Tissue Factor Pathway Inhibitor Expression Research Articles

Tissue factor pathway inhibitor‑2 is specifically expressed in ovarian clear cell carcinoma tissues in the nucleus, cytoplasm and extracellular matrix.

Tissue factor pathway inhibitor-2 (TFPI-2) is a promising candidate as a serum biomarker of ovarian clear cell carcinoma (OCCC), a lethal histological subtype of epithelial ovarian cancer (EOC). TFPI-2 is a secreted serine protease inhibitor that suppresses cancer progression through the inhibition of matrix protease activities. Previous studies have also identified TFPI-2 in the nucleus, and a possible function of nuclear TFPI-2 as a transcriptional repressor of matrix metalloproteinase-2 (MMP-2) was recently demonstrated. We are currently establishing TFPI-2 as a serum biomarker for OCCC patients; however, TFPI-2 expression in OCCC tissues has not been previously investigated. In the present study, we examined TFPI-2 expression and its localization in 11 OCCC cell lines by western blotting and enzyme-linked immune assay. Four cell lines expressed TFPI-2 in the nucleus, cytoplasm and culture plate–attached extracellular fraction, while four other cell lines expressed TFPI-2 only in the extracellular fraction. In the remaining three cell lines, TFPI-2 was not identified in any fraction. The amount of secreted soluble TFPI-2 showed similar trends to that of the plate-attached fraction. We next investigated the expression levels and distribution of TFPI-2 in surgically resected EOC tissues by immunohistochemistry. In 52 of the 77 (67.5%) OCCC tumors, TFPI-2 expression was detected in at least one of the nuclear, cytoplasmic and extracellular matrix fractions. In contrast, we did not identify TFPI-2 in the other EOC subtypes (n=65). TFPI-2-positive expression distinguished CCC from the other EOC tissues with a sensitivity of 67.5% and specificity of 100%. Although the inherent tumor suppressor function, statistical analyses failed to demonstrate correlations between TFPI-2 expression and clinical parameters, including 5-year overall survival, except for the patient age. In conclusion, we identified TFPI-2 expression in the nucleus, cytoplasm and extracellular matrix in OCCC tissues. The high specificity of TFPI-2 may support its use for diagnosis of OCCC in combination with existing markers.

Retracted] Epigenetic silencing of miR‑130a ameliorates hemangioma by targeting tissue factor pathway inhibitor2 through FAK/PI3K/Rac1/mdm2 signaling.

Following the publication of this paper, it was drawn to the authors' attention by an interested reader that some tumours featured in Fig.6A of the above paper were strikingly similar to those featured in Fig.8A of an article appearing in the same journal [Fan F‑Y, Deng R, Yi H, Sun H‑P, Zeng Y, HeG‑C and SuY: The inhibitory effect of MEG3/miR‑214/AIFM2 axis on the growth of T‑cell lymphoblastic lymphoma. Int JOncol 51:316‑326, 2017]. Furthermore, flow cytometric images featured in Fig.2G of the above paper were strikingly similar to data featured in the following article [ZhangH‑j, Wei Q‑f, WangS‑j, Zhang H‑j, Zhang X‑y, Geng Q, CuiY‑h and WangX‑h:LncRNA HOTAIR alleviates rheumatoid arthritis by targeting miR‑138 and inactivating NF‑κB pathway. Int Immunopharmacol 50:283‑290, 2017]. The Editor asked the authors for an explanation to account for the appearance of strikingly similar data in their paper independently, and they responded to request that the paper be retracted from International Journal of Oncology. All the authors agreed that the article should be retracted. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in International Journal of Oncology 50: 1821‑1831, 2017; DOI: 10.3892/ijo.2017.3943].

Sirtuin 5 promotes arterial thrombosis by blunting the fibrinolytic system.

