TRIB1 regulates LDL metabolism through CEBPα-mediated effects on the LDL receptor in hepatocytes.

Genetic variants near the TRIB1 gene are highly significantly associated with plasma lipid traits and coronary artery disease. While TRIB1 is likely causal of these associations, the molecular mechanisms are not well understood. Here we sought to investigate how TRIB1 influences low density lipoprotein cholesterol (LDL-C) levels in mice. Hepatocyte-specific deletion of Trib1 (Trib1Δhep) in mice increased plasma cholesterol and apoB and slowed the catabolism of LDL-apoB due to decreased levels of LDL receptor (LDLR) mRNA and protein. Simultaneous deletion of the transcription factor CCAAT/enhancer-binding protein alpha (CEBPα) with TRIB1 eliminated the effects of TRIB1 on hepatic LDLR regulation and LDL catabolism. Using RNA-seq, we found that activating transcription factor 3 (Atf3) was highly upregulated in the livers of Trib1Δhep but not Trib1Δhep CebpaΔhep mice. ATF3 has been shown to directly bind to the CEBPα protein, and to repress the expression of LDLR by binding its promoter. Blunting the increase of ATF3 in Trib1Δhep mice reduced the levels of plasma cholesterol and partially attenuated the effects on LDLR. Based on these data, we conclude that deletion of Trib1 leads to a posttranslational increase in CEBPα, which increases ATF3 levels, thereby contributing to the downregulation of LDLR and increased plasma LDL-C.

Tanshinol alleviates ulcerative colitis by promoting the expression of VLDLR.

Tanshinol (TAN) is a widely used Chinese medicine ingredient with anti-inflammatory activity. The therapeutic effect of TAN in ulcerative colitis (UC) deserves further investigation. DSS induced UC model for mice, and TAN of different concentrations was used for in vivo therapy. Colons length was measured; expression of VLDLR in colonic mucosal tissue was evaluated by qRT-PCR, Western blot and histochemical staining. Besides, normal colorectal mucosal cell line (FHC) was treated with LPS to imitate the inflammatory process of UC in vitro. Different concentrations of TAN treated UC cell model. ELISA and qRT-PCR were applied to examine the concentrations of inflammatory cytokines (TNF-α, IL-6, IL-8, or IL-1ß). Flow cytometry and MTT was used to identify the apoptosis and viability of FHC cells, respectively. Afterwards, Western blot was performed to detect the expressions of Bax, Bcl-2, Cleaved caspase-3, and Cleaved caspase-9 in FHC cells. VLDLR was low-expressed in UC tissues as compared to the normal tissue. TAN could alleviate DSS-induced colons length shortening, colonic tissue structure destruction, inflammatory response, and VLDLR expression decrease in vivo. Further study found that TAN could alleviate LPS-induced inflammatory response, apoptosis, and viability decrease of FHC cells, and siVLDLR could partially offset the effect of TAN. TAN alleviates LPS-induced viability decrease, apoptosis, and inflammatory response in FHC cells by promoting VLDLR expression.

Low-density lipoprotein receptor (LDLR) is an independent adverse prognostic factor in acute myeloid leukaemia.

The low-density lipoprotein receptor (LDLR) is a membrane receptor that mediates the endocytosis of low-density lipoprotein (LDL). Uptake of LDL has been proposed to contribute to chemotherapy resistance of acute myeloid leukaemia (AML) cell lines in vitro. In the present study, we analysed LDLR expression and survival using bone marrow biopsies from 187 intensively treated patients with AML. Here, increasing LDLR expression was associated with decreasing overall (58·4%, 44·2%, and 24·4%; P = 0·0018), as well as event-free survival (41·7%, 18·1%, and 14·3%; P = 0·0077), and an increasing cumulative incidence of relapse (33·9%, 55·1%, and 71·4%; P = 0·0011). Associations of LDLR expression with survival were confirmed in 557 intensively treated patients from two international validation cohorts. In the analytic and validation cohorts, LDLR expression remained associated with outcome in multivariable regression analyses including the European LeukemiaNet genetic risk classification. Thus, LDLR predicts outcome of patients with AML beyond existing risk factors. Furthermore, we found low expression levels of LDLR in most healthy tissues, suggesting it as a promising target for antibody-based pharmacodelivery approaches in AML.

Deep learning reveals 3D atherosclerotic plaque distribution and composition.

Complications of atherosclerosis are the leading cause of morbidity and mortality worldwide. Various genetically modified mouse models are used to investigate disease trajectory with classical histology, currently the preferred methodology to elucidate plaque composition. Here, we show the strength of light-sheet fluorescence microscopy combined with deep learning image analysis for characterising and quantifying plaque burden and composition in whole aorta specimens. 3D imaging is a non-destructive method that requires minimal ex vivo handling and can be up-scaled to large sample sizes. Combined with deep learning, atherosclerotic plaque in mice can be identified without any ex vivo staining due to the autofluorescent nature of the tissue. The aorta and its branches can subsequently be segmented to determine how anatomical position affects plaque composition and progression. Here, we find the highest plaque accumulation in the aortic arch and brachiocephalic artery. Simultaneously, aortas can be stained for markers of interest (for example the pan immune cell marker CD45) and quantified. In ApoE-/- mice we observe that levels of CD45 reach a plateau after which increases in plaque volume no longer correlate to immune cell infiltration. All underlying code is made publicly available to ease adaption of the method.

Loss of core fucosylation reduces low-density lipoprotein receptor expression in hepatocytes by inducing PCSK9 production.

Fucosylation is a type of glycosylation, a form of post-transcriptional regulation of proteins, involved in cancer and inflammation. It involves the attachment of a fucose residue to N-glycans, O-glycans, and glycolipids, which is catalyzed by a family of enzymes called fucosyltransferases (Futs). Among the many Futs, α-1,6-fucosyltransferase (Fut8) is the only enzyme that produces α-1,6-fucosylated oligosaccharides (core fucose). In the human liver, the expression and activity of Fut8 are frequently elevated during progression of chronic liver diseases. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a well-known negative regulator of the low-density lipoprotein receptor (LDLR). Here, we found that loss of core fucose in immortalized hepatocytes led to LDLR downregulation through a dramatic induction of PCSK9. We used the immortalized hepatocytes derived from Fut8 knockout mice or a Fut8 knockdown AML12 hepatocyte cell line. Using these cells, we investigated the effects of Fut8 on hepatocyte cholesterol influx. Both cell lines had reduced LDLR protein levels, resulting from marked increases in PCSK9 expression. Intracellular cholesterol levels were significantly lower and LDL cholesterol uptake was suppressed in Fut8-KO cells. Hepatocyte nuclear factor 1α accumulated in nuclei of Fut8-KO hepatocytes, which mediated increases in PCSK9 mRNA expression. Our findings demonstrated that loss of core fucosylation promoted degradation of LDLR and impaired cholesterol uptake, which is a novel mechanism that regulates cholesterol influx, suggesting that Fut8 might be a novel causative gene for familial hypercholesterolemia.

Association between SNPs of Circulating Vascular Endothelial Growth Factor Levels, Hypercholesterolemia and Metabolic Syndrome.

