Transfection of inducible nitric oxide synthase gene causes apoptosis in vascular smooth muscle cells.
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.


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.
Iwashina
M
M
Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
Shichiri
M
M
Marumo
F
F
Hirata
Y
Y
D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D009569:Nitric Oxide / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D051381:Rats; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000032:analysis; D019323:omega-N-Methylarginine / Q000494:pharmacology
D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat