Suppression of arterial intimal hyperplasia by cilostamide, a cyclic nucleotide phosphodiesterase 3 inhibitor, in a rat balloon double-injury model

Abstract

The effects of cilostamide, a cyclic nucleotide phosphodiesterase 3 (PDE3) selective inhibitor, on vascular intimal hyperplasia were evaluated using a single-balloon injury model and a double-injury model in which the rat common carotid artery was subjected to a second injury at a site injured 14 days previously. In the double-injury model, the second balloon injury caused more severe intimal hyperplasia (intima/media (IM) ratio, 1.88+/-0.10) than in the single-injury model (1.09+/-0.08). Histopathological study revealed that vascular smooth muscle cells (VSMC) were the predominant cell-type in the affected neointimal area. Oral administration of cilostamide for 2 weeks after the second injury suppressed intimal hyperplasia in the double-injury model (30 mg kg(-1) bid, 83% inhibition in terms of the IM ratio, P<0.05; 100 mg kg(-1) bid, 69% inhibition, P<0.05). Similar effects were also observed in the single-injury model with oral administration of cilostamide for 2 weeks (100 mg kg(-1) bid, 36% inhibition, P<0.01). Cilostamide inhibited DNA synthesis of cultured VSMC stimulated by foetal calf serum or different kinds of growth factors, but did not affect their migration stimulated by platelet-derived growth factor (PDGF)-BB. Cilostamide significantly increased the cyclic AMP concentration of VSMC dose-dependently. These results indicate that cilostamide suppresses intimal hyperplasia both in the single- and double-injury models of rat, presumably by inhibiting proliferation rather than migration of VSMC. It is suggested that PDE3 inhibitors might find application in preventing intimal hyperplasia following angioplasty such as percutaneous transluminal coronary angioplasty (PTCA) or stent.

Figure 1

Microphotographs of cross sections from rat left carotid arteries after balloon injury, stained with Elastica van Gieson (left column), and immunohistochemically for PCNA (middle column) and HHF-35 (right column). Day 0 (A) is the day of balloon injury, Day 14 (B) and Day 28 (C) are days after the first injury. Day 14+1 (D), Day 14+3 (E), and Day14+14 (F) are days after the first injury + days after the second injury.

Figure 2

Time course of change in intimal hyperplasia in rat left carotid arteries after balloon injury in single- and double-injury models. (a) Medial area; (b) intimal area; (c) lumen area; (d) intima/media ratio. Each value is the mean±s.e.mean of data for 4–6 rats.

Figure 3

Time course of change in DNA content in rat left carotid arteries after balloon injury in single- and double-injury models. Each value is the mean±s.e.mean of data for five rats on days 0–14, six rats on day 21, and 10 rats on day 28 in the single-injury group. In the double-injury group, means are for five rats except for days 17 and 28 (mean of four rats each).

Figure 4

Inhibitory effects of cilostamide on DNA synthesis in VSMC. Quiescent cells were stimulated with FCS, EGF, FGF, PDGF-AA, PDGF-AB, PDGF-BB and TGF-β1 in the presence of various amounts of cilostamide. ^*P<0.05 and ^**P<0.01 compared with the control level.

Figure 5

Effects of cilostamide on VSMC migration stimulated by PDGF-BB. Migration assays were performed in Chemotaxicell cell culture chambers. PDGF-BB (10 ng ml⁻¹) was placed in the lower compartment together with the indicated concentration of cilostamide or carvedilol. VSMC (2×10⁵ cells) were loaded in the upper compartment and incubated for 6 h. The number of VSMC per x 100 high-power field (HPF) that had migrated to the lower surface of the filters was determined. Five HPFs were counted per filter. Experiments were performed either in duplicate or in triplicate.

Figure 6

Effects of cilostamide on cyclic AMP concentration in VSMC. VSMC were cultured with cilostamide for 3 h. Data are means from three experiments and the vertical lines represent s.e.mean values. ^**P<0.01 compared with the control.

Figure 7

Effects of cilostamide on [Ca²⁺]_i in VSMC. VSMC were pre-treated with 100 μM cilostamide for 15 min and stimulated with AVP. Data are means from five experiments and the vertical lines represent s.e.mean values.

Figure 8

Effects of cilostamide on LDH release from VSMC. Quiescent VSMC were incubated for 24 h in the presence of the indicated concentrations of cilostamide and nifedipine. Data are means from three experiments and the vertical lines are s.e.mean values. ^**P<0.01 compared with the control level.