BACKGROUND AND OBJECTIVES: Four single nucleotide polymorphisms (SNPs); rs6921438 and rs4416670 in LOC100132354-C6orf223, rs6993770 in ZFPM2, and rs10738760 in VLDLR-KCNV2 were reported to explain up to 50% of the heritability of vascular endothelial growth factor circulating levels. These SNPs were also studied for possible associations with circulating lipid levels in supposedly healthy European individuals and in a limited number of Iranian individuals with metabolic syndrome. To go further, the association of those four SNPs with plasma lipid parameters, hypercholesterolemia and metabolic syndrome (MetS) was assessed. MATERIALS AND METHODS: A cross-sectional study was conducted on 460 individuals chosen from the general population. Demographic and clinical data were collected and DNA was extracted and genotyped using Kompetitive allele specific PCR (KASP™). A meta-analysis followed, combining our participants with the Iranian individuals (n = 336). RESULTS: Whereas rs10738760 was associated with total cholesterol (Tchol) (p = 0.01), rs6993770 showed significant associations with both Tchol and low-density lipoprotein cholesterol (LDL-C) levels (p = 0.007 and p = 0.01 respectively). Using a multivariate logistic regression model adjusted for different confounding factors, we found that rs6993770 was associated with hypercholesterolemia, specifically high Tchol (p = 0.01) and LDL-C levels (p = 0.01). Furthermore, rs10738760 was positively associated with the risk of MetS in these individuals (p = 0.02) and in the meta-analysis (OR = 1.67, p = 0.01). CONCLUSION: Our results suggest that whereas rs6993770 in ZFPM2 was positively associated with hypercholesterolemia, rs10738760 (VLDLR-KCNV2) has a possible implication in MetS in two Middle Eastern populations.

Proteolysis of the low density lipoprotein receptor by bone morphogenetic protein-1 regulates cellular cholesterol uptake.

The development of cardiovascular disease is intimately linked to elevated levels of low-density lipoprotein (LDL) cholesterol in the blood. Hepatic LDL receptor (LDLR) levels regulate the amount of plasma LDL. We identified the secreted zinc metalloproteinase, bone morphogenetic protein 1 (BMP1), as responsible for the cleavage of human LDLR within its extracellular ligand-binding repeats at Gly171↓Asp172. The resulting 120 kDa membrane-bound C-terminal fragment (CTF) of LDLR had reduced capacity to bind LDL and when expressed in LDLR null cells had compromised LDL uptake as compared to the full length receptor. Pharmacological inhibition of BMP1 or siRNA-mediated knockdown prevented the generation of the 120 kDa CTF and resulted in an increase in LDL uptake into cells. The 120 kDa CTF was detected in the livers from humans and mice expressing human LDLR. Collectively, these results identify that BMP1 regulates cellular LDL uptake and may provide a target to modulate plasma LDL cholesterol.

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 and angiopoietin-like protein 4 are associated with the increase of lipoprotein lipase activity in epicardial adipose tissue from diabetic patients.

BACKGROUND AND AIMS: Epicardial adipose tissue (EAT) is a visceral AT, surrounding myocardium and coronary arteries. Its volume is higher in Type 2 diabetic (DM2) patients, associated with cardiovascular disease risk. Lipoprotein lipase (LPL) hydrolyses triglycerides (TG) from circulating lipoproteins, supplying fatty acids to AT, contributing to its expansion. We aimed to evaluate LPL expression and activity in EAT from DM2 and no DM2 patients, and its regulators ANGPTL4, GPIHBP1 and PPARγ levels, together with VLDLR expression and EAT LPL association with VLDL characteristics. METHODS: We studied patients undergoing coronary by-pass graft (CABG) divided into CABG-DM2 (n = 21) and CABG-noDM2 (n = 29), and patients without CABG (No CABG, n = 30). During surgery, EAT and subcutaneous AT (SAT) were obtained, in which LPL activity, gene and protein expression, its regulators and VLDLR protein levels were determined. Isolated circulating VLDLs were characterized. RESULTS: EAT LPL activity was higher in CABG-DM2 compared to CABG-noDM2 and No CABG (p=0.002 and p<0.001) and in CABG-noDM2 compared to No CABG (p=0.02), without differences in its expression. ANGPTL4 levels were higher in EAT from No CABG compared to CABG-DM2 and CABG-noDM2 (p<0.001). GPIHBP1 levels were higher in EAT from CABG-DM2 and CABG-noDM2 compared to No CABG (p= 0.04). EAT from CABG-DM2 presented higher PPARγ levels than CABG-noDM2 and No CABG (p=0.02 and p=0.03). No differences were observed in VLDL composition between groups, although EAT LPL activity was inversely associated with VLDL-TG and TG/protein index (p<0.05). CONCLUSIONS: EAT LPL regulation would be mainly post-translational. The higher LPL activity in DM2 could be partly responsible for the increase in EAT volume.

