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MicroRNAs (miRNAs) are a class of non-coding RNAs known to repress mRNA translation and subsequent protein production. miRNAs are predicted to modulate many targets and are involved in regulating various cellular processes. Identifying their role in cell function regulation may allow circulating miRNAs to be used as diagnostic or prognostic markers of various diseases. Increasing numbers of clinical studies have shown associations between circulating miRNA levels and platelet reactivity or the recurrence of cardiovascular events. However, these studies differed regarding population selection, sample types used, miRNA quantification procedures, and platelet function assays. Furthermore, they often lacked functional validation of the miRNA identified in such studies. The latter step is essential to identifying causal relationships and understanding if and how miRNAs regulate platelet function. This review describes recent advances in translational research dedicated to identifying miRNAs' roles in platelet function regulation.

Background Several platelet-derived microRNAs are associated with platelet reactivity (PR) and clinical outcome in cardiovascular patients. We previously showed an association between miR-204-5p and PR in stable cardiovascular patients, but data on functional mechanisms are lacking. Aims To validate miR-204-5p as a regulator of PR in platelet-like structures (PLS) derived from human megakaryocytes and to address mechanistic issues. Methods Human hematopoietic stem cells were differentiated into megakaryocytes, enabling the transfection of miR-204-5p and the recovery of subsequent PLS. The morphology of transfected megakaryocytes and PLS was characterized using flow cytometry and microscopy. The functional impact of miR-204-5p was assessed using a flow assay, the quantification of the activated form of the GPIIbIIIa receptor, and a fibrinogen-binding assay. Quantitative polymerase chain reaction and western blot were used to evaluate the impact of miR-204-5p on a validated target, CDC42. The impact of CDC42 modulation was investigated using a silencing strategy. Results miR-204-5p transfection induced cytoskeletal changes in megakaryocytes associated with the retracted protrusion of proPLS, but it had no impact on the number of PLS released. Functional assays showed that the PLS produced by megakaryocytes transfected with miR-204-5p were more reactive than controls. This phenotype is mediated by the regulation of GPIIbIIIa expression, a key contributor in platelet–fibrinogen interaction. Similar results were obtained after CDC42 silencing, suggesting that miR-204-5p regulates PR, at least in part, via CDC42 downregulation. Conclusion We functionally validated miR-204-5p as a regulator of the PR that occurs through CDC42 downregulation and regulation of fibrinogen receptor expression.

MicroRNAs (miRNAs) are small noncoding RNAs modulating protein production. They are key players in regulation of cell function and are considered as biomarkers in several diseases. The identification of the proteins they regulate, and their impact on cell physiology, may delineate their role as diagnostic or prognostic markers and identify new therapeutic strategies. During the last 3 decades, development of a large panel of techniques has given rise to multiple models dedicated to the study of miRNAs. Since plasma samples are easily accessible, circulating miRNAs can be studied in clinical trials. To quantify miRNAs in numerous plasma samples, the choice of extraction and purification techniques, as well as normalization procedures, are important for comparisons of miRNA levels in populations and over time. Recent advances in bioinformatics provide tools to identify putative miRNAs targets that can then be validated with dedicated assays. In vitro and in vivo approaches aim to functionally validate candidate miRNAs from correlations and to understand their impact on cellular processes. This review describes the advantages and pitfalls of the available techniques for translational research to study miRNAs with a focus on their role in regulating platelet reactivity.

Platelet reactivity (PR), a key pharmacodynamic (PD) component of the action of antiplatelet drugs in cardiovascular disease (CVD) patients, is highly variable. PR is associated with occurrence or recurrence of thrombotic and bleeding events, but this association is modulated by several factors. Conventional pharmacogenetics explains a minor part of this PR variability, and among determinants of PR, circulating microRNAs (miRNAs) have been the focus of attention during these last years as biomarkers to predict PR and clinical outcomes in CVD. This being said, the impact of miRNAs on platelet function and the mechanisms behind it are largely unknown. The level of a set of candidate miRNAs including miR-126-3p, miR-150-5p, miR-204-5p and miR-223-3p was quantified in plasma samples of stable CVD patients and correlated with PR as assessed by light-transmission aggregometry and in vivo thrombin generation markers. Finally, miRNA target networks were built based on genes involved in platelet function. We show that all candidate miRNAs were associated with platelet aggregation, while only miR-126-3p and miR-223-3p were positively correlated with in vivo thrombin generation markers. In silico analysis identified putative miRNA targets involved in platelet function regulation. Circulating miRNAs were associated with different aspects of platelet reactivity, including platelet aggregation and platelet-supported thrombin generation. This paves the way to a personalized antithrombotic treatment according to miRNA profile in CVD patients.

