Cholesterol Accumulation In Cells Research Articles

Abstract 3046: Macrophage-smooth Muscle Cell Interactions In Atherosclerotic Foam Cell Formation

Background: A hallmark of atherosclerosis is uncontrolled uptake of atherogenic lipoproteins by artery wall cells that accumulate large amounts of cholesteryl esters (“foam cells”). While smooth muscle cells (SMCs) and macrophages are in close proximity in lesions and contribute to cholesterol accumulation, whether these cells influence each other’s tendency to form foam cells is not clearly established. Hypothesis: Direct and indirect interactions between SMCs and macrophages promote foam cell formation by both cell types. Method: Human vascular SMCs were seeded onto macrophages to mimic direct macrophage-SMC interactions (n=5). Alternatively, conditioned media generated from human macrophages or SMCs with or without lipoprotein exposure was incubated with the alternate cell type (n=3). Aggregated lipoproteins were added in both models to induce cellular cholesterol accumulation. Intracellular cholesterol was quantified to compare lipoprotein uptake in cocultures relative to monocultures. Crosstalk based on CellChat analysis using single-cell RNA sequencing data from human coronary samples was performed to identify potential factors responsible for foam cell development (n=3). One-way ANOVA was used for statistical analysis. Result: SMCs showed a 4-fold (+/-0.3, p=0.002) increase in cell cholesterol accumulation when in direct contact with macrophages and a 3-fold (+/- 0.3, p<0.0001) increase when exposed to the conditioned media from macrophage foam cells. In contrast, macrophages showed a 3-fold reduction (+/- 0.3, p=0.001) in intracellular cholesterol accumulation when in direct contact with SMCs and a 2-fold reduction (+/-0.2, p=0.0005) when exposed to SMC foam cell-conditioned media. CellChat analysis suggested macrophage foam cells but not macrophage non-foam cells promote SMC foam cell formation by secretion of inflammatory cytokines, and that SMCs promote macrophage foam cell formation by secretion of extracellular matrix components. Conclusion: Our study provides novel understanding of how macrophage-SMC interactions influence macrophage and SMC foam cell formation. Further studies will be presented examining the role of SMC extracellular matrix proteins and macrophage-secreted cytokines on foam cell formation.

The Role of Cytokines in Cholesterol Accumulation in Cells and Atherosclerosis Progression

Atherosclerosis is the most common cardiovascular disease and is the number one cause of death worldwide. Today, atherosclerosis is a multifactorial chronic inflammatory disease with an autoimmune component, accompanied by the accumulation of cholesterol in the vessel wall and the formation of atherosclerotic plaques, endothelial dysfunction, and chronic inflammation. In the process of accumulation of atherogenic lipids, cells of the immune system, such as monocytes, macrophages, dendritic cells, etc., play an important role, producing and/or activating the production of various cytokines-interferons, interleukins, chemokines. In this review, we have tried to summarize the most important cytokines involved in the processes of atherogenesis.

Achilles tendon xanthoma thickness associates to serum lipoprotein functions alterations

Background and Aims : Achilles tendon xanthoma (ATX) formation involves the uptake of modified LDL by macrophages within the tendon similarly to that occurring in the atheroma. Macrophage cholesterol homeostasis strictly depends on serum lipoprotein functions, including the HDL capacity to promote cell cholesterol efflux (cholesterol efflux capacity, CEC) and the serum capacity to promote cell cholesterol accumulation (cholesterol loading capacity, CLC). In this study, we explored the serum lipoproteins functions comparing FH patients with ATX to those without ATX.

Inhibition of PCSK9 affects serum lipoprotein functions in patients with familial hypercholesterolemia

Background and Aims : PCSK9, beyond regulating plasma cholesterol levels, exerts several pleiotropic effects modulating lipid metabolism in extrahepatic cells, including macrophages. Macrophage cholesterol homeostasis strictly depends on serum lipoprotein functions, including the HDL capacity to promote cell cholesterol efflux (cholesterol efflux capacity, CEC) and the serum capacity to promote cell cholesterol accumulation (cholesterol loading capacity, CLC). The aim of the present study was to evaluate potential changes in HDL-CEC and serum CLC after six-month of treatment with PCSK9 inhibitors in Familial Hypercholesterolemia (FH) subjects.

