Tissue Factor Pathway Inhibitor Expression Research Articles

Future directions for xenotransplantation in lungs.

Advancements in preclinical xenotransplant studies have opened doors for clinical heart and kidney xenotransplantation. This review assesses recent progress in lung xenotransplantation research and its potential clinical implications. The efficacy of the humanized von Willebrand factor in reducing platelet sequestration in ex-vivo and in-vivo lung xenotransplant models was showcased. Combining human tissue factor pathway inhibitor and CD47 expression with selectin and integrin inhibition delayed neutrophil and platelet sequestration. Enhanced expression of human complement regulatory proteins and thrombomodulin in genetically engineered pig lungs improved graft survival by reducing platelet activation and modulating coagulation disruptions. Knocking out the CMAH gene decreased antibody-mediated inflammation and coagulation activation, enhancing compatibility for human transplantation. Furthermore, CMAH gene knockout in pigs attenuated sialoadhesin-dependent binding of human erythrocytes to porcine macrophages, mitigating erythrocyte sequestration and anemia. Meanwhile, in-vivo experiments demonstrated extended survival of xenografts for up to 31 days with multiple genetic modifications and comprehensive treatment strategies. Experiments have uncovered vital insights for successful xenotransplantation, driving further research into immunosuppressive therapy and genetically modified pigs. This will ultimately pave the way for clinical trials designed to improve outcomes for patients with end-stage lung disease.

The RNA-binding protein sorbin and SH3 domain-containing 2 are transcriptionally regulated by specificity protein 1 and function as tumor suppressors in bladder cancer by stabilizing tissue factor pathway inhibitor.

Sorbin and SH3 domain-containing 2 (SORBS2) is anRNA-binding protein and has been implicated in the development of some cancers. However, its role in bladder cancer (BC) is yet to be established. The expression of SORBS2 in BC tissues was determined from the Gene Expression Omnibusand Gene Expression Profiling Interactive Analysisdatabases and collected paired tumor/normal samples. The effects of SORBS2 on BC cells were detected by CCK-8, colony formation, Transwell, dual-luciferase, RNA immunoprecipitation, chromatin immunoprecipitation, and DNA pull-down assays. In vivo, BC cell growth and metastasis were studied by a xenograft subcutaneous model and a tail-vein metastasis model. The results showed that SORBS2 expression was significantly decreased in BC tissues and cells. SORBS2 overexpression inhibited cell proliferation, migration, invasion, and epithelial-mesenchymal transitionin vitro and tumor growth and metastasis in vivo, while silencing SORBS2 produced the opposite effect. Mechanistically, we found that SORBS2 enhanced the stability of tissue factor pathway inhibitor (TFPI) mRNA via direct binding to its 3' UTR. Restoration of TFPI expression reversed SORBS2 knockdown-induced malignant phenotypes of BC cells. In addition, SORBS2 expression was negatively regulated by the transcription factor specificity protein 1 (SP1). Conversely, SORBS2 can be transcriptionally regulated by SP1 and inhibit BC cell growth and metastasis via stabilization of TFPI mRNA, indicating SORBS2 may be a promising therapeutic target for BC.

Thrombin generation on vascular cells in the presence of factor VIII and/or emicizumab

BackgroundThe effect of factor VIII (FVIII) or emicizumab on thrombin generation is usually assessed in assays using synthetic phospholipids. Here, we assessed thrombin generation at the surface of human arterial cells (aortic endothelial cells [hAECs] and aortic vascular smooth muscle cells [hVSMCs]). ObjectivesTo explore the capacity of hAECs (resting or stimulated) and hVSMCs to support thrombin generation by FVIII or emicizumab. MethodsPrimary hVSMCs and hAECs were analyzed for tissue factor (TF)-activity and antigen, phosphatidylserine (PS)-exposure, tissue factor pathway inhibitor (TFPI)-content and thrombomodulin expression. Cells were incubated with FVIII-deficient plasma spiked with FVIII, emicizumab, activated prothrombin complex concentrate (APCC) or combinations thereof. ResultsTF activity and PS-exposure were present on both hVSMCs and hAECs. In contrast, thrombomodulin and TFPI were expressed on hAECs, while virtually lacking on hVSMCs, confirming the procoagulant nature of hVSMCs. Tumor necrosis factor α-mediated stimulation of hAECs increased not only TF antigen, TF activity, and PS-exposure but also TFPI and thrombomodulin expression. As expected, FVIII and emicizumab promoted thrombin generation on nonstimulated hAECs and hVSMCs, with more thrombin being generated on hVSMCs. Unexpectedly, FVIII and emicizumab increased thrombin generation to a lesser extent on stimulated hAECs compared with nonstimulated hAECs. Finally, adding emicizumab to FVIII did not further increase thrombin generation, whereas the addition of emicizumab to APCC resulted in exaggerated thrombin generation. ConclusionTumor necrosis factor stimulation of hAECs increases both pro- and anticoagulant activity. Unexpectedly, the increased anticoagulant activity is sufficient to limit both FVIII- and emicizumab-induced thrombin generation. This protective effect disappears when emicizumab is combined with APCC.

