Cholesterol esterification as a mediator of proliferation of vascular smooth muscle cells and peripheral blood mononuclear cells during atherogenesis.

BACKGROUND/AIMS: We determined growth rates, cholesterol esterification and mRNA levels for caveolin-1 (Cav-1), neutral cholesterol esters hydrolase (n-CEH) and ATP-binding cassette transporter (ABCA-1), in quiescent and growth-stimulated peripheral blood mononuclear cells (PBMCs) and intimal vascular smooth muscle cells (VSMCs) from blood and primary atherosclerotic plaques, respectively. These cells were cultured in the presence or absence of the mTOR inhibitor 40-O-(2-hydroxyethyl) rapamycin (RAD). METHODS: The rate of cell proliferation was determined by 3H-thymidine incorporation into DNA and that of lipid metabolism by utilizing 14C-acetate and 14C-oleate as precursors. Lipid deposit in the vascular cells was evaluated by Oil Red O staining and lipid mass by thin layer chromatography-linked enzymatic assay. RESULTS: Growth stimulation of PBMCs and VSMCs caused a rapid increase in intracellular cholesterol esterification and an accumulation of cholesterol esters (CEs) accompanied by a reduction of free cholesterol (FC) and Cav-1, ABCA-1 and n-CEH mRNAs. RAD reduced intracellular lipid accumulation in growth-stimulated cells and also increased expression of Cav-1, n-CEH and ABCA-1 genes. CONCLUSION: Collectively, these data provide evidence that the determination of CEs in PBMCs may be an easy prescreening test to identify subjects at risk for vascular proliferative disease and that FC, CE, Cav-1, n-CEH and ABCA-1 may be suitable targets for antiproliferative therapies.

Age-related macular degeneration and cognitive impairment show similarities in changes of neutral lipids in peripheral blood mononuclear cells.

Starting from previous studies showing that patients with cognitive deficit present neutral lipids (NLs) accumulation in cytoplasm of their peripheral blood mononuclear cells (PBMCs) and considering that there is epidemiological evidence linking age-related macular degeneration (AMD) to cognitive deficit, the first purpose of this study was to test whether neutral lipids also accumulated in PBMCs from AMD subjects. Moreover, the impact of statin use on AMD was explored and whether such use in AMD subjects was associated with NLs accumulation in PBMCs. The study was conducted on 222 subjects: 136 AMD (36 of which - 26.5% - using statins], 48 cognitive deficit (20 of which - 41.7% - using statins) and 38 healthy controls (4 of which -10.1% - using statins), AMD lesions were assessed from color fundus photographs. Mini-mental state examination (MMSE), demographics, lifestyle factors and medical history were collected at interview. MMSE score was categorized as normal (24-30), and impaired (<24), NLs content was evaluated by oil red 0 (ORO) staining method. ORO determination showed that neutral lipids were generally absent or very low (score between 0 and 1) in healthy controls while most of PBMCs from cognitive deficit and AMD had ORO staining levels scoring 2-4. Post hoc analysis (Bonferroni) in a one-way ANOVA revealed that ORO score was significantly higher in cognitive deficit and AMD subjects compared to healthy controls and in cognitive deficit compared to AMD. Bonferroni-test also showed that AMD subjects had significantly lower total cholesterol (TC) levels compared to healthy controls while high density lipoprotein-cholesterol (HDL-C) did not reach statistical significance. The results also revealed a significant higher number of statin-users in AMD compared to healthy controls. Likewise when cognitive deficit vs healthy controls was analyzed, the number of statin users were found to be significant higher in cognitive deficit than in healthy controls. There were no significant differences in statin use between AMD and cognitive deficit. Compared to healthy controls, statin use in cognitive deficit and AMD groups was significantly associated with ORO scores of 2-4. This data supports the hypothesis that AMD and cognitive deficit share similar complex pathophysiology and risk factors including NLs accumulation in their PBMCs, although this does not necessarily imply that one disease causes the other. In addition, they provide further evidence that statin use may increase the risk of AMD.

