Abstract 377: Phorbol 12-myristate 13-acetate Downregulates Tissue Factor Gene Expression in Human Pericytes

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Laura Sommerville ◽  
Stacey Snyder ◽  
Maureane Hoffman
Keyword(s):  
Gene Expression ◽  
Wound Healing ◽  
Tissue Factor ◽  
Primary Cultures ◽  
Factor Vii ◽  
Mrna Levels ◽  
Mrna Synthesis ◽  
Mrna Destabilization ◽  
Tf Gene

Tissue factor (TF) is a transmembrane receptor for factor VII/VIIa that plays a central role in hemostasis and angiogenesis. While TF is generally upregulated in pathologic conditions, we have reported that TF is downregulated in pericytes around vessels near a healing cutaneous wound. As the first demonstration of physiologic downregulation of TF, this finding suggested a potential means of modulating TF expression for therapeutic purposes. The goal of this study was to contribute to implementation of future therapies by elucidating mechanisms that regulate loss pericyte TF. To identify mediators of TF downregulation we utilized primary cultures of human placental pericytes to screen growth factors involved in wound healing including TGFβ, bFGF, VEGF, PDGF and ANG2. None significantly reduced pericyte TF expression as assessed by western blotting. We next tested agents that directly activate signaling pathways. Phorbol 12-myristate 13-acetate (PMA) triggered ~60% reduction in TF protein 4 hours after treatment. Complete loss of TF occurred by 8 hours (p<0.001) and remained until the experiment was terminated 24 hours after PMA addition. These results suggested that TF loss is mediated, at least in part, by downregulation of TF gene expression. We utilized qRT-PCR to determine the effects of PMA on synthesis of TF transcripts 4, 8, 12, and 24 hours after treatment. TF mRNA levels were unchanged 4 hours after treatment. However, TF transcripts decreased 4 and 6 fold 8 hours and 12 hours after PMA, respectively (p<0.01). 24 hours after treatment the amount of TF mRNA rebounded, yet remained 2 fold lower than the control (p<0.05). To determine if the decrease in TF transcripts is caused by mRNA destabilization, pericytes were treated with actinomycin D prior to PMA. Degradation of TF transcripts in PMA-treated cells was similar to that of DMSO-treated control cells at all time points (p=ns), indicating that PMA-mediated downregulation of TF expression occurs primarily through inhibition of mRNA synthesis rather than through destabilization of existing transcripts. In conclusion, our results demonstrate a role for transcriptional downregulation in PMA-mediated loss of pericyte TF protein, findings that pave the way for future in vivo studies of wound healing.

Acute hypoxia stimulates renin secretion and renin gene expression in vivo but not in vitro

1997 ◽  
Vol 272 (4) ◽  
pp. R1105-R1111 ◽  
Author(s):  
T. Ritthaler ◽  
K. Schricker ◽  
F. Kees ◽  
B. Kramer ◽  
A. Kurtz
Keyword(s):  
Gene Expression ◽  
Acute Hypoxia ◽  
Primary Cultures ◽  
Renin Secretion ◽  
Plasma Norepinephrine ◽  
Mrna Levels ◽  
Sprague Dawley ◽  
Renin Gene ◽  
Renin Mrna

This study aimed at examining the influence of acute hypoxia on renin secretion and renin gene expression in the kidney. To this end, male Sprague-Dawley rats were exposed to severe hypoxic stress (8% O2) or to carbon monoxide (0.1% CO) for 6 h, and plasma renin activity (PRA) and renal renin mRNA levels were determined. PRA values increased from 3 to 13 and 10 ng angiotensin I x h(-1) x ml(-1), and renin mRNA levels increased by 120 and 100% during hypoxia and CO, respectively. Lowering the PO2 from 150 to 20 or 7 mmHg in the gas atmosphere of primary cultures of renal juxtaglomerular cells had no influence on renin secretion and renin gene expression after 6 and 20 h. Our findings thus suggest that both arterial and venous hypoxia can be powerful stimulators of renin secretion and renin gene expression in vivo. Because renal denervation did not prevent stimulation of the renin system by hypoxia, the effect could be indirectly mediated via the baroreceptor-macula densa mechanism. Another potential mediator of the effect could be circulating catecholamines, since we found that plasma norepinephrine increased from 0.7 to 1.5 and 2.4 ng/ml and plasma epinephrine increased from 0.3 to 1.4 and 2.7 ng/ml during hypoxia and CO inhalation, respectively.

scholarly journals Modelization of Blood-Borne Hypercoagulability in Myeloma: A Tissue-Factor-Bearing Microparticle-Driven Process