Background Platelets are an abundant source of micro-ribonucleic acids (miRNAs) that may play a role in the regulation of platelet function. Some miRNAs, such as miR-126-3p, have been noted as potential biomarkers of platelet reactivity and the recurrence of cardiovascular events. However, the biological relevance of these associations remains uncertain, and the functional validation of candidate miRNAs on human-derived cells is lacking. Objective This article functionally validates miR-126-3p as a regulator of platelet reactivity in platelet-like structures (PLS) derived from human haematopoietic stem cells. Materials and Methods CD34+-derived megakaryocytes were transfected with miR-126-3p and differentiated in PLS. PLS reactivity was assessed using perfusion in a fibrinogen-coated flow chamber. miR-126-3p's selected gene targets were validated using quantitative polymerase chain reaction, protein quantification and a reporter gene assay. Results CD34+-derived megakaryocytes transfected with miR-126-3p generated PLS exhibiting 156% more reactivity than the control. These functional data were in line with those obtained analysing CD62P expression. Moreover, miR-126-3p transfection was associated with the down-regulation of a disintegrin and metalloproteinase-9 (ADAM9) messenger RNA (mRNA), a validated target of miR-126-3p, and of Plexin B2 (PLXNB2) mRNA and protein, an actin dynamics regulator. Silencing PLXNB2 led to similar functional results to miR-126-3p transfection. Finally, using a reporter gene assay, we validated PLXNB2 as a direct target of miR-126-3p. Conclusion We functionally validated miR-126-3p as a regulator of platelet reactivity in PLS derived from human haematopoietic stem cells. Moreover, PLXNB2 was validated as a new gene target of miR-126-3p in human cells, suggesting that miR-126-3p mediates its effect on platelets, at least in part, through actin dynamics regulation.

Aims Tissue-type plasminogen activator (t-PA) is produced by endothelial cells (EC) and is responsible for the removal of intravascular fibrin deposits. We investigated whether expression of t-PA by EC is under epigenetic control. Methods and results Methylation analysis of the proximal t-PA promoter revealed a stretch of unmethylated CpG dinucleotides from position −121 to +59, while upstream CpG dinucleotides were all methylated. In contrast, in human primary hepatocytes, which express t-PA at much lower levels than EC, the proximal promoter was partially methylated. Treatment of EC with the non-specific histone deacetylase (HDAC) inhibitors butyrate and trichostatin and with MS275, a specific inhibitor of class I HDAC, resulted in a time- and dose-dependent increase in t-PA expression. Garcinol and anacardic acid, inhibitors of the histone acetyl transferases CBP/p300 and PCAF, reduced basal and HDAC inhibitor-induced t-PA expression, whereas curcumin, an inhibitor of CBP/p300 only, had no effect. We performed chromosome immunoprecipitation analysis of the t-PA promoter using antibodies specific for acetylated histone H3 or H4 and observed an increase in H3 acetylation of 10 ± 3 and 44 ± 14-fold in EC treated with trichostatin or MS275, respectively, and in H4 acetylation of 7.7 ± 1.4 and 16 ± 3-fold, respectively. Conclusion The proximal t-PA promoter is unmethylated in human EC and partially methylated in human primary hepatocytes. Expression of t-PA by EC is repressed by HDACs in a mechanism that involves de-acetylation of histone H3 and H4

Expression of the tissue-type plasminogen activator gene (t-PA; gene name PLAT) is regulated, in part, by epigenetic mechanisms. We investigated the relationship between PLAT methylation and PLAT expression in five primary human cell types and six transformed cell lines. CpG methylation was analyzed in the proximal PLAT gene promoter and near the multihormone responsive enhancer (MHRE) -7.3 kilobase pairs upstream of the PLAT transcriptional start site (TSS, -7.3 kb). In Bowes melanoma cells, the PLAT promoter and the MHRE were fully unmethylated and t-PA secretion was extremely high. In other cell types the region from -647 to -366 was fully methylated, whereas an unmethylated stretch of DNA from -121 to +94 was required but not sufficient for detectable t-PA mRNA and t-PA secretion. DNA methylation near the MHRE was not correlated with t-PA secretion. Specific methylation of the PLAT promoter region -151 to +151, inserted into a firefly luciferase reporter gene, abolished reporter gene activity. The region -121 to + 94 contains two well-described regulatory elements, a PMA-responsive element (CRE) near -106 and a GC-rich region containing an Sp1 binding site near +59. Methylation of double-stranded DNA oligonucleotides containing the CRE or the GC-rich region had little or no effect on transcription factor binding. Methylated CpGs may attract co-repressor complexes that contain histone deacetylases (HDAC). However, reporter gene activity of methylated plasmids was not restored by the HDAC inhibitor trichostatin. In conclusion, efficient PLAT gene expression requires a short stretch of unmethylated CpG sites in the proximal promoter.