Nuclear localization of LDL receptor-related protein 1B in mammary gland carcinogenesis.

LRP1B intracellular domain is released and transported to the nucleus; however, pathological consequences of this nuclear transport are largely unclear. We aimed to unravel the pathobiological significance of nuclear localization of LRP1B intracellular domain in mammary gland carcinogenesis. Immunohistochemical staining using antibodies for LRP1B intracellular domain was performed to determine LRP1B expression in 92 invasive ductal breast carcinomas. LRP1B immunoreactivity was detected in the surface membrane and cytoplasm of 60 of 92 invasive ductal carcinomas and in the nucleus of 15 of 92 carcinomas. Nuclear LRP1B was significantly associated with poor patient prognosis, particularly luminal A type breast cancer, where it was significantly related to nodal metastasis. Doxycycline-dependent nuclear expression of LRP1B intracellular domain was established in cultured breast cancer cells. Enforced nuclear expression significantly increased Matrigel invasion activity in MCF-7 and T47D luminal A breast cancer cells. Moreover, enforced nuclear expression of LRP1B intracellular domain facilitated MCF-7 cells growth in mammary fat pad of nude mice, which was supplemented with estrogen. Comprehensive microarray-based analysis demonstrated that nuclear expression of LRP1B intracellular domain significantly increased long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) expression, which facilitates breast cancer invasion with poor prognosis. Nuclear-localized LRP1B intracellular domain promoted breast cancer progression with poor prognosis, possibly through the NEAT1 pathway. Nuclear transport of LRP1B intracellular domain could be a therapeutic target for breast cancer patients. KEY MESSAGES: Nuclear LRP1B was significantly associated with poor patient prognosis. Nuclear LRP1B increased Matrigel invasion activity of breast cancer cells. Nuclear expression of LRP1B intracellular domain increased NEAT1 expression.

Estudos da atividade do receptor da LDL em pacientes com Hipercolesterolemia Familial / Studies of LDL receptor activity in patients with familial hypercholesterolemia

A hipercolesterolemia familial (HF) é uma doença autossômica dominante considerada como uma das formas mais graves de hiperlipidemia, assim como, a principal causa de morbi-mortalidade por ser o principal fator desencadeante da aterosclerose. A alteração primária e mais freqüente da HF incide no gene do receptor da LDL (LDLr), sabe-se que mais de 1600 mutações são descritas na literatura e a principal consequência dessas alterações resultam no comprometimento da remoção da LDL, aumentando a concentração plasmática. Atualmente, o ultrasequenciamento genômico permite gerar muitos dados, que podem identificar novas mutações gênicas de forma eficiente, reprodutiva e rápida. No entanto, somente a validação da nova mutação por atividade funcional pode realmente estabelecer a associação com a doença. O presente estudo tem como objetivo realizar a análise da atividade do receptor da LDL, identificadas através do sequenciamento de alto rendimento, no gene LDLr realizado pelo nosso grupo de pesquisa e correlacionar com dados clínicos, in vitro, in silico e estrutural. Para cumprir esta meta, os linfócitos T dos portadores de HF foram isolados do sangue periférico, cultivados e submetidos a estímulo para a expressão de receptores da LDL, incubados com LDL marcada para avaliação de ligação e interiorização pelas células de cada paciente. Dos 30 pacientes selecionados para esse estudo, 63% apresentaram mutação no LDLR, sendo que quase todas as variantes (p.Gly373Asp, p.Asp601His, p.Ile488Thr, p.Gly549Asp, p.Gly592Glu e Gly681Asp) são localizadas no segundo domínio entre os éxons 7 ao 14. De acordo com o docking molecular a variante p.Gly592Glu (rs137929307), que já foi identificada na população polonesa, espanhola e brasileira, já relacionada com a HF, pode aumentar a interação do LDLr com a ApoB e consequentemente o modo de interação entre as proteínas, no estudo in vitro foi possível notar um aumento tanto na média de fluorescência da ligação e da ligação e interiorização em relação a quantidade de LDLr na superfície celular

