HUMAN HEPATIC ENDOTHELIAL CELLS AND HEPATOCYTES IN CULTURE: MORPHOLOGICAL FEATURES, AND PRODUCTION OF VON WILLEBRAND FACTOR AND FIBRINOGEN

1987 ◽  
Author(s):  
R Harrison
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Human Liver ◽  
Liver Cells ◽  
Mixed Population ◽  
Dependent Manner ◽  
Cell Type ◽  
Von Willebrand ◽  
Willebrand Factor

Liver cells were derived from cadaveric organ donors. Pieces of human liver 5 to 50 grams were minced, washed, and incubated in collagenase at 37 degrees C. After washing, the cell suspension was plated into culture vessels that had been briefly pre-treated with an extract derived from human liver. A mixed population of liver cells, including endothelial cells, hepatocytes, and Kupffer cells, attached within hours. At the end of 2 to 3 weeks there developed clusters of densely packed cells of two types. The most numerous cells were initially fusiform but grew as a monolayer even when densely packed. As density increased they assumed a polygonal form; cells with this morphological appearance stained immunocytochemically for von Willebrand factor antigen. They were relatively small and resembled cells derived from human umbilical vein except that the cytoplasm was more filmy in appearance. The second prominent cell type was significantly larger and likewise replicated to form clusters. These large cells sometimes contained multiple nuclei, exhibited a relatively low nuclear to cytoplasmic ratio, and immunocytochemically stained for human fibrinogen. A more distinct nuclear membrane and prominent nucleoli were characteristics of hepatocytes that were useful light microscopically in distinguishing these cells from sinusoidal endothelial cells. Ultrastructurally, endothelial cells were characterized by small size, holes in and among the cells that probably were the in vitro analogue of fenestrae, and numerous Weibel-Palade bodies in the cytoplasm, which otherwise was relatively bland. Hepatocytes, by contrast, had an active appearing cytoplasm containing more organelles. Canaliculi and typical tight junctions formed between adjacent hepatocytes. Levels of vWF and fibrinogen increased in a time dependent manner in media overlying this mixed population of cells. Human factor VIII has not yet been detected in the media overlying these mixed cells derived from human liver, and factor VIII antigen has not yet been demonstrable immunocytochemically in either cell type.

scholarly journals Differential regulation by cytokines of constitutive and stimulated secretion of von Willebrand factor from endothelial cells

Blood ◽  
1990 ◽  
Vol 75 (3) ◽  
pp. 688-695 ◽  
Author(s):  
EM Paleolog ◽  
DC Crossman ◽  
JH McVey ◽  
JD Pearson
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Messenger Rna ◽  
Cell Injury ◽  
Interleukin 1 ◽  
Tnf Alpha ◽  
Detectable Effect ◽  
Dependent Manner ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We examined the effect of cytokines on basal and agonist-stimulated release of von Willebrand factor (vWf) by human endothelial cells. Treatment of endothelial cells for up to 48 hours with human recombinant or purified interleukin 1 (IL-1) or human recombinant tumor necrosis factor-alpha (TNF-alpha) did not significantly affect constitutive secretion of vWf or intracellular levels of vWf, although basal prostacyclin (PGI2) production was markedly enhanced. In contrast, both IL-1 and TNF-alpha modulated vWf release in response to thrombin or phorbol ester. Pretreatment of endothelial cells for 2 hours with either cytokine enhanced by up to threefold the stimulatory effect of a subsequent 60-minute exposure to thrombin. Addition of cycloheximide (5 micrograms/mL) during the preincubation abolished this enhancement. Moreover, if the cytokine pretreatment time was extended to 24 hours, agonist-stimulated vWf release was significantly suppressed. Cytokine treatment for 2 or 24 hours had no detectable effect on levels of vWf messenger RNA. The effects of cytokines were not the result of contamination with bacterial lipopolysaccharide and were not attributable to endothelial cell injury. These results show that cytokines have little or no direct effect on vWf release from endothelial cells but can significantly modulate its acute release in response to other stimuli in a complex time- and dose-dependent manner.

