Troglitazone induces apoptosis via the p53 and Gadd45 pathway in vascular smooth muscle cells
Introduction
The proliferation and migration of vascular smooth muscle cells are critical factors in the progression of atherosclerosis and the development of restenosis after percutaneous transluminal angioplasty (Schwartz, 1997). Recently, vascular smooth muscle cell apoptosis has been identified in these lesions, suggesting that vascular smooth muscle cell apoptosis as well as vascular smooth muscle cell proliferation are involved in the etiology or pathogenesis of vascular remodeling Isner et al., 1995, Geng and Libby, 1995, Igase et al., 1999.
The thiazolidinediones, novel insulin-sensitizing agents, have been shown to significantly reduce hyperinsulinemia in insulin-resistant animals and humans Lee et al., 1994, Iwamoto et al., 1991, Nolan et al., 1994. Another functionally important property of thiazolidinediones is their ability to inhibit the growth and proliferation of vascular smooth muscle cells Dubey et al., 1993, Law et al., 1996, Goetze et al., 1999. One of the thiazolidinedione analogues, pioglitazone, inhibits the growth of vascular smooth muscle cells stimulated by insulin, epidermal growth factor, or serum (Dubey et al., 1993). Another analogue, troglitazone, suppresses the growth and migration of vascular smooth muscle cells induced by basic fibroblast growth factor or platelet-derived growth factor-BB (Law et al., 1996). Furthermore, thiazolidinediones have recently been reported to induce apoptosis in many types of cells, including endothelial cells (Bishop-Bailey and Hla, 1999), monocyte-derived macrophages (Chinetti et al., 1998), choriocarcinoma cells (Keelan et al., 1999), and gastric cancer cells (Takahashi et al., 1999). These effects have been thought to be produced primarily by the activation of peroxisome proliferator-activated receptor (PPAR)γ. PPAR belongs to a family of ligand-activated transcriptional factors and is composed of three members, PPARα, δ, and γ. Each PPAR member is specifically activated by each different ligand. The activation of PPAR is regulated by this restrictive ligand-receptor binding system, and therefore thiazolidinedione analogues are thought to be one of the specific ligands for PPARγ. However, recent reports have demonstrated that trogitazone has its own specific functions in addition to PPARγ activation Hattori et al., 1999, Ikeda and Watanabe, 1998, Wang et al., 1999, Sunaga et al., 1999, Ishizuka et al., 1998. Troglitazone, but not pioglitazone, has an inhibitory effect on platelet aggregation or cholesterol biosynthesis that is not due to PPARγ activation Wang et al., 1999, Ishizuka et al., 1998. Another troglitazone-specific action is an effect on the excitability, action potential configurations, and the membrane currents of ventricular myocytes (Ikeda and Watanabe, 1998).
In this study, we have evaluated the direct effects of a PPARγ activator, either pioglitazone or troglitazone, on vascular smooth muscle cell proliferation, and have also demonstrated that troglitazone-induced vascular smooth muscle cell apoptosis occurs through a pathway independent of PPARγ.
Section snippets
Materials
Troglitazone and pioglitazone were kindly provided by Sankyo Pharmaceutical (Tokyo, Japan) and Takeda Chemical Industries (Osaka, Japan), respectively. These agents were dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 0.1% in a culture medium. Caspase-3/7 inhibitor [Acetyl-l-Aspartyl-l-Glutamyl-l-Valyl-l-Aspart-1-al (Ac-DEVD-CHO)] was purchased from the Peptide Institute (Osaka, Japan). [α-32P]dCTP (110 TBq/mmol) was obtained from Amersham Pharmacia Biotech (Tokyo, Japan).
Direct effect of thiazolidinediones on vascular smooth muscle cell viability
After treatment with various concentrations of troglitazone or pioglitazone, the vascular smooth muscle cell viability was determined by a WST assay. Although 1 μM of troglitazone did not alter the cell viability, 5−30 μM troglitazone reduced the cell viability. In contrast, similar to the control, pioglitazone did not alter the viability in any concentration (Fig. 1A). Fig. 2 shows the typical cell morphology after incubation of two thiazolidinedione analogues for 48 h. Apparent morphological
Discussion
Both troglitazone and pioglitazone are active antidiabetic thiazolidinediones that increase tissue sensitivity to the actions of insulin though the activation of PPARγ Lee et al., 1994, Iwamoto et al., 1991, Nolan et al., 1994. Recent studies have also shown that PPARγ activation induces apoptosis in endothelial cells (Bishop-Bailey and Hla, 1999), monocyte-derived macrophages (Chinetti et al., 1998), JEG3 choriocarcinoma cells (Keelan et al., 1999), and gastric cancer cells (Takahashi et al.,
Acknowledgements
This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (No. 11838012), a Japan Heart Foundation Grant for research on Hypertension and Vascular Metabolism, and the Takeda Medical Foundation.
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