Parenteral nutrition emulsion inhibits CYP3A4 in an iPSC derived liver organoids testing platform.

OBJECTIVES: Parenteral nutrition (PN) is used for patients of varying ages with intestinal failure to supplement calories. Premature newborns with low birth weight are at a high risk for developing PN associated liver disease (PNALD) including steatosis, cholestasis, and gallbladder sludge/stones. To optimize nutrition regimens, models are required to predict PNALD. METHODS: We have exploited induced pluripotent stem cell derived liver organoids to provide a testing platform for PNALD. Liver organoids mimic the developing liver and contain the different hepatic cell types. The organoids have an early postnatal maturity making them a suitable model for premature newborns. To mimic PN treatment we used medium supplemented with either clinoleic (80% olive oil/20% soybean oil) or intralipid (100% soybean oil) for 7 days. RESULTS: Homogenous HNF4a staining was found in all organoids and PN treatments caused accumulation of lipids in hepatocytes. Organoids exhibited a dose dependent decrease in CYP3A4 activity and expression of hepatocyte functional genes. The lipid emulsions did not affect overall organoid viability and glucose levels had no contributory effect to the observed results. CONCLUSIONS: Liver organoids could be utilized as a potential screening platform for the development of new, less hepatotoxic PN solutions. Both lipid treatments caused hepatic lipid accumulation, a significant decrease in CYP3A4 activity and a decrease in the RNA levels of both CYP3A4 and CYP1A2 in a dose dependent manner. The presence of high glucose had no additive effect, while Clinoleic at high dose, caused significant upregulation of interleukin 6 and TLR4 expression.

FSAP Protects against Histone-Mediated Increase in Endothelial Permeability In Vitro.

Factor-VII-activating protease (FSAP) is involved in the regulation of hemostasis and inflammation. Extracellular histones play a role in inflammation and the conversion of latent pro-FSAP into active FSAP. FSAP has been shown to regulate endothelial permeability, but the mechanisms are not clear. Here, we have investigated the effects of FSAP on endothelial permeability in vitro. A mixture of histones from calf thymus stimulated permeability, and the wild-type (WT) serine protease domain (SPD) of FSAP blocked this effect. WT-SPD-FSAP did not influence permeability on its own, nor that stimulated by thrombin or vascular endothelial growth factor (VEGF)-A165. Histones induced a large-scale rearrangement of the junction proteins VE-cadherin and zona occludens-1 from a clear junctional distribution to a diffuse pattern. The presence of WT-SPD-FSAP inhibited these changes. Permeability changes by histones were blocked by both TLR-2 and TLR4 blocking antibodies. Histones upregulated the expression of TLR-2, but not TLR-4, in HUVEC cells, and WT-SPD-FSAP abolished the upregulation of TLR-2 expression. An inactive variant, Marburg I (MI)-SPD-FSAP, did not have any of these effects. The inhibition of histone-mediated permeability may be an important function of FSAP with relevance to sepsis, trauma, and stroke and the need to be investigated further in in vivo experiments.

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.

EPAS1/HIF-2 alpha-mediated downregulation of tissue factor pathway inhibitor leads to a pro-thrombotic potential in endothelial cells.

Neovascularization and hemorrhaging are evident in advanced atherosclerotic plaques due to hypoxic conditions, and mediate the accumulation of metabolic substrates, inflammatory cells, lipids, and other blood born factors inside the plaque. Tissue factor (TF) pathway inhibitor (TFPI) is mainly expressed by endothelial cells and is the endogenous inhibitor of the coagulation activator TF, which together with the downstream product thrombin can drive plaque progression and atherogenesis. We aimed to investigate the effect of hypoxic conditions on endothelial cell expression and activity of TFPI and TF that are important in coagulation initiation. Hypoxia was induced in primary human umbilical vein endothelial cells using chemicals or 1% oxygen tension, and mRNA and protein expressions were measured using qRT-PCR, ELISA, and Western blot analysis. Microscopy of fluorescence-labeled cells was used to visualize cell-associated TFPI. Cell-surface factor Xa (FXa) activity was measured using a two-stage chromogenic substrate method. We found that hypoxia reduced the TFPI mRNA and protein levels and increased the TF mRNA expression in a dose-dependent manner. The effect on TFPI was apparent on all the protein pools of TFPI, i.e., secreted TFPI, cell-surface associated TFPI, and intracellular TFPI, and seemed to be dependent upon hypoxia inducible factor-2α (HIF-2α). An increase in FXa activity was also observed on the endothelial cell surface, reflecting an increase in pro-thrombotic potential of the cells. Our findings indicate that hypoxic conditions may enhance the pro-coagulant activity of endothelial cells, which may promote atherogenesis in addition to clinical events and thus the severity of atherosclerotic disorders.

