[Show abstract][Hide abstract] ABSTRACT: Tumour are characterised by a high content of cholesteryl esters (CEs) stored in lipid droplets purported to be due to a high rate of intracellular esterification of cholesterol. To verify whether and which pathways involved in CE accumulation are essential in tumour proliferation, the effect of CE deprivation, from both exogenous and endogenous sources, on CEM-CCRF cells was investigated. Cholesterol synthesis, esterification and content, low-density lipoprotein (LDL) binding and high-density lipoprotein (HDL)-CE uptake were evaluated in cultured in both conventional and delipidated bovine serum with or without oleic or linoleic acids, cholesteryl oleate, LDL and HDL. High content of CEs in lipid droplets in this cell line was due to esterification of both newly synthesised cholesterol and that obtained from hydrolysis of LDL; moreover, a significant amount of CE was derived from HDL-CE uptake. Cell proliferation was slightly affected by either acute or chronic treatment up to 400 μM with Sz-58035, an acyl-cholesteryl cholesterol esterification inhibitor (ACAT); although when the enzyme activity was continuously inhibited, CE content in lipid droplets was significantly higher than those in control cells. In these cells, analysis of intracellular and medium CEs revealed a profile reflecting the characteristics of bovine serum, suggesting a plasma origin of CE molecules. Cell proliferation arrest in delipidated medium was almost completely prevented in the first 72 h by LDL or HDL, although in subsequent cultures with LDL, it manifested an increasing mortality rate. This study suggests that high content of CEs in CEM-CCRF is mainly derived from plasma lipoproteins and that part of CEs stored in lipid droplets are obtained after being taken up from HDL. This route appears to be up-regulated according to cell requirements and involved in low levels of c-HDL during cancer. Moreover, the dependence of tumour cells on a source of lipoprotein provides a novel impetus in developing therapeutic strategies for use in the treatment of some tumours.
[Show abstract][Hide abstract] ABSTRACT: Scrapie is a prion disease for which no means of ante-mortem diagnosis is available. We recently found a relationship between
cell susceptibility to scrapie and altered cholesterol homeostasis. In brains and in skin fibroblasts and peripheral blood
mononuclear cells from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype, the levels of cholesterol
esters were consistently higher than in tissues and cultures derived from animals with a scrapie-resistant genotype. Here
we show that intracellular accumulation of cholesterol esters (CE) in fibroblasts derived from scrapie-susceptible sheep was
accompanied by parallel alterations in the expression level of acyl-coenzymeA: cholesterol-acyltransferase (ACAT1) and caveolin-1
(Cav-1) that are involved in the pathways leading to intracellular cholesterol esterification and trafficking. Comparative
analysis of cellular prion protein (PrPc) mRNA, showed an higher expression level in cells from animals carrying a susceptible
genotype, with or without Scrapie. These data suggest that CE accumulation in peripheral cells, together with the altered
expression of some proteins implicated in intracellular cholesterol homeostasis, might serve to identify a distinctive lipid
metabolic profile associated with increased susceptibility to develop prion disease following infection.
Full-text · Article · Feb 2010 · Central European Journal of Biology
[Show abstract][Hide abstract] ABSTRACT: Intracellular cholesterol metabolism was reported to modulate amyloid-beta (Abeta) generation in Alzheimer's disease (AD). Results presented herein demonstrated that, like brain cells, cultured skin fibroblasts from AD patients contained more cholesterol esters than fibroblasts from healthy subjects. Particularly, Oil Red-O, Nile Red, and filipin staining highlighted higher levels of neutral lipids which responded to inhibitors of acyl-coenzyme A:cholesterol acyl-transferase (ACAT-1), associated with an increase in free cholesterol. ACAT-1 mRNA levels increased significantly in AD fibroblasts, whereas those of sterol regulatory element binding protein-2, neutral cholesterol ester hydrolase, and ATP-binding cassette transporter member 1 were markedly down-regulated. Instead, mRNA levels of low-density lipoprotein receptor, hydroxy-methyl-glutaryl-coenzyme A reductase, caveolin-1, and amyloid-beta protein precursor (AbetaPP) were virtually unchanged. Notably, mRNA levels of both beta-site AbetaPP-cleaving enzyme 1 (BACE1) and neprilysin were significantly down-regulated. An increase in Abeta(40) and Abeta(42) immunostaining and a decrease in BACE1 active form were also found in AD versus control fibroblasts. Altogether, these findings support the hypothesis that the derangement of cholesterol homeostasis is a systemic alteration involving central but also peripheral cells of AD patients, and point to cholesterol ester levels in AD fibroblasts as an additional metabolic hallmark useful in the laboratory and clinical practice.
