Disruption Of Cholesterol Homeostasis Research Articles

Defective triglyceride biosynthesis in CETP-deficient SW872 cells

We previously reported that reducing the expression of cholesteryl ester transfer protein (CETP) disrupts cholesterol homeostasis in SW872 cells and causes an ∼50% reduction in TG. The causes of this reduced TG content, investigated here, could not be attributed to changes in the differentiation status of CETP-deficient cells, nor was there evidence of endoplasmic reticulum (ER) stress. In short-term studies, the total flux of oleate through the TG biosynthetic pathway was not altered in CETP-deficient cells, although mRNA levels of some pathway enzymes were different. However, the conversion of diglyceride (DG) to TG was impaired. In longer-term studies, newly synthesized TG was not effectively transported to lipid droplets, yet this lipid did not accumulate in the ER, apparently due to elevated lipase activity in this organelle. DG, shown to be a novel CETP substrate, was also inefficiently transferred to lipid droplets. This may reduce TG synthesis on droplets by resident diacylglycerol acyltransferase. Overall, these data suggest that the decreased TG content of CETP-deficient cells arises from the reduced conversion of DG to TG in the ER and/or on the lipid droplet surface, and enhanced TG degradation in the ER due to its ineffective transport from this organelle.

Cholesterol and lipoprotein metabolism: Early Career Committee contribution.

Cholesterol and lipoprotein metabolism: Early Career Committee contribution.

Leelamine Mediates Cancer Cell Death through Inhibition of Intracellular Cholesterol Transport

Leelamine is a promising compound for the treatment of cancer; however, the molecular mechanisms leading to leelamine-mediated cell death have not been identified. This report shows that leelamine is a weakly basic amine with lysosomotropic properties, leading to its accumulation inside acidic organelles such as lysosomes. This accumulation leads to homeostatic imbalance in the lysosomal endosomal cell compartments that disrupts autophagic flux and intracellular cholesterol trafficking as well as receptor-mediated endocytosis. Electron micrographs of leelamine-treated cancer cells displayed accumulation of autophagosomes, membrane whorls, and lipofuscin-like structures, indicating disruption of lysosomal cell compartments. Early in the process, leelamine-mediated killing was a caspase-independent event triggered by cholesterol accumulation, as depletion of cholesterol using β-cyclodextrin treatment attenuated the cell death and restored the subcellular structures identified by electron microscopy. Protein microarray-based analyses of the intracellular signaling cascades showed alterations in RTK-AKT/STAT/MAPK signaling cascades, which was subsequently confirmed by Western blotting. Inhibition of Akt, Erk, and Stat signaling, together with abnormal deregulation of receptor tyrosine kinases, was caused by the inhibition of receptor-mediated endocytosis. This study is the first report demonstrating that leelamine is a lysosomotropic, intracellular cholesterol transport inhibitor with potential chemotherapeutic properties leading to inhibition of autophagic flux and induction of cholesterol accumulation in lysosomal/endosomal cell compartments. Importantly, the findings of this study show the potential of leelamine to disrupt cholesterol homeostasis for treatment of advanced-stage cancers.

Endoplasmic Reticulum Stress Mediates Amyloid β Neurotoxicity via Mitochondrial Cholesterol Trafficking

Disrupted cholesterol homeostasis has been reported in Alzheimer disease and is thought to contribute to disease progression by promoting amyloid β (Aβ) accumulation. In particular, mitochondrial cholesterol enrichment has been shown to sensitize to Aβ-induced neurotoxicity. However, the molecular mechanisms responsible for the increased cholesterol levels and its trafficking to mitochondria in Alzheimer disease remain poorly understood. Here, we show that endoplasmic reticulum (ER) stress triggered by Aβ promotes cholesterol synthesis and mitochondrial cholesterol influx, resulting in mitochondrial glutathione (mGSH) depletion in older age amyloid precursor protein/presenilin-1 (APP/PS1) mice. Mitochondrial cholesterol accumulation was associated with increased expression of mitochondrial-associated ER membrane proteins, which favor cholesterol translocation from ER to mitochondria along with specific cholesterol carriers, particularly the steroidogenic acute regulatory protein. In vivo treatment with the ER stress inhibitor 4-phenylbutyric acid prevented mitochondrial cholesterol loading and mGSH depletion, thereby protecting APP/PS1 mice against Aβ-induced neurotoxicity. Similar protection was observed with GSH ethyl ester administration, which replenishes mGSH without affecting the unfolded protein response, thus positioning mGSH depletion downstream of ER stress. Overall, these results indicate that Aβ-mediated ER stress and increased mitochondrial cholesterol trafficking contribute to the pathologic progression observed in old APP/PS1 mice, and that ER stress inhibitors may be explored as therapeutic agents for Alzheimer disease.

