Effects of thrombospondin antibody on the recovery of endothelial cells from hyperthermia

In addition to the increased synthesis of the classical heat-shock proteins (28,000, 71,000, 73,000, 90,000 and 100,000 Mr polypeptides) there is also an increase of thrombospondin in the growth medium of endothelial cells exposed to hyperthermia. The effect of a monoclonal antibody to thrombospondin on the recovery of endothelial cells from hyperthermia as it relates to cytoskeletal organization and cell spreading was assessed. The antibody interacts with the heparin-binding domain of thrombospondin in the extracellular matrix of cells. We report that during recovery from thermal insult at 37 degrees C, intermediate filaments, stress fibres and microtubules show distinct time-recovery characteristics in bovine aortic endothelial cells; that in the presence of this antibody the cytoskeleton is notably altered; that this antibody causes retraction of endothelial cell processes; and that the recovery of the cytoskeleton in endothelial cells exposed to hyperthermia is prevented by the thrombospondin antibody in the time frame examined. Our data suggest that the recovery of cells from heat shock requires the integrity of thrombospondin and its interactions.

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Effects of heat shock on the expression of thrombospondin by endothelial cells in culture.

The Journal of Cell Biology ◽

10.1083/jcb.106.3.893 ◽

1988 ◽

Vol 106 (3) ◽

pp. 893-904 ◽

Cited By ~ 17

Author(s):

N V Ketis ◽

J Lawler ◽

R L Hoover ◽

M J Karnovsky

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Heat Shock ◽

Heat Shock Proteins ◽

Mrna Levels ◽

Aortic Endothelial Cells ◽

Bovine Aortic Endothelial Cells ◽

Shock Proteins ◽

Intracellular Fluorescence ◽

Aortic Endothelial

Heat-shock proteins from confluent primary cultures of bovine aortic endothelial cells were analyzed by SDS-polyacrylamide gels. In addition to the increased synthesis of the classical heat-shock proteins, there is an increase of a 180,000-mol wt polypeptide in the growth media of heat-shocked cells. Immunoprecipitation with specific antiserum indicates that the 180,000-mol wt polypeptide is thrombospondin. Assay of mRNA levels coding for thrombospondin after brief hyperthermic treatment (45 degrees C, 10 min), followed by a recovery of 2 h at 37 degrees C, results in a twofold increase in mRNA abundance. In contrast, the activation level of the 71,000-mol wt heat-shock protein mRNA occurs at an earlier time than for thrombospondin mRNA. Immunofluorescence microscopy was used to study the intracellular and extracellular distribution of thrombospondin. Thrombospondin is localized to a prominent pattern of granules of intracellular fluorescence in a perinuclear distribution in cells not exposed to heat. Upon heat treatment, the pattern of granules of intracellular fluorescence appears more pronounced, and the fluorescence appears to be clustered more about the nucleus. There are at least three pools of extracellular forms of thrombospondin: (a) the fine fibrillar extracellular matrix thrombospondin; (b) the punctate granular thrombospondin; and (c) the thrombospondin found in the conditioned medium not associated with the extracellular matrix. When bovine aortic endothelial cells are exposed to heat, the extracellular matrix staining of a fibrillar nature is noticeably decreased, with an increase in the number and degree of fluorescence of focal areas where the punctate granule thrombospondin structures are highly localized. No gross morphological changes in extracellular matrix staining of fibronectin was noted. However, the intermediate filament network was very sensitive and collapsed around the nucleus after heat shock. We conclude that the expression of thrombospondin is heat-shock stimulated.

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Heparin-binding vitronectin up-regulates latent TGF-beta production by bovine aortic endothelial cells

Journal of Cell Science ◽

10.1242/jcs.108.4.1553 ◽

1995 ◽

Vol 108 (4) ◽

pp. 1553-1561

Author(s):

S.M. Ribeiro ◽

S. Schultz-Cherry ◽

J.E. Murphy-Ullrich

Keyword(s):

