The Role of Tissue Factor- β1- Integrin Complex Formation on Src Activity during the Apoptosis Mechanism in Endothelial Cells = دور العامل النسيجي البروتيني بيتا أنتجرين على نشاط بروتين الكاينيز في الخلايا السرطانية خلال آلية موت الخلايا المبرمج في الخلايا البطانية

2019 ◽  
Vol 4 (2) ◽  
pp. 124-138
Author(s):  
Alabdulmonem , Waleed ◽  
Alahaydib , Faisal
Keyword(s):  
Endothelial Cells ◽  
Complex Formation ◽  
Tissue Factor ◽  
Β1 Integrin

scholarly journals Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

APOPTOSIS ◽  
2019 ◽  
Vol 25 (1-2) ◽  
pp. 29-41 ◽  
Author(s):  
Ali M. Ethaeb ◽  
Mohammad A. Mohammad ◽  
Yahya Madkhali ◽  
Sophie Featherby ◽  
Anthony Maraveyas ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Kinase Activity ◽  
Β1 Integrin ◽  
Wild Type ◽  
Human Coronary Artery ◽  
Src Inhibitor ◽  
Cellular Apoptosis ◽  
Measured Activation ◽  
First Time

Abstract Accumulation of tissue factor (TF) within cells leads to cellular apoptosis mediated through p38 and p53 pathways. In this study, the involvement of Src1 in the induction of TF-mediated cell apoptosis, and the mechanisms of Src1 activation were investigated. Human coronary artery endothelial cell (HCAEC) were transfected with plasmids to express the wild-type TF (TFWt-tGFP), or a mutant (Ser253 → Ala) which is incapable of being released from cells (TFAla253-tGFP). The cells were then activated with PAR2-agonist peptide (SLIGKV-NH) and the phosphorylation of Src and Rac, and also the kinase activity of Src were assessed. Transfected cells were also pre-incubated with pp60c Src inhibitor, FAK inhibitor-14, or a blocking anti-β1-integrin antibody prior to activation and the phosphorylation of p38 as well as cellular apoptosis was examined. Finally, cells were co-transfected with the plasmids, together with a Src1-specific siRNA, activated as above and the cellular apoptosis measured. Activation of PAR2 lead to the phosphorylation of Src1 and Rac1 proteins at 60 min regardless of TF expression. Moreover, Src phosphorylation and kinase activity was prolonged up to 100 min in the presence of TF, with a significantly higher magnitude when the non-releasable TFAla253-tGFP was expressed in HCAEC. Inhibition of Src with pp60c, or suppression of Src1 expression in cells, reduced p38 phosphorylation and prevented cellular apoptosis. In contrast, inhibition of FAK had no significant influence on Src kinase activity or cellular apoptosis. Finally, pre-incubation of cells with an inhibitory anti-β1-integrin antibody reduced both Src1 activation and cellular apoptosis. Our data show for the first time that the over-activation of Src1 is a mediator of TF-induced cellular apoptosis in endothelial cells through a mechanism that is dependent on its interaction with β1-integrin.

Role of Tissue Factor in Adhesion of Mononuclear Phagocytes to and Trafficking Through Endothelium In Vitro

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4167-4177 ◽  
Author(s):  
Gwendalyn J. Randolph ◽  
Thomas Luther ◽  
Sybille Albrecht ◽  
Viktor Magdolen ◽  
William A. Muller
Keyword(s):  
Amino Acids ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Mononuclear Phagocytes ◽  
Adhesive Protein ◽  
Endothelial Surface ◽  
Apical Direction ◽  
Vitro Model

