title: A new constitutively active mutant of AMP-activated protein kinase inhibits anoxia-induced apoptosis of vascular endothelial cell.
abstract: The inhibition of apoptotic changes in vascular endothelial cells is important for preventing vascular damage from hypoxia. AMP-activated protein kinase (AMPK) has recently been identified as playing a role in vascular protection. Although the chemical reagent 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) has been used to stimulate AMPK activity, AICAR has been associated with several nonspecific reactions. We therefore constructed a new constitutively active mutant of AMPK alpha 1 (NcaAMPK), which lacks the autoinhibitory domain in AMPK alpha 1 and in which threonine 172 has been replaced with aspartate. We investigated whether NcaAMPK has an anti-apoptotic effect in vascular endothelial cells under anoxic conditions. NcaAMPK, or green fluorescent protein (GFP) as a control, was overexpressed in human umbilical vein endothelial cells (HUVECs). After HUVECs were incubated for 40 h under normoxic or anoxic conditions, we examined cell viability, caspase 3/7 activity, and expression and phosphorylation levels of apoptosis-related proteins. Cell viabilities under anoxic conditions were improved in NcaAMPK-overexpressing cells. Anoxia increased caspase 3/7 activity, but NcaAMPK reduced this increase significantly. NcaAMPK overexpression increased protein kinase B/Akt Ser473 and endothelial nitric oxide synthase Ser1177 phosphorylation, but pretreatment with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) did not decrease the viability of NcaAMPK-overexpressing HUVECs. Furthermore, co-expression of a dominant-negative Akt reduced the improvement in cell viability and the suppression of poly (ADP-ribose) polymerase cleavage by NcaAMPK under anoxic conditions. In conclusion, NcaAMPK inhibited anoxia-induced apoptosis in vascular endothelial cells through Akt activation, suggesting that activation of AMPK might protect against ischemic vascular injury.
authors.affiliation: Department of Molecular Research for Vascular Diseases, Graduate School of Medicine, University of Tokyo, Tokyo, Japan. dskngtendo0504-tky@umin.ac.jp
authors.forename: Daisuke
authors.initials: D
authors.lastname: Nagata
authors.forename: Arihiro
authors.initials: A
authors.lastname: Kiyosue
authors.forename: Masao
authors.initials: M
authors.lastname: Takahashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Satonaka
authors.forename: Kimie
authors.initials: K
authors.lastname: Tanaka
authors.forename: Masataka
authors.initials: M
authors.lastname: Sata
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
authors.forename: Ryozo
authors.initials: R
authors.lastname: Nagai
authors.forename: Yasunobu
authors.initials: Y
authors.lastname: Hirata
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D003432:Cross-Linking Reagents; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000494:pharmacology*; D017931:DNA Primers; D042783:Endothelial Cells / Q000187:drug effects*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D000860:Hypoxia / Q000473:pathology*; D009154:Mutation / Q000502:physiology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors; D051058:Oncogene Protein v-akt / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D013778:Tetrazolium Salts
chemical_list: C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D003432:Cross-Linking Reagents; D017931:DNA Primers; D004791:Enzyme Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D012265:Ribonucleotides; D013778:Tetrazolium Salts; D000620:Aminoimidazole Carboxamide; D052250:Nitric Oxide Synthase Type III; D011065:Poly(ADP-ribose) Polymerases; D051058:Oncogene Protein v-akt; D017868:Cyclic AMP-Dependent Protein Kinases; D053148:Caspase 3; D053179:Caspase 7; C031143:AICA ribonucleotide; D019331:NG-Nitroarginine Methyl Ester