Tissue cholesterol metabolism and prostate cancer aggressiveness: Ethno-geographic variations.

BACKGROUND: Prostate cancer (PCa) is more frequent and more aggressive in populations of African descent than in Caucasians. Since the fatty acid composition of peri-prostatic adipose tissue (PPAT) has been shown to differ according to the ethno-geographic origin and is involved in PCa aggressiveness, we aimed to analyze the cholesterol content of PPAT from Caucasian and African-Caribbean patients, in correlation with markers of disease aggressiveness and cholesterol metabolism in cancer tissues. METHODS: The quantification of cholesterol in PPAT was analyzed in 52 Caucasian and 52 African-Caribbean PCa patients, with in each group 26 indolent tumors (ISUP Group1 and pT2) and 26 potentially aggressive tumors (ISUP Group 3-5 and/or pT3). The expression of proteins involved in cholesterol metabolism was analyzed by immunohistochemistry on cancer tissue samples included in tissue microarrays. RESULTS: The amount of cholesterol esters was lower in PPAT from African-Caribbean patients compared with Caucasians, without any correlation with markers of disease aggressiveness. In cancer tissues from African-Caribbean patients, the expression of ABCA1 (involved in cholesterol efflux) was decreased, and that of SREBP-2 (involved in cholesterol uptake) was increased. In both groups of patients, SREBP-2 expression was strongly associated with that of Zeb1, a key player in the epithelial-to-mesenchymal transition (EMT) process. CONCLUSION: These results suggest that cholesterol metabolism differs according to the ethno-geographic origin, in both PPAT and cancer tissues. In African-Caribbeans, the orientation towards accumulation of cholesterol in cancer cells is associated with a more frequent state of EMT, which may promote PCa aggressiveness in this population.

Localisation and regulation of cholesterol transporters in the human hair follicle: mapping changes across the hair cycle.

Cholesterol has long been suspected of influencing hair biology, with dysregulated homeostasis implicated in several disorders of hair growth and cycling. Cholesterol transport proteins play a vital role in the control of cellular cholesterol levels and compartmentalisation. This research aimed to determine the cellular localisation, transport capability and regulatory control of cholesterol transport proteins across the hair cycle. Immunofluorescence microscopy in human hair follicle sections revealed differential expression of ATP-binding cassette (ABC) transporters across the hair cycle. Cholesterol transporter expression (ABCA1, ABCG1, ABCA5 and SCARB1) reduced as hair follicles transitioned from growth to regression. Staining for free cholesterol (filipin) revealed prominent cholesterol striations within the basement membrane of the hair bulb. Liver X receptor agonism demonstrated active regulation of ABCA1 and ABCG1, but not ABCA5 or SCARB1 in human hair follicles and primary keratinocytes. These results demonstrate the capacity of human hair follicles for cholesterol transport and trafficking. Future studies examining the role of cholesterol transport across the hair cycle may shed light on the role of lipid homeostasis in human hair disorders.

Therapeutic Intranasal Vaccine HB-ATV-8 Prevents Atherogenesis and Non-alcoholic Fatty Liver Disease in a Pig Model of Atherosclerosis.

BACKGROUND AND AIMS: Atherosclerosis as an inflammatory disease involved in the etiology of cardiovascular disease worldwide, in our days demands an array of different therapeutic approaches in order to soon be able to visualize an effective prevention. Based on an immunotherapeutic approach, we designed a non-invasive vaccine (HB-ATV-8), contained in a micellar nanoparticle composed of lipids and a peptide segment derived from the C-terminus of the cholesterol-ester transfer protein (CETP). Now we extend our successful proof of concept from the rabbit to a porcine model and investigated its effect in an attempt to undoubtedly establish the efficacy of vaccination in a model closer to the human. METHODS: A preclinical trial was designed to study the efficacy of vaccine HB-ATV-8 in pigs (Large White × Landrace). Male experimental animals were fed with standard diet (control), high fat diet (HFD) or the same HFD but treated with HB-ATV-8 (HFD + Vaccine) applied nasally for up to 7 months. All biochemical and enzymatic analyses were performed in peripheral venous blood and thoracic aorta and liver samples examined using conventional, two-photon excitation and second harmonic generation microscopy to identify atherosclerotic and hepatic lesions. mRNA concentrations for KLF2, ACTA2, SOD1, COL1A1 genes and protein levels for PPARα and ABCA1 were quantified in aorta and liver respectively using qPCR and Western blot analysis. RESULTS: The administration of vaccine HB-ATV-8 induced anti-CETP IgG antibodies and reduced atherosclerotic and hepatic lesions promoted by the high fat diet. In addition, plasma triglyceride levels of vaccine treated pigs fed the HFD were similar to those of control group, in contrast to high concentrations reached with animals exclusively fed with HFD. Moreover, HFD promotes a tendency to decrease hepatic PPARα levels and increase in aorta gene expression of KLF2, ACTA2, SOD1 and COL1A1, while vaccine application promotes recovery close to control values. CONCLUSIONS: Vaccine HB-ATV-8 administration constitutes a promissory preventive approach useful in the control of atherogenesis and fatty liver disease. The positive results obtained, the non-invasive characteristics of the vaccine, the simple design employed in its conception and its low production cost, support the novelty of this therapeutic strategy designed to prevent the process of atherogenesis and control the development of fatty liver disease.