Abstract An in vitro model consisting of endothelium grown on collagen was used to investigate how mononuclear phagocytes traverse endothelium in the basal-to-apical direction (reverse transmigration), a process that mimics their migration across vascular and/or lymphatic endothelium during atherosclerosis and resolution of inflammation, respectively. Monoclonal antibody (MoAb) VIC7 against tissue factor (TF) inhibited reverse transmigration by 77%. Recombinant tissue factor fragments containing at least six amino acids C-terminal to residue 202 also strongly inhibited reverse transmigration. TF was absent on resting monocytes but was induced on these cells after initial apical-to-basal transendothelial migration. Two additional observations suggest that TF is involved in adhesion between mononuclear phagocytes and endothelium: (1) when monocytes were incubated with lipopolysaccharide (LPS) to stimulate expression of TF before they were added to endothelium, VIC7 or soluble TF modestly inhibited their adhesion to the apical endothelial surface, each by about 35%; and (2) endothelial cells specifically bound to surfaces coated with TF fragments containing amino acids 202-219. This binding was blocked by anti-TF MoAb, suggesting that endothelial cells bear a receptor for TF. These data suggest that mononuclear phagocytes use TF, perhaps as an adhesive protein, to exit sites of inflammation.

Abstract 190: Purinergic Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: New AP-1 Site and Negative Regulator

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Yiwei Liu ◽  
Lingxin Zhang ◽  
Chuan Wang ◽  
Shama Roy ◽  
Jianzhong Shen
Keyword(s):  
Endothelial Cells ◽  
Coronary Artery ◽  
Tissue Factor ◽  
Promoter Activity ◽  
Purinergic Receptor ◽  
Consensus Sequence ◽  
Negative Regulator ◽  
Coagulation Cascade ◽  
Human Coronary Artery

Previously we reported that the P2Y2 receptor (P2Y2R) is one of the predominant purinergic receptors expressed in human coronary artery endothelial cells (HCAEC), and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R-TF axis remains unclear. Here we report a role of a newly identified AP-1 consensus sequence along with its new binding components in P2Y2R regulation of TF transcription. We identified with bioinformatics tools that a novel AP-1 site at -1363 bp of human TF promoter region is highly conserved across multiple species. P2Y2R activation increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion, and mutation of this new distal AP-1 site all significantly supressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c-Jun, ATF-2 and Fra-1, but not the typical c-Fos, bound to the new AP-1 site. In addition, loss-of-function studies using siRNAs confirmed a positive transactivation role of c-Jun and ATF-2, but unexpectedly revealed a strong negative role of Fra-1 in P2Y2R-induced TF up-regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation, leading to Fra-1 activation while JNK activated c-Jun and ATF-2. These findings reveal the basis for P2Y purinergic receptor regulation of endothelial TF expression and indicate that targeting the P2Y2R-Fra-1-TF pathway may be an attractive new strategy in control of vascular thrombogenicity and/or inflammation associated with endothelial dysfunction.

scholarly journals Integral Role of RhoA Activation in Monocyte Adhesion–Triggered Tissue Factor Expression in Endothelial Cells

2003 ◽  
Vol 23 (4) ◽  
pp. 681-687 ◽  
Author(s):  
Toshiyuki Ishibashi ◽  
Takayuki Sakamoto ◽  
Hiroshi Ohkawara ◽  
Kenji Nagata ◽  
Koichi Sugimoto ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Tissue Factor Expression ◽  
Monocyte Adhesion ◽  
Rhoa Activation ◽  
Factor Expression ◽  
Integral Role

The Role of Catalytic Cleft and Exosite Residues of Factor VIIa for Complex Formation with Tissue Factor Pathway Inhibitor

2001 ◽  
Vol 85 (03) ◽  
pp. 458-463 ◽  
Author(s):  
Alexei Iakhiaev ◽  
Wolfram Ruf ◽  
Vijaya Mohan Rao
Keyword(s):  
Complex Formation ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Viia ◽  
Cell Surface Receptor ◽  
Tissue Factor Pathway ◽  
Gla Domain ◽  
Catalytic Cleft ◽  
S2 Subsite

SummaryThe extrinsic coagulation pathway is initiated by the binding of plasma factor VIIa (VIIa) to the cell surface receptor tissue factor (TF). Formation of the TF-VIIa complex results in allosteric activation of VIIa as well as the creation of an extended macromolecular substrate binding exosite that greatly enhances proteolytic activation of substrate factor X. The catalytic function of the TF-VIIa complex is regulated by a specific Kunitz-type inhibitor, tissue factor pathway inhibitor (TFPI). TFPI inhibition of the TF-VIIa complex was enhanced by the presence of Xa. This study investigates the relative contribution of catalytic cleft and exosite residues in VIIa for inhibitory complex formation with TFPI. VIIa protease domain residues Q176, T239 and E296 are involved in the formation of stable inhibitor complex with free TFPI. Kinetic analysis further demonstrated a predominant role of the S2’ subsite residue Q176 for the initial complex formation with TFPI. In contrast, no significant reductions in inhibition by TFPI-Xa were found for each of the mutants in complex with phospholipid reconstituted TF. However, reduced rates of inhibition of the VIIa Gla-domain (R36) and Q176 mutant by TFPI-Xa were evident when TF was solubilized by detergent micelles. These data demonstrate docking of the TFPI-Xa complex with the macromolecular substrate exosite and the catalytic cleft, in particular the S2’ subsite. The masking of the mutational effect by the presence of phospholipid shows a critical importance of Xa Gla-domain interactions in stabilizing the quaternary TF-VIIa-Xa-TFPI complex.

Human Alternatively Spliced Tissue Factor Promotes Monocyte-Endothelial Interactions Via Upregulation of Adhesion Molecules In Microvascular Endothelial Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 343-343
Author(s):  
Ramprasad Srinivasan ◽  
Evgeny Ozhegov ◽  
Yascha van den Berg ◽  
Henri H Versteeg ◽  
Vladimir Bogdanov
Keyword(s):  
Endothelial Cells ◽  
Laminar Flow ◽  
Tissue Factor ◽  
Adhesion Molecules ◽  
Flow Chamber ◽  
Microvascular Endothelial Cells ◽  
Β1 Integrin ◽  
Chemokine Gradient ◽  
Alternatively Spliced

Abstract Abstract 343 Background: Alternatively spliced Tissue Factor (asTF) is a soluble form of Tissue Factor (TF) that lacks the transmembrane domain and exhibits low pro-coagulant activity compared to decrypted full-length TF (Bogdanov et al, Nat Med 2003). Human asTF was recently shown to ligate integrins α6β1 and αVβ3 on endothelial cell surfaces, which triggered neovascularization in vitro, ex vivo, and in vivo in fVIIa- and/or PAR-2 independent fashion (van den Berg et al., PNAS 2009). This principally novel, non-proteolytic biologic activity of asTF was found to activate multiple integrin-linked kinases in human and murine macrovascular endothelial cells (EC), including those comprising the PI3K/Akt pathway. Engagement of PI3K/Akt signaling is known to activate NFkB – a transcription factor involved in upregulation of multiple leukocyte adhesion molecules that play a major role in various disease states, most notably atherogenesis and tumorigenesis. In this study, we sought to determine whether human asTF acts as an agonist on microvascular EC – the endothelial sub-type most relevant to monocyte egress from the circulation. Results: Primary cardiac and retinal human microvascular endothelial cells (micro-EC) expressed α6, β1, and β3 mRNA. Both micro-EC sub-types rapidly adhered to the recombinant N-terminally His-tagged human asTF: a 20+ fold increase in the number of adherent micro-EC was observed as early as 30 min after the cells were added (n=3,100 ng asTF vs BSA, p<0.0001). In both sub-types of micro-EC, anti-β1 integrin antibody completely blocked adhesion to asTF whereas anti-β3 and/or anti-αvβ3 antibody had no effect, likely due to the known scarcity of non-β1 integrins on the surface of human micro-EC (Wilson et al., IOVS 2003). We then examined the changes in gene expression elicited by asTF in micro-EC using Affymetrix Gene 1.0 ST microarrays. A marked upregulation of several cell adhesion molecules (CAMs) was observed in cardiac and retinal micro-EC including VCAM-1 and E-selectin, which was confirmed at the protein level by western blotting. We proceeded to investigate the functional significance of the upregulation of CAMs by performing adhesion assays using pre-labeled THP-1 cells. Under orbital shear stress conditions, a ∼76% increase in THP-1 adhesion was observed for cardiac micro-EC treated with asTF over control (n=5, p<0.001), and a ∼62% for retinal micro-EC (n=5, p<0.005). Addition of polymyxin B and/or non-charged agarose beads elicited no effect, whereas pre-treatment of asTF samples with Ni-charged beads and heat denaturation eliminated the effect, confirming that the observed findings are asTF-specific and not caused by LPS contamination. In the static assay and the laminar flow chamber assay performed under the flow rates found in postcapillary venules, the increase in THP-1 adhesion was 40% (n=3, p<0.001) and 250% (n=3, p<0.05), respectively, for cardiac micro-EC treated with asTF and 83% (n=3, p<0.001) and 290% (n=3, p<0.05), respectively, for retinal micro-EC. While performing laminar flow chamber assays, we observed relocation of some of the adhered THP-1 cells under the EC monolayer, which indicates that asTF may cause monocyte emigration in the absence of a chemokine gradient. To determine whether monocyte egress through asTF-stimulated micro-EC can also occur under a chemokine gradient, we performed transendothelial migration assays using transwell inserts with MCP-1 placed in the lower chamber. Here, we observed that asTF potentiated THP-1 migration through cardiac micro-EC by ∼206% (n=3, p<0.0001), and through retinal micro-EC – by ∼90% (n=3, p<0.005). Conclusions: We show for the first time that (i) human asTF ligates β1 integrin(s) on primary human micro-EC, (ii) this leads to transcriptional upregulation of CAMs, and (iii) this upregulation promotes adhesion and transendothelial migration of monocytic cells under physiologically relevant conditions. We note that the concentrations of asTF used here were in the range found in cervical cancer tissue (van den Berg et al., PNAS 2009). Our results expand the potential scope of asTF's contribution to atherogenesis and tumorigenesis. Evaluation of these findings in vivo is highly warranted as monocyte recruitment triggered by asTF may represent a novel nexus in atherosclerotic progression and/or tumor growth. Disclosures: No relevant conflicts of interest to declare.