Changes in cholesterol metabolism-related gene expression in peripheral blood mononuclear cells from Alzheimer patients.

BACKGROUND: Cholesterol homeostasis dysfunction has been reported to have role in the pathogenesis of Alzheimer disease (AD). Therefore, changes in cholesterol metabolism in blood components may help to develop new potential AD biomarkers. In this study changes in cholesterol metabolism-related gene expression genes were evaluated in peripheral blood mononuclear cells (PBMCs) from AD subjects, their first degree relatives (FDR) and two groups of age matched controls (C1 > 80 years, C2 < 60 years). The expression of three genes related to APP processing was also determined. RESULTS: Results showed significantly different behavior (P = 0.000) in the expression of all analyzed genes among the 4 groups. An inverse correlation emerged between the age of controls and the propensity of their PBMCs to express selected genes. Moreover, when gene expression was evaluated in PBMCs from AD patients and compared with that of PBMCs from healthy subjects of the same age, LDL-R and APP mRNAs were most abundant in AD as compared C1 whereas SREBP-2 and particularly nCEH were present at much lower mRNA levels in AD-PBMCs. This study describes for the first time a differential expression profile of cholesterol and APP related genes in PBMCs from AD patients and their FDR. CONCLUSIONS: We suggest that the expressions of cholesterol homeostasis and APP processing related genes in PBMC could be proposed as possible biomarkers to evaluate AD risk. In addition, gene expression in PBMC could be also used for diagnosis and development of therapeutic strategies as well as for personalized prediction in clinical outcome of AD.

Thalassemia intermedia is associated with a proatherogenic biochemical phenotype.

OBJECTIVE: Unlike beta thalassemia major (ß-TM) in which cardiac siderosis represents the leading cause of mortality and morbidity, in beta thalassemia intermedia (ß-TI), pulmonary hypertension (PHT) and thrombosis seems to be the major cardiovascular complications. However, the mechanism underlying these complications in ß-TI is still unclear. Endothelial dysfunction, the key early event in atherogenesis, is now emerging as an important cardiovascular risk determiner in ß-TI patients. Among the factors known to affect endothelial function, iron and cholesterol merit particular consideration in ß-TI patients. Therefore, with the aim to extend our knowledge on the mechanisms connecting atherosclerosis to ß-TI, in this study, we compared lipid and iron metabolism in serum and in peripheral blood mononuclear cells (PBMCs) from ß-TI and ß-TM patients and controls. METHODS AND RESULTS: In this study the iron status and the lipid profile in serum and in peripheral blood mononuclear cells (PBMCs) of 22 adult ß-TI patients were examined, and compared with 70 adult ß-TM, and 50 age-matched controls. Despite the great variability, levels of serum iron and transferrin saturation were significantly higher in ß-TI compared to both controls and ß-TM. By contrast, transferrin and hepcidin levels were lower in ß-TI patients. Changes in serum indicators in ß-TI patients were associated with altered expressions in PBMCs of hepcidin and IL-1α, involved in some way in the regulation of iron homeostasis. In addition ß-TI exhibited a reduction of total and high density lipoprotein cholesterol in serum and of neutral cholesterol ester hydrolase in PBMCs, and an increase of cytoplasmic neutral lipids and mRNA levels of acetyl-coenzymeA:cholesterol acyltransferase. CONCLUSIONS: Taken together, these findings provide experimental support for the idea that ß-TI patients exhibit a proatherogenic biochemical phenotype which may contribute to enhance cardiovascular risk in these subjects.

Evidence for a proatherogenic biochemical phenotype in beta thalassemia minor and intermedia.

