Proteometabolomics of initial and recurrent glioblastoma highlights an increased immune cell signature with altered lipid metabolism.

BACKGROUND: There is an urgent need to better understand the mechanisms associated with the development, progression, and onset of recurrence after initial surgery in glioblastoma (GBM). The use of integrative phenotype-focused -omics technologies such as proteomics and lipidomics provides an unbiased approach to explore the molecular evolution of the tumor and its associated environment. METHODS: We assembled a cohort of patient-matched initial (iGBM) and recurrent (rGBM) specimens of resected GBM. Proteome and metabolome composition were determined by mass spectrometry-based techniques. We performed neutrophil-GBM cell coculture experiments to evaluate the behavior of rGBM-enriched proteins in the tumor microenvironment. ELISA-based quantitation of candidate proteins was performed to test the association of their plasma concentrations in iGBM with the onset of recurrence. RESULTS: Proteomic profiles reflect increased immune cell infiltration and extracellular matrix reorganization in rGBM. ASAH1, SYMN, and GPNMB were highly enriched proteins in rGBM. Lipidomics indicates the downregulation of ceramides in rGBM. Cell analyses suggest a role for ASAH1 in neutrophils and its localization in extracellular traps. Plasma concentrations of ASAH1 and SYNM show an association with time to recurrence. CONCLUSIONS: We describe the potential importance of ASAH1 in tumor progression and development of rGBM via metabolic rearrangement and showcase the feedback from the tumor microenvironment to plasma proteome profiles. We report the potential of ASAH1 and SYNM as plasma markers of rGBM progression. The published datasets can be considered as a resource for further functional and biomarker studies involving additional -omics technologies.

Peroxisome Proliferator-Activated Receptor α in Lipoprotein Metabolism and Atherosclerotic Cardiovascular Disease.

Peroxisome proliferator-activated receptors (PPARs) are a group of ligand-binding transcription factors with pivotal action in regulating pleiotropic signaling pathways of energetic metabolism, immune responses and cell proliferation and differentiation. A significant body of evidence indicates that the PPARα receptor is an important modulator of plasma lipid and lipoprotein metabolism, with pluripotent effects influencing the lipid and apolipoprotein cargo of both atherogenic and antiatherogenic lipoproteins and their functionality. Clinical evidence supports an important role of PPARα agonists (fibric acid derivatives) in the treatment of hypertriglyceridemia and/or low high-density lipoprotein (HDL) cholesterol levels, although the effects of clinical trials are contradictory and point to a reduction in the risk of nonfatal and fatal myocardial infarction events. In this manuscript, we provide an up-to-date critical review of the existing relevant literature.

Synthetic lipoproteins based on apolipoprotein E coupled to fullerenol have anti-atherosclerotic properties.

BACKGROUND: Apolipoprotein E (apoE) is an anti-atherosclerotic protein associated with almost all plasma lipoproteins. Fullerenol (Full-OH) contains the fullerene hydrophobic cage and several hydroxyl groups that could be derivatized to covalently bind various molecules. Herein, we aimed to produce fullerenol-based nanoparticles carrying apoE3 (Full-apoE) and test their anti-atherosclerotic effects. METHODS: Full-apoE nanoparticles were obtained from Full-OH activated to reactive cyanide ester fullerenol derivative that was further reacted with apoE protein. To test their effect, the nanoparticles were administered to apoE-deficient mice for 24 h or 3 weeks. ApoE part of the nanoparticles was determined by Western Blot and quantified by ELISA. Atherosclerotic plaque size was evaluated after Oil Red O staining and the gene expression was determined by Real-Time PCR. RESULTS: Full-apoE nanoparticles were detected mainly in the liver, and to a lesser extent in the kidney, lung, and brain. In the plasma of the Full-apoE-treated mice, apoE was found associated with very-low-density lipoproteins and high-density lipoproteins. Treatment for 3 weeks with Full-apoE nanoparticles decreased plasma cholesterol levels, increased the expression of apolipoprotein A-I, ABCA1 transporter, scavenger receptor-B1, and sortilin, and reduced the evolution of the atheromatous plaques in the atherosclerotic mice. CONCLUSIONS: In experimental atherosclerosis, the administration of Full-apoE nanoparticles limits the evolution of the atheromatous plaques by decreasing the plasma cholesterol level and increasing the expression of major proteins involved in lipid metabolism. Thus, they represent a novel promising strategy for atherosclerosis therapy.