Expression of tissue factor pathway inhibitor 2 in the follicles of chicken ovaries and its regulatory mechanism in cultured granulosa cells1

Tissue factor pathway inhibitor 2 (TFPI2) plays a key role in female reproduction. However, its expression and function in chickens are still unclear. In the present study, ovarian tissues from 30 w and 15 w laying chickens were analyzed using RNA-seq to identify the differentially expressed gene TFPI2. The full-length cDNA of TFPI2 was obtained from adult chicken ovaries by rapid-amplification of cDNA ends (RACE), and the putative TFPI2 protein was found to share a highly conserved amino acid sequence with known bird homologs. In addition, TFPI2 was widely expressed in the tissues of adult chicken follicles according to quantitative real-time PCR (qRT–PCR) and Western blotting. Immunohistochemistry suggested that the TFPI2 protein existed in chicken ovary follicles at different developmental states, such as primordial follicles, the ovarian stroma, and the granulosa and theca layers of prehierarchical follicles (6-8 mm) and preovulatory follicles (F1). In vitro, Follicle stimulating hormone or Luteinizing hormone (FSH/LH) stimulated the expression of TFPI2 in chicken granulosa cells. FSH-/LH-induced TFPI2 mRNA expression was mediated by signaling pathways such as the PKA, PKC, PI3K, and mTOR pathways. Functionally, TFPI2 promoted the proliferation and viability of cultured granulosa cells and decreased the secretion of Progesterone (P4) and Estrogen (E2) and the mRNA abundance of key steroidogenic enzymes (STAR, Cyp17a1, Cyp19a1 and 3B-HSD) as well as MMPs (MMP2, 7, 9 and 11). Mechanistically, TFPI2 inhibited the expression of MMP7 via the Wnt signaling pathway. These findings indicate that TFPI2 may play an important role in regulating chicken follicular development and ovulation and suggest the molecular regulation mechanisms.

Immunohistochemical Analysis of the Tissue Factor Pathway Inhibitor-2 in Endometrial Clear Cell Carcinoma: A Single-center Retrospective Study.

The tissue factor pathway inhibitor-2 (TFPI2) was recently identified as a diagnostic serum marker for ovarian clear cell carcinoma. Moreover, the immunohistochemical expression of TFPI2 in ovarian clear cell carcinoma was recently reported. This single-center retrospective study aimed to evaluate whether TFPI2 can be a specific biomarker for immunohistological diagnosis of endometrial clear cell carcinoma (ECCC). Immunohistochemical staining of TFPI2 in 55 endometrial carcinomas was evaluated at Nara Medical University Hospital. Thirteen ECCC samples were included as cases and 38 samples were included as a control (endometrioid carcinoma grade 1, 11 cases; grade 2, 11 cases; grade 3, 10 cases; serous carcinoma, 10 cases). The mean ± SD TFPI2 histoscore for diagnosing ECCC was 115.4 ± 87.9, which was significantly higher than that of non-ECCC (21.3 ± 45.9, P = 0.002). The best TFPI2 histoscore value obtained from the analyses of receiver operating characteristic curves for immunohistochemical diagnosis of ECCC was 15. With TFPI2 histoscores ≥15.0 as positive and <15.0 as negative, all 13 ECCC cases (100%) were positive for TFPI2, whereas 11 (26.2%) non-ECCC cases were positive for TFPI2. The sensitivity and specificity of TFPI2 for diagnosing ECCC were 100% and 73.8%, respectively. TFPI2 is expressed in ECCC and is useful for histopathological diagnosis.

FTO overexpression expedites wound healing and alleviates depression in burn rats through facilitating keratinocyte migration and angiogenesis via mediating TFPI-2 demethylation.

