A knowledge base for the discovery of function, diagnostic potential and drug effects on cellular and extracellular miRNAs.

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs that play an important role in the regulation of various biological processes through their interaction with cellular mRNAs. A significant amount of miRNAs has been found in extracellular human body fluids (e.g. plasma and serum) and some circulating miRNAs in the blood have been successfully revealed as biomarkers for diseases including cardiovascular diseases and cancer. Released miRNAs do not necessarily reflect the abundance of miRNAs in the cell of origin. It is claimed that release of miRNAs from cells into blood and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. Moreover, miRNAs play a significant role in pharmacogenomics by down-regulating genes that are important for drug function. In particular, the use of drugs should be taken into consideration while analyzing plasma miRNA levels as drug treatment. This may impair their employment as biomarkers. DESCRIPTION: We enriched our manually curated extracellular/circulating microRNAs database, miRandola, by providing (i) a systematic comparison of expression profiles of cellular and extracellular miRNAs, (ii) a miRNA targets enrichment analysis procedure, (iii) information on drugs and their effect on miRNA expression, obtained by applying a natural language processing algorithm to abstracts obtained from PubMed. CONCLUSIONS: This allows users to improve the knowledge about the function, diagnostic potential, and the drug effects on cellular and circulating miRNAs.

MiR-492 impairs the angiogenic potential of endothelial cells.

Endothelial cells growing in high glucose-containing medium show reduced cell proliferation and in vitro angiogenesis. Evidence suggests that the molecular pathways leading to these cellular responses are controlled by microRNAs, endogenous post-transcriptional regulators of gene expression. To identify the microRNAs and their targeted genes involved in the glucose responses, we performed the miRNA signature of Human Umbelical Vein Endothelial Cells (HUVECs) exposed and unexposed to high glucose. Among differentially expressed microRNAs, we analysed miR-492 and showed that its overexpression was able to reduce proliferation, migration and tube formation of HUVEC. These effects were accompanied by the down-regulation of eNOS, a key regulator of the endothelial cell function. We showed that eNOS was indirectly down-regulated by miR-492 and we discovered that miR-492 was able to bind mRNAs involved in proliferation, migration, tube formation and regulation of eNOS activity and expression. Moreover, we found that miR-492 decreased VEGF expression in HUVEC and impaired in vivo angiogenesis in a tumour xenograft model, suggesting a role also in modulating the secretion of pro-angiogenic factors. Taken together, the data indicate that miR-492 exerts a potent anti-angiogenic activity in endothelial cells and therefore miR-492 seems a promising tool for anti-angiogenic therapy.

Fludarabine treatment favors the retention of miR-485-3p by prostate cancer cells: implications for survival.

BACKGROUND: Circulating microRNAs (miRNAs) have been found in many body fluids and represent reliable markers of several physio-pathological disorders, including cancer. In some cases, circulating miRNAs have been evaluated as markers of the efficacy of anticancer treatment but it is not yet clear if miRNAs are actively released by tumor cells or derive from dead tumor cells. RESULTS: We showed that a set of prostate cancer secretory miRNAs (PCS-miRNAs) were spontaneously released in the growth medium by DU-145 prostate cancer cells and that the release was greater after treatment with the cytotoxic drug fludarabine. We also found that the miRNAs were associated with exosomes, implying an active mechanism of miRNA release. It should be noted that in fludarabine treated cells the release of miR-485-3p, as well as its association with exosomes, was reduced suggesting that miR-485-3p was retained by surviving cells. Monitoring the intracellular level of miR-485-3p in these cells, we found that miR-485-3p was stably up regulated for several days after treatment. As a possible mechanism we suggest that fludarabine selected cells that harbor high levels of miR-485-3p, which in turn regulates the transcriptional repressor nuclear factor-Y triggering the transcription of topoisomerase IIα, multidrug resistance gene 1 and cyclin B2 pro-survival genes. CONCLUSIONS: Cytotoxic treatment of DU-145 cells enhanced the release of PCS-miRNAs with the exception of miR-485-3p which was retained by surviving cells. We speculate that the retention of miR-485-3p was a side effect of fludarabine treatment in that the high intracellular level of miR-485-3p plays a role in the sensitivity to fludarabine.