The purpose of this study was to focus on pathophysiological mechanisms linking ß-thalassemia intermedia (ß-TI) and minor (ß-TMI) with cardiovascular risk. Iron status, prooxidant-antioxidant balance and lipid profiles in serum, and lipid content in peripheral blood mononuclear cells (PBMCs) were evaluated in 20 ß-TMI subjects, 22 ß-TI patients and in 30 nonthalassemic blood donors. The mRNA levels of some genes involved in the regulation of iron and cholesterol metabolism were also determined. In ß-TI and in ß-TMI, serum iron, prooxidant-antioxidant ratio, transferrin saturation and erythropoietin levels were higher, while transferrin and hepcidin were lower compared to controls. Hepcidin and interleukin-1α mRNA levels were found to be reduced in ß-TI- and ß-TMI-PBMCs, while those of tumor necrosis factor alpha were increased. A reduction in high-density lipoprotein cholesterol in serum and an accumulation of neutral lipids coupled with increased mRNA levels of acetyl-coenzyme A:cholesterol acyltransferase and decreased neutral cholesterol ester hydrolase in PBMCs were also observed in ß-TI and ß-TMI compared to controls. Taken together, these findings provide experimental support for the idea that not only ß-TI patients but also ß-TMI have a proatherogenic biochemical phenotype which may contribute to increase their cardiovascular disease risk.

Accumulation of neutral lipids in peripheral blood mononuclear cells as a distinctive trait of Alzheimer patients and asymptomatic subjects at risk of disease.

BACKGROUND: Alzheimer's disease is the most common progressive neurodegenerative disease. In recent years, numerous progresses in the discovery of novel Alzheimer's disease molecular biomarkers in brain as well as in biological fluids have been made. Among them, those involving lipid metabolism are emerging as potential candidates. In particular, an accumulation of neutral lipids was recently found by us in skin fibroblasts from Alzheimer's disease patients. Therefore, with the aim to assess whether peripheral alterations in cholesterol homeostasis might be relevant in Alzheimer's disease development and progression, in the present study we analyzed lipid metabolism in plasma and peripheral blood mononuclear cells from Alzheimer's disease patients and from their first-degree relatives. METHODS: Blood samples were obtained from 93 patients with probable Alzheimer's disease and from 91 of their first-degree relatives. As controls we utilized 57, cognitively normal, over-65 year-old volunteers and 113 blood donors aged 21-66 years, respectively. Data are reported as mean +/- standard error. Statistical calculations were performed using the statistical analysis software Origin 8.0 version. Data analysis was done using the Student t-test and the Pearson test. RESULTS: Data reported here show high neutral lipid levels and increased ACAT-1 protein in about 85% of peripheral blood mononuclear cells freshly isolated (ex vivo) from patients with probable sporadic Alzheimer's disease compared to about 7% of cognitively normal age-matched controls. A significant reduction in high density lipoprotein-cholesterol levels in plasma from Alzheimer's disease blood samples was also observed. Additionally, correlation analyses reveal a negative correlation between high density lipoprotein-cholesterol and cognitive capacity, as determined by Mini Mental State Examination, as well as between high density lipoprotein-cholesterol and neutral lipid accumulation. We observed great variability in the neutral lipid-peripheral blood mononuclear cells data and in plasma lipid analysis of the subjects enrolled as Alzheimer's disease-first-degree relatives. However, about 30% of them tend to display a peripheral metabolic cholesterol pattern similar to that exhibited by Alzheimer's disease patients. CONCLUSION: We suggest that neutral lipid-peripheral blood mononuclear cells and plasma high density lipoprotein-cholesterol determinations might be of interest to outline a distinctive metabolic profile applying to both Alzheimer's disease patients and asymptomatic subjects at higher risk of disease.

Oxidative imbalance in HIV-1 infected patients treated with antiretroviral therapy.

It is generally accepted that oxidative stress is involved in HIV infection. However, the role in oxidative balance of Highly Active Antiretroviral Therapy (HAART) is still debated. In our study we assessed serum oxidant and antioxidant levels in an HIV-1-infected population treated with HAART, and compared them with those of untreated HIV-1 patients and HIV-1-negative subjects. The study included 116 HIV-1-infected patients (86 HAART-treated and 30 untreated), and 46 HIV-negative controls. Serum oxidant levels were significantly higher in the HIV-1 treated group as compared to untreated and control groups. In addition, a decrease of serum total antioxidant status was observed in the HIV-1 treated group. To be noted is that patients who rigorously follow antiretroviral therapy (optimal HAART adherence) have significantly higher oxidative status than those who do not closely follow the therapy (poor HAART adherence). Analysis of variance revealed no significant further increase in oxidative status in HIV-1-infected patients taking antiretroviral and other drugs with the exception of psychiatric drugs (e.g. anxiolytics or antidepressants). Taken together, our results indicate that HAART may affect oxidative stress in HIV-1-infected patients and suggest that antiretroviral therapy plays an important role in the synergy of HIV infection and oxidative stress.