TH Open ◽  
2019 ◽  
Vol 03 (04) ◽  
pp. e340-e347
Author(s):  
Loula Papageorgiou ◽  
Kutaiba Alhaj Hussen ◽  
Sandrine Thouroude ◽  
Elisabeth Mbemba ◽  
Héléne Cost ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Plasma Cells ◽  
Annexin V ◽  
Factor Vii ◽  
Newly Diagnosed ◽  
Normal Human ◽  
Tf Gene

Abstract Introduction Hypercoagulability is a common blood alteration in newly diagnosed chemotherapy naïve patients with multiple myeloma. The identification of the procoagulant potential of cancer cells, which is principally related to tissue factor (TF) expression, attracts particular interest. The mechanisms by which myeloma plasma cells (MPCs) activate blood coagulation have been poorly investigated. Aim To identify the principal actors related with MPCs that boost thrombin generation (TG). Methods TF and annexin V expression by MPCs and MPC-derived microparticles (MPC-dMPs) was analyzed by flow cytometry. TF activity (TFa) and TF gene expression were also determined. TG in the presence of MPCs or MPC-dMPs was assessed with the calibrated automated thrombogram assay (CAT) in normal human PPP and in plasma depleted of factor VII or XII. TG was also assessed in plasma spiked with MPCs and MPC-dMPs. Results MPC-dMPs expressed approximately twofold higher levels of TF as compared with MPCs. The TFa expressed by MPC-dMPs was significantly higher compared with that expressed by MPCs. MPCs and MPC-dMPs enhanced TG of human plasma. TG was significantly higher with MPC-dMPs compared with MPCs. Conclusion MPCs indirectly induce blood-borne hypercoagulability through the release of MPC-dMPs rich in TF. Since MPCs, expressing low TFa, represent a weak procoagulant stimulus, the hypercoagulability at the microenvironment could be the resultant of MPC-dMPs rich in TF.

The Effect of Grp78 Inhibition on Tissue Factor Gene Expression, Procoagulant Activity, and Factor Xa Generation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1924-1924
Author(s):  
Gourab Bhattacharjee ◽  
Jasimuddin Ahamed ◽  
Brian Pedersen ◽  
Amr El-Sheikh ◽  
Cheng Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Surface ◽  
Tissue Factor ◽  
Factor Xa ◽  
Cell Types ◽  
Procoagulant Activity ◽  
Coagulation Cascade ◽  
Clotting Factor ◽  
Tf Gene

Abstract In vivo biopanning with phage displayed peptide libraries has generated a group of peptide probes which bind selectively to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds to the 78 kDa glucose regulated protein (Grp78). Grp78 has been demonstrated to play a role in numerous pathological processes as well as a possible role in the local cell surface regulation of the coagulation cascade. The goal of this study is to determine the role of Grp78 in coagulation including plasma clotting, factor Xa (Xa) generation, and tissue factor (TF) gene expression. siRNA mediated inhibition of Grp78 results in a marked increase in TF gene expression in bEND.3 endothelial cells and RAW macrophage-like cells. Antibody mediated inhibition of cell surface Grp78 results in increased TF procoagulant activity and TF-dependent Xa generation in both the endothelial and macrophage cell types. These studies are consistent with results from another laboratory demonstrating that Grp78 over-expression inhibits TF mediated initiation and support of the coagulation protease cascade. Thus, our work indicates that Grp78 suppresses TF at both the functional and molecular level by inhibiting both its thrombogenic potential and gene expression.

The TF-603A/G gene promoter polymorphism and circulating monocyte tissue factor gene expression in healthy volunteers

2004 ◽  
Vol 91 (02) ◽  
pp. 248-254 ◽  
Author(s):  
Jean-Luc Reny ◽  
Ingrid Laurendeau ◽  
Pierre Fontana ◽  
Ivan Bièche ◽  
Annabelle Dupont ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tissue Factor ◽  
Ex Vivo ◽  
Gene Promoter ◽  
Promoter Polymorphism ◽  
Insertion Polymorphism ◽  
Mrna Levels ◽  
Tf Gene ◽  
Over Time ◽  
Gene Promoter Polymorphism

