A Monoclonal Antibody, VM64, Reacts with a 130 kDa Glycoprotein Common to Platelets and Endothelial Cells: Heterogeneity in Antibody Binding to Human Aortic Endothelial Cells

1991 ◽  
Vol 66 (04) ◽  
pp. 494-499 ◽  
Author(s):  
Alexey V Mazurov ◽  
Dimitry V Vinogradov ◽  
Naile V Kabaeva ◽  
Galina N Antonova ◽  
Yuri A Romanov ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Human Platelets ◽  
Human Aorta ◽  
Vascular Endothelial ◽  
Aortic Endothelial Cells ◽  
Number Of Binding Sites ◽  
Typical Distribution

SummaryA new monoclonal antibody (mAb), VM64, reacts with a common antigen on the surface of human platelets and vascular endothelial cells (EC). Under nonreduced conditions it recognized in immunoblotting a protein of 130 kDa both in platelets and EC. VM64 precipitated the same 130 kDa protein from the lysate of surface radioiodinated platelets. Electrophoretic mobility of this protein was not altered by reduction and differed from the bands precipitated by reference mAb against platelet glycoproteins (GP) Ia–IIa, Ib, IIb–IIIa and GMP130. VM64 binding to platelets and EC was specific and saturable. The number of binding sites on platelets was 9.9 ± 3.5 × 103 per platelet and on the surface of EC monolayer – 2.40 ± 0.32 × 106 per cell. VM64 also binds to platelets from Glanzmann's thrombasthenia patients which lack GPIIb–IIIa. VM64 did not affect platelet aggregation induced by ADP, collagen, thrombin and ristocetin. In the monolayers of EC from umbilical vein and human aorta, VM64 stained the area at the periphery of the cells adjacent to the cell-cell boundaries. In preconfluent cultures preferential staining was observed at the active leading margins of the cells. Unlike EC cultures from umbilical vein, where all cells were positively stained, in aortic EC cultures some unstained or poorly stained cells were constantly present, indicating a heterogeneity of EC population related to the expression of VM64 antigen. The biochemical characteristics of VM64 antigen, its presence both on platelets and EC and typical distribution on the surface of EC suggested that this antigen is identical to PECAM (CD31) protein.

Modulation of calcium homeostasis in cultured rat aortic endothelial cells by intracellular acidification

1993 ◽  
Vol 265 (4) ◽  
pp. H1424-H1433 ◽  
Author(s):  
R. C. Ziegelstein ◽  
L. Cheng ◽  
P. S. Blank ◽  
H. A. Spurgeon ◽  
E. G. Lakatta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Cytosolic Calcium ◽  
Ph Sensitive ◽  
Vascular Endothelial ◽  
Intracellular Acidification ◽  
Cytosolic Ph ◽  
Aortic Endothelial Cells ◽  
Endothelial Monolayers ◽  
Aortic Endothelial

Acidosis produces vasodilation in a process that may involve the vascular endothelium. Because synthesis and release of endothelium-derived vasodilatory substances are linked to an increase in cytosolic calcium concentration ([Ca2+]i), we examined the effect of intracellular acidification on cultured rat aortic endothelial cells loaded either with the pH-sensitive probe carboxy-seminaphthorhodafluor-1 or the Ca(2+)-sensitive fluorescent probe indo 1. The basal cytosolic pH (pHi) of endothelial monolayers in a 5% CO2-HCO3- buffer was 7.27 +/- 0.02 and that in a bicarbonate-free solution was 7.22 +/- 0.03. Acidification was induced either by removal of NH4Cl (delta pHi = -0.10 +/- 0.02), changing from a bicarbonate-free to a 5% CO2-HCO3(-)-buffered solution at constant buffer pH (delta pHi = -0.18 +/- 0.03), or changing from a 5% to a 20% CO2-HCO3- solution (delta pHi = -0.27 +/- 0.07). Regardless of the method used, intracellular acidification increased [Ca2+]i as indexed by indo 1 fluorescence. The increase in [Ca2+]i induced by changing from a 5 to a 20% CO2-HCO3- solution was not significantly altered by removal of buffer Ca2+ either before or after depletion of bradykinin- and thapsigargin-sensitive intracellular Ca2+ stores. Thus intracellular acidification of vascular endothelial cells releases Ca2+ into the cytosol either from pH-sensitive intracellular buffer sites, mitochondria, or from bradykinin- and thapsigargin-insensitive intracellular stores. This Ca2+ mobilization may be linked to endothelial synthesis and release of vasodilatory substances during acidosis.

