Tissue Factor mRNA Research Articles

(−)-Epigallocatechin-3-gallate decreases thrombin/paclitaxel-induced endothelial tissue factor expression via the inhibition of c-Jun terminal NH2 kinase phosphorylation

Patients with paclitaxel-eluting stents are concerned with stent thrombosis caused by premature discontinuation of dual antiplatelet therapy or clopidogrel resistance. This study investigates the effect of (−)-epigallocatechin-3-gallate (EGCG) on the expression of thrombin/paclitaxel-induced endothelial tissue factor (TF) expressions in human aortic endothelial cells (HAECs). EGCG was nontoxic to HAECs at 6h up to a concentration of 25μmol/L. At a concentration of 25μmol/L, EGCG pretreatment potently inhibited both thrombin-stimulated and thrombin/paclitaxel-stimulated endothelial TF protein expression. Thrombin and thrombin/paclitaxel-induced 2.6-fold and 2.9-fold increases in TF activity compared with the control. EGCG pretreatment caused a 29% and 38% decrease in TF activity on thrombin and thrombin/paclitaxel treatment, respectively. Real-time polymerase chain reaction (PCR) showed that thrombin and thrombin/paclitaxel-induced 3.0-fold and 4.6-fold TF mRNA expressions compared with the control. EGCG pretreatment caused an 82% and 72% decrease in TF mRNA expression on thrombin and thrombin/paclitaxel treatment, respectively. The c-Jun terminal NH2 kinase (JNK) inhibitor SP600125 reduced thrombin/paclitaxel-induced TF expression. Furthermore, EGCG significantly inhibited the phosphorylation of JNK to 49% of thrombin/paclitaxel-stimulated HAECs at 60min. Immunofluorescence assay did not show an inhibitory effect of EGCG on P65 NF-κB nuclear translocation in the thrombin/paclitaxel-stimulated endothelial cells. In conclusion, EGCG can inhibit TF expression in thrombin/paclitaxel-stimulated endothelial cells via the inhibition of JNK phosphorylation. The unique property of EGCG may be used to develop a new drug-eluting stent by co-coating EGCG and paclitaxel.

Four SR Proteins Play Opposing Roles in the Regulated Biosynthesis of Tissue Factor in Human Monocytic Cells.

Abstract 2128Poster Board II-105Background. Circulating Tissue Factor (TF) is a major contributor to the etiology of thrombotic disorders. Blood monocytes are the primary source of circulating TF, which they express in two forms: full length TF (flTF), a transmembrane surface protein, and alternatively spliced TF (asTF), a secreted soluble protein. The presence or absence of the internal cassette exon 5 in the TF mRNA determines whether the encoded protein is flTF or asTF, respectively. While the procoagulant potential of flTF vastly exceeds that of asTF when assessed using conventional static assays, asTF exhibits unique angiogenic properties distinct from flTF. Investigation of the molecular mechanisms governing TF exon 5 processing is of much interest as it may afford novel approaches to modulate monocyte-mediated blood thrombogenicity as well as monocyte-induced angiogenesis. Using a splicing reporter system developed to study the processing of TF pre-mRNA, we previously determined that SR proteins ASF/SF2 and SRp55 are vital for TF exon 5 inclusion in human monocytes (Tardos et al, J Thromb Haemost 6:877-884, 2008). In the course of more recent experiments, we found that in contrast to ASF/SF2 and SRp55, two other SR proteins – SC35 and SRp40 – appear to promote exclusion of this variable exon: weakening of the binding motifs for SC35 (sites 33 and 81) and SRp40 (site 44), whose positions in exon 5 overlap with the binding sites for ASF/SF2 and SRp55, results in the decrease of the splicing event unique to asTF. The competition of these four SR proteins for binding to their overlapping binding sites may thus play a role in the maintenance of the asTF / flTF ratio; however, a physical association of SC35 and SRp40 with their putative sites in exon 5 was not demonstrated. Objective. To determine whether SC35 and/or SRp40 physically associate with their putative binding sites. Results. To evaluate interaction of the SR proteins SC35 and SRp40 with their putative binding sites in TF exon 5, we employed RNA mobility shift assay methodology using freshly prepared nuclear extracts of THP-1 cells, a monocytic cell line, and in vitro transcribed, uniformly labeled RNA probes comprising the two regions of interest – one spanning SC35 site 33 and SRp40 site 44, and the other spanning the SC35 site 81. As expected, these RNA probes yielded reproducible band shift products, confirming RNA-protein complex formation. We then developed three counterpart mutant RNA probes in which the SC35 and SRp40 binding motifs were selectively weakened by targeted site-directed mutagenesis, and performed RNA gel shift assays alongside the corresponding wild-type probes. Each mutant probe exhibited a significantly weaker interaction with the SR proteins compared to its wild-type counterpart: the SC35 site 33 mutant probe produced a 31.1% reduction in complex formation relative to wild-type (p = 0.011), the SRp40 site 44 mutant probe also produced a 31.1% reduction in complex formation relative to wild-type (p = 0.0001), and the SC35 site 81 mutant probe produced a 33.0% reduction in complex formation relative to wild-type (p = 0.0151). Conclusions. We show for the first time that SR proteins SC35 and SRp40 physically associate with functional binding sites within TF exon 5. The SC35 and SRp40 binding sites overlap with the binding sites for ASF/SF2 and SRp55, the SR proteins that promote exon 5 inclusion. The opposing effects of distinct SR proteins on TF exon 5 processing reveal a heretofore unknown mechanism governing regulated TF biosynthesis. Further studies of SR protein-mediated effects on the TF profile of human monocytes are likely to aid in the development of novel therapeutic strategies aimed at selective targeting of biologically distinct TF forms. Disclosures:No relevant conflicts of interest to declare.

