TF Protein Research Articles

Caffeine induces endothelial tissue factor expression via phosphatidylinositol 3-kinase inhibition

Tissue factor (TF) is the key activator of coagulation and is involved in acute coronary syndromes. Caffeine is often reported to increase cardiovascular risk; however, its effect on cardiovascular morbidity and mortality is controversial. Hence, this study was designed to investigate the impact of caffeine on endothelial TF expression in vitro. Caffeine concentration-dependently enhanced TF protein expression and surface activity in human endothelial cells stimulated by tumour necrosis factor (TNF)-α or thrombin. Caffeine inhibited phosphatidylinositol 3-kinase (PI3K) activity and this effect was comparable to that of the known PI3K inhibitor LY294002. Consistently, treatment of endothelial cells with LY294002 enhanced TNF-α induced TF expression to a similar extent as caffeine, and adenoviral expression of the active PI3K mutant (p110) reversed the effect of both caffeine and LY294002 on TF expression. Caffeine and LY294002 increased DNA binding capacity of the transcription factor nuclear factor κB, whereas the activation pattern of mitogen-activated protein kinases (MAPK) remained unaltered. Luciferase reporter assay revealed a caffeine dependent activation of the TF promoter, and RT-PCR revealed a dose dependent increase in TF mRNA levels when stimulated with caffeine in the presence of TNF-α. In conclusion, caffeine enhances TNF-α-induced endothelial TF protein expression as well as surface activity by inhibition of PI3K signalling. Since the caffeine concentrations applied in the present study are within the plasma range measured in humans, our findings indicate that caffeine enhances the prothrombotic potential of endothelial cells and underscore the importance of PI3K in mediating these effects.

Mycobacterium Tuberculosis infection and Tissue Factor Expression in Macrophages

Abstract 1198Tissue factor (TF), the primary initiator of the coagulation cascade, is normally absent from cells that regularly come in contact with blood such as endothelial cells and monocytes yet could be induced in these cells in a variety of pathological conditions. The aberrant expression of TF by cells of the monocyte/macrophage lineage is thought to be a major contributor to thrombotic disorders and inflammation. Macrophages play a central role in the innate immune response essential for host defense against pathogenic infections. Tuberculosis (TB), a fatal disease caused by Mycobacterium tuberculosis (Mtb), affects nearly one third of the world’s population. A number of studies have reported the presence of thrombotic complications, particularly disseminated intravascular coagulation (DIC) in TB patients. However, it is unclear how Mtb infection causes DIC or other thrombotic disorders as mycobacteria are not known to produce endotoxins or exotoxins that otherwise initiate the clotting cascade. In the present study, we have investigated whether Mtb infection induces TF expression in macrophages and various host and pathogenic factors responsible for TF expression. We have tested the effect of live virulent Mtb H37Rv, gamma-irradiated Mtb H37Rv (γ-Mtb) and various components derived from Mtb H37Rv on TF expression in macrophages. Exposure of human monocyte-derived macrophages (MDM) to live virulent Mtb H37Rv (at 1 to 5 bacteria/macrophage) or γ-Mtb H37Rv (10 μg/ml) markedly increased TF expression in MDM (5 to 20-fold increase compared to untreated MDM). TF expression in macrophages in response to Mtb is predominantly higher in CD14hi (CD14+) macrophages. Although CD14loCD16hi (CD16+) macrophages also expressed TF in response to Mtb, the level of TF induction in these cells was much lower. γ-Mtb induced TF expression in macrophages much more robustly than E.coli-derived LPS. The γ-Mtb-mediated induction of TF expression in macrophages peaked around 9 h and was sustained throughout 48 h, which markedly varies from the kinetics of LPS-induced TF expression, which peaks between 3–6 h and then reduced down to basal level by 18–24 h. To identify the potential macrophage receptor(s) responsible for the induction of TF by Mtb, MDM were pre-treated with antibodies against CD14 and Toll-like receptors (TLR) before they were exposed to γ-Mtb. Although CD14 antibodies markedly inhibited LPS-induced TF activity, they only suppressed Mtb-induced TF activity minimally. Pretreatment of MDMs with TLR2 or TLR4 antibodies had no significant effect on γ-Mtb-induced TF expression. However, combining the CD14 antibodies together with TLR2 and TLR4 antibodies showed nearly a 50% reduction in γ-Mtb-induced TF activity. These data indicate that a cooperative action of multiple receptors and signaling pathways may be responsible for the robust and sustained induction of TF expression. In order to identify the specific component(s) of γ-Mtb that are responsible for TF induction, we have treated MDMs with various subcellular fractions or purified components derived from Mtb H37Rv. The whole cell lysate of Mtb, cell wall, cell membrane and culture filterate proteins induced TF activity to varying degrees. Of all the purified components tested, the mycobacterial cell wall core component mycolyl arabinogalactan peptidoglycan (mAGP), phosphatidylinositol mannoside-6 (PIM6) and lipomannan (LM) elicited induction of TF protein and activity in the order of mAGP > PIM6 > LM. It is interesting to note that the treatment of MDMs with individual components, mycolic acid, arabinogalactan and peptidoglycan of mAGP complex did not induce TF expression, indicating that the structure of mAGP may be required for recognition by pattern recognition receptors on macrophages. In summary, our data show that Mtb induces TF expression in macrophages, probably through a novel mechanism. Although Mtb-induced TF expression in monocytes/macrohages may lead to systemic thrombotic disorders through the release of TF containing microparticles, it may be the localized and sustained expression of TF on macrophages that internalize the infectious bacteria at the site of infection in lung is responsible for granuloma formation in tuberculosis patients that prevents the spread of the bacteria. Disclosures:No relevant conflicts of interest to declare.

