Study on molecular marker in perturbed human endothelium

为了研究内皮细胞紊乱的分子标记，我们进行了生理实验。血流动力学力调节各种内皮细胞功能，甚至在基因调控下存在细胞因子。我们研究了剪切应力对受细胞因子干扰的培养人脐静脉内皮细胞（HUVEC）凝血和纤溶系统的影响，使用改进的锥形板粘度计，在该粘度计中产生良好控制和定义的剪切力。组织因子（TF）是一种跨膜糖蛋白，在血液凝固过程中起着核心作用，是内皮细胞凝血的重要调节因子。6-24 dynes/cm^2的剪切应力以剪切强度和暴露时间依赖的方式降低tnfalpha诱导的TF的表达。逆转录-聚合酶链反应显示，tnfalpha诱导的TF mRNA水平随流动而降低。在TNF刺激前15小时和刺激后6小时分别施加18达因/cm^2的剪切力，HTF-K180抗体在细胞表面表达量下降至1 / 3。然而，在没有或存在剪切应力的情况下，TF mRNA的稳定性没有改变。组织型纤溶酶原激活剂（t-PA）可将纤溶酶原转化为纤溶酶以降解纤维蛋白凝块，而抑制t-PA功能的纤溶酶原激活剂抑制剂-1 （PAI-1）在内皮细胞纤维蛋白溶解中起核心作用。静态条件下il -1 β或tnf - α处理细胞对t-PA分泌无影响，而PAI-1的释放增加。当细胞暴露于不断增加的剪切应力至24 dynes/cm^2时，t-PA和t-PA/PAI-1复合物的水平相对于剪切应力显著升高，而总PAI-1和活性PAI-1的分泌逐渐减少。在il -1 β或tnf - α的存在下，t-PA和t-PA/PAI-1复合物的产生增加进一步增强。用t-PA和PAI-1 cDNA探针对培养细胞进行斑点杂交分析，结果显示，静息或细胞因子刺激条件下，静态内皮细胞的总RNA中没有t-PA mRNA，但在剪切力为18 dynes/cm^2的细胞中检测到丰富的t-PA mRNA，并且在细胞因子的作用下进一步增加。相比之下，在静息和细胞因子刺激的非剪切内皮细胞中检测到PAI-1 mRNA，但暴露于剪切应激后，即使存在细胞因子，其水平也会下降。这些结果清楚地表明，剪切力是内皮细胞凝血和纤溶系统的重要调节因子，以协调维持血管管腔表面的抗血栓性。这些分子标记可能是内皮微扰非常有用的指标。少

To investigate molecular markers in perturbed endothelium, physiological experiments have been undertaken. Hemodynamic forces modulate various endothelial cell functions even in the presence of cytokines under gene regulation. We have investigated the effect of shear stress on coagulation and fibrinolysis system in cultured human umbilical vein endothelial cells (HUVEC) perturbed by cytokines, using modified cone-plate viscometer, in which well controlled and defined shear forces were geperated. Tissue factor (TF), a transmembrane glycoprotein that acts a central role in blood coagulation, are important regulators for coagulation in endothelium. Sheaur stress of 6-24 dynes/cm^2 decreased the expression of TNFalpha-induced TF in a shear intensity- and exposure time- dependent manner. Reverse transcriptase-polymerase chain reaction showed that TNFalpha-induced TF mRNA levels decreased in response to flow. TF expressed on cell surface measured by flow cytometry using an atnti-TF monoclona … More l antibody, HTF-K180, was decreased to one third by shear forces of 18 dynes/cm^2 applied for 15 hours before and 6 hours after TNF stimulation. The stability of TF mRNA,however, was not changed in the absence or presence of shear stress. Tissue plasminogen activator (t-PA), which convert plasminogen to plasmin to degrade fibrin clot, and plasminogen activator inhibitor-1 (PAI-1), which inhibit t-PA function, play central roles for fibrinolysis in endothelium. Treatment of the cells with IL-1beta or TNF-alpha under static conditions had no effect on t-PA secretion, while release of PAI-1 increased. When cells were exposed to increasing shear stress up to 24 dynes/cm^2, levels of t-PA and t-PA/PAI-1 complex significantly increased relative to shear stress, while total PAI-1 and active PAI-1 secretion decreased gradually. In the presence of IL-1beta or TNF-alpha, the increase in production of t-PA and t-PA/PAI-1 complex was further augmented. Dot blot hybridization analysis of cultured cells using t-PA and PAI-1 cDNA probes revealed no t-PA mRNA in 3 mug of total RNA from static endothelial cells under resting or cytokine-stimulated conditions but abundant t-PA mRNA was detected in cells subjected to a shear force of 18 dynes/cm^2 and the increase was further augmented by addition of cytokines. In contrast, PAI-1 mRNA was detected in resting and cytokine-stimulated, non-sheared endothelial cells, but levels decreased after exposure to shear stress, even in the presence of cytokines.These results clearly indicate that shear forces act as an important regulator of coagulation and fibrinolysis system in endothelium, to coordinately maintain aantithrombogenicity of luminal surface of blood vessels. These molecular markers might be very useful indicators for endothelial perturbation. Less