Dietary plant microRNAs as potential regulators of cellular cholesterol efflux.

AIM: Epidemiological evidence suggests adherence to vegetable-rich diets is associated to atheroprotective effects and bioactive components are most likely to play a relevant role. The notion of inter-kingdom regulation has opened a new research paradigm and perhaps microRNAs (miRNAs) from edible vegetables could influence consumer gene expression and lead to biological effects. We aimed to investigate the potential impact of broccoli-derived miRNAs on cellular cholesterol efflux in vitro. METHODS: Four miRNAs (miR159a, miR159b, miR166a and miR403) from Brassica oleracea var. italica (broccoli), a widely consumed cruciferous vegetable, were selected for further investigation, based on their high abundancy in this vegetable and their presence in other plants. Selected miRNAs were synthesized with a 3'-terminal 2'-O-methylation and their cellular toxicity, in vitro gastrointestinal resistance and cellular uptake were evaluated. Potential target genes within the mammalian transcriptome were assessed in silico following pathway analysis. In vitro cholesterol efflux was assessed in human THP-1-derived macrophages. RESULTS: miRNAs survival to in vitro GI digestion was around 1%, although some variation was seen between the four candidates. Cellular uptake by mammalian cells was confirmed, and an increase in cholesterol efflux was observed. Pathway analysis suggested these miRNAs are involved in biological processes related to phosphorylation, phosphatidylinositol and Wnt signaling, and to the insulin/IGF pathway. CONCLUSIONS: Health-promoting properties attributed to cruciferous vegetables, might be mediated (at least in part) through miRNA-related mechanisms.

Identification and Functional Analysis of APOB Variants in a Cohort of Hypercholesterolemic Patients.

Mutations in APOB are the second most frequent cause of familial hypercholesterolemia (FH). APOB is highly polymorphic, and many variants are benign or of uncertain significance, so functional analysis is necessary to ascertain their pathogenicity. Our aim was to identify and characterize APOB variants in patients with hypercholesterolemia. Index patients (n = 825) with clinically suspected FH were analyzed using next-generation sequencing. In total, 40% of the patients presented a variant in LDLR, APOB, PCSK9 or LDLRAP1, with 12% of the variants in APOB. These variants showed frequencies in the general population lower than 0.5% and were classified as damaging and/or probably damaging by 3 or more predictors of pathogenicity. The variants c.10030A>G;p.(Lys3344Glu) and c.11401T>A;p.(Ser3801Thr) were characterized. The p.(Lys3344Glu) variant co-segregated with high low-density lipoprotein (LDL)-cholesterol in 2 families studied. LDL isolated from apoB p.(Lys3344Glu) heterozygous patients showed reduced ability to compete with fluorescently-labelled LDL for cellular binding and uptake compared with control LDL and was markedly deficient in supporting U937 cell proliferation. LDL that was carrying apoB p.(Ser3801Thr) was not defective in competing with control LDL for cellular binding and uptake. We conclude that the apoB p.(Lys3344Glu) variant is defective in the interaction with the LDL receptor and is causative of FH, whereas the apoB p.(Ser3801Thr) variant is benign.

"MiR-7 controls cholesterol biosynthesis through posttranscriptional regulation of DHCR24 expression".

Dysregulation of cholesterol homeostasis is associated with several pathologies including cardiovascular diseases and neurological disorders such as Alzheimer's disease (AD). MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of cholesterol metabolism. We previously established the role of miR-7 in regulating insulin resistance and amyloidosis, which represents a common pathological feature between type 2 diabetes and AD. We show here an additional metabolic function of miR-7 in cholesterol biosynthesis. We found that miR-7 blocks the last steps of the cholesterol biosynthetic pathway in vitro by targeting relevant genes including DHCR24 and SC5D posttranscriptionally. Intracranial infusion of miR-7 on an adeno-associated viral vector reduced the expression of DHCR24 in the brain of wild-type mice, supporting in vivo miR-7 targeting. We also found that cholesterol regulates endogenous levels of miR-7 in vitro, correlating with transcriptional regulation through SREBP2 binding to its promoter region. In parallel to SREBP2 inhibition, the levels of miR-7 and hnRNPK (the host gene of miR-7) were concomitantly reduced in brain in a mouse model of Niemann Pick type C1 disease and in murine fatty liver, which are both characterized by intracellular cholesterol accumulation. Taken together, the results establish a novel regulatory feedback loop by which miR-7 modulates cholesterol homeostasis at the posttranscriptional level, an effect that could be exploited for therapeutic interventions against prevalent human diseases.

Connection between miRNA Mediation and the Bioactive Effects of Broccoli (Brassica oleracea var. italica): Exogenous miRNA Resistance to Food Processing and GI Digestion.

