Factor VII mRNA Research Articles

Factor VIII as a potential player in cancer pathophysiology

BackgroundTrousseau sign was the first demonstration of a close relationship between cancer and thrombosis. Currently, venous thromboembolism (VTE) is five to six times more likely to occur in cancer patients, whereas there is a greater risk of cancer diagnoses following thromboses. In considering novel players, factor VIII (FVIII), an essential coagulation cofactor with emerging extracoagulative functions, has been identified as an independent VTE risk factor in cancer; however, the basis of this increase is unknown. ObjectiveTo investigate the possible direct expression and secretion of FVIII by cancer cells. MethodsBladder cancer, with a high VTE risk, and normal bladder tissue and epithelium, were used to investigate FVIII. Factor VIII protein and secretion were examined in bladder cancer cell lines. Expanding to other cancers, the Cancer Cell line Encyclopedia database was used to analyze FVIII, tissue factor, FV, FVII, FIX, FX, and von Willebrand factor (VWF) mRNA in 811 cell lines subdivided according to origin. Factor VIII protein synthesis, secretion, and bioactivity were investigated in a profile of cancer cell lines of differing origins. Results and conclusionsAlthough expressed in the normal bladder epithelium, FVIII mRNA and protein were higher in matched bladder neoplasms, with synthesis and secretion of bioactive FVIII evident in bladder cancer cells. This can be extended to other cancer cell lines, with a pattern reflecting the tumor origin, and that is independent of VWF and other relevant players in the coagulation cascade. Here, evidence is provided of a possible independent role for FVIII in cancer‐related pathophysiology.

Effects of Shear Stress on Production of FVIII and vWF in a Cell-Based Therapeutic for Hemophilia A.

Microfluidic technology enables recapitulation of organ-level physiology to answer pertinent questions regarding biological systems that otherwise would remain unanswered. We have previously reported on the development of a novel product consisting of human placental cells (PLC) engineered to overexpress a therapeutic factor VIII (FVIII) transgene, mcoET3 (PLC-mcoET3), to treat Hemophilia A (HA). Here, microfluidic devices were manufactured to model the physiological shear stress in liver sinusoids, where infused PLC-mcoET3 are thought to lodge after administration, to help us predict the therapeutic outcome of this novel biological strategy. In addition to the therapeutic transgene, PLC-mcoET3 also constitutively produce endogenous FVIII and von Willebrand factor (vWF), which plays a critical role in FVIII function, immunogenicity, stability, and clearance. While vWF is known to respond to flow by changing conformation, whether and how shear stress affects the production and secretion of vWF and FVIII has not been explored. We demonstrated that exposure of PLC-mcoET3 to physiological levels of shear stress present within the liver sinusoids significantly reduced mRNA levels and secreted FVIII and vWF when compared to static conditions. In contrast, mRNA for the vector-encoded mcoET3 was unaltered by flow. To determine the mechanism responsible for the observed decrease in FVIII and vWF mRNA, PCR arrays were performed to evaluate expression of genes involved in shear mechanosensing pathways. We found that flow conditions led to a significant increase in KLF2, which induces miRNAs that negatively regulate expression of FVIII and vWF, providing a mechanistic explanation for the reduced expression of these proteins in PLC under conditions of flow. In conclusion, microfluidic technology allowed us to unmask novel pathways by which endogenous FVIII and vWF are affected by shear stress, while demonstrating that expression of the therapeutic mcoET3 gene will be maintained in the gene-modified PLCs upon transplantation, irrespective of whether they engraft within sites that expose them to conditions of shear stress.

Restore the Secretion and Activity of Factor VIII Mutants By Proteostasis Regulator