scholarly journals Citrus bergamiaRisso Elevates Intracellular Ca2+in Human Vascular Endothelial Cells due to Release of Ca2+from Primary Intracellular Stores

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Purum Kang ◽  
Seung Ho Han ◽  
Hea Kyung Moon ◽  
Jeong-Min Lee ◽  
Hyo-Keun Kim ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Channel Blocker ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Concentration Dependent Manner ◽  
Carbonyl Cyanide ◽  
Intracellular Ca ◽  
Umbilical Vein Endothelial Cells

The purpose of the present study is to examine the effects of essential oil ofCitrus bergamiaRisso (bergamot, BEO) on intracellular Ca2+in human umbilical vein endothelial cells. Fura-2 fluorescence was used to examine changes in intracellular Ca2+concentration[Ca2+]i. In the presence of extracellular Ca2+, BEO increased[Ca2+]i, which was partially inhibited by a nonselective Ca2+channel blocker La3+. In Ca2+-free extracellular solutions, BEO increased[Ca2+]iin a concentration-dependent manner, suggesting that BEO mobilizes intracellular Ca2+. BEO-induced[Ca2+]iincrease was partially inhibited by a Ca2+-induced Ca2+release inhibitor dantrolene, a phospholipase C inhibitor U73122, and an inositol 1,4,5-triphosphate (IP3)-gated Ca2+channel blocker, 2-aminoethoxydiphenyl borane (2-APB). BEO also increased[Ca2+]iin the presence of carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial Ca2+uptake. In addition, store-operated Ca2+entry (SOC) was potentiated by BEO. These results suggest that BEO mobilizes Ca2+from primary intracellular stores via Ca2+-induced and IP3-mediated Ca2+release and affect promotion of Ca2+influx, likely via an SOC mechanism.

Intracellular pathways of insulin transport across vascular endothelial cells

1988 ◽  
Vol 255 (4) ◽  
pp. C459-C464 ◽  
Author(s):  
H. L. Hachiya ◽  
P. A. Halban ◽  
G. L. King
Keyword(s):  
Endothelial Cells ◽  
Insulin Receptor ◽  
Vascular Endothelial Cells ◽  
Insulin Degradation ◽  
Vascular Endothelial ◽  
Aortic Endothelial Cells ◽  
Lysosomal Protease ◽  
Insulin Transport ◽  
Bovine Aortic Endothelial Cells ◽  
Aortic Endothelial

Processing and transport of hormones across vascular endothelial cells may modulate hormone action at subendothelial tissue sites. Insulin was transported across cultured rat capillary and bovine aortic endothelial cells, after a delay of 5-10 min, at a constant rate for 60 min at 37 degrees C. 125I-labeled insulin transport was inhibited by 88 +/- 11% (SE, n = 4) and 75 +/- 18% (SE, n = 4) in the presence of anti-insulin receptor antibody and unlabeled insulin (at 10(-7) M), respectively. Reverse phase high-performance liquid chromatography showed 88% of the 125I-insulin transported over 60 min was indistinguishable from the 125I-insulin added to the cells at 4 degrees C. In aortic endothelial cells preincubated with 2.3 x 10(-9) M of insulin for 24 h, insulin receptor binding was downregulated by 67%, and 125I-insulin transport was decreased by 52 +/- 11%. The proton ionophore monensin (0.05 mM) increased the internalized insulin in bovine aortic endothelial cells by 78%, with a corresponding decrease in 125I-insulin released by 76 +/- 2% (SE, n = 4). 125I-insulin transport across the aortic endothelial cell monolayer was similarly decreased (54 +/- 12%, SE, n = 4) by monensin. In contrast, the lysosomal protease inhibitor leupeptin had no effect. Degradation and transport were similarly dissociated by low temperature. At 15 degrees C, no significant insulin degradation was detected, whereas 125I-insulin release from the cells continued at 30 +/- 3% of the rate at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Stereoselectivity of ectonucleotidases on vascular endothelial cells

1983 ◽  
Vol 214 (3) ◽  
pp. 975-981 ◽  
Author(s):  
N J Cusack ◽  
J D Pearson ◽  
J L Gordon
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Thiol Group ◽  
Side Chain ◽  
Vascular Endothelial ◽  
Nucleotide Analogues ◽  
Aortic Endothelial Cells ◽  
Nucleotide Analogue ◽  
High Degree ◽  
Aortic Endothelial