Opposing and uncoupling effects of mTOR and S6K1 in the regulation of endothelial tissue factor expression

Rapamycin has been reported to enhance tissue factor (TF) expression. The present study investigated roles of mammalian target of rapamycin (mTOR) and its downstream S6K1 in this process. We showed here that, consistent with rapamycin, knocking-down mTOR enhanced thrombin-induced TF mRNA and protein levels, whereas silencing S6K1 mitigated up-regulation of TF protein but not TF mRNA level. The enhanced TF protein level upon mTOR-silencing was further augmented by over-expression of a constitutively active S6K1 mutant and reduced by blocking RhoA, p38 mapk or NF-κB. The results reveal an opposing and uncoupling effect of mTOR and S6K1 in regulating TF expression.

Increased Tissue Factor Expression Is Associated with Reduced Survival in Non–Small Cell Lung Cancer and with Mutations of TP53 and PTEN

Tissue factor (TF), the main initiator of blood coagulation, is also a signaling protein that regulates cancer progression. TF synthesis was recently shown to be affected by tumor suppressor genes (TSGs) in tumor cell lines. We therefore studied TF gene (F3) expression and the status of genes coding for tumor protein p53 (TP53), phosphatase and tensin homolog (PTEN), and serine/threonine kinase 11 (STK11) in non-small cell lung cancer (NSCLC). Heparanase (HPSE) gene expression was also measured because this endo-beta-D-glucuronidase was recently shown to enhance TF gene expression. TF and heparanase mRNA expression was measured by real-time PCR in 53 NSCLC tumors. Exons 5-8 of TP53 were sequenced from genomic DNA. Mutations of PTEN and STK11 were screened by multiplex ligation-dependent probe amplification. TF mRNA levels were significantly higher in T(3)-T(4) tumors (P = 0.04) and in stages III-IV of NSCLC (P = 0.03). Mutations of TP53, STK11, and PTEN were identified in 20 (37.7%), 21 (39%), and 20 (37.7%) of tumors, respectively. TF expression was higher in mutated TP53 (TP53(Mut)) (P = 0.02) and PTEN(Mut) (P = 0.03) samples. Moreover, TF mRNA increased from 2700 copies (no mutation) to 11 6415 when 3 TSG were mutated. Heparanase gene expression did not differ according to TF gene (F3) expression or TSG mutation. The median survival time was shorter in patients with tumor TF mRNA levels above median values (relative risk 2.2; P = 0.03, multivariate analysis) and when TP53 was mutated (relative risk 1.8; P = 0.02). These results provide clear evidence that combined oncogene events affecting TSG dramatically increase TF gene expression in lung tumors. Moreover, this study suggests that TF gene expression could be used as a prognostic marker in NSCLC.

PDGF-CC induces tissue factor expression: role of PDGF receptor α/β

Tissue factor (TF) is the principal trigger of the coagulation cascade and involved in arterial thrombus formation. Platelet-derived growth factor CC (PDGF-CC) is a recently discovered member of the PDGF family released upon platelet activation. This study assesses the impact of PDGF-CC on TF expression in human cells. PDGF-CC concentration-dependently induced TF expression by 2.5-fold in THP-1 cells, by 2.0-fold in human peripheral blood monocytes, by 1.4-fold in vascular smooth muscle cells, and by 2.6-fold in microvascular endothelial cells, but did not affect TF expression in aortic endothelial cells. A similar pattern was observed with PDGF-BB. In contrast, PDGF-AA did not alter TF expression in THP-1 cells. TF whole cell activity was induced following stimulation with PDGF-BB and PDGF-CC in THP-1 cells. Real-time polymerase chain reaction revealed that PDGF-CC induced TF mRNA. PDGF-CC transiently activated p42/44 MAP kinase [extracellular signal-regulated kinase (ERK)], while phosphorylation of the MAP kinases c-Jun NH(2)-terminal kinase (JNK) and p38 remained unaffected. PD98059, a specific inhibitor of ERK phosphorylation, but not the p38 inhibitor SB203580 or the JNK inhibitor SP600125 prevented PDGF-CC induced TF expression in a concentration-dependent manner. The effect of PDGF-CC was antagonized by both PDGF receptor alpha and PDGF receptor beta neutralizing antibodies; in contrast, PDGF-BB was only inhibited by PDGF receptor beta blocking antibody. PDGF receptor alpha and PDGF receptor beta inhibition prevented PDGF-CC-induced ERK phosphorylation. PDGF-CC induces TF expression via activation of alpha/beta receptor heterodimers and an ERK-dependent signal transduction pathway.

Mechanical Stimulation of HUVEC: Different Flow Patterns have Differential Effects on TF Expression

Human umbilical vein endothelial cells (HUVECs) are known to respond differentially to various mechanical stimuli, and these different responses maybe involved in atherogenesis. Here we investigated the differential effects of different flow patterns, such as laminar flow (LF) and orbital shear stress (OSS), on the expression of tissue factor (TF). HUVECs were seeded on fibronectin coated glass slides and selectively in the periphery of gelatin coated culture dishes. When HUVECs on slides reached confluence, they were exposed to two, four, or six hours of LF (14 dynes/cm2). HUVECs in culture dishes were exposed to the same time course of low OSS (4 dynes/cm2) or high OSS (11 dynes/cm2). OSS was generated by placing dishes on an orbital shaker. TF mRNA levels were quantified with reverse transcription polymerase chain reaction (RT-PCR), with b-actin as the reference gene. Fold changes are expressed as the mean ± SE. Statistical significance between treated groups and controls was evaluated using the Mann-Whitney test. Analysis of variance (ANOVA) and Tamhane's post-hoc analysis were employed for between group analysis and P < .05 was considered significant. Fold changes as compared with controls are summarized in the Table and Fig (P values are in brackets). LF resulted in significant increases in TF expression throughout the six-hour time course when compared with static controls. However no significant differences were noted between the two, four, and six-hour LF groups. Both HOSS and LOSS reduced TF mRNA levels with significance attained at four and six hours respectively. No significant differences were observed between the different OSS groups.TableTF expression fold changes compared with controlsN = 6HOSSLOSSLF2 hrs1.17 ± 0.15 (.80)1.34 ± 0.37 (.21)10.44 ± 6.63 (.002)4 hrs0.67 ± 0.05 (<.001)0.64 ± 0.1 (.08)9.34 ± 3.8 (.002)6 hrs0.73 ± 0.15 (.07)0.69 ± 0.18 (.03)5.00 ± 1.32 (.056)HOSS, High orbital sheer stress; LF, laminar flow; LOSS, low orbital sheer stress; TF, tissue factor. Open table in a new tab HOSS, High orbital sheer stress; LF, laminar flow; LOSS, low orbital sheer stress; TF, tissue factor. LF induced a significant increase of TF mRNA levels. Interestingly both high and low levels of OSS seemed to decrease TF expression after two hours. These data demonstrate that different flow patterns have differential effects on the expression of TF in HUVEC, indicating the involvement of different intracellular signaling pathways. Current efforts are focused on the interaction of flow patterns with chemical stimulation, as a means of elucidating the intracellular signal conduction pathway.

