Grammas Paula, Ottman Todd, Reimann-Philipp Ulrich, Larabee Jason, Weigel Paul H
Department of Pathology, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA.
J Alzheimers Dis. 2004 Jun;6(3):275-81. doi: 10.3233/jad-2004-6308.
The multifunctional serine protease thrombin has been shown to be neurotoxic in vitro and in vivo and is demonstrable in the Alzheimer disease (AD) brain. We have documented that in AD the cerebral microvasculature is a source of inflammatory and neurotoxic proteins. The objective of this study was to determine if injured brain endothelial cells could be a source of neurotoxic thrombin. Brain endothelial cells were incubated with either sodium nitroprusside (SNP, 10 microM), inflammatory proteins (IL-1beta, IL-6, TNFalpha, LPS, IFNgamma) or the PKC inhibitor bisindolymaleimide (1 microM) for 24 h and conditioned media collected. Endothelial cell conditioned medium was incubated with purified apolipoprotein E4 (apoE4) for 24 h, and then analyzed for neurotoxic activity against primary cortical cultures and for apoE4 fragments by western blot. Endothelial cell conditioned medium collected after treatment with either SNP, inflammatory proteins, or the PKC inhibitor bisindolymaleimide, demonstrated a significant (p < 0.005) level of thrombin activity, the presence of apoE4 fragments, and was capable of evoking neuronal cell death. These data demonstrate that endothelial cell injury results in thrombin release and suggest that the brain microcirculation could be a source of neurotoxic factors in AD.
多功能丝氨酸蛋白酶凝血酶已被证实在体外和体内具有神经毒性，且在阿尔茨海默病（AD）患者的大脑中也可检测到。我们已记录到，在AD中，脑微血管是炎症和神经毒性蛋白的一个来源。本研究的目的是确定受损的脑内皮细胞是否可能是神经毒性凝血酶的一个来源。将脑内皮细胞与硝普钠（SNP，10微摩尔）、炎症蛋白（白细胞介素-1β、白细胞介素-6、肿瘤坏死因子α、脂多糖、干扰素γ）或蛋白激酶C抑制剂双吲哚马来酰亚胺（1微摩尔）孵育24小时，然后收集条件培养基。将内皮细胞条件培养基与纯化的载脂蛋白E4（apoE4）孵育24小时，然后分析其对原代皮质培养物的神经毒性活性，并通过蛋白质免疫印迹法检测apoE4片段。在用SNP、炎症蛋白或蛋白激酶C抑制剂双吲哚马来酰亚胺处理后收集的内皮细胞条件培养基，显示出显著（p < 0.005）水平的凝血酶活性、apoE4片段的存在，并且能够引起神经元细胞死亡。这些数据表明内皮细胞损伤会导致凝血酶释放，并提示脑微循环可能是AD中神经毒性因子的一个来源。