Tissue factor pathway inhibitor attenuates ER stress-induced inflammation in human M2-polarized macrophages.

Endoplasmic reticulum (ER) stress has been shown to play a key role during the initiation and clinical progression of the cardiovascular diseases, such as atherosclerosis. We have recently shown that expression of tissue factor pathway inhibitor (TFPI) in human monocyte-derived macrophages (MDMs) was induced by cholesterol crystals (CC). In the present study we aimed to determine the role of TFPI under ER stress conditions using human MDMs. qRT-PCR and immunohistochemistry analysis were performed to determine the presence of the ER stress marker CCAAT/enhancer binding protein homologous protein (CHOP) and TFPI in human carotid plaque material and also in human MDMs polarized into pro-inflammatory M1 or anti-inflammatory M2 populations. CHOP mRNA levels were upregulated in the plaques compared to healthy vessels, and CHOP protein was localized in the same area as TFPI in the plaques. Both CHOP and TFPI mRNA levels were upregulated after CC treatment, especially in the M2 phenotype, and the ER stress inhibitor 4-phenylbutyric acid (PBA) reversed this effect. Furthermore, CC treatment increased the levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-8, which for TNF-α and IL-8 was inhibited by PBA, and reduced the levels of the anti-inflammatory cytokine IL-10 in M2-polarized macrophages. Knockdown of TFPI prior to CC treatment exacerbated TNF-α and IL-6 levels, but reduced IL-8 and IL-10 levels. Our results show that CC induce TFPI and cytokine expression in M2-polarized macrophages through activation of the ER stress pathway and that TFPI has a protective effect against TNF-α and IL-6 mediated inflammation. These mechanisms may have implications for the pathogenesis of atherosclerosis.

LIGHT/TNFSF14 is increased in patients with type 2 diabetes mellitus and promotes islet cell dysfunction and endothelial cell inflammation in vitro.

AIMS/HYPOTHESIS: Activation of inflammatory pathways is involved in the pathogenesis of type 2 diabetes mellitus. On the basis of its role in vascular inflammation and in metabolic disorders, we hypothesised that the TNF superfamily (TNFSF) member 14 (LIGHT/TNFSF14) could be involved in the pathogenesis of type 2 diabetes mellitus. METHODS: Plasma levels of LIGHT were measured in two cohorts of type 2 diabetes mellitus patients (191 Italian and 40 Norwegian). Human pancreatic islet cells and arterial endothelial cells were used to explore regulation and relevant effects of LIGHT in vitro. RESULTS: Our major findings were: (1) in both diabetic cohorts, plasma levels of LIGHT were significantly raised compared with sex- and age-matched healthy controls (n = 32); (2) enhanced release from activated platelets seems to be an important contributor to the raised LIGHT levels in type 2 diabetes mellitus; (3) in human pancreatic islet cells, inflammatory cytokines increased the release of LIGHT and upregulated mRNA and protein levels of the LIGHT receptors lymphotoxin ß receptor (LTßR) and TNF receptor superfamily member 14 (HVEM/TNFRSF14); (4) in these cells, LIGHT attenuated the insulin release in response to high glucose at least partly via pro-apoptotic effects; and (5) in human arterial endothelial cells, glucose boosted inflammatory response to LIGHT, accompanied by an upregulation of mRNA levels of HVEM (also known as TNFRSF14) and LTßR (also known as LTBR). CONCLUSIONS/INTERPRETATION: Our findings show that patients with type 2 diabetes mellitus are characterised by increased plasma LIGHT levels. Our in vitro findings suggest that LIGHT may contribute to the progression of type 2 diabetes mellitus by attenuating insulin secretion in pancreatic islet cells and by contributing to vascular inflammation.

