Adenovirus-mediated expression of p35 prevents hypoxia/reoxygenation injury by reducing reactive oxygen species and caspase activity.
OBJECTIVE
This study aimed to examine the effects of adenovirus-mediated expression of p35, a baculovirus gene, on apoptosis induced by hypoxia/reoxygenation (H/R) in cardiomyocytes.


METHODS
Neonatal rat cardiomyocytes were infected with recombinant adenoviral vectors expressing p35 (Ad2/CMVp35) or no transgene (Ad2/CMVEV) and were then subjected to H/R. Separate groups of non-infected cardiomyocytes were treated with pharmacological caspase inhibitors or antioxidants. Cell viability, apoptosis, caspase activity, and cellular reactive oxygen species (ROS) were measured using various assays.


RESULTS
H/R decreased cell viability and increased cellular ROS levels, caspase activity, and cell apoptosis. Infection with Ad2/CMVp35 effectively inhibited the increase in cellular ROS levels, the activities of caspases 3 and 8, apoptosis, and cell death following H/R, whereas Ad2/CMVEV had no effect. Despite its ability to abolish the increase in caspase activity and partially inhibit apoptosis, the pan-caspase inhibitor ZVAD-fmk (100 microM) failed to significantly reduce cell death induced by H/R. N-acetyl-L-cysteine, an antioxidant, completely inhibited H/R-induced increase in cellular ROS levels, but reduced apoptosis and cell death by 30% only.


CONCLUSIONS
Adenovirus-mediated expression of p35 effectively inhibits H/R-induced cardiomyocyte apoptosis by reducing cellular ROS levels and inhibiting caspase activity.
Genzyme Corporation, 31 New York Avenue, Framingham, MA 01701, USA.
Taro
T
Date
Adam J
AJ
Belanger
Seibu
S
Mochizuki
Jennifer A
JA
Sullivan
Louis X
LX
Liu
Abraham
A
Scaria
Seng H
SH
Cheng
Richard J
RJ
Gregory
Canwen
C
Jiang
D000256:Adenoviridae / Q000235:genetics; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D018929:Cell Culture Techniques; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D015687:Cell Hypoxia / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D014764:Viral Proteins
D000590:Amino Acid Chloromethyl Ketones; D000975:Antioxidants; D001425:Bacterial Outer Membrane Proteins; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D008074:Lipoproteins; D017382:Reactive Oxygen Species; D014764:Viral Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C492234:p35 protein, Baculovirus; D013482:Superoxide Dismutase; D020169:Caspases