MicroRNAs mediate metabolic stresses and angiogenesis.

MicroRNAs are short endogenous RNA molecules that are able to regulate (mainly inhibiting) gene expression at the post-transcriptional level. The MicroRNA expression profile is cell-specific, but it is sensitive to perturbations produced by stresses and diseases. Endothelial cells subjected to metabolic stresses, such as calorie restriction, nutrients excess (glucose, cholesterol, lipids) and hypoxia may alter their functionality. This is predictive for the development of pathologies like atherosclerosis, diabetes, and hypertension. Moreover, cancer cells can activate a resting endothelium by secreting pro-angiogenic factors, in order to promote neoangiogenesis, which is essential for tumor growth. Endothelial altered phenotype is mirrored by altered mRNA, microRNA, and protein expression, with a microRNA being able to control pathways by regulating the expression of multiple mRNAs. In this review we will consider the involvement of microRNAs in modulating the response of endothelial cells to metabolic stresses and their role in promoting or halting angiogenesis.

An RbAp48-like gene regulates adult stem cells in planarians.

Retinoblastoma-associated proteins 46 and 48 (RbAp46 and RbAp48) are factors that are components of different chromatin-modelling complexes, such as polycomb repressive complex 2, the activity of which is related to epigenetic gene regulation in stem cells. To date, no direct findings are available on the in vivo role of RbAp48 in stem-cell biology. We recently identified DjRbAp48 - a planarian (Dugesia japonica) homologue of human RBAP48 - expression of which is restricted to the neoblasts, the adult stem cells of planarians. In vivo silencing of DjRbAp48 induces lethality and inability to regenerate, even though neoblasts proliferate and accumulate after wounding. Despite a partial reduction in neoblast number, we were always able to detect a significant number of these cells in DjRbAp48 RNAi animals. Parallel to the decrease in neoblasts, a reduction in the number of differentiated cells and the presence of apoptotic-like neoblasts were detectable in RNAi animals. These findings suggest that DjRbAp48 is not involved in neoblast maintenance, but rather in the regulation of differentiation of stem-cell progeny. We discuss our data, taking into account the possibility that DjRbAp48 might control the expression of genes necessary for cell differentiation by influencing chromatin architecture.

Enhancement of gene targeting in human cells by intranuclear permeation of the Saccharomyces cerevisiae Rad52 protein.

The introduction of exogenous DNA in human somatic cells results in a frequency of random integration at least 100-fold higher than gene targeting (GT), posing a seemingly insurmountable limitation for gene therapy applications. We previously reported that, in human cells, the stable over-expression of the Saccharomyces cerevisiae Rad52 gene (yRAD52), which plays the major role in yeast homologous recombination (HR), caused an up to 37-fold increase in the frequency of GT, indicating that yRAD52 interacts with the double-strand break repair pathway(s) of human cells favoring homologous integration. In the present study, we tested the effect of the yRad52 protein by delivering it directly to the human cells. To this purpose, we fused the yRAD52 cDNA to the arginine-rich domain of the TAT protein of HIV (tat11) that is known to permeate the cell membranes. We observed that a recombinant yRad52tat11 fusion protein produced in Escherichia coli, which maintains its ability to bind single-stranded DNA (ssDNA), enters the cells and the nuclei, where it is able to increase both intrachromosomal recombination and GT up to 63- and 50-fold, respectively. Moreover, the non-homologous plasmid DNA integration decreased by 4-fold. yRAD52tat11 proteins carrying point mutations in the ssDNA binding domain caused a lower or nil increase in recombination proficiency. Thus, the yRad52tat11 could be instrumental to increase GT in human cells and a 'protein delivery approach' offers a new tool for developing novel strategies for genome modification and gene therapy applications.

MicroRNAs couple cell fate and developmental timing in retina.

