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혈관평활근세포에서 산화에너지대사 억제에 의한 아밀로이드전구단백질 대사의 변화
한문구,최웅,김헌식,안희열,한설희 대한치매학회 2002 Dementia and Neurocognitive Disorders Vol.1 No.1
Background: A reduction in the activity of cytochrome c oxidase(COX) has been recently identified in mitochondria from platelets and postmortem brain tissue of AD patients Sodium azide (NaN₃). a COX inhibitor, is an effective chemical agent producing energy shortage and oxidative stress both in vitro and in vivo system Furthermore it has been suggested that vascular compromise could be either directly involved AD pathogenesis or indirectly associated with triggering pathogenetic events leading to AD This study was performed to investigate amyloid precursor protein (APP) metabolism by inhibition of mitochondrial energy metabolism in cultured vascular smooth muslce cells (VSMCs) Materials and Methods: VSMCs isolated from the aorta of seven weeks old Spraque-Dawley rat were treated with NaN₃in a low concentration (100-500μM) or in a high concentration (1-100mM) Cellular proliferation and viability were determined by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenly)-2H-tetrazolium) assay Cellular APP was detected with N-terminal specific antibody 22C11. Celldeath was determined by observation of morphology and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling stain (TUNEL) We used ginkgo biloba extract(EGb761) and melatonin as anti-oxidants to investigate the mechanism of latered APP metabolism Results: The viability of VSMCs was increased after treatment with 1 mM and 10mM NaN₃(p<0.05) unitl 3 hr and then dimnished Many TUNEL positive cells were found in 10mM and 100mM treatment group. but were not apoptotic in nature 22C11 immunoreactivity was not changed at 3 hr, 6hr, 12hr Anti-oxidants reduced cellular proliferation (p<0.05). but did not block TUNEL positivities and did not influence the 22C11 immunoreactivity In a low concentration NaN₃ treatment group the viability of VSMCs was increased concentration dependently(p<0.05) Immunoblot with 22C11 showed the concentration dependent decrease at 145 kDa, 125 kDa. and high molecular weight range (>160kDa) TUNEL staining showed DNA fragmentations and condensations of nuclear chromatin suggesting apoptosis After treatment with anti-oxidants, the cellular proliferation was more decreased (p<0.05), and TUNEL positive cell deaths were blocked Immunoreactivities of 125 kDa (immature APP). 145 kDa (mature APP). and higher molecular weight bands were recovered below 400μM of NaN₃ Immunoreactivity of 145 kDa was recovered in 100 μM NaN₃ treated group Conclusions: The presumed mechanism of low concentration COX inhibitor is the overproduction of reactive oxygen species resulting from a depression of the mitochondiral electron transport chain. whereas potential consequence of high concentration COX inhibitor might be related to decreassion of ATP synthesis and bioenergetic impairment Reactive oxygen radicals in response to low concentration COX inhibitor alter the processing of APP in VSMCs This investigation demonstrated analtered APP metabolism as a peripheral marker of AD Therefore VSMCs treated with low concentration COX inhibitor could be concsidered as a novel in vitro model of AD.
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