Burn injury is a serious traumatic injury that leads to severe physical and psychosocial impairment. Wound healing after burn injury is a substantial challenge in medical community. This study investigated the biological effects of the demethylase fat mass and obesity-associated protein (FTO) on burn injury. FTO protein level in burn skin tissues of patients was measured with Western blot assay. Keratinocytes (HaCaT cells) were given heat stimulation to induce an in vitro burn injury model, and then transfected with overexpression plasmids of FTO (pcDNA-FTO) or small interfering RNA against FTO (si-FTO). Cell proliferation, migration, and angiogenesis in keratinocytes were evaluated with CCK-8, Transwell, and tube formation assays, respectively. Tissue factor pathway inhibitor-2 (TFPI-2) m6A methylation level was detected with MeRIP‑qPCR assay. Then rescue experiments were conducted to explore the effects of FTO/TFPI-2 axis on keratinocyte functions. Lentivirus carrying FTO overexpression plasmids was injected into a burn rat model to detect its effects on wound healing and depressive-like behaviors in burn rats. FTO was downregulated in burn skin and heat-stimulated keratinocytes. FTO prominently augmented proliferation, migration and angiogenesis in heat-stimulated keratinocytes, while FTO knockdown showed the opposite results. FTO inhibited TFPI-2 expression by FTO-mediated m6A methylation modification. TFPI-2 overexpression abrogated FTO mediated enhancement of proliferation, migration and angiogenesis in keratinocytes. Additionally, FTO overexpression accelerated wound healing and improved depressive-like behaviors in burn rat model. FTO prominently augmented proliferation, migration and angiogenesis in heat-stimulated keratinocytes though inhibiting TFPI-2, and then improved wound healing and depressive-like behaviors.

Abstract B043: Tissue Factor Pathway Inhibitor (TFPI), a novel invasion and therapy target in glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is a deadly brain cancer with a 4-10% five-year survival rate. Even with the advent of temozolomide and tumor treating fields, at best GBM patients can hope for a median survival of approximately 25 months. The major cause of poor survivorship is the recurrence of inoperable therapy resistant secondary tumors. With the poor survivorship, there is an urgent need for novel therapy regimens. We propose a novel therapy and metastatic target for GBM treatment. Tissue Factor Pathway Inhibitor (TFPI) is a serine proteinase inhibitor. Classically, TFPI inhibits FXa and/or TF-VIIa to reduce blood coagulation. Recently, TFPI expression have been correlated with solid tumors such as breast, pancreatic, colorectal and brain tumors. However, it is unknown how TFPI contributes to cancer disease. We observed that TFPI increased expression correlated with GBM tumor samples. Additionally, GBM patients with high expression of TFPI had a poorer survivorship than those with low expression of TFPI. Using live cell imaging, we found that TFPI is required for cell motility, an initial and required step in cancer cell invasion. Additionally, we found that loss of TFPI increased 53bp1 foci formation. Increased 53bp1 foci formation is an indication of defunct DNA double stranded break repair. We found that loss of TFPI sensitizes cells to ionizing radiation another indication of a dysfunctional DNA double stranded break repair program. We found that loss of TFPI sensitizes cells to Parp-1 inhibition, a hallmark of a defunct homologous recombination repair program. Homologous recombination and non-homologous end-joining are the two major repair pathways for DNA double stranded breaks. Based on these preliminary data, we hypothesize that TFPI plays role in motility/invasion and DNA repair, which leads to therapy resistance and metastasis in GBM. TFPI is an attractive radio-sensitizer for GBM treatment since radiation is a first-line therapy modality. Additionally, Parp-1 inhibition is currently in clinical trials for GBM treatments. Lastly, there is an FDA approved inhibitor for TFPI, ANDEXXA and several TFPI antibodies in clinical trials. Our future endeavors are to elucidate the mechanisms by which TFPI regulates DNA repair and cancer cell invasion. These findings will lay down the foundation for using TFPI inhibitors as a concurrent treatment plan to standard of care. Citation Format: Tuyen T. Dang, Julio C. Morales. Tissue Factor Pathway Inhibitor (TFPI), a novel invasion and therapy target in glioblastoma multiforme [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B043.

A vasculogenic mimicry prognostic signature associated with immune signature in human gastric cancer.

