MicroRNA-663 regulates human vascular smooth muscle cell phenotypic switch and vascular neointimal formation

Abstract

Rationale: Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. MicroRNAs (miRNAs) have emerged as important regulators for VSMC function, and we recently identified miR-663 as critical for controlling human aortic smooth muscle cell proliferation.

Objective: To investigate whether miR-663 plays a role in human VSMC phenotypic switch and the development of neointima formation.

Methods and results: By using quantitative reverse-transcription polymerase chain reaction, we found that miR-663 was significantly downregulated in human aortic VSMCs on platelet-derived growth factor treatment, whereas expression was markedly increased during VSMC differentiation. Furthermore, we demonstrated that overexpression of miR-663 increased expression of VSMC differentiation marker genes, such as smooth muscle 22α, smooth muscle α-actin, calponin, and smooth muscle myosin heavy chain, and potently inhibited platelet-derived growth factor-induced VSMC proliferation and migration. We identified the transcription factor JunB and myosin light chain 9 as downstream targets of miR-663 in human VSMCs, because overexpression of miR-663 markedly inhibited expression of JunB and its downstream molecules, such as myosin light chain 9 and matrix metalloproteinase 9. Finally, we showed that adeno-miR-663 markedly suppressed the neointimal lesion formation by ≈50% in mice after vascular injury induced by carotid artery ligation, specifically via decreased JunB expression.

Conclusions: These results indicate that miR-663 is a novel modulator of human VSMC phenotypic switch by targeting JunB/myosin light chain 9 expression. These findings suggest that targeting miR-663 or its specific downstream targets in human VSMCs may represent an attractive approach for the treatment of proliferative vascular diseases.

Keywords: miR-663; migration; proliferation; vascular remodeling; vascular smooth muscle cells.

Figure 1. MicroRNA (miR)-663 is significantly downregulated in proliferating vascular smooth muscle cells (VSMCs)

A, Platelet-derived growth factor-BB (PDGF-BB; 20 ng/mL) caused a time-dependent decrease in miR-663 expression in human aortic VSMCs, as determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) (n=4). *P<0.05 compared with 0 h group. B, PDGF-BB caused a dose-dependent decrease in miR-663 expression in human aortic VSMCs at 24 h after PDGF treatment, as determined by qRT-PCR (n=4). *P<0.05 compared with that in VSMCs without PDGF-BB. C, In situ hybridization (violet) shows miR-663 was significantly decreased in PDGF-BB–stimulated (20 ng/mL) VSMCs. D, Serum (5%) caused a time-dependent decrease in miR-663 expression in human aortic VSMCs, as demonstrated by qRT-PCR (n=4). *P<0.05 compared with that without 5% fetal bovine serum treatment.

Figure 2. MicroRNA (miR)-663 is upregulated in differentiated human aortic smooth muscle cells (SMCs)

A, Vascular SMCs (VSMCs) cultured in SMC differentiation medium (DM) for 48 h caused a significantly increased expression of VSMC differentiation marker genes, such as smooth muscle α-actin, smooth muscle 22α, calponin, and smooth muscle myosin heavy chain, as determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) (n=5).*P<0.05 compared with SMC growth medium (smGM). B, Human aortic SMCs were cultured in SMC differentiation medium to induce SMC differentiation and the expression of miR-663 was measured by qRT-PCR at different time points (n=5). *P<0.05 compared with 0 h group. C, Human aortic SMCs were treated with all transretinoic acid (atRA; 1 mmol/L) or dimethyl sulfoxide (DMSO) and the expression of SMC differentiation marker genes was determined by qRT-PCR after 72 h of atRA treatment (n=5). *P<0.05 compared with control D, miR-663 was significantly upregulated in atRA-treated VSMCs, as determined by qRT-PCR miRNA assay (n=5). *P<0.05 compared with 0 h.