Abstract 466: Stigmasterol Accumulation in ABCG8 Knockout Mice Does Not Account for Xenosterol Toxicity

Xenosterol accumulation mice deficient in sterolin function leads to significant toxicity, with infertility, decreased body fat accumulation, macrothrombocytopenia, cardiac fibrosis and premature death1, 2. The predominant xenosterols are phytosterols derived from the diet and are a mixture of sitosterol (typically 70%), campesterol (~20%) and stigmasterol (~5-10%). Although sitosterol has been shown to have some biological effect in tissue culture, only stigmasterol has been shown to have a potent biological effect, by activating the transcriptional factor, Lxr3. To delineate whether all of the toxic biological effects were mediated by stigmasterol accumulation, we designed a diet that was supplemented with highly enriched stigmasterol (>80% purity) and fed this to Abcg8 knockout mice. Stigmasterol accumulation in the plasma and tissues was comparable to the levels observed in mice fed a mixed phytosterol-enriched diet that had been shown to result in toxicity. Over a 12-week period, both male and female Abcg8 knockout mice gained normal amounts of weight, body fat, showed no disturbances in tail-cuff measured blood pressure, and plasma analyses showed no abnormalities of platelet counts or volumes, blood glucose, plasma cholesterol, despite accumulation of stigmasterol in the plasma and tissues. Fertility testing showed no abnormalities. Gene expression analyses of livers did not show any consistent patterns, although Lxr target genes were not up-regulated. These data do not support the concept that stigmasterol accumulation, at levels of 5-10mg/dL in plasma, account for the xenosterol-mediated toxicity observed. 1. McDaniel, A.L., H.M. Alger, J.K. Sawyer, K.L. Kelley, N.D. Kock, J.M. Brown, R.E. Temel, and L.L. Rudel, Phytosterol feeding causes toxicity in ABCG5/G8 knockout mice. Am J Pathol, 2013. 182(4): p. 1131-8. 2. Solca, C., G.S. Tint, and S.B. Patel, Dietary xenosterols lead to infertility and loss of abdominal adipose tissue in sterolin-deficient mice. J Lipid Res, 2013. 54(2): p. 397-409. 3. Yang, C., L. Yu, W. Li, F. Xu, J.C. Cohen, and H.H. Hobbs, Disruption of cholesterol homeostasis by plant sterols. J. Clin. Invest., 2004. 114(6): p. 813-22.

Lipid rafts: a signaling platform linking cholesterol metabolism to synaptic deficits in autism spectrum disorders