Arterial thrombosis as a result of plaque rupture or erosion is a key event in acute cardiovascular events. Sirtuin 5 (SIRT5) belongs to the lifespan-regulating sirtuin superfamily and has been implicated in acute ischaemic stroke and cardiac hypertrophy. This project aims at investigating the role of SIRT5 in arterial thrombus formation. Sirt5 transgenic (Sirt5Tg/0) and knock-out (Sirt5-/-) mice underwent photochemically induced carotid endothelial injury to trigger arterial thrombosis. Primary human aortic endothelial cells (HAECs) were treated with SIRT5 silencing-RNA (si-SIRT5) as well as peripheral blood mononuclear cells from acute coronary syndrome (ACS) patients and non-ACS controls (case-control study, total n = 171) were used to increase the translational relevance of our data. Compared to wild-type controls, Sirt5Tg/0 mice displayed accelerated arterial thrombus formation following endothelial-specific damage. Conversely, in Sirt5-/- mice, arterial thrombosis was blunted. Platelet function was unaltered, as assessed by ex vivo collagen-induced aggregometry. Similarly, activation of the coagulation cascade as assessed by vascular and plasma tissue factor (TF) and TF pathway inhibitor expression was unaltered. Increased thrombus embolization episodes and circulating D-dimer levels suggested augmented activation of the fibrinolytic system in Sirt5-/- mice. Accordingly, Sirt5-/- mice showed reduced plasma and vascular expression of the fibrinolysis inhibitor plasminogen activator inhibitor (PAI)-1. In HAECs, SIRT5-silencing inhibited PAI-1 gene and protein expression in response to TNF-α. This effect was mediated by increased AMPK activation and reduced phosphorylation of the MAP kinase ERK 1/2, but not JNK and p38 as shown both in vivo and in vitro. Lastly, both PAI-1 and SIRT5 gene expressions are increased in ACS patients compared to non-ACS controls after adjustment for cardiovascular risk factors, while PAI-1 expression increased across tertiles of SIRT5. SIRT5 promotes arterial thrombosis by modulating fibrinolysis through endothelial PAI-1 expression. Hence, SIRT5 may be an interesting therapeutic target in the context of atherothrombotic events.

TFPI-2 inhibits the invasion and metastasis of bladder cancer cells

Bladder cancer metastasis seriously affects the prognosis of patients, but its molecular mechanism is unclear. This study sought to explore the roles of tissue factor pathway inhibitor-2 (TFPI-2) gene overexpression in the infiltration and metastasis of bladder cancer. Firstly, real-time PCR and immunohistochemistry were used to compare themRNA and protein expression levels, respectively, of TFPI-2 and matrix metalloproteinase-1 (MMP-1) in adjacent non-tumoral tissues, muscle-invasive bladder cancer (MIBC) tissues, and non-muscle-invasive bladder cancer (NMIBC) tissues. BIU-87-TFPI-2 cells that stably expressed TFPI-2 were generated by transfection with pcDNA3.1-TFPI-2. Real-time PCR and western blotting were performed to determine the mRNA and protein expression levels, respectively, of TFPI-2 and MMP-1 in BIU-87-TFPI-2 cells. The invasion and migration abilities of BIU-87-TFPI-2 cells were investigated using the Transwell chamber method. TFPI-2 was found to be significantlydownregulatedin bladder cancer tissue. The expression of MMP-1 was increased with the progression of bladder cancer. BIU-87 cells that overexpressed TFPI-2 were successfully generated by transfection with pcDNA3.1-TFPI-2. TFPI-2 overexpression in BIU-87 cells significantly inhibited cancer cell invasion and metastasis. Furthermore, the mRNA and protein expression levels of MMP-1 were significantly reduced in TFPI-2-overexpressing cells. Decreased TFPI-2 expression in bladder tissue was correlated with invasion and metastasis in bladder cancer. TFPI-2 overexpression could inhibit bladder cancer cell invasion andmigration in vitroby inhibiting MMP-1 protein expression. 3.

Overexpression of tissue factor pathway inhibitor 2 attenuates trophoblast proliferation and invasion in preeclampsia.

Pre-eclampsia (PE) is a disorder of pregnancy characterized by proteinuria and high blood pressure, affecting 2-8% of pregnancies worldwide. Previous studies have shown that PE is closely associated with trophoblast cell dysfunction. Here, we investigated the role of tissue factor pathway inhibitor-2 (TFPI-2) in regulating the biological processes of trophoblast cells. The TFPI-2 levels in plasma samples and placental tissues were tested by ELISA, immunohistochemistry, qRT-PCR, and western blot. HTR8/Svneo cell line was used to simulate the primary trophoblast cells and H/R culture was applied to mimic the oxidative stress state of PE. MTT assay, Annexin V/propidium iodide (PI) apoptosis assay, and transwell assay were used to determine the cell proliferation, apoptosis, and invasion. The expression levels of matrix metalloproteinases (MMPs) were evaluated by western blot. The expression of TFPI-2 was remarkably up-regulated in both the serum and placenta of PE patients. Hypoxia/reoxygenation increased the expression of TFPI-2 in HTR-8/SVneo cell line. TFPI-2 promoted that cell proliferation and inhibited the cell apoptosis of HTR8/SVneo cells in H/R condition. In addition, downregulation of TFPI-2 increased the cell invasion and the expression of MMP2 and MMP9. This study reveals that TFPI-2 plays a crucial role in monitoring the biological function of trophoblast cells, which might provide theoretical basis and therapeutic targets for the treatment of PE.

