Electroimmunochemical Characterization of Endothelial Cell Proteins: Antigenic Relationship with Platelet and Erythrocyte Membrane Proteins

1987 ◽  
Vol 58 (02) ◽  
pp. 686-693 ◽  
Author(s):  
Tone Børsum ◽  
Inger Hagen ◽  
Ole J Bjerrum
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Primary Cultures ◽  
Rabbit Antiserum ◽  
Human Platelets ◽  
Erythrocyte Membranes ◽  
Antigenic Relationship ◽  
Platelet Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryHuman endothelial cells isolated from umbilical cords and cultured in primary cultures were solubilized in Triton X-100 and examined by crossed immunoelectrophoresis using rabbit antiserum against endothelial cells. Endogeneous labelling of the endothelial cell proteins with 35S-methionine or 14C-mannose followed by crossed immunoelectrophoresis and autoradiography revealed about 30 or 8 immunoprecipitates, respectively. Antigenic relationship between endothelial cell proteins and proteins in human platelets or erythrocyte membranes was demonstrated by use of the corresponding antisera and by antigen addition experiments. One of the endothelial cell proteins cross-reacted with antiserum against erythrocyte membranes and showed a partial antigenic identity reaction with the band 3 protein complex of erythrocyte membranes. The same protein showed antigenic relationship also with a platelet protein. In addition, endothelial cells contain at least 7 proteins antigenically related to platelet proteins, of which at least 5 were labelled with 14C-mannose and thus were glycoproteins. Three of these glycoproteins were antigenically related to proteins from isolated platelet membranes and three were related to the release products obtained after thrombin treatment of platelets. The present study demonstrated numerous platelet and endothelial cell proteins that were antigenically related, more than previously anticipated.

Immunoelectrophoretic Analysis of Membrane Glycoproteins in Cultured Human Endothelial Cells

1990 ◽  
Vol 63 (02) ◽  
pp. 303-311
Author(s):  
Tone Børsum
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Rabbit Antiserum ◽  
Platelet Glycoprotein ◽  
Membrane Glycoproteins ◽  
Human Endothelial Cells ◽  
Immunoelectrophoretic Analysis ◽  
Glycoprotein Complex ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryHuman endothelial cells isolated from umbilical cordswere solubilized in Triton X-100 and examined by crossedimmunoelec-trophoresis using rabbit antiserum against endothelial cells. Endogenous labelling of the endothelialcell proteins with 14Cmannose followed by crossed immunoelectrophoresis and autoradiography revealed about 10 immunoprecipitates. Four of these endothelial cell glycoproteins were labelled by lactoperoxidase catalyzed iodination and thus were surface located. Three of the surface located glycoproteins showed reduced electrophoretic mobility after incubation of the endothelial cells with neuraminidase and were therefore sialoglycoproteins. Amphiphilicity of endothelial cell glycoproteins was studied by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose in the intermediate gel. Amphiphilic proteins also show increasing electrophoretic migration velocity with decreasing concentration of Triton X-100 in the first dimension gels. Five of the endothelial cell glycoproteins were shown to be amphiphilic using these two techniques.Two monoclonal antibodies against the platelet glycoprotein complex Ilb-IIIa and glycoprotein IlIa, respectively, reacted with the same precipitate of endothelial cells. When a polyclonal antibody against the platelet glycoprotein complex Ilb-IIIa was incorporated into the intermediate gel the position of two endothelial cell precipitates were lowered. One of these was a sialoglycoprotein.

scholarly journals Loss of 111Indium as indicator of platelet injury

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 350-353 ◽  
Author(s):  
JH Joist ◽  
RK Baker
Keyword(s):  
Small Molecules ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Metabolic Energy ◽  
Platelet Factor ◽  
Platelet Protein ◽  
Threshold Rate ◽  
Immune Injury ◽  
Triton X

Abstract We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

Plasticity of endothelial cells: rapid dedifferentiation of freshly isolated high endothelial venule endothelial cells outside the lymphoid tissue microenvironment

Blood ◽  
2004 ◽  
Vol 103 (11) ◽  
pp. 4164-4172 ◽  
Author(s):  
Delphine-Armelle Lacorre ◽  
Espen S. Baekkevold ◽  
Ignacio Garrido ◽  
Per Brandtzaeg ◽  
Guttorm Haraldsen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Lymphoid Tissue ◽  
Ex Vivo ◽  
Primary Cultures ◽  
High Endothelial Venule ◽  
Tissue Specific ◽  
Tissue Microenvironment ◽  
Cell Phenotypes