Cell identity is acquired in different brain structures according to a stereotyped timing schedule, by accommodating the proliferation of multipotent progenitor cells and the generation of distinct types of mature nerve cells at precise times. However, the molecular mechanisms coupling the identity of a specific neuron and its birth date are poorly understood. In the neural retina, only late progenitor cells that divide slowly can become bipolar neurons, by the activation of otx2 and vsx1 genes. In Xenopus, we found that Xotx2 and Xvsx1 translation is inhibited in early progenitor cells that divide rapidly by a set of cell cycle-related microRNAs (miRNAs). Through expression and functional screenings, we selected 4 miRNAs--mir-129, mir-155, mir-214, and mir-222--that are highly expressed at early developmental stages in the embryonic retina and bind to the 3' UTR of Xotx2 and Xvsx1 mRNAs inhibiting their translation. The functional inactivation of these miRNAs in vivo releases the inhibition, supporting the generation of additional bipolar cells. We propose a model in which the proliferation rate and the age of a retinal progenitor are linked to each other and determine the progenitor fate through the activity of a set of miRNAs.

Immortalized mouse embryo fibroblasts are resistant to miR-290-induced senescence regardless of p53 status.

The prosenescence role of miR-290 and nocodazole has been documented in primary mouse embryo fibroblasts (MEF), while it is not clear whether immortal murine fibroblasts are still responsive to these senescence inducing stimuli. To establish this point, immortal murine fibroblasts with functional (NIH3T3) or nonfunctional p53 (I-MEF) and low levels of miR-290 were tested for their capability to undergo senescence after exposure to either nocodazole or miR-290. Our results clearly indicate that nocodazole induces senescence only in NIH3T3 cells with a functional p53 but not in I-MEF lacking a functional p53. miR-290 overexpression is unable to address any of the tested immortalized clones toward senescence, regardless of the p53 status, suggesting that the prosenescence role of miR-290 is specific for primary but not for immortal murine fibroblasts. Moreover our findings suggest that the mere downregulation of a potential tumor suppressor miRNA in a given cell type does not necessarily imply that it behaves as a tumor suppressor.

MicroRNA (miRNA)-mediated interaction between leukemia/lymphoma-related factor (LRF) and alternative splicing factor/splicing factor 2 (ASF/SF2) affects mouse embryonic fibroblast senescence and apoptosis.

Leukemia/lymphoma-related factor (LRF) is a transcriptional repressor, which by recruiting histone deacetylases specifically represses p19/ARF expression, thus behaving as an oncogene. Conversely, in mouse embryonic fibroblasts (MEF), LRF inhibition causes aberrant p19ARF up-regulation resulting in proliferative defects and premature senescence. We have recently shown that LRF is controlled by microRNAs. Here we show that LRF acts on MEF proliferation and senescence/apoptosis by repressing miR-28 and miR-505, revealing a regulatory circuit where microRNAs (miRNAs) work both upstream and downstream of LRF. By analyzing miRNA expression profiles of MEF transfected with LRF-specific short interfering RNAs, we found that miR-28 and miR-505 are modulated by LRF. Both miRNAs are predicted to target alternative splicing factor/splicing factor 2 (ASF/SF2), a serine/arginine protein essential for cell viability. In vertebrates, loss or inactivation of ASF/SF2 may result in genomic instability and induce G(2) cell cycle arrest and apoptosis. We showed that miR-28 and miR-505 modulate ASF/SF2 by directly binding ASF/SF2 3'-UTR. Decrease in LRF causes a decrease in ASF/SF2, which depends on up-regulation of miR-28 and miR-505. Alteration of each of the members of the LRF/miR-28/miR-505/ASF/SF2 axis affects MEF proliferation and the number of senescent and apoptotic cells. Consistently, the axis is coordinately modulated as cell senescence increases with passages in MEF culture. In conclusion, we show that LRF-dependent miRNAs miR-28 and miR-505 control MEF proliferation and survival by targeting ASF/SF2 and suggest a central role of LRF-related miRNAs, in addition to the role of LRF-dependent p53 control, in cellular homeostasis.

DeltaN133p53 expression levels in relation to haplotypes of the TP53 internal promoter region.

