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Journals / The Anatolian Journal of Cardiology / 2016 / Cilt: 16 - Sayı: 6

Everolimus (RAD001) inhibits the proliferation of rat vascular smooth muscle cells by up-regulating the activity of the p27/kip1 gene promoter

Boli RAN
Minfeng Lİ
Yeqing Lİ
Yang LİN
Weimin LİU
Qiulin LUO
Yongxin FU
Qianmei TANG
Ya YANG
Yunfei PU
Pages
385–391
DOI
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⚡ TL;DR(Too Long; Didn't Read)

RAD001 intervention increased p27/kip1 gene promoter activity 3.5-fold, promoted p27/kip1 mRNA and protein expression, increased G0-phase cells, reduced DNA synthesis, and, overall, inhibited PDGF-BB-stimulated cell proliferation.Conclusion: RAD001 inhibits PDGF-BB-stimulated proliferation of cultured VSMCs by upregulating p27/kip1 gene promoter activity and increasing p27/kip1 mRNA and protein expression.

📌 3 Key Takeaways

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Objective & Hypothesis:Objective: We investigated whether the inhibitory effect of the immunosuppressant everolimus (RAD001) on vascular smooth muscle cell (VSMC) proliferation is mediated by p27/kip1 gene promoter activity.Methods: In this experimental study, cultured rat VSMCs were transiently transfected with a recombinant plasmid (pXp27) containing p27/kip1 gene promoter sequence and a chloramphenicol acetyltransferase (CAT) reporter gene.
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Key Findings & Data:After stimulation with the mitogen platelet-derived growth factor (PDGF-BB, 10 ng/mL) in the presence or absence of RAD001 (10 nM), the promoter activity, mRNA expression, and protein expression of p27/kip1 were examined by CAT assay, RT-PCR, and immunoblotting, respectively.
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Conclusion & Impact:RAD001 intervention increased p27/kip1 gene promoter activity 3.5-fold, promoted p27/kip1 mRNA and protein expression, increased G0-phase cells, reduced DNA synthesis, and, overall, inhibited PDGF-BB-stimulated cell proliferation.Conclusion: RAD001 inhibits PDGF-BB-stimulated proliferation of cultured VSMCs by upregulating p27/kip1 gene promoter activity and increasing p27/kip1 mRNA and protein expression.
🔬Design:Research Article
📍Scope:Comprehensive Sample
⚙️Variables:Domain Parameters

Abstract

Objective: We investigated whether the inhibitory effect of the immunosuppressant everolimus (RAD001) on vascular smooth muscle cell (VSMC) proliferation is mediated by p27/kip1 gene promoter activity.Methods: In this experimental study, cultured rat VSMCs were transiently transfected with a recombinant plasmid (pXp27) containing p27/kip1 gene promoter sequence and a chloramphenicol acetyltransferase (CAT) reporter gene. After stimulation with the mitogen platelet-derived growth factor (PDGF-BB, 10 ng/mL) in the presence or absence of RAD001 (10 nM), the promoter activity, mRNA expression, and protein expression of p27/kip1 were examined by CAT assay, RT-PCR, and immunoblotting, respectively. Cell cycle-related changes were detected by flow cytometry. DNA synthesis was determined using 3H-TdR incorporation.Results: Compared with the non-stimulation group, PDGF-BB stimulation induced a significant proliferative response in the VSMCs as indicated by decreased p27/kip1 gene promoter activity, decreased p27/kip1 mRNA and protein expression, increased S-phase and G2/M-phase cells, and increased DNA synthesis. RAD001 intervention increased p27/kip1 gene promoter activity 3.5-fold, promoted p27/kip1 mRNA and protein expression, increased G0-phase cells, reduced DNA synthesis, and, overall, inhibited PDGF-BB-stimulated cell proliferation.Conclusion: RAD001 inhibits PDGF-BB-stimulated proliferation of cultured VSMCs by upregulating p27/kip1 gene promoter activity and increasing p27/kip1 mRNA and protein expression.

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Cite This Article

RAN, B., Lİ, M., Lİ, Y., LİN, Y., LİU, W., LUO, Q., FU, Y., TANG, Q., YANG, Y., PU, Y. (2016). Everolimus (RAD001) inhibits the proliferation of rat vascular smooth muscle cells by up-regulating the activity of the p27/kip1 gene promoter. The Anatolian Journal of Cardiology, Cilt: 16 - Sayı: 6, 385–391. https://ajindex.com/articles/365470

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