AUTISM AND CHOLESTEROL METABOLISM Autism spectrum disorders (ASDs) are a group of developmental psychiatric disorders characterized by impaired social interaction and communication, and by restricted, repetitive and stereotyped behaviors and interests (Zoghbi and Bear, 2012; Ebert and Greenberg, 2013; Huguet et al., 2013). Since the features of ASDs usually manifest at the early childhood when sensory experience is modifying the development and balance of excitatory and inhibitory synapses, it has been hypothesized that ASDs may be due to the disruption of experience dependent synaptic development and function, resulting in an imbalance between excitation and inhibition in brain (Auerbach et al., 2011; Zoghbi and Bear, 2012; Delorme et al., 2013; Wang and Doering, 2013). Although genetic causes have been identified in many individuals with ASDs, the details about how those causal genes converge on common pathways to alter synaptic homeostasis in ASDs still need to be investigated. Recently, Buchovecky et al. described disturbances in cholesterol homeostasis in animal model of the ASD Rett syndrome (RTT), giving rise to an exciting prospect that changes in cholesterol metabolism might underlie the development of ASDs (Buchovecky et al., 2013). Cholesterol, an essential cell membrane component, influences the establishment and maintenance of synaptic connection and glial cell development in the nervous system. Balanced cholesterol homeostasis is an important aspect of nervous system function (Mauch et al., 2001; Pfrieger, 2003; Linetti et al., 2010; Pfrieger and Ungerer, 2011; Mathews et al., 2014) (Figure 1A). Perturbed cholesterol homeostasis can affect neural development and synaptogenesis and result in synaptic dysfunction, thus may lead to disorders of nervous system (Simons and Ehehalt, 2002; Linetti et al., 2010; Pani et al., 2010; Karasinska and Hayden, 2011). Evidence from patients indicates that cholesterol homeostasis could be altered in autistic disorders. The Smith-Lemli-Opitz Syndrome (SLOS), a genetic condition of impaired cholesterol biosynthesis due to mutations of the 7-dehydrocholesterol reductase gene (DHCR7), has been found to be associated with autism, supporting genetic defects in cholesterol metabolism can cause autism (Sikora et al., 2006; Bukelis et al., 2007; Diaz-Stransky and Tierney, 2012). Abnormal cholesterol metabolism has also been observed in patients with the ASD Asperger syndrome and other nonsyndromic ASDs, suggesting different abnormalities of cholesterol metabolism may exist in ASDs (Tierney et al., 2006; Dziobek et al., 2007). However, little is known about the mechanisms that mediate the abnormal cholesterol metabolism in these ASD conditions. RTT is caused largely by mutations in the X-linked methyl CpG-binding protein 2 gene (MECP2) (Baker et al., 2013; Lyst et al., 2013; Xu and PozzoMiller, 2013). In Mecp2 mutant mice, Buchovecky et al. found that the expression of genes (Hmgcr, Sqle and Cyp46a1) which encode key enzymes (3-hydroxy-3methylglutaryl-CoA reductase, squalene epoxidase, CYP46A1) in the cholesterol metabolic pathway and cholesterol concentrations are altered in the brain in a development dependent manner (Buchovecky et al., 2013). Their study indicates that loss of Mecp2 disrupts cholesterol homeostasis, suggesting abnormal cholesterol metabolism might be involved in the pathogenesis of RTT (Nagy and Ackerman, 2013). It thus links the autism associated gene to cholesterol metabolism, providing further insights into the relationship between cholesterol metabolism and ASDs.

Plasma from rheumatoid arthritis patients promotes pro-atherogenic cholesterol transport gene expression in THP-1 human macrophages

Immunologic derangements in rheumatoid arthritis (RA) patients likely contribute to premature atherosclerotic cardiovascular disease (CVD). Traditional CVD risk factors do not reliably identify at-risk RA patients, probably because disease-associated mechanisms are not taken into account. The purpose of this study was to determine whether plasma from subjects with RA exhibits atheroma-promoting properties leading to disruption of cholesterol homeostasis in human monocytes/macrophages. Twenty-one healthy controls (HC) and 22 RA patients were enrolled in an IRB approved study at Winthrop University Hospital. Naïve THP-1 macrophages were exposed to plasma from each HC and RA patient. Following incubation, RNA and protein were isolated. QRT-PCR and Western blotting techniques were then used to measure expression of proteins responsible for cholesterol efflux (ATP binding cassette transporter (ABC)A1, ABCG1, 27-hydroxylase) and cholesterol uptake (CD36, ScR-A1, lectin oxidized low density lipoprotein receptor (LOX)-1, CXCL16). To confirm the pro-atherogenic effects of RA plasma on macrophages, foam cell formation was quantified. Results showed that RA plasma downregulates cholesterol efflux proteins and upregulates scavenger receptors CD36, LOX1 and CXCL16. These pro-atherogenic changes in gene expression in the presence of RA plasma are associated with augmented lipid accumulation and foam cell formation by THP-1 macrophages. RA plasma induces macrophage cholesterol overload. Demonstration of disrupted cholesterol homeostasis mediated by RA plasma provides further evidence of the involvement of the immune system in atherogenesis. Our data suggest that chronic exposure to RA plasma adversely affects the capacity of monocytes/macrophages in the arterial wall to metabolize cholesterol and maintain lipid homeostasis, thereby contributing to the development of premature atherosclerosis.