"iPSC-derived liver organoids and inherited bleeding disorders: Potential and future perspectives".

The bleeding phenotype of hereditary coagulation disorders is caused by the low or undetectable activity of the proteins involved in hemostasis, due to a broad spectrum of genetic alterations. Most of the affected coagulation factors are produced in the liver. Therefore, two-dimensional (2D) cultures of primary human hepatocytes and recombinant overexpression of the factors in non-human cell lines have been primarily used to mimic disease pathogenesis and as a model for innovative therapeutic strategies. However, neither human nor animal cells fully represent the hepatocellular biology and do not harbor the exact genetic background of the patient. As a result, the inability of the current in vitro models in recapitulating the in vivo situation has limited the studies of these inherited coagulation disorders. Induced Pluripotent Stem Cell (iPSC) technology offers a possible solution to overcome these limitations by reprogramming patient somatic cells into an embryonic-like pluripotent state, thus giving the possibility of generating an unlimited number of liver cells needed for modeling or therapeutic purposes. By combining this potential and the recent advances in the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology, it allows for the generation of autologous and gene corrected liver cells in the form of three-dimensional (3D) liver organoids. The organoids recapitulate cellular composition and organization of the liver, providing a more physiological model to study the biology of coagulation proteins and modeling hereditary coagulation disorders. This advanced methodology can pave the way for the development of cell-based therapeutic approaches to treat inherited coagulation disorders. In this review we will explore the use of liver organoids as a state-of-the-art methodology for modeling coagulation factors disorders and the possibilities of using organoid technology to treat the disease.

SARS-CoV-2 vaccines are not associated with hypercoagulability in apparently healthy people.

Background: SARS-CoV-2 adenoviral vector DNA vaccines have been linked to the rare but serious thrombotic postvaccine complication vaccine-induced immune thrombotic thrombocytopenia. This has raised concerns regarding the possibility of increased thrombotic risk after any SARS-CoV-2 vaccines. Objectives: To investigate whether SARS-CoV-2 vaccines cause coagulation activation leading to a hypercoagulable state. Methods: This observational study included 567 health care personnel; 521 were recruited after the first dose of adenoviral vector ChAdOx1-S (Vaxzevria, AstraZeneca) vaccine and 46 were recruited prospectively before vaccination with a messenger RNA (mRNA) vaccine, either Spikevax (Moderna, n = 38) or Comirnaty (Pfizer-BioNTech, n = 8). In the mRNA group, samples were acquired before and 1 to 2 weeks after vaccination. In addition to the prevaccination samples, 56 unvaccinated blood donors were recruited as controls (total n = 102). Thrombin generation, D-dimer levels, and free tissue factor pathway inhibitor (TFPI) levels were analyzed. Results: No participant experienced thrombosis, vaccine-induced immune thrombotic thrombocytopenia, or thrombocytopenia (platelet count <100 × 109/L) 1 week to 1 month postvaccination. There was no increase in thrombin generation, D-dimer level, or TFPI level in the ChAdOx1-S vaccine group compared with controls or after the mRNA vaccines compared with baseline values. Eleven of 513 (2.1%) participants vaccinated with ChAdOx1-S had anti-PF4/polyanion antibodies without a concomitant increase in thrombin generation. Conclusion: In this study, SARS-CoV-2 vaccines were not associated with thrombosis, thrombocytopenia, increased thrombin generation, D-dimer levels, or TFPI levels compared with baseline or unvaccinated controls. These findings argue against the subclinical activation of coagulation post-COVID-19 vaccination.

Scalable production of tissue-like vascularized liver organoids from human PSCs.