Broccoli (Brassica oleracea var. italica) and its bioactive compounds are associated with beneficial health effects, which might be enabled, at least in part, through miRNA regulation, despite recent controversial studies suggesting that exogenous dietary miRNAs may reach host circulation and target cells to regulate gene expression. Here, a computational analysis was performed to explore the processes and pathways associated with genes targeted either by (1) host-expressed miRNAs (endogenous) modulated by the bioactive compounds in broccoli or (2) miRNAs derived from broccoli (exogenous). In addition, the stability of exogenous miRNAs from broccoli was assessed after broccoli was subjected to the usual processing methods and in vitro digestion-simulating gastrointestinal (GI) conditions. Overall, bioinformatic results show that the anticarcinogenic and cancer-preventive properties attributed to cruciferous vegetables might be mediated, at least in part, through miRNA-related mechanisms. Moreover, results show that broccoli-derived miRNAs can survive common food-processing conditions and GI digestion.

Changes in Soluble TWEAK Concentrations, but Not Those in Amyloid-ß(1-40), Are Associated with a Decrease in Carotid Intima-Media Thickness after Bariatric Surgery in Obese Women.

BACKGROUND/AIM: Soluble tumor necrosis factor-like weak inducer of apoptosis (sTWEAK) and amyloid-ß(1-40) (Aß40) emerged as markers of cardiovascular risk because of their actions in the endothelium and their role in atherosclerotic progression. The aim of this study was to analyze the association of these two factors with the decrease in carotid intima-media thickness (cIMT) after bariatric surgery in obese women. METHODS: We studied 60 severely obese women, of whom 20 were submitted to laparoscopic Roux-en-Y gastric bypass (RYGB), 20 to sleeve gastrectomy (SG), and 20 to lifestyle modification therapy. Circulating sTWEAK, Aß40, high-sensitivity C-reactive protein, plasminogen activator inhibitor type 1, insulin resistance (HOMA-IR), and cIMT were measured at baseline and after 1 year of follow-up. RESULTS: sTWEAK increased similarly after both surgical procedures, whereas the increase observed after lifestyle intervention did not reach statistical significance. Aß40 showed no differences between groups of women, nor did it change during follow-up. The decrease in cIMT at 12 months correlated with the decrease in body mass index (BMI) (r = 0.45; p < 0.001) and fasting insulin (r = 0.30; p = 0.038), and also with the increase in sTWEAK (r = -0.43; p = 0.002). Multivariate linear regression showed that only the changes in BMI (ß = 0.389; p = 0.005) and sTWEAK (ß = -0.358; p = 0.009) were associated with the decrease in cIMT (R2 = 0.313; F = 9.348; p < 0.001). CONCLUSIONS: One year after bariatric surgery, RYGB and SG induced a similar increase in circulating sTWEAK that occurred in parallel to the decrease observed in cIMT.

First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro.

First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit ß (ß-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and ß-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes' internal milieu induced by haloperidol affects lysosomal functionality.

Disruption of the mevalonate pathway induces dNTP depletion and DNA damage.

The mevalonate pathway is tightly linked to cell division. Mevalonate derived non-sterol isoprenoids and cholesterol are essential for cell cycle progression and mitosis completion respectively. In the present work, we studied the effects of fluoromevalonate, a competitive inhibitor of mevalonate diphosphate decarboxylase, on cell proliferation and cell cycle progression in both HL-60 and MOLT-4 cells. This enzyme catalyzes the synthesis of isopentenyl diphosphate, the first isoprenoid in the cholesterol biosynthesis pathway, consuming ATP at the same time. Inhibition of mevalonate diphosphate decarboxylase was followed by a rapid accumulation of mevalonate diphosphate and the reduction of ATP concentrations, while the cell content of cholesterol was barely affected. Strikingly, mevalonate diphosphate decarboxylase inhibition also resulted in the depletion of dNTP pools, which has never been reported before. These effects were accompanied by inhibition of cell proliferation and cell cycle arrest at S phase, together with the appearance of γ-H2AX foci and Chk1 activation. Inhibition of Chk1 in cells treated with fluoromevalonate resulted in premature entry into mitosis and massive cell death, indicating that the inhibition of mevalonate diphosphate decarboxylase triggered a DNA damage response. Notably, the supply of exogenously deoxyribonucleosides abolished γ-H2AX formation and prevented the effects of mevalonate diphosphate decarboxylase inhibition on DNA replication and cell growth. The results indicate that dNTP pool depletion caused by mevalonate diphosphate decarboxylase inhibition hampered DNA replication with subsequent DNA damage, which may have important consequences for replication stress and genomic instability.

Curcumin promotes exosomes/microvesicles secretion that attenuates lysosomal cholesterol traffic impairment.