Modulation of cholesterol homeostasis by antiproliferative drugs in human pterygium fibroblasts.

PURPOSE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Role of cholesterol ester pathway in the control of cell cycle in human aortic smooth muscle cells.

Cholesterol esterification by acyl-CoA:cholesterol acyltransferase (ACAT) and proliferation of vascular smooth muscle cells (VSMC) are key events in vascular proliferative diseases. Here we performed experiments to ascertain the role of cholesterol ester pathway in the control of human aortic VSMC cycle progression. Results showed that serum-induced VSMC proliferation was preceded by an increased ability of the cells to esterify cholesterol as well as by an increased expression of ACAT and multidrug resistance (MDR1) mRNAs and extracellular related kinases 1/2 (ERK1/2), whereas caveolin-1 levels were markedly decreased. Cell cycle analyses performed in the presence of two inhibitors of cholesterol esterification, directly inhibiting ACAT (Sandoz 58-035) or the transport of cholesterol substrate from plasma membrane to endoplasmic reticulum (progesterone), indicate that each inhibitor suppressed the serum-induced DNA synthesis by accumulation of VSMCs in the G1 phase. The effect was associated with a rapid inhibition of ERK1/2 mitogenic signaling pathway; a down-regulation of cyclin D1, ACAT, and MDR1 mRNA; and an up-regulation of caveolin-1. These data provide a plausible link between cholesterol esterification and control of cell cycle G1/S transition, supporting the hypothesis that cholesterol esterification may accelerate the progression of human vascular proliferative diseases by modulating the rate of the VSMC proliferation.

Hyperexpression of low-density lipoprotein receptors and hydroxy-methylglutaryl-coenzyme A-reductase in human pinguecula and primary pterygium.

PURPOSE: There is now increasing evidence that pterygium and pinguecula are tumorlike tissues and that cell growth and DNA replication are closely linked to cholesterol metabolism. In this study, the expression of two main genes correlated to cholesterol metabolism--namely, the low-density lipoprotein receptor (LDL-R) gene and the hydroxy-methylglutaryl-coenzyme A-reductase (HMG-CoA-R) gene--were investigated in primary pterygium, pinguecula, and normal conjunctiva. METHODS: Pterygium, pinguecula, and normal conjunctiva samples were obtained from 30 eyes (50% men) at the time of surgery. Total RNA extracted from the specimens was subjected to semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Equal amounts of total RNA were reverse transcribed into cDNA. The cDNA was subsequently amplified by the PCR in the presence of specific primers for low-density lipoprotein receptor (LDL-R) and for hydroxy-methylglutaryl coenzyme A reductase (HMG-CoA-R). RESULTS: Semiquantitative RT-PCR analysis revealed that the mRNA levels of LDL-R and HMG-CoA-R were increased in pterygia, compared with levels in both pingueculae and normal conjunctivae. Differences were statistically significant (P <0.05), including pingueculae versus normal conjunctivae. CONCLUSIONS: This study indicates that pterygium and pinguecula have an altered metabolism of cholesterol-namely increased LDL-R and HMG-CoA-R mRNAs-as is characteristic of tumorlike tissues, and that the high expression of LDL receptors renders them amenable to be treated by photodynamic therapy with intravenously injected verteporfin.

Mononuclear cells in dementia.