The lack of physiological parity between 2D cell culture and in vivo culture has led to the development of more organotypic models, such as organoids. Organoid models have been developed for a number of tissues, including the liver. Current organoid protocols are characterized by a reliance on extracellular matrices (ECMs), patterning in 2D culture, costly growth factors and a lack of cellular diversity, structure, and organization. Current hepatic organoid models are generally simplistic and composed of hepatocytes or cholangiocytes, rendering them less physiologically relevant compared to native tissue. We have developed an approach that does not require 2D patterning, is ECM independent, and employs small molecules to mimic embryonic liver development that produces large quantities of liver-like organoids. Using single-cell RNA sequencing and immunofluorescence, we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, and a population of resident macrophages: Kupffer cells. The organoids exhibit key liver functions, including drug metabolism, serum protein production, urea synthesis and coagulation factor production, with preserved post-translational modifications such as N-glycosylation and functionality. The organoids can be transplanted and maintained long term in mice producing human albumin. The organoids exhibit a complex cellular repertoire reflective of the organ and have de novo vascularization and liver-like function. These characteristics are a prerequisite for many applications from cellular therapy, tissue engineering, drug toxicity assessment, and disease modeling to basic developmental biology.

Coagulation Factor V (F5) is an Estrogen-Responsive Gene in Breast Cancer Cells.

Most breast cancers express estrogen receptor (ER) where estrogen signaling plays an important role. Cancer contributes to activation of the coagulation system leading to an imbalance in the hemostatic system, and coagulation factor (F) V, which is a key regulator of blood coagulation, has been shown to be increased in breast tumors. Thus, the molecular association between estrogens and FV was explored. Stimulation with 17-ß-estradiol (E2) or 17-ß-ethinylestradiol (EE2) resulted in a time- and dose-dependent increase in F5 messenger RNA and FV protein in ERα-positive MCF-7 cells. Pretreatment with the ER antagonist fulvestrant or knockdown of ERα prior to stimulation with E2 counteracted this effect. Three ERα-binding half-sites were identified in the promoter region of the F5 gene in silico. Reporter gene analysis showed that all three half-sites were involved in the estrogen-induced gene regulation in vitro, as the effect was abolished only when all half-sites were mutated. High F5 levels in ER-positive breast tumors were associated with increased relapse-free survival of breast cancer patients.

Downregulation of TFPI in breast cancer cells induces tyrosine phosphorylation signaling and increases metastatic growth by stimulating cell motility.

BACKGROUND: Increased hemostatic activity is common in many cancer types and often causes additional complications and even death. Circumstantial evidence suggests that tissue factor pathway inhibitor-1 (TFPI) plays a role in cancer development. We recently reported that downregulation of TFPI inhibited apoptosis in a breast cancer cell line. In this study, we investigated the effects of TFPI on self-sustained growth and motility of these cells, and of another invasive breast cancer cell type (MDA-MB-231). METHODS: Stable cell lines with TFPI (both α and ß) and only TFPIß downregulated were created using RNA interference technology. We investigated the ability of the transduced cells to grow, when seeded at low densities, and to form colonies, along with metastatic characteristics such as adhesion, migration and invasion. RESULTS: Downregulation of TFPI was associated with increased self-sustained cell growth. An increase in cell attachment and spreading was observed to collagen type I, together with elevated levels of integrin α2. Downregulation of TFPI also stimulated migration and invasion of cells, and elevated MMP activity was involved in the increased invasion observed. Surprisingly, equivalent results were observed when TFPIß was downregulated, revealing a novel function of this isoform in cancer metastasis. CONCLUSIONS: Our results suggest an anti-metastatic effect of TFPI and may provide a novel therapeutic approach in cancer.

Accumulation and effects of natural mixtures of persistent organic pollutants (POP) in Zebrafish after two generations of exposure.

Effects of exposure to environmentally realistic mixtures of persistent organic pollutants (POP) harvested from aquatic ecosystems in Norway were studied in an in vivo zebrafish model. POP were extracted from burbot (Lota lota) liver from two separate lakes, Lake Losna and Lake Mjøsa, and exposed to zebrafish through the diet in a two-generation study. Effects on survival, growth, sex ratio, and timing of puberty were investigated. In addition, the biomarkers 7-ethoxyresorufin O-deethylase (EROD) and vitellogenin (Vtg) were measured. The ratios of contaminant levels in extracts collected from Lake Mjøsa:Lake Losna were 6, 10, and 270 for polychlorinated biphenyls (PCB), dichlorodiphenyltrichloroethanes (DDT), and polybrominated diphenyl ethers (PBDE), respectively. The concentration range of POP measured in zebrafish was lower than in burbot originating from Lake Mjøsa, but comparable to concentrations previously reported in humans and wildlife. The results showed that exposure to environmentally realistic mixtures of POP exerted a negative effect on survival of fish in both generations. The marked drop in survival during 9-20 days post fertilization (dpf) suggested that this period may be a critical window for development. In both generations an earlier onset of puberty was observed and a higher proportion of males than females was noted in exposed fish compared to controls. Suprising effects of exposure were found on body weight. In the first generation (F(0)), body weight was significantly higher in both exposure groups compared to controls, while in the next generation (F(1)) the same exposures were associated with a decrease in body weight. Zebrafish exposed to relatively low quantities of POP showed a significant induction of biomarkers (EROD and Vtg), while fish exposed to higher exposure doses did not demonstrate induction.