The transcription of the DeltaN133p53 isoform of the TP53 gene is controlled by an internal promoter region (IPR) containing eight polymorphisms in 11 common haplotypes, following a resequencing of 47 Caucasians. We assayed the functional effects of the commonest six haplotypes on the promoter activity with a luciferase reporter system, in HeLa and 293T cells. These studies showed that different IPR haplotypes are associated with differences in the promoter activity resulting in marked variation in the baseline expression of DeltaN133p53. In vivo quantitative-polymerase chain reaction (PCR) on human tissues confirmed that the baseline levels of DeltaN133p53 showed haplotype specific differences that paralleled those seen in vitro. When cell lines were treated with camptothecin, the fold-increase in DeltaN133p53 levels was dose-dependent but haplotype-independent (i.e., similar for all the haplotypes). Finally, we used an electrophoretic mobility shift assay to analyze the rs1794287 polymorphism and found changes in the pattern of protein binding. This partially confirmed our in silico analysis showing that the polymorphism rs1794287 can affect the function of the internal promoter by changing its affinity for several transcription factors. Thus, we showed that the expression of DeltaN133p53 is under genetic control, and suggested the presence of interindividual differences underlying this mechanism.

miR-20a and miR-290, multi-faceted players with a role in tumourigenesis and senescence.

Expression of microRNAs changes markedly in tumours and evidence indicates that they are causatively related to tumourigenesis, behaving as tumour suppressor microRNAs or onco microRNAs; in some cases they can behave as both depending on the type of cancer. Some tumour suppressor microRNAs appear to be an integral part of the p53 and Retinoblastoma (RB) network, the main regulatory pathways controlling senescence, a major tumour suppressor mechanism. The INK4a/ARF locus which codifies for two proteins, p19ARF and p16INK4a, plays a central role in senescence by controlling both p53 and RB. Recent evidence shows that the proto-oncogene leukaemia/lymphoma related factor, a p19ARF specific repressor, is controlled by miRNAs and that miRNAs, in particular miR-20a and miR-290, are causatively involved in mouse embryo fibroblasts (MEF) senescence in culture. Intriguingly, both miR-20a, member of the oncogenic miR-17-92 cluster, and miR-290, belonging to the miR-290-295 cluster, are highly expressed in embryonic stem (ES) cells. The pro-senescence role of miR-20a and miR-290 in MEF is apparently in contrast with their proliferative role in tumour and ES cells. We propose that miRNAs may exert opposing functions depending on the miRNAs repertoire as well as target/s level/s present in different cellular contexts, suggesting the importance of evaluating miRNAs activity in diverse genetic settings before their therapeutic use as tumour suppressors.

Adult stem cell plasticity: neoblast repopulation in non-lethally irradiated planarians.

Planarians are a model system for studying adult stem cells, as they possess the neoblasts, a population of pluripotent adult stem cells able to give rise to both somatic and germ cells. Although over the last years several efforts have been made to shed light on neoblast biology, only recent evidence indicate that this population of cells is heterogeneous. In this study we irradiated planarians with different non-lethal X-ray doses (1-5 Gy) and we identified subpopulations of neoblasts with diverse levels of tolerance to X-rays. We demonstrated that a dramatic reduction of neoblasts occurred soon after non-lethal irradiations and that de-novo proliferation of some radioresistant cells re-established the primary neoblast number. In particular, a strong proliferation activity occurred at the ventral side of irradiated animals close to the nervous system. The produced cells migrated towards the dorsal parenchyma and, together with some dorsal radioresistant cells, reconstituted the entire neoblast population demonstrating the extreme plasticity of this adult stem cell system.

miR-290 acts as a physiological effector of senescence in mouse embryo fibroblasts.