Figure 3. MicroRNA (miR)-663 regulates the expression of vascular smooth muscle cell (VSMC) contractile genes

A, Adenovirus-mediated overexpression of miR-663 (Ad-miR-663) with a multiplicity of infection (MOI) of 100 increases miR-663 levels in a dose-dependent manner (n=4). *P<0.05 vs control viruses expressing green fluorescent protein (Ad-GFP). B, Seventy-two hours after transduction of cell with Ad-miR-663 (MOI=100), the expression of VSMC differentiation marker genes in human aortic SMCs was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) (n=5). *P<0.05 compared with Ad-GFP group. C, Ad-miR-663 (MOI=100) increases the expression of SMC differentiation marker genes in human aortic SMCs in the presence and absence of platelet-derived growth factor (PDGF; 20 ng/mL) treatment, as determined by Western blot analysis. D, Densitometric analysis of SMC differentiation marker genes expression as determined by Western blot (n=5). *P<0.05 vs Ad-GFP without PDGF treatment; †P<0.05 vs Ad-GFP with PDGF (20 ng/mL) treatment.

Figure 4. Role of microRNAs (miR)-663 in vascular smooth muscle cell (VSMC) proliferation

A, Human aortic VSMCs were transfected with control miR mimics, miR-663 mimics, or miR-663 inhibitors, respectively, and then subjected to quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of miR-663 level (n=4). *P<0.05 vs CTRL miR; †P<0.05 vs control (CTRL) miR. B, miR-663 mimic inhibits platelet-derived growth factor-BB (PDGF-BB)-induced VSMC proliferation in a dose-dependent manner as measured by BrdU incorporation (n=4). *P<0.05 vs CTRL miR with PDGF treatment. C, Forty-eight hours after transfection with either anti-miR-663 (50 nmol/L) or CTRL anti-miR (50 nmol/L), SMCs were stimulated with or without PDGF-BB (2 ng/mL) for 24 h. Anti-miR-663 (50 nmol/L) significantly increased human aortic VSMCs proliferation stimulated by PDGF-BB (2 ng/mL) as determined by BrdU incorporation (n=5). *P<0.05 compared with CTRL miR treated with PDGF-BB (2 ng/mL) for 24 h.

Figure 5. Role of microRNA (miR)-663 in vascular smooth muscle cell (VSMC) migration

A, Forty-eight hours after transduction of adenovirus-mediated overexpression of miR-663 (Ad-miR-663; MOI=100), VSMCs were starved and cell migration was measured after platelet-derived growth factor (PDGF; 20 ng/mL) stimulation for 24 h by scratch-wound assay. Migrated cells were quantitated and the results are shown in (B). The data are means±SD of the number of migrated cells from 3 independent experiments. *P<0.05 vs control viruses expressing green fluorescent protein (Ad-GFP) without PDGF treatment; †P<0.05 vs Ad-GFP with PDGF treatment. C, Forty-eight hours after transfected with anti-miR-663 (50 nmol/L) or anti-miR control (CTRL) (50 nmol/L), smooth muscle cells (SMCs) were subjected to injury by scraping and then were stimulated with or without PDGF-BB (20 ng/mL) for 24 h. Migrated cells were quantitated using Image J software program and the results are shown in (D). The data are means±SD of the number of migrated cells from 3 independent experiments. *P<0.05 vs CTRL anti-miR without PDGF treatment; †P<0.05 vs CTRL anti-miR with PDGF treatment. E, Ad-miR-663 decreases matrix metalloproteinase 2 (MMP-2) and MMP-9 mRNA levels in human aortic SMCs as measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) (n=5).

Figure 6. Identification of JunB and myosin light chain 9 (Myl9) as target genes of microRNA (miR)-663 in vascular smooth muscle cells (VSMCs)