In Vitro Effects of Antiphospholipid Syndrome‐IgG Fractions and Human Monoclonal Antiphospholipid IgG Antibody on Human Umbilical Vein Endothelial Cells and Monocytes

It has been shown that stimulation of endothelial cells and monocytes by antiphospholipid antibodies leads to a prothrombotic state involving upregulation of tissue factor (TF). We examined the in vitro effects of IgG fractions from patients with antiphospholipid syndrome (APS) and of a beta-2-glycoprotein 1-independent human monoclonal antiphospholipid antibody (HL-5B) on human umbilical vein endothelial cells (HUVEC) in comparison to untreated cell controls and to exposure to monoclonal IgG control antibody. We also examined the effect of recombinant monocyte chemoattractant protein-1 (MCP-1) on peripheral blood monocytes. Stimulation of endothelial cells with APS IgG fractions or HL-5B resulted in time-dependent upregulation of MCP-1 mRNA and protein expression. Stimulation with HL-5B also led to time-dependent upregulation of interleukin (IL)-8 and intracellular adhesion molecule-1 (ICAM-1) mRNA and IL-8 protein expressions. Stimulation of monocytes with recombinant MCP-1 resulted in an upregulation of TF mRNA and TF protein. In conclusion these results might represent a mechanism for antiphospholipid antibody-mediated thrombosis in APS patients.

Investigation of the mechanisms of coagulation factor VIIa-induced colon cancer SW620 cell proliferation and migration

To investigate the mechanisms that coagulation factor VIIa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro. The expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor VIIa or protease activated receptor 2 agonist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR. Factor VIIa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, upregulated TF mRNA expression and TF activity, but down-regulated caspase-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor VIIa were not only blocked by anti-TF but also by anti-PAR2 antibodies. Factor VIIa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caspase-7 expression, thus promotes the tumor cell proliferation and migration. p38 MAPK may negatively regulate this process.

Tissue factor expression demonstrates severe sinusoidal endothelial cell damage during rejection after living-donor liver transplantation

Since it is well known that endothelial cells may be important targets during rejection after living-donor liver transplantation, in this study we investigated liver sinusoidal endothelial cell (SEC) damage during rejection by focusing on thrombomodulin (TM) and hyaluronic acid (HA) as plasma markers of SEC damage. We also examined tissue factor (TF) expression in SECs, because damage to endothelial cells leads to immediate activation of the coagulation system, with the damage being triggered mainly by TF. Living-donor liver transplantation was performed at Mie University Hospital between March 2002 and December 2007; there were 8 patients with rejection (4 with acute cellular rejection and 4 with chronic rejection) and 32 patients without rejection. Liver biopsy tissue was immunostained with an anti-TF antibody, and assessed for SEC damage. In addition, total RNA was extracted from liver biopsy tissue and tested for TF mRNA expression by reverse-transcription polymerase chain reaction (RT-PCR). The plasma TM level was significantly higher in the rejection group than in the non-rejection group. TF expression was observed in SECs, in infiltrating inflammatory cells, and in the vascular endothelium in the rejection group. TF mRNA expression was significantly higher in the rejection group than in the non-rejection group. We demonstrated that TF expression revealed severe SEC damage in grafted liver during both acute cellular rejection and chronic rejection.