Familial hypercholesterolemia (HF) is an autosomal dominant disease considered as one of the most severe forms of hyperlipidemia, as well as the main cause of morbidity and mortality because it is the main triggering factor for atherosclerosis. The primary and more frequent alteration of the HF affects the LDL receptor gene (LDLr), it is known that more than 1600 mutations are described in the literature and the main consequence of these alterations results in the compromise of the LDL removal, increasing the plasma concentration. Nowadays, genomic ultrasequencing allows the generation of many data, which can identify new gene mutations efficiently, reproductively and rapidly. However, only the validation of the new functional activity mutation can actually establish association with the disease. The aim of the present study was to analyze LDL receptor activity, identified by high-throughput sequencing, in the LDLr gene performed by our research group and to correlate with clinical, in vitro, in silico and structural data. To meet this goal, the T lymphocytes from the HF carriers were isolated from the peripheral blood, cultured and challenged for the expression of LDL receptors, incubated with labeled LDL for binding assessment and internalization by the cells of each patient. Of the 30 patients selected for this study, 63% had a mutation in LDLR, and almost all variants (p.Gly373Asp, p.Asp601His, p.Ile488Thr, p.Gly549Asp, p.Gly592Glu and Gly681Asp) are located in the second domain between exons 7 to 14. According to the molecular docking the variant p.Gly592Glu (rs137929307), which has already been identified in the Polish, Spanish and Brazilian population, already related to HF, can increase the interaction of LDLr with ApoB and consequently the mode of interaction between proteins, in the in vitro study it was possible to note an increase in both the mean fluorescence of binding and binding and internalization in relation to the amount of LDLr on the cell surface

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring.

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

Suppression of abdominal fat and anti-hyperlipidemic potential of Emblica officinalis: Upregulation of PPARs and identification of active moiety.

Since ancient time, Emblica officinalis (E. officinalis) is being used for the management of various ailments. Phytochemical analysis proves that fruit juice of E. officinalis contains high amount gallic acid, which could be responsible for medicinal potentials. Hence in this study, gallic acid and fruit juice of E. officinalis were evaluated for anti-hyperlipidemic potential in various experimental animal models. Experimentally, hyperlipidemia was induced through administration of poloxamer-407, tyloxapol and high-fat-diet supplement in rats. Treatment with gallic acid as well as fruit juice of E. officinalis decreased plasma cholesterol and reduced oil infiltration in liver and aorta. Mechanistically, E. officinalis increased peroxisome proliferator-activated receptors-α (PPARα) expression and increased activity of lipid oxidation through carnitine palmitoyl transferase (CPT) along with decreased activity of hepatic lipogenic enzymes i.e. glucose-6-phosphate dehydrogenase (G6PD), fatty acid synthase (FAS) and malic enzyme (ME). Additionally, E. officinalis increased cholesterol uptake through increased LDL-receptor expressions on hepatocytes and decreased LDL-receptor degradation due to decreased proprotein convertase subtilisin/kexin type 9 (PCSK9) expression. Simultaneously, E. officinalis showed ability to restore glucose homeostasis through increased Glut4 and PPARγ protein expression in adipose tissue. These findings exposed central role of gallic acid in E. officinalis arbitrated anti-hyperlipidemic action through upregulation of PPARs, Glut4 and lipogenic enzymes, and decreased expression of PCSK9 and lipogenic enzymes. Findings from this experiment demonstrated that E. officinalis is a potential therapy for management of hyperlipidemia and gallic acid could be a potential lead candidate.