ABCA1 affects placental function via trophoblast and macrophage.

AIMS: To study the potential impact of ABCA1 on the function of the placenta. MAIN METHODS: Trophoblasts and macrophages were isolated from the placenta with enzymatic digestion; Immunofluorescence assay was used to detect the location of ABCA1 in cells; RT-PCR and Western-blot were used to detect the expression of ABCA1; The cholesterol efflux assays of primary trophoblasts was detected by Amplex Red cholesterol assay kit (Invitrogen);Inflammatory factor secretion from primary macrophages was detected by Elisa. KEY FINDINGS: ABCA1 was mainly located on trophoblast membranes. Decreased ABCA1 expression in trophoblasts reduced the cholesterol efflux of trophoblasts (P<0.01). while increased ABCA1 expression in trophoblasts reduced the cholesterol efflux of trophoblasts (P<0.05). ABCA1 was uniformly expressed on the cell membrane, cytoplasm, and nucleus of macrophages. Decreased ABCA1 expression in macrophages, increased inflammatory factors but reduced IL-10 (P<0.01). While increased ABCA1 expression in macrophages, reduced inflammatory factors but increased IL-10 (P<0.01). SIGNIFICANCE: ABCA1 may be a potential target for the prevention of gestation diseases.

Leonurine Prevents Atherosclerosis Via Promoting the Expression of ABCA1 and ABCG1 in a Pparγ/Lxrα Signaling Pathway-Dependent Manner.

BACKGROUND/AIMS: Previous studies have demonstrated that leonurine, a unique alkaloid compound of Herba leonuri, can exert anti-oxidative and anti-inflammatory effects on the development of atherosclerosis (AS). This study was designed to investigate the effects of leonurine on cholesterol efflux from THP-1 macrophage-derived foam cells and development of atherosclerotic lesions in apoE-/- mice, and further determine the potential mechanisms. METHODS: Human THP-1 cells were fully differentiated into foam cells by the pre-treatment with phorbol-12-myristate-13-acetate (PMA) and oxidized density lipoproteins (ox-LDL). After cells were incubated with various concentrations of leonurine, Oil Red O staining and high-performance liquid chromatography (HPLC) assays were utilized to detect cellular lipid accumulation and cholesterol content, respectively. Cellular cholesterol efflux was determined by liquid scintillation counting. The mRNA and protein levels of ATP-binding cassette transporter A1/G1 (ABCA1/G1), peroxisome proliferator-activated receptor γ (PPARγ) and liver X receptor α (LXRα) in foam cells were assessed using real-time quantitative PCR (RT-qPCR) and western blot analyses, respectively. Plasma triglyceride (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) levels in apoE-/- mice were evaluated using enzymatic methods. The atherosclerotic lesion sizes and collagen contents in aortic roots were determined by Oil Red O and Masson's trichrome staining, respectively. RESULTS: Oil Red O staining and liquid scintillation counting assays showed that leonurine significantly inhibited lipid accumulation and promoted 3H-cholesterol efflux in human THP-1 macrophage-derived foam cells in a concentration-dependent manner. Besides, both the mRNA and protein levels of ABCA1/G1, PPARγ and LXRα were enhanced by leonurine, which were attenuated by LXRα siRNA or PPARγ siRNA transfection. Finally, leonurine improved plasma lipid profile, decreased atherosclerotic lesion sizes, increased collagen contents and amplified PPARγ, LXRα and ABCA1/G1 expressions in aortic roots of apoE-/- mice. CONCLUSIONS: Leonurine can promote cholesterol efflux and alleviate cellular lipid accumulation by magnifying the expression of ABCA1/G1 in a PPARγ/LXRα signaling pathway-dependent manner in human THP-1 macrophage-derived foam cells and abate atherogenesis in apoE-/- mice, which may offer a promising therapeutic intervention of leonurine in protecting against AS.

Heparin-derived oligosaccharide inhibits vascular intimal hyperplasia in balloon-injured carotid artery.