Signaling pathways for induction of platelet aggregation by SAS tongue cancer cells – a mechanism of hematogenous metastasis

Tongue cancer metastasis is mainly through blood stream and possibly associated with tumor cell-induced platelet aggregation (TCIPA). Platelet aggregation was induced by different amounts of SAS tongue cancer cells with/without inhibitors and the latent period for induction of platelet aggregation was recorded. Gene expression was analyzed by reverse transcriptase-polymerase chain reaction. SAS cells (4 x 10(4) to 1 x 10(6) cells/ml) induced platelet aggregation in a cell density-dependent manner. The latent period for induction of platelet aggregation reduced from 11.3 min (2 x 10(5) cells/ml) to 0.9 min (5 x 10(5) cells/ml). The extent of platelet aggregation increased from 39% to 76% by 2 x 10(5) and 5 x 10(5) SAS cells. Pre-treatment of SAS cells with aspirin showed little effect on its induction of platelet aggregation. SAS cells expressed tissue factor (TF) mRNA and the SAS cells-induced TCIPA was inhibited by TF neutralization antibody (5-20 microg/ml), heparin (5-10 U/ml), Hirudin fragment 54-65 (50 microg/ml) and D-Phenylalanyl-L-prolyl-L-arginine chloromethyl ketone. But areca nut (AN, a betel quid component known to generate reactive oxygen species (ROS)) extract showed little effect on TF expression in SAS cells. Pre-treatment with U73122 and 2-aminoethoxydiphenylborate inhibited SAS-induced TCIPA. Interestingly, catalase suppressed SAS cells-induced TCIPA, whereas AN extract enhanced this event. These results suggest that tongue cancer cells may induce TCIPA and enhance tumor metastasis. SAS-induced TCIPA is related to TF secretion, thrombin generation and associated with Phospholipase C-Inositol triphosphate signaling and ROS production. Betel quid chewing may potentially promote tongue cancer metastasis.

Regulation of LPS‐induced tissue factor expression in human monocytic THP‐1 cells by curcumin

Tissue factor (TF) is a transmembrane receptor, which initiates thrombotic episodes associated with various diseases. In addition to membrane‐bound TF, we have reported alternatively spliced form of human TF mRNA which was later confirmed that this form of TF mRNA expresses a soluble protein circulating in blood. Curcumin, a natural polyphenol, has been shown to inhibit the induction of TF activity in cultured endothelial cell. Since monocytes are the only circulating blood cells that can be induced to express TF, we tested the effect of curcumin on TF expression in human monocytic THP‐1 cell. We found that curcumin dose‐dependently reduced LPS‐induced procoagulant activity in intact THP‐1 cells, which indicates decreased membrane‐bound TF. However, curcumin at low concentration (10 µM) enhanced LPS‐induced TF protein in the whole cell lysate, and majority of the increased TF protein is a soluble form as indicated in Western blotting. TF mRNA level was also increased at 10 µM curcumin treatment; whereas high concentration of curcumin (40 µM) completely inhibited LPS‐induced TF mRNA and protein expression. Overall, our data show that curcumin could suppress the LPS‐induced procoagulant activity in THP‐1 monocytic cells, but it requires further investigation to understand how the two forms of TF are regulated by curcumin. (Supported by USDA agreement #58‐1950‐7‐707)

Progestin and Thrombin Regulate Tissue Factor Expression in Human Term Decidual Cells

Perivascular cell membrane-bound tissue factor (TF) initiates hemostasis via thrombin generation. The identity and potential regulation of TF-expressing cells at the human maternal-fetal interface that confers hemostatic protection during normal and preterm delivery is unclear. The objective of the study were to identify TF-expressing cells at the maternal-fetal interface in term and preterm decidual sections by immunohistochemistry and evaluate progestin, thrombin, TNF-alpha, and IL-1beta effects on TF expression by cultured human term decidual cells (DCs). Serial placental sections were immunostained for TF. Leukocyte-free term DC monolayers were incubated with 10(-8) M estradiol (E2) or E2 plus 10(-7) M medroxyprogestrone acetate (MPA) +/- thrombin or TNF-alpha or IL-1beta. ELISA and Western blotting assessed TF in cell lysates. Quantitative real-time RT-PCR measured TF mRNA levels. Immunolocalized TF in DC membranes in preterm and term placental sections displayed higher Histologic Scores than villous mesenchymal cells (P < 0.05). TF was undetected in interstitial or extravillous trophoblasts. Compared with DCs incubated with E2, MPA and 2.5 U/ml thrombin each doubled TF levels (P < 0.05) and E2 + MPA + thrombin further doubled TF levels (P < 0.05), whereas TNF-alpha and IL-1beta were ineffective. Western blotting confirmed the ELISA results. Quantitative RT-PCR revealed corresponding changes in TF mRNA levels. In human term placental sections, DC-expressed TF exceeds that of other cell types at the maternal-fetal interface and is localized at the cell membranes in which it can bind to factor VII and meet the hemostatic demands of labor and delivery via thrombin formation. Unlike the general concept that TF is constitutive in cells that highly express it, MPA and thrombin significantly enhanced TF expression in term DC monolayers.