Abstract Endothelial cells display remarkable heterogeneity in different organs and vascular beds. Although many studies suggest that tissues “speak” to endothelial cells, endothelial cell diversity remains poorly characterized at the molecular level. Here, we describe a novel strategy to characterize tissue-specific endothelial cell phenotypes and to identify endothelial cell genes that are under the control of the local microenvironment. By comparing post-capillary high endothelial venule endothelial cells (HEVECs), freshly isolated from human tonsils without any cell culture step, with HEVECs cultured for 2 days, we found that HEVECs rapidly lost their specialized characteristics when isolated from the lymphoid tissue microenvironment. Striking changes occurred as early as after 48 hours, with complete loss of the postcapillary venule–specific Duffy antigen receptor for chemokines (DARCs) and the HEV-specific fucosyltransferase Fuc-TVII. DNA microarray analysis identified several other candidate HEV genes that were rapidly down-regulated ex vivo, including type XV collagen, which we characterized as a novel, abundant HEV transcript in situ. Together, our results demonstrate that blood vessel type–specific and tissue-specific characteristics of endothelial cells are under the control of their microenvironment. Therefore, even short-term primary cultures of human endothelial cells may not adequately mimic the differentiated endothelial cell phenotypes existing in vivo.

scholarly journals Perturbations in the fibrinolytic pathway abolish cyst formation but not capillary-like organization of cultured murine endothelial cells

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3206-3217 ◽  
Author(s):  
N Dubois-Stringfellow ◽  
A Jonczyk ◽  
VL Bautch
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Basement Membrane ◽  
Organizational Behavior ◽  
Cell Lines ◽  
Primary Cultures ◽  
Cyst Formation ◽  
Fibrin Gels

Abstract Fibrinolytic activity and its relation to morphogenesis was investigated in several transformed murine endothelial cell lines and primary cultures of endothelial cells. Two in vitro systems, fibrin gels and Matrigel (Collaborative Research, Bedford, MA), were used. Fibrin gels model a fibrin-rich extracellular matrix that frequently supports neovascularization in vivo, and Matrigel models the basement membrane surrounding quiescent endothelial cells in vivo. The transformed endothelial cell lines have higher levels of plasminogen activator (PA) mRNA than primary cultures of endothelial cells, and an increased PA-mediated proteolytic activity was correlated with formation of cysts in fibrin gels. Addition of neutralizing anti- urokinase antibodies, plasminogen depletion, or addition of a plasmin inhibitor prevented cyst formation. Addition of plasminogen restored the ability to form cysts in the plasminogen-depleted system. Normal endothelial cells organized into capillary-like structures in fibrin gels regardless of manipulations affecting the fibrinolytic pathway. In Matrigel, both transformed and primary cultures of endothelial cells rapidly formed a capillary-like network that was not affected by plasminogen depletion or addition of plasmin inhibitors. Thus, elements of the fibrinolytic pathway necessary for cyst formation are not critical in capillary-like structure formation on a reconstituted basement membrane. These results suggest that plasmin is essential for hemangioma formation but is not critical to the organizational behavior of normal endothelial cells.

Incorporation of Some Eicosaenoic Acids into Endothelial Cells – Effect on Platelet Inhibitory Activity and Prostacyclin Production

1985 ◽  
Vol 53 (02) ◽  
pp. 264-267 ◽  
Author(s):  
Béatrice Sicard ◽  
Michel Lagarde
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Endothelial Cell ◽  
Beneficial Effect ◽  
Primary Cultures ◽  
Eicosatrienoic Acid ◽  
Dihomogammalinolenic Acid ◽  
Prostacyclin Production ◽  
Prostacyclin Synthesis ◽  
Platelet Functions

SummaryPrimary cultures of endothelial cells from human umbilical veins were grown until confluency. Then, dihomogammalinolenic acid (DHLA or 20:3n-6) and eicosapentaenoic acid (EPA or 20:5n-3), precursors of monoenoic and trienoic prostanoids, respectively, as well as 5,8,11-eicosatrienoic acid (20:3n-9), and isomer of DHLA, were incorporated into endothelial lipids. DHLA-rich endothelial cells had a decreased capacity of prostacyclin production. By contrast EPA- or 20:3n-9-rich endothelial cells were comparable to controls in this respect. DHLA and EPA were efficiently acylated into cell phospholipids and triglycerides at the opposite of 20:3n-9. It is suggested that both DHLA and EPA could alter the liberation of endogenous arachidonic acid for prostacyclin synthesis but this might be counterbalanced in EPA-rich endothelial cells by PGI3 production. We conclude that DHLA enrichment of endothelial cell lipids may impair the possible beneficial effect of the acid upon platelet functions whereas that of EPA would not be modified.