SummaryThree single nucleotide polymorphisms (-603A/G, -1322C/T, -1812C/T) and one deletion/insertion polymorphism (-1208D/I) are present in the tissue factor (TF) gene promoter sequence. These polymorphisms are in complete linkage disequilibrium, determining two haplotypes with almost equal frequency. The -603A/-1208D/-1322C/-1812C haplotype, presently defined as TF-603A, has been linked to venous thromboembolic disease, with a potentially protecting effect. The effects of the TF-603A/G gene polymorphism on monocyte gene expression and on a whole-blood clotting time (WBCT) are not known. We determined the WBCT in basal conditions (H0) and after 4 hours of LPS stimulation ex vivo (H4LPS) on blood samples from 100 young healthy caucasian male subjects on 2 visits, 7 days apart. Monocyte TF mRNA was quantified at H0 and H4LPS by real-time quantitative reverse-transcription PCR. The monocyte TF mRNA values determined at the first and second visits were concordant. In H4LPS samples, TF mRNA levels were increased 70-fold. The TF-603A haplotype was associated with 40%-lower TF mRNA levels at H0 (P=0.0002) and this association followed the same trend but was no longer significant at H4LPS. At H4LPS, TF mRNA levels were associated with WBCT shortening (P=0.0003). WBCT at H0 was not concordant over time, precluding any genotype-phenotype analysis. WBCT at H4LPS was concordant over time but was not related to the TF-603A/G polymorphism. The TF-603A/G gene promoter polymorphism thus significantly influences constitutive TF gene expression in human monocytes but has no major effect on TF gene expression or on WBCT in LPS stimulated conditions.

scholarly journals Increased Tissue Factor Expression Is Associated with Reduced Survival in Non–Small Cell Lung Cancer and with Mutations of TP53 and PTEN

2009 ◽  
Vol 55 (10) ◽  
pp. 1834-1842 ◽  
Author(s):  
Sandra Regina ◽  
Jean-Baptiste Valentin ◽  
Sébastien Lachot ◽  
Etienne Lemarié ◽  
Jérôme Rollin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lung Cancer ◽  
Relative Risk ◽  
Small Cell Lung Cancer ◽  
Tissue Factor ◽  
Cell Lung Cancer ◽  
Small Cell ◽  
Mrna Levels ◽  
Small Cell Lung ◽  
Tf Gene

Abstract Background: Tissue factor (TF), the main initiator of blood coagulation, is also a signaling protein that regulates cancer progression. TF synthesis was recently shown to be affected by tumor suppressor genes (TSGs) in tumor cell lines. We therefore studied TF gene (F3) expression and the status of genes coding for tumor protein p53 (TP53), phosphatase and tensin homolog (PTEN), and serine/threonine kinase 11 (STK11) in non–small cell lung cancer (NSCLC). Heparanase (HPSE) gene expression was also measured because this endo-β-D-glucuronidase was recently shown to enhance TF gene expression. Methods: TF and heparanase mRNA expression was measured by real-time PCR in 53 NSCLC tumors. Exons 5–8 of TP53 were sequenced from genomic DNA. Mutations of PTEN and STK11 were screened by multiplex ligation-dependent probe amplification. Results: TF mRNA levels were significantly higher in T3–T4 tumors (P = 0.04) and in stages III–IV of NSCLC (P = 0.03). Mutations of TP53, STK11, and PTEN were identified in 20 (37.7%), 21 (39%), and 20 (37.7%) of tumors, respectively. TF expression was higher in mutated TP53 (TP53Mut) (P = 0.02) and PTENMut (P = 0.03) samples. Moreover, TF mRNA increased from 2700 copies (no mutation) to 11 6415 when 3 TSG were mutated. Heparanase gene expression did not differ according to TF gene (F3) expression or TSG mutation. The median survival time was shorter in patients with tumor TF mRNA levels above median values (relative risk 2.2; P = 0.03, multivariate analysis) and when TP53 was mutated (relative risk 1.8; P = 0.02). Conclusions: These results provide clear evidence that combined oncogene events affecting TSG dramatically increase TF gene expression in lung tumors. Moreover, this study suggests that TF gene expression could be used as a prognostic marker in NSCLC. .

scholarly journals Reduction of hepatic glucocorticoid receptor and hexose-6-phosphate dehydrogenase expression ameliorates diet-induced obesity and insulin resistance in mice

2008 ◽  
Vol 41 (2) ◽  
pp. 53-64 ◽  
Author(s):  
Yanjun Liu ◽  
Yuichi Nakagawa ◽  
Ying Wang ◽  
Limei Liu ◽  
Hongwei Du ◽  
...  
Keyword(s):  
Gene Expression ◽  
Insulin Resistance ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Primary Cultures ◽  
Mrna Levels ◽  
Insulin Resistant ◽  
Beneficial Effects ◽  
Tissue Sensitivity

Intracellular glucocorticoid (GC) receptor (GR) function determines tissue sensitivity to GCs and strongly affects the development of type 2 diabetes and obesity. 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) mediates intracellular steroid exposure to mouse liver GR by prereceptor reactivation of GCs and is crucially dependent on hexose-6-phosphate dehydrogenase (H6PDH)-generating NADPH system. Pharmacological inhibition of 11β-HSD1 improves insulin intolerance and obesity. Here, we evaluated the potential beneficial effects of 11β-HSD1 inhibitor carbenoxolone (CBX) in diet-induced obese (DIO) and insulin-resistant mice by examining the possible influence of CBX on the expression of GR, 11β-HSD1, and H6PDH in vivo and in vitro in hepatocytes. Treatment of DIO mice with CBX markedly reduced hepatic GR mRNA levels and reduced weight gain, hyperglycemia, and insulin resistance. The reduction of hepatic GR gene expression was accompanied by CBX-induced inhibition of both 11β-HSD1 and H6PDH activity and mRNA in the liver. Moreover, CBX treatment also suppressed the expression of both phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase enzyme (G6Pase) mRNA and improved hepatic [1, 2-3H] deoxy-d-glucose uptake in DIO mice. In addition, the treatment of primary cultures of hepatocytes with increasing concentrations of CBX led to a dose-dependent downregulation of GR mRNA levels, which correlated with the suppression of both 11β-HSD1 and H6PDH activity and their gene expression. Addition of CBX to primary hepatocytes also resulted in suppression of both PEPCK and G6Pase mRNA levels. These findings suggest that CBX exerts some of its beneficial effects, at least in part, by inhibiting hepatic GR and H6PDH expression.

scholarly journals Monocyte tissue factor induction by lipopolysaccharide (LPS): dependence on LPS-binding protein and CD14, and inhibition by a recombinant fragment of bactericidal/permeability-increasing protein

Blood ◽  
1994 ◽  
Vol 83 (9) ◽  
pp. 2516-2525 ◽  
Author(s):  
K Meszaros ◽  
S Aberle ◽  
R Dedrick ◽  
R Machovich ◽  
A Horwitz ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Binding Protein ◽  
Mononuclear Phagocytes ◽  
Factor Vii ◽  
Peripheral Blood Mononuclear ◽  
Recombinant Fragment

Abstract Mononuclear phagocytes, stimulated by bacterial lipopolysaccharide (LPS), have been implicated in the activation of coagulation in sepsis and endotoxemia. In monocytes LPS induces the synthesis of tissue factor (TF) which, assembled with factor VII, initiates the blood coagulation cascades. In this study we investigated the mechanism of LPS recognition by monocytes, and the consequent expression of TF mRNA and TF activity. We also studied the inhibition of these effects of LPS by rBPI23, a 23-kD recombinant fragment of bactericidal/permeability increasing protein, which has been shown to antagonize LPS in vitro and in vivo. Human peripheral blood mononuclear cells, or monocytes isolated by adherence, were stimulated with Escherichia coli O113 LPS at physiologically relevant concentrations (&gt; or = 10 pg/mL). The effect of LPS was dependent on the presence of the serum protein LBP (lipopolysaccharide-binding protein), as shown by the potentiating effect of human recombinant LBP or serum. Furthermore, recognition of low amounts of LPS by monocytes was also dependent on CD14 receptors, because monoclonal antibodies against CD14 greatly reduced the LPS sensitivity of monocytes in the presence of serum or rLBP. Induction of TF activity and mRNA expression by LPS were inhibited by rBPI23. The expression of tumor necrosis factor showed qualitatively similar changes. Considering the involvement of LPS-induced TF in the potentially lethal intravascular coagulation in sepsis, inhibition of TF induction by rBPI23 may be of therapeutic benefit.

Transferrin gene expression and transferrin immunolocalization in developing foetal rat lung

1991 ◽  
Vol 99 (3) ◽  
pp. 651-656 ◽  
Author(s):  
S.J. Skinner ◽  
C.E. Somervell ◽  
S. Buch ◽  
M. Post
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Chondroitin Sulphate ◽  
Primary Cultures ◽  
Basement Membranes ◽  
Lung Cells ◽  
Intense Staining ◽  
Tf Gene ◽  
Foetal Lung