VASOACTIVE INTESTINAL PEPTIDE AND PITUARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE INHIBIT TISSUE FACTOR EXPRESSION IN MONOCYTE IN VITRO AND IN VIVO

Tissue factor (TF), which is expressed on the surface of activated monocytes, is the major procoagulant that initiates thrombus formation in sepsis. Two endogenous neuropeptides, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), are attractive candidates for the development of therapies against septic shock. The purpose of this study was to examine whether VIP or PACAP inhibit the LPS-induced TF expression in monocytes. Treatment of freshly isolated human monocytes or cultured monocytic THP-1 cells with VIP or PACAP leads to reduced LPS-induced TF protein, mRNA expression and activity, as demonstrated by Western blot, real-time polymerase chain reaction, and TF activity assay, respectively. In an endotoxemic model, VIP blunts the increase of LPS-induced TF expression in blood cells at the transcriptional level, as demonstrated by real-time polymerase chain reaction. However, neither neuropeptide affects the expression of TF pathway inhibitor in monocytes. In vitro, LPS increases the migration of c-Rel/p65 into the nucleus and the phosphorylation of p38 and JNK, all of which are essential for LPS-induced TF expression. In addition, interestingly, VIP and PACAP block both the migration of c-Rel/p65 and the phosphorylation of p38 and JNK, as demonstrated by Western blot analysis. These data indicate that VIP and PACAP inhibit LPS-induced TF expression in monocytes in vitro and in vivo, confirming these peptides as candidates for the multitarget therapy of septic shock.

Human peripheral blood endothelial progenitor cells synthesize and express functionally active tissue factor

Introduction Endothelial progenitor cells are circulating cells able to home to sites of vascular damage and to contribute to the revascularization of ischemic areas. We evaluated whether endothelial progenitor cells synthesize tissue factor, a procoagulant protein also involved in angiogenesis. Materials and Methods Endothelial progenitor cells were obtained from the peripheral blood mononuclear fraction of normal donors and cultured in endothelial medium supplemented with specific growth factors. The procoagulant activity expressed by cells disrupted by freeze-thaw cycles was assessed by a one stage clotting assay. Tissue factor mRNA expression was evaluated by RT-PCR. Results Endothelial progenitor cells do not express procoagulant activity in baseline conditions. However, lipopolysaccharide induces the expression of procoagulant activity. The effect is dose-dependent and reaches statistical significance at 100 ng/mL lipopolysaccharide. Inhibition with an anti-tissue factor antibody and amplification of cDNA with primers based on the tissue factor sequence confirm the identity of this activity with tissue factor. The kinetics of tissue factor expression by endothelial progenitor cells is identical to that of human umbilical vein endothelial cells showing maximal activity within 4 hours, and then decreasing; in contrast, tissue factor expression by mononuclear cells lasts for longer times. Both 5,6-dichloro-beta D-ribofuranosyl-benzimidazole and cycloheximide prevented the expression of procoagulant activity. Stimulation of endothelial progenitor cells with tumor necrosis factor-α did not elicit any detectable procoagulant activity. Conclusions Endothelial progenitor cells can be stimulated by lipopolysaccharide to synthesize tissue factor. This protein might be involved in thrombotic phenomena and might contribute to endothelial progenitor cells related neovascularization.

