Exogenous Nitric Oxide-Induced Apoptosis in Cultured Rat Vascular Smooth Muscle Cells

Chae, In Ho; Kim, Hyo Soo; Sohn, Dae Won; Oh, Byung Hee; Lee, Myoung Mook; Park, Young Bae; Choi, Yun Shik; Seo, Jung Don; Lee, Young Woo

Abstract

BACKGROUND
Atherosclerosis is the most important disease that may cause ischemic syndrome in many organs including heart. It is supposed that apoptosis of vascular smooth muscle cells(VSMCs) is closely related to the progression and rupture of atheromatous plaque. Recent studies have documented evidence for elevated level of nitric oxide(NO) within advanced human atheroma and evidence of regression of atheroma by NO. So this study is designed to evaluate whether exogenous NO from NO donors can induce apoptosis of cultured rat VSMCs and which proapoptotic gene(s) is involved in this type of apoptosis.
METHODS
Rat VSMCs were cultured and used for experiment at passage 5 through 7. For NO donor, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine(SNAP) of 0.5, 1, 2, 4mM were exposed to subconfluent VSMCs. The cells were harvested at 6, 12, 24, 48, 72hours after exposure of NO donors. Apoptosis was to be identified by 4, 6-diamidino-2-phenylindole dihydrochloride(DAPI) staining of nuclei and in-situ nick end labeling(TUNEL). The amount of fragmented DNA was analyzed semiquantitatively by diphenylamine(DPA) assay. Immunocytochemical(ICC) staining and western bolt analyses were designed to detect apoptosisrelated gene products, such as Bax-a, Fas and Bcl-2.
RESULTS
1) Decreased mitotic activity was shown after 12 hours exposure of exogenous NO donors, and condensation and margination of chromatin was identified agter 24 hours exposure, by DAPI staining. 2) Percent DNA fragmentation assessed by DPA method was 0,2,9,48,45% at 0,6,12,24,48 hours after exposure of 2mM of NO donors respectively. 3) The expression of Bax-a and Bcl-2 proteins was demonstrated in apoptotic cells by ICC staining. 4) The expression of Bax-a protein in cells under 24 hours exposure of NO donors was elevated by more than 18% of control level on densitometric analysis of western blot. The level of Bcl-2 was suppressed by 26% of control. So, Bax-a/Bcl-2 ratio in cells under exposure of NO donors was elevated to 2.0 from 1.2 of control level.
CONCLUSIONS
Exogenous NO from NO donors can induce apoptosis of cultured rat VSMCs, and it is considered that bax-a and bcl-2 genes are involved in this type of apoptosis.