title: Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ßI and prooxidant enzyme NADPH oxidase.
abstract: Blood-brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2 (•-) generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2 (•-) by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2 (•-) production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase.
authors.affiliation: Stroke, Division of Clinical Neuroscience, School of Medicine, University of Nottingham, UK.
authors.forename: Beili
authors.initials: B
authors.lastname: Shao
authors.affiliation: Stroke, Division of Clinical Neuroscience, School of Medicine, University of Nottingham, UK.
authors.forename: Ulvi
authors.initials: U
authors.lastname: Bayraktutan
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001812:Blood-Brain Barrier / Q000187:drug effects; D001921:Brain / Q000166:cytology; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006943:Hyperglycemia / Q000401:mortality / Q000473:pathology; D055806:Microvessels / Q000166:cytology*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D064546:Protein Kinase C beta / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D034741:RNA, Small Interfering; D051028:bcl-2-Associated X Protein; D013481:Superoxides; D019255:NADPH Oxidases; C579054:PRKCB protein, human; D064546:Protein Kinase C beta; D053148:Caspase 3; D053179:Caspase 7; D013755:Tetradecanoylphorbol Acetate
keywords: Apoptosis; Endothelial cell; Hyperglycaemia; NADPH oxidase; Protein kinase C