Quantitative or qualitative deficiency in factor VIII (FVIII) result in hemophilia A. FVIII deficiency is caused by broad spectrum mutations, and missense mutation is the most common type of mutation, which lead to various defects of FVIII from decreased secretion to impaired function. Cellular protein homeostasis is disturbed by FVIII mutants, and thus excessive misfolding and degradation of FVIII mutant result in FVIII deficiency. We hypothesized that folding and/or trafficking of FVIII can be enhanced by proteostasis regulators, leading to increased coagulation factor levels sufficient to restore hemostasis. We performed a cell-based screening using a FDA-approved drug library and a natural compound library looking for compounds that enhance secretion of FVIII with a B domain deletion. We identified SAHA (Suberanilohydroxamic acid) as a compound that increased both wild-type (WT) and a missense mutant FVIII. SAHA is a histone deacetylase inhibitor that was also reported to enhance proteostaisis of mutant CFTR, a-1 antitrypsin, lysosomal glucocerebrosidase and the GABA receptor. Here we show that SAHA can increase the secretion and activity of FVIII mutants by enhancing their folding and trafficking as well transcription. To confirm the ability of SAHA to enhance FVIII mutant secretion, we first used the WT FVIII A2 domain and the M614T mutant A2 domain to detect the proper working time and concentration of SAHA in HEK 293T cells. M614T is a missense mutation that was shown to cause secretion defect of FVIII in our previous study. Western blot showed a dose-dependent increase in levels of both A2-WT and A2-M614T in cell culture media between 0 and 2.5ug/ml of SAHA after 24-hour treatment. Levels of both WT and mutant A2 domain dropped when SAHA concentration exceeded 5 ug/ml due to cellular toxicity. SAHA enhanced the secretion of A2-M614T much more than that of A2-WT. Of note, protein levels of both A2-WT and A2-M614T in cell lysate were also slightly elevated by SAHA. Time course study showed that the maximum effect on secretion was achieved after 24 hours of SAHA treatment. We next studied the effect of SAHA on activity and secretion of several full-length FVIII mutants (A469S, R527W, R531G, I566T, N582D, S584T, R593C, M614T, R1997G, R2150C), which were shown to decrease FVIII secretion due to various defects in intracellular trafficking in our previous studies. Except for N582D, activity levels of all FVIII mutants in cell culture media were elevated 3-14 folds after a 24-hour SAHA treatment (P<0.05), whereas the activity level of FVIII WT only increased by ~2 folds (P<0.05). Antigen levels of FVIII mutants in both cell lysates and cell culture media increased by ~3 folds following SAHA treatment (P<0.05). The increase in activities of several FVIII mutants (I566T, S584T, R593C, R1997G, R2150C) is higher than the increase in antigen levels of the same mutants in cell culture media, which indicates that, in addition to enhancing secretion, SAHA treatment also increased the proportions of functional proteins in secreted FVIII mutants in cell culture media. To investigate the mechanism, we studied the interactions of WT FVIII and several responsive mutant FVIII (I566T, S584T, R593C and R1997C) with endoplasmic reticulum (ER) chaperones BiP and calreticulin by co-Immunoprecipitation. The protein levels of BiP and calreticulin in HEK 293T cells were not affected by SAHA. However, the interactions of BiP with both WT FVIII and FVIII mutants were remarkably enhanced by SAHA, while no significant differences in the interactions of calreticulin with FVIII WT or FVIII mutants were observed. The effect of SAHA on FVIII secretion can be blocked by simultaneously treating cells with a BiP inhibitor SubAB. We next measured mRNA levels of BiP as well as FVIII in cells expressing FVIII WT and FVIII mutants with or without SAHA treatment via real time PCR. Similar to protein levels, BiP mRNA levels were not changed by SAHA. However, FVIII mRNA levels were elevated by 1.2 to 3.1 folds (P<0.05) by SAHA, in keeping with the histone deacetylase inhibitor activity of SAHA. In summary, we have shown that SAHA increases FVIII antigen and activity levels in cell culture media by increasing FVIII mRNA levels and by enhancing its folding and trafficking via specifically promoting its interaction with BiP. SAHA is a potential proteostaisis regulator that can increase the secretion and restore the function of certain FVIII missense mutants. Disclosures No relevant conflicts of interest to declare.

Inhibition of the extrinsic or intrinsic coagulation pathway during pneumonia-derived sepsis.

Pneumonia is the most frequent cause of sepsis, and Klebsiella pneumoniae is a common pathogen in pneumonia and sepsis. Infection is associated with activation of the coagulation system. Coagulation can be activated by the extrinsic and intrinsic routes, mediated by factor VII (FVII) and factor XII (FXII), respectively. To determine the role of FVII and FXII in the host response during pneumonia-derived sepsis, mice were treated with specific antisense oligonucleotide (ASO) directed at FVII or FXII for 3 wk before infection with K. pneumoniae via the airways. FVII ASO treatment strongly inhibited hepatic FVII mRNA expression, reduced plasma FVII to ~25% of control, and selectively prolonged the prothrombin time. FXII ASO treatment strongly suppressed hepatic FXII mRNA expression, reduced plasma FXII to ~20% of control, and selectively prolonged the activated partial thromboplastin time. Lungs also expressed FVII mRNA, which was not altered by FVII ASO administration. Very low FXII mRNA levels were detected in lungs, which were not modified by FXII ASO treatment. FVII ASO attenuated systemic activation of coagulation but did not influence fibrin deposition in lung tissue. FVII ASO enhanced bacterial loads in lungs and mitigated sepsis-induced distant organ injury. FXII inhibition did not affect any of the host response parameters measured. These results suggest that partial inhibition of FVII, but not of FXII, modifies the host response to gram-negative pneumonia-derived sepsis.

Human Placenta-Derived Mesenchymal Stem Cells Reduce Mortality and Hematoma Size in a Rat Intracerebral Hemorrhage Model in an Acute Phase.

Intracerebral hemorrhage (ICH) is a critical disease, highly associated with mortality and morbidity. Several studies have demonstrated the beneficial effect of mesenchymal stem cells (MSCs) on ICH, mostly focused on their mid-to-long-term effect. Acute hematoma expansion is one of the most important prognostic factors of ICH. We hypothesized that MSCs would decrease mortality and hematoma size in acute ICH, based on the findings of a few recent researches reporting their effect on blood-brain barrier and endothelial integrity. Rat ICH models were made using bacterial collagenase. One hour after ICH induction, the rats were randomly divided into MSC-treated and control groups. Mortality, hematoma volume, ventricular enlargement, brain edema, and degenerating neuron count were compared at 24 hours after ICH induction. Expression of tight junction proteins (ZO-1, occludin) and coagulation factor VII mRNA was also compared. Mortality rate (50% versus 8.3%), hematoma size, ventricular size, hemispheric enlargement, and degenerating neuron count were significantly lower in the MSC-treated group (p = 0.034, 0.038, 0.001, 0.022, and <0.001, resp.), while the expression of ZO-1 and occludin was higher (p = 0.007 and 0.012). Administration of MSCs may prevent hematoma expansion in the hyperacute stage of ICH and decrease acute mortality by enhancing the endothelial integrity of cerebral vasculature.

