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Title: The role of calcium in blood-brain barrier damage during oxygen-glucose deprivation and reperfusion : implication of protein kinase C, NADPH oxidase, plasminogen activators and matrix metalloproteinases
Author: Rakkar, Kamini
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2016
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Abstract:
Background and purpose - The blood-brain barrier is an extremely important structure which serves to maintain brain homeostasis. Any alteration to its permeability can therefore have damaging consequences to the central nervous system. Ischaemic stroke is characterised by a loss of blood supply to the brain which results in a cascade of destructive mechanisms leading to blood-brain barrier damage and cell death. Reperfusion of the occluded vessel and downstream tissue, either endogenously through the plasminogen-plasmin system or through medicated intervention with alteplase, can add to this damage by further increasing oxidative stress and blood-brain barrier permeability. Furthermore, the plasminogen-plasmin system can activate downstream matrix metalloproteinases, which can degrade the extracellular matrix leading to increased barrier degradation. One of the initial damaging mechanisms is an increase in intracellular calcium in endothelial cells. It can activate protein kinase C and induce reactive oxygen species generation and apoptosis, promoting blood-brain barrier permeability and leading to the formation of brain oedema. Brain oedema and haemorrhagic transformation are the leading cause of death after an ischaemic stroke and can be the results of reperfusion injury. As impairment of the blood-brain barrier accounts for much of the oedema formation and reperfusion injury, the role of calcium and its relationship with protein kinase C, pro-oxidant nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, plasminogen activators and matrix metalloproteinase-2 and matrix metalloproteinase-9 was explored. Methods - Human brain microvascular endothelial cells were exposed to oxygen and glucose deprivation alone or followed by reperfusion. NADPH oxidase, matrix metalloproteinase-2, matrix metalloproteinase-9, plasminogen activator and caspase-3/7 activities and intracellular calcium, and superoxide anion levels were measured. Stress fibre formation and apoptotic nuclei were also viewed. Blood-brain barrier permeability was also measured using an in vitro model comprising human brain microvascular endothelial cells and human astrocytes and was studied by transendothelial electrical resistance and paracellular flux of Evan's blue tagged albumin and sodium fluorescein. Protein levels of the tight junction proteins claudin-5, occludin and zonula occluden-1, plasminogen activators and subunits of NADPH oxidase, p22-phox and gp91-phox were also measured under the above conditions. Similar studies were conducted in the absence or presence of inhibitors for urokinase plasminogen activator (amiloride), NADPH oxidase (apocynin), intracellular calcium (1,2-Bis (2- aminophenoxy) ethane-N.N.N'.N'-tetraacetic acid tetrakis-acetoxymethyl ester, [BAPTA-AM]), protein kinase C (bisindolylmaleimide), matrix metalloproteinase-2 (MMP-2 Inhibitor III) and protein kinase C-alpha (RO-32- 0432). Endothelial cells with protein kinase C-alpha knockdown, achieved by small interfering RNA, were also exposed to oxygen and glucose deprivation alone or followed by reperfusion and the above parameters measured. In a subset of experiments endothelial cells were exposed to phorbol myristate acetate (a protein kinase C activator), pyrogallol (a superoxide generator) or transfected with p22-phox (a N ADPH oxidase subunit) cDNA to directly and independently observe the roles of protein kinase C, superoxide anion and NADPH oxidase respectively, in blood-brain barrier permeability. Results - Oxygen and glucose deprivation alone, and followed by 20 hours of reperfusion, significantly increased protein kinase C, NADPH oxidase, plasminogen activator and caspase-3/7 activities, intracellular calcium, and superoxide anion levels, stress fibre formation, and blood-brain barrier permeability. Matrix metalloproteinase-2 activity was increased during reperfusion only and matrix metalloproteinase-9 could not be detected. Claudin- 5, occludin and zonula occluden-1 protein levels were significantly decreased and plasminogen activator protein levels were increased in the above conditions. Treatment with the above inhibitors prevented or attenuated the changes in parameters observed. Exposure to phorbol myristate acetate or pyrogallol increased blood-brain barrier permeability. Transfection with p22-phox cDNA increased plasminogen activator protein levels and activities and blood-brain barrier permeability. Conclusions - This study reveals increases in intracellular calcium levels and protein kinase C, NADPH oxidase and plasminogen activator activities are pivotal mechanisms in oxygen and glucose deprivation and reperfusion- mediated impairment of the blood-brain barrier. Calcium may mediate blood- brain barrier dysfunction through a novel protein kinase C-NADPH oxidase- plasminogen activator pathway and selective targeting of any component may protect blood-brain barrier integrity and function by concomitantly attenuating eytoskeletal reorganisation, oxidative stress, basement membrane degradation and apoptosis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.716473  DOI: Not available
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