Low abundance of TFPI-2 by both promoter methylation and miR-27a-3p regulation is linked with poor clinical outcome in gastric cancer.

The tumor suppressor role of tissue factor pathway inhibitor 2 (TFPI-2) has been reported in various tumors. The present study aimed to improve the understanding of the oncogenic properties of TFPI-2 in gastric cancer. Relative expression of TFPI-2 was determined by a real-time polymerase chain reaction (PCR) and western blotting, respectively. Cell viability was measured via a cell counting kit-8 assay and proliferation was evaluated by a colony formation assay. Cell apoptosis was assessed with a caspase-3 activity kit and invasion was evaluated by a transwell chamber assay. The methylation level of TFPI-2 promoter was assayed by methylation-specific PCR. The regulatory effect of miR-27a-3p on TFPI-2 was analyzed with a luciferase reporter assay. The direct association between miR-27a-3p and TFPI-2 was shown by biotin-labelling pulldown. TFPI-2 was down-regulated in gastric cancer, which associated with an unfavorable prognosis clinically. Ectopic introduction of TFPI-2 greatly compromised cell viability, colony formation and invasive capacity, and also induced cell apoptosis simultaneously. The promoter region of TFPI-2 was extensively methylated in gastric cancer tissues compared to normal tissues, suggesting the epigenetic inhibition of TFPI-2 expression. We further identified that TFPI-2 functioned as sponge RNA against miR-27a-3p. Most importantly, miR-27a-3p-specific inhibitor significantly exerted a tumor suppressor function akin to TFPI-2 itself, and the anti-tumoral activities were completely abolished by TFPI-2 knockdown. We found that the epigenetically suppressed TFPI-2 compromised sponging effects with respect to miR-27a-3p in gastric cancer, which consequently and mechanistically contributed to the tumor biology of gastric cancer.

Tissue factor pathway inhibitor 2 suppresses the growth of thyroid cancer cells through by induction of apoptosis.

Tissue factor pathway inhibitor 2 (TFPI-2) has been recently identified as a tumor suppressor gene in several human cancers, whereas its role in thyroid cancer has been unclear. The TFPI-2 protein level in thyroid cancer tissues and cell lines (8305C and B-CPAP) were examined using immunohistochemistry and immunoblotting. The TFPI-2 promoter methylation was examined using methylation-specific polymerase chain reaction (MSP). Lentivirus containing TFPI-2 cDNA (Lenti-TFPI-2) was constructed to elevate TFPI-2 expression in 8305C and B-CPAP cells. The effects of Lenti-TFPI-2 on cell proliferation in vitro and in vivo were evaluated by MTT assay and mouse xenograft model. Annexin V/PI double staining assay was performed to detect the effect of Lenti-TFPI-2 on cell apoptosis. TFPI-2 protein level were decreased in cancer tissues and lymph node metastasis, and TFPI-2 protein level is positively associated with survival time. The promoter of TFPI-2 is hypermethylated in cancer tissues. TFPI-2 mRNA and protein levels were abundant in normal human thyroid follicular cell line Nthy-ori 3-1 cells, whereas they were decreased in 8305C and B-CPAP cells. pcDNA-TFPI-2 elevated TFPI-2 mRNA and protein in 8305C and B-CPAP cells. TFPI-2 overexpression suppressed proliferation and induced apoptosis of 8305C and B-CPAP cells. TFPI-2 inactivation may play a role in thyroid cancer tumorigenesis and development. TFPI-2 overexpression suppressed cell proliferation through induction of cell apoptosis, suggesting that TFPI-2 may serve as a novel and effective target for thyroid cancer therapy.

2182Sirtuin 5 regulates arterial thrombosis by modulating endothelial plasminogen activator inhibitor-1