Genetic diagnosis in Hemophilia A from southern China: five novel mutations and one preimplantation genetic analysis.

As there is currently no complete cure for hemophilia A (HA), the identification of pathogenic mutations in factor VIII (FVIII) gene from HA patients and carriers, which can contribute to genetic counseling prenatal diagnosis, and preimplantation genetic diagnosis (PGD), is an important step to prevent HA. A total of 14 unrelated Chinese HA subjects (FVIII activity <40%), 20 carrier subjects, three fetuses, and one PGD were included in this study. We first screened for the presence of FVIII intron 22 and intron 1 inversions. Second, the coding region of the FVIII gene was sequenced. For the novel mutations, FVIII mRNA expression was detected by real-time PCR and the protein structures were analyzed by bioinformatic tools. Five novel mutations (c.1A>C, c.304_305insA, c.1594T>A, c.6045G>A, and c.2645_2646insG) were found. The real-time PCR showed that the expression of FVIII mRNAs was lower in HA patients than in normal subjects. Prenatal diagnosis and PGD were successfully performed: Two of three fetuses and four of eight blastomeres were confirmed to be normal. In conclusion, genetic diagnosis of 14 unrelated HA subjects, 20 carrier subjects, three fetuses, and one PGD was successfully performed in our study.

Bioengineering Coagulation Factor VIII through Ancestral Protein Reconstruction

The development of transformative hemophilia A therapeutics has been hindered by the size, instability, immunogenicity and biosynthetic inefficiency of coagulation factor VIII (FVIII). Through the study of FVIII orthologs from existing vertebrate species, we discovered unique molecular, cellular and biochemical properties that can overcome the limitations of human FVIII. This approach facilitated the development of recombinant porcine FVIII for acquired hemophilia A and has enabled low resolution mapping and bioengineering of functional sequence determinants into human FVIII. To further extend this bioengineering approach, we employed a novel methodology termed ancestral protein reconstruction that provides certain advantages over 'rational design' approaches including a priori confidence that each ancestral FVIII is hemostatically functional. First, a mammalian FVIII phylogenetic tree with corresponding ancestral node (An) sequences was constructed through Bayesian inference using both DNA and amino acid-based models in PAML Version 4.1 (Figure 1). The limited availability of non-mammalian sequences precluded accurate ancestor prediction outside of this class. Initially, we selected 14 An-FVIII sequences for reconstruction and subsequent molecular, cellular, biochemical and immunological characterization. Each An-FVIII displayed activity in coagulation assays utilizing human hemophilia A plasma as a substrate thus demonstrating evolutionary mammalian compatibility. Infusion of highly purified preparations of several An-FVIIIs into hemophilia A mice also corrected the bleeding phenotype following a tail transection bleeding challenge confirming in vivo functionality. To study biosynthetic efficiency, secreted FVIII activity and mRNA transcript levels were analyzed following transfection of An-FVIII plasmids into HEK293 and BHK-M cell lines. An-53, common ancestor to rodents and primates, and An-68, ancestor to a subset of current rodents, displayed the highest FVIII biosynthetic efficiencies that were 12 and 15 fold greater than human FVIII, respectively (P = 0.002; Mann Whitney U test). These two An-FVIII sequences share 95 and 87% amino acid identity to human FVIII, respectively. In contrast, intermediate ancestors between An-53 and human FVIII, designated An-55, -56 and -57, do not display enhanced biosynthetic efficiency suggesting that the functional sequence determinant of high expression was lost during primate evolution. Predicting that high expression ancestral FVIIIs would be enabling to gene therapy approaches, An-53, An-68 and human FVIII cDNAs were placed in an AAV expression cassette under the control of a potent liver-specific promoter and the resulting plasmid DNA was infused hydrodynamically into hemophilia A mice. An-53 and An-68, but not human FVIII vector treated animals, achieved sustained, therapeutic plasma FVIII activity levels over 4 weeks (0.1 - 0.6 IU/ml versus <0.01 IU/ml, respectively). Recombinant An-FVIIIs were expressed, purified and biochemically characterized by SDS-PAGE, specific activity, decay following thrombin activation and inhibitor recognition. Early mammalian and all primate lineage thrombin-activated An-FVIII(a) displayed half-lives between 1.5 - 2.2 min that were not distinguishable from human FVIII. We have shown previously that modern murine, porcine, and ovine FVIIIa display significantly longer half-lives and thus this property may have evolved under positive selection. Supporting this conclusion, An-68 and An-78 display prolonged half-lives of 16 and 7 min, respectively. Lastly, the immune recognition of An-FVIIIs by a panel of A2 and C2 domain targeting inhibitory murine monoclonal antibodies as well as hemophilia A inhibitor patient plasmas was examined and many examples of reduced reactivity were revealed, which may enable the development of less immunogenic FVIII products. Herein, we report molecular discoveries that enhance our understanding of FVIII structure/function and provide a blueprint for bioengineering novel FVIII molecules with enhanced properties. These studies also show 'proof of concept' for ancestral protein reconstruction as a powerful approach to studying the biochemistry, molecular biology and evolution of the vertebrate coagulation system, which should enable identification of other new hematological drug targets and candidate biotherapeutics. [Display omitted] DisclosuresSpencer:Expression Therapeutics: Equity Ownership. Doering:Expression Therapeutics: Equity Ownership; Bayer Healthcare: Consultancy, Honoraria, Research Funding.