According to the World Health Organization statistics, dementias are the largest contributors to disease burden in advanced market economies, and the leading cause of disability and dependence among older people worldwide. So far, several techniques have been developed to identify dementias with reasonable accuracy while the patient is still alive, however, no single of them has proven to be ideal, especially if you need to have a satisfactory early diagnosis. Studies of early onset dementia are largely limited by the inaccessibility to direct examination of the living human brain: it appears therefore that for a correct biochemical and molecular characterization of dementias, potential surrogate tissues must be identified. In this context, peripheral blood mononuclear cells (PBMCs) appear particularly attractive because they can be obtained in a minimally invasive manner and can be easily analyzed. This review focuses on the most representative methodologies and strategies in detecting and quantifying fluctuation in dementia that are currently being developed. In addition it provides a comprehensive evaluation of the diagnostic sensitivity of PBMCs in patients with dementia. Finally, it discusses the data supporting the use of the determination of neutral lipids (NLs) in PBMCs by Oil Red O (ORO) staining, which is a minimally invasive, cheap, easy and fast procedure, as the promising method for early detection of dementia and to search for new effective treatments.

Cognitive decline and depressive symptoms in late-life are associated with statin use: evidence from a population-based study of Sardinian old people living in their own home.

OBJECTIVES: This study was designed to provide further insights into the effects of dyslipidemia (Dys-y) and use of statins (St-y) on cognitive functions and mood in older people. METHODS: Three hundred and twenty-nine subjects aged > or = 65 years were screened for cognitive dysfunction using mini mental state examination (MMSE). The geriatric depression scale (GDS) was used to detect depression. Interview questionnaires surveyed activities of daily living (ADL) and instrumental ADL (IADL), as well as other functional disabilities. The presence of neutral lipids (NLs) in cytoplasm of peripheral blood mononuclear cells (PBMCs) was determined with the Oil red O (ORO) staining. RESULTS: There was no significant difference in MMSE and GDS scores between normal (Dys-n) and Dys-y. However, when Dys-y subjects were divided into St-y and non-statin users (St-n), significant differences emerged in the scores of MMSE and GDS: St-y had lower MMSE and higher GDS than St-n. Multiple correspondence analysis and logistic regression provided further evidence that elderly St-y were much more likely to suffer of cognitive impairment and depression than St-n. Another interesting finding was that the intensity of NL-PBMCs measured by ORO staining was greater in subjects with altered MMSE compared with cognitively normal subjects. In addition St-y had higher ORO score than St-n. DISCUSSION: This is an observational study and cannot, therefore, prove a causal relationship between St-y in the elderly and a higher cognitive decline, nevertheless it provides substantial indications that caution should be exercised in the provision of statins in elderly subjects to avoid accelerated memory loss.

Altered proteolysis in fibroblasts of Alzheimer patients with predictive implications for subjects at risk of disease.

There is great interest in developing reliable biomarkers to support antemortem diagnosis of late-onset Alzheimer's disease (AD). Early prediction and diagnosis of AD might be improved by the detection of a proteolytic dysfunction in extracts from cultured AD fibroblasts, producing altered isoelectrophoretic forms of the enzyme transketolase (TK-alkaline bands). The TK profile and apolipoprotein E (APOE) genotype were examined in fibroblasts from 36 clinically diagnosed probable late-onset sporadic AD patients and 38 of their asymptomatic relatives, 29 elderly healthy individuals, 12 neurological non-AD patients, and 5 early-onset AD patients. TK alterations occurred in (i) several probable AD patients regardless of age-of-onset and severity of disease; (ii) all early-onset AD patients and APOE ε 4/4 carriers; and (iii) nearly half of asymptomatic AD relatives. Normal subjects and non-AD patients were all negative. Notably, culture conditions promoting TK alterations were also effective in increasing active BACE1 levels. Overall, the TK assay might represent a low-cost laboratory tool useful for supporting AD differential diagnosis and identifying asymptomatic subjects who are at greater risk of AD and who should enter a follow-up study. Moreover, the cultured fibroblasts were confirmed as a useful in vitro model for further studies on the pathogenetic process of AD.

Cognitive Impairment and Age-Related Vision Disorders: Their Possible Relationship and the Evaluation of the Use of Aspirin and Statins in a 65 Years-and-Over Sardinian Population.