The Influence of Brain Death on Tissue Factor Expression in the Pancreatic Tissues and Isolated Islets in Rats

Introduction Tissue factor (TF) in islets has been identified as the main trigger of the instant blood-mediated inflammatory reaction. Because the crucial events that directly induce TF remain to be determined, we focused on the influence of brain death (BD) on TF expression in pancreatic tissues and isolated islets. Materials and Methods BD was induced in male Lewis rats weighing 250–300 g by inflation of a Fogarty catheter placed intracranially. The rats were mechanically ventilated for 6 hours until removal of the pancreas. The expression of TF protein in pancreatic tissues was examined using Western blotting assay. Messenger RNA (mRNA) expressions of TF in pancreatic tissue and isolated islets were analyzed using real-time polymerase chain reaction (PCR) assay. The influence of BD on the isolation outcome was evaluated by islet yield, purity, viability, and function. Results TF protein and mRNA levels in the pancreatic tissues were similar between the groups. However, TF mRNA in the isolated islets of the BD group was significantly greater than that of the control group ( P = .04). Islet yield was considerably lower, and purity significantly lower in the BD than the control group ( P = .002). Unexpectedly, ATP/DNA ratio and respiratory activity were comparable between the groups. Conclusions Although BD per se was not sufficient to induce TF expression in pancreatic tissues, BD combined with subsequent warm ischemic damage during isolation procedures remarkably up-regulated TF expression in isolated islets, suggesting that BD is of great importance as an initiator of TF induction in the islet grafts. The present study demonstrated that the expression of inflammatory mediators rather than islet viability is more susceptible to BD.

The TF-603A/G gene promoter polymorphism and circulating monocyte tissue factor gene expression in healthy volunteers

Three single nucleotide polymorphisms (-603A/G, -1322C/T, -1812C/T) and one deletion/insertion polymorphism (-1208D/I) are present in the tissue factor (TF) gene promoter sequence. These polymorphisms are in complete linkage disequilibrium, determining two haplotypes with almost equal frequency. The -603A/-1208D/-1322C/-1812C haplotype, presently defined as TF-603A, has been linked to venous thromboembolic disease, with a potentially protecting effect. The effects of the TF-603A/G gene polymorphism on monocyte gene expression and on a whole-blood clotting time (WBCT) are not known. We determined the WBCT in basal conditions (H0) and after 4 hours of LPS stimulation <i>ex vivo</i> (H4LPS) on blood samples from 100 young healthy caucasian male subjects on 2 visits, 7 days apart. Monocyte TF mRNA was quantified at H0 and H4LPS by real-time quantitative reverse-transcription PCR. The monocyte TF mRNA values determined at the first and second visits were concordant. In H4LPS samples, TF mRNA levels were increased 70-fold. The TF-603A haplotype was associated with 40%-lower TF mRNA levels at H0 (<i>P</i>=0.0002) and this association followed the same trend but was no longer significant at H4LPS. At H4LPS, TF mRNA levels were associated with WBCT shortening (<i>P</i>=0.0003). WBCT at H0 was not concordant over time, precluding any genotype-phenotype analysis. WBCT at H4LPS was concordant over time but was not related to the TF-603A/G polymorphism. The TF-603A/G gene promoter polymorphism thus significantly influences constitutive TF gene expression in human monocytes but has no major effect on TF gene expression or on WBCT in LPS stimulated conditions.

Coagulation and fibrinolysis related cytokine imbalance in preeclampsia: the role of placental trophoblasts

Cytokine imbalance might have a pivotal role in hypercoagulability seen in preeclampsia. Our objective was to determine the relationship of blood coagulation related factors in placental tissue and peripheral blood in preeclamptic and normal pregnancies. We compared mRNA and protein levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), and tissue factor (TF) in the placenta of normal and preeclamptic pregnancies. Placental and peripheral blood t-PA and PAI-1 levels were examined. Trophoblasts were used to study the effects of hypoxia, hypoxia-reperfusion, and inflammatory cytokines on t-PA, PAI-1, tissue factor pathway inhibitor (TFPI), and TF. PAI-1 and TF mRNA and protein levels were higher in placental tissue of preeclamptic pregnancies and in the peripheral blood of patients with preeclampsia. mRNA and protein secretion of TF, TFPI, PAI-1, but not t-PA, was increased in trophoblast cell culture under hypoxia and hypoxia-reoxygenation. Cell cultures with high levels of tumor necrosis factor-alpha (TNF-alpha) exhibited increased expression and secretion of TF and PAI-1, decreased TFPI, and no significant change of t-PA. Imbalanced synthesis of t-PA, PAI-1, TFPI, and TF in trophoblasts may contribute to hypercoagulability in patients with preeclampsia.