Junctional Adhesion Molecule-A/F11 Receptor (JAM-A/F11R) Is a Novel Biomarker and a Potential Treatment Target In Multiple Myeloma Tumor and Its Microvascular Milieu

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 450-450
Author(s):  
Bani M Azari ◽  
Danielle F Joseph ◽  
Marc J Braunstein ◽  
H. Uwe Klueppelberg ◽  
Eric LP Smith ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Tumor Cells ◽  
Β1 Integrin ◽  
Mrna Levels ◽  
Treatment Target

Abstract Abstract 450 Background: Multiple myeloma (MM) is a disease of clonal plasma cells that accumulate in the bone marrow (BM), causing monoclonal immunoglobulin (IG) production, BM failure, osteolytic lesions, and kidney disease. Although initially treatable, tumor cells ultimately become resistant to drug-treatment, and the disease is invariably fatal. Therefore, novel treatment targets need to be identified. The tumor microenvironment, and vascular endothelial cells in particular, play a key role in the adhesion and migration of MM cells and thus govern tumor survival and growth, as well as the acquisition of drug-resistance. Hence, the adhesion/migration systems of MM cells are key potential therapeutic targets. The cell membrane protein JAM-A/F11R is an endothelial cell (EC) adhesion molecule of the IG superfamily, and its expression is upregulated by TNF-a through NF-κB signaling. F11R also alters EC migration and paracellular permeability via stabilization of β1 integrin. We have previously shown that F11R gene expression and serum levels are upregulated in patients with MM compared to healthy controls. In this study, we further explored the functions of F11R within MM cells in order to gain insight into the potential role of this molecule in the progression and treatment of MM. Methods: The MM cell line RPMI-8266 (RPMI) was examined for functional studies in vitro. Informed consent was obtained from all subjects. Primary BM tumor cells were enriched to > 95% CD138+ cells by positive selection using anti-CD138 MACS MicroBeads. The CD138– fraction was used for outgrowth of confluent EPCs (> 98% vWF/CD133/KDR+). Human umbilical vein endothelial cells (HUVECs) served as controls. F11R mRNA levels were assessed by Affymetrix GeneChip analysis and by F11R probe-based real-time PCR compared to a standard curve normalized to GAPDH mRNA levels. F11R protein levels were measured by immunofluorescence (IF) and flow cytometry. The role of F11R in MM cell migration and survival was quantified by examining these functions in RPMI cells in which F11R was knocked down by siRNA silencing and comparing them with control untransfected RPMI cells or cells transfected with a non-targeting siRNA or lipofectamine. Tumor migration and survival were determined by the Millipore QCM Chemotaxis assay (using a 5 micron pore size) and an Promega Cell Proliferation Assay, respectively. Each assay was performed in triplicate and replicated at least twice. Statistical analyses were performed using Student's t-test, two-tailed; P≤.05 was considered significant. Results: Inhibition of F11R gene expression by siRNA resulted in 70% cell death compared to control untransfected (P<.001), non-targeting siRNA (P=.04), or lipofectamine-treated (P=.003) MM cells (RPMI). Moreover, migration of MM cells was also inhibited by 23% after silencing of F11R expression compared to cells transfected with control siRNA (P=.008). Elevated F11R mRNA levels in MM cell lines and patient-derived tumor endothelial progenitor cells (EPCs) was confirmed by IF and flow cytometry using a specific monoclonal antibody, and showed increased expression of both membrane and cytoplasmic F11R compared to controls. Gene expression profiles from 20 patients' corresponding BM tumor cells and EPCs showed that F11R mRNA levels in tumor cells were higher than MM in EPCs by 12.62 fold, (P=1×10-4). However, F11R had a higher level of expression in MM EPCs compared to healthy control EPCs by 2.41 fold (P=.001), reflecting a complex regulatory role of F11 signaling in MM, similar to breast cancer cells (Naik et al., 2008). Conclusion: We show, for the first time, that targeted inhibition of F11R/JAM-A expression bears key anti-myeloma consequences, defined by inhibition of tumor migration and survival. Taken together with elevated gene and protein expression of F11R/JAM-A expression, these results underscore the importance of this receptor as a tumor biomarker and a potential MM treatment target that warrants further validation. Future studies: Under investigation are the in vivo effect of F11R silencing in combination with other anti-myeloma strategies in a murine myeloma model; and also, whether F11R effects on MM cell migration involve stabilization of β1 integrin, as recently described in cardiovascular disease by Azari BM et al. 2010. Disclosures: No relevant conflicts of interest to declare.

Role of Tissue Factor in Adhesion of Mononuclear Phagocytes to and Trafficking Through Endothelium In Vitro

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4167-4177 ◽  
Author(s):  
Gwendalyn J. Randolph ◽  
Thomas Luther ◽  
Sybille Albrecht ◽  
Viktor Magdolen ◽  
William A. Muller
Keyword(s):  
Amino Acids ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Mononuclear Phagocytes ◽  
Adhesive Protein ◽  
Endothelial Surface ◽  
Apical Direction ◽  
Vitro Model

An in vitro model consisting of endothelium grown on collagen was used to investigate how mononuclear phagocytes traverse endothelium in the basal-to-apical direction (reverse transmigration), a process that mimics their migration across vascular and/or lymphatic endothelium during atherosclerosis and resolution of inflammation, respectively. Monoclonal antibody (MoAb) VIC7 against tissue factor (TF) inhibited reverse transmigration by 77%. Recombinant tissue factor fragments containing at least six amino acids C-terminal to residue 202 also strongly inhibited reverse transmigration. TF was absent on resting monocytes but was induced on these cells after initial apical-to-basal transendothelial migration. Two additional observations suggest that TF is involved in adhesion between mononuclear phagocytes and endothelium: (1) when monocytes were incubated with lipopolysaccharide (LPS) to stimulate expression of TF before they were added to endothelium, VIC7 or soluble TF modestly inhibited their adhesion to the apical endothelial surface, each by about 35%; and (2) endothelial cells specifically bound to surfaces coated with TF fragments containing amino acids 202-219. This binding was blocked by anti-TF MoAb, suggesting that endothelial cells bear a receptor for TF. These data suggest that mononuclear phagocytes use TF, perhaps as an adhesive protein, to exit sites of inflammation.

Sign in / Sign up

Export Citation Format

Share Document