Regulating action of iron regulatory protein-2 in iron metabolism of lung cancer

To discuss the regulating mechanism of iron regulatory protein-2 (IRP2) in the iron metabolism of lung cancer. The cultured A549 cells were divided into 3 groups: liposome group (including liposomes 20 mg/L), random oligonucleotide group (SCODN group) and antisense oligonucleotide group (ASODN group). And the liposome-mediated transfection was employed with the liposome and SCODN groups as controls. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to examine the mRNA and protein expressions of iron metabolism-related transferring (Tf), transferrin receptor (TfR) and ferritin (Fn) genes, etc. After a 48-hour transfection, the mRNA expression of Tf had no statistically significant difference among three groups (F = 2.18, P = 0.078); the mRNA expression of TfR in the ASODN group was significantly lower than that in the liposome and SCODN groups (P < 0.05). The expression of Fn mRNA in the ASODN group (0.56 ± 0.06) was higher than that in the liposome (0.36 ± 0.05) and SCODN groups (0.39 ± 0.03) (P < 0.05). After a 48-hour transfection, the IRP2 protein expression of the ASODN group was significantly lower than those of the liposome and SCODN groups (P < 0.05). The Tf protein expression was not statistically different in three groups (F = 2.67, P = 0.088). The TfR protein expression of the ASODN group was lower than those of the liposome and SCODN groups (P < 0.05). And the Fn protein expression of the ASODN group was higher than those of the liposome and SCODN groups (P < 0.05). IRP2 may affect the expressions of TfR and Fn in lung adenocarcinoma A549 cells by changing the amount of protein and regulating the iron metabolism.

Raman detection of localized transferrin-coated gold nanoparticles inside a single cell

We investigated the cellular uptake behavior of non-fluorescent metal nanoparticles (NPs) by use of surface-enhanced Raman scattering (SERS) combined with dark-field microscopy (DFM). The uptake of Au NPs inside a single cell could also be identified by DFM first and then confirmed by z-depth-dependent SERS at micrometer resolution. Guided by DFM for the location of Au NPs, an intracellular distribution assay was possible using Raman dyes with unique vibrational marker bands in order to identify the three-dimensional location inside the single cell by obtaining specific spectral features. Au NPs modified by 4-mercaptobenzoic acid (MBA) bearing its -COOH surface functional group were used to conjugate transferrin (Tf) protein using the 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) reaction. The protein conjugation reaction on Au surfaces was examined by means of color change, absorption spectroscopy, and SERS. Our results demonstrate that DFM techniques combined with SERS may have great potential for monitoring biological processes with protein conjugation at the single-cell level.

Pre-analytical and analytical variables affecting the measurement of plasma-derived microparticle tissue factor activity

Introduction Elevated levels of tissue factor positive (TF +) microparticles (MPs) are observed in plasma from a variety of patients with an increased risk of thrombosis. We and others have described the measurement of TF activity in MPs isolated from plasma. The aim of this study was to investigate the effects of pre-analytical and analytical variables on TF activity of MPs isolated from blood of healthy volunteers either untreated or treated ex vivo with bacterial lipopolysaccharide. Materials and methods We evaluated the following parameters: use of different centrifugation speeds to isolate the MPs; comparison of TF activity of MPs isolated from platelet poor plasma versus platelet free plasma; effect of freeze/thaw on MP TF activity; and comparison of the MP TF activity assay with the measurement of TF protein by ELISA or flow cytometry. Results MPs prepared from platelet poor plasma by centrifugation at 20,000 × g or 100,000 × g for 15 minutes had similar levels of TF activity. However, significantly less TF activity was found in MPs isolated from platelet free plasma compared with platelet poor plasma. Interestingly, freeze/thawing of the plasma showed donor to donor variation in MP TF activity, with a moderate increase in some individuals. Conclusion TF + MPs can be quantitatively isolated from platelet poor or platelet free plasma by centrifugation at 20,000 × g for 15 minutes. Measurement of MP TF activity in plasma may be used to detect a prothrombotic state in patients with various diseases.

