SIGNAL-TRANSDUCTION PATHWAYS OF BACTERIAL LIPOPOLYSACCHARIDE-STIMULATED BOVINE VASCULAR ENDOTHELIAL-CELLS

Increased procoagulant activity of vascular endothelial cells may be a n important component in the pathogenesis of intravascular coagulation associated with gram-negative bacterial diseases. Two bovine endothel ial cell (BEC) lines isolated from pulmonary arteries (ENS-2 and ENT-1 8) were used in this study to investigate procoagulant signal transduc tion pathways of endotoxin (lipopolysaccharide, LPS) stimulated BECs. The endothelial cell line ENS-2 was sensitive to LPS as demonstrated b y tissue factor (TF) expression, but in contrast, the ENT-18 endotheli al cell line was unusually resistant to the effects of LPS. No remarka ble quantitative difference in binding of radiolabeled LPS was detecte d between the two endothelial cell lines. A protein kinase C (PKC) act ivator (phorbol 12-myristate 13-acetate, PMA) failed to induce TF expr ession in either cell line at concentrations ranging from 0.05 to 1.00 mu M when used as a sole stimulus for the endothelial cells. However, when PMA was used in combination with LPS, PMA enhanced the stimulato ry effect of LPS on the endothelial cells. In parallel experiments, PK C inhibitors (H-7 and GF 109203X) interfered with the stimulatory effe ct of LPS on the cells by decreasing tissue factor expression. We also found that an activator of adenylate cyclase, forskolin, similarly in hibited LPS-induced tissue factor activity. In contrast, protein tyros ine kinase inhibitors (genistein, lavendustin A) had no inhibitory eff ect on LPS-induced endothelial cell tissue factor expression. Our resu lts collectively suggest that activation of PKC is an important step i n stimulation of endothelial cells by LPS, and that LPS and phorbol es ters may synergize to produce an enhanced stimulatory effect. Our resu lts also suggest participation of cAMP in controlling LPS-mediated sti mulation of endothelial cells, but fail to demonstrate a role for prot ein tyrosine kinase activity.