The Effect of Polyunsaturated Fatty Acids on Endothelial Cells and Their Production of Prostacyclin, Thromboxane and Platelet Inhibitory Activity

1983 ◽  
Vol 50 (04) ◽  
pp. 762-767 ◽  
Author(s):  
Jan H Brox ◽  
Arne Nordøy
Keyword(s):  
Fatty Acids ◽  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Endothelial Cell ◽  
Polyunsaturated Fatty Acids ◽  
Linolenic Acid ◽  
Inhibitory Activity ◽  
Primary Cultures ◽  
Cell Monolayers ◽  
Docosahexaenoic Acids

SummaryPrimary cultures of human endothelial cell monolayers were incubated with albumin-bound fatty acids of the ω-3 and ω-6 families for a maximum of 24 hrs, to investigate the production of 6-keto-PGF1α, TXB2 and platelet inhibitory activity (PIA). Arachidonic acid was a potent stimulator of all parameters. The release of 6-keto-PGF1α was significantly reduced by equimolar concentrations of linoleic, dihomogamma linolenic and eicosapentaenoic acids, but not by linolenic acid. PIA was not similarity affected.Dihomogamma linolenic add was also a weak stimulator of 6- keto-PGF1α and PIA, but reduced the content of both in the cells after 24 hrs. Eicosapentaenoic and docosahexaenoic acids both depressed 6-keto-PGF1α production but PIA was maintained after 24 hrs. Indomethacin always blocked 6-keto-PGF1α and PIA production. None of the effects correlated to release of 51CR from prelabelled cells.

Human Platelet-Secreted Proteins (LA-PF4, βTG) Do Not Modify Prostacyclin (PGI2) Production By Bovine Aortic Endothelial Cells

1981 ◽  
Author(s):  
A Poggi ◽  
S Niewiarowski ◽  
J C Holt ◽  
A Dall’Olio ◽  
G J Stewart ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Human Platelet ◽  
Primary Cultures ◽  
Human Platelets ◽  
Secreted Proteins ◽  
Experimental Conditions ◽  
Aortic Endothelial Cells ◽  
Bovine Endothelial Cells ◽  
Ec Cells ◽  
Dose Dependent

It has been proposed that platelets may counteract the antithrombotic activity of the vessel wall by releasing proteins with inhibitory activity on vascular PGI2. The aim of our study has been to evaluate the effect of LA-PF4 and βTG, two human platelet-secreted proteins which may be increased in conditions associated with thrombosis, on the production of PGI2 by bovine endothelial cells (EC). Cells obtained by mild collagenase digestion of calf aortas, were plated in plastic dishes and cultured for 5-6 days in minimum essential medium. Only primary cultures were used. Confluent cells (6-10x105)5/dish) were washed twice with PBS and incubated in the same buffer at 37°C for 5-10 minutes in the presence or absence of the platelet-secreted proteins. LA-PF4 was prepared from washed human platelets aggregated with thrombin after chromatography on CM-sephadex and heparin- sepharose. βTG was obtained by partial trypsinization of LA-PF4. LA-PF4 or βTG at the doses of 5-50 ug/ml did not modify the production by EC of 6-keto PGF1α(measured by a radioimmunoassay). EC indeed released 8.9 ± 3.8 pmoles/ml of 6-keto PGF1α, when incubated with buffer, and 13.6 ± 2.3 or 4.1 ± 0.9 when incubated with LA-PF4 or βTG (10 ug/ml). A dose-dependent release of 6-keto-PGF1α was found after stimulation of EC with arachidonic acid (AA, 1-50 um). After addition of AA (2 uM) the generation of 6-keto-PGF1α was 153.5 ± 45.0 pmoles/ml in control samples, 86.7 ± 15.2 and 163.3 ± 15.2 in samples incubated with LA-PF4 or βTG, respectively. Similarly, no effect was found when PGI2 production by EC was measured by bioassay of its antiaggregatory activity. The tested proteins did not modify either the production of TXB2 (2-10 pmoles per ml of incubation medium) by the same EC. These data indicate that, in our experimental conditions, LA-PF4 and βTG did not show potentially thrombogenic effects on prostaglandin production by EC.

scholarly journals Cultured human endothelial cells synthesize a plasma membrane protein complex immunologically related to the platelet glycoprotein IIb/IIIa complex

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1176-1180 ◽  
Author(s):  
OC Leeksma ◽  
J Zandbergen-Spaargaren ◽  
JC Giltay ◽  
JA van Mourik
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Membrane Protein ◽  
Protein Complex ◽  
Platelet Glycoprotein ◽  
Plasma Membrane Protein ◽  
Human Endothelial Cells ◽  
Membrane Protein Complex ◽  
Crossed Immunoelectrophoresis

