RESUMEN
Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.
Asunto(s)
Ensamble y Desensamble de Cromatina/efectos de los fármacos , Cromatina/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Factores de Transcripción de Tipo Kruppel/biosíntesis , Quinolinas/farmacología , Cromatina/genética , Regulación de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Óxido Nítrico Sintasa de Tipo III/biosíntesis , Óxido Nítrico Sintasa de Tipo III/genética , Elementos de Respuesta , Trombomodulina/biosíntesis , Trombomodulina/genéticaRESUMEN
BACKGROUND: K-134 is a more potent antiplatelet drug with a selective inhibitory effect on phosphodiesterase 3 (PDE3) compared with its analogue, cilostazol. OBJECTIVES: This study was performed to compare the ameliorating effects of K-134 and cilostazol on brain damage in an experimental photothrombotic cerebral infarction model. METHODS AND RESULTS: We investigated the effects of oral preadministration of PDE3 inhibitors in a rat stroke model established by photothrombotic middle cerebral artery (MCA) occlusion. K-134 significantly prolonged MCA occlusion time at doses >10 mg/kg, and reduced cerebral infarct size at 30 mg/kg in the stroke model (nâ=â12, 87.5±5.6 vs. 126.8±7.5 mm(3), P<0.01), indicating its potent antithrombotic effect. On the other hand, the effects of cilostazol on MCA occlusion time and cerebral infarct size are relatively weak even at the high dosage of 300 mg/kg. Furthermore, K-134 blocked rat platelet aggregation more potently than cilostazol in vitro. Also in an arteriovenous shunt thrombosis model, K-134 showed an antithrombotic effect greater than cilostazol. CONCLUSIONS: These findings suggest that K-134, which has strong antithrombotic activity, is a promising drug for prevention of cerebral infarction associated with platelet hyperaggregability.
Asunto(s)
Encéfalo/efectos de los fármacos , Infarto Cerebral/tratamiento farmacológico , Inhibidores de Fosfodiesterasa 3/uso terapéutico , Quinolinas/uso terapéutico , Trombosis/prevención & control , Urea/análogos & derivados , Animales , Encéfalo/metabolismo , Encéfalo/patología , Infarto Cerebral/patología , Masculino , Ratones , Ratones Endogámicos ICR , Ratas , Ratas Sprague-Dawley , Urea/uso terapéuticoRESUMEN
OBJECTIVE: Cilostazol is a phosphodiesterase (PDE)3 inhibitor used to treat peripheral arterial disease with intermittent claudication, as there is clinical evidence that it improves treadmill exercise capacity. However, details of the mechanism underlying this enhanced walking capacity remain to be elucidated. METHODS: Based on the hypothesis that PDE3 inhibitors improve peripheral microcirculation in the hindlimbs via vascular smooth muscle relaxation and antiplatelet effects, we examined the effects of a more potent and selective PDE3 inhibitor, K-134, in rat models of peripheral arterial disease (PAD). RESULTS: In a hindlimb ischemia model established by bilateral femoral artery occlusion, oral administration of K-134 for 27 days significantly increased blood flow in hindlimb skeletal muscle after exercise induced by electrical stimulation of the sciatic nerve. Moreover, K-134 enlarged the luminal area of intramuscular arteries and prevented rarefaction of capillary density in the gastrocnemius muscle. These effects were observed without pre-administration on the day following the last administration, suggesting that vasodilatory, antiplatelet and angiogenic activities of K-134 were indirectly responsible for the long-term beneficial effects. In fact, K-134 dose-dependently induced relaxation of rat femoral arteries in vitro, and inhibited rat platelet aggregation ex vivo. Interestingly, in a laurate-induced peripheral vascular injury model, oral administration of K-134 for 6 days prevented progression of hindlimb necrosis. CONCLUSION: These findings suggest that the beneficial effects of PDE3 inhibitors on walking capacity are due to increased hindlimb skeletal muscle blood flow via intramuscular artery enlargement, and that K-134 is a promising drug for PAD associated with platelet hyperaggregability.