Synthesis of heterocyclic ring-fused analogs of HMG499 as novel degraders of HMG-CoA reductase that lower cholesterol

HMG-CoA reductase (HMGCR) is the rate-limiting enzyme in cholesterol de novo biosynthesis and its degradation may bring therapeutic benefits for the treatment of cardiovascular disease (CVD) and nonalcoholic steatohepatitis (NASH). Before, we disclosed compound HMG499 as a potent HMGCR degrader, which could be a promising agent for treating CVD, however its side-effect of promoting cholesterol accumulation in cells should be eliminated before progression. Herein, a series of novel heterocyclic ring-fused analogs of HMG499 were synthesized and investigated for their activities of stimulating HMGCR degradation using a HMGCR (TM1-8)-GFP reporting system. Among them, the most active compound 29 (QH536) showed an EC50 of 0.22 μΜ in promoting HMGCR degradation, which was about 2 times more potent than HMG499 (EC50 = 0.43 μM). Interestingly, 29 was different from HMG499, it had no side-effect of inducing cholesterol accumulation in cells. Mechanistic studies disclosed that 29 could significantly decrease statin-induced accumulation of HMGCR protein via ubiquitination and degradation of HMGCR through ubiquitin-proteasome pathway and inhibit the cholesterol biosynthesis in cells. Therefore, these heterocyclic ring-fused analogs could be used as promising leads for the development of new types of agents against CVD. Furthermore, 29 also lowered cholesterol levels and suppressed TGFβ1-induced proliferation of LX-2 hepatic stellate cells in a dose-dependent manner. In particular, 29 not only decreased the NASH associated fibrotic mRNA and protein expression of α-SMA, COL1A1, TIMP1 and TGFβ1 but also suppressed cholesterol levels and inflammatory genes of TNF-α, IL-6 an IL-1β in RAW264.7 macrophage cells, indicating that 29 may bring therapeutic benefit to treat NASH.

Emerging investigator series: long-term exposure of amorphous silica nanoparticles disrupts the lysosomal and cholesterol homeostasis in macrophages

Long-term ASiNP exposure led to lysosomal density and size increments as well as protein changes, and autophagosome and cholesterol accumulation in cells.

Effects of Aster B-mediated intracellular accumulation of cholesterol on inflammatory process and myocardial cells in acute myocardial infarction

BackgroundTo investigate the effect of cholesterol accumulation in cells on the inflammatory process of acute myocardial infarction and cardiomyocytes and its mechanism. MethodsBlood samples of 15 patients with myocardial infarction were clinically collected to detect enzyme levels of cholesterol and related myocardial parameters in the serum. Correlation analysis was carried out. At the cellular level, simulation of cholesterol entry and exit from cells was conducted by a liposome-loaded cholesterol model in this study, and BNP and inflammatory factors were detected with enzyme-linked immunosorbent assay. Moreover, to investigate the molecular mechanism of myocardial damage caused by cholesterol, Gramd1b and Prkaca of HL-1 were knocked down with small interference RNA technique. Then, inhibitor C3 was used to weaken RhoA activity to explore the level of cardiac muscle cell BNP in order to identify key protein target sites that may be involved in the process of cholesterol damage to cardiac muscle cells. ResultsSerum cholesterol concentration showed a significantly positive correlation with the levels of AST, CK, and LD in serum of patients with myocardial infarction. Cholesterol accumulation in cardiac muscle cells significantly increased the levels of BNP, inflammatory factors (IL-1β, IL-6, TNF-α, and CCL-2) in cardiac muscle cells, which exacerbated cardiomyocyte damage. Conversely, cholesterol excretion caused significant downregulation of BNP and inflammatory factors. Moreover, after knocking down Gramd1b, the accumulation of cholesterol in myocardial cells decreased, the levels of BNP and inflammatory factors significantly reduced, and the degree of myocardial cell damage was weakened. Knockdown of Prkaca inhibited RhoA activity and reversed cholesterol-induced elevation of BNP and inflammatory factors. ConclusionASTER B-mediated intracellular accumulation of cholesterol in cardiac muscle cells may cause cardiomyocyte damage and inflammatory factor infiltration through PKA-Ca2+-RhoA pathways.

