Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-βI and prooxidant enzyme {NADPH} oxidase

摘要

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-α, PKC-β or PKC-βI 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-βI 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-βI and {NADPH} oxidase.