Apolipoprotein A-II, a Player in Multiple Processes and Diseases.

Apolipoprotein A-II (apoA-II) is the second most abundant apolipoprotein in high-density lipoprotein (HDL) particles, playing an important role in lipid metabolism. Human and murine apoA-II proteins have dissimilar properties, partially because human apoA-II is dimeric whereas the murine homolog is a monomer, suggesting that the role of apoA-II may be quite different in humans and mice. As a component of HDL, apoA-II influences lipid metabolism, being directly or indirectly involved in vascular diseases. Clinical and epidemiological studies resulted in conflicting findings regarding the proatherogenic or atheroprotective role of apoA-II. Human apoA-II deficiency has little influence on lipoprotein levels with no obvious clinical consequences, while murine apoA-II deficiency causes HDL deficit in mice. In humans, an increased plasma apoA-II concentration causes hypertriglyceridemia and lowers HDL levels. This dyslipidemia leads to glucose intolerance, and the ensuing high blood glucose enhances apoA-II transcription, generating a vicious circle that may cause type 2 diabetes (T2D). ApoA-II is also used as a biomarker in various diseases, such as pancreatic cancer. Herein, we provide a review of the most recent findings regarding the roles of apoA-II and its functions in various physiological processes and disease states, such as cardiovascular disease, cancer, amyloidosis, hepatitis, insulin resistance, obesity, and T2D.

Treatment with Mesenchymal Stromal Cells Overexpressing Fas-Ligand Ameliorates Acute Graft-versus-Host Disease in Mice.

Allogeneic hematopoietic cell transplantation (allo-HCT) has the potential to cure malignant and non-malignant hematological disorders, but because of the serious side effects of this intervention its applications are limited to a restricted number of diseases. Graft-versus-host disease (GvHD) is the most frequent complication and the leading cause of mortality and morbidity following allo-HCT. It results from the attack of the transplanted T cells from the graft against the cells of the recipient. There is no clear treatment for this severe complication. Due to their immunomodulatory properties, mesenchymal stromal cells (MSC) have been proposed to treat GvHD, but the results did not meet expectations. We have previously showed that the immunomodulatory effect of the MSC was significantly enhanced through adenoviral-mediated overexpression of FasL. In this study, we have tested the properties of FasL-overexpressing MSC in vivo, in a mouse model for acute GvHD. We found that treatment with FasL-overexpressing MSC delayed the onset of the disease and increased survival of the mice.

An Efficient Method for Adenovirus Production.

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by "negative" and "positive" PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

K2 Transfection System Boosts the Adenoviral Transduction of Murine Mesenchymal Stromal Cells.

Adenoviral vectors are important vehicles for delivering therapeutic genes into mammalian cells. However, the yield of the adenoviral transduction of murine mesenchymal stromal cells (MSC) is low. Here, we aimed to improve the adenoviral transduction efficiency of bone marrow-derived MSC. Our data showed that among all the potential transduction boosters that we tested, the K2 Transfection System (K2TS) greatly increased the transduction efficiency. After optimization of both K2TS components, the yield of the adenoviral transduction increased from 18% to 96% for non-obese diabetic (NOD)-derived MSC, from 30% to 86% for C57BL/6-derived MSC, and from 0.6% to 63% for BALB/c-derived MSC, when 250 transduction units/cell were used. We found that MSC derived from these mouse strains expressed different levels of the coxsackievirus and adenovirus receptors (MSC from C57BL/6≥NOD>>>BALB/c). K2TS did not increase the level of the receptor expression, but desensitized the cells to foreign DNA and facilitated the virus entry into the cell. The expression of Stem cells antigen-1 (Sca-1) and 5'-nucleotidase (CD73) MSC markers, the adipogenic and osteogenic differentiation potential, and the immunosuppressive capacity were preserved after the adenoviral transduction of MSC in the presence of the K2TS. In conclusion, K2TS significantly enhanced the adenoviral transduction of MSC, without interfering with their main characteristics and properties.

Adenovirus-Mediated FasL Minigene Transfer Endows Transduced Cells with Killer Potential.