Relationship between coagulation factor VII and progressive hemorrhage of brain contusion in mice

Objective To study the correlation between the coagulation factor Ⅶ (FⅦ) and progressive hemorrhage after brain contusion in mice and provide the experimental evidence for the clinical application of recombinant human FⅦa. Methods Twelve male BALB/c mice were given liposome-encapsulated FⅦsiRNA via tail vein at doses of 1, 3, 5 and 10 mg/kg with 3 mice per dosage. The other 3 mice received equivalent volume of normal saline as controls. Two days after the injection, mice blood sampling was used to detect FⅦ mRNA expression in liver using real-time PCR, level of plasma FⅦ using ELISA method, and activity of plasma FⅦ using chromogenic substrate assay. The optimal dose at which FⅦ expression was inhibited was determined. Thirty BALB/c male mice were assigned to two groups (n=15 per group) according to the random number table: FⅦ-suppressing group, mice were injected with FⅦsiRNA at the optimal dose and control group, mice were injected with same volume of negative control vector. The model of brain contusion was established in both groups. Volume of hemorrhage following brain contusion was measured at 3, 24 and 72 h postinjury, and hematoma volume at 24 and 48 h postinjury. Results Liposome-encapsulated siRNA delivery down-regulated FⅦ expression in the mouse liver. Level and activity of plasma FⅦ were also reduced significantly. The optimal siRNA dose was 3 mg/kg. At 3, 24 and 72 h postinjury, relative volume of brain hemorrhage in FⅦ-suppressing group was 1.46±0.10, 1.82±0.23 and 2.28±0.15 respectively, significantly higher than that in control group(1.00±0.25, 1.20±0.31 and 1.20±0.22 respectively)(P<0.05). At 24 and 48 h postinjury, volume of hematoma in FⅦ-suppressing group was (6.7±1.5)mm3 and (9.8±1.0)mm3, significantly higher than that in control group [(5.2±1.2)mm3 and (5.5±1.5)mm3](P<0.01). Conclusions Level of FⅦ in vivo relates closely to the progressive hemorrhage of brain contusion in mice. Administration of FⅦ is effective to reduce the incidence of progressive hemorrhage. Key words: Factor Ⅶ; Brain injuries; Cerebral hemorrhage

The production of coagulation factor VII by adipocytes is enhanced by tumor necrosis factor-α or isoproterenol.

A relationship has been reported between blood concentrations of coagulation factor VII (FVII) and obesity. In addition to its role in coagulation, FVII has been shown to inhibit insulin signals in adipocytes. However, the production of FVII by adipocytes remains unclear. We herein investigated the production and secretion of FVII by adipocytes, especially in relation to obesity-related conditions including adipose inflammation and sympathetic nerve activation. C57Bl/6J mice were fed a low- or high-fat diet and the expression of FVII messenger RNA (mRNA) was then examined in adipose tissue. 3T3-L1 cells were used as an adipocyte model for in vitro experiments in which these cells were treated with tumor necrosis factor-α (TNF-α) or isoproterenol. The expression and secretion of FVII were assessed by quantitative real-time PCR, Western blotting and enzyme-linked immunosorbent assays. The expression of FVII mRNA in the adipose tissue of mice fed with high-fat diet was significantly higher than that in mice fed with low-fat diet. Expression of the FVII gene and protein was induced during adipogenesis and maintained in mature adipocytes. The expression and secretion of FVII mRNA were increased in the culture medium of 3T3-L1 adipocytes treated with TNF-α, and these effects were blocked when these cells were exposed to inhibitors of mitogen-activated kinases or NF-κB activation. The β-adrenoceptor agonist isoproterenol stimulated the secretion of FVII from mature adipocytes via the cyclic AMP/protein kinase A pathway. Blockade of secreted FVII with the anti-FVII antibody did not affect the phosphorylation of Akt in the isoproterenol-stimulated adipocytes. Obese adipose tissue produced FVII. The production and secretion of FVII by adipocytes was enhanced by TNF-α or isoproterenol via different mechanisms. These results indicate that FVII is an adipokine that plays an important role in the pathogenesis of obesity.

Ribavirin-induced intracellular GTP depletion activates transcription elongation in coagulation Factor VII gene expression

Coagulation FVII (Factor VII) is a vitamin K-dependent glycoprotein synthesized in hepatocytes. It was reported previously that FVII gene (F7) expression was up-regulated by ribavirin treatment in hepatitis C virus-infected haemophilia patients; however, its precise mechanism is still unknown. In the present study, we investigated the molecular mechanism of ribavirin-induced up-regulation of F7 expression in HepG2 (human hepatoma cell line). We found that intracellular GTP depletion by ribavirin as well as other IMPDH (inosine-5'-monophosphate dehydrogenase) inhibitors, such as mycophenolic acid and 6-mercaptopurine, up-regulated F7 expression. FVII mRNA transcription was mainly enhanced by accelerated transcription elongation, which was mediated by the P-TEFb (positive-transcription elongation factor b) complex, rather than by promoter activation. Ribavirin unregulated ELL (eleven-nineteen lysine-rich leukaemia) 3 mRNA expression before F7 up-regulation. We observed that ribavirin enhanced ELL3 recruitment to F7, whereas knockdown of ELL3 diminished ribavirin-induced FVII mRNA up-regulation. Ribavirin also enhanced recruitment of CDK9 (cyclin-dependent kinase 9) and AFF4 to F7. These data suggest that ribavirin-induced intracellular GTP depletion recruits a super elongation complex containing P-TEFb, AFF4 and ELL3, to F7, and modulates FVII mRNA transcription elongation. Collectively, we have elucidated a basal mechanism for ribavirin-induced FVII mRNA up-regulation by acceleration of transcription elongation, which may be crucial in understanding its pleiotropic functions invivo.