Cholesterol efflux is differentially regulated in neurons and astrocytes: Implications for brain cholesterol homeostasis

Disruption of cholesterol homeostasis in the central nervous system (CNS) has been associated with neurological, neurodegenerative, and neurodevelopmental disorders. The CNS is a closed system with regard to cholesterol homeostasis, as cholesterol-delivering lipoproteins from the periphery cannot pass the blood–brain-barrier and enter the brain. Different cell types in the brain have different functions in the regulation of cholesterol homeostasis, with astrocytes producing and releasing apolipoprotein E and lipoproteins, and neurons metabolizing cholesterol to 24(S)-hydroxycholesterol. We present evidence that astrocytes and neurons adopt different mechanisms also in regulating cholesterol efflux. We found that in astrocytes cholesterol efflux is induced by both lipid-free apolipoproteins and lipoproteins, while cholesterol removal from neurons is triggered only by lipoproteins. The main pathway by which apolipoproteins induce cholesterol efflux is through ABCA1. By upregulating ABCA1 levels and by inhibiting its activity and silencing its expression, we show that ABCA1 is involved in cholesterol efflux from astrocytes but not from neurons. Furthermore, our results suggest that ABCG1 is involved in cholesterol efflux to apolipoproteins and lipoproteins from astrocytes but not from neurons, while ABCG4, whose expression is much higher in neurons than astrocytes, is involved in cholesterol efflux from neurons but not astrocytes. These results indicate that different mechanisms regulate cholesterol efflux from neurons and astrocytes, reflecting the different roles that these cell types play in brain cholesterol homeostasis. These results are important in understanding cellular targets of therapeutic drugs under development for the treatments of conditions associated with altered cholesterol homeostasis in the CNS.

APP/PS1 transgenic mice overexpressing SREBP2 as a new model to study the pathogenic role of cholesterol in Alzheimer's disease (AD)

Disruption of cholesterol homeostasis is associated with AD. Increased brain cholesterol triggers amyloid precursor-BACE1 clustering in lipid rafts stimulating the synthesis and aggregation of toxic amyloid beta peptides (Ab). Moreover, we have recently described that the trafficking of cholesterol to mitochondria sensitizes to Ab-mediated neurotoxicity by depletion of mitochondrial glutathione (mGSH). The aims of this work were to examine the regulatory effect of cholesterol in AD progression and to evaluate Ab-induced ER stress as a causal mechanism of the altered cholesterol metabolism in brain. We generated APP/PS1 transgenic mice that overexpress the sterol regulatory element binding protein 2 (Tg-APP/PS1-SREBP2) and analysed several pathological hallmarks of AD at 4, 7 and 10 month of age. The increase (1-5-2 fold) of total brain and mitochondrial cholesterol levels and subsequent selective depletion of mGSH were accelerated in Tg-APP/PS1-SREBP2 mice compared to either Tg-APP/PS1 or Tg-SREBP2. Both alterations were detectable in 10-month old Tg-APP/PS1 mice and associated with elevated expression of SREBP2. The incidence of high Ab levels raised with the overexpression of SREBP2 and correlated with enhanced activity of BACE1. Seven-month old Tg-APP/PS1-SREBP2 mice displayed brain oxidative damage and neuroinflammation, features that were unnoticeable in Tg-APP/PS1 mice up to 10 month of age. The increased brain oxidative stress in Tg-APP/PS1-SREBP2 mice promoted the activation of GSK3-beta, ERK1/2 and CDK5 that led to enhanced TAU phosphorilation and neurofibrillary tangle formation. Furthermore, overexpression of SREBP2 accelerated synaptic degeneration and neuronal death. The analyses of GADD153 and GRP78 expression indicated an early onset of ER stress. Moreover, treatment of 10-month old Tg-APP/PS1 mice with the chaperon-like compound sodium 4-phenylbutyrate (4-PBA) significantly reduced the levels of the ER stress markers and prevented the induction of SREBP2 expression as well as the increase of proteins involved in mitochondrial cholesterol transport, including STAR and MLN64. The administration of 4-PBA restored the mitochondrial cholesterol and mGSH content to control levels. Increased brain cholesterol by SREBP2 activation potentiates Ab-induced neuronal damage/neurofibrillary tangle formation, accelerating the AD phenotype of Tg-APP/PS1 mice. ER stress and regulation of mitochondrial cholesterol trafficking underlie the pathology progression observed in old Tg-APP/PS1 mice.

