From the Second Department of Internal Medicine, Tokyo Medical and Dental
University, Tokyo, Japan.
Correspondence to Yukio Hirata, MD, Second Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail mshichiri.med2{at}med.tmd.ac.jp
Background—Excess production
of nitric oxide (NO) by inducible NO synthase (iNOS) has been
implicated in a variety of physiological processes
including vascular remodeling. To elucidate whether
endogenous NO generated by iNOS is involved in the
programmed cell death (apoptosis) of the vasculature, iNOS
cDNA– expressing construct was transfected into rat and human vascular
smooth muscle cells (VSMCs) by lipofection.
Methods and Results—VSMCs transiently transfected with iNOS cDNA
functionally expressed 130 kd iNOS protein with full catalytic activity
to generate massive NO in proportion to the doses of cDNA used; its
enzymatic activity as well as NO production was completely
blocked by an NOS inhibitor,
NG-monomethyl-L-arginine
(LNMMA). Overexpression of iNOS led to a marked inhibition of DNA
synthesis as well as induction of apoptosis in VSMCs. Evidence
for apoptotic cell death was provided by internucleosomal DNA
fragmentation by agarose gel electrophoresis, positive staining for
TdT-mediated dUTP biotin nick end-labeling, and appearance of
hypodiploid cells by flow cytometry analysis. Apoptosis
after transfection with iNOS cDNA was abrogated by LNMMA. Transfection
of iNOS cDNA caused accumulation of the tumor suppressor gene
p53 but not of bcl-2, which was also
blocked by LNMMA.
Conclusions—These results demonstrate that massive generation of
endogenous NO derived from iNOS overexpression leads to a
marked apoptosis in VSMCs, thus suggesting an important role of
NO as a proapoptotic factor for VSMCs in the process of
vascular remodeling.
© 1998 American Heart Association, Inc.
Basic Science Reports
Transfection of Inducible Nitric Oxide Synthase Gene Causes Apoptosis in Vascular Smooth Muscle Cells
Key Words: vasculature • apoptosis • endothelium-derived factors • atherosclerosis • genes
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