Gastric cancer (GC) is one of the most lethal malignant tumors worldwide with poor outcomes. Vascular mimicry (VM) is an alternative blood supply to tumors that is independent of endothelial cells or angiogenesis. Previous studies have shown that VM was associated with poor prognosis in patients with GC, but the underlying mechanisms and the relationship between VM and immune infiltration of GC have not been well studied. In this study, expression profiles from VM-related genes were retrieved from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Cox regression was performed to identify key VM-related genes for survival. Subsequently, a novel risk score model in GC named VM index and a nomogram was constructed. In addition, the expression of one key VM-related gene (serpin family F member 1, SERPINF1) was validated in 33 GC tissues and 23 paracancer tissues using immunohistochemistry staining. Univariate and multivariate Cox regression suggested that SERPINF1 and tissue factor pathway inhibitor 2 (TFPI2) were independent risk factors for the prognosis of patients with GC. The AUC (> 0.7) indicated the satisfactory discriminative ability of the nomogram. SsGESA and ESTIMATE showed that higher expression of SERPINF1 and TFPI2 is associated with immune infiltration of GC. Immunohistochemistry staining confirmed that the expression of SERPINF1 protein was significantly higher in GC tissues than that in paracancer tissues. A VM index and a nomogram were constructed and showed satisfactory predictive performance. In addition, VM was confirmed to be widely involved in immune infiltration, suggesting that VM could be a promising target in guiding immunotherapy. Taken together, we identified SERPINF1 and TFPI2 as immunologic and prognostic biomarkers related to VM in GC.

Exosomal miR-195 in hUC-MSCs alleviates hypoxia-induced damage of trophoblast cells through tissue factor pathway inhibitor 2

MicroRNA-195 (miR-195) was decreased in the patients with pre-eclampsia (PE), which was implicated to modulate PE. Moreover, tissue factor pathway inhibitor 2 (TFPI2), which was highly expressed in the placenta of PE patients, was negatively correlated with miR-195 levels. This study aimed to explore the role of miR-195 in the cell therapy for the treatment of PE and the underlying mechanisms. Human umbilical cord mesenchymal stem cells (hUC-MSCs) were transfected with miR-195 mimic or mimic negative control to extract exosomes. HTR8/SVneo was incubated under hypoxia condition to induce cell damage, and co-co-cultured with exosomes derived from hUC-MSCs to evaluate its effect. Hypoxia time-dependently caused a decrease on miR-195 level with an increase on TFPI2 expression in HTR8/SVneo. MiR-195 directly bind to TFPI2 and inhibited TFPI2 expression in hUC-MSCs. Moreover, hypoxia-induced cell damage in HTR8/SVneo was significantly attenuated by co-culture with hUC-MSC-derived exosomes. Exosomes extracted from miR-195-overexpressed hUC-MSCs, could further ameliorate hypoxia-induced cell damage, due to the excessive amount of miR-195 delivered by exosomes. Exosomal miR-195 in hUC-MSCs alleviated hypoxia-induced cell damage through TFPI2, which might provide a potential therapeutic approach for pre-eclampsia.

Effects of Aging and Hypertension on the Antithrombotic Function of Atrial Endocardium in Rats.

We have previously reported that atrial endocardial remodeling is induced by atrial fibrillation (AF), and the endocardial dysfunction may be partly responsible for the thrombus formation in the left atrium associated with AF. However, the relationship between the endocardial dysfunction and the epidemiologically determined risk factors of AF-related strokes, including aging, hypertension, and diabetes mellitus, is yet to be elucidated.To test the hypothesis that aging, hypertension, and diabetes mellitus individually impair the atrial endocardial function in conjunction with AF, we have analyzed the expression of the tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) in the atrial endocardium in 30-week-old Wister-Kyoto (WKY), 60-week-old WKY, 30-week-old spontaneously hypertensive rats (SHR), and 30-week Goto-Kakizaki (GK) rats during normal sinus rhythm and after rapid atrial pacing at 1200 bpm for 8 hours, using Western blotting and immunohistochemical analysis. Even during sinus rhythm, the TFPI and TM expressions were noted to be remarkably downregulated in the atrial endocardium among 60-week-old WKY rats. In contrast, in SHR rats, only the TFPI expression has significantly decreased, while TM was preserved to the same level of control 30-week-old WKY rats. Rapid atrial pacing significantly reduced the TM and TFPI expression similarly in each model, thereby augmenting the endocardial dysfunction during normal sinus rhythm individually induced by the risk factors themselves prior to AF.Aging and hypertension, both of which are epidemiologically well-known risk factors for strokes in AF, have been associated with a specific atrial endocardial impairment prior to AF that could additionally disturb the antithrombotic function of the atrial endocardium.