Molecular Characterization of Two Homozygous Factor VII Variants Associated with Intracranial Bleeding.

Clinical parameters have been extensively studied in factor (F) VII deficiency, but the knowledge of molecular mechanisms of this disease is scarce. We report on three probands with intracranial bleeds at an early age, one of which had concomitant high titer of FVII inhibitor. The aim of the present study was to identify the causative mutations and to elucidate the underlying molecular mechanisms. All nine F7 exons were sequenced in the probands and the closest family members. A homozygous deletion in exon 1, leading to a frame shift and generation of a premature stop codon (p.C10Pfs*16), was found in proband 1. Probands 2 and 3 (siblings) were homozygous for a missense mutation in exon 8, resulting in a glycine (G) to arginine (R) substitution at amino acid 240 (p.G240R). All probands had severely reduced FVII activity (FVII:C < 1 IU/dL). Treatment consisted of recombinant FVIIa and/or plasma concentrate, and proband 1 developed a FVII inhibitor shortly after initiation of treatment. The FVII variants were overexpressed in mammalian cell lines. No FVII protein was produced in cells expressing the p.C10Pfs*16 variant, and the inhibitor development in proband 1 was likely linked to the complete absence of circulating FVII. Structural analysis suggested that the G to R substitution in FVII found in probands 2 and 3 would destabilize the protein structure, and cell studies demonstrated a defective intracellular transport and increased endoplasmic reticulum stress. The molecular mechanism underlying the p.G240R variant could be reduced secretion caused by protein destabilization and misfolding.

Functional characterization of polymorphisms in the human TFPI gene.

Tissue factor pathway inhibitor (TFPI) is the primary physiological inhibitor of tissue factor (TF) induced coagulation. Low plasma TFPI levels have been shown to be associated with increased risk of arterial and venous thrombosis. Several clinical studies have reported that single nucleotide polymorphisms (SNPs) in the regulatory regions of the gene, such as the -287T/C, the -399C/T, and the -33T/C SNPs, may affect plasma TFPI levels. However, molecular studies investigating the functionality of the polymorphisms are lacking. In this study, we found that the -287C and -399T alleles affected the activity of the promoter using a reporter gene system. This was also the case for the -33T/C polymorphism. An association regarding the transcriptional activity of the reporter gene was detected between the -287C allele and the -33T/C polymorphism. Analysis of the polymorphic sites with electrophoretic mobility shift assay (EMSA) showed that all three polymorphisms potentially alter DNA-protein interactions. Based on these findings, we speculate that the -287C and the -33C alleles can be associated with lowered risk of thrombosis.

Overexpression of both TFPIα and TFPIß induces apoptosis and expression of genes involved in the death receptor pathway in breast cancer cells.

Thrombosis is a major complication and an important cause of death in cancer patients. Tumor cells may trigger coagulation and induce a prothrombotic phenotype, which in return may enhance angiogenesis, tumor growth, and metastasis. Tissue factor pathway inhibitor (TFPI) has been reported to reduce tumor growth and metastasis in vivo and to induce apoptosis and inhibit proliferation in normal cells in vitro. However, no effect has so far been observed in cancer cells. We therefore aimed to characterize the functional effects of ectopic overexpression and endogenous downregulation of TFPI in cancer cells, and to elucidate possible mechanisms involved. The tumor derived breast cancer cells SK-BR-3 and Sum102 were used to construct stable cell lines overexpressing TFPIα and TFPIß, and with TFPI knocked down, respectively. Effects of altered TFPI expression were evaluated by measuring apoptosis and proliferation of the cells, and gene expressions were analyzed using PCR arrays. Increased DNA fragmentation and Caspase 3 activity was observed in SK-BR-3 cells overexpressing TFPIα and TFPIß, while a decrease in apoptosis was seen in Sum102 cells with TFPI expression knocked down. An increase and reduction in expression of pro- and anti-apoptotic genes, respectively, were seen in TFPI overexpressing cells, and the majority of the upregulated genes encoded proteins involved in the death receptor pathway, among them the death receptor ligand TNF-α. In conclusion, TFPIα and TFPIß induced apoptosis in breast cancer cells and increased expression of apoptotic genes indicating a possible involvement of the death receptor pathway.

Natural mixtures of persistent organic pollutants (POP) increase weight gain, advance puberty, and induce changes in gene expression associated with steroid hormones and obesity in female zebrafish.

In the present study, developmental and reproductive effects of lifelong exposure to environmental relevant concentrations of two natural mixtures of persistent organic pollutants (POP) were investigated using classical and molecular methods in a controlled zebrafish model. The mixtures used were extracted from burbot (Lota lota) liver originating from freshwater systems in Norway: one mixture with high levels and one mixture with background levels of polybrominated diphenyl ethers (PBDE), polychlorinated biphenyls (PCB), and dichlorodiphenyltrichloroethane metabolites (DDT). The concentration of POP measured in the zebrafish ranged from levels detected in wild fish from Lake Mjøsa to concentrations reported in human and wildlife populations, indicating that the experimental fish were exposed to concentrations comparable with wild fish. Phenotypic effects observed in both exposure groups included earlier onset of puberty, increased male/female sex ratio, and differences in body weight at 5 mo of age. Interestingly, genome-wide transcription profiling showed changes in regulation of genes involved in endocrine signaling and growth. The transcriptomics changes include key regulator genes for steroid hormone functions (ncoa3), and growth (c/ebp, ncoa3). The effects observed in the experimental zebrafish model raise the question whether chemical pollution represents a risk to reproductive health of wild fish inhabitating the freshwater system.

Coagulation factor V is a marker of tumor-infiltrating immune cells in breast cancer.

Background: Factor (F) V is an essential cofactor in blood coagulation, however, F5 expression in breast tumors has also been linked to tumor aggressiveness and overall survival. The specific role of FV in breast cancer is yet unknown. We therefore aimed at dissecting the biological relevance of FV in breast cancer. Methods: Gene expression data from a Scandinavian breast cancer cohort (n = 363) and the cancer genome atlas (TCGA) (n = 981) and 12 replication cohorts were used to search for F5 co-expressed genes, followed by gene ontology analysis. Pathological and bioinformatic tools were used to evaluate immune cell infiltration and tumor purity. T cell activation, proliferation and migration were studied in FV treated Jurkat T cells. Results: F5 co-expressed genes were mainly associated with immune system processes and cell activation. Tumors with high expression of F5 were more infiltrated with both lymphoid (T cells, NK cells, and B cells) and myeloid cells (macrophages and dendritic cells), and F5 expression was negatively correlated with tumor purity (ρ = -0.32). Confirming a prognostic role, data from the Kaplan-Meier plotter showed that high F5 expression was associated with improved relapse-free survival. The strongest association was observed in basal-like breast cancer (HR = 0.55; 95% CI, 0.42-0.71). Exogenous FV did not substantially affect activation, proliferation or migration of human T cells. Conclusions: F5 was identified as a novel marker of immune cell infiltration in breast cancer, and the prognostic role of F5 was verified. FV emerge as an interesting immunological biomarker with potential therapeutic relevance for the cancer-inflammation-thrombosis circuit.

Indirect regulation of TFPI-2 expression by miR-494 in breast cancer cells.