Characterization of the gilthead seabream ( Sparus aurata L.) transferrin gene: Genomic structure, constitutive expression and SNP variation

Transferrin (Tf) is a multi-function protein with a central role in iron metabolism, and it is this function that is associated with a role in the innate immune system response. The clear link between Tf and immune defense mechanism lead to propose Tf as a candidate gene for disease resistance. In this study, genomic and cDNA sequences of Tf gene in gilthead seabream ( Sparus aurata L.) ( SaTf gene), were identified and characterized. SaTf gene structure consists of a coding region of 2076 nucleotides divided into 17 exons and a no-coding region that includes 16 introns and spans 5495 nucleotides. The deduced Tf protein for gilthead seabream is composed of 691 amino acids and consists of an initial peptide and two lobes (N- and C-lobes). This gene structure is similar to that of previously described Tf genes in other fish species. RT-PCR analyses carried out in different tissues and two developmental stages showed tissue-and stage-specific Tf expression in gilthead seabream. Finally, by sequencing the transferrin genomic sequences of 20 unrelated seabreams, 31 SNPs were identified. These data allowed the estimation of the frequency of nucleotide substitution in the SaTf gene as 1SNP per 253 bp. SNPs were detected in different regions of the genomic sequence but they were mainly localized in non-coding regions, specifically, SNP frequency in non-coding regions was fifteen-fold higher than within coding regions.

Three novel SNPs of the bovine Tf gene in Chinese native cattle and their associations with milk production traits

Transferrin (Tf) is a β-globulin protein that transports iron ions in mammalian cells. It contributes to innate immunity to microbial pathogens, primarily by limiting microbial access to iron. Thus, polymorphisms present in bovine Tf could potentially underlie inherited differences in mastitis resistance and milk production traits. We detected three novel single-nucleotide polymorphisms of the Tf gene in Chinese native cattle by screening for genetic variation of Tf in 751 individuals of three Chinese cattle breeds, namely China Holstein, Luxi Yellow and Bohai Black, using PCR-RFLP and DNA sequencing techniques. The three new SNPs, g.-1748G>A ss250608649, g.13942T>C ss250608650, and g.14037A>G ss250608651, had allele frequencies of 85.9, 86.3 and 92.5%, 64.5, 73.3 and 65.0%, and 67.6, 73.7 and 60.0%, respectively. SNP g.-1748G>A was located in the 5' flanking region of Tf. SNP g.14037A>G was located in intron 8 of Tf. SNP g.13942T>C, located in exon 8 of Tf, was a synonymous mutation (TTA > CTA), encoding a leucine (326 aa) in the Tf protein. Associations of the Tf SNPs with milk traits were also analyzed. Significant (P < 0.05) relationships among the Tf polymorphisms, somatic cell scores (SCS), and milk productive traits were observed. Cows with genotypes TT (g.13942T>C), GG (g.-1748G>A) and AG (g.14037A>G) had a lower SCS and higher protein levels and 305-day milk yield. Nineteen combinations of different haplotypes from the three SNPs were identified in Chinese Holstein cattle. The haplotype combination ATA/GCA, GCA/GCA and GCG/ GTA was dominant in cows with a lower SCS, a higher protein level and a higher 305-day milk yield, respectively. Moreover, the gene expression level of Tf was higher in mastitis-affected mammary tissues than in normal mammary tissues. These results suggest that the Tf gene affects milk production, as well as mastitis-resistance traits, in Chinese Holsteins.

Members of the CREB/ATF and AP1 family of transcription factors are involved in the regulation of SOX18 gene expression

The SOX18 transcription factor plays an important role in endothelial cell specification, angiogenesis and atherogenesis. By profiling transcription factor interactions (TranSignal TM TF Protein Array) we identified several transcription factors implicated in angiogenesis that have the ability to bind to the SOX18 optimal promoter region in vitro. In this report we focused our attention on distinct transcription factors identified by the array as belonging to AP-1 and CREB/ATF protein families. In particular, we analyzed the effects of CREB, JunB, c-Jun and ATF3 on SOX18 gene expression. Functional analysis revealed that CREB acts as a repressor, while JunB, c-Jun and ATF3 act as activators of SOX18 promoter activity. Our findings indicate that a transcriptional network that includes CREB, JunB, c-Jun and ATF3 could be involved in angiogenesis-related transcriptional regulation of the SOX18 gene.

