RESUMO
OBJECTIVE: Beraprost sodium (BPS), a prostacyclin (PGI(2)) analogue, has been reported to exhibit beneficial effects on atherosclerosis in both human and animal models. To clarify the underlying mechanism, we investigated the effects of BPS on neointimal formation after balloon injury in the canine coronary artery. Furthermore, we determined its anti-atherosclerotic effects in cultured smooth muscle cells (SMCs). METHODS: Adult beagle dogs (10-12 kg) were fed on a high-cholesterol diet (10 g/day) and underwent balloon-denudation of the coronary artery. The dogs were divided into two groups: a BPS-treated group (20 microg/kg per day) and a control group. Twenty-eight days after injury, the dogs were killed and the coronary arteries were examined morphometrically. Three days after injury, the proliferative activity in the medial layer of the coronary artery was evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation, and p27(Kip1), a cyclin-dependent kinase (cdk) inhibitor, expression was examined by immunohistochemistry. We also examined the effects of BPS on SMC proliferation based on BrdU incorporation and cell cycle analysis. In addition, p27(Kip1) regulation was evaluated in primary-cultured SMCs. RESULTS: BPS administration decreased the intima/media ratio (I/M) by 88% in the control group. Three days after injury, BPS attenuated the proliferation rate of the cells in the media of the coronary artery by 35%, and maintained p27(Kip1) expression, which declined in the control cells. In the cultured proliferating SMC, BPS prevented the down-regulation of p27(Kip1). The 8-bromo-cyclic adenosine monophosphate (8-br-cAMP), a cAMP analogue, had similar actions as BPS in the regulation of p27(Kip1). The proliferation of cultured SMC was inhibited in a dose-dependent manner, and cell cycle arrest in the G1 phase was induced by BPS. CONCLUSIONS: Our data suggest that BPS inhibits neointimal formation after balloon denudation in the coronary artery through its inhibitory effect on SMC proliferation by preventing p27(Kip1) down-regulation.
Assuntos
Angioplastia Coronária com Balão/efeitos adversos , Reestenose Coronária/patologia , Epoprostenol/análogos & derivados , Epoprostenol/farmacologia , Músculo Liso Vascular/patologia , Transdução de Sinais/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Reestenose Coronária/tratamento farmacológico , Vasos Coronários , AMP Cíclico/metabolismo , Cães , Epoprostenol/uso terapêutico , Feminino , Citometria de Fluxo , Modelos Animais , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Vasodilatadores/uso terapêuticoRESUMO
Migration and proliferation of smooth muscle cells (SMC) contribute to neointimal formation after arterial injury. However, the relation between migration and proliferation in these cells is obscure. To discriminate between migration and proliferation, we employed a migration assay of SMC at different phases of the cell cycle. Serum-deprived SMC were synchronized in different phases of the cell cycle by addition of serum for various periods of time. Migration induced by platelet-derived growth factor B-chain homodimer was maximal in SMC that were predominantly in the late G(1) (G(1b)) phase. In addition, in nonsynchronized SMC, 65-75% of SMC that had migrated were in the G(1b) phase. Phosphorylated myosin light chain was enriched around the cell periphery in SMC in the G(1b) phase compared with SMC in the other cell cycle phases. Interestingly, the Triton X-100-insoluble fraction of myosin was remarkably decreased in G(1b)-enriched SMC. These findings suggest that migratory activity of SMC may be coupled with the G(1b) phase. The phosphorylation and retention of myosin might explain some of the properties responsible for increased migration.
Assuntos
Movimento Celular/fisiologia , Fase G1/fisiologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Actinas/química , Actinas/metabolismo , Animais , Becaplermina , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , DNA/metabolismo , Fibronectinas/metabolismo , Citometria de Fluxo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Interfase/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Octoxinol/química , Octoxinol/farmacologia , Paxilina , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-sis , RNA/metabolismo , Coelhos , Solubilidade/efeitos dos fármacosRESUMO
Smooth muscle cell (SMC) migration plays an important role in restenosis after angioplasty. Myosin phosphorylation is necessary for cell migration. Fasudil is an inhibitor of protein kinases, including myosin light chain kinase and Rho associated kinase, thereby inhibiting myosin phosphorylation, and it has been clinically used to prevent vasospasm following subarachnoid hemorrage. Based on these findings, we examined the anti-migrative action of fasudil. In SMC (SM-3), fasudil (1-100 microM) inhibited SMC migration in a dose-dependent manner (p < 0.001). Fasudil suppressed actin stress fiber formation dose dependently. In rabbit carotid artery, fasudil (10 mg/kg/day) markedly reduced intimal hyperplasia 14 days following balloon injury. Cell kinetic study showed that fasudil did not affect proliferation but enhanced cell loss in the media after injury. We concluded that fasudil reduced neointimal formation after balloon injury through both inhibiting migration and enhancing cell loss of medial SMC.