High laminar shear stress (HLSS) is vasculoprotective partly through induction of Kruppel-like factor 2 (KLF2). Connexin37 (Cx37) is highly expressed in endothelial cells (ECs) of healthy arteries, but not in ECs overlying atherosclerotic lesions. Moreover, Cx37 deletion in apolipoprotein E-deficient (ApoE(-/-)) mice increases susceptibility to atherosclerosis. We hypothesized that shear stress, through KLF2 modulation, may affect Cx37 expression in ECs. Cx37 expression and gap-junctional intercellular (GJIC) dye transfer are prominent in the straight portion of carotid arteries of ApoE(-/-) mice, but are reduced at the carotid bifurcation, a region subjected to oscillatory flow. Shear stress-modifying vascular casts were placed around the common carotid artery of ApoE(-/-) mice. Whereas Cx37 expression was conserved in HLSS regions, it was downregulated to ~50% in low laminar or oscillatory flow regions. To study the mechanisms involved, HUVECs or bEnd.3 cells were exposed to flow in vitro. Cx37 and KLF2 expression were increased after 24h of HLSS. Interestingly, shear-dependent Cx37 expression was significantly reduced after silencing of KLF2. Moreover after exposure to simvastatin, a well-known KLF2 inducer, KLF2 binds to the Cx37 promoter region as shown by ChIP. Finally, GJIC dye transfer was highly reduced after KLF2 silencing and was increased after exposure to simvastatin. HLSS upregulates the expression of Cx37 in ECs by inducing its transcription factor KLF2, which increases intercellular communication. Therefore, this effect of shear stress on Cx37 expression may contribute to the synchronization of ECs and participate in the protective effect of HLSS.

Objective Cx40 is a gap junction protein important for cell-cell communication in the endothelium. Polymorphisms in the promoter region of the human Cx40 gene, −44G>A and +71A>G, were shown to reduce Cx40 transcription by half. As mice with an endothelial-specific deletion of Cx40 are more susceptible to atherosclerosis, this study was designed to discover a correlation between these polymorphisms and atherosclerosis in European populations. Methods and results 803 patients referred to the Geneva University Hospitals for elective coronary angiography were divided according to the number of significantly stenosed vessels (from 0 to 3) and were genotyped for the Cx40 polymorphisms. Genotype distribution in the control group was −44GG/+71AA=59.8%, −44AG/+71AG=35.1% and −44AA/+71GG=5.2%. Surprisingly, this distribution was similar in the CAD group, with −44GG/+71AA=58.5%, −44AG/+71AG=37.6% and −44AA/+71GG=3.8% ( p =0.67). Moreover, no significant association between histological carotid plaque composition of culprit lesions and Cx40 polymorphisms could be detected in 583 Dutch patients of the Athero-Express study. Conclusions Despite a clear antiatherogenic role of Cx40 in mice, our study could not detect an association of Cx40 promoter polymorphisms and CAD in human. Moreover, a correlation with atherosclerotic plaque stability or hypertension could not be demonstrated either. Connexin polymorphisms affecting channel function may be of greater importance for cardiovascular disease than polymorphisms affecting the expression level of the protein.

Background: Endothelial progenitor-derived cells (EPC) are a cell therapy tool in peripheral arterial disease and for re-endothelialization of bypasses and stents. Objective: To assess EPC behavior under flow conditions normally found in vivo. Results: EPC were isolated from human cord blood, cultured on compliant tubes and exposed in an in vitro flow system mimicking hemodynamic environments normally found in medium and large arteries. EPC exposed for 24 h to unidirectional (0.3 ± 0.1 or 6 ± 3 dynes/cm2) shear stress oriented along flow direction, while those exposed to bidirectional shear stress (0.3 ± 3 dynes/cm2) or static conditions had random orientation. Under bidirectional flow, tissue factor (TF) activity and mRNA expression were significantly increased (2.5- and 7.0-fold) compared to static conditions. Under low shear unidirectional flow TF mRNA increased 4.9 ± 0.5-fold. Similar flow-induced increases were observed for TF in mature umbilical vein-derived endothelial cells. Expression of tissue-type plasminogen activator (t-PA), urokinase (u-PA) and monocyte chemotactic protein 1 (MCP1) were reduced by 40–60% in late outgrowth endothelial progenitor-derived cells (LO-EPC) exposed to any flow environment, while MCP1, but not t-PA or u-PA, was decreased in HUVEC. Conclusions: Flow, in particular bidirectional, modifies the hemostatic balance in LO-EPC with increased TF and decreased plasminogen activator expression.

The presence of antiphospholipid antibodies (aPLAs) is associated with arterial or venous thrombosis and/or recurrent fetal loss. The proposed pathogenic mechanisms for aPLA effects include the inflammatory activation of monocytes and endothelial cells. Toll-like receptors (TLRs) are candidate signaling intermediates. The aim of this study was to investigate the relative contribution of TLR2 and TLR4 in cell activation by aPLAs. Of 32 patient-derived aPLAs, 19 induced an inflammatory activation of human monocytes and umbilical vein endothelial cells (HUVECs). In HUVECs, inflammatory responses to these aPLAs were increased by TNF pretreatment, which increases the expression of TLR2 but not TLR4. Anti-TLR2 but not anti-TLR4 antibodies reduced the aPLA-induced activation of monocytes and HUVECs. aPLAs activated TLR2-expressing human embryonic kidney 293 (HEK293) cells but not TLR4-expressing cells. Binding studies demonstrated an interaction between aPLAs and TLR2 but not TLR4. A role for CD14, a coreceptor for TLR2 and TLR4, can be inferred by observations that anti-CD14 antibodies reduced responses to aPLAs in monocytes, and that responses in HEK293 cells expressing TLR2 and CD14 were greater than in HEK293 cells expressing TLR2 alone. Our results demonstrate a role for TLR2 and CD14 in human endothelial cell and monocyte activation by aPLAs.