We have investigated the stereoselectivity of ectonucleotidases (nucleoside triphosphatase, EC 3.6.1.15; nucleoside diphosphatase, EC 3.6.1.6; 5′-nucleotidase, EC 3.1.3.5) on pig aortic endothelial cells using two classes of nucleotide analogue. In experiments with nucleotide enantiomers in which the natural D-ribofuranosyl moiety is replaced by an L-ribofuranosyl moiety, the rate of catabolism of 100 microM-L-ATP was one-fifth that of D-ATP, the rate of catabolism of 100 microM-L-ADP was one-fifteenth that of D-ADP and there was no detectable catabolism of 100 microM-L-AMP. Each of the L-enantiomers inhibited, apparently competitively, the catabolism of the corresponding D-enantiomer; Ki values were approx. 0.6 mM, 1.0 mM and 3.9 mM for L-ATP, L-ADP and L-AMP respectively. Experiments with adenosine 5′-[beta, gamma-imido]triphosphate and with D- and L-enantiomers of adenosine 5′-[beta, gamma-methylene]triphosphate revealed modest ectopyrophosphatase activity, undetectable in experiments with natural nucleotides, which was also stereoselective. Use of phosphorothioate nucleotide analogues demonstrated that ATP catabolism was virtually stereospecific with respect to the geometry of the thiol group substituted on the beta-phosphate: the Rp isomer was degraded, whereas there was little or no breakdown of the Sp isomer. ADP catabolism was also stereospecific with respect to the geometry of the thiol group substituted on the alpha-phosphate: the Sp isomer but not the Rp isomer was degraded. The geometry of thiol-group substitution on the alpha-phosphate had no effect on ATP catabolism to ADP. There was no detectable catabolism of analogues with thiol-group substitution on the terminal phosphate. Each of the phosphorothioate analogues that was catabolized broke down at a rate similar to that of the natural nucleotide from which it was derived. These results demonstrate that the ectonucleotidases on pig aortic endothelial cells exhibit a high degree of stereoselectivity, characteristic for each enzyme, both with respect to the ribofuranosyl moiety and to the phosphate side chain.

scholarly journals Oxidized polyamines and the growth of human vascular endothelial cells. Prevention of cytotoxic effects by selective acetylation

1987 ◽  
Vol 242 (2) ◽  
pp. 347-352 ◽  
Author(s):  
D M L Morgan
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Amine Oxidase ◽  
Umbilical Vein ◽  
Polyamine Oxidase ◽  
Oxidation Products ◽  
Vascular Endothelial ◽  
Cytotoxic Effects ◽  
Naturally Occurring ◽  
Aminopropyl Group

The responses of human umbilical-vein vascular endothelial cells in culture to the naturally occurring polyamines spermine, spermidine and putrescine, their acetyl derivatives and oxidation products were examined. In the absence of human polyamine oxidase, exposure of cells to polyamines (up to 160 microM) had no adverse effects. In the presence of polyamine oxidase, spermine and spermidine were cytotoxic, but putrescine was not. Acetylation of the aminopropyl group of spermidine or both aminopropyl groups of spermine prevented this cytotoxicity. The amino acids corresponding to the polyamines, representing a further stage of oxidation, were also without effect. The cytotoxic effects were irreversible. Use of bovine serum amine oxidase in place of the human enzyme gave qualitatively similar results.

Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism

2003 ◽  
Vol 284 (6) ◽  
pp. H1978-H1984 ◽  
Author(s):  
M. Ursula Norman ◽  
Shane B. Reeve ◽  
Vincent Dive ◽  
A. Ian Smith ◽  
Rebecca A. Lew
Keyword(s):  
Endothelial Cells ◽  
High Performance ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Hybrid Cell ◽  
Fluorescent Substrate ◽  
Intact Cells ◽  
Aortic Endothelial Cells

The closely related metalloendopeptidases EC 3.4.24.15 (EP24.15; thimet oligopeptidase) and 24.16 (EP24.16; neurolysin) cleave a number of vasoactive peptides such as bradykinin and neurotensin in vitro. We have previously shown that hypotensive responses to bradykinin are potentiated by an inhibitor of EP24.15 and EP24.16 (26), suggesting a role for one or both enzymes in bradykinin metabolism in vivo. In this study, we have used selective inhibitors that can distinguish between EP24.15 and EP24.16 to determine their activity in cultured endothelial cells (the transformed human umbilical vein endothelial hybrid cell line EA.hy926 or ovine aortic endothelial cells). Endopeptidase activity was assessed using a specific quenched fluorescent substrate [7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-d-Lys(2,4-dinitrophenyl)], as well as the peptide substrates bradykinin and neurotensin (assessed by high-performance liquid chromatography with mass spectroscopic detection). Our results indicate that both peptidases are present in endothelial cells; however, EP24.16 contributes significantly more to substrate cleavage by both cytosolic and membrane preparations, as well as intact cells, than EP24.15. These findings, when coupled with previous observations in vivo, suggest that EP24.16 activity in vascular endothelial cells may play an important role in the degradation of bradykinin and/or other peptides in the circulation.