Androgen-dependent tissue factor pathway inhibitor regulating protein: a review of its peripheral actions and association with cardiometabolic diseases

The first genome-wide association study on coronary artery disease (CAD) in the Han Chinese population identified C6orf105 as a susceptibility gene. The C6orf105 gene was later found to encode for a protein that regulates tissue factor pathway inhibitor (TFPI) expression in endothelial cells in an androgen-dependent manner, and the novel protein was thus termed androgen-dependent TFPI-regulating protein (ADTRP). Since the identification of ADTRP, there have been several studies associating genetic variants on the ADTRP gene with CAD risk, as well as research providing mechanistic insights on this novel protein and its functional role. ADTRP is a membrane protein, whose expression is upregulated by androgen, GATA-binding protein 2, oxidized low-density lipoprotein, peroxisome proliferator-activated receptors, and low-density lipoprotein receptors. ADTRP regulates multiple downstream targets involved in coagulation, inflammation, endothelial function, and vascular integrity. In addition, ADTRP functions as a fatty acid esters of hydroxy fatty acid (FAHFA)-specific hydrolase that is involved in energy metabolism. Current evidence suggests that ADTRP may play a role in the pathogenesis of atherosclerosis, CAD, obesity, and metabolic disorders. This review summarizes the current literature on ADTRP, with a focus on the peripheral actions of ADTRP, including expression, genetic variations, signaling pathways, and function. The evidence linking ADTRP and cardiometabolic diseases will also be discussed.

CD248 enhances tissue factor procoagulant function, promoting arterial and venous thrombosis in mouse models

BackgroundCD248 is a pro‐inflammatory, transmembrane glycoprotein expressed by vascular smooth muscle cells (VSMC), monocytes/macrophages, and other cells of mesenchymal origin. Its distribution and properties are reminiscent of those of the initiator of coagulation, tissue factor (TF). ObjectiveWe examined whether CD248 also participates in thrombosis. MethodsWe evaluated the role of CD248 in coagulation using mouse models of vascular injury, and by assessing its functional interaction with the TF‐factor VIIa (FVIIa)‐factor X (FX) complex. ResultsThe time to ferric chloride‐induced occlusion of the carotid artery in CD248 knockout (KO) mice was significantly longer than in wild‐type (WT) mice. In an inferior vena cava (IVC) stenosis model of thrombosis, lack of CD248 conferred relative resistance to thrombus formation compared to WT mice. Levels of circulating cells and coagulation factors, prothrombin time, activated partial thromboplastin time, and tail bleeding times were similar in both groups. Proximity ligation assays revealed that TF and CD248 are <40 nm apart, suggesting a potential functional relationship. Expression of CD248 by murine and human VSMCs, and by a monocytic cell line, significantly augmented TF‐FVIIa‐mediated activation of FX, which was not due to differential expression or encryption of TF, altered exposure of phosphatidylserine or differences in tissue factor pathway inhibitor expression. Rather, conformation‐specific antibodies showed that CD248 induces allosteric changes in the TF‐FVIIa‐FX complex that facilitates FX activation by TF‐FVIIa. ConclusionCD248 is a newly uncovered protein partner and potential therapeutic target in the TF‐FVIIa‐FX macromolecular complex that modulates coagulation.

Knockdown screening of chromatin binding and regulatory proteins in zebrafish identified Suz12b as a regulator of tfpia and an antithrombotic drug target

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits factor VIIa and Xa in the coagulation cascade. It has been shown that forkhead box P3 protein is a TFPI transcriptional repressor. However, there are no studies on chromatin remodeling that control TFPI expression. We hypothesized that the genome-wide knockdowns of the chromatin binding and regulatory proteins (CBRPs) in zebrafish could identify novel tfpia gene regulators. As an initial step, we selected 69 CBRP genes from the list of zebrafish thrombocyte-expressed genes. We then performed a 3-gene piggyback knockdown screen of these 69 genes, followed by quantification of tfpia mRNA levels. The results revealed that knockdown of brd7, ing2, ing3, ing4, and suz12b increased tfpia mRNA levels. The simultaneous knockdown of these 5 genes also increased tfpia mRNA levels. We also performed individual gene and simultaneous 5-gene knockdowns on the 5 genes in zebrafish larvae. We found that after laser injury, it took a longer time for the formation of the thrombus to occlude the caudal vessel compared to the control larvae. We then treated the larvae and adults with a chemical UNC6852 known to proteolytically degrade polycomb repressor complex 2, where SUZ12 is a member, and observed prolongation of time to occlude (TTO) the caudal vein after laser injury and increased tfpia mRNA levels in larvae and adults, respectively. In summary, our results have identified novel epigenetic regulators for tfpia and exploited this information to discover a drug that enhances tfpia mRNA levels and prolongation of TTO. This discovery provides the basis for testing whether UNC6852 could be used as an antithrombotic drug. This approach could be used to study the regulation of other plasma proteins, including coagulant and anticoagulant factors.