Von Willebrand Factor Binds to the Endothelial Growth Factor Angiopoietin-2 Both within Endothelial Cells and Upon Release From Weibel Palade Bodies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 698-698 ◽  
Author(s):  
Thomas A J Mckinnon ◽  
Richard D Starke ◽  
Kushani Ediriwickrema ◽  
Anna Maria Randi ◽  
Michael Laffan
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Extracellular Matrix Proteins ◽  
Matrix Proteins ◽  
Dependent Manner ◽  
Platelet Receptor ◽  
Angiopoietin 2 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 698 Von Willebrand Factor (VWF) is a large multimeric plasma glycoprotein essential for homeostasis, also involved in inflammation and angiogenesis. The majority of VWF is synthesised by endothelial cells (EC) and is either constitutively secreted or stored in Weibel-Palade bodies (WPB), ready to be released in response to endothelial stimulation. Several studies have shown that formation of WPB is dependent on the presence of VWF, and deletion of VWF in human umbilical vein EC (HUVEC) results in loss of WPB. Amongst the other proteins shown to co-localise to WPB is angiopoietin-2 (Ang2), a ligand of the receptor tyrosine kinase Tie-2. Ang2 regulates endothelial cell survival, vascular stability and maturation, by destabilizing quiescent endothelium and facilitating the response to inflammatory and angiogenic stimuli. VWF is required for storage of Ang2, and release of Ang-2 from EC is increased in VWF-deficient HUVEC. Recently, we have shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by Ang-2. In the present study we investigated the interaction between Ang2 and VWF. Binding analysis demonstrated that recombinant human Ang2 bound to purified plasma-derived VWF in a pH and calcium dependent manner, with optimal binding occurring at pH 6.5 and 10mM calcium, indicative of binding within the Golgi body. Generation of binding isotherms established that Ang2 bound to VWF with high affinity (KD∼3nM); furthermore binding affinity was not dependent on VWF conformation. Using an array of VWF constructs we determined that Ang2 bound predominantly to the VWF A1 domain, which also contains binding sites to the platelet receptor GPIb and extracellular matrix proteins. Co-immunoprecipitation experiments performed on TNFα- and ionomycin-stimulated HUVECs, to induce WPB exocytosis, confirmed that a portion of Ang2 remained bound to secreted VWF. Moreover, immunofluorescence staining of histamine-stimulated HUVECs to induce VWF release demonstrated the presence of Ang2 on VWF strings secreted from ECs. Finally we demonstrated that Ang2 bound to VWF was still able to interact with Tie-2. These data demonstrate that binding of Ang2 to VWF occurs within the cell; we propose that this is the mechanism mediating storage of Ang2 in WPB. Moreover, the finding that the Ang2-VWF interaction is preserved following secretion raises the intriguing possibility VWF may affect Ang2 function, possibly by localising Ang2 to the Tie 2 receptor under the shear forces experienced in flowing blood. Similarly, Ang-2 binding to VWF may modulate its interaction with receptors and extracellular matrix proteins, and ultimately influence the role of VWF in the angiogenic processes. Disclosures: No relevant conflicts of interest to declare.

Intracellular interaction of von Willebrand factor and factor VIII depends on cellular context: lessons from platelet-expressed factor VIII