The culture-induced senescence of mouse embryo fibroblasts (MEF) correlates with reduction of cell proliferation. In this work we found that the accumulation of cells with 4C DNA content and the transcriptional change of several microRNAs (miRNAs or miRs) are relevant events in culture senescence. By comparing the miRNA expression profiles of physiologically senescent MEF and that of senescent MEF induced by the downregulation of leukemia-related factor, we identified miR-290 as a common upregulated miRNA. When miR-290 was transfected in presenescent MEF, SA-beta-gal(+) cells and p16, two markers of culture senescence, increased compared with control, indicating that miR-290 is causally involved in senescence. Interestingly, nocodazole (NCZ), which induces G2/M block, increased the percentage of senescent cells as well as the expression of miR-290 and of the tumor suppressor p16, thus mimicking culture senescence. As miR-290 was overexpressed in NCZ-treated cells and it was able to induce senescence in proliferating MEF, we investigated whether miR-290 and NCZ could share common mechanisms of culture senescence. Whereas the induction of SA-beta-gal(+) by miR-290 was not strengthened by coupling its transfection with NCZ treatment, the transfection of the antagomir 290 (d-290) plus NCZ treatment, while blocking cells at G2/M, suppressed SA-beta-gal(+) and p16 induction. On the basis of these findings we conclude that miR-290 might act as a physiological effector of NCZ induced as well as culture senescence via p16 regulation expanding the role of this miRNA from embryonic stem to differentiated cells.

TSPO over-expression increases motility, transmigration and proliferation properties of C6 rat glioma cells.

Gliomas are one of the most malignant cancers. The molecular bases regulating the onset of such tumors are still poorly understood. The translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, is a mitochondrial permeability transition (MPT)-pore protein robustly expressed in gliomas and involved in the regulation of apoptosis and cell proliferation. TSPO expression levels have been correlated with tumor malignancy. Here we describe the production of C6 rat glioma cells engineered to over-express the TSPO protein with the aim of providing the first direct evidence of a correlation between TSPO expression level and glioma cell aggressiveness. We observed that TSPO potentiates proliferation, motility and transmigration capabilities as well as the ability to overcome contact-induced cell growth inhibition of glioma cells. On the whole, these data demonstrate that TSPO density influences metastatic potential of glioma cells. Since several data suggest that TSPO ligands may act as chemotherapeutic agents, in this paper we also demonstrate that TSPO ligand-induced cell death is dependent on TSPO density. These findings suggest that the use of TSPO ligands as chemotherapeutic agents could be effective on aggressive tumor cells with a high TSPO expression level.

Role of E2F1-cyclin E1-cyclin E2 circuit in human coronary smooth muscle cell proliferation and therapeutic potential of its downregulation by siRNAs.

Aberrant coronary vascular smooth muscle cell (CSMC) proliferation is a pivotal event underlying intimal hyperplasia, a phenomenon impairing the long-term efficacy of bypass surgery and angioplasty procedures. Consequently research has become focused on efforts to identify molecules that are able to control CSMC proliferation. We investigated downregulation of CSMC growth by small interfering RNAs (siRNAs) targeted against E2F1, cyclin E1, and cyclin E2 genes, whose contribution to CSMC proliferation is only now being recognized. Chemically synthesized siRNAs were delivered by two different transfection reagents to asynchronous and synchronous growing human CSMCs cultivated either in normo- or hyperglycemic conditions. The depletion of each of the three target genes affected the expression of the other two genes, demonstrating a close regulatory control. The clearest effects associated with the inhibition of the E2F1-cyclin E1/E2 circuit were the reduction in the phosphorylation levels of the retinoblastoma protein pRB and a decrease in the amount of cyclin A2. At the phenotypic level the downmodulation of CSMC proliferation resulted in a decrease of S phase matched by an increase of G1-G0 phase cell amounts. The antiproliferative effect was cell-donor and transfectant independent, reversible, and effective in asynchronous and synchronous growing CSMCs. Importantly, it was also evident in hyperglycemia, a condition that underlies diabetes. No significant aspecific cytotoxicity was observed. Our data demonstrate the interrelation among E2F1-cyclin E1-cyclin E2 and the pivotal role this circuit exerts in CSMC proliferation. Additionally, our work validates the concept of utilizing anti-E2F1-cyclin E1-cyclin E2 siRNAs to develop a potential novel therapy to control intimal hyperplasia.