Abstract Introduction Arterial thrombosis as a result of plaque rupture or erosion is a crucial event in myocardial infarction and stroke. Oxidative stress and inflammation promote endothelial dysfunction and play a pivotal role in destabilization of the atherosclerotic plaque. Sirtuin 5 (SIRT5) is a member of the sirtuin protein family with function as a NAD+-dependent protein desuccinylase and demalonylase. Being implicated in the regulation of different pathophysiological processes among which production of reactive oxygen species and transcription of inflammatory mediators, SIRT5 plays a role in the development of several cardiovascular diseases such as myocardial infarction and stroke. To date, the possible involvement of SIRT5 as a mediator of arterial thrombosis remains to be investigated. Purpose In this study we investigate the putative role of this protein in arterial thrombosis by using an established in vivo mouse model. The translational value of animal findings as well as the molecular mechanism underlying the observed effect will be investigated also in primary human aortic endothelial cells (HAECs). Methods SIRT5 knockout (KO) as well as SIRT5 transgenic (TG) animals were used for in vivo experiments. HAECs treated with SIRT5 silencing RNA (si-SIRT5) and stimulated with tumor necrosis factor (TNF)-α were used for in vitro assays. Results When compared to WT animals, SIRT5 KO mice display blunted carotid artery thrombus formation as underlined by delayed time to occlusion in a photochemical injury model. Oppositely, in SIRT5 TG mice the formation of an occlusive thrombus is accelerated (Fig 1). Mechanistically, SIRT5 KO and WT animals show no difference in terms of vascular tissue factor (TF) activity, TF concentration in plasma and expression of TF pathway inhibitor (TFPI) in the aorta. In line with the observed reduced thrombogenicity, SIRT5 KO animal express reduced level of the pro-thrombotic plasminogen activator-1 (PAI-1), as assessed by western blot in aorta lysate. Of interest, SIRT5 genetic deletion does not affect platelet aggregation, as assessed by ex-vivo collagen-induced aggregometry. In HAECs, SIRT5-silencing inhibits PAI-1 expression in response to TNF-α. Real-time polymerase chain reaction revealed that inhibition of PAI-1 expression occurs at the mRNA level. This effect is mediated by reduced activation of the MAP kinase Erk 1/2, but not JNK (Fig 1). Conclusions SIRT5 mediates arterial thrombosis by increasing endothelial PAI-1 expression. Hence, SIRT5 may be an effective therapeutic target in the context of atherothrombosis.

LongShengZhi Capsule reduces carrageenan-induced thrombosis by reducing activation of platelets and endothelial cells

Formation of thrombosis is associated with activation of platelets and endothelial cells. The effect of LongShengZhi Capsule (LSZ), a traditional Chinese medicine used for treatment of vascular diseases, on thrombosis was investigated in this study. BALB/c mice were induced thrombosis by injection of carrageenan while receiving pre or simultaneous LSZ treatment. We also compared the therapeutic effects of LSZ and clopidogrel on formed thrombi. LSZ inhibited carrageenan-induced thrombi in mouse tissue vessels. In addition, LSZ but not clopidogrel reduced formed thrombi with a short time window. The reduction of thrombi by LSZ was associated with reduced serum P-selectin, reduced expression of TNF-α and P-selectin and activated matrix metalloproteinase 2 expression in tissues. In vitro, LSZ decreased thrombin-induced human platelet clot retraction which was associated with inactivation of AKT and ERK1/2. LSZ also reduced adhesion of platelets or THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein or lipopolysaccharide. The anti-adherent actions of LSZ was attributed to reduction of oxidative stress, expression of platelet receptors (P2Y12, PAR4 and CD36) and AKT activity in platelets. LSZ also reduced adhesion molecules or tissue factor but activated tissue factor pathway inhibitor expression in HUVECs. Taken together, our study demonstrates the antithrombotic properties of LSZ by reducing activation of platelets and endothelial cells, and suggests its potential application in clinics.

Transcription factor FOXP3: A repressor of the TFPI gene?

Single nucleotide polymorphisms (SNPs) may play an important role in the risk of certain diseases. We have previously shown that the -287T/C SNP of the tissue factor pathway inhibitor (TFPI) gene promoter region exerts differential impact on TFPI mRNA expression; the C allele being associated with higher TFPI expression, which in turn is associated with reduced risk of thrombosis. In the present study, we aimed to reveal the underlying molecular mechanisms using human embryonic kidney 293 (HEK293) and Michigan Cancer Foundation-7 (MCF7) cells that both express TFPI. Transfecting the cells with luciferase reporter gene constructs containing the TFPI promoter with either the T or the C allele of -287T/C resulted in increased luciferase activity with the C allele relative to the T allele. Three potential candidate transcription factors for binding to the two -287 alleles were predicted using the ALGGEN PROMO software, and results from electrophoretic mobility shift assays indicated that forkhead box protein 3 (FOXP3), initially identified as a functional marker of T regulator cells, bound more specifically to the T allele compared with the C allele. By chromatin immunoprecipitation assays analysis it was confirmed that FOXP3 was able to bind to the DNA region that contains the SNP. Knockdown or overexpression of FOXP3 resulted in increased or decreased TFPI levels, respectively, in both cell types. In conclusion, this study indicates that FOXP3 most likely is involved in the increased levels of TFPI observed with the -287C allele and also that FOXP3 might be a repressor for TFPI expression.