Regulatory effect of Phikud Navakot extract on HMG-CoA reductase and LDL-R: potential and alternate agents for lowering blood cholesterol.

BACKGROUND: For decades, various cardiovascular symptoms have been relieved by the use of Ya-Hom Navakot, which is a formulation comprising 54 herbal medicines. The Thailand Ministry of Public Health listed Ya-Hom Navakot's nine active principle and nomenclative herbal ingredients and termed them 'Phikud Navakot' (PN). Several reports have confirmed that PN has cardiovascular benefits similar to Ya-Hom Navakot. However, whether PN facilitates lipid-lowering activity remains unclear. METHODS: The present study investigated an in vitro model for examining the gene expression levels of 3-hydroxyl-3-methylglutaryl-CoA reductase (HMGCR) and low-density lipoprotein receptor (LDL-R) in HepG2 cells using qRT-PCR. The ethanol and water extractions of Ya-Hom Navakot, PN and Ya-Hom Navakot without PN were compared. RESULTS: One mg/ml of both NYEF and NYWF were found to significantly lower cholesterol by either the up-regulation of LDL-R or down-regulation of HMGCR compared with negative controls and 1 mg/ml simvastatin (p < 0.05). PNEF also up-regulated LDL-R gene expression, even more than NYEF (p < 0.05). In addition, the ethanol and water extracts of PN significantly down-regulated HMGCR gene expression compared with those of Ya-Hom Navakot without PN (p < 0.05). CONCLUSION: The use of Ya-Hom Navakot or PN may provide an alternative treatment to lower cholesterol through HMGCR gene inhibition and LDL-R gene enhancement.

Transplantation of genetically modified hepatocytes after liver preconditioning in Watanabe heritable hyperlipidemic rabbit.

BACKGROUND: Hepatocyte transplantation is a potentially less invasive alternative to liver transplantation for treating inherited metabolic liver diseases. We developed an autotransplantation protocol of ex vivo genetically modified hepatocytes combining lentiviral transduction and transplantation after liver preconditioning by partial portal vein embolization. We investigated the metabolic efficiency of this approach in Watanabe rabbits, animal model of familial hypercholesterolemia. METHODS: Our autotransplantation experimental protocol was used in two groups of rabbits (n = 10), experimental and sham, receiving transduced and control hepatocytes, respectively. Isolated hepatocytes from left liver lobes were transduced using recombinant lentiviruses. Median lobe portal branches were embolized under fluoroscopic control. Functional measurement of low-density lipoprotein (LDL) receptor expression was assessed by LDL internalization assays. Cholesterol level evolution was monitored. Rabbits were killed 20 wk after the procedure. RESULTS: Three rabbits of each group died several hours after hepatocyte transplantation; autopsy revealed portal vein thrombosis in two rabbits from each group. The protocol was therefore modified with hepatocytes being transplanted through splenic injection. Lentiviral hepatocyte transduction efficacy was 64.5%. Fluorescence microscopy revealed Dil-LDL internalization of transduced hepatocytes. Seven rabbits in each group were considered for lipid analysis. Four weeks after autotransplantation, median total cholesterol level decreased in the experimental group, without reaching statistical significance (8.9 [8.0-9.8] g/L versus 6.3 [0.5-8.3]; P = 0.171). In the experimental group, enzyme-linked immunosorbent assay detected significant antibody expression against human low-density lipoprotein receptor. CONCLUSIONS: Autotransplantation protocol allowed a nonstatistically significant improvement of the lipid profile in Watanabe rabbits. Further experiments involving a larger number of animals are necessary to confirm or refute our findings.

Blackcurrant anthocyanins stimulated cholesterol transport via post-transcriptional induction of LDL receptor in Caco-2 cells.