Platelet Tissue Factor Activity In Patients with Hypercholesterolemia: Modulation of Procoagulant Activity by Statins

AbstractAbstract 156Human platelets store TF that expresses procoagulant activity (PCA) associated with platelet activation (Panes O. et al, Blood 2007;109:5242-50). We have also observed that platelet TF-induced PCA is specifically and rapidly induced by activation of GPIb complex by VWF-Ristocetin (Panes O. et al. Blood 2008;112:A113). Hypercholesterolemia has been associated with increased platelet function and hypercoagulability, but the mechanisms are still unknown. The cholesterol content in cell membranes is directly related with the plasma concentration of the sterol. Inhibition of HMG-CoA reductase by statins reduces cardiovascular risk by decreases in plasma cholesterol and by other independent (“pleiotropic”) effects.We studied the relationship between total and LDL plasma cholesterol with platelet TF content and TF-dependent PCA of washed platelets activated for 5 min with VWF-Ristocetin (VWF-R). We also examined these variables after atorvastatin or rosuvastatin intake (80 and 20 mg/day × 1 month, respectively) in 25 subjects with hypercholesterolemia. TF was measured by ELISA and PCA by FXa generation (chromogenic assay) in washed platelet suspensions before and after activation. We found no significant differences between both statins regarding the decrease in plasma LDL-cholesterol levels and platelet TF-dependent PCA; both statins had any significant effects on the levels of blood inflammatory markers (usCRP, fibrinogen and VWF). Thus, we analyzed the pooled data of patients receiving either one or the other statin. Total and LDL-Chol dropped from 271±41 and 185±40 to 167±30 and 87±23 mg dL-1, respectively. This was associated with decrease in basal PCA (non-stimulated platelets) from 34±27 to 22±14 nmol of FXa/2*107platelets (p=0.02). After VWF-R stimulation, a mean 3.48-fold increase in platelet PCA was observed in hypercholesterolemic patients before taking statins. Surprisingly, a mean 20-fold increase in FXa generation in activated platelets was observed after 1 month on statin therapy, and this difference was statistically significant (p=0.018). These changes in PCA were not associated with significant changes in TF content of platelet membranes (621±481 to 555±300 pg/mg prot). We also found that plasma LDL-Chol was negatively correlated with platelet PCA induced by VWF-R activation (r = −0.239, p = 0.026) and with the ratio of PCA between activated and non-activated platelets (r = −0.27, p = 0.011). The striking post-statin increase of platelet-PCA without change in the total platelet TF protein suggests that PCA depends more on the fraction of platelet TF available for activation than on the total mass of platelet TF. The decrease in basal PCA of non-stimulated platelets after statin therapy suggests that lower concentration of cholesterol in platelet membranes makes the platelets more stable during the isolation procedure. The higher increase after stimulation would reflect a better hemostatic response to a specific stimulus. Thus, changes in plasma cholesterol, possibly through decrease in platelet membrane cholesterol, modulates the PCA of platelets, either by making platelets more stable under resting conditions and more responsive after specific activation. Disclosures:No relevant conflicts of interest to declare.

Effect of epigallocatechin-3-gallate on iron overload in mice with alcoholic liver disease

Iron has long been related to the pathological process of alcoholic liver disease (ALD). Liver iron overload is known to accelerate the development of ALD. In the present study we aimed to examine the effect of epigallocatechin-3-gallate (EGCG) on iron overload of ALD and to explore the potential mechanisms involved in its protection against ALD in mice. Male C57BL/6J mice were given alcohol by intragastric administration for 12 weeks. At the end of 8th week, ALD mice were treated for 4 weeks for 10, 20 and 30 mg kg(-1) EGCG by intraperitoneal injection. Liver injuries were assessed by histopathologic examination and Serum Alanine Aminotransferase (ALT) levels. Serum iron content, hepatic iron concentration and liver malondialdehyde (MDA) contents were examined. In addition, hepcidin mRNA levels and transferrin (Tf) and transferrin receptor 1 (TfR1) protein levels of liver tissue were also evaluated. Compared with model group, treatment of ALD mice with EGCG ameliorated liver injuries, decreased serum iron level, hepatic iron levels and liver MDA contents, increased hepcidin mRNA level and decreased Tf and TfR1 protein expression in the liver. The results of our study explain a new point of view that the protective effect of EGCG on ALD is associated with its iron-chelating property. The possible mechanisms are that EGCG affects hepatic iron uptake and inhibits iron absorption in the small intestinal.

Cryptic Vs. Active Tissue Factor: Cystine186-Cystine 209 Disulfide Bond Switch Is Not the Answer.