Neurological disorders (Alzheimer's disease, vascular and mixed dementia) and visual loss (cataract, age-related macular degeneration, glaucoma, and diabetic retinopathy) are among the most common conditions that afflict people of at least 65 years of age. An increasing body of evidence is emerging, which demonstrates that memory and vision impairment are closely, significantly, and positively linked and that statins and aspirin may lessen the risk of developing age-related visual and neurological problems. However, clinical studies have produced contradictory results. Thus, the intent of the present study was to reliably establish whether a relationship exist between various types of dementia and age-related vision disorders, and to establish whether statins and aspirin may or may not have beneficial effects on these two types of disorders. We found that participants with dementia and/or vision problems were more likely to be depressed and displayed worse functional ability in basic and instrumental activities of daily living than controls. Mini mental state examination scores were significantly lower in patients with vision disorders compared to subjects without vision disorders. A closer association with macular degeneration was found in subjects with Alzheimer's disease than in subjects without dementia or with vascular dementia, mixed dementia, or other types of age-related vision disorders. When we considered the associations between different types of dementia and vision disorders and the use of statins and aspirin, we found a significant positive association between Alzheimer's disease and statins on their own or in combination with aspirin, indicating that these two drugs do not appear to reduce the risk of Alzheimer's disease or improve its clinical evolution and may, on the contrary, favor its development. No significant association in statin use alone, aspirin use alone, or the combination of these was found in subjects without vision disorders but with dementia, and, similarly, none in subjects with vision disorders but without dementia. Overall, these results confirm the general impression so far; namely, that macular degeneration may contribute to cognitive disorders (Alzheimer's disease in particular). In addition, they also suggest that, while statin and aspirin use may undoubtedly have some protective effects, they do not appear to be magic pills against the development of cognitive impairment or vision disorders in the elderly.

Cholesterol homeostasis: a key to prevent or slow down neurodegeneration.

Neurodegeneration, a common feature for many brain disorders, has severe consequences on the mental and physical health of an individual. Typically human neurodegenerative diseases are devastating illnesses that predominantly affect elderly people, progress slowly, and lead to disability and premature death; however they may occur at all ages. Despite extensive research and investments, current therapeutic interventions against these disorders treat solely the symptoms. Therefore, since the underlying mechanisms of damage to neurons are similar, in spite of etiology and background heterogeneous, it will be of interest to identify possible trigger point of neurodegeneration enabling development of drugs and/or prevention strategies that target many disorders simultaneously. Among the factors that have been identified so far to cause neurodegeneration, failures in cholesterol homeostasis are indubitably the best investigated. The aim of this review is to critically discuss some of the main results reported in the recent years in this field mainly focusing on the mechanisms that, by recovering perturbations of cholesterol homeostasis in neuronal cells, may correct clinically relevant features occurring in different neurodegenerative disorders and, in this regard, also debate the current potential therapeutic interventions.

Cholesterol esterification during differentiation of mouse erythroleukemia (Friend) cells.

Cholesterol is an essential constituent of all mammalian cell membranes and its availability is therefore a prerequisite for cellular growth and other functions. Several lines of evidence are now indicating an association between alterations of cholesterol homeostasis and cell cycle progression. However, the role of cholesterol in cell differentiation is still largely unknown. To begin to address this issue, in this study we examined changes in cholesterol metabolism and in the mRNA levels of proteins involved in cholesterol import and esterification (multi-drug resistance, MDR-3) and acylCoA: cholesterol acyltransferase (ACAT) and cholesterol export (caveolin-1) in Friend virus-induced erythroleukemia cells (MELC), in the absence or in the presence of the chemical inducer of differentiation, hexamethylene bisacetamide (HMBA). FBS-stimulated growth of MELC was accompanied by an immediate elevation of cholesterol synthesis and cholesterol esterification, and by an increase in the levels of MDR-3 and ACAT mRNAs. A decrease in caveolin-1 expression was also observed. However, when MELC were treated with HMBA, the inhibition of DNA synthesis caused by HMBA treatment, was associated with a decrease in cholesterol esterification and in ACAT and MDR-3 mRNA levels and an increase in caveolin-1 mRNA. Detection of cytoplasmic neutral lipids by staining MELC with oil red O, a dye able to evidence CE but not FC, revealed that HMBA-treatment also reduced growth-stimulated accumulation of cholesterol ester to approximately the same extent as the ACAT inhibitor, SaH. Overall, these results indicate for the first time a role of cholesterol esterification and of some related genes in differentiation of erythroid cells.