Ozone and cardiovascular injury.

Air pollution is increasingly recognized as an important and modifiable determinant of cardiovascular diseases in urban communities. The potential detrimental effects are both acute and chronic having a strong impact on morbidity and mortality. The acute exposure to pollutants has been linked to adverse cardiovascular events such as myocardial infarction, heart failure and life-threatening arrhythmias. The long-terms effects are related to the lifetime risk of death from cardiac causes. The WHO estimates that air pollution is responsible for 3 million premature deaths each year. The evidence supporting these data is very strong nonetheless, epidemiologic and observational data have the main limitation of imprecise measurements. Moreover, the lack of clinical experimental models makes it difficult to demonstrate the individual risk. The other limitation is related to the lack of a clear mechanism explaining the effects of pollution on cardiovascular mortality. In the present review we will explore the epidemiological, clinical and experimental evidence of the effects of ozone on cardiovascular diseases. The pathophysiologic consequences of air pollutant exposures have been extensively investigated in pulmonary systems, and it is clear that some of the major components of air pollution (e.g. ozone and particulate matter) can initiate and exacerbate lung disease in humans 1. It is possible that pulmonary oxidant stress mediated by particulate matter and/or ozone (O3) exposure can result in downstream perturbations in the cardiovasculature, as the pulmonary and cardiovascular systems are intricately associated, and it is well documented that specific environmental toxins (such as tobacco smoke 2) introduced through the lungs can initiate and/or accelerate cardiovascular disease development. Indeed, several epidemiologic studies have proved that there is an association between PM and O3 and the increased incidence of cardiovascular morbidity and mortality 3. Most of the evidence comes from studies of ambient particles concentrations. However, in Europe and elsewhere, the air pollution profile has gradually changed toward a more pronounced photochemical component. Ozone is one of the most toxic components of the photochemical air pollution mixture. Indeed, the biological basis for these observations has not been elucidated. In the present review, the role of ozone as chemical molecule will be firstly considered. Secondly, pathogenetic mechanisms connecting the atmospheric ozone level and cardiovascular pathology will be examined. Thirdly, the literature relating hospitalization frequency, morbidity and mortality due to cardiovascular causes and ozone concentration will be studied. The correlation between ozone level and occurrence of acute myocardial infarction will be eventually discussed.

PK 11195 differentially affects cell survival in human wild-type and 18 kDa translocator protein-silenced ADF astrocytoma cells.

Gliomas are the most common brain tumours with a poor prognosis due to their aggressiveness and propensity for recurrence. The 18 kDa translocator protein (TSPO) has been demonstrated to be greatly expressed in glioma cells and its over-expression has been correlated with glioma malignance grades. Due to both its high density in tumours and the pro-apoptotic activity of its ligands, TSPO has been suggested as a promising target in gliomas. With the aim to evidence if the TSPO expression level alters glioma cell susceptibility to undergo to cell death, we analysed the effects of the specific TSPO ligand, PK 11195, in human astrocytoma wild-type and TSPO-silenced cell lines. As first step, TSPO was characterised in human astrocytoma cell line (ADF). Our data demonstrated the presence of a single class of TSPO binding sites highly expressed in mitochondria. PK 11195 cell treatment activated an autophagic pathway followed by apoptosis mediated by the modulation of the mitochondrial permeability transition. In TSPO-silenced cells, produced by siRNA technique, a reduced cell proliferation rate and a decreased cell susceptibility to the PK 11195-induced anti-proliferative effect and mitochondrial potential dissipation were demonstrated respect to control cells. In conclusion, for the first time, PK 11195 was demonstrated to differentially affect glioma cell survival in relation to TSPO expression levels. These results encourage the development of specific-cell strategies for the treatment of gliomas, in which TSPO is highly expressed respect to normal cells.

Effect of the administration of n-3 polyunsaturated fatty acids on circulating levels of microparticles in patients with a previous myocardial infarction.

BACKGROUND: Increased levels of microparticles exposing tissue factor circulate in the blood of patients with coronary heart disease, possibly disseminating their pro-thrombotic and pro-inflammatory potential. Because diets rich in n-3 (polyunsaturated) fatty acids have been associated with reduced incidence of coronary heart disease-related events, we investigated the in vivo effects of treatments with n-3 fatty acids on levels of circulating microparticles and their tissue factor- dependent procoagulant activity in patients with a previous myocardial infarction. DESIGN AND METHODS: Forty-six post-myocardial infarction patients were assigned to receive either 5.2 g of n-3 fatty acids daily (n=23) or an olive oil placebo (n = 23) for 12 weeks. Circulating microparticles were isolated from peripheral blood. The number of microparticles, their cellular source and tissue factor antigen were determined by flow cytometry, and their procoagulant potential assayed by a fibrin generation test. RESULTS: The total number of microparticles, endothelium-derived microparticles and microparticle tissue factor antigen were not significantly different between the two groups. However, the number of platelet-derived microparticles [from a median of 431 (126-1796, range) x 10(6)/L to a median of 226 (87-677, range)] x 10(6)/L and monocyte-derived microparticles [from a median of 388 (9-1681, range) x 10(6)/L to a median of 265 (7-984, range) x 10(6)/L] in plasma were significantly (p < 0.05) decreased by n-3 fatty acids, while they were unchanged in the placebo group. Total microparticle tissue factor-procoagulant activity was also reduced in the n-3 fatty acid group compared to that in the placebo group. CONCLUSIONS: Treatment with n-3 fatty acids after myocardial infarction exerts favorable effects on levels of platelet- and monocyte-derived microparticles, thus possibly explaining some of the anti-inflammatory and anti-thrombotic properties of these natural compounds.

Parallel decrease of tissue factor surface exposure and increase of tissue factor microparticle release by the n-3 fatty acid docosahexaenoate in endothelial cells.

Tissue factor (TF) is expressed on the endothelium in response to inflammatory mediators, giving endothelial cells a pro-thrombotic phenotype. Since fish-derived n-3 fatty acids (FA) have been associated with reduced incidence of myocardial infarction, we investigated the endothelial effects of the most abundant n-3 FA, docosahexaenoate (DHA), on TF expression. Human umbilical vein endothelial cells were pre-incubated with DHA (or stearate and arachidonate as controls) for 48-72 hours, and then stimulated with bacterial lipopolysaccharide (LPS) or tumor necrosis factor-alpha. Pre-incubation of endothelial cells with DHA (but not stearate or arachidonate) concentration-dependently reduced surface protein exposure, independent of TF mRNA or total protein expression regulation. Conversely, DHA treatment in conjunction with activating stimuli, induced the release of endothelial TF-exposing microparticles from endothelial cells, quantitatively accounting for the decreased TF cell surface exposure. In conclusion, DHA treatment, with a time-course consistent with its incorporation in membrane phospholipids, increases the release of TF-exposing microparticles from endothelial cells, accounting for decreased endothelial cell TF surface exposure, thus potentially modifying the overall endothelial control of microparticle-related effects.

Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52.

When exogenous DNA is stably introduced in mammalian cells, it is typically integrated in random positions, and only a minor fraction enters a pathway of homologous recombination (HR). The complex Rad51/Rad52 is a major player in the management of exogenous DNA in eukaryotic organisms and plays a critical role in the choice of repair system. In Saccharomyces cerevisiae, the pathway of choice is HR, mediated by Rad52 (ScRad52), which differs slightly from its human homologue. Here, we present an approach that utilizes ScRad52 to enhance HR in human cells containing a specific substrate for recombination. Clones of HeLa cells were produced expressing functional ScRad52. These cells showed enhanced resistance to DNA damaging treatments and revealed a different distribution of Rad51 foci (a marker of recombination complex formation). More significantly, ScRad52 expression resulted in an up to 37-fold increase in gene targeting by HR. In the same cells, random integration of exogenous DNA was significantly reduced, consistent with the view that HR and non-homologous end joining are alternative competing pathways. Expression of ScRad52 could offer a major improvement for experiments requiring gene targeting by HR, both in basic research and in gene therapy studies.