Biomedical subjects

Hiroki Aoki

Publications and source records attributed to Hiroki Aoki.

3 recordsLinked to original sources

PubMed2005

Gene silencing using adenoviral RNAi vector in vascular smooth muscle cells and cardiomyocytes.

RNA interference (RNAi) is a new and rapidly progressing technology for facilitating functional gene silencing. To perform highly efficient RNAi in cardiomyocytes, vascular smooth muscle cells, and vascular endothelial cells, which are known to have very low transfection efficiency, adenovirus-mediated RNAi was employed. The effects of RNAi on GAPDH transcripts were successfully reduced by nearly 90% in the primary cultured cells, indicating that adenovirus-mediated gene silencing is a promising technique for gene silencing in cardiovascular studies. This chapter describes general guidelines for selecting RNAi target sites, construction of a shuttle vector encoding short hairpin RNAi, and generation of recombinant adenovirus.

Adenoviridae↗

PubMed2003

Proteasome-dependent decrease in Akt by growth factors in vascular smooth muscle cells.

Akt is activated by growth factors to regulate various aspects of vascular smooth muscle cell function. Platelet-derived growth factor (PDGF) and insulin-like growth factor-1 activated Akt in vascular smooth muscle cells with a rapid reduction of total Akt protein that lasted for several hours. The downregulation of Akt required phosphatidylinositol 3-kinase activity, but not intrinsic Akt activity. The downregulation of Akt was abrogated by MG-132, a proteasome inhibitor, but not by inhibitors of calpain or cathepsins. Akt was found in ubiquitin immune complex after PDGF treatment. Proteasome-dependent degradation of Akt may provide a counter-regulatory mechanism against overactivation of Akt.

Animals↗

PubMed2002

Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes.

Although oxidative stress causes activation of c-Jun N-terminal kinase (JNK) and apoptosis in many cell types, how the JNK pathway is connected to the apoptosis pathway is unclear. The molecular mechanism of JNK-mediated apoptosis was investigated in adult rat cardiac myocytes in culture as a model system that is sensitive to oxidative stress. Oxidative stress caused JNK activation, cytochrome c release, and apoptosis without new protein synthesis. Oxidative stress-induced apoptosis was abrogated by dominant negative stress-activated protein kinase/extracellular signal-regulated kinase kinase-1 (SEK1)-mediated inhibition of the JNK pathway, whereas activation of the JNK pathway by constitutively active SEK1 was sufficient to cause apoptosis. Inhibition of caspase-9, an apical caspase in the mitochondrial apoptosis pathway, suppressed oxidative stress-induced apoptosis, whereas inhibition of caspase-8 had no effect, indicating that both the JNK pathway and the mitochondrial apoptosis machinery are central to oxidative stress-induced apoptosis. Both JNK and SEK1 localized on mitochondria where JNK was activated by oxidative stress. Furthermore, active JNK caused the release of apoptogenic factors such as cytochrome c from isolated mitochondria in a cell-free assay. These findings indicate that the JNK pathway is a direct activator of mitochondrial death machinery without other cellular components and provide a molecular linkage from oxidative stress to the mitochondrial apoptosis machinery.

Adenoviridae↗