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

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

Targeted Transfection Using PEGylated Cationic Liposomes Directed Towards P-Selectin Increases siRNA Delivery into Activated Endothelial Cells

: The progress in small-interfering RNA (siRNA) therapeutics depends on the development of suitable nanocarriers to perform specific and effective delivery to dysfunctional cells. In this paper, we questioned whether P-selectin, a cell adhesion molecule specifically expressed on the surface of activated endothelial cells (EC) could be employed as a target for nanotherapeutic intervention. To this purpose, we developed and characterized P-selectin targeted PEGylated cationic liposomes able to efficiently pack siRNA and to function as efficient vectors for siRNA delivery to tumour necrosis factor-α (TNF-α) activated EC. Targeted cationic liposomes were obtained by coupling a peptide with high affinity for P-selectin to a functionalized PEGylated phospholipid inserted in the liposomes’ bilayer (Psel-lipo). As control, scrambled peptide coupled cationic liposomes (Scr-lipo) were used. The lipoplexes obtained by complexation of Psel-lipo with siRNA (Psel-lipo/siRNA) were taken up specifically and at a higher extent by TNF-α activated b.End3 endothelial cells as compared to non-targeted Scr-lipo/siRNA. The Psel-lipo/siRNA delivered with high efficiency siRNA into the cells. The lipoplexes were functional as demonstrated by the down-regulation of the selected gene (GAPDH). The results demonstrate an effective targeted delivery of siRNA into cultured activated endothelial cells using P-selectin directed PEGylated cationic liposomes, which subsequently knock-down the desired gene

<i>In vivo</i> cell-specific effect of dexamethasone on apoE expression in macrophages and liver of C57BL/6J mice.

<p>Representative images show the expression of apoE and GAPDH genes in mouse peritoneal macrophages <b>(A)</b> and liver extracts <b>(B)</b>, as detected by RT-PCR (upper panels). Quantification of apoE gene expression normalized to GAPDH in peritoneal macrophages and liver extracts in dexamethasone-treated mice, as compared to control mice, is depicted in the lower panels. Five-day dexamethasone (30mg/kg body weight) treatment of C57BL/6J mice significantly increases apoE mRNA levels in mouse peritoneal macrophages (p<0.001), but does not affect hepatic apoE gene expression (p>0.05).</p

In macrophages glucocorticoid receptors (GRs) bind to a consensus sequence in apoE proximal promoter and upregulate its activity.

<p>(A) <i>In silico</i> analysis of apoE proximal promoter revealing a GRα binding site in the region -111→-104. (B) In transiently transfected RAW 264.7 macrophages, dexamethasone significantly enhances the activity of [-500/+73] apoE promoter and of the [-200/+73] fragment, but not the activities of the [-100/+73] and [-55/+73] deletion fragments or the activity of the pGL4 vector. (C) Chromatin immunoprecipitation assay shows that GR proteins are recruited to the human apoE promoter in PMA-differentiated THP1 macrophages treated with dexamethasone (lane 4). No bands were observed in untreated macrophages (lane 2) or when the antibodies were omitted (lanes 1 and 3). PCR using the input as template and primers for apoE promoter generated the expected bands (lanes 5 and 6, respectively). Lane 8 represents the no template control PCR (NTC). Specific 258 bp PCR product is indicated by the arrow. (D) GR proteins bind efficiently to oligonucleotides corresponding to the -115/-75 region of apoE promoter as revealed by DNA pull-down assays (lane 4). GR proteins also bind to control oligonucleotides containing a known glucocorticoid responsive element (lane 2), but do not bind to the mutated glucocorticoid responsive element (lane 3). Whole cell extract was used as positive control (lane 1); there is no binding of GRs to uncoupled Dynabeads, used as negative control (lane 5). The symbol *** is for p<0.001.</p

Macrophage-specific enhancement of apoE protein expression by dexamethasone.

<p><b>(A)</b> Dexamethasone (100–1000 nM, 48 hours) significantly increases the expression of apoE protein in mouse peritoneal macrophages (MPM), and mifepristone attenuates this effect. <b>(B)</b> No alteration in apoE protein expression is detected in murine primary hepatocytes (MPH) under dexamethasone treatment. Western blots results were normalized to β-actin. Upper panels represent the quantification of three independent experiments, and lower panels are representative images. The symbol ** is for p<0.01.</p

Glucocorticoid receptor (GR) expression and intracellular distribution in macrophages and hepatocytes upon dexamethasone treatment.