Ribavirin-Induced Intracellular GTP Depletion Upregulates Factor VII Expression

AbstractAbstract 1113In this study, we investigated the molecular basis of upregulation of factor VII (FVII) gene expression by ribavirin, and found that intracellular GTP depletion induced by ribavirin activated FVII gene transcription and modulated transcription elongation.In 2006, Yamamoto et al. reported that anti-hepatitis C virus (HCV) agent ribavirin elevated the activity of FVII in HCV-infected hemophilia patients; however, the precise mechanisms were still unknown. In addition, the anti-HCV mechanisms of ribavirin were not yet fully elucidated, although the extended studies have been done. We investigated the effects of ribavirin in vitro and confirmed the approximately 4-fold upregulation of FVII mRNA by ribavirin treatment in HepG2 cells. FVII mRNA was increased in a dose-dependent manner up to 100μg/mL of ribavirin at a lower concentration than therapeutic concentration of 150μg/mL. FVII mRNA induction by ribavirin was also observed in a time-dependent manner from 24 h to 72 h after treatment.Ribavirin metabolite ribavirin 5'-monophosphate is one of the IMP dehydrogenase (IMPDH) inhibitors, and the other IMPDH inhibitors mycophenolic acid (MPA) and 6-mercaptupurine (6-MP) also induced FVII upregulation. It is well known that inhibition of IMPDH causes intracellular GTP depletion, and guanosine supplementation to salvage GTP could reverse FVII mRNA increase in ribavirin-treated cells. These results indicated that cellular GTP reduction associated with FVII gene upregulation.The mechanisms of gene upregulation by GTP depletion were not elucidated. The promoter activities and mRNA stability of FVII were analyzed under ribavirin treatment. The FVII gene promoter activity was enhanced up to 1.5-fold by ribavirin treatment; however the activation did not reach 4-fold induction of FVII mRNA increase. There was no significant change of FVII mRNA half-life in ribavirin-treated cells. Since the promoter activation might display transcription initiation capacity, the contribution of transcription elongation stage was further investigated.Transcription elongation was regulated by phosphorylation of carbo-terminal domain (CTD) of RNA polymerase II (PolII). Transcription elongation factor P-TEFb (positive-transcription elongation factor b), which consists as a complex of CDK9 and cyclin T, phosphorylates Ser of PolII CTD. The kinase activity of P-TEFb could be inhibited by 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB). In FVII gene upregulation, DRB completely canceled ribavirin-induced FVII mRNA increase. We also performed nuclear run-on assay to verify the potential transcription elongation capacity of paused PolII, and observed a dramatic increase of FVII mRNA in ribavirin-treated cells. These results suggested that ribavirin-induced FVII gene upregulation was caused not only by transcription initiation but also by accelerated transcription elongation rate.There are various transcription factor associated with transcription elongation in addition to P-TEFb, such as elongin, ELL (eleven nineteen-lysine rich leukemia). We found that ELL3, a member of ELL family protein, was upregulated by ribavirin treatment. A ELL3 mRNA increase occurred prior to FVII mRNA upregulation, and the ELL3 upregulation was also canceled by guanosine supplementation. These results indicated ELL3 induction by ribavirin was also a response to cellular GTP depletion. To confirm the contribution of ELL3 protein to FVII gene transcription elongation, we used siRNAs specific to ELL3 and as expected, knockdown of ELL3 resulted in diminished FVII upregulation. A chromatin immunoprecipitation (ChIP) revealed ELL3 recruitment to the FVII gene, and the recruitments of PolII and CDK9 were also enhanced by ribavirin treatment.Taken together, FVII gene upregulation by ribavirin was associated with intracellular GTP depletion. The GTP reduction mainly modulates transcription elongation rate rather than transcription initiation, though the relationships between cellular GTP depletion and enhanced transcription elongation must be investigated. This study uncovered candidate mechanisms of ribavirin and the other IMPDH inhibitors and highlights a development of novel pharmaceutical therapies for hemophilia. Disclosures:No relevant conflicts of interest to declare.

Norethisterone acetate alters coagulation gene expression in vitro in human cell culture

IntroductionBoth oestrogen and progestin and the route of administration have been implicated in cardiovascular and thromboembolic risk in post menopausal hormone users. Transdermal preparations have been reported as safer indicating that liver derived metabolites of oestrogen may be important. The aim of our study was to investigate the in vitro effects of 17β-estradiol, its metabolites, and Norethisterone acetate (NETA) on the expression of coagulation genes in cultured human cells. MethodsHuman hepatocytes and human umbilical vein endothelial cells(HUVECS) were treated with 17β-estradiol, estrone, 2-hydroxyestradiol (2-OH), NETA and NETA/17β-estradiol (10nM) for 24hours. Fibrinogen, factor VII, prothrombin and Plasminogen Activator Inhibitor −1 (PAI-1) mRNA expression was determined in hepatocyte cultures using TaqMan PCR. Tissue Factor (TF), Tissue Factor Pathway Inhibitor (TFPI), tissue plasminogen activator (tPA) and PAI-1 expression was determined in HUVECS. Expression of estrogen receptors was also determined. ResultsFibrinogen and factor VII mRNA expression was upregulated 2–4 fold by estradiol and estrone. Addition of NETA downregulated fibrinogen and prothrombin. PAI-1 expression in hepatocytes was upregulated by estrone, 2-OH, NETA and NETA/17β-estradiol. In HUVECS, TF, TFPI and PAI-1 expression was upregulated by estrone but not by 17β-estradiol. NETA upregulated TF, TFPI and tPA expression. Estrogen receptor status was unaffected by the addition of NETA. ConclusionsThis data suggests a role for progestins in modifying the effects of oestrogen and its metabolites on coagulation gene expression which may contribute to the reduced thrombotic risk associated with transdermal preparations.