[Show abstract][Hide abstract] ABSTRACT: Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefold-higher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC(50)] range, 1.4 to 40 microM) were comparable to those of antiprion reference compounds (EC(50) range, 0.6 to 10 microM). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.
Full-text · Article · Dec 2007 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: The authors have previously shown that the growth of cultured fibroblasts obtained from primary pterygia was associated with an increase in cholesterol esterification, suggesting that alterations of cholesterol homeostasis may be involved in the development and progression of this disorder. This investigation was conducted to determine whether antiproliferative agents such as pioglitazone (PIO) and everolimus (EVE) may inhibit proteins involved in the cholesterol ester cycle and the proliferation of pterygium fibroblasts (PF).
Quiescent normal conjunctival fibroblasts and PFs were treated with or without inhibitors of cell proliferation (PIO and EVE) or with inhibitors of cholesterol esterification-progesterone (Pg) and Sandoz compound (SaH)-and then were stimulated to growth by 10% fetal calf serum (FCS). Cell proliferation was assessed by counting cells. Trypan blue uptake was used to determine cell viability. mRNA and protein levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively.
PIO and EVE significantly abolished the increase in cholesterol esters, acyl-coenzyme A cholesterol acyltransferase (ACAT1), and multidrug resistance protein (MDR1) mRNA observed in growing cells. Each inhibitor upregulated ATP-binding cassette-A1 (ABCA1), neutral cholesterol ester hydrolase (NCEH) mRNA, and caveolin-1 expression in a manner similar to that of specific inhibitors of cholesterol esterification such as Pg and SaH.
Intracellular modifications of cholesterol homeostasis may be relevant to pterygium development. Moreover, antiproliferative agents such as PIO and EVE may represent a potential topical medication in the prevention and inhibition of pterygium growth at an early stage, probably by modulation of cholesterol ester metabolism.
[Show abstract][Hide abstract] ABSTRACT: The effects of ageing on the metabolism of cholesterol were examined in three different organs (liver, aorta and brain) of 6-, 12- and 24-month-old male Sprague-Dawley rats. Ageing was associated with a significant increase in intracellular cholesterol esters in all three organs. Steady state mRNA levels of multidrug resistance protein (MDR) and acylCoA:cholesterol acyl transferase (ACAT), enzymes involved in cholesterol import and esterification, were also increased. By contrast, expression of mRNA for neutral cholesterol ester hydrolase (nCEH) and caveolin-1, proteins involved in cholesterol ester hydrolysis and export, were significantly reduced. Dietary restriction is the only intervention shown to extend lifespan and retard age-related declines in function in mammals. To further explore the possible correlation between changes in cholesterol esterification and ageing, we analysed cholesterol metabolism in liver, aorta, and brain of aged rats exposed to two dietary restriction regimens: intermittent (alternate-day) fasting (IF) and food intake restriction (60% of ad libitum feeding). Both dietary regimens attenuated the age-related changes in cholesterol esters and in the expression of genes involved in cholesterol metabolism. These results provide evidence that distinctive age-associated changes in intracellular cholesterol metabolism occur in rats. Furthermore, these modifications can be partially reversed by dietary restriction, a condition known to affect the ageing process. Age-related changes in cholesterol metabolism may play a role in triggering and/or aggravating senescence-related disorders characterized by altered cholesterol homeostasis.
No preview · Article · Jun 2005 · Mechanisms of Ageing and Development
[Show abstract][Hide abstract] ABSTRACT: Background. Previous epidemiological and experimental studies indicated cholesterol as a central player in Alzheimer disease (AD). Here, we utilized skin fibroblasts and PBMC as possible ex vivo models for the study of dysfunctions of cholesterol homeostasis which may be related to AD development.
