in ovarian/endometrial cancer progression - An invitro study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4416. doi:10.1158/1538-7445.AM2017-4416

LDL-C levels in older people: Cholesterol homeostasis and the free radical theory of ageing converge

The cardiovascular disease (CVD) risk factor, low density lipoprotein cholesterol (LDL-C) increases with age, up until the midpoint of life in males and females. However, LDL-C can decrease with age in older men and women. Intriguingly, a recent systematic review also revealed an inverse association between LDL-C levels and cardiovascular mortality in older people; low levels of LDL-C were associated with reduced risk of mortality. Such findings are puzzling and require a biological explanation. In this paper a hypothesis is proposed to explain these observations. We hypothesize that the free radical theory of ageing (FRTA) together with disrupted cholesterol homeostasis can account for these observations. Based on this hypothesis, dysregulated hepatic cholesterol homeostasis in older people is characterised by two distinct metabolic states. The first state accounts for an older person who has elevated plasma LDL-C. This state is underpinned by the FRTA which suggests there is a decrease in cellular antioxidant capacity with age. This deficiency enables hepatic reactive oxidative species (ROS) to induce the total activation of HMG-CoA reductase, the key rate limiting enzyme in cholesterol biosynthesis. An increase in cholesterol synthesis elicits a corresponding rise in LDL-C, due to the downregulation of LDL receptor synthesis, and increased production of very low density lipoprotein cholesterol (VLDL-C). In the second state of dysregulation, ROS also trigger the total activation of HMG-CoA reductase. However, due to an age associated decrease in the activity of cholesterol-esterifying enzyme, acyl CoA: cholesterol acyltransferase, there is restricted conversion of excess free cholesterol (FC) to cholesterol esters. Consequently, the secretion of VLDL-C drops, and there is a corresponding decrease in LDL-C. As intracellular levels of FC accumulate, this state progresses to a pathophysiological condition akin to nonalcoholic fatty liver disease. It is our conjecture this deleterious state has the potential to account for the inverse association between LDL-C level and CVD risk observed in older people.

ATR-101, a Selective and Potent Inhibitor of Acyl-CoA Acyltransferase 1, Induces Apoptosis in H295R Adrenocortical Cells and in the Adrenal Cortex of Dogs.

ATR-101 is a novel, oral drug candidate currently in development for the treatment of adrenocortical cancer. ATR-101 is a selective and potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase 1 (ACAT1), an enzyme located in the endoplasmic reticulum (ER) membrane that catalyzes esterification of intracellular free cholesterol (FC). We aimed to identify mechanisms by which ATR-101 induces adrenocortical cell death. In H295R human adrenocortical carcinoma cells, ATR-101 decreases the formation of cholesteryl esters and increases FC levels, demonstrating potent inhibition of ACAT1 activity. Caspase-3/7 levels and terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeled-positive cells are increased by ATR-101 treatment, indicating activation of apoptosis. Exogenous cholesterol markedly potentiates the activity of ATR-101, suggesting that excess FC that cannot be adequately esterified increases caspase-3/7 activation and subsequent cell death. Inhibition of calcium release from the ER or the subsequent uptake of calcium by mitochondria reverses apoptosis induced by ATR-101. ATR-101 also activates multiple components of the unfolded protein response, an indicator of ER stress. Targeted knockdown of ACAT1 in an adrenocortical cell line mimicked the effects of ATR-101, suggesting that ACAT1 mediates the cytotoxic effects of ATR-101. Finally, in vivo treatment of dogs with ATR-101 decreased adrenocortical steroid production and induced cellular apoptosis that was restricted to the adrenal cortex. Together, these studies demonstrate that inhibition of ACAT1 by ATR-101 increases FC, resulting in dysregulation of ER calcium stores that result in ER stress, the unfolded protein response, and ultimately apoptosis.

Free-cholesterol-mediated autophagy of ORMDL1 stimulates sphingomyelin biosynthesis

Cholesterol confers unique biophysical properties to the plasma membrane bilayer that are essential for maintaining optimal membrane fluidity, which in turn regulate multiple physiological functions required to promote cellular integrity and viability. Conversely, excessive cholesterol causes pathological conditions such as atherosclerosis that can lead to heart attacks. Human atheroma macrophages carry a large burden of free cholesterol (FC) in addition to cholesterol esters. It is recognized that sterols can modulate the levels of other lipids to attain lipid homeostasis; thus, excess FC may play a role in modulating compensatory sphingolipid pathways. Recent studies have shown that excess lipids can cause ER stress and apoptosis. In contrast, autophagy may play a protective role by clearing excess lipids from macrophage foam cell lipid droplets. Interestingly, a macrophage study using a TLR4-specifc agonist showed that de novo sphingolipid biosynthesis is essential for autophagy induction, suggesting links between sphingolipid biosynthesis and autophagy. While the role of autophagy in removing excess lipids has been the focus of many studies, its role in fine-tuning cellular lipid homeostasis remains largely unexplored.