In previous studies we have shown that transferrin (Tf) specifically stimulates dermatan- and chondroitin-sulphate proteoglycan accumulation around lung cells, and in the extracellular matrix of lung tissue, in vitro. The aim of this study was to determine whether the gene for Tf was activated in specific lung cells during development, and whether the protein product showed evidence of association with extracellular matrix. The expression of the gene in developing lung was shown by the hybridization of a Tf cDNA to a 2.4 kb (kilobase) mRNA species in total RNA extracts of foetal lung. The expression of the Tf gene in comparison to a control gene (GAPD, glyceraldehyde phosphate dehydrogenase) was greatest in 19, 20 and 21 day foetal lung, rising from low levels on day 18 and decreasing markedly at term (day 22). Extracts of RNA from primary cultures of mesenchymal fibroblasts and type II epithelial cells were also analysed for Tf mRNA. These experiments indicated that Tf gene expression was predominantly confined to the mesenchymal compartment. The presence of Tf in histological sections of foetal lung was demonstrated by immunohistochemistry and showed a distinct pattern, with intense staining of the alveolar and the capillary basement membranes. The matrix surrounding the mesenchymal fibroblasts was stained in a diffuse network while epithelial cells were unstained. The staining was low from days 12–16 of gestation, increased to a maximum at days 19–20 but decreased markedly toward term. The Tf staining did not co-localize with transferrin receptor, also demonstrated by immunohistochemistry. These results suggest that Tf is not only present at specific sites in the developing lung, but also is synthesized according to a strict developmental schedule of gene expression.

scholarly journals Activation of human factor VII during clotting in vitro

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 218-226 ◽  
Author(s):  
LV Rao ◽  
SP Bajaj ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Glass Tube ◽  
Factor Vii ◽  
Clotting Factor ◽  
Activate Factor ◽  
Factor Xii ◽  
Factor X ◽  
Normal Plasma

Abstract We have studied factor VII activation by measuring the ratio of factor VII clotting to coupled amidolytic activity (VIIc/VIIam) and cleavage of 125I-factor VII. In purified systems, a low concentration of Xa or a higher concentration of IXa rapidly activated 125I-factor VII, yielding a VIIc/VIIam ratio of 25 and similar gel profiles of heavy and light chain peaks of VIIa. On further incubation, VIIa activity diminished and a third 125I-peak appeared. When normal blood containing added 125I- factor VII was clotted in a glass tube, the VIIc/VIIam ratio rose fivefold, and 20% of the 125I-factor VII was cleaved. Clotting normal plasma in an activated partial thromboplastin time (APTT) system yielded a VIIc/VIIam ratio of 25 and over 90% cleavage of 125I-factor VII. Clotting factor XII-deficient plasma preincubated with antibodies to factor X in an APTT system with added XIa yielded a VIIc/VIIam ratio of 19 and about 60% cleavage, which indicates that IXa, at a concentration achievable in plasma, can effectively activate factor VII. Clotting normal plasma with undiluted tissue factor yielded a VIIc/VIIam ratio of 15 to 20 and 60% cleavage of 125I-factor VII, whereas clotting plasma with diluted tissue factor activated factor VII only minimally. We conclude that both Xa and IXa can function as significant activators of factor VII in in vitro clotting mixtures but believe that only small amounts of factor VII may be activated in vivo during hemostasis.

scholarly journals The 12-HHT/BLT2/NO Axis Is Associated to the Wound Healing and Skin Condition in Different Glycaemic States

2019 ◽  
Vol 7 (4) ◽  
pp. 65
Author(s):  
Leguina-Ruzzi ◽  
Ortiz Diban ◽  
Velarde
Keyword(s):  
Nitric Oxide ◽  
Wound Healing ◽  
Tight Junction ◽  
Skin Condition ◽  
Inos Mrna ◽  
Nitric Oxide Production ◽  
Mrna Levels ◽  
Oxide Production

Type 2 diabetes affects over 340 million people worldwide. This condition can go unnoticed and undiagnosed for years, leading to a late stage where high glycaemia produces complications such as delayed wound healing. Studies have shown that 12-HHT through BLT2, accelerates keratinocyte migration and wound healing. Additionally, evidence has shown the role of nitric oxide as a pro-regenerative mediator, which is decreased in diabetes. Our main goal was to study the association between the 12-HHT/BLT2 axis and the nitric oxide production in wound healing under different glycaemia conditions. For that purpose, we used in vivo and in vitro models. Our results show that the skin from diabetic mice showed reduced BLT2 and iNOS mRNA, TEER, 12-HHT, nitrites, and tight junction levels, accompanied by higher MMP9 mRNA levels. Furthermore, a positive correlation between BLT2 mRNA and nitrites was observed. In vitro, HaCaT-BLT2 cells showed higher nitric oxide and tight junction levels, and reduced MMP9 mRNA levels, compared to mock-keratinocytes under low and high glucose condition. The wound healing capacity was associated with higher nitric oxide production and was affected by the NOS inhibition. We suggest that the BLT2 expression improves the keratinocyte response to hyperglycaemia, associated with the production of nitric oxide.

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