Abstract 333▪▪This icon denotes an abstract that is clinically relevant.Tissue factor (TF) plays the key role in initiation of blood coagulation by allosterically activating coagulation factor VIIa (FVIIa). TF is essential for hemostasis but aberrant expression would lead to thrombotic disorders. TF on cell surfaces resides mostly in a cryptic state. At present it is not entirely clear how cryptic TF differs from procoagulant active TF and how de-encryption occurs. Recent studies have suggested that cryptic and active TF may have different conformations, i.e., cryptic TF contains unpaired cysteine thiols at Cys 186 and Cys 209 in the membrane proximal domain whereas procoagulant active TF contains the oxidized Cys186-Cys209 disulfide bond. It has been suggested that the catalytic switching of this disulfide bond between reduced and oxidized states by protein disulfide isomerase (PDI) might modulate the activity status of TF. Although the validity of this hypothesis has been questioned, more recent reports have supported the contention that PDI-catalyzed disulfide bond formation of Cys186-Cys209 in TF plays a role in regulation of TF activity both in vitro and in vivo. To reconcile the conflicting data in the recent literature, we re-examined in the present study the central dogma on which the hypothesis was originated, i.e., a TF mutant lacking Cys186-Cys 209 disulfide bond is functionally inactive (cryptic) and that TF activation requires formation of the Cys186-Cys209 disulfide bond. CHO cells were transfected with wild type TF or TF mutants that selectively preclude Cys186-Cys209 disulfide bond formation, i.e., TFC186S, TFC209S, or TFC186S/C209S, and TF procoagulant and antigen expression levels at the cell surface were quantified accurately by measuring TF procoagulant activity in a factor × activation assay and TF antigen levels in radioligand binding studies utilizing two different TF mAb (10H10 and 5G9) and FVIIa. CHO cells transfected with TFC186S, TFC209S or TFC186S/C209S expressed very little procoagulant activity (less than 1% of CHO cells transfected with wild-type TF under identical experimental conditions). However, TF mAb and FVIIa binding studies revealed that CHO cells transfected with TFC186S, TFC209S or TFC186S/C209S expressed very little TF protein on the cell surface (∼ 1 to 2% of CHO cells transfected with wild-type TF). Similar results were obtained whether we mutated Cys 186 or Cys 209 to Ser or Ala or whether we used transient or stable transfections. To confirm that the lower expression of TFC186S, TFC209S or TFC186S/C209S is neither an artifact nor limited to CHO cell model system, we extended these studies to endothelial cells. Human umbilical vein endothelial cells (HUVECs) were transduced with adenovirus particles (25 moi/cell) encoding wild-type TF, TFC186S, TFC209S or TFC186S/C209S and TF antigen levels at the cell surface and in cell lysates were measured using multiple methods - confocal microscopy, FACS, TF antigen assay, immuno blot analysis, TF mAb binding and FVIIa binding. HUVEC transduced with adenoviral particles encoding TFC186S, TFC209S or TFC186S/C209S expressed very low levels of TF antigen both at the cell surface ( 1 to 5%) as well as in total (10 to 15%) (TFC186S > TFC209S >TFC186S/C209S) compared to HUVEC transduced with the same number of adenovirus particles expressing wild-type TF. Cell surface TF activity measured in the presence of saturating concentrations of FVIIa revealed that the TF procoagulant activity of the mutants correlate well with the amount of TF antigen present on the cell surface, i.e., there were no significant differences in the specific activity of TF mutants and wild-type TF (TF procoagulant activity/amount of TF antigen, pM FXa formed/femto mole TF: wild-type TF, 68 ± 6; TFC186S, 55 ± 10; TFC209S, 57 ± 17; and TFC186S/C209S, 64 ± 0.2). More importantly, treatment of HUVEC with the thiol-oxidizing agent HgCl2 or with ionomycin increased the cell surface TF activity to the same extent (200 to 400% increase over HUVEC treated with control vehicle) in HUVEC expressing TFC186S/C209S or wild-type TF. In summary the present data provide clear evidence for that TF lacking Cys186-Cys209 bond is coagulantly active and a lower TF procoagulant activity in cells expressing the mutant TF that precludes Cys186-Cys209 disulfide bond formation is due to severe impairment in TF protein synthesis/processing and not because the mutant TF assumes the cryptic conformation. Our data also show that TF de-encryption does not require the formation of Cys186-Cys209 disulfide bond. Disclosures:No relevant conflicts of interest to declare.

Controlled release of siRNA nanoparticles loaded in a novel external stent prepared by emulsion electrospinning attenuates neointima hyperplasia in vein grafts