The serine protease tissue-type plasminogen activator (t-PA) is involved in both vital physiological brain processes, such as synaptic plasticity, and pathophysiological conditions, such as neurodegeneration and ischemic stroke. Recent data suggest that epigenetic mechanisms play an important role in the regulation of t-PA in human endothelial cells. However, there are limited data on epigenetic regulation of t-PA in human brain-derived cells. We demonstrate that treatment of cultured human neurons and human astrocytes with the histone deacetylase inhibitors trichostatin A (TSA) and MS-275 resulted in a two- to threefold increase in t-PA mRNA and protein expression levels. Next, we performed a chromatin immunoprecipitation assay on treated astrocytes with antibodies directed against acetylated histones H3 and H4 (both markers of gene activation). Treatment with MS-275 and TSA for 24 hours resulted in a significant increase in H3 acetylation, which could explain the observed increase in t-PA gene activity after the inhibition of histone deacetylation. Furthermore, DNA methylation analysis of cultured human neurons and astrocytes, as well as human postmortem brain tissue, revealed a stretch of unmethylated CpG dinucleotides in the proximal t-PA promoter, whereas more upstream CpGs were highly methylated. Taken together, these results implicate involvement of epigenetic mechanisms in the regulation of t-PA expression in the human brain.

SummaryAntiphospholipid antibodies are a risk factor for venous and arterial thrombosis and may contribute to the development of atherosclerosis. The aim of this study was to investigate whether antibodies to human β2-glycoprotein 1 (β2 GP1), as a model of antiphospholipid antibodies, modify the phenotype of atherosclerotic lesions. LDL receptor-deficient mice were immunized with human β2 GP1, human serum albumin (HSA), or not immunized, and fed a high-cholesterol diet for 14 weeks. Some mice also received pravastatin. Immunization with human β2 GP1 or HSA resulted in formation of autoantibodies recognizing murine β2 GP1 or murine albumin, respectively. We quantified atherosclerotic lesion development and mRNA levels of inflammationassociated proteins in the thoraco-abdominal aorta as well as lesion development, cellular composition and collagen content in the aortic roots. Immunization with β2 GP1 or HSA had no effect on lesion size, but modified the expression in plaque areas of several inflammation-associated proteins. Expression of matrix metalloproteinase-9, tissue factor, interferon-gamma and CD25 was highest in the thoraco-abdominal aorta of β2 GP1-immunized mice, lowest in non-immunized mice and intermediate in HSA-immunized animals. Immunization with β2 GP1, but not HSA, resulted in a lower smooth muscle cell and collagen content of lesions in aortic roots. Statin treatment partially reversed the effects of β2 GP1 immunization. We conclude that immunization with β2 GP1, and to a lesser extent with HSA, leads to modifications in the cellular and protein composition of atherosclerotic plaques, which are associated with a more inflammatory phenotype. Statin treatment partially prevents these changes.

Peripheral blood– derived endothelial progenitor cells (EPCs) have considerable potential for the autologous therapy of vascular lesions or ischemic tissues. By introducing stable genetic modifications into these cells, this potential might be further enhanced. We investigated to what extent transgene expression can be controlled by using different transgene promoters. This was investigated in early- or late-outgrowth human EPCs obtained by culturing blood mononuclear cells for 1 or 4 weeks on type 1 collagen in medium containing endothelial growth supplements. A large fraction of these cells were stably transduced using lentiviral vectors for expression of the enhanced green fluorescent protein (EGFP). Transgene expression in vitro or in vivo after injection into nude mice was highest when under the control of the cytomegalovirus (CMV) promoter, intermediate with the EF1α promoter, and lowest with the phosphoglycerate kinase promoter. When blood mononuclear cells were cultured for 1 week in the absence of endothelial growth supplements, CMV promoter– driven expression of EGFP was two orders of magnitude lower than in similarly transduced EPCs. Our results show that lentiviral vectors are useful tools for the stable introduction of exogenous genes into EPCs and for their expression at desired levels using the appropriate gene promoter.

