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KMID : 0960920020010010013
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Dementia and Neurocognitive Disorders
2002 Volume.1 No. 1 p.13 ~ p.21
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Inhibition of Oxidative Energy Metabolism Alters Amyloid Precursor Protein Metabolism in Cultured Vascular Smooth Muscle Cells
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Han Moon-Ku
Choi Woong
Kim Hun-Sik
Ahn Hee-Yul
Han Seol-Heui
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Abstract
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Background: A reduction in the activity of cytochrome c oxidase(COX)has been recently idenified 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. Futhermore, it has been suggested that vascular compromise could be either directly involved in AD pathogenesis or indirectly associated with triggering pathogenetic events leading to AD. This study was preformed to investigate amyloid precursor protein(APP) metabolism by inhibition of mitochondrial energy metabolism in cultured vascular smooth muslce cells(VSMCs).
Materials and Methods: VSMCs is isolated from the aorta of seven weeks old Sprague-Dawley rat were treated with NaN3 in 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-sulfopheny)-2H-tetrazolium) assay. Cellular APP was detected with N-terminal specific antibody 22C11. Cell death was determined by observation of morphology and terminal deoxynucleotidyl transferasemediated dUTP-fluorescein nick end labelling 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 1mM and 10mM NaN3(p<0.05) until 3 hr and then diminished. Many TUNEL positive cells were found in 10mM and 100mM treatement group, but were not apoptotic in nature. 22C11 immunoreacivity was not changed at 3 hr, 6 hr, 12 hr, Anti-oxidants reduced cellular proliferation(p<0.05), but did not block TUNEL positiveities and did not influence the 22C11 immunoreacivity. 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(>160 kDa). TUNEL staining showed DNA fragmentations and condensation of nuclear chromatin suggesing 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 uin olny 100μM 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 depression of ATP synthesis and bioenergetic impairment. Reactivity oxygen radicals in response to low concentration COX inhibitor alters the processing of APP in VSMCs. This investigation demonstrated an altered APP metabolism as a peripheral marker of AD. Therefore VSMCs treated with low concentration COX inhibitor could be considered as a novel in vitro model of AD.
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KEYWORD
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Vasclar smooth muscle cells, Sodium azide, Reactive oxygen species, Amyloid precursor protein, Alzheimer’s disease
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