Tissue Factor Pathway Inhibitor Enhances Transendothelial Migration of Chronic Lymphocytic Leukemia Cells through Binding to Glypican-3

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. Cell trafficking and homing of CLL cells play a critical role in organ infiltration and contribute to the clinical course of CLL. Interaction between CLL cells and endothelial cells affects gene expression in CLL cells and further regulates cell trafficking. Endothelial cells are the main source of tissue factor (TF) pathway inhibitor (TFPI), which is the primary inhibitor of TF. However, the role of TFPI in leukemic cell trafficking has not been explored before. CXCL12 and its receptor CXCR4 play an important role in CLL cell trafficking and homing. To assess the effect of TFPI on CXCL12-mediated migration of leukemic cells, we investigated transendothelial migration (TEM) of B cells from CLL patients by using CXCL12 as a chemoattractant. We found that full-length recombinant TFPI (rTFPI) increased CXCL12-mediated migration of CLL cells dose-dependently by using MACS flow cytometry to count the migrated cells. Accordingly, Western blot showed the binding of rTFPI to the CLL cells after 24 h treatment. However, the chemokine receptor CXCR4 expression in CLL cells was not increased or even slightly decreased by rTFPI treatment, suggesting that TFPI enhances leukemic cell migration probably through interacting with other factor(s) than overexpressing CXCR4 in CLL cells. Surface expression and intracellular trafficking of TFPI is regulated by glycosylphosphatidylinositol (GPI)-anchored co-receptor(s). Glypican-3, a member of proteoglycan family, binds to the plasma membrane by a GPI anchor and its heparan sulfate (HS)-type glycosaminoglycan chains are considered to mediate the interaction with other cell membrane proteins. To determine whether TFPI interacts with glypican-3, immunofluorescence staining and co-immunoprecipitation were performed in K562 and HL60 cells. Immunofluorescence analysis revealed colocalization of TFPI and glypican-3 on the surface of K562 and HL60 cells. Co-immunoprecipitation confirmed the binding between TFPI and glypican-3 in K562 cells. Moreover, co-localized TFPI and glypican-3 were found in bone marrow biopsies of leukemia patients. We further employed an antibody against HS chains of glypican-3, which inhibits the activity of glypican-3. Western blot showed that rTFPI treatment failed to increase TFPI protein in CLL cells when glypican-3 inhibitor HS20 was applied. In addition, TEM assay showed that HS20 treatment abolished the effect of TFPI on cell migration, which suggests that inhibition of glypican-3 blocks the binding of TFPI to CLL cells and then abolishes the effect of TFPI on cell migration. It indicates that TFPI affects CLL cell migration through binding to glypican-3. Using a factor Xa generation assay, we found that HS20 suppressed TF activity on the surface of HL60 cells, suggesting that the inhibition of glypican-3 impaired the procoagulant activity of leukemic cells. In addition, Western blot and RT-PCR showed that HS20 downregulated TF mRNA and protein expression in CLL cells. Finally, we explored the role of Wnt/β-catenin signaling, which has been shown to regulate tumor progression and metastasis. We found a dose-dependent increase of activated β-catenin in the CLL cells after rTFPI treatment. Inhibition of Wnt/β-catenin signaling by a potent inhibitor IWP4 strongly impaired the migration of CLL cells induced by rTFPI. In this study, we show that TFPI enhances CLL cell migration through binding to glypican-3 and activating Wnt/β-catenin signaling pathway. Our findings suggest a novel molecular mechanism regulating the transmigration of leukemic cells. It may open a new window for therapeutic targeting in the treatment of organ infiltration of CLL patients. DisclosuresNo relevant conflicts of interest to declare.

Indoleamine 2,3‐dioxygenase 1 in coronary atherosclerotic plaque enhances tissue factor expression in activated macrophages