Fas ligand (First apoptosis signal ligand, FasL, also known as CD95L) is the common executioner of apoptosis within the tumor necrosis factor (TNF) superfamily. We aimed to induce functional FasL expression in transduced cells using an adenovirus vector, which has the advantage of strong and transient induction of the gene included in the adenoviral genome. Here, we report that the adenovirus carrying a truncated FasL gene, named FasL minigene, encoding the full-length FasL protein (Ad-gFasL) is more efficient than the adenovirus carrying FasL cDNA (Ad-cFasL) in the induction of FasL expression in transduced cells. FasL minigene (2887 bp) lacking the second intron and a part of the 3'-UTR was created to reduce the gene length due to the size limitation of the adenoviral genome. The results show that, in transduced hepatocytes, strong expression of mRNA FasL appeared after 10 h for Ad-gFasL, while for Ad-cFasL, a faint expression appeared after 16 h. For Ad-gFasL, the protein expression was noticed starting with 0.5 transfection units (TU)/cell, while for Ad-cFasL, it could not be revealed. FasL-expressing endothelial cells induced apoptosis of A20 cells in co-culture experiments. FasL-expressing cells may be exploitable in various autoimmune diseases such as graft-versus-host disease, chronic colitis, and type I diabetes.

Enhanced Suppression of Immune Cells In Vitro by MSC Overexpressing FasL.

Mesenchymal stromal cells (MSC) display several mechanisms of action that may be harnessed for therapeutic purposes. One of their most attractive features is their immunomodulatory activity that has been extensively characterized both in vitro and in vivo. While this activity has proven to be very efficient, it is transient. We aimed to enhance it by transforming MSC to overexpress a first apoptosis signal (Fas) ligand (FasL). In this study, our goal was to induce FasL overexpression through adenoviral transduction in MSC to improve their immunomodulatory activity. We characterized the impact of FasL overexpression on the morphology, proliferation, viability, phenotype, multilineage differentiation potential and immunomodulation of MSC. Moreover, we determined their suppressive properties in mixed reactions with A20 cells, as well as with stimulated splenocytes. Our findings demonstrate that FasL-overexpressing MSC exhibit improved immunosuppressive properties, while maintaining their MSC-characteristic features. In conclusion, we establish, in a proof-of-concept set-up, that FasL-overexpressing MSC represent good candidates for therapeutic intervention targeted at autoimmune disorders.

Simple and dual cross-linked chitosan millicapsules as a particulate support for cell culture.

The chitosan hydrochloride (Cs·HCl) was obtained as a polymer soluble in physiological solutions to be used as potential support for safely cell culture or cell encapsulation. Viability tests showed that concentrations between 0.16 and 5 mg/mL of Cs·HCl were not toxic for the HEK293 cells. In parallel, aldehyde-functionalized pullulan (Pul-CHO) was synthesized as the macromolecular cross-linker. Cs·HCl was dissolved in 0.9% NaCl and injected (INJECTOMAT SEP 21S PLUS) through a needle to obtain small droplets in a sodium tripolyphosphate solution in the absence and presence of 0.1% (w/v) Pul-CHO. Simple and dual cross-linked millicapsules were obtained with pore size ranging from 50 µm to 5 µm, respectively. FITC-Dextran with molecular weights of 4000 and 70,000 g/mol was encapsulated during microcapsule synthesis as macromolecular models to check the permeability of Cs millicapsules. The results show that FITC-Dextran 4000 and 70,000 diffuses quickly from simple cross-linked millicapsules while dual cross-linked millicapsules release slowly both FITC-dextrans. Microscopy experiments show that HEK 293 cells adhere to the surface of millicapsules. Taken together the data reveal that Cs millicapsules allow the cell growth on their surface, and thus, they offer new perspectives for cell encapsulation strategy.

The Mechanism of Bisphenol A Atherogenicity Involves Apolipoprotein A-I Downregulation through NF-κB Activation.

