Sandra Dessì

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.

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.

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.

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.

Changes in serum and hepatic cholesterol in lead-induced liver hyperplasia.

Lead nitrate when injected intravenously as a single dose to male Wistar rats causes a strong hepatic proliferative response followed by reabsorption of excess tissue within 10-14 days. The rate of cell proliferation in this hyperplastic model was positively correlated with hepatic de novo synthesis of cholesterol, stimulation of the hexose monophosphate shunt pathway of glucose metabolism and with alterations in serum lipoproteins.

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

BackgroundCholesterol 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.ResultsResults 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.ConclusionsWe 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.

Cell growth and cholesterol metabolism in human glucose-6-phosphate dehydrogenase deficient lymphomononuclear cells

Abstract. Atherosclerosis is an inflammatory‐fibroproliferative response of the arterial wall involving a complex set of interconnected events where cell proliferation (lymphomonocytes, and endothelial and smooth‐muscle cells) and substantial perturbations of intracellular cholesterol metabolism are considered to be among the main features. Glucose‐6‐phosphate dehydrogenase (G6PD), the key enzyme of the hexose‐monophosphate shunt pathway, is an essential enzyme involved in both cell growth and cholesterol metabolism, raising the question as to whether G6PD deficiency may have metabolic and growth implications in a deficient population. In the present study, we investigated cell growth and cholesterol metabolism in peripheral blood lymphomononuclear cells (PBMC) from G6PD‐normal (n = 5) and ‐deficient (n = 5) subjects stimulated with lectins (phytohaemoagglutinin and Concanavalin A). G6PD activity, DNA ([3H]‐thymidine incorporation) cholesterol synthesis and esterification ([14C]‐acetate and [14C]‐oleate incorporation), and G6PD, HMGCoA reductase and low density lipoprotein (LDL) receptor mRNA levels (RT‐PCR) all increased following lectin stimulation in both normal and G6PD‐deficient cells. However, these parameters were significantly lower in G6PD‐deficient cells (P < 0.05). It is of interest that G6PD‐deficient PBMC, which showed lower expression of G6PD and higher expression of the LDL receptor gene than normal PBMC under basal conditions, exhibited an opposite pattern after stimulation: G6PD and HMGCoA reductase being expressed at significantly higher levels in deficient than in normal cells (P < 0.05). We conclude that the reduced capability of G6PD‐deficient cells to respond to mitogenic stimuli and to synthesize cholesterol esters may represent favourable conditions for reducing the risk of cardiovascular diseases.

MDR1 gene expression in normal and atherosclerotic human arteries(1).

Recent studies have shown that a membrane p-glycoprotein, encoded by MDR1 gene, is involved in the transport of free cholesterol from the plasma membrane to endoplasmic reticulum, the site of cholesterol esterification by acyl-CoA:cholesterol acyltransferase (ACAT). Moreover, results deriving from our previous studies have shown that the rate of cell proliferation was positively correlated with cholesteryl ester levels as well as with ACAT and MDR1 gene expression. In this study, lipid content and the expression of the genes involved in cholesterol metabolism such as hydroxy-methylglutaryl coenzyme A reductase (HMGCoA-R), low-density lipoprotein receptor (LDL-R), ACAT and MDR1 have been investigated in control and atherosclerotic arteries. The results have shown that the levels of cholesteryl ester increase with the age of cadaveric donors in arteries prone to atherosclerosis (abdominal aorta, superficial femoral artery) and become predominant in advanced atherosclerotic lesions. The mRNA levels of ACAT and MDR1 showed the same age correlation, reaching the highest values in atherosclerotic specimens. These results suggest that MDR1 may be involved in the accumulation of intracellular cholesterol ester levels found in atherosclerotic lesions. Moreover, the levels of HMGCoA-R, LDL-R and ACAT gene expressions progressively increased with the age of cadaveric donors; conversely, in atherosclerotic specimens, the mRNA levels of HMGCoA-R and LDL-R drastically decreased while ACAT gene expression reached its maximum. These findings suggest a reactivation of normal homeostatic regulation of cholesterol in advanced and complicated lesions.

Production of Inflammatory Molecules in Peripheral Blood Mononuclear Cells from Severely Glucose-6-Phosphate Dehydrogenase-Deficient Subjects

Objective: We have previously demonstrated that Mediterranean glucose-6-phosphate dehydrogenase (G6PD)-deficient peripheral blood mononuclear cells (PBMC) respond to mitogenic stimuli with a reduced cholesterol synthesis and growth. In the present study, we have investigated the release of inflammatory molecules by PBMC following a mitogenic stimulus, as well as the transformation to foam cells of monocyte-derived macrophages from severely G6PD-deficient and normal subjects. Methods and Results: PBMC from G6PD-deficient subjects produced interleukin (IL)-1β and IL-6 to a lower extent compared with normal subjects. 5-Hydroxyeicosatetraenoic acid, a primary product of 5-lipoxygenase, was slightly decreased. Tumour necrosis factor-α and IL-1β secretion was significantly reduced in monocyte-derived macrophages. No difference was found in IL-10 secretion, whereas transforming growth factor-β was invariably found to be significantly higher in G6PD-deficient cells. In cells incubated with acetylated low-density lipoprotein, cholesterol esterification and its storage in lipid droplets were lower than in normal G6PD cells. Conclusions: We conclude that by reducing the secretion of inflammatory molecules by PBMC and increasing the secretion of transforming growth factor-β and the capability of monocyte-derived macrophages to accumulate lipid droplets and convert into foam cells, G6PD deficiency may confer a partial protection against atherosclerosis leading to the reduced risk of cardiovascular diseases reported in G6PD-deficient subjects.