To evaluate a strategy of using TF siRNA loaded in a novel external stent prepared by hybrid ultrafine fibrous membrane consisting of PLGA/Chitosan nanoparticles as a therapy for vein graft disease. Hybrid ultrafine fibrous membranes consisting of PLGA/Chitosan nanoparticles were fabricated via a specially designed electrospinning setup. After soaking in chloroform to dissolve PLGA, the amount of chitosan in the hybrid membranes was determined. The water uptake of the hybrid ultrafine fibrous membranes was investigated by incubation in phosphate buffer solution. Right jugular vein-carotid artery interposition grafting models in Sprague-Dawley rats were randomly divided into five groups:Group A (external stent consisting of PLGA/CS-TFsiRNA nanoparticles), Group B (external stent consisting of PLGA/CS-Stealth(TM) RNAi negative control nanoparticles), Group C (external stent consisting of PLGA/CS blank nanoparticles), Group D (external stent consisting of PLGA), Group E (without perivenous external stent). BLOCK-iT(TM) Fluorescent Oligo was used to confirm its stability and successful transfer into the vein graft wall. The vein grafts were harvested at 1, 3, 7, 14, 28 d after operation, respectively. The TF protein expression of vein grafts was analyzed by Western blot and immunochemistry at 1, 3, 7 d after operation, respectively. The expression of proliferating cell nuclear antigen (PCNA) was identified by immunochemistry methods. The thickness of neointima at 28 d was calculated by computer imaging analysis system. The PLGA and CS amount in PLGA/Chitosan nanoparticles membranes could be well controlled by adjusting the flow rate for electrospinning of PLGA and chitosan nanoparticles, respectively. Because of the introduction of chitosan, which is a naturally hydrophilic polymer, the hybrid membranes exhibited good water absorption properties. BLOCK-iT(TM) Fluorescent Oligo could be detected in the graft wall even 12 days after operation. The expression of TF protein in Group A was significantly less than that in control groups at 3 d after operation (P < 0.05, 0.40 +/- 0.03 vs 0.75 +/- 0.01, 0.75 +/- 0.05, 0.77 +/- 0.07) and at 7 d after operation (P < 0.05, 0.30 +/- 0.03 vs 0.84 +/- 0.05, 0.86 +/- 0.06, 0.85 +/- 0.06). The expression of PCNA in Group A decreased significantly in comparison with control groups at 14 d after operation (P < 0.01, 13.0% +/- 2.6% vs 25.0% +/- 2.8%, 24.2% +/- 3.9%, 24.0% +/- 4.1%, 44.8% +/- 3.7%). The thickness of neointima at 28 d after grafting in Group A was significantly less than the untreated group (P < 0.01, 18.8 microm +/- 2.9 microm vs 38.7 microm +/- 5.0 microm, 37.3 microm +/- 3.6 microm, 37.2 microm +/- 2.6 microm, 67.5 microm +/- 4.8 microm). The novel external stent prepared by hybrid ultrafine fibrous membrane consisting of PLGA/Chitosan nanoparticles inhibits early neointima formation in rat vein grafts. This strategy may be a practicable and promising form of gene delivery against vein graft failure.

The role of transferrin receptor 1 and 2 in transferrin-bound iron uptake in human hepatoma cells

Transferrin receptor (TFR) 1 and 2 are expressed in the liver; TFR1 levels are regulated by cellular iron levels while TFR2 levels are regulated by transferrin saturation. The aims of this study were to 1) determine the relative importance of TFR1 and TFR2 in transferrin-bound iron (TBI) uptake by HuH7 human hepatoma cells and 2) characterize the role of metal-transferrin complexes in the regulation of these receptors. TFR expression was altered by 1) incubation with metal-transferrin (Tf) complexes, 2) TFR1 and TFR2 small interfering RNA knockdown, and 3) transfection with a human TFR2 plasmid. TBI uptake was measured using (59)Fe-(125)I-labeled Tf and mRNA and protein expression by real-time PCR and Western blot analysis, respectively. Fe(2)Tf, Co(2)Tf, and Mn(2)Tf increased TFR2 protein expression, indicating that the upregulation was not specifically regulated by iron-transferrin but also other metal-transferrins. In addition, Co(2)Tf and Mn(2)Tf upregulated TFR1, reduced ferritin, and increased hypoxia-inducible factor-1alpha protein expression, suggesting that TFR1 upregulation was due to a combination of iron deficiency and chemical hypoxia. TBI uptake correlated with changes in TFR1 but not TFR2 expression. TFR1 knockdown reduced iron uptake by 80% while TFR2 knockdown did not affect uptake. At 5 microM transferrin, iron uptake was not affected by combined TFR1 and TFR2 knockdown. Transfection with a hTFR2 plasmid increased TFR2 protein expression, causing a 15-20% increase in iron uptake and ferritin levels. This shows for the first time that TFR-mediated TBI uptake is mediated primarily via TFR1 but not TFR2 and that a high-capacity TFR-independent pathway exists in hepatoma cells.

Transferrin D protein variants in the diagnosis of congenital disorders of glycosylation (CDG)

Congenital disorders of glycosylation are a rapidly growing group of inherited (neuro)metabolic disorders characterized by defects in glycosylation of proteins and lipids. This study discusses an analytical problem in the differentiation between hypoglycosylation and transferrin (Tf) protein variants. Analysis of serum Tf by isoelectric focusing is used as a common method suitable for screening 19 out of a total of 22 types of glycosylation defects identified so far. In three members of a family, several indicators showed evidence of a Tf protein variant, however, routine neuraminidase-based demonstration failed to confirm this result. On the assumption that we should be able to exclude Tf protein variants at the screening-level of the diagnostic algorithm, our concern is a possible cause of our failure to confirm some of the Tf D variants (in contrast to the other C, B and D allelic combinations that are commonly well identified). Several explanations are discussed.