Methods. We analyzed cholesterol homeostasis using colorimetric, thin layer chromatography (TLC), and histologic technique in ex vivo cultures of skin fibroblasts and PBMCs from patients with probable AD and their first-degree relatives. Additionally, healthy age-matched individuals served as controls.
Findings. As compared to controls, skin fibroblasts and PBMCs from AD patients, displayed an evident alteration of cholesterol metabolism; namely an anomalous accumulation of cholesterol esters in their cytoplasm. No change in intracellular free cholesterol was observed. Cellular overloading of cholesterol esters was dramatically increased after specific growth stimulation of the different cell types. Cholesterol ester accumulation was negatively correlated to plasma levels of high density lipoprotein cholesterol (HDL-C) and positively correlated with severity of cognitive symptoms measured by Mini-Mental State Examination (MMSE). Inhibitors of cholesterol esterification, such as progesterone and SaH, as well as a potent inhibitor of cell proliferation, RAD, were able to prevent accumulation of cholesterol esters.
Interpretation. Changes of cholesterol esters in the peripheral compartment may be indicative of a systemic alteration of intracellular cholesterol homeostasis, which in turn might create a cellular milieu favourable to the production of ß-amyloid in the brain. Pathways that control cholesterol esterification might represent promising targets for novel diagnostic and therapeutic AD approaches.
[Show abstract][Hide abstract] ABSTRACT: Recent studies in both animal and cell models of Alzheimer's disease (AD) indicated that sub-cellular cholesterol distribution seems to regulate amyloid-beta (A[beta]) generation in the brain. In particular, cholesterol-esters (CE), rather than total cholesterol levels, appear directly correlated with A[beta] production. Here we observed that, similarly to brain cells, skin fibroblasts obtained from AD patients produce and accumulate more CE than skin fibroblasts from age-matched healthy controls do. AD fibroblasts also exhibited a 2 fold increase in the expression of ACAT1, in addition to lower levels of SREBP2, nCEH, Caveolin-1 and ABCA1 mRNA levels, all of which are involved in the CE cycle. HMGCoA-reductase and LDL-receptor mRNAs levels did not show statistically significant changes in AD, compared to non-AD, cells. Furthermore, although APP mRNA did not significantly vary, neprilysin (NEP), the most important enzyme in the proteolysis of A[beta], was expressed at very low levels in skin fibroblasts of sporadic AD patients. Our results contribute to the concept that AD may be the consequence of a basic and systemic defect in the CE cycle. Moreover, our results identify new possible targets for the diagnosis, prevention, and cure or, at least, amelioration of the symptoms of AD.
[Show abstract][Hide abstract] ABSTRACT: Scrapie is an infective ovine neurodegenerative disease; the only identified component of the infectious agent being an aberrant isoform (PrPSc) of the cellular prion protein (PrPC). So far, no means for ante-mortem diagnosis are available for Scrapie as well as for any other mammal Transmissible Spongiform Encephalopaties. We recently found a strong relationship between cell susceptibility to scrapie-infection and intracellular cholesterol homeostasis alterations. In brain tissues as well as in ex vivo cultures of skin fibroblasts and PBMCs from healthy and scrapie-affected sheep carrying a scrapie-susceptible (ARQ/ARQ) genotype, the levels of cholesterol esters were consistently higher than in tissues and cultures derived from animals with a scrapie-resistant (ARR/ARR) genotype. Moreover, both uninfected and scrapie-affected ARQ/ARQ sheep showed abnormally low levels of high density lipoprotein-cholesterol (HDL-C) in their plasma, as compared to ARR/ARR animals. We now show that intracellular accumulation of cholesterol esters in fibroblasts derived from scrapie-susceptible sheep was accompanied by parallel alterations in the expression level of genes and gene products (ACAT1 and Cav-1) that are involved in the pathways leading to intracellular cholesterol esterification and trafficking. Comparative analysis of PrPc mRNA, showed an higher expression level in cells from animals carrying susceptible genotype, with or without Scrapie. Preliminary experiments also revealed the presence of PK-resistant PrP isoforms in the latter cultures. The data reported in the present paper suggest that accumulation of cholesterol esters in peripheral cells, together with the altered expression of some proteins implicated in intracellular cholesterol homeostasis, might serve to identify a distinctive lipid metabolic profile associated with increased susceptibility to develop prion disease following infection.