SCOPE: Exosomes/microvesicles are originated from multivesicular bodies that allow the secretion of endolysosome components out of the cell. In the present work, we investigated the effects of curcumin, a polyphenol, on exosomes/microvesicles secretion in different cells lines, using U18666A as a model of intracellular cholesterol trafficking impairment. METHODS AND RESULTS: In both HepG2 hepatocarcinoma cells and THP-1 differentiated macrophages, treatment with curcumin affected the size and the localization of endosome/lysosomes accumulated by U18666A, and reduced the cholesterol cell content. To ascertain the mechanism, we analyzed the incubation medium. Curcumin stimulated the release of cholesterol and the lysosomal ß-hexosaminidase enzyme, as well as the exosome markers, flotillin-2 and CD63. Electron microscopy studies demonstrated the presence of small vesicles similar to exosomes/microvesicles in the secretion fluid. These vesicles harbored CD63 on their surface, indicative of their endolysosomal origin. These effects of curcumin were particularly intense in cells treated with U18666A. CONCLUSION: These findings indicate that curcumin ameliorates the U18666A-induced endolysosomal cholesterol accumulation by shuttling cholesterol and presumably other lipids out of the cell via exosomes/microvesicles secretion. This action may contribute to the potential of curcumin in the treatment of lysosomal storage diseases.

Atypical antipsychotics alter cholesterol and fatty acid metabolism in vitro.

Haloperidol, a typical antipsychotic, has been shown to inhibit cholesterol biosynthesis by affecting Δ(7)-reductase, Δ(8,7)-isomerase, and Δ(14)-reductase activities, which results in the accumulation of different sterol intermediates. In the present work, we investigated the effects of atypical or second-generation antipsychotics (SGA), such as clozapine, risperidone, and ziprasidone, on intracellular lipid metabolism in different cell lines. All the SGAs tested inhibited cholesterol biosynthesis. Ziprasidone and risperidone had the same targets as haloperidol at inhibiting cholesterol biosynthesis, although with different relative activities (ziprasidone > haloperidol > risperidone). In contrast, clozapine mainly affected Δ(24)-reductase and Δ(8,7)-isomerase activities. These amphiphilic drugs also interfered with the LDL-derived cholesterol egress from the endosome/lysosome compartment, thus further reducing the cholesterol content in the endoplasmic reticulum. This triggered a homeostatic response with the stimulation of sterol regulatory element-binding protein (SREBP)-regulated gene expression. Treatment with SGAs also increased the synthesis of complex lipids (phospholipids and triacylglycerides). Once the antipsychotics were removed from the medium, a rebound in the cholesterol biosynthesis rate was detected, and the complex-lipid synthesis further increased. In this condition, apolipoprotein B secretion was also stimulated as demonstrated in HepG2 cells. These effects of SGAs on lipid homeostasis may be relevant in the metabolic side effects of antipsychotics, especially hypertriglyceridemia.

Inhibition of cholesterol biosynthesis disrupts lipid raft/caveolae and affects insulin receptor activation in 3T3-L1 preadipocytes.

Lipid rafts are plasma membrane microdomains that are highly enriched with cholesterol and sphingolipids and in which various receptors and other proteins involved in signal transduction reside. In the present work, we analyzed the effect of cholesterol biosynthesis inhibition on lipid raft/caveolae composition and functionality and assessed whether sterol precursors of cholesterol could substitute for cholesterol in lipid rafts/caveolae. 3T3-L1 preadipocytes were treated with distal inhibitors of cholesterol biosynthesis or vehicle (control) and then membrane rafts were isolated by sucrose density gradient centrifugation. Inhibition of cholesterol biosynthesis with either SKF 104976, AY 9944, 5,22-cholestadien-3beta-ol or triparanol, which inhibit different enzymes on the pathway, led to a marked reduction in cholesterol content and accumulation of different sterol intermediates in both lipid rafts and non-raft domains. These changes in sterol composition were accompanied by disruption of lipid rafts, with redistribution of caveolin-1 and Fyn, impairment of insulin-Akt signaling and the inhibition of insulin-stimulated glucose transport. Cholesterol repletion abrogated the effects of cholesterol biosynthesis inhibitors, reflecting they were specific. Our results show that cholesterol is required for functional raft-dependent insulin signaling.

Effects of red grape juice polyphenols in NADPH oxidase subunit expression in human neutrophils and mononuclear blood cells.