Association of ApoE and LRP mRNA levels with dementia and AD neuropathology

Inheritance of the ε4 allele of apolipoprotein E (ApoE) is the only confirmed and consistently replicated risk factor for late onset Alzheimer's disease (AD). ApoE is also a key ligand for low-density lipoprotein (LDL) receptor-related protein (LRP), a major neuronal low-density lipoprotein receptor. Despite the considerable converging evidence that implicates ApoE and LRP in the pathogenesis of AD, the precise mechanism by which ApoE and LRP modulate the risk for AD remains elusive. Moreover, studies investigating expression of ApoE and LRP in AD brain have reported variable and contradictory results. To overcome these inconsistencies, we studied the mRNA expression of ApoE and LRP in the postmortem brain of persons who died at different stages of dementia and AD-associated neuropathology relative to controls by quantitative polymerase chain reaction (qPCR) and Western blotting analyses. Clinical dementia rating scores were used as a measure of dementia severity, whereas, Braak neuropathological staging and neuritic plaque density were used as indexes of the neuropathological progression of AD. ApoE and LRP mRNA expression was significantly elevated in the postmortem inferior temporal gyrus (area 20) and the hippocampus from individuals with dementia compared with those with intact cognition. In addition to their strong association with the progression of cognitive dysfunction, LRP and ApoE mRNA levels were also positively correlated with increasing neuropathological hallmarks of AD. Additionally, Western blot analysis of ApoE protein expression in the hippocampus showed that the differential expression observed at the transcriptional level is also reflected at the protein level. Given the critical role played by LRP and ApoE in amyloid beta (Aβ) and cholesterol trafficking, increased expression of LRP and ApoE may not only disrupt cholesterol homeostasis but may also contribute to some of the neurobiological features of AD, including plaque deposition.

Disruption of cellular cholesterol transport and homeostasis as a novel mechanism of action of membrane-targeted alkylphospholipid analogues.

Alkylphospholipid (APL) analogues constitute a new class of synthetic anti-tumour agents that act directly on cell membranes. We have previously demonstrated that hexadecylphosphocholine (HePC) alters intracellular cholesterol traffic and metabolism in HepG2 cells. We now extended our studies to analyse the effects of other clinically relevant APLs, such as edelfosine, erucylphosphocholine and perifosine on intracellular cholesterol homeostasis. Using radiolabelled substrates we determined the effect of APLs on cholesterol metabolism and cholesterol traffic from the plasma membrane to the endoplasmic reticulum (ER). Protein levels and gene expression of the main proteins involved in cholesterol homeostasis were analysed by Western blot and RT-PCR respectively. Membrane raft and non-raft fractions were isolated from HepG2 cells by a detergent-free method. All APLs inhibited the transport of cholesterol from the plasma membrane to the ER, which induced a significant cholesterogenic response in HepG2 cells. This response involved an increased gene expression and higher levels of several proteins related to the biosynthesis and the receptor-mediated uptake of cholesterol. Cell exposure to the APL-representative HePC enhanced the content of cholesterol mainly in the membrane raft fractions, compared with the untreated cells. Membrane-targeted APLs exhibited a novel and common mechanism of action, through disruption of cholesterol homeostasis, which in turn affected specific lipid microdomains of cellular membranes.

Alterations in the homeostasis of phospholipids and cholesterol by antitumor alkylphospholipids

The alkylphospholipid analog miltefosine (hexadecylphosphocholine) is a membrane-directed antitumoral and antileishmanial drug belonging to the alkylphosphocholines, a group of synthetic antiproliferative agents that are promising candidates in anticancer therapy. A variety of mechanisms have been suggested to explain the actions of these compounds, which can induce apoptosis and/or cell growth arrest. In this review, we focus on recent advances in our understanding of the actions of miltefosine and other alkylphospholipids on the human hepatoma HepG2 cell line, with a special emphasis on lipid metabolism. Results obtained in our laboratory indicate that miltefosine displays cytostatic activity and causes apoptosis in HepG2 cells. Likewise, treatment with miltefosine produces an interference with the biosynthesis of phosphatidylcholine via both CDP-choline and phosphatidylethanolamine methylation. With regard to sphingolipid metabolism, miltefosine hinders the formation of sphingomyelin, which promotes intracellular accumulation of ceramide. We have demonstrated for the first time that treatment with miltefosine strongly impedes the esterification of cholesterol and that this effect is accompanied by a considerable increase in the synthesis of cholesterol, which leads to higher levels of cholesterol in the cells. Indeed, miltefosine early impairs cholesterol transport from the plasma membrane to the endoplasmic reticulum, causing a deregulation of cholesterol homeostasis. Similar to miltefosine, other clinically-relevant synthetic alkylphospholipids such as edelfosine, erucylphosphocholine and perifosine show growth inhibitory effects on HepG2 cells. All the tested alkylphospholipids also inhibit the arrival of plasma-membrane cholesterol to the endoplasmic reticulum, which induces a significant cholesterogenic response in these cells, involving an increased gene expression and higher levels of several proteins related to the pathway of biosynthesis as well as the receptor-mediated uptake of cholesterol. Thus, membrane-targeted alkylphospholipids exhibit a common mechanism of action through disruption of cholesterol homeostasis. The accumulation of cholesterol within the cell and the reduction in phosphatidylcholine and sphingomyelin biosyntheses certainly alter the ratio of choline-bearing phospholipids to cholesterol, which is critical for the integrity and functionality of specific membrane microdomains such as lipid rafts. Alkylphospholipid-induced alterations in lipid homeostasis with probable disturbance of the native membrane structure could well affect signaling processes vital to cell survival and growth.