Harnessing a Physiologic Mechanism for siRNA Delivery With Mimetic Lipoprotein Particles

Therapeutics based on RNA interference (RNAi) have emerged as a potential new class of drugs for treating human disease by silencing the target messenger RNA (mRNA), thereby reducing levels of the corresponding pathogenic protein. The major challenge for RNAi therapeutics is the development of safe delivery vehicles for small interfering RNAs (siRNAs). We previously showed that cholesterol-conjugated siRNAs (chol-siRNA) associate with plasma lipoprotein particles and distribute primarily to the liver after systemic administration to mice. We further demonstrated enhancement of silencing by administration of chol-siRNA pre-associated with isolated high-density lipoprotein (HDL) or low-density lipoprotein (LDL). In this study, we investigated mimetic lipoprotein particle prepared from recombinant apolipoprotein A1 (apoA) and apolipoprotein E3 (apoE) as a delivery vehicle for chol-siRNAs. We show that apoE-containing particle (E-lip) is highly effective in functional delivery of chol-siRNA to mouse liver. E-lip delivery was found to be considerably more potent than apoA-containing particle (A-lip). Furthermore, E-lip–mediated delivery was not significantly affected by high endogenous levels of plasma LDL. These results demonstrate that E-lip has substantial potential as delivery vehicles for lipophilic conjugates of siRNAs.

Endogenous Expression of Functional Factor VIII by Human Mesenchymal Stem Cells In Culture

AbstractAbstract 2216The liver is considered to be the primary site of factor VIII (FVIII) production in the body; however, evidence is mounting that suggests there are secondary sites in which considerable synthesis of FVIII takes place. Studies of FVIII mRNA expression in various human tissues have revealed that FVIII message can be found throughout the body. Additionally, acute liver failure correlates with an increase in circulating FVIII levels. Some reports have identified endothelial cells as a significant extra-hepatic source of FVIII, possibly explaining both the widespread presence of FVIII mRNA and the increase in FVIII levels upon liver failure. However, the possibility exists that other cell types present throughout the body also produce FVIII and contribute to circulating FVIII levels. Mesenchymal Stem Cells (MSCs) represent a potential alternative; they are a diverse group of stromal cells which can be found in the perivascular regions of multiple tissues throughout the body. Previous studies demonstrated that MSCs are capable of efficiently producing and secreting high levels of FVIII in vitro when transduced with FVIII-encoding viral vectors, but to date, the innate ability of MSCs to produce FVIII has not been explored. As such, we investigated the potential for MSCs to produce endogenous FVIII message and secrete functional protein. MSCs isolated based on Stro-1 positivity from human lung, liver, brain, and bone marrow (BM) were grown in cell culture and assayed for production of FVIII message by both microarray analysis and RT-PCR. Microarray data showed that there were significant amounts of FVIII message in all four cell types tested and that the amount of message in BM MSCs was three-fold higher than each of the other three cell types. RT-PCR analysis confirmed the presence of FVIII message in all four MSC populations. Secretion of functional FVIII protein was subsequently measured using a chromogenic assay. MSC culture supernatants were collected for either 24 or 48 hours, and FVIII activity was determined using pooled normal human plasma as a control to create a standard curve. FVIII activity in the supernatants of MSCs was in the range of 0.6 to 2.0 mU/1×10^6 cells/ 24hr. Moreover, MSCs continued to express and produce FVIII during time in culture until our last evaluation at passage 20, indicating that there is an innate ability of these cells to continually produce FVIII. Taken together, these data demonstrate that human MSCs are capable of producing and secreting functional FVIII in vitro, and given their widespread location throughout the body, this finding raises the possibility that, in vivo, these cells might significantly contribute to the total FVIII pool. This is the first report, to our knowledge, that implicates MSCs as a potential endogenous source for circulating FVIII. Further studies of in vivo FVIII expression by MSCs are warranted and may provide a clearer understanding of extra-hepatic FVIII production in the body while aiding in the discovery of novel therapies for hemophilia A. Disclosures:No relevant conflicts of interest to declare.

Expression and purification of recombinant human coagulation factor VII fused to a histidine tag using Gateway technology.

