Kjell Sverre Galdal

Cellular cooperation in endothelial cell thromboplastin synthesis

Summary. Endothelial cells from human umbilical veins produce a procoagulant identified as thromboplastin (tissue factor, factor III) when stimulated with the phorbol ester 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA), phytohaemagglutinin (PHA) or endotoxin. Inducible thromboplastin synthesis (i.e. synthesis of the protein component of thromboplastin, apoprotein III) was totally inhibited by cycloheximide and actinomycin D, indicating that de novo protein and RNA syntheses are necessary. Serum enhanced the induced apoprotein synthesis.

LDL-induced cytotoxicity and its inhibition by anti-oxidant treatment in cultured human endothelial cells and fibroblast

Low density lipoproteins (LDL) isolated by ultracentrifugation induce cytotoxic changes in cultured human endothelial cells (EC) and fibroblasts if the ratio between LDL cholesterol and the final protein concentration of the culture medium exceeds 0.10-0.12 mmol/g protein. In order to investigate if reactive oxygen species could contribute to the cytotoxicity, LDL were prepared in the presence of the antioxidants butylated hydroxytoluene (BHT) or superoxide dismutase (SOD), while routinely prepared LDL from the same donors served as control (N-LDL). A radiochromium release assay was used to evaluate cellular injury. BHT treatment of the LDL fraction virtually abolished LDL-induced cytotoxicity in cultured human EC and fibroblasts. SOD-LDL offered partial protection against LDL cytotoxicity. A positive correlation between the cytotoxicity of the various fractions and their content of malondialdehyde (MDA) further supports our conclusion that lipid peroxides in the LDL fractions mediate the cytotoxic effect on cultured cells.

Thrombin-induced shape changes of cultured endothelial cells: Metabolic and functional observations

Cultured human umbilical vein endothelial cells (EC) incubated in the presence of 2-deoxy-D-glucose (20 mmol/l) or at 4 degrees C lost their ability to undergo shape changes when exposed to thrombin (1 N.I.H. u/ml). Drugs blocking Ca++-flux (verapamil and nifedipin), microfilament disrupting agents (cytochalasin B and D) and microtubule disrupting agents (colchicine and colcemid) did not prevent thrombin-induced shape changes. None of the agents tested inhibited the accelerated thrombin-induced 51Cr-release from the cells. Pretreatment of EC with thrombin did not influence their ability to mediate clot retraction.

Thromboplastin Synthesis in Endothelial Cells

Endothelial cells (EC) in culture produce a procoagulant identified as thromboplastin when stimulated with 12-O-tetradecanoyl-phorbol-13-acetate, phytohemagglutinin, endotoxin, thrombin, histamine or epinephrine. Inducible thromboplastin generation most likely depends on de novo synthesis of RNA and the protein component of thromboplastin, apoprotein III. It is further probably regulated by intracellular mechanisms involving cAMP, transmethylation and calcium ions. The EC thromboplastin response is enhanced in lymphocyte-EC and platelet-EC cocultures and thromboplastin becomes partly available on the cell surface. Levels of procoagulant activity are reached which clearly may be of clinical importance if similar levels appear in vivo.

Effects of divalent cations and various vasoactive and haemostatically active agents on the integrity of monolayers of cultured human endothelial cells

Abstract Injury to human endothelial cells in primary culture was evaluated by a 51 Cr-release assay, phase contrast microscopy and the trypan blue exclusion test. Over a period of 24 hours incubation with either normal human serum or standard culture medium, the normal integrity of the confluent endothelium was maintained and total 51 Cr-release showed a slow, linear increase. If Tris-buffered saline was used as incubation medium considerable contraction and detachment of cells and accelerated 51 Cr-release were observed. This evidence of injury was markedly reduced by the addition of small amounts of Ca ++ ions, while Mg ++ had no effect. Among 4 haemostatically active agents investigated (ADP, thrombin, endotoxin and sodium polyanethol sulfonate) only thrombin induced morphological evidence of injury accompanied by accelerated release of 51 Cr. These effects of thrombin were inhibited by hirudin. Five vasoactive agents (adrenaline, noradrenaline, histamine, bradykinin and serotonin) had no appreciable effect on endothelial morphology and did not accelerate 51 Cr-release.

Actin pools and actin microfilament organization in cultured human endothelial cells after exposure to thrombin

Human umbilical vein endothelial cells (HUVEC) in primary confluent cultures lost their normal polygonal shape and assumed a ‘contracted’ appearance as judged by phase contrast microscopy when exposed to highly purified bovine thrombin (2 N.I.H. u/ml). Total actin in thrombin‐exposed cells did not differ from that of control cells, as measured by the deoxyribonuclease I inhibition assay. However, the monomeric actin pool (unpolymerized actin) in thrombin‐treated HUVEC was c. 15% smaller (P<0.01) than in control HUVEC (in which it represented approximately 50% of total actin). Transmission electron microscopy showed that thrombin‐stimulated HUVEC contained more and thicker bundles of filamentous actin than control cells. Polymerization of actin and reorganization of actin microfilaments may contribute to the shape changes of HUVEC induced by thrombin.

The effect of thrombin on fibronectin in cultured human endothelial cells

Cultures of human endothelial cells (EC) incubated for periods up to 24 h with highly purified thrombin (2 NIH u/ml) contained considerably less cell-associated fibronectin fibrils than corresponding controls. The loss of fibronectin fibrils was evident after 4 h and was accompanied by a 2-3 fold increase in the concentration of fibronectin in the incubation medium. Hirudin inhibited the effects of thrombin. Thrombin also induced characteristic shape changes of EC. These shape changes were reversible within a 4-6 h period and could not be reinvoked by new additions of thrombin. Thus, structural refractoriness to thrombin coincided temporally with a period when EC-associated fibronectin fibrils were markedly reduced.

Inhibition of the thromboplastin response of endothelial cells in vitro.

Confluent monolayers of human umbilical vein endothelial cells in culture responded with a 5-fold increase in thromboplastin (tissue factor) synthesis when exposed to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (50 ng/ml) or endotoxin (ETX) (25 micrograms/ml) for 16 hr. This induced thromboplastin synthesis was markedly inhibited by exposure of the cells to two different phosphodiesterase inhibitors, methylisobutylxanthine (MIX) and rac-4(3-butoxy-4-methoxybenzyl)-2-imidazole idinone (RO-20-1724) and to the transmethylation inhibitors 3-deazaadenosine (DZA) and 1-homocysteine thiolactone (Hcy) in combination. It was slightly (TPA) or not at all (ETX) inhibited upon exposure of the cells to the intracellular calcium antagonist 8-(N,N-diethylamino)-octyl 3,4, 5-trimethoxybenzoate hydrochloride (TMB-8). However, in the presence of MIX TMB-8 had a moderate additional inhibitory effect on TPA-induced thromboplastin response. The thromboplastin response of endothelial cells in vitro thus probably depends on transmethylation events for its full expression. It is also strongly modulated by the intracellular level of cAMP.

Injury to cultured human fibroblasts induced by low density lipoproteins: Potentiating and protective factors

Low density lipoproteins (LDL) injured cultured fibroblasts provided a certain ratio existed between lipoproteins and infranatant proteins. The mechanism of action appeared similar to the mechanism whereby LDL injure endothelial cells in culture. Enrichment of normal fibroblasts with cholesterol esters by pretreatment with chloroquine and LDL potentiated injury inflicted by subsequent LDL exposure. Pretreatment with chloroquine alone offered partial protection against LDL-induced damage while indomethacin and sulphinpyrazone added prior to or together with LDL had no effect.