On the evolutionary significance and metal-binding characteristics of a monolobal transferrin fromCiona intestinalis

Transferrins are a family of proteins that bind and transport Fe(III). Modern transferrins are typically bilobal and are believed to have evolved from an ancient gene duplication of a monolobal form. A novel monolobal transferrin, nicatransferrin (nicaTf), was identified in the primitive ascidian species Ciona intestinalis that possesses the characteristic features of the proposed ancestral Tf protein. In this work, nicaTf was expressed in Pichia pastoris. Extensive solution studies were performed on nicaTf, including UV-vis, fluorescence, CD, EPR and NMR spectroscopies, and electrospray time-of-flight mass spectrometry. The expressed protein is nonglycosylated, unlike the protein isolated from the organism. This property does not affect its ability to bind Fe(III). However, Fe(III)-bound nicaTf displays important spectral differences from other Fe(III)-bound transferrins, which are likely the consequence of differences in metal coordination. Coordination differences could also account for the weaker affinity of nicaTf for Fe(III) (log K = 18.5) compared with bilobal human serum transferrin (HsTf) (log K = 22.5 and 21.4). The Fe-nicaTf complex is not labile, as indicated by slow metal removal kinetics by the high-affinity chelator tiron at pH 7.4. The protein alternatively binds up to one equivalent of Ti(IV) or V(V), which suggests that it may transport nonferric metals. These solution studies provide insight into the structure and function of the primitive monolobal transferrin of C. intestinalis for comparison with higher order bilobal transferrins. They suggest that a major advantage for the evolution of modern transferrins, dominantly of bilobal form, is stronger Fe(III) affinity because of cooperativity.

Adiponectin inhibits tissue factor expression and enhances tissue factor pathway inhibitor expression in human endothelial cells

Tissue factor (TF) plays a pivotal role in thrombus formation and atherogenesis in acute coronary syndrome. Tissue factor pathway inhibitor (TFPI) is a specific physiological inhibitor of TF/FVIIa complex that regulates TF-induced coagulation. Adiponectin (Adp) is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Adp inhibits inflammatory cytokine and adhesion molecules expression, and it can prevent endothelial dysfunction. In this study, we investigated the effects of Adp on tumor necrosis factor-alpha (TNF-alpha)-induced expression of TF and TFPI in human umbilical vein endothelial cells (HUVECs), and the signaling transduction pathways involved. It was found that Adp significantly inhibited both TF protein expression and activity in TNF-alpha-stimulated HUVECs. In the meanwhile, it increased TFPI protein expression and activity for about two folds. Adp also inhibited TF mRNA expression induced by TNF-alpha, but had no effect on TFPI mRNA expression. The inhibitory effect of Adp on TNF-alpha-induced TF expression was prevented by pretreatment with Rp-cAMPs, a PKA inhibitor. Adp increased intracellular cAMP content and PKA activity levels in a dose-dependent manner. Phosphorylation of IkappaB-alpha was decreased by Adp, but phosphorylation of p44/42 MAPK, SAPK/JNK, and p38 MAPK were not affected. These results suggested that Adp inhibits TF expression through inhibition of a PKA dependent nuclear factor-kappaB (NF-kappaB) signaling pathway. It was also found that adiponectin promoted Akt and AMP-activated protein kinase phosphorylation. The inhibitory effect of Adp on TNF-alpha-induced TF synthesis was abrogated in part by pretreatment with the PI3kinase inhibitor LY294002, suggesting that Akt activation might inhibit TF expression induced by TNF-alpha. The inhibitory effect of Adp is almost completely abrogated by inhibition of both the cAMP/PKA pathway and PI3K/Akt pathway. In conclusion, our data indicated that inhibition of NF-kappaB through stabilization of IkappaB-alpha and activation of Akt phosphorylation may mediate the inhibitory effect of Adp on TF expression; but the enhancement effect of Adp on the TFPI production might occur via translational rather than transcriptional regulation.

Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA

The results of administering escalating, i.v. doses of targeted nanoparticles containing a siRNA targeting the M2 subunit of ribonucleotide reductase to non-human primates are reported. The nanoparticles consist of a synthetic delivery system that uses a linear, cyclodextrin-containing polycation, transferrin (Tf) protein targeting ligand, and siRNA. When administered to cynomolgus monkeys at doses of 3 and 9 mg siRNA/kg, the nanoparticles are well tolerated. At 27 mg siRNA/kg, elevated levels of blood urea nitrogen and creatinine are observed that are indicative of kidney toxicity. Mild elevations in alanine amino transferase and aspartate transaminase at this dose level indicate that the liver is also affected to some extent. Analysis of complement factors does not reveal any changes that are clearly attributable to dosing with the nanoparticle formulation. Detection of increased IL-6 levels in all animals at 27 mg siRNA/kg and increased IFN-gamma in one animal indicate that this high dose level produces a mild immune response. Overall, no clinical signs of toxicity clearly attributable to treatment are observed. The multiple administrations spanning a period of 17-18 days enable assessment of antibody formation against the human Tf component of the formulation. Low titers of anti-Tf antibodies are detected, but this response is not associated with any manifestations of a hypersensitivity reaction upon readministration of the targeted nanoparticle. Taken together, the data presented show that multiple, systemic doses of targeted nanoparticles containing nonchemically modified siRNA can safely be administered to non-human primates.

Activation, Proliferation and Commitment of Endogenous Stem/Progenitor Cells to the Oligodendrocyte Lineage by TS1 in a Rat Model of Dysmyelination

Wild-type and myelin-deficient rats received a single intraparenchymal injection of TS1, a specific combination of IGF-1 and transferrin (Tf), into their corpus callosum at postnatal day 4. The fate of endogenous stem cells in the brain was examined by the expression of the stem cell marker nestin, together with Tf, neurofilaments and glial fibrillary acidic protein from 2 to 14 days after injection. Treated mutants lacked nestin expression in the ventricular wall and had an increase in nestin-labeled radial cell processes in the subventricular regions, and extended into the parenchyma. The subventricular zone was populated by healthy new oligodendrocytes (OLs). BrdU incorporation showed that these cells originated by proliferation and were identified as OLs based upon Tf expression. Thus, TS1 is an effective treatment to promote endogenous subventricular zone progenitor proliferation, migration and OL lineage specification. This strategy offers for the first time the possibility of myelin restoration to treat myelin disorders.

Manganese and iron transport across pulmonary epithelium

Pathways mediating pulmonary metal uptake remain unknown. Because absorption of iron and manganese could involve similar mechanisms, transferrin (Tf) and transferrin receptor (TfR) expression in rat lungs was examined. Tf mRNA was detected in bronchial epithelium, type II alveolar cells, macrophages, and bronchus-associated lymphoid tissue (BALT). Tf protein levels in lung and bronchoalveolar lavage fluid did not change in iron deficiency despite increased plasma levels, suggesting that lung Tf concentrations are regulated by local synthesis in a manner independent of body iron status. Iron oxide exposure upregulated Tf mRNA in bronchial and alveolar epithelium, macrophages, and BALT, but protein was not significantly increased. In contrast, TfR mRNA and protein were both upregulated by iron deficiency. To examine potential interactions with lung Tf, rats were intratracheally instilled with (54)Mn or (59)Fe. Unlike (59)Fe, interactions between (54)Mn and Tf in lung fluid were not detected. Absorption of intratracheally instilled (54)Mn from the lungs to the blood was unimpaired in Belgrade rats homozygous for the functionally defective G185R allele of divalent metal transporter-1, indicating that this transporter is also not involved in pulmonary manganese absorption. Pharmacological studies of (54)Mn uptake by A549 cells suggest that metal uptake by type II alveolar epithelial cells is associated with activities of both L-type Ca(2+) channels and TRPM7, a member of the transient receptor potential melastatin subfamily. These results demonstrate that iron and manganese are absorbed by the pulmonary epithelium through different pathways and reveal the potential role for nonselective calcium channels in lung metal clearance.

Differential expression of tissue factor mRNA and protein expression in murine sepsis

Tissue factor (TF) is a transmembrane protein, which is essential for initiation of the coagulation cascade. TF has been reported to play an important role in the progression of endotoxin (lipopolysaccharide, LPS)-mediated endotoxemia, being induced in numerous tissues, such as kidney, spleen and lung. We developed and validated a rabbit anti-murine TF peptide antiserum to localize TF protein in a murine sepsis model. TF protein distribution was compared to localization of TF mRNA and fibrin deposits, the ultimate resultant of procoagulant TF activity. Evident LPS mediated TF mRNA induction was observed in the tubular area at the cortico-medullar junction in the kidney, and TF activity was increased after 6 hours of endotoxemia. In the spleen, however, TF mRNA was induced in the interfollicular region upon LPS injection, corresponding to increased TF protein in the same area. The clusters of TF-protein positive cells in the spleen are predominantly granulocytes, but no TF mRNA expression was observed within these cells. Based on these observations and the presence of TF-protein positive granulocytes after splenectomy, we hypothesize that granulocytes take-up TF for transport to other locations in order to initiate fibrin formation or to induce pro-inflammatory gene expression upon interaction with factor VIIa.