Factor VII (FVII) is a plasma glycoprotein that participates in the coagulation process leading to the generation of fibrin. The aim of this study was to construct, express and purify recombinant FVII fused to a polyhistidine (his) tag using Gateway technology. To construct the entry clone, blunt-end FVII cDNA and subsequent polymerase chain reaction (PCR) product isolated from a HepG2 cell line was TOPO-cloned into a pENTR TOPO vector. To construct the expression clone, a LR recombination reaction was carried out between the entry clone and destination vector, pDEST26. Chinese hamster ovary (CHO) cells were transfected with 1 microg of DNA of PDEST26-FVII using the FuGENE HD transfection reagent. Two cell lines that permanently expressed recombinant FVII were established. The expression of recombinant FVII was confirmed by reverse transcriptase PCR and enzyme-linked immunosorbent assay. Culture medium containing his-tagged FVII was added to the nickel-nitrilotriacetic acid resin column and bound protein was eluted. The purified protein was detected by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The biological activity of the recombinant FVII was determined by a prothrombin time assay using FVII-depleted plasma. The results showed that human recombinant FVII was successfully cloned and the accuracy of the nucleotide sequence of the gene and its frame in the vector were confirmed by DNA sequencing. Stable clones transfected with the construct expressed FVII mRNA and related protein but no expression was detected in the CHO cells containing an empty vector. A protein of about 52 KDa was detected in SDS-PAGE and was further confirmed by western blot analysis. A three-fold decrease in clotting time was observed using this recombinant FVII. As far as we are aware, this is the first report of expression of recombinant FVII fused with a his-tag through Gateway technology. The next steps, including large scale expression, purification, activation and stabilisation, are underway.

Establishment of a cell line expressing recombinant factor VII and its subsequent conversion to active form FVIIa through hepsin by genetic engineering method

Factor VII (FVII) is a plasma glycoprotein that participates in the coagulation process leading to generation of fibrin. It is converted to factor VIIa that plays an important role in the coagulation cascade. The aim of this study was isolating and cloning the genes of human factor VII and hepsin and subsequent co-transfection of the constructs to Chinese hamster ovary (CHO) cell line to obtain rFVIIa. Factor VII and hepsin cDNAs were isolated from HepG2 cell line and cloned into pcDNA3.1 (+) vector. The constructs were co-transfected to CHO cell line. A cell line that permanently expressed recombinant factor VII (rFVII) and hepsin was established. The expression of rFVII was confirmed by reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay and Western blot analysis. Biological activity of rFVII was evaluated by prothrombin time assay. The results showed that the genes of FVII and hepsin were successfully cloned and expressed. Stable CHO cells co-transfected with pcNDA3.1-FVII and pcNDA3.1-hepsin expressed FVII and hepsin mRNA, but there was no expression in the CHO cells transfected with insert free pcDNA3.1. FVIIa protein was secreted to medium of CHO cells co-transfected with pcNDA3.1-FVII and pcNDA3.1-hepsin. The expected band of rFVII was detected in Western blot analysis. A three- to fourfold decrease in clotting time was observed when human FVII-depleted plasma was used in combination with human thromboplastin in the presence of rFVII, confirming the biological activity of rFVII. As we are aware, this is the first report of establishing a cell line expressing FVIIa using genetic engineering methods.

A Frame Shift-Induced Stop Codon Causes Hemophilia a in Sheep.

Hemophilia A (HA), or Factor VIII (FVIII) deficiency, is the most common severe hereditary coagulation disorder, affecting 1 in 5000 males. We have successfully re-established and characterized a line of sheep with a bleeding disorder that closely mimics severe human HA. These animals have almost non-existent levels of FVIIIc and an extremely prolonged PTT, with normal levels of platelets, fibrinogne, FVII, FIX, and vWF. All animals that survived birth have exhibited prolonged tail and nail cuticle bleeding, multiple episodes of severe spontaneous bleeding including hemarthroses, muscle hematomas, and hematuria, all of which have responded to recombinant human FVIII. Thus far, low-titer inhibitors to human FVIII have been detected in 3 treated animals, further validating the clinical relevance of this model. Unfortunately, almost nothing is known about the sheep FVIII gene or about the nature of the mutation causing HA in these animals. We thus undertook to characterize the normal sheep FVIII mRNA and elucidate the nature of the hemophilia-inducing mutation. RT-PCR of mRNA from the spleen of normal sheep, followed by overlapping sequence analysis allowed us to walk along the mRNA and obtain the sequence of the complete coding sequence for factor VIII. The coding sequence for ovine FVIII is 6765 nucleotides, which is translated into a protein consisting of 2254 amino acids (a.a.). BLAST alignment of this sequence to that of human FVIII at the a.a. level revealed a high degree of identity in all regions except the B domain (which in humans is known to be dispensable for clotting activity). Specifically, the A1 domain (ovine a.a. 1–332) showed 81% identity, A2 (a.a. 333–709) 88%, A3 (a.a. 1596–1924) 87%, C1 (a.a. 1925–2074) 90%, and C2 (a.a. 2075–2254) 86%, while the B domain (a.a. 710–1595) exhibited only 47% identity. A comparison of these sequences taking into consideration conservative a.a. changes that maintain the chemical properties of sheep and human FVIII proteins indicated even higher levels of homology, with the A1 domain exhibiting 87% positivity, A2 94%, A3 93%, C1 94%, and C2 93%. Even with this approach, however, there was still very low conservation (59%) of the B domain between the two species. Analysis of mRNA isolated from the spleen of a deceased HA sheep revealed several conservative point mutations in the hemophiliac and identified 11bp in exon 14 that differed between the wild type and the hemophiliac. Importantly, this difference included a frame-shift that introduced a premature stop codon in exon 14, as is seen in some human patients with HA. Using a PCR-based RFLP analysis, we can unequivocally distinguish sheep that are wild-type, heterozygous, or homozygous for the HA mutation, and thus confirm the genotype of all 26 existing carriers and 8 hemophiliac sheep. This PCR-based assay will greatly facilitate studies using these sheep, since it is now possible to either screen embryos prior to implantation or screen fetuses in utero using a small volume of blood or amniotic fluid, as is done for prenatal diagnosis of human genetic diseases. These studies thus defined the molecular basis for the HA in these sheep that are a valuable model of human disease. Given the close physiologic similarity between sheep and humans, the high degree of identity in their FVIII protein, and the decades of experience using the sheep to study both normal physiology and a wide array of diseases, we hope that this large animal model will contribute to a better understanding of HA and the development of novel treatments such as stem cell transplantation and gene therapy-based approaches that can directly translate to human patients with hemophilia.