Impact of Methyl-β-Cyclodextrin and Apolipoprotein A-I on The Expression of ATP-Binding Cassette Transporter A1 and Cholesterol Depletion in C57BL/6 Mice Astrocytes.

Objective Dysregulation of cholesterol metabolism in the brain is responsible for many lipid storage disorders, including Niemann-Pick disease type C (NPC). Here, we have investigated whether cyclodextrin (CD) and apolipoprotein A-I (apoA-I) induce the same signal to inhibit cell cholesterol accumulation by focusing on the main proteins involved in cholesterol homeostasis in response to CD and apoA-I treatment. Materials and Methods In this experimental study, astrocytes were treated with apoA-I or CD and then lysed in RIPA buffer. We used Western blot to detect protein levels of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) and ATP-binding cassette transporter A1 (ABCA1). Cell cholesterol content and cholesterol release in the medium were also measured. Results ApoA-I induced a significant increase in ABCA1 and a mild increase in HMGCR protein level, whereas CD caused a significant increase in HMGCR with a significant decrease in ABCA1. Both apoA-I and CD increased cholesterol release in the medium. A mild, but not significant increase, in cell cholesterol content was seen by apoA-I; however, a significant increase in cell cholesterol was detected when the astrocytes were treated with CD. ConclusionCD, like apoA-I, depletes cellular cholesterol. This depletion occurs in a different way from apoA-I that is through cholesterol efflux. Depletion of cell cholesterol with CDs led to reduced protein levels of ABCA1 along with increased HMGCR and accumulation of cell cholesterol. This suggested that CDs, unlike apoA-I, could impair the balance between cholesterol synthesis and release, and interfere with cellular function that depends on ABCA1.

Properties of Lipid Rafts in Two Epigenetically Distinct Subtypes of the Oncogenic Cell‐line SW13

IntroductionMutant GOF p53 is an epigenetic regulator which promotes oncogenesis. Many cancers with a GOF mutation accumulate cholesterol and inhibition of cholesterol synthesis with statin treatment reverses some of the pro‐oncogenic properties of GOF p53. Our central hypothesis is that lipid raft signaling is altered by cholesterol accumulation in cells expressing a GOF p53. To address the role of cholesterol in promoting oncogenesis, we use the SW13 cell line bearing a GOF p53 (H193Y). This cell line has two epigenetically distinct cell subtypes that differentially express genes coding for GPI anchors, cholesterol biosynthesis, and sphingolipids, all of which are components of lipid rafts. In addition, the SW13 subtypes have different oncogenic profiles. The SW13(Vim−) subtype has an epithelial morphology and is highly proliferative. The SW13(Vim+) subtype has a mesenchymal like phenotype and a higher metastatic potential. When treated with histone deacetylase inhibitors (HDACi), SW13(Vim−) appear to adopt a SW13(Vim+) phenotype. Therefore, we treat with HDACi to control the subtype conversion and shRNA to knockdown GOF p53 expression. This experimental approach allows the composition of raft fraction to be correlated to GOF p53 expression and oncogenic properties of each SW13 subtype, with and without p53 expression.MethodsProliferation rate, MMP expression, and migration of SW13(Vim−), SW13(Vim+), shRNA SW13(Vim−), and shRNA SW13(Vim+) were quantitated by EdU assays and in situ zymography respectively. We have isolated lipid raft fractions by the detergent resistant enrichment technique from SW13 cells of each subtype, with and without GOF p53 expression knockdown by shRNA. Proteins were identified by mass spectrometry analysis. Fluorescent cholesterol assays and p53 western blots were used to quantify cholesterol levels and p53 protein expression, respectively.ResultsData suggest that there is a shift from planar rafts in the SW13(Vim−) to caveolar rafts in the SW13(Vim+) line. Although the contribution of GOF p53 to this shift is under study, evidence suggests that raft composition is altered in the shRNA lines, in which GOF p53 has been knocked down by ~90%. In addition, p53 knockdown increases the proliferation rate in SW13(Vim−) (p = 0.01) but does not affect SW13(Vim+). In contrast, p53 knockdown decreases MMP activity in SW13(Vim+) (p = 0.05) but not SW13(Vim−).ConclusionsOur data support the hypothesis that lipid raft signaling is significantly altered between the epigenetically distinct SW13 subtypes and that these differences may correlate to oncogenic potential. In addition, statin treatment, which reverses some of the pro oncogenic properties of GOF p53, may do so, in part, by altering raft composition and signaling.

