Trapidil inhibits platelet-derived growth factor-induced migration via protein kinase A and RhoA/Rho-associated kinase in rat vascular smooth muscle cells
Introduction
Restenosis is the major limitation for the long-term efficacy of angioplasty. Injury to the arterial wall induces endothelial denudation, vascular smooth muscle cell (VSMC) migration and proliferation, resulting in the formation of neointimal hyperplasia (Schwartz et al., 1992). Among several growth factors capable of stimulating VSMC migration and proliferation, platelet-derived growth factor (PDGF) plays a critical role in the development of restenosis (Ferns et al., 1991). Trapidil (triazolopyrimidine), an anti-platelet drug with broad biological activities, has been demonstrated to reduce restenosis after angioplasty in animals as well as in humans (Maresta et al., 1994, Ohnishi et al., 1982). It inhibits PDGF-stimulated proliferation of VSMCs both in vivo and in vitro (Ohnishi et al., 1982, Hoshiya and Awazu, 1998). Its mechanism of action has previously been considered to be the competitive blockade at the receptor level (Gesualdo et al., 1994). A study from our laboratory, however, showed that trapidil did not affect tyrosine kinase activity of PDGF β-receptor in VSMCs (Hoshiya and Awazu, 1998). Trapidil's mechanism of action was shown to be the inhibition of extracellular signal-regulated kinase (ERK), a key enzyme in a wide range of cellular processes including proliferation. Stimulation of Raf-1, an upstream activator of ERK, by PDGF was also attenuated by trapidil. These actions of trapidil were accompanied by an increase in cellular generation of cAMP. In view of the evidences that cAMP/protein kinase A (PKA) inhibits Raf-1 (Graves et al., 1993), trapidil may antagonize mitogenic action of PDGF through cAMP/PKA.
While VSMC proliferation is an important event in restenosis, VSMC migration precedes proliferation. RhoA, a member of Rho family small GTPases, has been shown to be important in mediating migration in various cell types (Takai et al., 1995). Upon stimulation, RhoA translocates from the cytosolic to the membrane fraction and stimulates downstream targets including Rho-associated kinase (ROCK). Recent evidence suggests that RhoA/ROCK plays an important role in neointimal formation after vascular injury (Sawada et al., 2000, Shibata et al., 2001).
In the present study, we examined whether trapidil inhibits PDGF-stimulated VSMC migration. Since trapidil's antiproliferative effect is mediated by cAMP/PKA, we compared the effects of trapidil with an adenylyl cyclase activator forskolin. We also examined the effects of trapidil and forskolin on PDGF-induced RhoA activation and proliferation, events important in neointimal formation. The role of ROCK in PDGF-stimulated migration and proliferation was also examined using a specific ROCK inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632). The results will allow us to identify a therapeutic target for the prevention of restenosis.
Section snippets
Materials
Human recombinant PDGF-BB and bovine serum albumin were purchased from Sigma (St. Louis, MO). 4-(2-Aminoethyl)-benzenesulphonyl fluoride (AEBSF) was from Molecular Probes, Inc. (Eugene, OR). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum, penicillin, streptomycin, trypsin–ethylene diamine tetraacetic acid (EDTA) and Hank's balanced salt solution (HBSS) were from Gibco Laboratories (Grand Island, NY). Trapidil was a gift from Mochida Pharmaceutical Co. (Tokyo, Japan). Rabbit
Trapidil or forskolin inhibits PDGF-induced migration
Trapidil 400 μg/ml and forskolin 10 μM inhibited PDGF-stimulated VSMC migration to the same extent (20 ± 2% and 20 ± 2% of PDGF alone, respectively, Fig. 1). A specific ROCK inhibitor Y-27632 also inhibited PDGF-induced migration (28 ± 3% of PDGF alone), which is in agreement with previous studies (Sawada et al., 2000). In the presence of a PKA inhibitor H89, the inhibition by forskolin was alleviated to 74 ± 7% of PDGF alone, but that of trapidil was reduced to only 52 ± 4% of PDGF alone (P < 0.05 vs.
Discussion
The present study demonstrates that trapidil may inhibit PDGF-induced VSMC migration by activating PKA and inhibiting RhoA/ROCK. Thus, trapidil 400 μg/ml and an adenylyl cyclase activator forskolin 10 μM inhibited VSMC migration to the same extent. This dose of forskolin is known to exert a maximal effect on PDGF-induced VSMC migration (Yasunari et al., 1997, Sun et al., 2002). In the presence of a PKA inhibitor H89, trapidil inhibited VSMC migration to a greater degree than forskolin. In the
Acknowledgements
This study was supported by grants from the Ministry of Education, Science and Culture, Japan (070362, 10670757, 12770610, 13770404, 14570772), Pharmacia-Upjohn Fund for Growth and Development Research.
References (28)
- et al.
Regulation of intracellular Ca2+ levels in cultured vascular smooth muscle cells: reduction of Ca2+ by atriopeptin and 8-bromo-cyclic GMP is mediated by cyclic GMP-dependent protein kinase
J. Biol. Chem.
(1989) - et al.
Inhibition of PKA blocks fibroblast migration in response to growth factors
Exp. Cell Res.
(2001) - et al.
The major catalytic subunit isoforms of cAMP-dependent protein kinase have distinct biochemical properties in vitro and in vivo
J. Biol. Chem.
(1996) - et al.
Trapidil inhibits human mesangial cell proliferation: effect on PDGF β-receptor binding and expression
Kidney Int.
(1994) - et al.
Platelet-derived growth factor stimulates protein kinase A through a mitogen-activated protein kinase-dependent pathway in human arterial smooth muscle cells
J. Biol. Chem.
(1996) - et al.
Effect of trapidil on cardiovascular events in patients with coronary artery disease (results from the Japan Multicenter Investigation for Cardiovascular Diseases-Mochida [JMIC-M])
Am. J. Cardiol.
(2003) - et al.
Effects of trapidil on thromboxane A2-induced aggregation of platelets, ischemic changes in heart and biosynthesis of thromboxane A2
Prostaglandins Med.
(1981) - et al.
An evidence for ‘response to injury’ hypothesis
Life Sci.
(1982) - et al.
The restenosis paradigm revisited: an alternative proposal for cellular mechanisms
J. Am. Coll. Cardiol.
(1992) Cyclic nucleotide-dependent protein kinases
Pharmacol. Ther.
(1991)