Evidence for an Overlapping Role of CLOCK and NPAS2 Transcription Factors in Liver Circadian Oscillators

The mechanisms underlying the circadian control of gene expression in peripheral tissues and influencing many biological pathways are poorly defined. Factor VII (FVII), the protease triggering blood coagulation, represents a valuable model to address this issue in liver since its plasma levels oscillate in a circadian manner and its promoter contains E-boxes, which are putative DNA-binding sites for CLOCK-BMAL1 and NPAS2-BMAL1 heterodimers and hallmarks of circadian regulation. The peaks of FVII mRNA levels in livers of wild-type mice preceded those in plasma, indicating a transcriptional regulation, and were abolished in Clock(-/-); Npas2(-/-) mice, thus demonstrating a role for CLOCK and NPAS2 circadian transcription factors. The investigation of Npas2(-/-) and Clock(Delta19/Delta19) mice, which express functionally defective heterodimers, revealed robust rhythms of FVII expression in both animal models, suggesting a redundant role for NPAS2 and CLOCK. The molecular bases of these observations were established through reporter gene assays. FVII transactivation activities of the NPAS2-BMAL1 and CLOCK-BMAL1 heterodimers were (i) comparable (a fourfold increase), (ii) dampened by the negative circadian regulators PER2 and CRY1, and (iii) abolished upon E-box mutagenesis. Our data provide the first evidence in peripheral oscillators for an overlapping role of CLOCK and NPAS2 in the regulation of circadianly controlled genes.

Long-Term Phenotypic Correction of Hemophilia A Mice Following Intravenous Injection of miRNA-Regulated Lentiviral Vectors.

Hemophilia A is an excellent candidate disorder for the use of gene therapy as a treatment modality. To date, although lentiviral delivery of the factor VIII (FVIII) transgene has the potential to provide sustained therapeutic correction of the hemophilia A phenotype, this has not been achieved in adult animals because of the anti-FVIII immune response. We have used lentiviral vectors to deliver the canine FVIII transgene to hemophilia A neonates and although no anti-FVIII immune response occurred, and indeed the treated mice displayed long-term tolerance to the canine FVIII antigen, this strategy did not provide sustained therapeutic levels of plasma FVIII. To overcome these limitations, we modified our lentiviral vector and the protocol for viral delivery to enhance transduction of hepatocytes and direct transgene expression away from antigen presenting cells. We engineered lentiviral vectors that encode the B-domain deleted canine FVIII cDNA under the transcriptional control of either a non-viral ubiquitous promoter or two different liver-restricted promoters. However, no plasma FVIII was detected in any of the adult hemophilia A mice after intravenous injection of the various lentiviral vectors because of an anti-canine FVIII immune response. An alternate pseudotype (GP64) was used to enhance transduction of hepatocytes and a target sequence for a hematopoietic-specific microRNA was incorporated into the transgene to prevent FVIII expression in antigen presenting cells that may arise from promoter trapping. When hemophilia A mice received intravenous infusions of these modified vectors, where the cFVIII trangene is under the control of either of the liver-restricted promoters, all treated mice (n=4) showed sustained FVIII expression (mean FVIII levels 28.2±2.4 mU/mL) for more than 150 days (last time analyzed) without developing anti-FVIII antibodies. Moreover, temporary depletion of Kuppfer cells prior to viral administration resulted in a 3-fold elevation of levels of plasma FVIII (mean FVIII levels 83.3±2.1mU/mL; n=4). Analysis of the biodistribution of the integrated FVIII transgene and expression of canine FVIII mRNA indicate an enhanced restriction of FVIII expression in hepatocytes with the use of the modified lentiviral vectors. These results demonstrate, for the first time, the long-term therapeutic potential of modified lentiviral vectors for treating adult pre-clinical animal models of hemophilia A.

CCAAT/Enhancer-binding Protein-β Participates in Insulin-responsive Expression of the Factor VII Gene

Expression of the human coagulation factor VII (FVII) gene by hepatoma cells was modulated in concert with levels of glucose and insulin in the culture medium. In low glucose medium without insulin, amounts of both FVII mRNA and secreted FVII protein were coordinately increased; in the presence of glucose with insulin, both were decreased. Analysis of the FVII promoter showed that these effects could be reproduced in a reporter-gene system, and a small promoter element immediately upstream of the translation start site of the gene, which mediated these effects, was identified. Mutation of this element largely abrogated the glucose/insulin-responsive change in expression of the reporter gene. Several members of the CCAAT/enhancer-binding protein family were found to be capable of binding the identified sequence element but not the mutated element. The expression of a FVII minigene directed by a segment of the native FVII promoter responded to co-expressed activating and inhibiting forms of CCAAT/enhancer-binding protein beta.