Epothilone B Inhibits Neointimal Formation after Rat Carotid Injury through the Regulation of Cell Cycle-Related Proteins

Abstract

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial walls is an important pathogenetic factor of vascular disorders such as atherosclerosis and restenosis after angioplasty. Epothilone B, a novel potential antitumor compound, has a potent effect on preventing postangioplasty restenosis. Therefore, we established an in vivo rat carotid injury model and examined the potential effects of epothilone B on cardiovascular disease. We found that epothilone B potently prevented neointimal formation and in vivo VSMCs proliferation. In addition, we also showed that epothilone B significantly inhibited 5% fetal bovine serum (FBS)-and 50 ng/ml plateletderived growth factor (PDGF)-BB-induced proliferation and cell cycle progression in rat aortic VSMCs. Furthermore, FBS and PDGF-BB induced the activations of extracellular signal-regulated kinases 1 and 2, Akt, phospholipase C ␥ 1, and PDGFreceptor ␤ chain tyrosine kinase were not changed by epothilone B. However, epothilone B treatment caused a significant decrease in the level of cyclin-dependent protein kinase (CDK) 2, whereas it caused no change in the levels of cyclin E and down-regulated the phosphorylation of retinoblastoma, which plays a critical role in cell cycle regulation. Furthermore, levels of p27, an inhibitor of cyclin E/CDK2 complex, were significantly increased in VSMCs treated with epothilone B, indicating that this might be a major molecular mechanism for the inhibitory effects of epothilone B on the proliferation and cell cycle of VSMCs. These findings suggest that epothilone B can inhibit neointimal formation via the cell cycle arrest by the regulation of the cell cycle-related proteins in VSMCs.Proliferation and migration of vascular smooth muscle cells (VSMCs) play a pivotal role in the development of restenosis and in the progression of atherosclerosis (Ross, 1993). Arterial injury results in the migration of VSMCs into the intimal layer of the arterial wall, where they proliferate and synthesize extracellular matrix components. Many growth factors induce the proliferation of VSMCs in vitro and in vivo. Among them, platelet-derived growth factor (PDGF) and basic fibroblast growth factor are important regulators of VSMC behavior through their well defined actions as potent chemoattractants and strong mitogens. Administration of these growth factors enhances intimal thickening after angioplasty in rat, whereas injection of antibodies or use of antisense technology to block signal transduction by either of these growth factors potently inhibits postinjury intimal hyperplasia in rat and restenosis in pig, suggesting that VSMC growth plays an important role in the pathogenesis of these models.Arterial injury during percutaneous transluminal coronary angioplasty induces multiple signaling pathways that activate VSMC migration and proliferation. Immediately after injury, VSMCs leave their quiescent state and enter the cell

DNA Enzyme ED5 Depletes Egr-1 and Inhibits Neointimal Hyperplasia in Rats

“…PDGF-BB initiates a multitude of biological effects through the activation of intracellular signal transduction pathways that contribute to VSMC proliferation, migration, and collagen synthesis [17,18]. Animal experiments demonstrated that increasing levels of PDGF after arterial injury correlated with neointimal cellular proliferation [19,20,21]. Targeting PDGF with an anti-PDGF antibody [22], targeting the expression of the receptor with antisense oligonucleotides [23], or using inhibitors of PDGF signal transduction [24] have all been shown to attenuate neointima development.…”

Inhibitory effects of OD 78 [3-(4-bromo-phenoxy)-4,5-dihydroxybenzoic acid-methyl ester] on the proliferation and migration of TNF-α-induced rat aortic smooth muscle cells

“…RASMCs were isolated by enzymatic dispersion according to a previously reported method (Lim et al, 2007), with some modifications. Cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS, 100 IU/mL penicillin, 100 µg/ mL streptomycin, 8 mM HEPES and 2 mM L-glutamine at 37 o C in a humidified atmosphere of 95% air and 5% CO 2 .…”

“…DNA synthesis was determined by a [ 3 H]-thymidine incorporation assay as previously described (Lim et al, 2007). In brief, RASMCs were seeded in 24-well plates.…”

“…The cell cycle was determined as previously described (Lim et al, 2007). In brief, RASMCs in a 60-mm 2 cell culture dish were incubated in DMEM medium without serum (Gibco-BRL) in the presence or absence of various concentrations of OD 78 (1, 2, and 4 µM) for 24 h. RASMCs were then stimulated with 100 ng/mL TNF-α for another 24 h. The cells were trypsinized and centrifuged at 1,500 × g for 7 min.…”

“…Immunoblotting was performed as previously described (Lim et al, 2007). Briefly, RASMCs were seeded in 6-well culture plates at 4 × 10 4 cells/mL and cultured in DMEM with 10% FBS at 37 o C for 24 h. When cells reached 70% confluence, the medium was replaced with serum-free medium, and the cells were incubated for 24 h. The medium was then changed to serum-free medium containing various OD 78 concentrations, and the cells were incubated for 24 h.…”