Blood ◽  
2005 ◽  
Vol 105 (12) ◽  
pp. 4674-4676 ◽  
Author(s):  
Helen Yarovoi ◽  
Alan T. Nurden ◽  
Robert R. Montgomery ◽  
Paquita Nurden ◽  
Mortimer Poncz
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Blood Clotting ◽  
Immunogold Electron Microscopy ◽  
Bleeding Diathesis ◽  
Cell Type ◽  
Carotid Artery Injury ◽  
Cellular Context ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract We have previously reported that ectopically expressed factor VIII (FVIII) is stored within platelets and is released upon platelet activation. Studies by others in various cell lines have suggested that having von Willebrand factor (VWF) coexpression is necessary for FVIII granular storage and for its secretion. We tested the importance of VWF coexpression for ectopic storage of FVIII in platelets and for its bioavailability. Transgenic mice expressing platelet-specific FVIII were crossed onto a VWF-/- background. Antigenic levels of platelet FVIII in these mice were nearly unchanged whether VWF was present or not. Whole-blood clotting times and FeCl3 carotid artery injury correction demonstrated that platelet FVIII demonstrably improved the bleeding diathesis in FVIIInull mice independent of the platelets' VWF status. Immunogold electron microscopy demonstrated that platelet FVIII is stored in platelet α-granules independent of the presence of VWF. It appears that FVIII's interaction with VWF and its intracellular transportation, storage, and secretion differ greatly depending on the cell type. The molecular basis for these differences now needs to be elucidated. (Blood. 2005;105:4674-4676)

scholarly journals Localization of a factor VIII binding domain on a 34 kilodalton fragment of the N-terminal portion of von Willebrand factor

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1679-1682 ◽  
Author(s):  
Y Takahashi ◽  
M Kalafatis ◽  
JP Girma ◽  
K Sewerin ◽  
LO Andersson ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Solid Phase ◽  
Binding Domain ◽  
Phase System ◽  
Dependent Manner ◽  
Terminal Portion ◽  
Binding Domains ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Factor VIII (F.VIII) was tested for its ability to bind in solid phase system to von Willebrand Factor (vWF) or fragments obtained with Staphylococcus aureus V-8 protease, ie, SpIII (N-terminal), SpI (central), and SpII (C-terminal). Bound F.VIII was estimated in situ by clotting and chromogenic assays. F.VIII bound in a dose-dependent manner to immobilized vWF and SpIII but not to SpII or SpI. Binding was inhibited by 0.25 mol/L CaCl2 as well as by an excess of vWF or SpIII. Accordingly, immobilized F.VIII specifically bound 125I-vWF and SpIII but not SpII or SpI. Twelve monoclonal antibodies (MoAbs) directed towards SpIII, specifically blocking binding of F.VIII to vWF or SpIII, were used for the mapping of plasmic or tryptic fragments of vWF or SpIII. We thus established that a F.VIII binding domain of vWF is located on a 34 kilodalton (kd) fragment of the N-terminal portion of vWF, between residues 1 and 910, and that it is distinct from the GPIb and collagen binding domains.

Cell type–specific regulation of von Willebrand factor expression by the E4BP4 transcriptional repressor

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1531-1539 ◽  
Author(s):  
Christine Hough ◽  
Carla D. Cuthbert ◽  
Colleen Notley ◽  
Christine Brown ◽  
Carol Hegadorn ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Promoter Activity ◽  
Transcriptional Repressor ◽  
Binding Motif ◽  
Cell Type ◽  
Response Element ◽  
Cell Type Specific ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractMechanisms of tissue-restricted patterns of von Willebrand factor (VWF) expression involve activators and repressors that limit expression to endothelial cells and megakaryocytes. The relative transcriptional activity of the proximal VWF promoter was assessed in VWF-producing and -nonproducing cells, and promoter activity was highest in endothelial cells followed by megakaryocytes. Only basal VWF promoter activity was seen in nonendothelial cells. Here we identify a negative response element located at nucleotides (nts) +96/+105 and demonstrate, using chromatin immunoprecipitation (ChIP) analysis, that in vivo this sequence interacts with the E4BP4 transcriptional repressor. Differences in size and relative abundance of nuclear E4BP4 were observed. In HepG2 cells, low levels of larger forms of E4BP4 are present that directly interact with the negative response element. In VWF-expressing cells, high levels of smaller forms predominate with no evidence of direct DNA binding. However, in endothelial cells, mutation of the VWF E4BP4 binding motif not only restores but also further elevates VWF promoter activity, suggesting that E4BP4 may be part of a coordinated binding complex. These observations implicate this binding motif in repressing both activated and basal levels of VWF transcription by different cell type–specific mechanisms, and support the hypothesis that E4BP4 sequesters negative regulators of transcription, thereby enhancing activated gene expression.

