Heme induces mRNA expression and activation of tissue factor by TLR4 dependent mechanisms

AbstractIntroductionHemolytic diseases such as Sickle Cell Disease (SCD) are characterized by a natural propensity for both arterial and venous thrombosis. Evidence showing that heme can induce tissue factor (TF) expression in endothelial cells and TF-dependent coagulation activation in animal models of SCD suggest that heme can contribute to hypercoagulability in this condition. We recently demonstrated that heme can induce coagulation activation in whole blood of healthy volunteers in a TF-dependent fashion.MethodsHerein, we aimed to evaluate whether this heme-induced coagulation activity was dependent on the expression and/or activation of hematopoietic TF in human mononuclear cells. TF mRNA expression was evaluated by qPCR and TF procoagulant activity was evaluated using a 2-stage assay based on the generation of FXa.ResultsHeme was capable of inducing TF expression and activation in a TLR4-dependent pathway. This activity was further amplified after TNF-α-priming.ConclusionOur results provide additional evidences on the mechanisms by which heme is involved in the pathogenesis of hypercoagulability in hemolytic diseases.

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Adiponectin inhibits tissue factor expression and enhances tissue factor pathway inhibitor expression in human endothelial cells

Thrombosis and Haemostasis ◽

10.1160/th08-02-0124 ◽

2008 ◽

Vol 100 (08) ◽

pp. 291-300 ◽

Cited By ~ 20

Author(s):

Yi-Jian Chen ◽

Li-Qun Zhang ◽

Guang-Ping Wang ◽

Hui Zeng ◽

Ben Lü ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Expression ◽

Mrna Expression ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Inhibitory Effect ◽

Dependent Manner ◽

Tnf Α ◽

Tissue Factor Pathway ◽

Expression And Activity

SummaryTissue factor (TF) plays a pivotal role in thrombus formation and atherogenesis in acute coronary syndrome. Tissue factor pathway inhibitor (TFPI) is a specific physiological inhibitor of TF/ FVIIa complex that regulates TF-induced coagulation. Adiponectin (Adp) is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Adp inhibits inflammatory cytokine and adhesion molecules expression, and it can prevent endothelial dysfunction. In this study, we investigated the effects of Adp on tumor necrosis factor-α (TNF-α)-induced expression of TF and TFPI in human umbilical vein endothelial cells (HU-VECs), and the signaling transduction pathways involved. It was found that Adp significantly inhibited both TF protein expression and activity in TNF-α-stimulated HUVECs. In the meanwhile, it increased TFPI protein expression and activity for about two folds. Adp also inhibited TF mRNA expression induced by TNF-α, but had no effect on TFPI mRNA expression. The inhibitory effect of Adp onTNF-α-inducedTF expression was prevented by pretreatment with Rp-cAMPs, a PKA inhibitor. Adp increased intracellular cAMP content and PKA activity levels in a dose-dependent manner. Phosphorylation of IκB-α was decreased by Adp, but phosphorylation of p44/42MAPK, SAPK/ JNK, and p38MAPK were not affected. These results suggested that Adp inhibits TF expression through inhibition of a PKA dependent nuclear factor- κB (NF-κB) signaling pathway. It was also found that adiponectin promoted Akt and AMP-activated protein kinase phosphorylation. The inhibitory effect of Adp on TNF-α-induced TF synthesis was abrogated in part by pretreatment with the PI3kinase inhibitor LY 294002, suggesting that Akt activation might inhibit TF expression induced by TNF-α. The inhibitory effect of Adp is almost completely abrogated by inhibition of both the cAMP/PKA pathway and PI3K/Akt pathway. In conclusion, our data indicated that inhibition of NF-κB through stabilization of IκB-α and activation of Akt phosphorylation may mediate the inhibitory effect of Adp on TF expression; but the enhancement effect of Adp on the TFPI production might occur via translational rather than transcriptional regulation.

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Abstract 191: NF-?B p65 Methylation by Protein Arginine Methyltransferase 5 (PRMT5) is Necessary for the Transcriptional Induction of CXCL10 by TNF-a

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a191 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Daniel P Harris

Keyword(s):

Endothelial Cells ◽

Mrna Expression ◽

Chromatin Immunoprecipitation ◽

Proximal Promoter ◽

Protein Arginine Methyltransferase ◽

Arginine Methyltransferase ◽

Expression Of Genes ◽

Tnf Α ◽

Affect Expression ◽

Transcriptional Induction

TNF-α initiates the expression of genes involved in the recruitment, adhesion, and transmigration of leukocytes to sites of inflammation. Here, we report that the protein arginine methyltransferase PRMT5 is required for the transcriptional induction of the pro-inflammatory chemokine CXCL10 (IP-10) in endothelial cells. Depletion of PRMT5 by siRNA results in significantly diminished TNF-α-induced CXCL10 mRNA expression, but does not affect expression of other chemokines, such as MCP-1 or IL-8. Chromatin immunoprecipitation experiments of the CXCL10 proximal promoter show the presence of symmetrical dimethylated arginine (sDMA)-containing proteins upon exposure to TNF-α. This methylation is completely lost when PRMT5 is removed from cells by siRNA. Using immunoprecipitation, we show that PRMT5 enhances CXCL10 expression by methylating the RelA (p65) subunit of NF-κB. In summary, we have identified that PRMT5 is a novel regulator of CXCL10 expression. Further, we have discovered that PRMT5 methylates NF-κB, a finding which may further knowledge of the post-translational code governing NF-κB regulation and target specificity.

