scholarly journals Degradation of Sulphated Glycosaminoglycans at the Surface of Cultured Human Endothelial Cells by a Platelet Heparitinase

1977 ◽  
Author(s):  
C. Busch ◽  
B. Glimelius ◽  
Å Wastesson ◽  
B. Westermark
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Incubation Medium ◽  
Chondroitin Sulphate ◽  
Coagulation Factors ◽  
Platelet Lysate ◽  
Human Endothelial Cells ◽  
Endothelial Cell Surface ◽  
Sulphated Glycosaminoglycans

The non-thrombogenic property of the endothelial cell surface is a prerequisite for maintainance of blood circulation. The nature of this property is poorly understood. Recent advances in culturing techniques of endothelial cells in vitro may facilitate studies of the surface biochemistry. Human endothelial cells (EC) isolated from umbilical veins were shown to synthesize and secrete sulphated glycosaminoglycans (GAG). The recent finding of a platelet enzyme capable of degrading heparin sulphate (HS) raised the question:Can platelet lysate or a purified heparitinase detach and degrade endothelial HS? EC cultured in the presence of 35S-sulphate, produce 35S-labelled GAG which was isolated from the incubation medium from a cell associated trypsin labile pool and from a cellular pool not liberated by trypsin. After 48 hours of incorporation about 95% of 35S-GAG was found in the medium fraction, 5% in the trypsin fraction and negligible amounts in the cell fraction. In the trypsin pool (“surface fraction”) heparin sulphate comprised about 85%, while the remaining 15% consisted of chondroitin sulphate and/or dermatan sulphate. Incubation of 35S-labelled EC with platelet lysate or a partially purified preparation of the enzyme from the same source caused a marked release of cell-surface associated HS to the incubation medium as oligosaccharides. These effects could be ascribed to heparitinase activity and may alter the properties of the EC-surface and influence the interaction between these cells on one hand and blood cells or plasma components, e.g., coagulation factors on the other.

CULTURED HUMAN ENDOTHELIAL CELLS BIND AND INTERNALIZE HIGH MOLECULAR WEIGHT KININOGEN

1987 ◽  
Author(s):  
Freek van Iwaarden ◽  
G Philip ◽  
de Groot ◽  
Bonno N Bouma
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
High Molecular Weight ◽  
Binding Sites ◽  
High Molecular Weight Kininogen ◽  
Human Endothelial Cells ◽  
Endothelial Cell Surface ◽  
Coagulation Pathway

The presence of High Molecular Weight kininogen (HMWK) was demonstrated in cultured human endothelial cells (EC) by immunofluorescence techniques. Using an enzyme linked immunosorbent assay a concentration of 58 ng HMWK/10 cells was determined. Immunoprecipitation studies performed with lysed metabolically labelled endothelial cells and mono-specific antisera directed against HMWK suggested that HMWK is not synthesized by the endothelial cells. Endothelial cells cultured in the presence of HMWK-depleted serum did not contain HMWK. This, suggests that endothelial cells can internalize HMWK. Using 125I-HMWK it was demonstrated that cultured endothelial cells bind HMWK in a time-dependent, specific and saturable.way. The cells were found to internalize 125I-HMWK, since I-HMWK was detected in solubilized endothelial cells after the cell bound 125I-HMWK had been eluted with dextran sulphate.The binding of I-HMWK required the presence of zinc ions. Optimal binding of 125I-HMWK was observed at 50 μM Zn++ . Calcium ions inhibited the Zn++ dependent binding of 125I-HMWK |25EC. In the presence of 3 mM CaCl2 the total binding of 125I-HMWK was significantly decreased, and a .concentration of 200 μM Zn++ was Required for the binding of 125I-HMWK to thecells. Higher,. Ca concentrations did not further decrease the binding of 125I-HMWK. Analysis of tl^e binding data by the ligand computer program indicated 3.2 x 10 binding sites per cell for HMWK with a Kd of 35 nM at 50 μM ZnCl2 and 1 mM CaCl2. Specify binding of HMWK did also occur at physiological plasma Zn++ concentrations. Half maximal binding was observed at HMWK concentrations of ± 105 nM at 10 μM ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 pM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway. M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway. M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway. M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway.M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 16 molecules of HMWK bound per cell and at 80 nM with 2.8 x 106 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway.

Endothelial adhesion molecules and their role in inflammation

1993 ◽  
Vol 71 (1) ◽  
pp. 76-87 ◽  
Author(s):  
C. Wayne Smith
Keyword(s):  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Adhesion Molecules ◽  
Surrounding Tissue ◽  
Intercellular Adhesion Molecule ◽  
Intercellular Adhesion Molecule 1 ◽  
Endothelial Cell Surface

The emigration of leukocytes such as neutrophils into inflammatory sites requires adhesion to the endothelium of small venules. The initial adhesive event is margination characterized by rolling of neutrophils along the luminal surface of the endothelium. Each member of the selectin family of adhesion molecules has been shown to support neutrophil rolling under conditions of flow. E-selectin is synthesized by endothelial cells following cytokine stimulation, P-selectin is rapidly mobilized from Weibel–Palade bodies to the endothelial cell surface following stimulation with agents such as histamine, and L-selectin is constitutively expressed on the surface of leukocytes. Each selectin functions primarily as a lectin, recognizing carbohydrate structures on the leukocyte or endothelial cell surface. Once the marginated neutrophil forms a stationary adhesion with endothelial cells, it is stimulated by chemotactic factors to downregulate the selectin-based adhesion and upregulate adherence dependent on β2-integrins, principally CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1). These adhesion molecules interact with intercellular adhesion molecule 1 (ICAM-1) and possibly other structures on the endothelial cell, and the leukocyte rapidly emigrates into surrounding tissue. Transendothelial migration in vitro is markedly inhibited by monoclonal antibodies against CD18 integrins or ICAM-1. Monoclonal antibodies against the selectins, CD18, CD11a, CD11b, and ICAM-1 have all been shown to significantly reduce the influx of neutrophils into sites of inflammation in various animal models.Key words: adhesion, integrins, selectins, leukocytes, endothelial cells.

Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

1979 ◽  
Author(s):  
S. Korach ◽  
D. Ngo
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Vascular Endothelial Cells ◽  
Intercellular Junctions ◽  
Cell Types ◽  
Endothelial Damage ◽  
Antibody Concentration ◽  
High Yields ◽  
Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

Vascular repair utilising immobilised heparin conjugate for protection against early activation of inflammation and coagulation

2015 ◽  
Vol 113 (06) ◽  
pp. 1312-1322 ◽  
Author(s):  
Sofia Nordling ◽  
Jaan Hong ◽  
Karin Fromell ◽  
Fredrik Edin ◽  
Johan Brännström ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Thrombus Formation ◽  
Innate Immune ◽  
Human Endothelial Cells ◽  
Coagulation Activation ◽  
Ischaemia Reperfusion Injury ◽  
Novel Approach ◽  
One Step ◽  
Chamber Model

SummaryIschaemia-reperfusion injury (IRI) poses a major challenge in many thrombotic conditions and in whole organ transplantation. Activation of the endothelial cells and shedding of the protective vascular glycocalyx during IRI increase the risk of innate immune activation, cell infiltration and severe thrombus formation, promoting damage to the tissue. Here, we present a novel one-step strategy to protect the vasculature by immobilisation of a unique multi-arm heparin conjugate to the endothelium. Applying a new in vitro blood endothelial cell chamber model, the heparin conjugate was found to bind not only to primary human endothelial cells but also directly to the collagen to which the cells adhered. Incubation of hypoxic endothelial cells with freshly drawn human blood in the blood chambers elicited coagulation activation reflected by thrombin anti-thrombin formation and binding of platelets and neutrophils. Immobilisation of the heparin conjugate to the hypoxic endothelial cells created a protective coating, leading to a significant reduction of the recruitment of blood cells and coagulation activation compared to untreated hypoxic endothelial cells. This novel approach of immobilising multi-arm heparin conjugates on the endothelial cells and collagen of the basement membrane ensures to protect the endothelium against IRI in thrombotic disorders and in transplantation.

scholarly journals MMP-12, Secreted by Pro-Inflammatory Macrophages, Targets Endoglin in Human Macrophages and Endothelial Cells

2019 ◽  
Vol 20 (12) ◽  
pp. 3107 ◽  
Author(s):  
Mikel Aristorena ◽  
Eunate Gallardo-Vara ◽  
Matej Vicen ◽  
Mateo de Las Casas-Engel ◽  
Luisa Ojeda-Fernandez ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Macrophage Polarization ◽  
Cell Surface Receptor ◽  
Soluble Form ◽  
Specific Marker ◽  
Good Resolution ◽  
Angiogenic Activity ◽  
Soluble Endoglin

Upon inflammation, monocyte-derived macrophages (MΦ) infiltrate blood vessels to regulate several processes involved in vascular pathophysiology. However, little is known about the mediators involved. Macrophage polarization is crucial for a fast and efficient initial response (GM-MΦ) and a good resolution (M-MΦ) of the inflammatory process. The functional activity of polarized MΦ is exerted mainly through their secretome, which can target other cell types, including endothelial cells. Endoglin (CD105) is a cell surface receptor expressed by endothelial cells and MΦ that is markedly upregulated in inflammation and critically involved in angiogenesis. In addition, a soluble form of endoglin with anti-angiogenic activity has been described in inflammation-associated pathologies. The aim of this work was to identify components of the MΦ secretome involved in the shedding of soluble endoglin. We find that the GM-MΦ secretome contains metalloprotease 12 (MMP-12), a GM-MΦ specific marker that may account for the anti-angiogenic activity of the GM-MΦ secretome. Cell surface endoglin is present in both GM-MΦ and M-MΦ, but soluble endoglin is only detected in GM-MΦ culture supernatants. Moreover, MMP-12 is responsible for the shedding of soluble endoglin in vitro and in vivo by targeting membrane-bound endoglin in both MΦ and endothelial cells. These data demonstrate a direct correlation between GM-MΦ polarization, MMP-12, and soluble endoglin expression and function. By targeting endothelial cells, MMP-12 may represent a novel mediator involved in vascular homeostasis.

Centrifugal enhancement of Kaposi's sarcoma-associated virus infection of human endothelial cells in vitro

2008 ◽  
Vol 154 (1-2) ◽  
pp. 160-166 ◽  
Author(s):  
Seung-Min Yoo ◽  
Ae-Kyung Ahn ◽  
Taegun Seo ◽  
Hyo Bong Hong ◽  
Myung-Ae Chung ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Virus Infection ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Human Endothelial Cells
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