Yang Z, Bochsler P N, Carroll R C, Carter C D, Khemlani L S, Breider M A
Department of Pathobiology, University of Tennessee, Knoxville.
Inflammation. 1994 Apr;18(2):221-33. doi: 10.1007/BF01534563.
Increased procoagulant activity of vascular endothelial cells may be an important component in the pathogenesis of intravascular coagulation associated with gram-negative bacterial diseases. Two bovine endothelial cell (BEC) lines isolated from pulmonary arteries (ENS-2 and ENT-18) were used in this study to investigate procoagulant signal transduction pathways of endotoxin (lipopolysaccharide, LPS)--stimulated BECs. The endothelial cell line ENS-2 was sensitive to LPS as demonstrated by tissue factor (TF) expression, but in contrast, the ENT-18 endothelial cell line was unusually resistant to the effects of LPS. No remarkable quantitative difference in binding of radiolabeled LPS was detected between the two endothelial cell lines. A protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate, PMA) failed to induce TF expression in either cell line at concentrations ranging from 0.05 to 1.00 microM when used as a sole stimulus for the endothelial cells. However, when PMA was used in combination with LPS, PMA enhanced the stimulatory effect of LPS on the endothelial cells. In parallel experiments, PKC inhibitors (H-7 and GF 109203X) interfered with the stimulatory effect of LPS on the cells by decreasing tissue factor expression. We also found that an activator of adenylate cyclase, forskolin, similarly inhibited LPS-induced tissue factor activity. In contrast, protein tyrosine kinase inhibitors (genistein, lavendustin A) had no inhibitory effect on LPS-induced endothelial cell tissue factor expression. Our results collectively suggest that activation of PKC is an important step in stimulation of endothelial cells by LPS, and that LPS and phorbol esters may synergize to produce an enhanced stimulatory effect. Our results also suggest participation of cAMP in controlling LPS-mediated stimulation of endothelial cells, but fail to demonstrate a role for protein tyrosine kinase activity.
血管内皮细胞促凝活性增加可能是革兰氏阴性菌相关疾病中血管内凝血发病机制的一个重要组成部分。本研究使用从肺动脉分离的两种牛内皮细胞系（ENS - 2和ENT - 18）来研究内毒素（脂多糖，LPS）刺激的牛内皮细胞的促凝信号转导途径。内皮细胞系ENS - 2对LPS敏感，表现为组织因子（TF）表达增加，但相比之下，ENT - 18内皮细胞系对LPS的作用异常抵抗。在两种内皮细胞系之间未检测到放射性标记LPS结合的显著定量差异。当作为内皮细胞的唯一刺激物时，蛋白激酶C（PKC）激活剂（佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯，PMA）在0.05至1.00微摩尔浓度范围内未能诱导任何一种细胞系中的TF表达。然而，当PMA与LPS联合使用时，PMA增强了LPS对内皮细胞的刺激作用。在平行实验中，PKC抑制剂（H - 7和GF 109203X）通过降低组织因子表达来干扰LPS对细胞的刺激作用。我们还发现腺苷酸环化酶激活剂福斯可林同样抑制LPS诱导的组织因子活性。相反，蛋白酪氨酸激酶抑制剂（染料木黄酮、拉文达ustin A）对LPS诱导的内皮细胞组织因子表达没有抑制作用。我们的结果共同表明，PKC激活是LPS刺激内皮细胞的重要步骤，并且LPS和佛波酯可能协同产生增强的刺激作用。我们的结果还表明cAMP参与控制LPS介导的内皮细胞刺激，但未能证明蛋白酪氨酸激酶活性的作用。