Apolipoprotein A-I (apoA-I) is the major protein component of high-density lipoproteins (HDL), mediating many of its atheroprotective properties. Increasing data reveal the pro-atherogenic effects of bisphenol A (BPA), one of the most prevalent environmental chemicals. In this study, we investigated the mechanisms by which BPA exerts pro-atherogenic effects. For this, LDLR-/- mice were fed with a high-fat diet and treated with 50 µg BPA/kg body weight by gavage. After two months of treatment, the area of atherosclerotic lesions in the aorta, triglycerides and total cholesterol levels were significantly increased, while HDL-cholesterol was decreased in BPA-treated LDLR-/- mice as compared to control mice. Real-Time PCR data showed that BPA treatment decreased hepatic apoA-I expression. BPA downregulated the activity of the apoA-I promoter in a dose-dependent manner. This inhibitory effect was mediated by MEKK1/NF-κB signaling pathways. Transfection experiments using apoA-I promoter deletion mutants, chromatin immunoprecipitation, and protein-DNA interaction assays demonstrated that treatment of hepatocytes with BPA induced NF-κB signaling and thus the recruitment of p65/50 proteins to the multiple NF-κB binding sites located in the apoA-I promoter. In conclusion, BPA exerts pro-atherogenic effects downregulating apoA-I by MEKK1 signaling and NF-κB activation in hepatocytes.

Apolipoprotein C1: Its Pleiotropic Effects in Lipid Metabolism and Beyond.

Apolipoprotein C1 (apoC1), the smallest of all apolipoproteins, participates in lipid transport and metabolism. In humans, APOC1 gene is in linkage disequilibrium with APOE gene on chromosome 19, a proximity that spurred its investigation. Apolipoprotein C1 associates with triglyceride-rich lipoproteins and HDL and exchanges between lipoprotein classes. These interactions occur via amphipathic helix motifs, as demonstrated by biophysical studies on the wild-type polypeptide and representative mutants. Apolipoprotein C1 acts on lipoprotein receptors by inhibiting binding mediated by apolipoprotein E, and modulating the activities of several enzymes. Thus, apoC1 downregulates lipoprotein lipase, hepatic lipase, phospholipase A2, cholesterylester transfer protein, and activates lecithin-cholesterol acyl transferase. By controlling the plasma levels of lipids, apoC1 relates directly to cardiovascular physiology, but its activity extends beyond, to inflammation and immunity, sepsis, diabetes, cancer, viral infectivity, and-not last-to cognition. Such correlations were established based on studies using transgenic mice, associated in the recent years with GWAS, transcriptomic and proteomic analyses. The presence of a duplicate gene, pseudogene APOC1P, stimulated evolutionary studies and more recently, the regulatory properties of the corresponding non-coding RNA are steadily emerging. Nonetheless, this prototypical apolipoprotein is still underexplored and deserves further research for understanding its physiology and exploiting its therapeutic potential.

The Opposite Effect of c-Jun Transcription Factor on Apolipoprotein E Gene Regulation in Hepatocytes and Macrophages.

Apolipoprotein E (apoE) is mainly secreted by hepatocytes and incorporated into most plasma lipoproteins. Macrophages, which accumulate cholesterol and are critical for the development of the atherosclerotic plaque, are also an important, albeit smaller, apoE source. Distal regulatory elements control cell-specific activity of the apoE promoter: multienhancers (ME.1/2) in macrophages and hepatic control regions (HCR-1/2) in hepatocytes. A member of AP-1 cell growth regulator, c-Jun regulates the transcription of various apolipoproteins and proinflammatory molecules implicated in atherosclerosis. We aimed to investigate the effect of c-Jun on apoE expression in macrophages versus hepatocytes and to reveal the underlying molecular mechanisms. Herein we show that c-Jun had an opposite, cell-specific effect on apoE expression: downregulation in macrophages but upregulation in hepatocytes. Transient transfections using ME.2 deletion mutants and DNA pull-down (DNAP) assays showed that the inhibitory effect of c-Jun on the apoE promoter in macrophages was mediated by a functional c-Jun binding site located at 301/311 on ME.2. In hepatocytes, c-Jun overexpression strongly increased apoE expression, and this effect was due to c-Jun binding at the canonical site located at -94/-84 on the apoE proximal promoter, identified by transient transfections using apoE deletion mutants, DNAP, and chromatin immunoprecipitation assays. Overall, the dual effect of c-Jun on apoE gene expression led to decreased cholesterol efflux in macrophages resident in the atherosclerotic plaque synergized with an increased level of systemic apoE secreted by the liver to exacerbate atherogenesis.

Preferential Association of Lissencephaly-1 Gene Expression with CD133+ Glioblastoma Cells.

