title: NADPH oxidase mediates TNF-α-evoked in vitro brain barrier dysfunction: roles of apoptosis and time.
abstract: The pro-inflammatory cytokine TNF-α severely perturbs the integrity of the blood-brain barrier (BBB). This study explored the specific roles of NADPH oxidase and associated downstream effectors by using human brain microvascular endothelial cells (HBMECs) and human astrocytes (HAs), the key components of BBB, alone or in co-cultures to mimic human BBB. Exposure to TNF-α (6h) impaired BBB integrity as evidenced by marked decreases in transendothelial electrical resistance and concurrent increases in paracellular flux which appeared to subside with time (24h). Increased barrier dysfunction concurred with increases in endothelial NADPH oxidase activity, O2(-) production, actin stress fibre formation, MMP-2/9 activities and concomitant decreases in antioxidant (CuZn-SOD and catalase) and tight junction (claudin-5 and occludin) protein expressions. Conversely, TNF-α did not affect astrocytic MMP activities and antioxidant enzyme expressions. Unlike BBB damage, rates of HBMEC and HA apoptosis increased by time. Suppression of NADPH oxidase by apocynin or diphenyleneiodonium prevented TNF-α-evoked morphological changes and apoptosis, attenuated endothelial MMP activity and helped retain usual tight junction protein expression and barrier function. In conclusion, HBMECs constitute the main source of oxidative stress and basement-membrane degrading endopeptidases in inflammatory conditions associated with excessive release of TNF-α where targeting NADPH oxidase may prove extremely beneficial in maintaining proper barrier activity through prevention of cytoskeletal and tight junction reorganisations.
authors.affiliation: Stroke, Division of Clinical Neuroscience, University of Nottingham, UK.
authors.forename: Zuraidah
authors.initials: Z
authors.lastname: Abdullah
authors.affiliation: Stroke, Division of Clinical Neuroscience, University of Nottingham, UK. Electronic address: ulvi.bayraktutan@nottingham.ac.uk.
authors.forename: Ulvi
authors.initials: U
authors.lastname: Bayraktutan
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001253:Astrocytes / Q000187:drug effects*; D002374:Catalase / Q000378:metabolism; D018920:Coculture Techniques; D045304:Cytochromes c / Q000378:metabolism; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D000098:Acetophenones; D004791:Enzyme Inhibitors; D009861:Onium Compounds; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; C007517:diphenyleneiodonium; D045304:Cytochromes c; C056165:acetovanillone; D002374:Catalase; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D010100:Oxygen
keywords: Blood–brain barrier; Cytoskeleton; MMP; NADPH oxidase; TNF-α