miR-210 is a key player of cell response to hypoxia, modulating cell survival, VEGF-driven endothelial cell migration, and the ability of endothelial cells to form capillary-like structures. A crucial step in understanding microRNA (miRNA) function is the identification of their targets. However, only few miR-210 targets have been identified to date. Here, we describe an integrated strategy for large-scale identification of new miR-210 targets by combining transcriptomics and proteomics with bioinformatic approaches. To experimentally validate candidate targets, the RNA-induced silencing complex (RISC) loaded with miR-210 was purified by immunoprecipitation along with its mRNA targets. The complex was significantly enriched in mRNAs of 31 candidate targets, such as BDNF, GPD1L, ISCU, NCAM, and the non-coding RNA Xist. A subset of the newly identified targets was further confirmed by 3-untranslated region (UTR) reporter assays, and hypoxia induced down-modulation of their expression was rescued blocking miR-210, providing support for the approach validity. In the case of 9 targets, such as PTPN1 and P4HB, miR-210 seed-pairing sequences localized in the coding sequence or in the 5-UTR, in line with recent data extending miRNA targeting beyond the "classic" 3-UTR recognition. Finally, Gene Ontology analysis of the targets highlights known miR-210 impact on cell cycle regulation and differentiation, and predicts a new role of this miRNA in RNA processing, DNA binding, development, membrane trafficking, and amino acid catabolism. Given the complexity of miRNA actions, we view such a multiprong approach as useful to adequately describe the multiple pathways regulated by miR-210 during physiopathological processes.miRNAs are 21-23-nucleotide non-protein coding RNA molecules that regulate the stability and/or the translational efficiency of target messenger RNAs (1-3). Mature miRNAs are loaded into the RNA-induced silencing complex (RISC) 3 and mediate the translational inhibition of target mRNA, albeit a few opposing examples have been described as well (4 -6). The rules that guide miRNA-mRNA interaction are very complex and still under investigation. However, the current paradigm states that a Watson-Crick pairing between the mRNA and the 5Ј-region of the miRNA centered on nucleotides 2-7, termed "seed sequence," is required for miRNA-mediated inhibition (7). RISC-miRNA complexes can move the mRNAs they bind to the P-bodies, which are specialized cytoplasmic compartments where translational repression and mRNA turnover is thought to occur (8). Because P-bodies contain many enzymes involved in mRNA exonucleolitic degradation, miRNAs may also have a secondary quantitative inhibitory effect on mRNAs. A role for miRNAs in mRNA destabilization is also suggested by studies reporting robust correlations between the levels of miRNAs and the message of multiple predicted or validated targets (9 -11).miR-210 is currently regarded as "master miRNA" of hypoxic response, because it was found up-regulated by hypoxia in all the ce...

High plasma levels of VLDL are associated with increased risk for atherosclerosis. Here we show that VLDL (75 to 150 microg/mL) activates nuclear factor-kappaB (NF-kappaB), a transcription factor known to play a key role in regulation of inflammation. Oxidation of VLDL reduced its capacity to activate NF-kappaB in vitro, whereas free fatty acids such as linoleic and oleic acid activated NF-kappaB to the same extent as did VLDL. Intravenous injection of human VLDL (6 mg protein per kg) into rats resulted in arterial activation of NF-kappaB as assessed by electrophoretic mobility shift assay. Aortic endothelial cells showed positive nuclear staining for the activated RelA (p65) subunit of NF-kappaB at 6 to 24 hours after injection. There was also a parallel expression of the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, as well as the cytokine tumor necrosis factor-alpha. Pretreatment of the rats with diet containing 1% of the antioxidant probucol for 8 weeks did not inhibit arterial activation of NF-kappaB in response to injection of VLDL. Moreover, injection of triglycerides (10% Intralipid, 5 mL/kg) activated arterial expression of NF-kappaB to the same extent as VLDL. Our results suggest that VLDL may promote the development of atherosclerotic lesions by activation of the proinflammatory transcription factor NF-kappaB. The effect appears to be mediated by a release of VLDL fatty acids but not to involve VLDL oxidation.

