Fabrizio Angius

Cannabinoid self-administration in rats: sex differences and the influence of ovarian function

We recently demonstrated the existence of strain differences in self‐administration of the cannabinoid CB1 receptor agonist WIN55,212‐2 (WIN) by Long Evans (LE) and Lister Hooded (LH) but not Sprague‐Dawley (SD) male rats. This follow‐up study is aimed at verifying whether sex and ovarian hormones might also be critical factors in the initiation, retention and extinction of WIN self‐administration.

Hydrophobic characterization of intracellular lipids in situ by Nile Red red/yellow emission ratio

Nile Red (9-diethylamino-5H-benzo [alpha] phenoxazine-5-one) is a fluorescent lipophilic dye characterized by a shift of emission from red to yellow according to the degree of hydrophobicity of lipids. Polar lipids (i.e., phospholipids) which are mostly present in membranes, are stained in red whereas neutral lipids (esterified cholesterol and triglycerides) which are present in lipid droplets, are stained in yellow. Besides this marked, qualitative contrast between polar and neutral lipids, small differences of the hydrophobic strength could be assessed by the quantitative ratio of red and yellow emissions, in order to extend the discrimination of lipids within the groups of neutral and polar lipids. On the other hand, ratiometric data of red and yellow emissions have not yet been evaluated in the numerous previous light microscopy investigations which used Nile Red. In this work we show that the Nile Red red/yellow ratio enables discrimination of different lipids (monooleine>oleic acid>phosphatidylcholine>free cholesterol>trioleine>oleyl cholesteryl ester). We also show changes in the Nile Red red/yellow emission ratio of lipid droplets of 3T3 mouse fibroblasts induced by drugs interfering with the cholesterol cycle.

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 Alzheimers 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.

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

BackgroundAlzheimers disease is the most common progressive neurodegenerative disease. In recent years, numerous progresses in the discovery of novel Alzheimers 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 Alzheimers disease patients. Therefore, with the aim to assess whether peripheral alterations in cholesterol homeostasis might be relevant in Alzheimers disease development and progression, in the present study we analyzed lipid metabolism in plasma and peripheral blood mononuclear cells from Alzheimers disease patients and from their first-degree relatives.MethodsBlood samples were obtained from 93 patients with probable Alzheimers 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.ResultsData 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 Alzheimers disease compared to about 7% of cognitively normal age-matched controls. A significant reduction in high density lipoprotein-cholesterol levels in plasma from Alzheimers 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 Alzheimers disease-first-degree relatives. However, about 30% of them tend to display a peripheral metabolic cholesterol pattern similar to that exhibited by Alzheimers disease patients.ConclusionWe 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 Alzheimers disease patients and asymptomatic subjects at higher risk of disease.

Lipid droplet changes in proliferating and quiescent 3T3 fibroblasts

Lipid droplets (LDs) are fat-storing organelles present in virtually all eukaryotic cells and involved in many aspects of cell biology related to lipid metabolism and cholesterol homeostasis. In this study, we investigated the presence of LDs in proliferating and quiescent (contact-inhibited) 3T3 fibroblasts to verify a correlation with cell growth. LDs were characterized by Nile red staining, positivity to adipophilin and negativity to perilipin. LDs were numerous in proliferating cells, but very few in quiescent cells. However, the fraction of quiescent cells, which resumed proliferation after scratch-wound assay, also resumed the formation of LDs. In proliferating cells, the number of LDs correlated with the DNA content, suggesting a continuous accumulation of LDs during cell growth. These findings were supported by biochemical data showing much higher rates of cholesterol esterification and triglyceride synthesis in proliferating cells. Both filipin staining and the fluorescent cholesterol analog dehydroergosterol revealed the presence of an intense traffic of free cholesterol, mediated by acidic vesicles, in proliferating cells. Nile red ratiometric measurements revealed a different lipid composition of LDs in proliferating and quiescent cells. Changes in the number and composition of LDs were also found in growing cells treated with inhibitors of cholesterol esterification (Sandoz 58-035), endosomal cholesterol efflux (U18666A) and V-ATPase (bafilomycin-A1).

Liposomes and MTT cell viability assay: an incompatible affair.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is commonly used to evaluate the cytotoxicity potential of drugs vehicled by liposomes. However, liposome delivering drugs could produce inconsistent values of MTT absorbance. On the basis of previous experiments demonstrating the MTT affinity for lipid droplets, this paper aims to show that empty-liposomes interfere, per se, on MTT assay due to its lipidic nature. This brings into question the use of MTT testing cytotoxicity when liposomes are involved in delivering drugs.

