Jacobi Jeanna, Sela Shifra, Cohen Hector I, Chezar Judith, Kristal Batya
Eliachar Research Laboratory, Western Galilee Hospital, Nahariya, Israel.
Am J Physiol Heart Circ Physiol. 2006 May;290(5):H2051-8. doi: 10.1152/ajpheart.01040.2005. Epub 2005 Dec 30.
Peripheral polymorphonuclear leukocytes (PMNL) in hemodialysis (HD) patients are primed, continually releasing and exposing the vascular endothelium to soluble factors such as reactive oxygen species and inflammatory mediators. To mimic the close proximity between PMNL and the endothelial monolayer and to monitor and characterize the influence of soluble mediators released from PMNL, we developed a novel cocultivation system using primary human umbilical vein endothelial cell (HUVEC) cultures and PMNL, with a sieve separating the two cell types to prevent direct adhesive effects. PMNL (10(6)) from HD patients or from healthy normal controls were cocultivated with HUVEC (10(5)) for 15 min, and endothelial cell injury was assessed by HUVEC morphology, cell detachment, and apoptosis. Proinflammatory changes were estimated by expression of HUVEC adhesion molecule P-selectin and by endothelial IL-8 and endothelial nitric oxide synthase mRNA. The levels of intracellular tissue factor reflected the procoagulant state, whereas NADPH oxidase activity served as an indicator for prooxidative changes in HUVEC. Mediators released from the primed PMNL triggered activation/dysfunction of endothelial cells, causing 1) an increase in endothelial cell detachment and apoptosis, 2) a proinflammatory state manifested by increased IL-8 mRNA expression and P-selectin on the endothelial surface, 3) activation of endothelial NADPH oxidase, 4) an increase in endothelial cell tissue factor that directly correlated with PMNL priming index, and 5) a decrease in endothelial nitric oxide synthase mRNA. Our data support a pathogenic link between PMNL priming and endothelial dysfunction, suggesting that PMNL priming is a potential new nontraditional risk factor for the development of atherosclerosis.
血液透析（HD）患者的外周多形核白细胞（PMNL）处于预激活状态，持续释放并使血管内皮暴露于活性氧和炎症介质等可溶性因子中。为模拟PMNL与内皮单层细胞的紧密接近程度，并监测和表征PMNL释放的可溶性介质的影响，我们开发了一种新型共培养系统，该系统使用原代人脐静脉内皮细胞（HUVEC）培养物和PMNL，用筛网分隔两种细胞类型以防止直接黏附作用。将HD患者或健康正常对照的PMNL（10⁶）与HUVEC（10⁵）共培养15分钟，并通过HUVEC形态、细胞脱离和凋亡评估内皮细胞损伤。通过HUVEC黏附分子P-选择素的表达以及内皮IL-8和内皮一氧化氮合酶mRNA评估促炎变化。细胞内组织因子水平反映促凝状态，而NADPH氧化酶活性作为HUVEC中促氧化变化的指标。预激活的PMNL释放的介质引发内皮细胞的激活/功能障碍，导致1）内皮细胞脱离和凋亡增加，2）以内皮表面IL-8 mRNA表达增加和P-选择素增加为表现的促炎状态，3）内皮NADPH氧化酶激活，4）内皮细胞组织因子增加，且与PMNL预激活指数直接相关，5）内皮一氧化氮合酶mRNA减少。我们的数据支持PMNL预激活与内皮功能障碍之间的致病联系，表明PMNL预激活是动脉粥样硬化发展的潜在新的非传统危险因素。