BackgroundRecent clinical studies have found that changes in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism are associated with cardiovascular events. However, the roles of the Kyn pathway on vascular wall thrombogenicity remain unknown. Indoleamine 2,3‐dioxygenase 1 (IDO1) is a rate‐limiting enzyme of the Kyn pathway. ObjectiveThe present study aimed to localize IDO1 in human coronary atherosclerotic plaques from patients with angina pectoris and define its role in plaque thrombogenicity. MethodsImmunohistochemical methods were applied to localize IDO1 in coronary atherosclerotic plaques from patients with stable (SAP) and unstable (UAP) angina pectoris. The role of IDO1 in tissue factor (TF) expression was investigated in THP‐1 macrophages activated by interferon (IFN)γ and tissue necrosis factor (TNF)α. ResultsWe localized IDO1 mainly in CD68‐positive macrophages within atherosclerotic plaques, and in close association with TF. Areas that were immunopositive for IDO1, TF, and CD3‐positive T lymphocytes were significantly larger in plaques from patients with UAP than SAP. Macrophages activated by IFNγ and TNFα upregulated IDO1 expression, increased the Kyn/Trp ratio and enhanced TF expression and activity, but not TF pathway inhibitor expression. The IDO1 inhibitor epacadostat significantly reduced the Kyn/Trp ratio, TF expression and activity, as well as NF‐κB (p65) binding activity in activated macrophages. Inhibition of the aryl hydrocarbon receptor that binds to Kyn, also reduced Kyn‐induced TF expression in activated macrophages. ConclusionIndoleamine 2,3‐dioxygenase 1 expressed in coronary atherosclerotic plaques might contribute to thrombus formation through TF upregulation in activated macrophages.

Lysine‑specific demethylase 2 contributes to the proliferation of small cell lung cancer by regulating the expression of TFPI‑2.

The present study aimed to investigate the effect of lysine-specific demethylase 2 (LSD2) in small cell lung cancer (SCLC) and explore its underlying regulatory mechanism. Cell growth was tested by MTT assay and mRNA and protein expression was determined by quantitative polymerase chain reaction (q-PCR) and western blot analysis, respectively. Chromatin immunoprecipitation (ChIP) was used to investigate the degree of H3K4me2 enrichment in the promoter region of tissue factor pathway inhibitor-2 (TFPI-2). SCLC tissues and cell lines presented significantly higher expression of LSD2 and DNA methyltransferase 3B (DNMT3B) and lower expression of TFPI-2 compared with the controls. In H1417 cells LSD2 overexpression increased the mRNA and protein expression of DNMT3B, while inhibiting the mRNA and protein expression of TFPI-2. Following transfection with short interfering (si) RNA-DNMT3B, the expression of TFPI-2 increased in H1417 cells. The results of ChIP demonstrated that compared with the controls, H3K4me1 enrichment in the TFPI-2 promoter region was to a lower degree in the H1417 cells with LSD2 overexpression and a higher degree in the H1417 cells with LSD2 silencing. MTT assays revealed that LSD2 overexpression significantly promoted the growth of H69, DMS-114 and H1417 cells, which was contradictory to the effect on LSD2 silencing. Compared with the LSD2 overexpression cells, SCLC cells with simultaneous overexpression of LSD2 and TFPI-2 demonstrated a decreased proliferation. These results suggest that LSD2 achieves a promoting effect on SCLC by indirectly regulating TFPI-2 expression through the mediation of DNMT3B expression or through the regulation of the demethylation of H3K4me1 in the promoter region of the TFPI-2 gene.

Involvement of MAFB and MAFF in Retinoid-Mediated Suppression of Hepatocellular Carcinoma Invasion.

Retinoids exert antitumor effects through the retinoic acid receptor α (RARα). In the present study, we sought to identify the factors involved in the RARα-mediated transcriptional regulation of the tumor suppressor gene and the tissue factor pathway inhibitor 2 (TFPI2) in hepatocellular carcinoma (HCC). All-trans-retinoic acid (ATRA) was used in the in vitro experiments. Cell invasiveness was measured using trans-well invasion assay. ATRA significantly increased TFPI2 expression through RARα in a human HCC cell line known as HuH7. TFPI2 was vital in the ATRA-mediated suppression of HuH7 cell invasion. The musculo-aponeurotic fibrosarcoma oncogene homolog B (MAFB) significantly enhanced the activation of the TFPI2 promoter via RARα while MAFF inhibited it. The knockdown of RARα or MAFB counteracted the ATRA-mediated suppression of HuH7 cell invasion while the knockdown of MAFF inhibited the invasion. TFPI2 expression in HCC tissues was significantly downregulated possibly due to the decreased expression of RARβ and MAFB. Patients with HCC expressing low MAFB and high MAFF levels showed the shortest disease-free survival time. These results suggest that MAFB and MAFF play critical roles in the antitumor effects of retinoids by regulating the expression of retinoid target genes such as TFPI2 and can be promising for developing therapies to combat HCC invasion.