Chronic Alcohol Consumption Disrupted Cholesterol Homeostasis in Rats: Down‐Regulation of Low‐Density Lipoprotein Receptor and Enhancement of Cholesterol Biosynthesis Pathway in the Liver

Chronic alcohol consumption causes alcoholic liver disease, which is associated, or initiated, with dysregulated lipid metabolism. Very recent evidence suggested that dysregulated cholesterol metabolism plays an important role in the pathogenesis of alcoholic fatty liver diseases, however, the effects of chronic alcohol exposure on cholesterol homeostasis have not been well studied and underlying mechanisms behind are still elusive. Male Sprague-Dawley rats weighing 250 +/- 5.5 g (mean +/- SEM) divided into 2 groups (8 rats per group) and pair-fed with liquid diets containing (in percent of energy intake) 18% protein, 35% fat, 12% carbohydrate, and 35% either ethanol (ethanol diet) or an isocaloric maltose-dextrin mixture (control diet), according to Lieber and De Carli, for 4 weeks. Long-term excessive alcohol feeding to rats caused fatty liver and liver injury, which was associated with disrupted cholesterol homeostasis, characterized by increased hepatic cholesterol levels and hypercholesterolemia. Hepatic cholesterol increases were concomitant with constantly activated sterol regulatory element-binding protein-2 (SREBP-2) in the liver and increased expression of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, a rate-limiting enzyme for cholesterol de novo synthesis, indicating enhanced cholesterol biosynthesis. Alcohol-induced hypercholesterolemia was accompanied by decreased LDL receptor (LDLr) levels in the liver. Further investigations revealed that chronic alcohol exposure increased hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) contents to down-regulate LDLr via a post-translational mechanism. Moreover, alcohol feeding suppressed extracellular signal-regulated kinase (ERK) activation in the liver. In vitro studies showed that inhibition of ERK activation was associated with decreased LDLr expression in HepG2 cells. Our study provides the first evidence that both increased PCSK9 expression and suppressed ERK activation in the liver contributes to alcohol-induced hypercholesterolemia in rats.

Effects of inflammation on cholesterol metabolism: Impact on systemic lupus erythematosus

Inflammation and dysregulated cholesterol metabolism are key components in the pathogenesis of atherosclerosis. Premature atherosclerosis is a characteristic feature of systemic lupus erythematosus. Although the cellular and molecular mechanisms underlying accelerated atherogenesis in lupus are not thoroughly understood, inflammation associated with the rheumatic disease state may promote atherosclerosis. Increasing evidence indicates that the systemic inflammatory load in lupus disrupts cholesterol homeostasis, increasing vulnerability to cholesterol accumulation in cells of the artery wall, including macrophages and endothelium. The relationship between the inflammatory state and dyslipidemia in lupus is complex, involving lipoproteins, cholesterol transporters, scavenger receptors, and oxysterols. The impact of lupus on each of these components of the cholesterol flux pathways is discussed. The formation of autoantibodies against epitopes within lipoprotein particles and their controversial role in atherogenesis is addressed.