The aims of the present study were to determine the effects of heparin-derived oligosaccharides (HDOs) on vascular intimal hyperplasia (IH) in balloon-injured carotid artery and to elucidate the underlying mechanisms of action. An animal model was established by rubbing the endothelia within the common carotid artery (CCA) in male rabbits. The rabbits were fed a high-cholesterol diet. Arterial IH was determined by histopathological changes to the CCA. Serum lipids were detected using an automated biochemical analysis. Expressions of mRNAs for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1), scavenger receptor class B type I (SR-BI), and ATP-binding cassette transporter A1 (ABCA-1) were analyzed using reverse transcription polymerase chain reaction assays. Expressions of VEGF, VCAM-1, MCP-1, SR-BI and ABCA-1 proteins were analyzed by Western blotting. Enzyme-linked immunosorbent assays were used to quantify expression levels of VEGF and bFGF. Our results showed that administration of HDO significantly inhibited CCA histopathology and restenosis induced by balloon injury. The treatment with HDOs significantly decreased the mRNA and protein expression levels of VEGF, bFGF, VCAM-1, MCP-1, and SR-BI in the arterial wall; however, ABCA-1 expression level was elevated. HDO treatment led to a reduction in serum lipids (total cholesterol, triglycerides, high-density and low-density lipoproteins). Our results from the rabbit model indicated that HDOs could ameliorate IH and underlying mechanism might involve VEGF, bFGF, VCAM-1, MCP-1, SR-BI, and ABCA-1.

MiR-33a Decreases High-Density Lipoprotein-Induced Radiation Sensitivity in Breast Cancer.

PURPOSE: We previously showed that high-density lipoprotein (HDL) radiosensitizes inflammatory breast cancer (IBC) cells in vitro and is associated with better local control after radiation therapy in IBC patients. The microRNA miR-33 family negatively regulates the adenosine triphosphate binding cassette transporter subfamily A member 1. We hypothesized that variations in miR-33a expression in IBC cancer cells versus non-IBC cells would correlate with radiation sensitivity following exposure to HDL in vitro. METHODS AND MATERIALS: MiR-33a expression was analyzed by reverse transcriptase-polymerase chain reaction in 4 cell lines representing common clinical breast cancer subtypes. Overexpression and knockdown of miR-33a was demonstrated via transfection of an miR-33a mimic or an anti-miR-33a construct in high- and low-expressing miR-33a cell lines. Clonogenic survival in vitro in these cells was quantified at baseline and following HDL treatment. MiR-33a expression on distant relapse-free survival (DRFS) of 210 cases downloaded from the Oxford breast cancer dataset was determined. RESULTS: Expression levels of miR-33a were lower in IBC cell lines and IBC tumor samples than in non-IBC cell lines and normal breast tissue. Cholesterol concentrations in the cell membranes were higher in IBC cells than in non-IBC cells. Clonogenic survival following 24 hours of HDL treatment was decreased in response to irradiation in the low-miR-33a-expressing cell lines SUM149 and KPL4, but survival following HDL treatment decreased in the high-miR-33a-expressing cell lines MDA-MB-231 and SUM159. In the high-miR-33a-expressing cell lines, anti-miR-33a transfection decreased radiation resistance in clonogenic assays. Conversely, in the low-miR-33a-expressing cell lines, the miR-33a mimic reversed the HDL-induced radiation sensitization. Breast cancer patients in the top quartile based on miR-33a expression had markedly lower rates of DRFS than the bottom quartile (P=.0228, log-rank test). For breast cancer patients treated with radiation, high miR-33a expression predicted worse overall survival (P=.06). CONCLUSIONS: Our results reveal miR-33a negatively regulates HDL-induced radiation sensitivity in breast cancer.

[Effect of ferulic acid on cholesterol efflux in macrophage foam cell formation and potential mechanism].

The formation of macrophage-derived foam cells is a typical feature of atherosclerosis (AS). Reverse cholesterol efflux (RCT) is one of important factors for the formation of macrophage foam cells. In this study, macrophage form cells were induced by oxidized low density lipoprotein (ox-LDL) and then treated with different concentrations of ferulic acid, so as to observe the effect of ferulic acid on the intracellular lipid metabolism in the ox-LDL-induced macrophage foam cell formation, the cholesterol efflux and the mRNA expression and protein levels of ATP binding cassette transporter A1 (ABCA1) and ATP binding cassette transporter G1 (ABCG1) that mediate cholesterol efflux, and discuss the potential mechanism of ferulic acid in resisting AS. According to the findings, compared with the control group, the ox-LDL-treated group showed significant increase in intracellular lipid content, especially for the cholesterol content; whereas the intracellular lipid accumulation markedly decreased, after the treatment with ferulic acid. The data also demonstrated that the mRNA and protein expressions of ABCA1 and ABCG1 significantly increased after macrophage foam cells were treated with different concentrations of ferulic acid. In summary, ferulic acid may show the anti-atherosclerosis effect by increasing the surface ABCA1 and ABCG1 expressions of macrophage form cells and promoting cholesterol efflux.