Abstract 158: Indoleamine 2,3-dioxygenase 1 is Expressed in Macrophages in Human Coronary Atherosclerotic Plaque and is Associated With Tissue Factor Expression in Activated THP-1 Macrophages

Background: Recently, several clinical studies have found that changes in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism are associated with cardiovascular events. Indoleamine 2,3-dioxygenase 1 (IDO1) is a rate-limiting enzyme of the Kyn pathway and is induced by cytokines, particularly IFNγ. However, the roles of the Kyn pathway on vascular wall thrombogenicity remain unknown. Objective: The present study aimed to localize IDO1 in human coronary atherosclerotic plaques from patients with stable angina pectoris (SAP) and unstable angina pectoris (UAP) and define its role in plaque thrombogenicity. Methods: Immunohistochemical methods were applied to localize IDO1 in coronary atherosclerotic plaques from patients with SAP and UAP. We examined the role of IDO1 in tissue factor (TF) expression in THP-1 macrophages activated by interferon (IFN)γ and tissue necrosis factor (TNF)α. Results: We localized IDO1 mainly in CD68-positive macrophages in atherosclerotic plaques, and in close association with TF. Areas that were immunopositive for IDO1, TF, and CD3-positive T lymphocytes were significantly larger in plaques from patients with UAP than SAP. THP-1 macrophages activated by IFNγ and TNFα upregulated IDO1 expression, increased the Kyn/Trp ratio and enhanced TF expression and activity, but not TF pathway inhibitor expression. The IDO1 inhibitor epacadostat significantly reduced the Kyn/Trp ratio, TF expression and activity, as well as NF-κB (p65) binding activity in activated THP-1 macrophages. Inhibition of the aryl hydrocarbon receptor (AHR) that binds to Kyn, also reduced Kyn-induced TF expression in activated THP-1 macrophages. Kynurenine dose-dependently increased TF expression under IDO1 inhibition whereas hydroxyanthranilic and quinolinic acids did not. Conclusion: The IDO1 is expressed in coronary atherosclerotic plaques and might contribute to thrombus formation through TF upregulation via NF-κB (p65) and AHR in activated macrophages.

Targeting anticoagulant protein S to improve hemostasis in hemophilia

AbstractImproved treatments are needed for hemophilia A and B, bleeding disorders affecting 400 000 people worldwide. We investigated whether targeting protein S could promote hemostasis in hemophilia by rebalancing coagulation. Protein S (PS) is an anticoagulant acting as cofactor for activated protein C and tissue factor pathway inhibitor (TFPI). This dual role makes PS a key regulator of thrombin generation. Here, we report that targeting PS rebalances coagulation in hemophilia. PS gene targeting in hemophilic mice protected them against bleeding, especially when intra-articular. Mechanistically, these mice displayed increased thrombin generation, resistance to activated protein C and TFPI, and improved fibrin network. Blocking PS in plasma of hemophilia patients normalized in vitro thrombin generation. Both PS and TFPIα were detected in hemophilic mice joints. PS and TFPI expression was stronger in the joints of hemophilia A patients than in those of hemophilia B patients when receiving on-demand therapy, for example, during a bleeding episode. In contrast, PS and TFPI expression was decreased in hemophilia A patients receiving prophylaxis with coagulation factor concentrates, comparable to osteoarthritis patients. These results establish PS inhibition as both controller of coagulation and potential therapeutic target in hemophilia. The murine PS silencing RNA approach that we successfully used in hemophilic mice might constitute a new therapeutic concept for hemophilic patients.

Endocardial Changes in Nonvalvular Atrial Fibrillation Without Atrial Thrombus-Thrombomodulin and Tissue Factor Pathway Inhibitor.

Atrial fibrillation (AF) is a well-known cause for thromboembolism. Although blood stasis in the left atrium and hypercoagulable state of the blood have been emphasized as important mechanisms, limited attention has been paid to the endocardial changes in maintaining the balance of local coagulation, which may also contribute to the thrombus formation in AF. In the present study, left atrial appendage samples were obtained at heart surgery from nonvalvular AF and non-AF patients without atrial thrombus. Immunohistochemistry for endocardial markers including thrombomodulin (TM) and tissue factor pathway inhibitor (TFPI) was performed and semiquantitatively graded. In immunohistochemistry analysis, decreased expression of TM was found in patients with nonvalvular AF compared with those without AF (P < .001). There was no difference in TFPI expression between the 2 groups (P = .213). Patients with TM score of 0 or 1 seemed to have larger left atrial diameter (LAD) than those with TM score of 2 or 3 (44.0 (7.9) vs 40.6 (3.9); P = .009), while no relationship between LAD and TFPI expression was found (43.4 (7.0) vs 42.9 (7.8); P = .485). In conclusion, TM expression in the atrial endocardium is decreased in nonvalvular AF without atrial thrombus, while TFPI expression is not. Downregulated TM expression might be associated with enlarged LAD.

