Role of contaminating platelets in thromboxane synthesis in primary cultures of human umbilical vein endothelial cells.
Previous studies suggested that cultured human endothelial cells metabolize arachidonic acid to thromboxane A2. When primary cultures of human umbilical vein endothelial cells were incubated with 14C-arachidonic acid and the 14C-metabolites resolved by reverse phase high pressure liquid chromatography, radioactive products were observed that comigrated with 6-keto-prostaglandin F1alpha and thromboxane B2, the degradation products of prostacyclin and thromboxane A2, respectively. Since platelets synthesize thromboxane A2, the present study examined the hypothesis that adherent platelets may contaminate the primary cultures of human umbilical vein endothelial cells and be responsible for thromboxane B2 production. Confluent primary cultures or passaged cells were stimulated with histamine (10(-5) M). Incubation buffer was analyzed by specific radioimmunoassays for 6-keto-prostaglandin F1alpha and thromboxane B2. The production of thromboxane B2 decreased in the passaged cells (207 +/- 44 pg/ml versus 65 +/- 12 pg/ml; primary versus passaged cells). A moderate decrease in the yield of 6-keto-prostaglandin F1alpha was measured in the passaged cells compared to the primary cultures (3159 +/- 356 pg/ml versus 1678 +/- 224 pg/ml, primary versus passaged cells). If the primary cultures were incubated with human platelet-rich plasma for 30 min prior to stimulation with histamine, the amount of thromboxane B2 increased approximately 10-fold. In an additional experiment, sub-confluent primary cells were incubated with platelet-rich plasma for 30 min, washed to remove non-adherent platelets, and allowed to reach confluency. Confluent cells were then passaged and stimulated with histamine. The amount of thromboxane B2 was not significantly different from that obtained with passaged cells that had not been incubated with platelet-rich plasma during the primary culture (83 +/- 15 pg/ml versus 65 +/- 12 pg/ml, respectively). If the cyclooxygenase inhibitor indomethacin was included in the incubations, the amounts of both thromboxane B2 and 6-keto-prostaglandin F1alpha decreased. In contrast, the thromboxane A2 synthase inhibitor dazoxiben blocked thromboxane production and had no effect on the amount of 6-keto-prostaglandin F1alpha. Light microscopy revealed the presence of adherent platelets in primary cultures with and without platelet-rich plasma but no platelets were observed in any group of passaged cells. Histofluorescence for platelet serotonin indicated the presence of platelets only in primary cultures of human umbilical vein endothelial cells or in cultures pre-incubated with platelet-rich plasma. These studies suggest that primary cultures of human umbilical vein endothelial cells contain adherent platelets that contribute to thromboxane synthesis.
Pfister
Sandra L
SL
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee 53226, USA. spfister@mcw.edu
Hughes
Miranda J
MJ
Rosolowsky
Mark
M
Campbell
William B
WB
D015121:6-Ketoprostaglandin F1 alpha / Q000096:biosynthesis; D016718:Arachidonic Acid / Q000378:metabolism; D001792:Blood Platelets / Q000502:physiology*; D000001:Calcimycin / Q000494:pharmacology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008858:Microscopy, Phase-Contrast; D013928:Thromboxane A2 / Q000096:biosynthesis*; D014471:Umbilical Veins
D016718:Arachidonic Acid; D000001:Calcimycin; D013928:Thromboxane A2; D015121:6-Ketoprostaglandin F1 alpha