A BRIEF COMMUNICATION

Premature atherosclerotic cardiovascular disease (ASCVD) is a common and devastating complication of systemic lupus erythematosus (SLE). It is likely that immunologic derangements contribute to premature ASCVD in these patients, possibly by disrupting homeostatic mechanisms that orchestrate cholesterol balance in monocytes/macrophages in the artery wall. CD36, a macrophage scavenger receptor responsible for recognition and internalization of oxidized lipids, is a major participant in atherosclerotic foam cell formation. We hypothesized that lupus plasma would affect CD36 expression in a pro-atherogenic manner in THP-1 human monocytes and differentiated macrophages. SLE patient plasma markedly stimulated expression of CD36 message in a dose-dependent fashion in THP-1 human monocytes. A 50% volume/volume concentration of plasma derived from SLE patients increased CD36 mRNA by 71 +/- 8% (n = 3, P < 0.001) above 50% normal human plasma. 50% SLE patient plasma increased CD36 mRNA expression to 290 +/- 12% of no-plasma control (n = 3, P < 0.001), compared with only 118 +/- 3.7% of control in the presence of 50% normal human plasma (n = 3, not significant). 50% lupus plasma also upregulated CD36 protein expression by 482.3 +/- 76.2% (n = 4, P < 0.05), whereas the presence of 50% normal human plasma increased the CD36 protein level by only 239.8 +/- 61.9% (n = 4, P < 0.05). To our knowledge, this is the first demonstration that CD36 expression is enhanced by plasma from patients with an autoimmune disorder. Premature atherosclerosis is common in SLE patients. Upregulation of CD36 may contribute to this pathological process by increasing vulnerability to cholesterol overload. Demonstration of disrupted cholesterol homeostasis in this select group of patients provides further evidence of the involvement of the immune system in atherogenesis and may inform us of the role of CD36 in the general atherogenic process. CD36 may provide a novel therapeutic target in the treatment of ASCVD in SLE patients.

Hyaluronan Accumulation Is Elevated in Cultures of Low Density Lipoprotein Receptor-deficient Cells and Is Altered by Manipulation of Cell Cholesterol Content

The extracellular matrix molecule hyaluronan (HA) accumulates in human atherosclerotic lesions. Yet the reasons for this accumulation have not been adequately addressed. Because abnormalities in lipid metabolism promote atherosclerosis, we have asked whether disrupted cholesterol homeostasis alters HA accumulation in low density lipoprotein receptor-deficient cell cultures. Cultured aortic smooth muscle cells (ASMC) from Watanabe heritable hyperlipidemic (WHHL) rabbits and skin fibroblasts from homozygous patients with familial hypercholesterolemia accumulated 2-4-fold more HA than corresponding cells from age- and sex-matched normolipidemic rabbits and individuals. This occurred in both cell-associated and secreted HA fractions and was independent of cell density or medium serum concentration. WHHL ASMC cultures synthesized twice the proportion of high molecular mass HA (>2x10(6) Da) as normal rabbit ASMC but showed a lower capacity to degrade exogenous [3H]HA. Most importantly, cholesterol depletion or blocking cholesterol synthesis markedly reduced HA accumulation in WHHL ASMC cultures, whereas cholesterol replenishment or stimulation of cholesterol synthesis restored elevated HA levels. We conclude the following: 1) maintaining normal HA levels in cell cultures requires normal cell cholesterol homeostasis; 2) HA degradation may contribute to but is not the predominant mechanism to increase high molecular mass HA accumulation in low density lipoprotein receptor-deficient WHHL ASMC cultures; and 3) elevated accumulation of HA depends on cellular or membrane cholesterol content and, potentially, intact cholesterol-rich microdomains.

Hexadecylphosphocholine interferes with the intracellular transport of cholesterol in HepG2 cells

We have shown, in a previous publication, that nontoxic concentrations of hexadecylphosphocholine exert an antiproliferative effect on HepG2 cells. Hexadecylphosphocholine also interferes with the biosynthesis of cholesterol and phosphatidylcholine. We have now extended our studies to try to establish the molecular mechanism by which hexadecylphosphocholine disrupts cholesterol homeostasis. Using radiolabelled substrates we determined the effect of hexadecylphosphocholine on cholesterol synthesis, the destiny of cholesterol from low-density lipoprotein and the transport of cholesterol between the plasma membrane and the endoplasmic reticulum. Protein levels and gene expression of the main proteins involved in cholesterol homeostasis were analysed by western blotting and RT-PCR, respectively. HepG2 cells exposed to hexadecylphosphocholine showed an increase in cholesterol biosynthesis when acetate, but not mevalonate, was used as a substrate. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34) and low-density lipoprotein receptor, as well as the corresponding mRNA expression, increased after 24 h of treatment with hexadecylphosphocholine. Cholesteryl linoleate in low-density lipoprotein uptake and further hydrolysis of these esters increased but the cholesterol esterification was reduced after 6 h of treatment with alkylphosphocholine. Cholesterol transport from the plasma membrane to the endoplasmic reticulum was impaired by hexadecylphosphocholine. In conclusion, hexadecylphosphocholine interfered with the transport of cholesterol from the cell surface to the endoplasmic reticulum, leading to a depletion of cholesterol in the endoplasmic reticulum and a deregulation of cholesterol biosynthesis. The accumulation of cholesterol within the cell and the reduction in phosphatidylcholine synthesis produces an alteration in the phosphatidylcholine/cholesterol ratio that may well be responsible for the antiproliferative activity exhibited by hexadecylphosphocholine in HepG2 cells.