Summary. Background: Inhibitors of HMG-CoA reductase are widely used to prevent atherosclerosis progression. The expression of adhesion molecules on activated endothelial cells (EC) is an important step in the initiation and progression of atherosclerosis. Objectives: We investigated whether adhesion molecule expression on activated EC is influenced by simvastatin, fluvastatin and pravastatin and, if so, by which mechanisms. Methods: Human EC from umbilical veins or saphenous veins were pretreated overnight with statins with or without mevalonate, and also for simvastatin or fluvastatin with the isoprenoid intermediates, farnesyl pyrophosphate (FPP), or geranylgeranyl pyrophosphate (GGPP). After 4–6 h activation with tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS), surface adhesion molecule expression was evaluated by ELISA and by flow cytometry. The same experiments were performed with selective inhibitors of geranylgeranyltransferase (GGTI-286) and farnesyltransferase (FTI-277). Results: Pretreatment with simvastatin, fluvastatin or pravastatin potentiated the TNF-α and LPS-induced expression of E-selectin and VCAM-1, and mevalonate reversed the potentiating effect of these statins. GGPP also reversed the potentiating effect of simvastatin or fluvastatin on adhesion molecule expression, while FPP only partially reversed this effect. Furthermore, GGTI-286, but not FTI-277, mimicked the effect of simvastatin by increasing the TNF-α-mediated overexpression of E-selectin. Conclusions: Statins increase E-selectin- and VCAM-1-induced expression on vascular endothelial cells stimulated with TNF-α or LPS. The inhibition of geranylgeranylated proteins could contribute to this effect.

SummaryAntiphospholipid antibodies (aPLA) are associated with thrombophilia and recurrent pregnancy loss. They bind directly to anionic phospholipids or via phospholipid-binding proteins such as β2-glycoprotein 1 (β2GP1). The underlying mechanisms by which aPLA induce a thrombophilic phenotype are not well understood.The present work was done to determine whether antibodies to β2GP1 activate endothelial cells (EC) and whether NFκB is involved in this activation. Incubation of EC with these antibodies resulted in a redistribution of NFκB from the cytoplasm to the nucleus after a delay of several hours. This was accompanied by an increased expression of tissue factor and of the leukocyte adhesion molecules ICAM-1, VCAM-1 and E-selectin. Inhibition of the nuclear translocation of NFκB abolished the response to these antibodies. In comparison to anti-β2GP1 antibodies, incubation of EC with TNF resulted in a more rapid (within 30 minutes) redistribution of NFκB and a more pronounced expression of tissue factor and of the leukocyte adhesion molecules. The slower response to the antibodies as compared to TNF suggests that the NFκB response to anti-β2GP1 antibodies is indirect.Taken together our results imply that NFκB is an essential intermediate in the activation of EC by anti-β2GP1 antibodies.

SummaryAntiphospholipid antibodies (APLA) are associated with thrombophilia and recurrent pregnancy loss. Different mechanisms have been proposed to explain their pathogenic effects and among them, we have previously shown that APLA accumulate in late endosomes of human umbilical vein endothelial cells (HUVEC) leading to a redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR). Because many APLA are directed towards β2-glycoprotein 1 (β2GP1)-phospholipid complexes, we investigated the localisation of β2GP1 in HUVEC. By immunofluorescence analysis, using monoclonal and polyclonal anti- β2GP1 antibodies, we detected β2GP1 at the cell surface and in late endosomes. Incubation of HUVEC with anti- β2GP1 antibodies resulted in antibody accumulation at the cell surface and within late endosomes and in a redistribution of the CI-M6PR from the Golgi apparatus to late endosomes. The anti- β2GP1 antibodies remained detectable in late endosomes even after several days of incubation in antibody-free medium. The accumulation of anti- β2GP1 antibodies in late endosomes of endothelial cells and the resulting modification of intracellular protein trafficking may contribute to the pathogenic effects of these antibodies.

Hornbeam (Carpinus sp.) flowering occurs generallysimultaneously with birch anthesis. While birch pollenis known as the major tree-allergen in central andnorthern Europe, the quantitative and qualitative roleof hornbeam pollen in allergy has not been evaluated.In some regions of Switzerland, the annual ratiohornbeam SPI / birch SPI reaches 20–30% on average,and 50–180% some years. The relative importance ofhornbeam pollen hypersensitivity has been evaluated inmore than 200 patients suffering from spring allergiesto pollen during the last five years (1993–1997) inthe region of Geneva. Among symptomatic patientsduring the March-April period, 73.5% werehypersensitive to Betulaceae pollen. Overall, 71.7%of the patients allergic to Betulaceae pollen werefound to be sensitive to hornbeam, and 12.5% of thesame group of patients were sensitive to hornbeampollen only. Among this last group, half of thepatients, besides allergic rhino-conjunctivitis, werealso suffering from asthma. Hornbeam pollen isabundant and appears to have a significativeimportance in spring allergies. In some cases,specific immunotherapy to hornbeam pollen shouldperhaps be considered.