Statin use and non-alcoholic steatohepatitis in at risk individuals

Excess hepatic free cholesterol contributes to the pathogenesis of non-alcoholic steatohepatitis, and statins reduce cholesterol synthesis. Aim of this study was to assess whether statin use is associated with histological liver damage related to steatohepatitis. The relationship between statin use, genetic risk factors, and liver damage was assessed in a multi-center cohort of 1201 European individuals, who underwent liver biopsy for suspected non-alcoholic steatohepatitis. Statin use was recorded in 107 subjects, and was associated with protection from steatosis, NASH, and fibrosis stage F2-F4, in a dose-dependent manner (adjusted p<0.05 for all). In 100 treated patients matched 1:1 for modality of recruitment, gender, presence of IFG or type 2 diabetes, PNPLA3 I148M risk alleles, TM6SF2 E167K variant, age, and BMI, statin use remained associated with protection from steatosis (OR 0.09, 95% C.I. 0.01-0.32; p=0.004), steatohepatitis (OR 0.25, 95% C.I. 0.13-0.47; p<0.001), and fibrosis stage F2-F4 (OR 0.42, 95% C.I. 0.20-0.8; p=0.017). Results were confirmed in a second analysis, where individuals were matched within recruitment center (p<0.05 for all). The protective effect of statins on steatohepatitis was stronger in subjects not carrying the I148M PNPLA3 risk variant (p=0.02 for interaction), as statins were negatively associated with steatohepatitis in patients negative (p<0.001), but not in those positive for the I148M variant (p=n.s.). Statin use was associated with protection towards the full spectrum of liver damage in individuals at risk of non-alcoholic steatohepatitis. However, the I148M PNPLA3 risk variant limited this beneficial effect.

Lysosomal acid lipase: at the crossroads of normal and atherogenic cholesterol metabolism.

Unregulated cellular uptake of apolipoprotein B-containing lipoproteins in the arterial intima leads to the formation of foam cells in atherosclerosis. Lysosomal acid lipase (LAL) plays a crucial role in both lipoprotein lipid catabolism and excess lipid accumulation as it is the primary enzyme that hydrolyzes cholesteryl esters derived from both low density lipoprotein (LDL) and modified forms of LDL. Evidence suggests that as atherosclerosis progresses, accumulation of excess free cholesterol in lysosomes leads to impairment of LAL activity, resulting in accumulation of cholesteryl esters in the lysosome as well as the cytosol in foam cells. Impaired metabolism and release of cholesterol from lysosomes can lead to downstream defects in ATP-binding cassette transporter A1 regulation, needed to offload excess cholesterol from plaque foam cells. This review focuses on the role LAL plays in normal cholesterol metabolism and how the associated changes in its enzymatic activity may ultimately contribute to atherosclerosis progression.

Concentration-Dependent Diversifcation Effects of Free Cholesterol Loading on Macrophage Viability and Polarization

Background/Aims: The accumulation of free cholesterol in atherosclerotic lesions has been well documented in both animals and humans. In studying the relevance of free cholesterol buildup in atherosclerosis, contradictory results have been generated, indicating that free cholesterol produces both pro- and anti-atherosclerosis effects in macrophages. This inconsistency might stem from the examination of only select concentrations of free cholesterol. In the present study, we sought to investigate the implication of excess free cholesterol loading in the pathophysiology of atherosclerosis across a broad concentration range from (in µg/ml) 0 to 60. Methods: Macrophage viability was determined by measuring formazan formation and flow cytometry viable cell counting. The polarization of M1 and M2 macrophages was differentiated by FACS (Fluorescence-Activated Cell Sorting) assay. The secretion of IL-1β in macrophage culture medium was measured by ELISA kit. Macrophage apoptosis was detected by flow cytometry using a TUNEL kit. Results: Macrophage viability was increased at the treatment of lower concentrations of free cholesterol from (in µg/ml) 0 to 20, but gradually decreased at higher concentrations from 20 to 60. Lower free cholesterol loading induced anti-inflammatory M2 macrophage polarization. The activation of the PPARγ (Peroxisome Proliferator-Activated Receptor gamma) nuclear factor underscored the stimulation of this M2 phenotype. Nevertheless, higher levels of free cholesterol resulted in pro-inflammatory M1 activation. Moreover, with the application of higher free cholesterol concentrations, macrophage apoptosis and secretion of the inflammatory cytokine IL-1β increased significantly. Conclusion: These results for the first time demonstrate that free cholesterol could render concentration-dependent diversification effects on macrophage viability, polarization, apoptosis and inflammatory cytokine secretions, thereby reconciling the pros and cons of free cholesterol buildup in macrophages to the pathophysiology of atherosclerosis.

The natural compound berberine positively affects macrophage functions involved in atherogenesis

Background and aimsWe investigated the effect of berberine (BBR), an alkaloid showing antiatherogenic properties beyond the cholesterol lowering capacity, on macrophage cholesterol handling upon exposure to human serum and on macrophage responses to excess free cholesterol (FC) loading. Methods and resultsMouse and human macrophages were utilized as cellular models. Cholesterol content was measured by a fluorimetric assay; cholesterol efflux, cytotoxicity and membrane FC distribution were evaluated by radioisotopic assays. Monocyte chemotactic protein-1 (MCP-1) secretion was measured by ELISA; membrane ruffling and macropinocytosis were visualized by confocal microscopy. Exposure of cholesterol-enriched MPM to serum in the presence of 1 μM BBR resulted in a reduction of intracellular cholesterol content twice greater than exposure to serum alone (−52%; p < 0.01 and −21%; p < 0.05), an effect not mediated by an increase of cholesterol efflux, but rather by the inhibition of cholesterol uptake from serum. Consistently, BBR inhibited in a dose-dependent manner cholesterol accumulation in human macrophages exposed to hypercholesterolemic serum. Confocal microscope analysis revealed that BBR inhibited macropinocytosis, an independent-receptor process involved in LDL internalization. Macrophage FC-enrichment increased MCP-1 release by 1.5 folds, increased cytotoxicity by 2 fold, and induced membrane ruffling; all these responses were markedly inhibited by BBR. FC-enrichment led to an increase in plasma membrane cholesterol by 4.5 folds, an effect counteracted by BBR. ConclusionWe showed novel potentially atheroprotective activities of BBR in macrophages, consisting in the inhibition of serum-induced cholesterol accumulation, occurring at least in part through an impairment of macropinocytosis, and of FC-induced deleterious effects.