scholarly journals Differential regulation by cytokines of constitutive and stimulated secretion of von Willebrand factor from endothelial cells

Blood ◽  
1990 ◽  
Vol 75 (3) ◽  
pp. 688-695 ◽  
Author(s):  
EM Paleolog ◽  
DC Crossman ◽  
JH McVey ◽  
JD Pearson
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Messenger Rna ◽  
Cell Injury ◽  
Interleukin 1 ◽  
Tnf Alpha ◽  
Detectable Effect ◽  
Dependent Manner ◽  
Von Willebrand ◽  
Willebrand Factor

We examined the effect of cytokines on basal and agonist-stimulated release of von Willebrand factor (vWf) by human endothelial cells. Treatment of endothelial cells for up to 48 hours with human recombinant or purified interleukin 1 (IL-1) or human recombinant tumor necrosis factor-alpha (TNF-alpha) did not significantly affect constitutive secretion of vWf or intracellular levels of vWf, although basal prostacyclin (PGI2) production was markedly enhanced. In contrast, both IL-1 and TNF-alpha modulated vWf release in response to thrombin or phorbol ester. Pretreatment of endothelial cells for 2 hours with either cytokine enhanced by up to threefold the stimulatory effect of a subsequent 60-minute exposure to thrombin. Addition of cycloheximide (5 micrograms/mL) during the preincubation abolished this enhancement. Moreover, if the cytokine pretreatment time was extended to 24 hours, agonist-stimulated vWf release was significantly suppressed. Cytokine treatment for 2 or 24 hours had no detectable effect on levels of vWf messenger RNA. The effects of cytokines were not the result of contamination with bacterial lipopolysaccharide and were not attributable to endothelial cell injury. These results show that cytokines have little or no direct effect on vWf release from endothelial cells but can significantly modulate its acute release in response to other stimuli in a complex time- and dose-dependent manner.

Effects of inflammatory cytokines on the release and cleavage of the endothelial cell–derived ultralarge von Willebrand factor multimers under flow

Blood ◽  
2004 ◽  
Vol 104 (1) ◽  
pp. 100-106 ◽  
Author(s):  
Aubrey Bernardo ◽  
Chalmette Ball ◽  
Leticia Nolasco ◽  
Joel F. Moake ◽  
Jing-fei Dong
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Cytokines ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Dependent Manner ◽  
Induce Platelet Aggregation ◽  
Tnf Α ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract ADAMTS13 cleaves ultralarge and hyperreactive von Willebrand factor (ULVWF) freshly released from activated endothelial cells to smaller and less active forms. This process may be affected by the amount of ULVWF released and the processing capacity of ADAMTS13, contributing to the development of thrombotic diseases. We examined the effects of inflammatory cytokines on the release and cleavage of ULVWF to evaluate potential links between inflammation and thrombosis. Human umbilical vein endothelial cells were treated with interleukin 6 (IL-6), IL-8, or tumor necrosis factor α (TNF-α), and the formation of platelet-decorated ULVWF strings was quantitated. IL-8 and TNF-α significantly stimulated the release of ULVWF in a dose-dependent manner. IL-6 induced ULVWF release only when it was in complex with the soluble IL-6 receptor. IL-6, but not IL-8 nor TNF-α, inhibited the cleavage of ULVWF strings by ADAMTS13 under flowing, but not static, conditions. These results suggest that inflammatory cytokines may stimulate the ULVWF release (IL-8 and TNF-α) and inhibit the ULVWF cleavage (IL-6), resulting in the accumulation of hyperreactive ULVWF in plasma and on the surface of endothelial cells to induce platelet aggregation and adhesion on the vascular endothelium. The findings describe a potential linkage between inflammation and thrombosis that may be of therapeutic importance.

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