Vaspin Can Not Inhibit TNF-.ALPHA.-Induced Inflammation of Human Umbilical Vein Endothelial Cells

Visceral adipose tissue-derived serine protease inhibitor (vaspin) has been recently identified as an adipocytokine in a rat model of type 2 diabetes. Adipocytokines may directly influence the function of endothelial cells (ECs) and modulate inflammatory states. We therefore assessed the effects of vaspin on basal and TNF-alpha-stimulated human umbilical vein ECs. Vaspin (10-100 ng/ml, 24 hr) had no effects on both basal ECs morphology and TNF-alpha-induced (10 ng/ml, 24 hr) morphological damages. Vaspin did not inhibit the TNF-alpha (20 min) activation of JNK, p38 and NF-kappaB, but only slightly inhibited Akt. Furthermore, vaspin did not decrease the TNF-alpha (24 hr) induction of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, endothelial selectin, and cyclooxygenase-2 protein expression as well as monocyte chemotactic protein-1, tissue factor, and plasmogen activator inhibitor-1 mRNA expression. The present results indicate that vaspin has no effects on normal ECs, and can not prevent TNF-alpha-induced inflammatory injury.

Role of the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway in Regulating Alternative Splicing of Tissue Factor mRNA in Human Endothelial Cells

Tissue factor (TF) is the primary initiator of blood coagulation. In response to tumor necrosis factor (TNF)-alpha human umbilical vein endothelial cells (HUVECs) express 2 TF isoforms: a soluble alternatively spliced isoform (asHTF) and membrane-bound "full length" (fl)TF. How the differential TF isoform expression is regulated is still unknown. This study compared the impact of PI3K/Akt pathway inhibition on alternative splicing of TF in HUVECs, to the influence of transcriptional regulation by inhibiting nuclear factor kappa B (NFkappaB). The mRNA expression of TF isoforms was assessed by real-time PCR, the thrombogenic activity was measured by a chromogenic TF activity assay and the phosphorylation state of serine/arginine-rich (SR) proteins was analyzed by western blotting. Transfection of HUVECs was done 72 h before the inhibition experiments were performed. PI3K/Akt pathway inhibition reduced the mRNA expression of asHTF but not flTF. Inhibition of NFkappaB reduced the expression of both isoforms. Moreover, the PI3K/Akt pathway inhibition, but not that of NFkappaB, modified the phosphorylation of the SR proteins SRp75, SRp55 and SF2/ASF. Additionally, overexpression of SF2/ASF and SRp75 influenced the differential TF-isoform expression in HUVECs. The PI3K/Akt pathway modulates alternative splicing of TF in HUVECs, distinct from transcriptional regulation.

Ultrafine particles from diesel engines induce vascular oxidative stress via JNK activation

Exposure to particulate air pollution is linked to increased incidences of cardiovascular diseases. Ambient ultrafine particles (UFP) from diesel vehicle engines have been shown to be proatherogenic in ApoE knockout mice and may constitute a major cardiovascular risk in humans. We posited that circulating nano-sized particles from traffic pollution sources induce vascular oxidative stress via JNK activation in endothelial cells. Diesel UFP were collected from a 1998 Kenworth truck. Intracellular superoxide assay revealed that these UFP dose-dependently induced superoxide (O 2 − ) production in human aortic endothelial cells (HAEC). Flow cytometry showed that UFP increased MitoSOX red intensity specific for mitochondrial superoxide. Protein carbonyl content was increased by UFP as an indication of vascular oxidative stress. UFP also up-regulated heme oxygenase-1 (HO-1) and tissue factor (TF) mRNA expression, and pretreatment with the antioxidant N-acetylcysteine significantly decreased their expression. Furthermore, UFP transiently activated JNK in HAEC. Treatment with the JNK inhibitor SP600125 and silencing of both JNK1 and JNK2 with siRNA inhibited UFP-stimulated O 2 − production and mRNA expression of HO-1 and TF. Our findings suggest that JNK activation plays an important role in UFP-induced oxidative stress and stress response gene expression.