Specific and selective AT2-R stimulation has beneficial effects on the pathological events occurring in SHRSPs. These data indicate a new avenue for the pharmacological treatment of diseases in which modulation of the renin-angiotensin system is required.

Microparticles (MP) are small membrane vesicles that are released from cells upon activation or during apoptosis. Cellular MP in body fluids constitute a heterogeneous population, differing in cellular origin, numbers, size, antigenic composition and functional properties. MP support coagulation by exposure of tissue factor (TF), the initiator of coagulation in vivo. Moreover, MP may transfer bioactive molecules to other cells, thereby stimulating them to produce cytokines, cell-adhesion molecules, growth factors and TF, and modulate endothelial functions. However, a comprehensive characterization of the antigenic composition of MP has been poorly defined. This study describes the protein composition of endothelial cell (EC)-derived MP (EMP) using a proteomic approach. MS analysis indicated the presence of newly described protein such as metabolic enzymes, proteins involved in adhesion and fusion processes, members of protein folding event, cytoskeleton associated proteins and nucleosome. In conclusion, circulating EMP behave as an actual storage pool, able to disseminate blood-borne TF activity and other bioactive effectors, as confirmed by our experiments showing an increased procoagulant activity of EC exposed to EMP.

Abstract-In vivo studies have demonstrated a strong positive correlation between plasma very low density lipoprotein (VLDL) triglyceride and plasma plasminogen activator inhibitor-1 (PAI-1) activity levels. Furthermore, VLDL has been shown to induce PAI-1 secretion from cultured endothelial cells. In contrast, no or variable effects on PAI-1 secretion have been reported for native low density lipoprotein. It could be speculated that fatty acids derived from VLDL triglycerides are the actual mediators, resulting in an enhanced secretion of PAI-1. In the present study, we have analyzed the effects of both saturated and unsaturated fatty acids on PAI-1 expression and secretion by endothelial cells. Addition of 0 to 50 mol/L of either palmitic acid or stearic acid had no effect on PAI-1 secretion from human umbilical vein endothelial cells or EA.hy926 cells. In contrast, addition of oleic acid, linoleic acid, linolenic acid, and eicosapentaenoic acid resulted in a significant increase in PAI-1 secretion from both cell types. Northern blot analysis of PAI-1 mRNA levels was in agreement with these findings. Transfection experiments demonstrated that addition of linolenic acid and eicosapentaenoic acid significantly increased PAI-1 transcription. The fatty acid response region was localized to a previously described VLDL-inducible region of the PAI-1 promoter. Electromobility shift assays demonstrated that unsaturated fatty acids induced the same complex as did VLDL, whereas saturated fatty acids had no effect. Furthermore, it was demonstrated that the activation procedure did not involve fatty acid oxidation to any significant extent. In conclusion, the present study demonstrates that unsaturated fatty acids increase PAI-1 transcription and secretion by endothelial cells in vitro. The effect appears to be mediated by a previously described VLDL-inducible transcription factor. (Arterioscler Thromb Vasc Biol. 1998;18:1679-1685.)

The effect of VLDL on plasminogen activator inhibitor type 1 biosynthesis in HepG2 cells was investigated. Exposure of HepG2 cells to VLDL (range, 10 to 100 micrograms protein per milliliter) for 16 hours resulted in an enhanced release of PAI-1 antigen and PAI activity into conditioned medium, accompanied by the accumulation of intracellular triglycerides. By using a monoclonal antibody (IgG C7) specific to the LDL receptor, we showed that the effect of VLDL is mediated by its interaction with the LDL receptor. Enhanced PAI-1 release was due to increased biosynthesis: PAI-1 mRNA was doubled, mainly because of the effect on the 2.2-kb PAI-1 mRNA rather than the 3.2-kb transcript. Addition of insulin with the VLDL further enhanced PAI-1 antigen release and PAI-1 mRNA accumulation. The effect of VLDL on steady state levels of PAI-1 mRNA was apparently not due to an increase of gene transcription but to stabilization of both PAI-1 mRNA transcripts. The enhancing effect of VLDL on PAI-1 biosynthesis in HepG2 cells may raise PAI-1 antigen levels not only in hypertriglyceridemic states but also in those conditions in which both insulin and VLDL are elevated.