Lissencephaly-1 (Lis1) protein is a dynein-binding protein involved in neural stem cell division, morphogenesis and motility. To determine whether Lis1 is a key factor in glioblastoma, we evaluated its expression and function in CD133+ glioblastoma cells. Global, Lis1 gene expression is similar in glioblastoma and normal samples. Interestingly, immunohistochemistry data indicate increased Lis1 expression colocalized with CD133 in a subset of glioma cells, including the tumor cells with perivascular localization. Lis1 gene expression is increased up to 60-fold in CD133 positive cells isolated from primary cultures of glioblastoma and U87 glioblastoma cell line as compared to CD133 negative cells. To investigate the potential role of Lis1 in CD133+ glioblastoma cells, we silenced Lis1 gene in U87 cell line obtaining shLis1-U87 cells. In shLis1-U87 cell culture we noticed a significant decrease of CD133+ cells fraction as compared with control cells and also, CD133+ cells isolated from shLis1-U87 were two times less adhesive, migratory and proliferative, as compared with control transfected U87 CD133+ cells. Moreover, Lis1 silencing decreased the proliferative capacity of irradiated U87 cells, an effect attributable to the lower percentage of CD133+ cells. This is the first report showing a preferential expression of Lis1 gene in CD133+ glioblastoma cells. Our data suggest a role of Lis1 in regulating CD133+ glioblastoma cells function.

Apolipoprotein E - A Multifunctional Protein with Implications in Various Pathologies as a Result of Its Structural Features.

Apolipoprotein E (apoE), a 34 kDa glycoprotein, mediates hepatic and extrahepatic uptake of plasma lipoproteins and cholesterol efflux from lipid-laden macrophages. In humans, three structural different apoE isoforms occur, with subsequent functional changes and pathological consequences. Here, we review data supporting the involvement of apoE structural domains and isoforms in normal and altered lipid metabolism, cardiovascular and neurodegenerative diseases, as well as stress-related pathological states. Studies using truncated apoE forms provided valuable information regarding the regions and residues responsible for its properties. ApoE3 renders protection against cardiovascular diseases by maintaining lipid homeostasis, while apoE2 is associated with dysbetalipoproteinemia. ApoE4 is a recognized risk factor for Alzheimer's disease, although the exact mechanism of the disease initiation and progression is not entirely elucidated. ApoE is also implicated in infections with herpes simplex type-1, hepatitis C and human immunodeficiency viruses. Interacting with both viral and host molecules, apoE isoforms differently interfere with the viral life cycle. ApoE exerts anti-inflammatory effects, switching macrophage phenotype from the proinflammatory M1 to the anti-inflammatory M2, suppressing CD4+ and CD8+ lymphocytes, and reducing IL-2 production. The anti-oxidative properties of apoE are isoform-dependent, modulating the levels of various molecules (Nrf2 target genes, metallothioneins, paraoxonase). Mimetic peptides were designed to exploit apoE beneficial properties. The "structure correctors" which convert apoE4 into apoE3-like molecules have pharmacological potential. Despite no successful strategy is yet available for apoE-related disorders, several promising candidates deserve further improvement and exploitation.

Differential action of glucocorticoids on apolipoprotein E gene expression in macrophages and hepatocytes.

Apolipoprotein E (apoE) has anti-atherosclerotic properties, being involved in the transport and clearance of cholesterol-rich lipoproteins as well as in cholesterol efflux from cells. We hypothesized that glucocorticoids may exert anti-inflammatory properties by increasing the level of macrophage-derived apoE. Our data showed that glucocorticoids increased apoE expression in macrophages in vitro as well as in vivo. Dexamethasone increased ~6 fold apoE mRNA levels in cultured peritoneal macrophages and RAW 264.7 cells. Administered to C57BL/6J mice, dexamethasone induced a two-fold increase in apoE expression in peritoneal macrophages. By contrast, glucocorticoids did not influence apoE expression in hepatocytes, in vitro and in vivo. Moreover, dexamethasone enhanced apoE promoter transcriptional activity in RAW 264.7 macrophages, but not in HepG2 cells, as tested by transient transfections. Analysis of apoE proximal promoter deletion mutants, complemented by protein-DNA interaction assays demonstrated the functionality of a putative glucocorticoid receptors (GR) binding site predicted by in silico analysis in the -111/-104 region of the human apoE promoter. In hepatocytes, GR can bind to their specific site within apoE promoter but are not able to modulate the gene expression. The modulatory blockade in hepatocytes is a consequence of partial involvement of transcription factors and other signaling molecules activated through MEK1/2 and PLA2/PLC pathways. In conclusion, our study indicates that glucocorticoids (1) differentially target apoE gene expression; (2) induce a significant increase in apoE level specifically in macrophages. The local increase of apoE gene expression in macrophages at the level of the atheromatous plaque may have therapeutic implications in atherosclerosis.