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Protocatechuic aldehyde inhibits TNF-α-induced fibronectin expression in human umbilical vein endothelial cells via a c-Jun N-terminal kinase dependent pathway

Experimental and Therapeutic Medicine ◽

10.3892/etm.2015.2896 ◽

2015 ◽

Vol 11 (1) ◽

pp. 277-282 ◽

Cited By ~ 12

Author(s):

YUE-FENG TONG ◽

YONG LIU ◽

ZHI-XING HU ◽

ZHE-CHENG LI ◽

AGULA A

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Human Umbilical Vein ◽

Protocatechuic Aldehyde ◽

Tnf Α ◽

Fibronectin Expression ◽

Umbilical Vein Endothelial Cells ◽

Dependent Pathway

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Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes

Blood ◽

10.1182/blood.v87.1.331.bloodjournal871331 ◽

1996 ◽

Vol 87 (1) ◽

pp. 331-340

Author(s):

WE Kaminski ◽

E Jendraschak ◽

K Baumann ◽

R Kiefl ◽

S Fischer ◽

...

Keyword(s):

Gene Expression ◽

Mrna Expression ◽

Time Course ◽

Cell Activation ◽

Mononuclear Cells ◽

Constitutive Expression ◽

Ionophore A23187 ◽

Mrna Levels ◽

Rt Pcr ◽

Human Mononuclear Cells

Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12- LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

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Visfatin, TNF-α and IL-6 mRNA Expression is Increased in Mononuclear Cells from Type 2 Diabetic Women

Hormone and Metabolic Research ◽

10.1055/s-2007-990288 ◽

2007 ◽

Vol 39 (10) ◽

pp. 758-763 ◽

Cited By ~ 31

Author(s):

P. Tsiotra ◽

C. Tsigos ◽

E. Yfanti ◽

E. Anastasiou ◽

M. Vikentiou ◽

...

Keyword(s):

Mrna Expression ◽

Mononuclear Cells ◽

Tnf Α ◽

Type 2 Diabetic

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Abstract 805: Differential Regulation Of Cytokine-induced Alternative Splicing Of The TF Gene By Serine/Arginine Rich Protein Kinases

Circulation ◽

10.1161/circ.116.suppl_16.ii_155-c ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Ursula Rauch ◽

Andreas Eisenreich ◽

Wolfgang Poller ◽

Heinz-Peter Schultheiss

Keyword(s):

Alternative Splicing ◽

Protein Phosphorylation ◽

Mrna Expression ◽

Tissue Factor ◽

Splice Variant ◽

Sr Protein ◽

Phosphorylation State ◽

Post Induction ◽

Tnf Α ◽

Tf Gene

Background: Higher eukaryotes control gene expression and increase protein diversity by alternative splicing of pre-mRNA. The Cdc2-like kinase (Clk) family, DNA topoisomerase I (DNA topo I) or Akt kinase are involved in splicing control by regulating the phosphorylation state of serine/arginine rich (SR) proteins. We recently showed that alternatively spliced human tissue factor (asHTF), a soluble isoform of tissue factor (TF), the primary initiator of coagulation, is expressed in HUVECs in response to inflammatory cytokines. This study investigated the role of Clks, DNA topo I and the PI3K-Pathway in regulation of TF-splicing in TNF-α induced HUVECs. Methods: HUVECs were incubated with inhibitors of Clks, DNA-topo I or PI3K and were then stimulated with TNF-α. The SR protein phosphorylation state was determined 2 min post induction. The full length (fl) TF and asHTF mRNA were assessed 60 min post induction by Real-Time PCR. Proteins were measured 5 and 8 hours after stimulation by Western blots and the cell thrombogenicity was analyzed via a chromogenic assay. Results: TNF-α inceased the mRNA expression of asHTF and flTF in HUVECs. The Clk-inhibitor completely inhibited the TNF-α induced expression of asHTF and reduced flTF by 30 %. Inhibition of DNA topo I increased asHTF expression and reduced the flTF expression. Inhibition of the PI3K/Akt-pathway had no effect on TF mRNA expression. Reduced Clk-inhibition the TF activity by 50 % whereas DNA topo I inhibition significantly decreased the procoagulant TF activity 8 hours post TNF-α induction. The Clk- and DNA-topo I-inhibitors altered the SR-protein phosphorylation pattern post TNF-α-induction. Additionally resulted inhibition of Clks in the generation of a third TF mRNA-splice variant, TF-A. Conclusion: Selective inhibition of Clks or DNA topo I leads to alterations of SR-protein phosphorylation and affects the differential expression of TF isoforms, thereby modulating the thrombogenicity of HUVECs. The inhibition of Clks contributes to the generation of a third TF splice variant. The inhibition of these kinases gives new insights into the regulation of the TF gene splicing process, which may result in new therapeutic strategies for modulating cellular thrombogenicity.