Exosome-Mediated Transfer of Anti-miR-33a-5p from Transduced Endothelial Cells Enhances Macrophage and Vascular Smooth Muscle Cell Cholesterol Efflux.

Atherosclerosis is a disease of large- and medium-sized arteries that is caused by cholesterol accumulation in arterial intimal cells, including macrophages and smooth muscle cells (SMC). Cholesterol accumulation in these cells can be prevented or reversed in preclinical models-and atherosclerosis reduced-by transgenesis that increases expression of molecules that control cholesterol efflux, including apolipoprotein AI (apoAI) and ATP-binding cassette subfamily A, member 1 (ABCA1). In a previous work, we showed that transduction of arterial endothelial cells (EC)-with a helper-dependent adenovirus (HDAd) expressing apoAI-enhanced EC cholesterol efflux in vitro and decreased atherosclerosis in vivo. Similarly, overexpression of ABCA1 in cultured EC increased cholesterol efflux and decreased inflammatory gene expression. These EC-targeted gene-therapy strategies might be improved by concurrent upregulation of cholesterol-efflux pathways in other intimal cell types. Here, we report modification of this strategy to enable delivery of therapeutic nucleic acids to cells of the sub-endothelium. We constructed an HDAd (HDAdXMoAntimiR33a5p) that expresses an antagomiR directed at miR-33a-5p (a microRNA that suppresses cholesterol efflux by silencing ABCA1). HDAdXMoAntimiR33a5p contains a sequence motif that enhances uptake of anti-miR-33a-5p into exosomes. Cultured EC release exosomes containing small RNA, including miR-33a-5p. After transduction with HDAdXMoAntimiR33a5p, EC-derived exosomes containing anti-miR-33a-5p accumulate in conditioned medium (CM). When this CM is added to macrophages or SMC, anti-miR-33a-5p is detected in these target cells. Exosome-mediated transfer of anti-miR-33a-5p reduces miR-33a-5p by ∼65-80%, increases ABCA1 protein by 1.6-2.2-fold, and increases apoAI-mediated cholesterol efflux by 1.4-1.6-fold (all p ≤ 0.01). These effects were absent in macrophages and SMC incubated in exosome-depleted CM. EC transduced with HDAdXMoAntimiR33a5p release exosomes that can transfer anti-miR-33a-5p to other intimal cell types, upregulating cholesterol efflux from these cells. This strategy provides a platform for genetic modification of intimal and medial cells, using a vector that transduces only EC.

Abstract 4295: Differential lipid raft composition correlates with altered oncogenic properties in the SW13 cell line

Abstract Introduction: Mutant GOF p53 is an epigenetic regulator which promotes oncogenesis. Many cancers with a GOF mutation accumulate cholesterol and inhibition of cholesterol synthesis with statin treatment reverses some of the pro-oncogenic properties of GOF p53. Our central hypothesis is that lipid raft signaling is altered by cholesterol accumulation in cells expressing a GOF p53. To address the role of cholesterol in promoting oncogenesis, we use the SW13 cell line bearing a GOF p53 (H193Y). This cell line has two epigenetically distinct cell subtypes that differentially express genes coding for GPI anchors, cholesterol biosynthesis, and sphingolipids, all of which are components of lipid rafts. In addition, the SW13 subtypes have different oncogenic profiles. The SW13(Vim-) subtype has an epithelial morphology and is highly proliferative. The SW13(Vim+) subtype has a mesenchymal-like phenotype and a higher metastatic potential. When treated with histone deacetylase inhibitors (HDACi), SW13(Vim-) appear to adopt a SW13(Vim+) phenotype. Therefore, we treat with HDACi to control the subtype conversion and shRNA to knock-down GOF p53 expression. This experimental approach allows the composition of raft fraction to be correlated to GOF p53 expression and oncogenic properties of each SW13 subtype, with and without p53 expression. Methods: Proliferation rate, MMP expression, and migration of SW13(Vim-), SW13(Vim+), shRNA SW13(Vim-), and shRNA SW13(Vim+) were quantitated by EdU assays and in situ zymography respectively. We have isolated lipid raft fractions by the detergent-resistant enrichment technique from SW13 cells of each subtype, with and without GOF p53 expression knock-down by shRNA. Proteins by mass spectrometry analysis. Fluorescent cholesterol assays and p53 western blots were used to quantify cholesterol levels and p53 protein expression, respectively. Results: Data suggest that there is a shift from planar rafts in the SW13(Vim-) to caveolar rafts in the SW13(Vim+) line. Although the contribution of GOF p53 to this shift is under study, evidence suggests that raft composition is altered in the shRNA lines, in which GOF p53 has been knocked down by ~90%. In addition, p53 knock-down increases the proliferation rate in SW13(Vim-) (p = 0.01) but does not affect SW13(Vim+). In contrast, p53 knock-down decreases MMP activity in SW13(Vim+) (p = 0.05) but not SW13(Vim-). Conclusions: Our data support the hypothesis that lipid raft signaling is significantly altered between the epigenetically distinct SW13 subtypes and that these differences may correlate to oncogenic potential. In addition, statin treatment, which reverses some of the pro-oncogenic properties of GOF p53, may do so, at least in part, by altering lipid raft composition and signaling. Citation Format: Jacob Moore, Margaux Baatz, Luis Espejo, Kathryn J. Leyva, Elizabeth E. Hull. Differential lipid raft composition correlates with altered oncogenic properties in the SW13 cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4295.