TFPI-2 has been shown to be involved in breast cancer pathogenesis by inhibiting extracellular matrix degradation, and low levels are associated with disease progression. As microRNA-494 (miR-494) protects against breast cancer progression, we investigated whether miR-494 is involved in the regulation of TFPI-2 in MCF-7 breast cancer cells. TFPI-2 mRNA and protein levels increased after transfection with miR-494 mimic, and TFPI-2 mRNA and miR-494 levels correlated positively in tumors from breast cancer patients. No specific binding sites for miR-494 in the 3'-untranslated region (UTR) of TFPI2 were identified; however, miR-494 was predicted in silico to bind 3'-UTR of the transcription factors AHR and ELF-1, which have potential binding sites in the TFPI2 promoter. ELF-1 mRNA was downregulated whereas AHR mRNA levels were upregulated after transfection with miR-494 mimic. Knockdown of ELF-1 and AHR increased and reduced TFPI-2 mRNA levels, respectively. Increased luciferase activity was seen when TFPI-2 promoter constructs containing the potential AHR or ELF-1 binding sites were co-transfected with miR-494 mimic. In conclusion, TFPI-2 mRNA levels were upregulated by miR-494 in MCF-7 breast cancer cells most likely by an indirect association where miR-494 targeted the transcription factors AHR and ELF-1. This association was supported in a breast cancer cohort.

Transcriptional regulation in liver and testis associated with developmental and reproductive effects in male zebrafish exposed to natural mixtures of persistent organic pollutants (POP).

Persistent organic pollutants (POP) occur as mixtures in nature and it is difficult to predict the toxicity of such mixtures based on knowledge about toxicity and mechanisms of action for single compounds. The present knowledge on the combined toxic effects and modes of actions of exposure to mixtures is limited. Thus, the scientifically based hazard and risk assessment of POP requires analytical and toxicological data from studies with environmental mixtures of POP. The application of genome wide transcription profiling in toxicology, in combination with classical endpoints, will improve the current understanding of the mechanisms of toxic processes. Furthermore, gene expression data may be useful in establishing new hypothesis and discovering new biomarkers for known toxicity as well as not yet recognized toxicity endpoints. In the present study, developmental and reproductive effects of lifelong exposure to environmental relevant concentrations of two natural mixtures of POP were investigated using classical and molecular methods in a controlled zebrafish model. The mixtures used were extracted from burbot (Lota lota) liver originating from freshwater systems in Norway: one mixture with high levels and one mixture with background levels of polybrominated diphenyl ethers (PBD), polychlorinated biphenyls (PCB), and DDT. The concentration of POP in the zebrafish ranged from levels detected in wild fish from Lake Mjøsa, to concentrations reported in human and wildlife populations. Phenotypic effects observed in both exposure groups included (1) reduced survival, (2) earlier onset of puberty, (3) increased male/female sex ratio, and (4) differences in body weight at 5 mo of age. Interestingly, genome-wide transcription profiling showed changes in regulation of genes involved in endocrine signaling and growth. The transcriptomics changes included (1) key regulator genes for steroid and thyroid hormone functions (cga, ncoa3), (2) insulin signaling and metabolic homeostasis (pik3r1, pfkfb3, ptb1), and (3) p53 activation (mdm4). The effects observed in the experimental zebrafish model raise the question of whether chemical pollution represents a risk to the reproductive health of wild fish inhabiting the freshwater system.

Estrogen induced expression of tissue factor pathway inhibitor-2 in MCF7 cells involves lysine-specific demethylase 1.

Hormone-sensitive cancers can be influenced by estrogens, a process usually mediated through the estrogen receptor (ER). Tissue factor pathway inhibitor type 2 (TFPI-2) is a Kunitz-type serine protease inhibitor involved in regulating the extracellular matrix. The present study demonstrates that the expression of TFPI-2 can be induced by estrogens. Breast cancer data from GOBO displayed increased levels of TFPI-2 and increased survival in patients with ERα+ tumors. Treatment of MCF7 cells (ERα+) with 17ß-estradiol (E2) or 17α-ethinyl estradiol (EE2) increased TFPI-2 mRNA and protein levels. This effect was mitigated with fulvestrant and by knocking down ERα, indicating that estrogen mediated TFPI-2 induction was through ERα. Upon knock down of DNA cytosine-5 methyltransferase 1 (DNMT1) or lysine-specific demethylase 1 (LSD1) in MCF7 cells, reduced effect of E2 on TFPI-2 mRNA levels was observed. Our data thus suggest that estrogen induced TFPI-2 expression in MCF7 cells is mediated by ERα and also by the action of LSD1.