PURPOSES: We previously showed that polyphenol-rich blackcurrant extract (BCE) showed a hypocholesterolemic effect in mice fed a high fat diet. As direct cholesterol removal from the body via the intestine has been recently appreciated, we investigated the effect of BCE on the modulation of genes involved in intestinal cholesterol transport using Caco-2 cells as an in vitro model. METHODS: Caco-2 cells were treated with BCE to determine its effects on mRNA and protein expression of genes important for intestinal cholesterol transport, low-density lipoprotein (LDL) uptake, cellular cholesterol content, and cholesterol transport from basolateral to apical membrane of Caco-2 cell monolayers. Cells were also treated with anthocyanin-rich or -poor fraction of BCE to determine the role of anthocyanin on BCE effects. RESULTS: BCE significantly increased protein levels of LDL receptor (LDLR) without altering its mRNA, which consequently increased LDL uptake into Caco-2 cells. This post-transcriptional induction of LDLR by BCE was markedly attenuated in the presence of rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). In addition, BCE altered genes involved in cholesterol transport in the enterocytes, including apical and basolateral cholesterol transporters, in such a way that could enhance cholesterol flux from the basolateral to apical side of the enterocytes. Indeed, BCE significantly increased the flux of LDL-derived cholesterol from the basolateral to the apical chamber of Caco-2 monolayer. LDLR protein levels were markedly increased by anthocyanin-rich fraction, but not by anthocyanin-free fraction. CONCLUSION: mTORC1-dependent post-transcriptional induction of LDLR by BCE anthocyanins drove the transport of LDL-derived cholesterol to the apical side of the enterocytes. This may represent a potential mechanism for the hypocholesterolemic effect of BCE.

Dysregulation of the DKK1 gene in pediatric B-cell acute lymphoblastic leukemia.

BACKGROUND/AIM: The canonical Wingless-type (WNT) pathway is involved in normal hematopoietic cell development and deregulated WNT signaling is implicated in the development of hematological malignancies. Dickkopf 1 (DKK1) acts as a modulator of the ß-catenin regulated canonical pathway. Activation of DKK1 leads to apoptosis and growth suppression, whereas silencing by promoter hypermethylation results in abnormal WNT activation. The secreted inhibitor Dickkopf can antagonize WNT signaling by competitively binding to low density lipoprotein receptors (LRPs) 5 and 6. MATERIALS AND METHODS: We studied DKK1 gene promoter methylation and investigated DKK1, ß-catenin, LRP5, and LRP6 mRNA levels in B-cell acute lymphoblastic leukemia (B-ALL) patients (n = 90). Methylation-specific PCR and bisulfite sequencing were used for methylation profiling and quantitative real-time PCR was used for mRNA detection. RESULTS: The DKK1 gene was examined for its promoter methylation and only 33% of patients were found methylated. On the other hand, B-ALL cases showed high expression of DKK1 (P = 0.01), LRP5 (P = 0.04), and LRP6 (P = 0.02) compared to normal bone marrow cells. CONCLUSION: DKK1 methylation exists in some of cases but is not sufficient for WNT pathway activation alone in pediatric B-ALL.

Circular RNA circ-LDLRAD3 as a biomarker in diagnosis of pancreatic cancer.

