Nitric oxide-induced apoptosis is mediated by Bax/Bcl-2 gene expression, transition of cytochrome c, and activation of caspase-3 in rat vascular smooth muscle cells

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Abstract

Background: In contrast to the anti-apoptotic action of nitric oxide (NO) on endothelial cells, NO exerts a pro-apoptotic effect on vascular smooth muscle cells (VSMCs). This study was designed to elucidate the mechanism underlying NO-induced apoptosis in rat VSMCs. Methods and results: (1) Using the terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay and fluorescence activated cell sorter (FACS) analyses, apoptosis of rat VSMCs were confirmed after exposure to sodium nitroprusside (SNP) (0.5 to 4 mmol/l), an exogenous NO donor. The effects of SNP were blocked by hemoglobin. (2) A universal caspase inhibitor, z-VAD-fmk, dose-dependently inhibited NO-induced apoptosis. VSMCs degraded Ac-DEVD-pNA rather than Ac-WHED-pNA after exposure to SNP, which suggested that the activation of caspase 3 rather than caspase 1 was involved in the process. Immunoblot analysis confirmed the activation of caspase-3. (3) Exposure to SNP induced the release of cytochrome c from the mitochondria to the cytosol, which was detected by immunoblot analysis of mitochondrial and cytosol fractions. (4) SNP exposure increased the ratio of Bax/Bcl-2 protein expression twofold by immunoblot analysis. Conclusions: The mechanism of NO-induced apoptosis in rat VSMCs involves an increase in the ratio of Bax/Bcl-2 gene expression, which leads to the release of cytochrome c from the mitochondria to the cytosol, finally activating caspase-3 and resultant apoptosis.

Introduction

Apoptosis has been identified as an important process in various vascular pathophysiologies, such as, coronary atherosclerosis and plaque rupture, as well as remodelling and restenosis after angioplasty [1], [2], [3]. Among the various vasoactive substances that modulate such conditions, nitric oxide (NO) has been known to be one of the key molecules in terms of the control of vascular tone and vascular cell proliferation. Recently, NO has emerged as a bifunctional regulator of apoptosis. On one hand, it has been shown to exert an anti-apoptotic action in vascular endothelial cells [4], hepatocytes [5], eosinophils [6], and splenocytes [7]. On the other, it has also been shown that NO induces apoptosis in vascular smooth muscle cells (VSMCs) [8], macrophages [9], pancreatic islet cells [10], thymocytes [11], and neurons [12].

Although the mechanism behind its action has been investigated extensively, the downstream mediators of NO-induced apoptosis in VSMCs are not well known [1]. In some studies, the cGMP-dependent pathway was targeted as the key mechanism behind NO-induced apoptosis of VSMCs [13], [14], but in others, investigators have shown that NO-induced apoptosis works through a cGMP-independent pathways [15], [16]. Recently, it was shown in pulmonary vascular smooth muscle cells that NO may induce apoptosis by activating K+ channels, resulting in K+ efflux and cytosolic K+ loss [17]. Still others have pointed to the importance of the protein kinase C signaling pathway and the regulation of NF-kB binding activity in NO-induced apoptosis [18]. Among the apoptosis-related genes, Fas antigen [19] and CPP32 protease [20], [21] have been reported to be involved in the NO-induced apoptosis of VSMCs. In short, previous studies have only been able to show fragmented data concerning the mechanism, and the integrated picture of NO-induced apoptosis in VSMCs has not yet been elucidated.

In this study, we investigated the mechanism of NO-induced apoptosis in VSMCs and found that NO promotes cytochrome c release from mitochondria into the cytoplasm by modulating Bax/Bcl-2 gene expression, thereby activating caspase-3 which induces apoptosis of cultured rat VSMCs.

Section snippets

Cell culture

VSMCs were isolated enzymatically from thoracic aortas of male Sprague–Dawley rats (body weight 150–180 g), and cultured in DMEM/F12 supplemented with 10% heat-inactivated fetal bovine serum (GIBCO-BRL), and antibiotics (penicillin G sodium 100 units/ml, streptomycin sulfate 100 μg/ml, and amphotericin B 0.25 μg/ml). Cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C and were used between the fifth and sixth passage.

Twenty-four hours before exposure to sodium nitroprusside

Caspase and NO-induced apoptosis

Apoptosis of VSMCs exposed to SNP was identified by immunofluorescent microscopy, electron microscopy, and FACS analysis (Fig. 1A,B). When hemoglobin, a scavenger of nitric oxide, was added with SNP, apoptosis was prevented (Fig. 1C), which suggested that SNP-induced apoptosis of VSMCs was indeed due to nitric oxide. In addition, the number of viable cells decreased as the concentration of SNP and the exposure time was increased (Fig. 1D,E).

To see whether caspase is involved in NO-induced

Discussion

Since the downstream cell signals of NO-induced apoptosis in VSMCs are not fully understood, we investigated possible mechanisms of NO-induced apoptosis in cultured rat aortic VSMCs. In the present study, we found that NO modulates the balance of bax and bcl-2 gene expression, which induces the release of cytochrome c from the mitochondria to the cytoplasm. The increase of the cytosoplasmic cytochrome c, in turn, activates caspase-3 rather than caspase-1, which finally results in the apoptosis

Acknowledgments

This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (02-PJ10-PG8-EC01-0026 [Dr. Hyo-Soo Kim]) and a grant from the Korea Science and Engineering Foundation (KOSEF) to Dr. Hyo-Soo Kim through the aging and apoptosis research center at Seoul National University, Republic of Korea.

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    In-Ho Chae and Kyung-Woo Park contributed equally to the study.

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