Endothelial Cells ◽

Conditioned Media ◽

Extracellular Matrix Protein ◽

Beta Activity ◽

Heparin Binding ◽

Tgf Beta ◽

Free System ◽

Aortic Endothelial Cells ◽

Bovine Aortic Endothelial Cells ◽

Aortic Endothelial

Vitronectin, a serum and extracellular matrix protein, is present in vivo in two different conformations: a native form, which does not bind heparin, and a heparin-binding conformer, which results from interactions of native vitronectin with either the thrombin-antithrombin III complex or the terminal complement complex, C5b-9. We found that vitronectin stimulates the activity of the growth regulatory peptide, TGF-beta, in the conditioned media of bovine aortic endothelial cells as a result of increased production of latent TGF-beta. This effect is specific for the denatured, heparin-binding, form of vitronectin, since native vitronectin has no effect on the production of latent TGF-beta by those cells. Stimulation is time and concentration-dependent, but is independent of protease activity. Stimulation is dependent on the presence of cells, since there was no increase in TGF-beta activity observed when vitronectin was added to the conditioned media after removal from cells. Furthermore, incubation of recombinant latent TGF-beta with vitronectin in a cell-free system does not result in increased TGF-beta activity. Assays of total TGF-beta levels in heat-treated conditioned media showed that vitronectin treatment elevates the levels of total TGF-beta in the conditioned media. These results were further confirmed by western blot analysis of the conditioned media with antibodies specific for latent TGF-beta. These data suggest that vitronectin regulates expression and/or secretion of TGF-beta by bovine aortic endothelial cells. This cellular response to the heparin-binding form of vitronectin seems to be mediated by alpha v beta 3 integrins.(ABSTRACT TRUNCATED AT 250 WORDS)

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Ornithine Decarboxylase Activity in Cultured Endothelial Cells Stimulated by Serum, Thrombin and Serotonin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646709 ◽

1978 ◽

Vol 39 (02) ◽

pp. 496-503 ◽

Cited By ~ 8

Author(s):

P A D’Amore ◽

H B Hechtman ◽

D Shepro

Keyword(s):

Endothelial Cells ◽

Ornithine Decarboxylase ◽

Decarboxylase Activity ◽

Aortic Endothelial Cells ◽

Ornithine Decarboxylase Activity ◽

Rate Limiting Step ◽

Bovine Aortic Endothelial Cells ◽

Limiting Step ◽

Rate Limiting ◽

Serum Serotonin

SummaryOrnithine decarboxylase (ODC) activity, the rate-limiting step in the synthesis of polyamines, can be demonstrated in cultured, bovine, aortic endothelial cells (EC). Serum, serotonin and thrombin produce a rise in ODC activity. The serotonin-induced ODC activity is significantly blocked by imipramine (10-5 M) or Lilly 11 0140 (10-6M). Preincubation of EC with these blockers together almost completely depresses the 5-HT-stimulated ODC activity. These observations suggest a manner by which platelets may maintain EC structural and metabolic soundness.

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Effects of Dipyrone on Prostaglandin Production by Human Platelets and Cultured Bovine Aortic Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657338 ◽

1983 ◽

Vol 49 (02) ◽

pp. 132-137 ◽

Cited By ~ 16

Author(s):

A Eldor ◽

G Polliack ◽

I Vlodavsky ◽

M Levy

Keyword(s):

Endothelial Cells ◽

Arachidonic Acid ◽

Competitive Inhibition ◽

Inhibitory Effect ◽

Human Platelets ◽

Ionophore A23187 ◽

Aortic Endothelial Cells ◽

Bovine Aortic Endothelial Cells ◽

Aortic Endothelial

SummaryDipyrone and its metabolites 4-methylaminoantipyrine, 4-aminoantipyrine, 4-acetylaminoantipyrine and 4-formylaminoan- tipyrine inhibited the formation of thromboxane A2 (TXA2) during in vitro platelet aggregation induced by ADP, epinephrine, collagen, ionophore A23187 and arachidonic acid. Inhibition occurred after a short incubation (30–40 sec) and depended on the concentration of the drug or its metabolites and the aggregating agents. The minimal inhibitory concentration of dipyrone needed to completely block aggregation varied between individual donors, and related directly to the inherent capacity of their platelets to synthesize TXA2.Incubation of dipyrone with cultured bovine aortic endothelial cells resulted in a time and dose dependent inhibition of the release of prostacyclin (PGI2) into the culture medium. However, inhibition was abolished when the drug was removed from the culture, or when the cells were stimulated to produce PGI2 with either arachidonic acid or ionophore A23187.These results indicate that dipyrone exerts its inhibitory effect on prostaglandins synthesis by platelets or endothelial cells through a competitive inhibition of the cyclooxygenase system.

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