The role of TFPI2 hypermethylation in the detection of gastric and colorectal cancer.

Gastrointestinal cancer is a prevalent disease with high morbidity and mortality. Tissue factor pathway inhibitor 2 (TFPI2) gene could protect the extracellular matrix of cancer cells from degradation and tumor invasion. The goal of our study was to estimate the diagnostic value of TFPI2 hypermethylation in gastric cancer (GC) and colorectal cancer (CRC). TFPI2 methylation was measured by quantitative methylation-specific polymerase chain reaction (qMSP) method in 114 GC and 80 CRC tissues and their paired non-tumor tissues. Our results showed that TFPI2 methylation was significantly higher in tumor tissues (GC: 29.940% vs. 12.785%, P < 0.001; CRC: 26.930% vs. 5.420%, P < 0.001). The methylation level of TFPI2 in colorectal tumor tissues was significantly higher than that in colorectal normal tissues (26.930% versus 0.002%, P < 0.00001). In GC, TFPI2 hypermethylation yielded an area under the curve (AUC) of 0.762 (95% CI: 0.696–0.828) with a sensitivity of 68% and a specificity of 83%. In CRC, TFPI2 hypermethylation yielded an AUC of 0.759 (95% CI: 0.685–0.834) with a sensitivity of 61% and a specificity of 84%. Similarly, TCGA data also supported TFPI2 hypermethylation was a promising diagnostic marker for GC and CRC. Moreover, the dual-luciferase reporter assay showed TFPI2 fragment could upregulate gene expression (fold change = 5, P = 0.005). Data mining further indicated that TFPI2 expression in CRC cell lines was significantly increased after 5’-AZA-deoxycytidine treatment (fold change > 1.37). In conclusion, TFPI2 hypermethylation might be a promising diagnostic biomarker for GC and CRC.

Epigallocatechin gallate inhibits the growth and promotes the apoptosis of bladder cancer cells.

Epigallocatechin gallate (EGCG) has been revealed to inhibit the proliferation and induce the apoptosis of several types of tumor, in addition to inhibiting DNA methyltransferase activity, leading to CpG demethylation. Tissue factor pathway inhibitor 2 (TFPI-2) expression is downregulated in bladder cancer. The present study revealed that this downregulation was partly due to hypermethylation of the TFPI-2 gene promoter, which was decreased by EGCG treatment. In addition, the present study demonstrated that EGCG could inhibit the viability and invasion, and induce the apoptosis, of bladder cancer T24 cells. Furthermore, western blot analysis and reverse transcription-quantitative polymerase chain reaction analyses demonstrated that EGCG could upregulate the expression of TFPI-2. These results suggest that EGCG inhibits the growth and induces the apoptosis of bladder cancer cells through restoring TFPI-2 expression. Thus, EGCG is a potential therapeutic candidate for the treatment of bladder cancer.

A novel hypoxia response element regulates oxygen-related repression of tissue factor pathway inhibitor in the breast cancer cell line MCF-7

BackgroundHypoxia is one of the most pervasive physiological stresses in solid tumors. We have previously demonstrated that tissue factor (TF) pathway inhibitor (TFPI) expression was transcriptionally repressed by the activation of hypoxia inducible factor (HIF)-1α under hypoxic conditions. However, the role of HIF-2α, also known as endothelial PAS domain-containing protein 1 (EPAS1), on TFPI expression remains unclear. AimTo explore the role of HIF-2α/EPAS1 in the regulation of TFPI expression under hypoxia in breast cancer cells. Methods and resultsQuantitative RT-PCR showed that total TFPI mRNA and protein levels were decreased by the overexpression of HIF-2α/EPAS1 in MCF7 cells. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay indicated a HIF-2α/EPAS1 responsive region located in the TFPI promoter region at −170 to +21 relative to the transcriptional start site. Subsequent mutagenesis demonstrated a functional hypoxia response element (HRE) 5′-AAACAGGA-3′ for HIF-2α/EPAS1 within the TFPI promoter located at −45 to −38. In breast cancer patients, a positive correlation between HIF-2α/EPAS1 and total TFPI mRNA expression was observed by using gene expression analysis. ConclusionsThis study provides evidence that HIF-2α/EPAS1 is involved in the regulation of TFPI gene expression in breast cancer cells, suggesting that the activation of coagulation and the increased risk of thrombosis observed in breast cancer patients may correlate with local hypoxic regulation of coagulation factors and their inhibitors.