Abstract We have previously demonstrated that endothelial cells synthesize a plasma membrane protein indistinguishable from platelet glycoprotein (GP) IIa. The present study provides evidence for a further analogy between the platelet and the endothelial cell membrane by showing that cultured endothelial cells also synthesize a membrane protein complex immunologically related to the platelet GP IIb/GP IIIa complex. This evidence is based on the following observations: (1) C17, a murine monoclonal antiplatelet GP IIIa antibody, consistently precipitates two proteins, apparent molecular weights, respectively, 115,000 and 125,000 reduced and 95,000 and 135,000 nonreduced, from metabolically (35S- methionine) as well as surface 125I-labeled cultured human endothelial cells; (2) upon crossed immunoelectrophoresis of solubilized endothelial cells against a polyclonal rabbit antiplatelet antiserum and 125I-labeled C17 IgG, a single precipitate of the protein(s) recognized by C17 is observed. As judged by their mobility in 9% polyacrylamide gels, both endothelial proteins appear to have a somewhat larger molecular weight than their platelet counterparts. Patterns obtained by crossed immunoelectrophoresis are also indicative of a difference in electrophoretic behavior of the platelet GP IIb/IIIa complex and the endothelial cell protein complex.

Extracellular ATP signaling and P2X nucleotide receptors in monolayers of primary human vascular endothelial cells

2002 ◽  
Vol 282 (2) ◽  
pp. C289-C301 ◽  
Author(s):  
Lisa M. Schwiebert ◽  
William C. Rice ◽  
Brian A. Kudlow ◽  
Amanda L. Taylor ◽  
Erik M. Schwiebert
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Blood Vessels ◽  
Vascular Tone ◽  
Vascular Endothelial Cells ◽  
Purinergic Signaling ◽  
Primary Cultures ◽  
Atp Release ◽  
Vascular Endothelial ◽  
Multiple Blood

ATP and its metabolites regulate vascular tone; however, the sources of the ATP released in vascular beds are ill defined. As such, we tested the hypothesis that all limbs of an extracellular purinergic signaling system are present in vascular endothelial cells: ATP release, ATP receptors, and ATP receptor-triggered signal transduction. Primary cultures of human endothelial cells derived from multiple blood vessels were grown as monolayers and studied using a bioluminescence detection assay for ATP released into the medium. ATP is released constitutively and exclusively across the apical membrane under basal conditions. Hypotonic challenge or the calcium agonists ionomycin and thapsigargin stimulate ATP release in a reversible and regulated manner. To assess expression of P2X purinergic receptor channel subtypes (P2XRs), we performed degenerate RT-PCR, sequencing of the degenerate P2XR product, and immunoblotting with P2XR subtype-specific antibodies. Results revealed that P2X4and P2X5are expressed abundantly by endothelial cell primary cultures derived from multiple blood vessels. Together, these results suggest that components of an autocrine purinergic signaling loop exist in the endothelial cell microvasculature that may allow for “self-regulation” of endothelial cell function and modulation of vascular tone.

scholarly journals Light and electron microscopic localization of D-galactosyl residues in capillary endothelial cells of the canine cerebral cortex.

1986 ◽  
Vol 34 (5) ◽  
pp. 641-648 ◽  
Author(s):  
D Z Gerhart ◽  
M S Zlonis ◽  
L R Drewes
Keyword(s):  
Endothelial Cells ◽  
Cerebral Cortex ◽  
Endothelial Cell ◽  
Primary Cultures ◽  
Complex Method ◽  
Lectin Receptors ◽  
Ultrastructural Studies ◽  
Capillary Endothelial Cells ◽  
Cerebral Capillary ◽  
Galactosyl Residues

Ricinus communis agglutinin I (RCA-I), a lectin that binds to D-galactosyl residues, intensely stained capillaries in cryostat sections of canine cerebral cortex when evaluated by the avidin-biotin-peroxidase complex method. Of seven lectins tested, only RCA-I gave strong staining of vessels and capillaries with little staining of other cortical cells. Ultrastructural studies using ferritin-, biotin-, and peroxidase-labeled RCA-I indicated that this lectin was bound to the luminal membrane of the cerebral capillary endothelial cell and that lectin receptors were distributed continuously along this membrane. Plasmalemma invaginations that bound RCA-I were also present in endothelial cells. Primary cultures of cerebral capillary endothelial cells grown on plastic or gelatin-coated glass substrates demonstrated staining of the cell membrane and perinuclear structures which appeared to be the Golgi complex and secondary lysosomes. These staining characteristics were retained when the cells were subcultured and were confirmed by ultrastructural studies. In contrast, light microscopy showed that fibronectin was more widely distributed in the cytoplasm, a finding consistent with its occurrence in the endoplasmic reticulum. This work provides support for the concept that lectins may be useful endothelial cell markers in both intact tissue and cell culture.

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