SummaryStatins are cholesterol-lowering drugs that exert pleiotropic effects which include changes in the plasminogen activation (PA) system of endothelial cells (EC). It was the objective of this study to investigate the signal transduction pathways by which statins increase the expression of tissue-type PA (t-PA) and decrease PA inhibitor type 1 (PAI-1) in human umbilical vein EC. Fluvastatin treatment increased t-PA expression more than 10-fold and reduced PAI-1 expression up to fivefold. This effect was mimicked by geranylgeranyl transferase inhibition. The role of geranylgeranylated small G-proteins of the Rho family was assessed by adenovirus-mediated expression of dominant negative (DN) RhoA, Cdc42 and Rac1 and by siRNA-mediated suppression of these proteins. DN-Cdc42 and DN-Rac1, as well as siRNA for Cdc42, increased t-PA expression, while DN-RhoA and DN-Rac1 decreased PAI-1 expression. Latrunculin B, an inhibitor of actin polymerisation, in-creased t-PA mRNA and reduced PAI-1 mRNA to the same extent as fluvastatin. Inhibition of p38, as well as p38α or p38β siRNA, reversed the effects of fluvastatin on t-PA expression. Treatment with p38β siRNA partially reversed the effect of fluvastatin on PAI-1, whereas p38α siRNA had no significant effect. Inhibition of jun kinase reduced basal and fluvastatin-induced t-PA expression to the same extent and increased PAI-1. MEK/ERK inhibition had no effect. In human EC, the fluvastatin-induced increase in t-PA is mediated by Cdc42 and, as with t-PA induced by inhibition of actin polymerisation, requires activation of p38MAP kinase. The mechanisms by which fluvastatin treatment reduces PAI-1 are different from those that increase t-PA.

Inflammatory activation of the vascular endothelium is a major contributory factor to ischemic cardiovascular disease. Endothelial progenitor cells (EPCs) are being investigated for the treatment of ischemic disease or to coat vein grafts for bypass surgery. As an inflammatory environment might reduce their therapeutic efficacy, we sought to generate EPCs that are less sensitive to inflammatory activation. EPCs were obtained from human umbilical cord blood and transduced with a lentiviral vector for stable expression of A20, an anti-inflammatory protein. Nontransduced and green-fluorescent-protein-transduced cells were used as controls. Expression of A20 by EPCs did not modify cell morphology or expression of a panel of 20 proteins known to contribute to angiogenesis. Also, A20 had no effect on the capacity of EPCs to form tube-like structures in Matrigel™. A20 expression reduced EPC activation by tumor necrosis factor-α and interleukin-1β as determined from changes in vascular cell adhesion molecule 1 and E-selectin expression and decreased monocyte transmigration through a monolayer of EPCs. In conclusion, EPCs can be genetically modified to overexpress A20 in a stable fashion. These cells become less sensitive to inflammatory stimuli. This may be of interest in cell-based therapeutic approaches for clinical settings where inflammation is an important pathogenic factor.

Vessel wall trauma induces vascular remodeling processes including the development of intimal hyperplasia (IH). To assess the development of IH in human veins, we have used an ex vivo vein support system (EVVSS) allowing the perfusion of freshly isolated segments of saphenous veins in the presence of a pulsatile flow which reproduced arterial conditions regarding shear stress, flow rate and pressure during a period of 7 and 14 days. Compared to the corresponding freshly harvested human veins, histomorphometric analysis showed a significant increase in the intimal thickness which was already maximal after 7 days of perfusion. Expression of the endothelial marker CD31 demonstrated the presence of endothelium up to 14 days of perfusion. In our EVVSS model, the activity as well as the mRNA and protein expression levels of plasminogen activator inhibitor 1, the inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), were increased after 7 days of perfusion, whereas the expression levels of tPA and uPA were not altered. No major change was observed between 7 and 14 days of perfusion. These data show that our newly developed EVVSS is a valuable setting to study ex vivo remodeling of human veins submitted to a pulsatile flow.

Background Toll-like receptors (TLR) recognize pathogen-associated molecular patterns and are important mediators of the innate immune system. TLR1 and TLR6 are paralogs and located in tandem on the same chromosome in mammals. They form heterodimers with TLR2 and bind lipopeptide components of gram-positive and gram-negative bacterial cell walls. To identify conserved stretches in TLR1 and TLR6, that may be important for their function, we compared their protein sequences in nine mammalian species(Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus; Erinaceus europaeus, Bos Taurus, Sus scrofa and Canis familiaris). Results The N-terminal sequences of the orthologous proteins showed greater similarity than corresponding paralog sequences. However, we identified a region of 300 amino acids towards the C-terminus of TLR1 and TLR6, where paralogs had a greater degree of sequence identity than orthologs. Preservation of DNA sequence identity of paralogs in this region was observed in all nine mammalian species investigated, and is due to independent gene conversion events. The regions having undergone gene conversion in each species are almost identical and encode the leucine-rich repeat motifs 16 to 19, the C-terminal cap motif, the transmembrane domain and most of the intracellular Toll/interleukin-1 receptor (TIR) domain. Conclusion Our results show that, for a specific conserved region, divergence of TLR1 and TLR6 is limited by gene conversion, most likely because of the need for co-evolution with multiple intracellular and extracellular binding partners. Thus, gene conversion provides a mechanism for limiting the divergence of functional regions of protein paralogs, while allowing other domains to evolve diversified functions.