High levels of homocysteine downregulate apolipoprotein E expression via nuclear factor kappa B.

AIM: To investigate the effect of high homocysteine (Hcy) levels on apolipoprotein E (apoE) expression and the signaling pathways involved in this gene regulation. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot were used to assess apoE expression in cells treated with various concentrations (50-500 µmol/L) of Hcy. Calcium phosphate-transient transfections were performed in HEK-293 and RAW 264.7 cells to evaluate the effect of Hcy on apoE regulatory elements [promoter and distal multienhancer 2 (ME2)]. To this aim, plasmids containing the proximal apoE promoter [(-500/+73)apoE construct] alone or in the presence of ME2 [ME2/(-500/+73)apoE construct] to drive the expression of the reporter luciferase gene were used. Co-transfection experiments were carried out to investigate the downstream effectors of Hcy-mediated regulation of apoE promoter by using specific inhibitors or a dominant negative form of IKß. In other co-transfections, the luciferase reporter was under the control of synthetic promoters containing multiple specific binding sites for nuclear factor kappa B (NF-κB), activator protein-1 (AP-1) or nuclear factor of activated T cells (NFAT). Chromatin immunoprecipitation (ChIP) assay was accomplished to detect the binding of NF-κB p65 subunit to the apoE promoter in HEK-293 treated with 500 µmol/L Hcy. As control, cells were incubated with similar concentration of cysteine. NF-κB p65 proteins bound to DNA were immunoprecipitated with anti-p65 antibodies and DNA was identified by PCR using primers amplifying the region -100/+4 of the apoE gene. RESULTS: RT-PCR revealed that high levels of Hcy (250-750 µmol/L) induced a 2-3 fold decrease in apoE mRNA levels in HEK-293 cells, while apoE gene expression was not significantly affected by treatment with lower concentrations of Hcy (100 µmol/L). Immunoblotting data provided additional evidence for the negative role of Hcy in apoE expression. Hcy decreased apoE promoter activity, in the presence or absence of ME2, in a dose dependent manner, in both RAW 264.7 and HEK-293 cells, as revealed by transient transfection experiments. The downstream effectors of the signaling pathways of Hcy were also investigated. The inhibitory effect of Hcy on the apoE promoter activity was counteracted by MAPK/ERK kinase 1/2 (MEK1/2) inhibitor U0126, suggesting that MEK1/2 is involved in the downregulation of apoE promoter activity by Hcy. Our data demonstrated that Hcy-induced inhibition of apoE took place through activation of NF-κB. Moreover, we demonstrated that Hcy activated a synthetic promoter containing three NF-κB binding sites, but did not affect promoters containing AP-1 or NFAT binding sites. ChIP experiments revealed that NF-κB p65 subunit is recruited to the apoE promoter following Hcy treatment of cells. CONCLUSION: Hcy-induced stress negatively modulates apoE expression via MEK1/2 and NF-κB activation. The decreased apoE expression in peripheral tissues may aggravate atherosclerosis, neurodegenerative diseases and renal dysfunctions.

Thyroid hormones upregulate apolipoprotein E gene expression in astrocytes.