Prenatal Ethanol Exposure Up-Regulates the Cholesterol Transporters ATP-Binding Cassette A1 and G1 and Reduces Cholesterol Levels in the Developing Rat Brain.

AIMS: Cholesterol plays a pivotal role in many aspects of brain development; reduced cholesterol levels during brain development, as a consequence of genetic defects in cholesterol biosynthesis, leads to severe brain damage, including microcephaly and mental retardation, both of which are also hallmarks of the fetal alcohol syndrome. We had previously shown that ethanol up-regulates the levels of two cholesterol transporters, ABCA1 (ATP binding cassette-A1) and ABCG1, leading to increased cholesterol efflux and decreased cholesterol content in astrocytes in vitro. In the present study we investigated whether similar effects could be seen in vivo. METHODS: Pregnant Sprague-Dawley rats were fed liquid diets containing 36% of the calories from ethanol from gestational day (GD) 6 to GD 21. A pair-fed control groups and an ad libitum control group were included in the study. ABCA1 and ABCG1 protein expression and cholesterol and phospholipid levels were measured in the neocortex of female and male fetuses at GD 21. RESULTS: Body weights were decreased in female fetuses as a consequence of ethanol treatments. ABCA1 and ABCG1 protein levels were increased, and cholesterol levels were decreased, in the neocortex of ethanol-exposed female, but not male, fetuses. Levels of phospholipids were unchanged. Control female fetuses fed ad libitum displayed an up-regulation of ABCA1 and a decrease in cholesterol content compared with pair-fed controls, suggesting that a compensatory up-regulation of cholesterol levels may occur during food restriction. CONCLUSION: Maternal ethanol consumption may affect fetal brain development by increasing cholesterol transporters' expression and reducing brain cholesterol levels.

Immunolocalization of the cholesterol transporters ABCA1 and ABCG1 in canine reproductive tract tissues and spermatozoa.

The mammalian sperm membrane undergoes cholesterol efflux during maturation and fertilization. Although ATP-binding cassette (ABC) transporters are known to transport cholesterol through cell membranes in other organs, their presence in canine testis, epididymis and sperm has not been proven to date. Hence, the aim of the present study was to localize the ABC transporters ABCA1 and ABCG1 in canine testicular and epididymidal tissue as well as in spermatozoa membranes. To this end, semen samples from 12 dogs as well as testicles and epididymides of four young and healthy dogs were prepared for immunohistochemistry, respectively. Capacitation and acrosome reaction (AR) were induced in aliquots of the semen samples before immunostaining to assess changes in the expression of ABCA1 and ABCG1. Evaluation by confocal microscopy revealed the presence of both ABCA1 and ABCG1 in canine testicles and of ABCA1 in the epididymides. In spermatozoa, only ABCA1 immunoreactivity was detected, mainly in the region of the acrosome and midpiece. After induction of capacitation, ABCA1 signal persisted in the acrosome but disappeared after AR, indicating a loss of ABCA1 with the loss of the acrosome. We conclude that ABCA1 and ABCG1 are expressed in canine testis, whereas only ABCA1 is expressed in epididymis and spermatozoa membrane, both transporters probably contributing to the regulation of membrane cholesterol content.

An evaluation of the mechanism of ABCA7 on cellular lipid release in ABCA7-HEC293 cell.

BACKGROUND: ABCA7 is a member of the ABCA subfamily that shows a high degree of homology to ABCA1 and, like ABCA1, mediates cellular cholesterol and phospholipid release by apolipoproteins when transfected in vitro. However, expression of ABCA7 has been shown to be downregulated by increased cellular cholesterol while ABCA1 was upregulated. METHODS: The underlying mechanism for this effect was examined in ABCA1 or ABCA7-transfected HEC293. Lipid content in the medium and cells was determined by enzymatic assays. Gene expression was quantitated by real time PCR, and protein content was determined by Western blotting. RESULTS: While ABCA7 mRNA was decreased by 25-hydroxycholesterol treatment, ABCA1 was apparently increased. Treatment with the synthetic LXR agonist T0901317 (T09) upregulated ABCA1 expression and apoAI-mediated cellular lipid release in ABCA1-transfected HEC293 cells, but ABCA7 expression and cellular lipid release in ABCA7-transfected HEC293 cells showed no obvious changes. CONCLUSION: The ABCA7 gene is regulated by sterol in a direction opposite to that of ABCA1.