The NADPH oxidase enzyme system is the main source of superoxide anions in phagocytic and vascular cells. NADPH oxidase-dependent superoxide generation has been found to be abnormally enhanced in several chronic diseases. Evidence is accumulating that polyphenols may have the potential to improve cardiovascular health, although the mechanism is not fully established. Consumption of concentrated red grape juice, rich in polyphenols, has been recently shown to reduce NADPH oxidase activity in circulating neutrophils from human subjects. In the present work we studied whether red grape juice polyphenols affected NADPH oxidase subunit expression at the transcription level. For this, we used human neutrophils and mononuclear cells from peripheral blood, HL-60-derived neutrophils and the endothelial cell line EA.hy926.Superoxide production was measured with 2'7'-dichlorofluorescein diacetate or lucigenin, mRNA expression by real-time RT-PCR and protein expression by Western blot. Each experiment was performed at least three times. In all cell types tested, red grape juice, dealcoholised red wine and pure polyphenols decreased superoxide anion production. Red grape juice and dealcoholised red wine selectively reduced p47phox, p22phox and gp91phox expression at both mRNA and protein levels, without affecting the expression of p67phox. Pure polyphenols, particularly quercetin, also reduced NADPH oxidase subunit expression, especially p47phox, in all cell types tested. The present results showing that red grape juice polyphenols reduce superoxide anion production provide an alternative mechanism by which consumption of grape derivatives may account for a reduction of oxidative stress associated with cardiovascular and/or inflammatory diseases related to NADPH oxidase superoxide overproduction.

Effects of the antipsychotic drug haloperidol on the somastostatinergic system in SH-SY5Y neuroblastoma cells.

Antipsychotics are established drugs in schizophrenia treatment which, however, are not free of side effects. Lipid rafts are critical for normal brain function. Several G protein-coupled receptors, such as somatostatin (SRIF) receptors, have been shown to localize to lipid rafts. The aim of this study was to investigate whether haloperidol treatment affects the composition and functionality of lipid rafts in SH-SY5Y neuroblastoma cells. Haloperidol inhibited cholesterol biosynthesis, leading to a marked reduction in cell cholesterol content and to an accumulation of sterol intermediates, particularly cholesta-8,14-dien-3beta-ol. These changes were accompanied by a loss of flotillin-1 and Fyn from the lipid rafts. We next studied the functionality of the SRIF receptor. Treatment with haloperidol reduced the inhibitory effect of SRIF on adenylyl cyclase (AC) activity. On the other side, haloperidol decreased basal AC activity but increased forskolin-stimulated AC activity. Addition of free cholesterol to the culture medium abrogated the effects of haloperidol on lipid raft composition and SRIF signaling whereas the AC response to forskolin remained elevated. The results show that haloperidol, by affecting cholesterol homeostasis, ultimately alters SRIF signaling and AC activity, which might have physiological consequences.

Transepithelial transport across Caco-2 cell monolayers of antihypertensive egg-derived peptides. PepT1-mediated flux of Tyr-Pro-Ile.

This paper examines the in vitro transepithelial transport of antihypertensive peptides derived from egg proteins using Caco-2 cell monolayers. Ovokinin (FRADHPFL) was absorbed intact through the Caco-2 cell epithelium, although it was also susceptible to the action of brush-border aminopeptidases that yielded shorter fragments prior to their transport. The tripeptide YPI was resistant to cellular peptidases and transported through the monolayer, what suggests that the reduction in systemic blood pressure caused by this peptide may be mediated by effects at tissue level. Its pathway for transepithelial absorption was examined using inhibitors of the different mechanisms for oligopeptide transport in the intestinal tract. The main route involved in the transepithelial flux of YPI is probably the peptide H(+)-coupled transporter PepT1. These results highlight the potential of antihypertensive peptides to be used in the formulation of functional foods.

Cholesterol starvation induces differentiation of human leukemia HL-60 cells.

Cholesterol metabolism is particularly active in malignant, proliferative cells, whereas cholesterol starvation has been shown to inhibit cell proliferation. Inhibition of enzymes involved in cholesterol biosynthesis at steps before the formation of 7-dehydrocholesterol has been shown to selectively affect cell cycle progression from G(2) phase in human promyelocytic HL-60 cells. In the present work, we explored whether cholesterol starvation by culture in cholesterol-free medium and treatment with different distal cholesterol biosynthesis inhibitors induces differentiation of HL-60 cells. Treatment with SKF 104976, an inhibitor of lanosterol 14-alpha demethylase, or with zaragozic acid, which inhibits squalene synthase, caused morphologic changes alongside respiratory burst activity and expression of cluster of differentiation antigen 11c (CD11c) but not cluster of differentiation antigen 14. These effects were comparable to those produced by all-trans retinoic acid, which induces HL-60 cells to differentiate following a granulocyte lineage. In contrast, they differed from those produced by vitamin D(3), which promotes monocyte differentiation. The specificity of the response was confirmed by addition of cholesterol to the culture medium. Treatment with PD 98059, an inhibitor of extracellular signal-regulated kinase, abolished both the activation of NADPH oxidase and the expression of the CD11c marker. In sharp contrast, BM 15766, which inhibits sterol Delta(7)-reductase, failed to induce differentiation or arrest cell proliferation. These results show that changes in the sterol composition may trigger a differentiation response and highlight the potential of cholesterol pathway inhibition as a possible tool for use in cancer therapy.