Lnc-ing microRNA activity to atheroprotection.

Cholesterol accumulation in cells and blood vessels promotes the development of vascular diseases. The long non-coding RNA CHROME can help clear excess cholesterol and provide protection from atherosclerosis.

The cholesterol transport inhibitor U18666A inhibits type I feline coronavirus infection

Feline infectious peritonitis (FIP) is a feline coronavirus (FCoV)-induced fatal disease in wild and domestic cats. FCoV exists in two serotypes. Type I FCoV is the dominant serotype worldwide. Therefore, it is necessary to develop antiviral drugs against type I FCoV infection. We previously reported that type I FCoV is closely associated with cholesterol throughout the viral life cycle. In this study, we investigated whether U18666A, the cholesterol synthesis and transport inhibitor, shows antiviral effects against type I FCoV. U18666A induced cholesterol accumulation in cells and inhibited type I FCoV replication. Surprisingly, the antiviral activity of U18666A was suppressed by the histone deacetylase inhibitor (HDACi), Vorinostat. HDACi has been reported to revert U18666A-induced dysfunction of Niemann-Pick C1 (NPC1). In conclusion, these findings demonstrate that NPC1 plays an important role in type I FCoV infection. U18666A or other cholesterol transport inhibitor may be considered as the antiviral drug for the treatment of cats with FIP.

Some Structural and Chemical Changes in Endocardial Endothelium of Rats in Emotional and Pain Stress Complicated by Hypercholesterolemia

The objective of the research was to study the content of some neutral lipids of endocardial endothelium in rats in relation to structural changes occurring in it, in the co-existence of emotional and pain stress, as well as alimentary hypercholesterolemia. Materials and methods. The electric-impulse model was used for stress modeling. Alimentary hypercholesterolemia was modeled feeding animals an atherogenic diet. The concentration of triacylglycerols, free and esterified cholesterol were examined using the method of thin-layer chromatography performed on silica gel. The concentration of free fatty acids was determined using the radiochemical method. The state of endocardial endothelium was studied with the help of light microscopy; the impression smears obtained from macro preparations of ventricle were analyzed.Results. In co-existence of stress and hypercholesterolemia, significant increase in free cholesterol as well as free fatty acid concentration was noticed. This essentially exceeded the analogical indices under the action of stress only. Structural changes in the endocardium followed by desquamation of separate endotheliocytes were the result of stress reaction. In the action of both pathogenic factors, this process was intensified; layer-by-layer exfoliation of endotheliocytes was observed. Conclusions. In acute emotional and pain stress, changes in lipid spectrum of membrane structures of endocardial endotheliocytes the main manifestation of which is the accumulation of free cholesterol in cells and increase in the levels of free fatty acids take place. The increase in the number of desquamated endothelial cells is the result of stress action as well. Alimentary hypercholesterolemia significantly increases such pathological changes.