Apolipoprotein E (apoE), a protein mainly involved in lipid metabolism, is associated with several neurodegenerative disorders including Alzheimer's disease. Despite numerous attempts to elucidate apoE gene regulation in the brain, the exact mechanism is still uncovered. The mechanism of apoE gene regulation in the brain involves the proximal promoter and multienhancers ME.1 and ME.2, which evolved by gene duplication. Herein we questioned whether thyroid hormones and their nuclear receptors have a role in apoE gene regulation in astrocytes. Our data showed that thyroid hormones increase apoE gene expression in HTB14 astrocytes in a dose-dependent manner. This effect can be intermediated by the thyroid receptor ß (TRß) which is expressed in these cells. In the presence of triiodothyronine (T3) and 9-cis retinoic acid, in astrocytes transfected to overexpress TRß and retinoid X receptor α (RXRα), apoE promoter was indirectly activated through the interaction with ME.2. To determine the location of TRß/RXRα binding site on ME.2, we performed DNA pull down assays and found that TRß/RXRα complex bound to the region 341-488 of ME.2. This result was confirmed by transient transfection experiments in which a series of 5'- and 3'-deletion mutants of ME.2 were used. These data support the existence of a biologically active TRß binding site starting at 409 in ME.2. In conclusion, our data revealed that ligand-activated TRß/RXRα heterodimers bind with high efficiency on tissue-specific distal regulatory element ME.2 and thus modulate apoE gene expression in the brain.

Krüppel-like factor 4 synergizes with CREB to increase the activity of apolipoprotein E gene promoter in macrophages.

Krüppel-like factor 4 (KLF4) is a critical regulator of monocyte differentiation and macrophage polarization, and it also plays an important role in several vascular diseases, including atherosclerosis. Apolipoprotein E (apoE) is an essential anti-atherosclerotic glycoprotein involved in lipid metabolism, expressed by the liver, macrophages and other cell types. We hypothesized that KLF4 is involved in apoE gene regulation in macrophages. Our experiments showed that differentiation of THP-1 monocytes to macrophages using PMA was associated with a robust induction of both KLF4 and apoE genes. KLF4 bound to the apoE promoter, as revealed by chromatin immunoprecipitation and DNA pull-down (DNAP) assays, and transactivated the apoE promoter in a dose-dependent manner. Using a series of apoE promoter deletion mutants we revealed the biological activity of multiple KLF4 binding sites located in the [-500/-100] region of apoE promoter. Moreover, overexpression of cAMP-response-element-binding protein (CREB) further increased KLF4 up-regulatory effect on apoE promoter. Despite the fact that no putative CREB binding sites were predicted in silico, we found that in macrophages CREB bound to apoE proximal promoter in the region -200/+4 and even more strongly on -350/-274 region. In similar DNAP experiments using cell extracts obtained from monocytes (lacking KLF4), a very weak binding of CREB was detected, indicating that interaction of CREB with apoE promoter takes place indirectly. In conclusion our results show: (i) a robust synchronized induction of KLF4 and apoE expression during differentiation of monocytes to macrophages; (ii) KLF4 up-regulates apoE gene in a dose-dependent manner; (iii) biologically active KLF4 binding sites are present on apoE promoter and (iv) the interaction of KLF4 with CREB results in an enhanced up-regulatory effect of KLF4 on apoE promoter. Taken together these data provide novel knowledge on apoE gene regulation mechanism in macrophages, and offer insight into the therapeutic potential of KLF4 in atherosclerosis.

Metformin reduces the endotoxin-induced down-regulation of apolipoprotein E gene expression in macrophages.

The atheroprotective role of macrophage-derived apolipoprotein E (apoE) is well known. Our previous reports demonstrated that inflammatory stress down-regulates apoE expression in macrophages, aggravating atherogenesis. Metformin, extensively used as an anti-diabetic drug, has also anti-inflammatory properties, and thus confers vascular protection. In this study, we questioned whether metformin could have an effect on apoE expression in macrophages in normal conditions or under lipopolysaccharide (LPS)-induced stress. The results showed that metformin slightly increases the apoE expression only at high doses (5-10 mM). Low doses of metformin (1-3 mM) significantly reduce the LPS down-regulatory effect on apoE expression in macrophages. Our experiments demonstrated that LPS-induced NF-κB binds to the macrophage-specific distal regulatory element of apoE gene, namely to the multienhancer 2 (ME.2) and its 5'-deletion fragments. The NF-κB binding on ME.2 and apoE promoter has a down-regulatory effect. In addition, data revealed that metformin impairs NF-κB nuclear translocation, and thus, improves the apoE levels in macrophages under inflammatory stress. The positive effect of metformin in the inflammatory states, its clinical safety and low cost, make this drug a potential adjuvant in the therapeutic strategies for atherosclerosis.