| Vascular endothelial cell is a major target of tumor necrosis factor-α(TNF-α). The effects of TNF-αon the functions of endothelial cell play crucial role in the development of some central nervous system diseases. The tight junctions between brain microvascuiar endothelial cells (BMVECs) are the fundemantal structrure of blood-brain barrier, which has important role in maintaining the stability of brain tissue enviroment. The injury of BMVECs induced by TNF-αmight be a key point in the pathogenesis of central nervous system diseases(CNS). Therefore, it is important to investigate the effects of TNF-αon functions of BMVECs for understanding the role of TNF-αin the pathophysiology of CNS diseases so as to develop the new strategy for prevention and treatment of CNS diseases.Objective Using primary cultured human brain microvascular endothelial cell (HBMVECs), we investigated the effects of TNF-αon the functions of HBMVECs. such as the expression of intercellular adhesion molecule-1 (ICAM-1), the production of interleukin-6 (IL-6) and the activity of matrix metalloproteinase-2 (MMP-2), compared the effects of TNF-αand lippolysaccharide (LPS) on the coagulation and fibrinolytic activity of HBMVECs in vitro so as to analyze the potential mechanism underlying TNF-α-mediated immunopathology of CNS diseases.Methods HBMVECs were islolated from human brain tissue and purified by UEA-1 conjungated beads. The classical endothelial cell markers von Willebrand Factor (vWF),CD31 were identified by immunofluorescence staining. Positive cells of vWF were identified by flow cytometry. The level of uptake acetylated low density lipoprotein (Dil-Ac-LDL) were demonstrated by fluorescence microscopy. The tight junctions between HBMVECs were demonstrated by scanning and transmission electron microscopy. HBMVECs were stimulated with different doses of TNF-α, and then enzyme linked immunosorbent assay (ELISA) was used to detect the expression of ICAM-1, the production of IL-6 and the level of coagulation fibrinolytic activity(thrombomodulin, TM; plasminogen activator-inhibitor-1, PAI-1; tissue-type plasminogen activator, t-PA). Gelatin zymography was used to analyze the activity of MMP-2. The procoagulant activity (PCA) was assessed by one-stage recalcification clotting time assay. In addition, the mRNA expression of tissue factor(TF), TM, t-PA and PAI-1 were detected by real-time quantitative polymerase chain reaction (Real-time PCR)Results Purified HBMVECs presented characteristics of monolayer growth and contact inhibition. Cells expressed classic endothelial markers CD31,vWF(more than 95% of positive cells by flow cytometry)and uptake high level of Dil-Ac-LDL. By scanning electron microscopy, no gap was found between HBMVECs. By transmission electron microscopy, tight junctions between cells were demonstrated. The constitutive expression of ICAM-1 and IL-6 production were markedly increased on HBMVECs after TNF-a stimulation compared with controls, and significant dose-effect relationship was appeared. Zymography showed a significant increase of MMP-2 produced by HBMVECs after stimulated by TNF-a. In response to TNF-a or LPS, the procoagulant activity of HBMVECs were increased greatly shown as decreased level of TM,increased level of PCA and TF. Moreover, the fibrinolytic factors (t-PA and PAI-1) were also increased. But the effect of TNF-a was significantly stronger than LPS by comparation with the ratio of PAI-1/t-PA and the ratio of PCA/TM.Conclusions TNF-a may induce functional changes of HBMVECs, and result in dysfunction of HBMVECs, which might play a critical role in immunopathology of infectious central nervous system diseases.
|
PDF Full Text Request