Protective mechanism in NPC1 – the role of oxLDL

The Niemann Pick disease type C1 (NPC1) is a lysosomal storage disorder caused by a mutation in the NPC1 gene. The mutation disrupts the normal cholesterol traficking and induces cholesterol accumulation in cells of the liver and the brain. Till now, no therapy exists for NPC1 patients. But recently, pneumococci immunization has shown to reduce inflammation in Non-alcoholic steatohepatitis (NASH), another lipid storage disorder. Therefore, pneumococci immunization was also tested on NPC1mut mice. The results indicate an improved pathology in the NPC1 mice after the immunization

Cellular distribution and mechanism of delta-tocopherol on reduction of lysosomal cholesterol accumulation in cells with Niemann-Pick disease type C

Cellular distribution and mechanism of delta-tocopherol on reduction of lysosomal cholesterol accumulation in cells with Niemann-Pick disease type C

Effect of sex hormones on levels of mRNAs coding for proteins involved in lipid metabolism in macrophages

The effects of sex hormones estradiol (E2), testosterone (Te), and 5α-dihydrotestosterone (DT) on cholesterol accumulation induced by modified low density lipoproteins (LDL) in macrophages differentiated from human peripheral blood monocytes and on the levels of mRNAs coding for proteins involved in lipid metabolism have been studied. All three hormones at physiological concentrations (1 nM) are capable of reducing cholesterol accumulation in cells. The treatment of cells with modified and native (not inducing cholesterol accumulation) LDL results in similar alterations in the expression of several mRNAs aimed primarily at homeostatic regulation of lipid metabolism. These alterations depend on the sex of macrophage donors and in some cases are even reversed in cells obtained from male and female donors. The cells not treated with modified LDL have no significant gender differences in the expression of the examined mRNAs. Hormones, either independently or in combination with the modified LDL, influence the levels of some mRNAs, and each hormone shows an individual range of effects. Correlation analysis of changes in mRNA content in the cells showed that the hormones may interfere with coordination of gene expression. Hormone action leads to: (1) reduced coupling of the content of individual mRNAs with their initial levels in the control cells; (2) reduced coupling of different mRNA levels; (3) regrouping of mRNAs between the clusters; and (4) changes in the number of factors that determine the correlation links between mRNAs. The data show that sex hormones may have impact on the level of expression of certain genes and, in particular, on the coordination of gene expression in macrophages.

Mitofusin2 decreases intracellular cholesterol of oxidized LDL-induced foam cells from rat vascular smooth muscle cells

Mitofusin2 (Mfn2) plays a pivotal role in the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). The purpose of this study was to investigate the effects of Mfn2 on the trafficking of intracellular cholesterol in the foam cells derived from rat VSMCs (rVSMCs) and also to investigate the effects of Mfn2 on the expression of adenosine triphosphate-binding cassette subfamily A member 1 (ABCA1), adenosine triphosphate-binding cassette subfamily G member 1 (ABCG1) and peroxisome proliferator-activated receptor gamma (PPARγ). The rVSMCs were co-cultured with oxidized low density lipoprotein (LDL, 80 μg/mL) to produce foam cells and cholesterol accumulation in cells. Before oxidized LDL treatment, different titers (20, 40 and 60 pfu/cell) of recombinant adenovirus containing Mfn2 gene (Adv-Mfn2) were added into the culture medium for 24 h to transfect the Mfn2 gene into the rVSMCs. Then the cells were harvested for analyses. The protein expression of Mfn2 was significantly higher in Adv-Mfn2-transfected group than in untransfected group (P<0.05), and the expression levels significantly increased when the titer of Adv-Mfn2 increased (P<0.05). At 24 or 48 h after oxidized LDL treatment, rVSMCs became irregular and their nuclei became larger, and their plasma abounded with red lipid droplets. However, the number of red lipid droplets was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group. At 48 h after oxidized LDL treatment, the intracellular cholesterol in rVSMCs was significantly increased (P<0.05), but it was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05), and it also significantly decreased when the titer of Adv-Mfn2 increased (P<0.05). The mRNA and protein expression levels of ABCA1 and ABCG1 were significantly increased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05). Though the mRNA and protein expression levels of PPARγ was not significantly increased (P>0.05), the phosporylation levels of PPARγ were significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05). These results suggest that the transfection of Adv-Mfn2 can significantly reduce intracellular cholesterol in oxidized LDL-induced rVSMCs possibly by decreasing PPARγ phosporylation and then increasing protein expression levels of ABCA1 and ABCG1, which may be helpful to suppress the formation of foam cells.