Regulated secretion of EC (endothelial cell) vWF (von Willebrand factor) is part of the haemostatic response. It occurs in response to secretagogues that raise intracellular calcium or cAMP. Statins are cholesterol-lowering drugs used for the treatment of cardiovascular disease. We studied the effect of fluvastatin on regulated secretion of vWF from HUVEC (human umbilical-vein ECs). Secretion in response to thrombin, a protease-activated receptor-1 agonist peptide, histamine, forskolin and adrenaline (epinephrine) was inhibited. This inhibition was reversed by mevalonate or geranylgeranyl pyrophosphate, and mimicked by a geranylgeranyl transferase inhibitor, demonstrating that the inhibitory mechanism includes inhibition of protein geranylgeranylation. To investigate this mechanism further, calcium handling and NO (nitric oxide) regulation were studied in fluvastatin-treated HUVEC. Intracellular calcium mobilization did not correlate with vWF secretion. Fluvastatin increased eNOS [endothelial NOS (NO synthase)] expression, but NOS inhibitors failed to reverse the effect of fluvastatin on vWF secretion. Exogenous NO did not inhibit thrombin-induced vWF secretion. Many small GTPases are geranylgeranylated and some are activated by secretagogues. We overexpressed DN (dominant negative) Rho GTPases, RhoA, Rac1 and Cdc42 (cell division cycle 42), in HUVEC. DNCdc42 conferred inhibition of thrombin- and forskolin-induced vWF secretion. We conclude that, via inhibition of protein geranylgeranylation, fluvastatin is a broadspectrum inhibitor of regulated vWF secretion. Geranylgeranylated small GTPases with functional roles in regulated secretion, such as Cdc42, are potential targets for the inhibitory activity of fluvastatin.

Summary. Background: Blood-derived endothelial progenitor cells (EPC) have been used to treat ischemic disease. However, the number of EPC that can be obtained from adult blood is limited. Objective: To characterize endothelial-like cells obtained from human bone marrow and determine their ability to stimulate new blood vessel formation in vivo. Methods: Mononuclear cells (MNC) were isolated from human bone marrow or umbilical cord blood and cultured in endothelial growth medium (EGM-2). Mesenchymal stem cells (MSC) were isolated from bone marrow and induced to differentiate into endothelial-like cells (MSCE), or adipocytes or osteocytes by growth in EGM-2, adipogenic or osteogenic medium. Results: Cells obtained by culturing bone marrow MNC in EGM-2 formed cord- or tube-like structures when grown on MatrigelTM and expressed several endothelial marker proteins. However, cell morphology and the profile of endothelial marker protein expression were different from those of cord blood-derived EPC (cbEPC). Cells with a similar phenotype were obtained by differentiation of MSC into MSCE, which was accompanied by an increase of endothelial marker proteins and a diminished capacity to differentiate into adipocytes. Subcutaneous implantation of MSCE in collagen plugs in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice resulted in formation of functional blood vessels that had incorporated the MSCE. Conclusions: Our results show that MSCE and cbEPC are different cell types. The formation of functional blood vessels by MSCE, combined with high yields and a reduced capacity to differentiate into other cell types compared with MSC, makes these cells potentially useful for autologous therapy of ischemic disease.

Antiphospholipid antibodies (APLAs) promote inflammatory and procoagulant responses in endothelial cells and monocytes. Previous studies have shown that MyD88, TRAF6, and NF-κB mediate cell activation by APLAs. These intermediates are also used by toll-like receptors (TLRs). We investigated the role of TLRs in the cellular response to APLAs. IgGs were isolated from the plasma of 5 patients with antiphospholipid syndrome along with immunopurified anti–β2-glycoprotein 1 IgG from a sixth patient. Control IgG was obtained from a pool of healthy donor plasmas negative for APLAs. Wild-type mouse embryonic fibroblasts (EFs) and EFs deficient in TLR1, TLR2, TLR4, or TLR6 were incubated with APLAs, anti–β2-glycoprotein 1 IgG, or control IgG. On incubation with the patient IgG, but not control IgG, a significant increase in mRNA levels of the inflammatory marker proteins MCP-1, ICAM-1, and IL-6 as well as IL-6 secretion was observed in wild-type EFs, whereas TLR2-deficient EFs did not respond. Responses in TLR1- and TLR6-deficient EFs were decreased and those in TLR4-deficient EFs comparable to those in wild-type EFs. Overexpression of human TLR2 in the TLR2-deficient EFs restituted the response to patient IgG. Our results imply that TLR2 plays a role in mouse fibroblast activation by APLAs.