Thrombin receptor 14-amino acid peptide binds to endothelial cells and stimulates calcium transients

1992 ◽  
Vol 263 (5) ◽  
pp. L595-L601 ◽  
Author(s):  
C. Tiruppathi ◽  
H. Lum ◽  
T. T. Andersen ◽  
J. W. Fenton ◽  
A. B. Malik
Keyword(s):  
Endothelial Cells ◽  
Intracellular Calcium ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Calcium Transients ◽  
Cell Protein ◽  
Affinity Constant ◽  
Thrombin Receptor ◽  
Vascular Endothelial ◽  
Amino Terminal

We examined the binding characteristics of the recently described thrombin receptor amino-terminal peptide, SFLLRNPNDKYEPF (T. K. H. Vu, D. T. Hung, V. I. Wheaton, and S. R. Coughlin. Cell 64: 1057-1068, 1991), termed TRP-14, and its effect in activating intracellular calcium transients in pulmonary vascular endothelial cells. Binding of 125I-labeled TRP-14 was found to be saturable with a affinity constant of 2 microM and maximum binding of 41 pmol/mg of cell protein. The 125I-labeled TRP-14 also interacted with bovine pulmonary microvessel endothelial cells, human umbilical vein endothelial cells, and porcine pulmonary artery smooth muscle cells. Binding of 125I-labeled diisopropylphosphoryl (DIP)-alpha-thrombin, which is catalytically inactive but binds to thrombin receptors, was not inhibited by TRP-14 or vice versa, indicating that TRP-14 did not compete for the alpha-thrombin binding site(s) on the endothelial cell surface. TRP-14 (> 1 microM) increased the concentration of intracellular calcium ([Ca2+]i) in endothelial cells with kinetics similar to the increase in [Ca2+]i triggered by alpha-thrombin. In contrast, DIP-alpha-thrombin did not increase [Ca2+]i and also did not prevent the rise in [Ca2+]i induced by the subsequent challenge with either TRP-14 or alpha-thrombin. Because the generation of TRP-14 by the proteolytically active forms of thrombin stimulated a rise in endothelial [Ca2+]i, TRP-14 may be the agonist responsible for the activation of the alpha-thrombin receptor in pulmonary vascular endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Bovine brain and pituitary fibroblast growth factors: comparison of their abilities to support the proliferation of human and bovine vascular endothelial cells.

1983 ◽  
Vol 97 (6) ◽  
pp. 1677-1685 ◽  
Author(s):  
D Gospodarowicz ◽  
J Cheng ◽  
M Lirette
Keyword(s):  
Endothelial Cells ◽  
Growth Factors ◽  
Cell Cultures ◽  
Vascular Endothelial Cells ◽  
Fibroblast Growth Factors ◽  
Low Density ◽  
Vascular Endothelial ◽  
Fibroblast Growth ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells

The mitogenic effects of brain and pituitary fibroblast growth factors (FGF) on vascular endothelial cells derived from either human umbilical vein or bovine aortic arch have been compared. Both brain and pituitary FGF are mitogenic for low density human umbilical endothelial (HUE) cell cultures maintained on either fibronectin- or laminin-coated dishes or on biomatrices produced by cultured cells such as bovine corneal endothelial cells or the teratocarcinoma cell line PF-HR-9. Pituitary FGF triggered the proliferation of HUE cells at concentrations as low as 0.25 ng/ml, with a half-maximal response at 0.55 ng/ml and optimal effect at 2.5 to 5 ng/ml. It was 50,000-fold more potent than commercial preparations of endothelial cell growth factor and 40 times more potent than commercial preparations of pituitary FGF. Similar results were observed when the effect of pituitary FGF was tested on low density cultures of adult bovine aortic endothelial cells. When the activity of brain and pituitary FGF on low density HUE cell cultures was compared, both mitogens were active. To confirm the presence in brain extract of both acidic and neutral, as well as of basic mitogen, for HUE cells, brain tissues were extracted at acidic (4.5), neutral (7.2), and basic (8.5) pH. The three types of extracts were equally potent in supporting the proliferation of either HUE or adult bovine aortic endothelial cells. When the various extracts were absorbed at pH 6.0 on a carboxymethyl Sephadex C-50 column, the neutral and basic extracts had an activity after adsorption similar to that of unadsorbed extracts. In contrast, extracts prepared at pH 4.5 lost 90-95% of their activity which was recovered in the adsorbed fraction containing FGF.

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