Abstract 4121: Glycine N-methyltransferase deficiency affects hepatic cholesterol homeostasis and steatosis-associated inflammation

Abstract Non-alcoholic fatty liver disease (NAFLD) is a metabolic disorder with excess accumulation of lipid within hepatocyte. Severe NAFLD is associated with inflammation and contributes to the development of hepatocellular carcinoma (HCC). Previously, we reported that glycine N-methyltransferase knockout (Gnmt-/-) mice develop chronic hepatitis and HCC. The aim of this study was to understand the role of GNMT and its interactive proteins play in the pathogenesis of NAFLD. Firstly, we found that Gnmt-/- mice had hyperlipidemia and steatohepatitis and GNMT was down-regulated in the liver tissues from patients with NAFLD as well as from mice fed with high fat diet, high cholesterol diet or methionine/choline deficient diet. Besides, Q-PCR showed that the liver tissues of Gnmt-/- mice had higher levels of TNF-α, IL-6, IL-12p40 and IL-1α than the wild-type mice. Single photon emission computed tomography images demonstrated that Gnmt-/- mice had slower hepatic cholesterol uptake and excretion rates than the wild-type mice. Yeast two-hybrid screenings and co-immunoprecipitation assays elucidated that the Niemann-Pick type C2 protein (NPC2) interacts with GNMT. Confocal microscopy demonstrated that NPC2 was released from lysosomes and interacts with GNMT in the cytosol when cells were treated with low density lipoprotein. Over-expression of GNMT prolonged the half-lives of both NPC2 isoforms and reduced cholesterol accumulation in cells. In conclusion, we demonstrated that GNMT regulates the homeostasis of cholesterol metabolism and steatosis-associated inflammation. This study provides novel targets for the prevention and treatment of steatohepatitis and HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4121. doi:1538-7445.AM2012-4121

Glycine N-Methyltransferase Deficiency Affects Niemann-Pick Type C2 Protein Stability and Regulates Hepatic Cholesterol Homeostasis

Nonalcoholic fatty liver disease (NAFLD) is associated with the development of metabolic syndromes and hepatocellular carcinoma (HCC). Cholesterol accumulation is related to NAFLD, whereas its detailed mechanism is not fully understood. Previously, we reported that glycine N-methyltransferase (GNMT) knockout (Gnmt(-/-)) mice develop chronic hepatitis and HCC. In this study, we showed that Gnmt(-/-) mice had hyperlipidemia and steatohepatitis. Single photon emission computed tomography images of mice injected with (131)I-labeled 6β-iodocholesterol demonstrated that Gnmt(-/-) mice had slower hepatic cholesterol uptake and excretion rates than wild-type mice. In addition, genes related to cholesterol uptake (scavenger receptor class B type 1 [SR-B1] and ATP-binding cassette A1 [ABCA1]), intracellular trafficking (Niemann-Pick type C1 protein [NPC1] and Niemann-Pick type C2 protein [NPC2]) and excretion (ATP-binding cassette G1 [ABCG1]) were downregulated in Gnmt(-/-) mice. Yeast two-hybrid screenings and coimmunoprecipitation assays elucidated that the C conserved region (81-105 amino acids) of NPC2 interacts with the carboxyl-terminal fragment (171-295 amino acids) of GNMT. Confocal microscopy demonstrated that when cells were treated with low-density lipoprotein, NPC2 was released from lysosomes and interacts with GNMT in the cytosol. Overexpression of GNMT doubled the half-lives of both NPC2 isoforms and reduced cholesterol accumulation in cells. Furthermore, GNMT was downregulated in the liver tissues from patients suffering with NAFLD as well as from mice fed a high-fat diet, high-cholesterol diet or methionine/choline-deficient diet. In conclusion, our study demonstrated that GNMT regulates the homeostasis of cholesterol metabolism, and hepatic cholesterol accumulation may result from downregulation of GNMT and instability of its interactive protein NPC2. Novel therapeutics for steatohepatitis and HCC may be developed by using this concept.