TFPI2 Promotes Perivascular Migration in an Angiotropism Model of Melanoma

PurposeAngiotropism is the process by which cancer cells attach to and migrate along blood vessels to acquire vasculature, disseminate, and metastasize. However, the molecular basis for such vessel–tumor interactions has not been fully elucidated, partly due to limited experimental models. In this study, we aimed to observe and explore the molecular mechanism underlying angiotropism in melanoma.MethodsTo monitor the interactions of human melanoma cells with the vasculature in vivo, a murine coxenograft model was employed by co-injecting highly and poorly invasive melanoma cells subcutaneously. To identify key pathways and genes involved in the angiotropic phenotype of melanoma, analysis of differentially expressed genes (DEGs) and gene set enrichment analysis (GSEA) were performed. The role of tissue factor pathway inhibitor 2 (TFPI2) in angiotropism was evaluated by immunostaining, adhesion assay, shRNA, and in vivo tumorigenicity. Angiotropism and TFPI2 expression were examined in surgical specimens of melanoma by immunohistochemical staining. Data from The Cancer Genome Atlas (TCGA) were analyzed to explore the expression and prognostic implications of TFPI2 in uveal and cutaneous melanoma.ResultsHighly invasive melanoma cells spread along the branches of intratumor blood vessels to the leading edge of invasion in the coxenograft model, resembling angiotropic migration. Mechanisms underlying angiotropism were primarily associated with molecular function regulators, regulation of cell population proliferation, developmental processes, cell differentiation, responses to cytokines and cell motility/locomotion. TFPI2 downregulation weakened the perivascular migration of highly invasive melanoma cells. High levels of TFPI2 were correlated with worse and better survival in uveal and cutaneous melanoma, respectively.ConclusionThese results provide a straightforward in vivo model for the observation of angiotropism and suggest that TFPI2 could inhibit the angiotropic phenotype of melanoma.

Circ_0015382 is associated with preeclampsia and regulates biological behaviors of trophoblast cells through miR-149-5p/TFPI2 axis

IntroductionCirc_0015382 expression was found to be up-regulated in preeclampsia (PE) placenta tissues, while the role and molecular mechanisms of circ_0015382 in PE remain unclear. MethodsThe expression of circ_0015382, microRNA (miR)-149-5p, and tissue factor pathway inhibitor 2 (TFPI2) was measured using quantitative real-time polymerase chain reaction and Western blot. Cell proliferation, migration, invasion, apoptosis, and cell cycle, were detected using cell counting kit-8, transwell, and flow cytometry assays, respectively. The direct interaction between miR-149-5p and circ_0015382 or TFPI2 was analyzed using the dual-luciferase reporter assay. ResultsCirc_0015382 was highly expressed in placental tissues of PE. Overexpression of circ_0015382 suppressed trophoblast cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT), but induced apoptosis and cell cycle progression, while circ_0015382 knockdown showed inverse effects. MiR-149-5p was confirmed to be a target of circ_0015382, and silencing miR-149-5p reversed the regulatory effects of circ_0015382 knockdown on trophoblast cell biological behaviors. MiR-149-5P was expressed at lower levels in placental tissues of PE, while the expression of its target TFPI2 was higher. Importantly, circ_0015382 could regulate TFPI2 expression via miR-149-5p. Additionally, miR-149-5p was shown to promote trophoblast cell growth, migration, invasion and EMT through TFPI2. DiscussionCirc_0015382 was associated with the onset and development of PE through suppressing trophoblast cell growth, migration, invasion and EMT via miR-149-5p/TFPI2 axis, revealing a new insight into the pathogenesis of PE and a potential therapeutic target for PE treatment.

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.