<p>(A) Macrophages (MPM, RAW 264.7) and hepatocytes (MPH, HepG2) express similar levels of GRs independently of dexamethasone treatment, as evaluated by RT-PCR with GAPDH used as a reference gene. (B) Treatment of RAW 264.7 macrophages and HepG2 cells with 1 μM dexamethasone for 24 hours leads to GR translocation into the cell nucleus, as detected by Western Blot; actin and TFIID were used as protein load controls for the cytoplasmic and nuclear fractions, respectively.</p

In hepatocytes, glucocorticoid receptors (GRs) bind to apoE proximal promoter but are not capable to regulate apoE gene expression due to the interaction with other signaling pathways.

<p>(A) Dexamethasone induces GR recruitment to apoE promoter in HepG2 hepatocytes as revealed by ChIP assay (lane 4). In the absence of the ligand, GRs do not bind to apoE promoter (lane 2). In controls, the chromatin of dexamethasone-treated or untreated cells was processed omitting the antibodies (lanes 3 and 1, respectively). PCR using the input prepared from treated or untreated cells as template produced the expected bands (lanes 5 and 6, respectively). The arrow indicates the specific 258 bp band corresponding to apoE promoter, that is absent in the no template control reaction (lane 7, NTC). In lane 1 (M), marker DNA ladder is shown. (B) In transiently transfected HepG2 cells, exposure to dexamethasone does not significantly enhance the activity of [-500/+73] apoE promoter, its deletion fragments or pGL4 vector, used as a control (p>0.05). (C) DNA pull down experiments show that GR bind to the -115/-75 apoE promoter region in untransfected cells (lane 1) and binding is enhanced by overexpression of GRα (lane 3). A partial (~30%) inhibition of GR binding was detected in cells overexpressing both GR and c-Jun (lane 5). No binding occurs to non-specific oligonucleotides when the cells express only endogenous GR (lane 2), overexpress GR (lane 4) or overexpress both GR and c-Jun (lane 6). Western blots probed for GRα of whole extracts prepared from cells expressing only the endogenous protein or overexpressing it are illustrated in lanes 7 and 8, respectively. (D-F) The effects of various inhibitors on dexamethasone-treated HepG2 cells transiently transfected with plasmids containing luciferase under apoE proximal promoter. (D) Dexamethasone does not significantly modulate apoE promoter activity in the presence or absence of the JNK inhibitor, SP600125 (E) Exposure of HepG2 cells to dexamethasone (1 μM, for 24 hours) in the presence of increasing concentrations of MEK1/2 inhibitor (U0126) induces a dose-dependent enhancement of apoE promoter activity (F). Dexamethasone treatment in the presence of PLA2/PLC inhibitor U73122 increases apoE promoter activity as a function of the inhibitor concentration. The symbols are: *** p<0.001, **p<0.01 and *p<0.05.</p

The sequences of the primers used for RT-PCR, DNA pull-down assays and chromatin immunoprecipitation (ChIP) experiments.

<p>The sequences of the primers used for RT-PCR, DNA pull-down assays and chromatin immunoprecipitation (ChIP) experiments.</p

Nano-Polyplexes Mediated Transfection of Runx2-shRNA Mitigates the Osteodifferentiation of Human Valvular Interstitial Cells

Calcific aortic valve disease (CAVD) is a progressive disorder that increases in prevalence with age. An important role in aortic valve calcification is played by valvular interstitial cells (VIC), that with age or in pathological conditions acquire an osteoblast-like phenotype that advances the disease. Therefore, pharmacological interventions aiming to stop or reverse the osteoblastic transition of VIC may represent a therapeutic option for CAVD. In this study, we aimed at developing a nanotherapeutic strategy able to prevent the phenotypic switch of human aortic VIC into osteoblast-like cells. We hypothesize that nanocarriers designed for silencing the Runt-related transcription factor 2 (Runx2) will stop the progress or reverse the osteodifferentiation of human VIC, induced by high glucose concentrations and pro-osteogenic factors. We report here the potential of fullerene (C60)-polyethyleneimine (PEI)/short hairpin (sh)RNA-Runx2 nano-polyplexes to efficiently down-regulate Runx2 mRNA and protein expression leading subsequently to a significant reduction in the expression of osteogenic proteins (i.e., ALP, BSP, OSP and BMP4) in osteoblast-committed VIC. The data suggest that the silencing of Runx2 could represent a novel strategy to impede the osteoblastic phenotypic shift of VIC and the ensuing progress of CAVD