Spontaneously hypertensive stroke-prone rats (SHRSP) develop hypertension and systemic inflammation, with subsequent brain and renal disorders and early death. We tested the hypothesis that valsartan, an angiotensin II type 1 (AT1) receptor antagonist, exerts protective effects in SHRSP through its anti-inflammatory properties, even in the absence of a blood pressure-lowering effect. SHRSP fed a high-salt diet were treated with vehicle or valsartan (1-10 mg/kg/day). The vehicletreated rats developed hypertension, proteinuria, progressive kidney disease, and, 40 Ϯ 5 days from the beginning of the treatment, brain damage as visualized by magnetic resonance imaging. Rats treated with 1 mg/kg/day valsartan developed brain damage after 61 Ϯ 3 days (p Ͻ 0.01 versus vehicletreated rats). No damage showed after 100 days in 80% of the rats treated with 10 mg/kg/day. Valsartan treatment preserved renal structure, by preventing the infiltration of inflammatory cells, and lowered renal expression of monocyte chemoattractant protein-1, transforming growth factor-␤1, and interleukin-1␤, compared with vehicle-treated SHRSP. Urinary excretion of acute-phase proteins increased in the latter but remained negligible in the drug-treated animals. Furthermore, valsartan exerted protective effects also when given after established proteinuria. In SHRSP, blockade of AT1 receptor with valsartan prevents the development of proteinuria, delays the appearance of brain damage, preserves renal structure, and increases survival under stressful conditions. Valsartan exerts its beneficial effects independently of any blood pressure fall and by means of broad anti-inflammatory actions both at local and at systemic levels. These observations indicate that the administration of AT1 receptor antagonists may be useful in pathological situations in which an anti-inflammatory effect is required.

Impairment of the fibrinolytic system, caused primarily by increases in the plasma levels of plasminogen activator inhibitor (PAI) type 1, are frequently found in diabetes and the insulin-resistance syndrome. Among the factors responsible for the increases of PAI-1, insulin has recently attracted attention. In this study, we analyzed the effects of insulin on PAI-1 biosynthesis in HepG2 cells, paying particular attention to the signaling network evoked by this hormone. Experiments performed in CHO cells overexpressing the insulin receptor indicate that insulin increases PAI-1 gene transcription through interaction with its receptor. By using inhibitors of the different signaling pathways evoked by insulinreceptor binding, it has been shown that the biosynthesis of PAI-1 is due to phosphatidylinositol (PI) 3-kinase activation, followed by protein kinase C and ultimately by mitogen-activated protein (MAP) kinase activation and extracellular signal-regulated kinase 2 phosphorylation. We also showed that this pathway is Ras-independent. Transfection of HepG2 cells with several truncations of the PAI-1 promoter coupled to a CAT gene allowed us to recognize two major response elements located in the regions between ؊804 and ؊708 and between ؊211 and ؊54. Electrophoretic mobility shift assay identified three binding sites for insulin-induced factors, all colocalized with putative Sp1 binding sites. Using supershifting antibodies, the binding of Sp1 could only be confirmed at the binding site located just upstream from the transcription start site of the PAI-1 promoter. A construct comprising four tandem repeat copies of the ؊93/؊62 region of the PAI-1 promoter linked to CAT was transcriptionally activated in HepG2 cells by insulin. These results outline the central role of MAP kinase activation in the regulation of PAI-1 induced by insulin. Diabetes