Cholesterol, Alzheimer's disease, prion disorders: a ménage à trois?

Aberrant folded proteins are hallmarks of amyloidogenic diseases. Examples are Alzheimer's disease (AD) and prion-related disorders (PrD). These disorders, although clinically different, have the same underlying pathogenetic mechanism: an altered protein conformer with high beta-sheet structure content: the amyloid beta peptide (Abeta) in the case of AD, and the aberrant prion protein, PrPsc, in PrD. Although the molecular processes that cause these proteins to adopt non-native structures in vivo and become cytotoxic are still largely unknown, there is good reason to expect prion research to profit from advances in the understanding of AD, and vice versa. Growing evidence indicates that the various pathways of lipid/lipoprotein metabolism play a key role in AD and PrD pathophysiology. These findings clearly highlight the possible involvement of cholesterol in misfolded protein generation. In this review, we focus on recent studies which provide evidence that membrane domains, called lipid rafts, directly promote protein misfolding, and that this process takes place only if changes occur in the fine regulation of intracellular cholesterol. In addition, we discuss the implications of these results to introduce the concept that pharmacological interventions restoring cholesterol homeostasis could have potential preventive/therapeutic value against the progression of misfolding disorders. The aim of the review is to provide researchers with a general understanding of cholesterol's involvement in protein folding/misfolding processes which maybe relevant for knowledge advancement regarding amyloidogenic proteins, and possible ways to prevent their pathological activity.

Glucose-6-phosphate-dehydrogenase deficiency as a risk factor for pterygium.

PURPOSE: Glucose-6-phosphate dehydrogenase (G6PD) is an important site of metabolic control in the pentose phosphate pathway (PPP), providing reducing power (NADPH) and pentose phosphates. The purpose of this study was to investigate the possible involvement of G6PD deficiency (G6PD-) in the pathogenesis of pterygium. METHODS: Erythrocyte G6PD activity was evaluated in 123 pterygium patients and in 112 age-matched control patients. Enzyme activity, mRNA, rate of growth, green autofluorescence, response to oxidative stress, and cholesterol metabolism were determined in pterygium fibroblasts (PFs) and in normal conjunctival fibroblasts (NCFs) isolated from G6PD normal (NCFs+ and PFs+) and G6PD- (NCFs- and PFs-) patients. RESULTS: Higher prevalence of G6PD- was found in patients affected by primary pterygium than in control subjects, both men and women, suggesting that this enzymatic defect may be a predisposing factor for pterygium. G6PD activity was significantly lower in NCFs- than in NCFs+, but not in PFs- than in PFs+. In PFs-, G6PD mRNA levels were significantly higher than in PFs+. Growth-stimulated NCFs- grew at half the rate of NCFs+, although PFs- and PFs+ grew at the same rate. Increased green autofluorescence and susceptibility to oxidative stress were observed in PFs (+/-) and in NCFs-, but not in NCFs+. Moreover, ex vivo PFs (+/-) accumulated more lipids than corresponding NCFs. CONCLUSIONS: The results of this study, although restricted to a limited group of subjects (i.e., those of Sardinian ancestry), suggest that G6PD- not only does not protect against pterygium, but may even be considered a risk factor for the development of this disorder.

Altered cholesterol ester cycle in skin fibroblasts from patients with Alzheimer's disease.

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.

Antiprion activity of cholesterol esterification modulators: a comparative study using ex vivo sheep fibroblasts and lymphocytes and mouse neuroblastoma cell lines.

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.