Role of HDL in Cholesteryl Ester Metabolism of Lipopolysaccharide-Activated P388D1 Macrophages

Infections share with atherosclerosis similar lipid alterations, with accumulation of cholesteryl esters (CEs) in activated macrophages and concomitant decrease of cholesterol-HDL (C-HDL). Yet the precise role of HDL during microbial infection has not been fully elucidated. Activation of P388D1 by lipopolysaccharide (LPS) triggered an increase of CEs and neutral lipid contents, along with a remarkable enhancement in 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate-HDL uptake. Similar results were found in human monocyte-derived macrophages and monocytes cocultured with phytohemagglutinin-activated lymphocytes. Inhibition of cholesterol esterification with Sandoz-58035 resulted in 80% suppression of CE biosynthesis in P388D1. However, only a 35% decrease of CE content, together with increased scavenger receptor class B member 1 (SR-B1) protein expression, was found after 72 h and thereafter up to 16 passages of continuous ACAT suppression. Chronic inhibition blunted the effect of LPS treatment on cholesterol metabolism, increased the ratio of free cholesterol/CE content and enhanced interleukin 6 secretion.These results imply that, besides de novo biosynthesis and acquisition by LDL, HDL contributes probably through SR-B1 to the increased CE content in macrophages, partly explaining the low levels of C-HDL during their activation. Our data suggest that in those conditions where more CEs are required, HDL rather than removing, may supply CEs to the cells.

Differential induction of dyskinesia and neuroinflammation by pulsatile versus continuous L-DOPA delivery in the 6-OHDA model of Parkinson's disease

Neuroinflammation is associated with l-DOPA treatment in Parkinsons disease (PD), suggesting a role in l-DOPA-induced dyskinesia (LID), however it is unclear whether increased inflammation is specifically related to the dyskinetic outcome of l-DOPA treatment. Diversely from oral l-DOPA, continuous intrajejunal l-DOPA infusion is associated with very low dyskinetic outcome in PD patients. We reproduced these regimens of administration in 6-OHDA-lesioned hemiparkinsonian rats, where dyskinetic responses and striatal neuroinflammation induced by chronic pulsatile (DOPAp) or continuous (DOPAc) l-DOPA were compared. Moreover, we investigated the contribution of a peripheral inflammatory challenge with lipopolysaccharide (LPS), to DOPAp-induced dyskinetic and neuroinflammatory responses. Rats 6-OHDA-infused in the medial forebrain bundle received two weeks treatment with DOPAp, DOPAc via subcutaneous osmotic minipumps, or DOPAp followed by DOPAc. l-DOPA plasma levels were measured in all experimental groups. An independent group of rats received one peripheral dose of LPS 24h before DOPAp treatment. Abnormal involuntary movements (AIMs) were evaluated as a rat model of LID. Immunoreactivity (IR) for OX-42, microglial and neuronal TNF-α, iNOS and GFAP was quantified in denervated and contralateral striatum. In addition, serum TNF-α was measured. The 6-OHDA denervation induced a mild microgliosis in the striatum two weeks after neurotoxin infusion, and increased TNF-α IR in microglia. Rats receiving the DOPAp treatment developed AIMs and displayed increased striatal OX-42, microglial TNF-α, iNOS and GFAP. Moreover, TNF-α IR was also increased in a subpopulation of striatal neurons. Conversely, DOPAc did not induce AIMs or inflammatory responses in either drug-naïve animals or rats that were previously dyskinetic when exposed to DOPAp. Serum TNF-α was not altered by any l-DOPA treatment. LPS pre-treatment increased the degree of DOPAp-induced AIMs and striatal IR for OX-42, TNF-α, iNOS and GFAP. Altogether the present findings indicate that in the 6-OHDA model, chronic l-DOPA induces striatal inflammatory responses, which however depend upon the administration regimen and the dyskinetic outcome of drug treatment. The potentiation of dyskinetic responses by LPS suggests a reciprocal causal link between neuroinflammation and LID.

Thalassemia intermedia is associated with a proatherogenic biochemical phenotype

OBJECTIVE Unlike beta thalassemia major (β-TM) in which cardiac siderosis represents the leading cause of mortality and morbidity, in beta thalassemia intermedia (β-TI), pulmonary hypertension (PHT) and thrombosis seems to be the major cardiovascular complications. However, the mechanism underlying these complications in β-TI is still unclear. Endothelial dysfunction, the key early event in atherogenesis, is now emerging as an important cardiovascular risk determiner in β-TI patients. Among the factors known to affect endothelial function, iron and cholesterol merit particular consideration in β-TI patients. Therefore, with the aim to extend our knowledge on the mechanisms connecting atherosclerosis to β-TI, in this study, we compared lipid and iron metabolism in serum and in peripheral blood mononuclear cells (PBMCs) from β-TI and β-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 β-TI patients were examined, and compared with 70 adult β-TM, and 50 age-matched controls. Despite the great variability, levels of serum iron and transferrin saturation were significantly higher in β-TI compared to both controls and β-TM. By contrast, transferrin and hepcidin levels were lower in β-TI patients. Changes in serum indicators in β-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 β-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 β-TI patients exhibit a proatherogenic biochemical phenotype which may contribute to enhance cardiovascular risk in these subjects.

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.