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.

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.

The involvement of the monocytes/macrophages in chronic inflammation associated with atherosclerosis.

Atherosclerosis is a progressive chronic disease of large and medium arteries, characterized by the formation of atherosclerotic plaques. Monocytes and macrophages are key factors in lesion development, participating to the processes that mediate the progression of the atherosclerotic plaque (lipid accumulation, secretion of pro-inflammatory and cytotoxic factors, extracellular matrix remodeling). The recruitment of the monocytes in the vascular wall represents a hallmark in the pathology of the atherosclerotic lesion. Monocyte adhesion and transmigration are dependent on the complementary adhesion molecules expressed on the endothelial surface, whose expression is modulated by chemical mediators. The atherosclerotic plaque is characterized by a heterogeneous population of macrophages reflecting the complexity and diversity of the micro-environment to which cells are exposed after entering the arterial wall. Within the atherosclerotic lesions, macrophages differentiate, proliferate and undergo apoptosis. Taking into account that their behavior has a direct and critical influence on all lesional stages, the development of therapeutic approaches to target monocytes/macrophages in the atherosclerotic plaque became a focal interest point for researchers in the field.

Macrophage-specific up-regulation of apolipoprotein E gene expression by STAT1 is achieved via long range genomic interactions.

In atherogenesis, macrophage-derived apolipoprotein E (apoE) has an athero-protective role by a mechanism that is not fully understood. We investigated the regulatory mechanisms involved in the modulation of apoE expression in macrophages. The experiments showed that the promoters of all genes of the apoE/apoCI/apoCIV/apoCII gene cluster are enhanced by multienhancer 2 (ME.2), a regulatory region that is located 15.9 kb downstream of the apoE gene. ME.2 interacts with the apoE promoter in a macrophage-specific manner. Transient transfections in RAW 264.7 macrophages showed that the activity of ME.2 was strongly decreased by deletion of either 87 bp from the 5' end or 131 bp from the 3' end. We determined that the minimal fragment of this promoter that can be activated by ME.2 is the proximal -100/+73 region. The analysis of the deletion mutants of ME.2 revealed the importance of the 5' end of ME.2 in apoE promoter transactivation. Chromatin conformational capture assays demonstrated that both ME.2 and ME.1 physically interacted with the apoE promoter in macrophages. Our data showed that phorbol 12-myristate 13-acetate-induced differentiation of macrophages is accompanied by a robust induction of apoE and STAT1 expression. In macrophages (but not in hepatocytes), STAT1 up-regulated apoE gene expression via ME.2. The STAT1 binding site was located in the 174-182 region of ME.2. In conclusion, the specificity of the interactions between the two multienhancers (ME.1 and ME.2) and the apoE promoter indicates that these distal regulatory elements play an important role in the modulation of apoE gene expression in a cell-specific manner.