Nooteboom A, Bleichrodt R P, Hendriks T
Department of Surgery, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands.
Clin Exp Immunol. 2006 May;144(2):362-9. doi: 10.1111/j.1365-2249.2006.03074.x.
The aim of this study was to elucidate the time course of the permeability response of endothelial monolayers after exposure to plasma obtained from lipopolysaccharide (LPS)-treated human whole blood; to investigate the role of apoptosis in monolayer permeability, and to inhibit the permeability increase, particularly after addition of the plasma stimulus. Human umbilical vein endothelial cells (HUVEC) were cultured on semiporous membranes and the permeability for albumin was measured after exposure, according to different schedules, to LPS-conditioned plasma. Apoptotic HUVEC were measured by both flow cytometry and ELISA. A variety of agents, including antibodies against cytokines, inhibitors of NF-kappaB, and a caspase inhibitor, were added to HUVEC, either prior to or after the stimulus. A maximum increase of the permeability was achieved after 4-6 h of exposure to LPS-conditioned plasma. This response was not accompanied by an increase in the number of apoptotic HUVEC. Administration of antibodies against both Tumour Necrosis Factor-alpha (TNF-alpha) and Interleukin-1beta (IL-1beta) to HUVEC within 1 h after stimulation significantly reduced the permeability increase. Similarly, pyrollidine di-thiocarbamate (PDTC), but not N-acetylcysteine, could prevent the permeability response, and was still effective when added within 2 h after LPS-conditioned plasma. The TNF-alpha/IL-1beta signal present in LPS-conditioned plasma appears to increase endothelial permeability through intracellular pathways that very likely involve the activation of NF-kappaB. Although poststimulatory inhibition of the permeability response proves to be possible with agents such as PDTC, the window of opportunity appears very small if placed in a clinical perspective.
本研究的目的是阐明内皮细胞单层在暴露于脂多糖（LPS）处理的人全血所获血浆后通透性反应的时间进程；研究细胞凋亡在单层通透性中的作用，并抑制通透性增加，尤其是在添加血浆刺激物之后。人脐静脉内皮细胞（HUVEC）培养于半透膜上，按照不同时间表在暴露于LPS处理的血浆后测量白蛋白的通透性。通过流式细胞术和酶联免疫吸附测定法检测凋亡的HUVEC。在刺激之前或之后，将多种试剂添加到HUVEC中，包括抗细胞因子抗体、NF-κB抑制剂和一种半胱天冬酶抑制剂。暴露于LPS处理的血浆4 - 6小时后，通透性达到最大增加。这种反应并未伴随凋亡的HUVEC数量增加。在刺激后1小时内向HUVEC施用抗肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）的抗体可显著降低通透性增加。同样，吡咯烷二硫代氨基甲酸盐（PDTC）而非N-乙酰半胱氨酸能够阻止通透性反应，并且在LPS处理的血浆后2小时内添加时仍然有效。LPS处理的血浆中存在的TNF-α/IL-1β信号似乎通过很可能涉及NF-κB激活的细胞内途径增加内皮通透性。尽管用PDTC等试剂在刺激后抑制通透性反应被证明是可能的，但从临床角度来看，机会窗口似乎非常小。