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Lysophosphatidylcholine Decreases the Synthesis of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614242 ◽

1998 ◽

Vol 79 (01) ◽

pp. 217-221 ◽

Cited By ~ 13

Author(s):

Koichi Kokame ◽

Toshiyuki Miyata ◽

Naoaki Sato ◽

Hisao Kato

Keyword(s):

Endothelial Cells ◽

Mrna Expression ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Umbilical Vein ◽

Mrna Levels ◽

Down Regulation ◽

Human Umbilical Vein ◽

Tissue Factor Pathway ◽

Umbilical Vein Endothelial Cells

SummaryThrombotic complications are frequently associated with atherosclerosis. Lysophosphatidylcholine (LPC), a component accumulated in oxidatively modified LDL (ox-LDL), is known to play a crucial role in the initiation and progression of atherosclerotic vascular lesions. Since a vascular anticoagulant, tissue factor pathway inhibitor (TFPI), has the function of regulating the initial reaction of tissue factor (TF)-induced coagulation, we investigated the effect of LPC on TFPI synthesis in cultured human umbilical vein endothelial cells (HUVEC). The treatment of HUVEC with LPC for 24 h decreased TFPI antigen levels in both the culture medium and the cell lysate in a dose-dependent manner. Northern blot analysis revealed that LPC caused a time-dependent decrease in the TFPI mRNA levels. The levels of TFPI antigen and mRNA were decreased to 72% and 38%, respectively, by the incubation with 50 μM LPC for 24 h. The down-regulation by LPC of TFPI mRNA expression was not observed in the presence of cycloheximide, suggesting that protein synthesis was involved in the suppression of TFPI mRNA expression. The TFPI mRNA levels in actinomycin D-treated cells were relatively stable, indicating that the down-regulation of TFPI mRNA by LPC would be partly explained by the enhanced mRNA destabilization. In contrast to the significant down-regulatory effects of LPC on TFPI expression, LPC did not induce TF mRNA expression in HUVEC. These results indicate that LPC accumulated in the atherosclerotic vascular wall would suppress endothelial TFPI synthesis, reducing the antithrombotic property of endothelial cells.

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Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes

Blood ◽

10.1182/blood.v87.1.331.331 ◽

1996 ◽

Vol 87 (1) ◽

pp. 331-340 ◽

Cited By ~ 12

Author(s):

WE Kaminski ◽

E Jendraschak ◽

K Baumann ◽

R Kiefl ◽

S Fischer ◽

...

Keyword(s):

Gene Expression ◽

Mrna Expression ◽

Time Course ◽

Cell Activation ◽

Mononuclear Cells ◽

Constitutive Expression ◽

Ionophore A23187 ◽

Mrna Levels ◽

Rt Pcr ◽

Human Mononuclear Cells

Abstract Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12- LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

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Gene Expression of Adhesion Molecules in Endothelial Cells from Patients with Peripheral Arterial Disease Is Reduced after Surgical Revascularization and Pharmacological Treatment

International Journal of Vascular Medicine ◽

10.1155/2013/412761 ◽

2013 ◽

Vol 2013 ◽

pp. 1-8 ◽

Cited By ~ 4

Author(s):

Franca Marino ◽

Luigina Guasti ◽

Matteo Tozzi ◽

Laura Schembri ◽

Luana Castiglioni ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Mrna Expression ◽

Adhesion Molecules ◽

Mononuclear Cells ◽

Venous Blood ◽

Statin Treatment ◽

Early Event ◽

Mrna Levels ◽

Peripheral Blood Mononuclear

Atherosclerosis is an inflammatory disease characterized by immunological activity, in which endothelial dysfunction represents an early event leading to subsequent inflammatory vascular damage. We investigated gene expression of the adhesion molecules (AMs) ICAM-1, VCAM-1, andβ1-integrin in endothelial cells (ECs) isolated from venous blood (circulating EC, cEC) and purified from femoral plaques (pEC) obtained from 9 patients with peripheral artery disease (PAD) submitted to femoral artery thrombendarterectomy (FEA). In addition, in peripheral blood mononuclear cells (PBMCs) of the same subjects, we investigated gene expression of IFN-γ, IL-4, TGF-β, and IL-10. Patients were longitudinally evaluated 1 month before surgery, when statin treatment was established, at the time of surgery, and after 2 and 5 months. All AM mRNA levels, measured by means of real-time PCR, in cEC diminished during the study, up to 41–50% of initial levels at followup. AM mRNA expression was significantly higher in pEC than in cEC. During the study, in PBMCs, TGF-βand IL-10 mRNA levels remained unchanged while IFN-γand IL-4 levels increased; however, the ratio IFN-γ/IL-4 showed no significant modification. In PAD patients, FEA and statin treatment induce a profound reduction of AM expression in cEC and affect cytokine mRNA expression in PBMCs.

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