AIM: To analyze the diagnostic value of a circular RNA (circRNA), circ-LDLRAD3, in pancreatic cancer. METHODS: Expression levels of circ-LDLRAD3 were tested in both cells and clinical samples; the latter included 30 paired pancreatic cancer tissues and adjacent non-tumorous tissues, 31 plasma samples from patients with pancreatic cancer, and 31 plasma samples from healthy volunteers. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to measure expression levels of circ-LDLRAD3 in cells and clinical samples; then, the relationship between clinicopathological factors of patient samples and expression of circ-LDLRAD3 in pancreatic cancer was analyzed. The diagnostic value of circ-LDLRAD3 was verified by receiver operating characteristic (ROC) curve analysis. RESULTS: Circ-LDLRAD3 was up-regulated in pancreatic cancer cell lines (P < 0.01), pancreatic cancer tissues (P < 0.01), and plasma samples from patients with pancreatic cancer (P < 0.01). High expression of circ-LDLRAD3 was significantly associated with venous invasion, lymphatic invasion, and metastasis. The area under the ROC curve of circ-LDLRAD3 alone or combination with CA19-9 was 0.67 and 0.87, respectively, with a sensitivity and specificity of 0.5738 (alone) and 0.7049 (alone), and 0.8033 (combination) and 0.9355 (combination), respectively. CONCLUSION: These data suggest that circ-LDLRAD3 may be a biomarker in the diagnosis of pancreatic cancer.

Reelin-Dab1 signaling system in human colorectal cancer.

Reelin is an extracellular matrix protein that plays a critical role in neuronal migration. Here we show that the mucosa of human colon expresses reelin, its receptors ApoER2 and VLDLR, and its effector protein Dab1. Immunohistochemical analyses reveal that reelin expression is restricted to pericryptal myofibroblasts; Dab1 is detected at myofibroblasts, the apical domain of surface epithelial and crypt cells, and a strong linear staining is observed at the basement membrane; VLDLR and ApoER2 are in the cytoplasm of surface epithelium and myofibroblasts, and VLDLR is also detected in the cytoplasm of the crypt cells. Human colorectal cancer downregulates reelin without change in vimentin or N-cadherin mRNA levels. Decreased Reelin mRNA expression is accompanied by decreased HIC1 mRNA levels, increased mRNA levels of ApoER2 and DNMT1, increased reelin hypermethylation and no change in either Cask or TGF-ß1 mRNAs, suggesting that reelin repression results from a DNMT1-mediated hypermethylation of the reelin gene promoter. Decreased HIC1 expression may repress reelin transcription via increasing ApoER2 transcription. We conclude that the mucosa of human colon expresses the reelin-Dab1 signaling system and that reelin is repressed in colorectal cancer before epithelial-mesenchymal transition has occurred. The significant down-regulation of reelin expression makes this gene a promising biomarker for colorectal cancers. © 2016 Wiley Periodicals, Inc.

Nanoformulated alpha-mangostin ameliorates Alzheimer's disease neuropathology by elevating LDLR expression and accelerating amyloid-beta clearance.

Alzheimer's disease (AD), the most common form of dementia, is now representing one of the largest global healthcare challenges. However, an effective therapy is still lacking. Accumulation of amyloid-beta (Aß) in the brain is supposed to trigger pathogenic cascades that eventually lead to AD. Therefore, Aß clearance strategy is being actively pursued as a promising disease modifying therapy. Here, we found that α-mangostin (α-M), a polyphenolic xanthone derivative from mangosteen, up-regulated low density lipoprotein receptor (LDLR) expression in microglia and liver cells, and efficiently facilitated Aß clearance. However, the in vivo application of α-M is limited due to its hydrophobic nature, poor aqueous solubility and stability, and thus low bioavailability and accumulation in the target organs. To overcome this limitation, α-M was encapsulated into the core of poly(ethylene glycol)-poly(l-lactide) (PEG-PLA) nanoparticles [NP(α-M)]. Such nanoencapsulation improved the biodistribution of α-M in both the brain and liver, enhanced the brain clearance of (125)I-radiolabeled Aß1-42 in an LDLR-dependent manner, reduced Aß deposition, attenuated neuroinflammatory responses, ameliorated neurologic changes and reversed behavioral deficits in AD model mice. These findings justified the concept that polyphenol-mediated modulation of LDLR expression might serve as a safe and efficient disease-modifying therapy for AD by accelerating Aß clearance. It also demonstrated the powerful capacity of nanotechnology in modulating the biodistribution behavior of drug to improve its therapeutic efficacy in AD.