Neuronal differentiation of human mesenchymal stem cells: Changes in the expression of the Alzheimer's disease-related gene seladin-1

Seladin-1 (SELective Alzheimer's Disease INdicator-1) is an anti-apoptotic gene, which is down-regulated in brain regions affected by Alzheimer's disease (AD). In addition, seladin-1 catalyzes the conversion of desmosterol into cholesterol. Disruption of cholesterol homeostasis in neurons may increase cell susceptibility to toxic agents. Because the hippocampus and the subventricular zone, which are affected in AD, are the unique regions containing stem cells with neurogenic potential in the adult brain, it might be hypothesized that this multipotent cell compartment is the predominant source of seladin-1 in normal brain. In the present study, we isolated and characterized human mesenchymal stem cells (hMSC) as a model of cells with the ability to differentiate into neurons. hMSC were then differentiated toward a neuronal phenotype (hMSC-n). These cells were thoroughly characterized and proved to be neurons, as assessed by molecular and electrophysiological evaluation. Seladin-1 expression was determined and found to be significantly reduced in hMSC-n compared to undifferentiated cells. Accordingly, the total content of cholesterol was decreased after differentiation. These original results demonstrate for the first time that seladin-1 is abundantly expressed by stem cells and appear to suggest that reduced expression in AD might be due to an altered pool of multipotent cells.

Hexadecylphosphocholine disrupts cholesterol homeostasis and induces the accumulation of free cholesterol in HepG2 tumour cells

Hexadecylphosphocholine (HePC) is a synthetic lipid belonging to the alkylphosphocholines (APC), a new group of antiproliferative agents that are proving to be promising candidates in anticancer therapy. We reported in a previous study that HePC interferes with phosphatidylcholine (PC) synthesis in HepG2 cells via both CDP-choline and phosphatidylethanolamine (PE) methylation. We have subsequently extended our studies to show that HePC interferes with sphingolipid metabolism by hindering the formation of sphingomyelin (SM), an effect accompanied by a substantial increase in the incorporation of the exogenous lipogenic precursors into ceramides. Interestingly, we demonstrate for the first time that HePC strongly inhibits the esterification of free cholesterol (FC) by acting at the level of acyl CoA:cholesterol acyltransferase (ACAT) (EC 2.3.1.26) activity. This effect is accompanied by a considerable increase in the synthesis of cholesterol, which leads to a rise in the levels of FC in cells. We are left in no doubt that the imbalance in the metabolism of membrane-lipid components vital to cell survival may well be responsible for the observed DNA fragmentation and activation of caspase-3, an enzyme involved in the cell apoptosis found in this study.

Ezetimibe normalizes metabolic defects in mice lacking ABCG5 and ABCG8

The ATP binding cassette transporters ABCG5 (G5) and ABCG8 (G8) limit the accumulation of neutral sterols by restricting sterol uptake from the intestine and promoting sterol excretion into bile. Humans and mice lacking G5 and G8 (G5G8-/-) accumulate plant sterols in the blood and tissues. However, despite impaired biliary cholesterol secretion, plasma and liver cholesterol levels are lower in G5G8-/- mice than in wild-type littermates. To determine whether the observed changes in hepatic sterol metabolism were a direct result of decreased biliary sterol secretion or a metabolic consequence of the accumulation of dietary noncholesterol sterols, we treated G5G8-/- mice with ezetimibe, a drug that reduces the absorption of both plant- and animal-derived sterols. Ezetimibe feeding for 1 month sharply decreased sterol absorption and plasma levels of sitosterol and campesterol but increased cholesterol in both the plasma (from 60.4 to 75.2 mg/dl) and the liver (from 1.1 to 1.87 mg/g) of the ezetimibe-treated G5G8-/- mice. Paradoxically, the increase in hepatic cholesterol was associated with an increase in mRNA levels of HMG-CoA reductase and synthase. Together, these results indicate that pharmacological blockade of sterol absorption can ameliorate the deleterious metabolic effects of plant sterols even in the absence of G5 and G8.