SummaryThe presence of antiphospholipid antibodies (APLA) is associated with an increased risk of recurrent thrombosis and pregnancy loss. APLA are able to activate endothelial cells (EC) and induce an increase in the expression of inflammatory marker proteins, such as leukocyte adhesion molecules, tissue factor or the monocyte chemoattractant protein-1 (MCP-1). Our objective was to investigate the effect of statins on EC activation induced by APLA in vitro. IgG was purified from the plasma of six patients with APLA and from healthy controls. EC were incubated with patient IgG or with control IgG, in the presence or absence of 5μM of fluvastatin, and expression of the leukocyte adhesion molecules, VCAM-1 and E-selectin, analyzed by flow cytometry and by quantitative reverse transcriptase-PCR (QRT-PCR).The expression of tissue factor and the chemokine MCP-1 was analyzed by QRT-PCR alone. Incubation of EC with patient IgG increased the expression of VCAM-1, E-selectin, tissue factor and MCP-1. Prior treatment of the cells with fluvastatin further increased the expression of these proteins. The fluvastatin effect was reversed by co-incubation with mevalonate or geranylgeranylpyrophosphate and mimicked by the geranylgeranyl transferase inhibitor GGTI-286. Our results show that in cultured human EC, statins increase the extent of inflammatory activation induced by APLA. This effect appears to be mediated by an inhibitory effect of statins on one or more geranylgeranylated protein(s).

Summary. Background : Antiphospholipid antibodies (APLA) have been shown to activate endothelial cells (EC) in vitro, as documented by an increased expression of tissue factor as well as leukocyte adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin. Currently, treatment of patients with the antiphospholipid syndrome includes aspirin, particularly for women with recurrent fetal loss. Objective : The present study was undertaken to investigate whether aspirin interferes with EC activation induced by APLA in vitro. Methods : IgG from 14 patients with APLA, and suffering from thrombotic complications and/or pregnancy morbidity, and control IgG were tested for their ability to modify the expression of VCAM-1 in human umbilical vein endothelial cells. VCAM-1 antigen was measured by flow cytometry and its mRNA by quantitative reverse transcriptase-polymerase chain reaction. Results : Incubation of EC with IgG from most of the patients led to a higher VCAM-1 expression compared with incubation with control IgG. The effect of aspirin was studied for the eight IgG samples that induced a more than 50% increase in VCAM-1. Aspirin (10 mm) treatment of the cells significantly reduced the VCAM-1 response to these APLA. Conclusions : Our results indicate that besides its antiplatelet properties, aspirin exerts a protective effect towards APLA at the EC level by decreasing leukocyte adhesion molecule expression at the cell surface.

Antiphospholipid antibodies (aPLA) are associated with thrombophilia and recurrent pregnancy loss. They bind directly to anionic phospholipids or via phospholipid-binding proteins such as beta(2)-glycoprotein 1 (beta(2)GP1). The underlying mechanisms by which aPLA induce a thrombophilic phenotype are not well understood. The present work was done to determine whether antibodies to beta(2)GP1 activate endothelial cells (EC) and whether NFkappaB is involved in this activation. Incubation of EC with these antibodies resulted in a redistribution of NFkappaB from the cytoplasm to the nucleus after a delay of several hours. This was accompanied by an increased expression of tissue factor and of the leukocyte adhesion molecules ICAM-1, VCAM-1 and E-selectin. Inhibition of the nuclear translocation of NFkappaB abolished the response to these antibodies. In comparison to anti-beta(2)GP1 antibodies, incubation of EC with TNF resulted in a more rapid (within 30 minutes) redistribution of NFkappaB and a more pronounced expression of tissue factor and of the leukocyte adhesion molecules. The slower response to the antibodies as compared to TNF suggests that the NFkappaB response to anti-beta(2)GP1 antibodies is indirect. Taken together our results imply that NFkappaB is an essential intermediate in the activation of EC by anti-beta(2)GP1 antibodies.

Idiopathic thrombocytopenic purpura (ITP) is characterized by the presence of autoantibodies reacting with platelet-specific antigens resulting in thrombocytopenia. In the antiphospholipid antibody syndrome (APS), clinical manifestations are associated with the presence of antiphospholipid

The characteristic hemorrhages of acute promyelocytic leukemia (APL) are caused in part by the high expression of tissue factor (TF) on leukemic cells, which also produce TNF and IL-1β, proinflammatory cytokines known to increase TF in various cell types. Exposure of NB4 cells, an APL cell line, to all-trans retinoic acid (ATRA) or arsenic trioxide (ATO) rapidly and strongly reduced TF mRNA. Both drugs also reduced TNF mRNA, but later, and moreover increased IL-1β mRNA. The effect on procoagulant activity of cells and microparticles, as measured with calibrated automated thrombography, was delayed and only partial at 24 h. TNF and IL-1β inhibition reduced TF mRNA and activity only partially. Inhibition of the inflammatory signaling intermediate p38 reduced TF mRNA by one third but increased TNF and IL-1β mRNA. NF-κB inhibition reduced, within 1 h, TF and TNF mRNA but did not change IL-1β mRNA, and rapidly and markedly reduced cell survival, with procoagulant properties still being present. In conclusion, although we provide evidence that TNF, IL-1β, and their signaling intermediates have a regulatory function on TF expression by NB4 APL cells, the effect of ATRA and ATO on TF can only partially be accounted for by their impact on these cytokines. Electronic supplementary material The online version of this article (doi:10.1007/s00277-017-2970-5) contains supplementary material, which is available to authorized users.