Olmesartan alleviates bleomycin-mediated vascular smooth muscle cell senescence via the miR-665/SDC1 axis

Abstract

Olmesartan (OMST) is a new angiotensin II receptor antagonist recently approved by the FDA to treat cardiovascular diseases. We investigated the molecular mechanisms by which OMST regulates vascular senescence. In the present study, bleomycin (BLM) was used to induce senescence in vascular smooth muscle cells (VSMCs); after which, the cells were treated with OMST. The effects of OMST on BLM-mediated cell senescence were evaluated using cell adhesion, NAD⁺/NADH, and Annevin V/PI double staining assays, as well as by immunofluorescence staining of γH2AX, Edu flow cytometry, and evaluations of senescence-associated β-gal activity. Differentially expressed microRNAs (DEMs) were identified by miRNA microarray assays, and subsequently validated by quantitative real time PCR. Bisulfite sequencing PCR (BSP) was used to detect the methylation status of the miR-665 promoter. The target genes of miR-665 were predicted and confirmed using luciferase reporter assays. We found that miR-665 was upregulated in VSMCs in response to BLM-induced cellular senescence. BSP studies revealed that CpG sites in the promoter region of the miR-665 gene underwent extensive demethylation during BLM-induced cellular senescence, and there was a concomitant up-regulation of miR-665 expression. SDC1 mRNA was identified as a direct target of miR-665. Either miR-665 overexpression or SDC1 knockdown significantly reversed the effects of OMST on BLM-induced VSMC senescence. Moreover, SDC1 overexpression partially reversed the changes that occurred in cells with BLM-induced senescence caused by miR-665 overexpression. Our findings suggest that the miR-665/SDC1 axis functions as a vital modulator of VSMC senescence, and may represent a novel biological target for treating atherosclerosis.

Keywords: Atherosclerosis; MiR-665; SDC1; olmesartan; vascular smooth muscle cell senescence.

Figure 1

OMST treatment counteracted BLM-induced cellular senescence in human VSMCs. A. Cell morphology was observed in the BLM and control groups. We observed thattreatment with BLM resulted in shrunken cells that contained increased amounts of pigment when compared to control cells. B. Cell adhesion assays were performed using VSMCs in the BLM and control groups. C. The NAD⁺/NADH ratio was determined by an NAD⁺/NADH assay. D. Representative immunofluorescence images of nuclear γH2AX (cell nuclei: blue; γH2AX: red) in VSMCs from the control, BLM, and BLM+OMST groups. E, F. Cell senescence was evaluated by staining for SA-β-gal-positive cells. Representative photomicrographs showing SA-β-gal-positive cells (blue) among the VSMCs. G, H. Cell proliferation was assessed by the Edu flow cytometry assay. I, J. Flow cytometry with Annexin V/PI staining was used to analyze VSMC apoptosis. **P < 0.01, ***P < 0.001 vs. control; #P < 0.05, ##P < 0.01 vs. BLM group. Abbreviations: VSMCs, vascular smooth muscle cells; BLM, bleomycin; OMST, olmesartan.

Figure 2

Identification of miR-665 expressed in response to BLM-induced senescence in VSMCs. (A, B) The expression levels of 8 miRNAs in VSMCs with BLM-induced senescence with and without OMST treatment as determined via microarray analysis. Histogram of (C) enriched GO terms and (D) KEGG signaling pathways of 8 miRNAs and their targets. (E) The 8 differentially expressed miRNAs were validated by quantitative real time PCR. **P < 0.01, ***P < 0.001 vs. control; #P < 0.05, ##P < 0.01 vs. BLM group.

Figure 3

Analysis of the miR-665 promoter in VSMCs with BLM-induced senescence. A, B. The CpG Island of the miR-665 promoter was predicted, and primers were designed using MethPrimer. C. BSP results and the relative methylation ratio of each CpG site in the miR-665 promoter region in BLM-induced VSMCs are shown. The solid circles represent methylated CpG sites, while hollow circles represent non-methylated CpG sites.

