Figure 2: Everolimus Inhibits SMC Proliferation Through Transcriptional Repression of Telomerase

(A) Cell proliferation assay in primary murine vascular smooth muscle cells (mSMC) isolated from wild-type (WT) or telomerase reverse transcriptase–overexpressing transgenic (TERTtg) mice. Cells were serum-deprived (−) and treated with vehicle or 10 μmol/l everolimus in 20% fetal bovine serum (FBS). Values are expressed as the mean ± SEM (n = 12 samples/group) relative to quiescent WT mSMC. Significant levels versus quiescent cells (−): FBS + vehicle (WT), *p = 0.008; FBS + vehicle (TERTtg), *p < 0.001; FBS + 10 μmol/l everolimus (TERTtg); *p < 0.001. Significance levels versus WT: quiescent cells, #p < 0.001; FBS + vehicle, #p < 0.001; FBS + 10 μmol/l everolimus, #p < 0.001. (B) Western blotting for TERT expression in nuclear extracts isolated from hSMC treated with everolimus. Densitometric data are expressed as TERT normalized to TATA binding protein (TBP) and presented as mean ± SEM fold-increase over vehicle from 3 independently performed experiments (n = 9 samples/group). Significance levels versus quiescent cells (−): growth medium + vehicle, ∗p < 0.001; growth medium + 0.1 μmol/l everolimus, ∗p < 0.001; growth medium + 1 μmol/l everolimus, ∗p < 0.001; growth medium + 10 μmol/l everolimus, ∗p < 0.001. Significance level versus growth medium + vehicle: growth medium + 10 μmol/l everolimus, #p = 0.022. (C) Immunostaining for TERT expression (green) in WT mSMC treated with 10 μmol/l everolimus. 4′-6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. Images are representative of 3 independently performed experiments. (D) TERT mRNA expression in hSMC treated with 10 μmol/l everolimus analyzed by real-time PCR. Values are normalized to the housekeeping gene TBP and expressed as mean ± SEM (n = 12 samples/group) relative to quiescent hSMC (∗p = 0.047 versus quiescent cells). (E) hSMC were transfected with a reporter construct driven by the full length TERT promoter. Following transfection, cells were treated with everolimus and analyzed for luciferase activity. Values are mean ± SEM (n = 12 samples/group) relative to luciferase activity of quiescent hSMC (∗p < 0.001 versus quiescent cells).

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