Figure 4

Up-regulation of miR-665 reversed the effects of OMST on BLM-induced VSMC senescence. BLM-induced VSMCs were treated with OMST with or without miR-665 mimic transfection. A. MiR-665 expression was determined by quantitative real time PCR. B. The NAD⁺/NADH ratio was determined by a NAD⁺/NADH assay. C, D. Cell senescence was evaluated by staining for SA-β-gal-positive cells. Representative photomicrographs showing SA-β-gal-positive cells (blue) among VSMCs. E. Representative immunofluorescence images of nuclear γH2AX (cell nuclei: blue; γH2AX: red) in VSMCs from the control, BLM, BLM+OMST, and BLM+OMST+miR-665 mimic groups. F-H. Cell proliferation and cell apoptopsis was assessed by the Edu flow cytometry assay and Annexin V/PI staining flow cytometry assay. ***P < 0.001 vs. control; #P < 0.05, ##P < 0.01 vs. BLM group; &P < 0.05 vs. BLM+OMST group.

Figure 5

SDC1 was a direct target of miR-665. A. A western blot analysis was performed to detect the expression of SDC1 protein in VMSCs after transfection with miR-665 mimics or the NC. B. The predicted binding site of miR-665 on the 3’-UTR of human SDC1 mRNA. A mutant binding site was constructed and the red letters indicate mutated nucleotides. C. Relative luciferase activity was evaluated. **P < 0.01, ***P < 0.001 vs. NC. Abbreviations: UTR, untranslated region; NC, negativecontrol; WT, wild type; MUT, mutant.

Figure 6

Downregulation of SDC1 reversed the effects of OMST on BLM-induced VSMC senescence. BLM-induced VSMCs were treated with OMST with or without siSDC1 transfection. A. SDC1 protein expression was detected by western blot analysis. B. SDC1 mRNA expression was determined by quantitative real time PCR. C. The NAD⁺/NADH ratio was determined by an NAD⁺/NADH assay. D, E. Cell senescence was evaluated by staining for SA-β-gal-positivecells. Representative photomicrographs showing SA-β-gal-positive cells (blue) among VSMCs. F. Representative immunofluorescence images of nuclear γH2AX (cell nuclei: blue; γH2AX: red) in VSMCs from the control, BLM, BLM+OMST, and BLM+OMST+siSDC1 groups. G-I. Cell proliferation and cell apoptopsis was assessed by the Edu flow cytometry assay and Annexin V/PI staining flow cytometry assay. **P < 0.01, ***P < 0.001 vs. control; #P < 0.05, ##P < 0.01 vs. BLM group; &P < 0.05 vs. BLM+OMST group.

Figure 7

OMST counteracted BLM-induced cellular senescence via miR-665 targeting of SDC1. BLM-induced VSMCs were treated with OMST plusmiR-665 mimics + SDC1 transfection. A. SDC1 protein expression was detected by western blotting. B. SDC1 mRNA expression was determined by quantitative real time PCR. C. The NAD⁺/NADH ratio was determined by a NAD⁺/NADH assay. D, E. Cell senescence was evaluated by staining for SA-β-gal-positive cells. Representative photomicrographs showing SA-β-gal-positive cells (blue) among VSMCs. F. Representative immunofluorescence images of nuclear γH2AX (cell nuclei: blue; γH2AX: red) in VSMCs from the control, BLM, BLM+OMST, BLM+OMST+miR-665 mimic, and BLM+OMST+miR-665 mimic+SDC1 groups. G-I. Cell proliferation and cell apoptopsis was assessed by the Edu flow cytometry assay and Annexin V/PI staining flow cytometry assay. ***P < 0.001 vs. control; #P < 0.05, ##P < 0.01 vs. BLM group; &P < 0.05 vs. BLM+OMST group; @P < 0.05 vs. BLM+OMST+miR-665 mimic group.

Figure 8

A diagram showing the mechanism for up-regulation of miR-665 in BLM-induced cellular senescence.