A, miR-663 mimic (50 nmol/L) or nontargeting control (50 nmol/L) was cotransfected with the luciferase reporter carrying WT-JunB 3′-UTR or mutated JunB 3′-UTR. Forty-eight hours after transfection, renilla luciferase activities were measured (n=5). *P<0.05 vs JunB-3′-UTR-WT transfected with mimic control; †P<0.05 vs JunB-3′-UTR-WT transfected with mimic control. B, Effect of miR-663 on JunB expression in platelet-derived growth factor-BB (PDGF-BB; 20 ng/mL)-stimulated VSMCs. C, Densitometric analysis of JunB protein levels as measured by Western blot (n=5). *P<0.05 vs control (CTRL) miR without PDGF treatment; †P<0.05 vs CTRL miR with PDGF treatment. D, Effect of miR-663 on Myl9 expression in PDGF-BB (20 ng/mL)-stimulated VSMCs. Densitometric analysis of Myl9 protein levels as measured by Western blot (n=5). *P<0.05 vs CTRL miR without PDGF treatment; †P<0.05 vs CTRL miR with PDGF treatment. E, miR-663 mimic (50 nmol/L) or nontargeting control (50 nmol/L) was cotransfected with the luciferase reporter carrying WT-Myl9 3′-UTR or mutated Myl9 3′-UTR. Forty-eight hours after transfection, renilla luciferase activities were measured (n=5). *P<0.05 vs Myl9-3′-UTR-WT transfected with mimic control; †P<0.05 vs Myl9-3′-UTR-WT transfected with mimic control. F, VSMCs were transduced with either Ad-LacZ or Ad-miR-663. Forty-eight hours after transduction, SMCs were starved for 48 h and then stimulated with 25 ng/mL PDGF-BB for 20 min. Stress fiber formation was examined by staining cells with Alexa Fluor 546 phalloidin.

Figure 7. JunB is critically implicated in vascular smooth muscle cell (VSMC) proliferation and migration

A, Silencing of JunB by specific small interfering RNA (JunB siRNA; 100 nmol/L) reduced JunB expression as determined by Western blot. *P<0.05 vs control (CTRL) siRNA without platelet-derived growth factor (PDGF) treatment; †P<0.05 vs CTRL siRNA with PDGF (20 ng/mL) treatment. B, Silencing JunB increases the expression of SMC marker genes smooth muscle 22α (SM22α) and smooth muscle α-actin (SMA), as determined by Western blot analysis. C, Densitometric analysis of SM22α and SMA protein levels as measured by Western blot (n=5). *P<0.05 vs CTRL siRNA without PDGF treatment; †P<0.05 vs CTRL siRNA with PDGF (20 ng/mL) treatment. D, JunB knockdown attenuated PDGF-BB (20 ng/mL)-induced proliferation in human aortic SMCs as determined by MTT assay (n=4). *P<0.05 vs control siRNA with PDGF-BB treatment. E, VSMCs were transfected with JunB siRNA (100 nmol/L). Forty-eight hours after transfection, VSMCs were starved and cell migration was measured after PDGF-BB (20 ng/mL) stimulation for 24 h by scratch-wound assay. F, Quantitation of migrated cells. The data are means±SD of the number of migrated cells from 3 independent experiments. *P<0.05 vs CTRL siRNA without PDGF treatment; †P<0.05 vs CTRL siRNA with PDGF treatment.

Figure 8. MicroRNA (miR)-663 attenuates neointimal formation in a mouse model of carotid artery ligation

A, Adenovirus-mediated overexpression of miR-663 (Ad-miR-663) significantly reduced neointimal formation in vivo. Representative hematoxylin and eosin (H&E)-stained carotid artery slices from mouse treated with Ad-LacZ or Ad-miR-663 at 14 days after carotid artery ligation. B and C, The effect of miR-663 on vascular neointimal lesion formation in mouse carotid arteries at 14 days after ligation injury as quantitated by neointimal area and neointima/media (N/M) ratio. *P<0.05 vs Ad-LacZ or control (CTRL) D, Representative immunofluorescent staining of PCNA in mouse carotid arteries at 14 days after ligation injury. Green is the PCNA that represents proliferating cells; blue is the cell nuclear staining by DAPI. E, Quantification of PCNA-positive cells showed that fewer cells were proliferating in the injured arteries treated with Ad-miR-663 compared with Ad-LacZ-treated vessels. *P<0.05 vs Ad-LacZ. F, Representative immunofluorescent staining of JunB in mouse carotid arteries at 14 days after ligation injury. Green is smooth muscle α-actin; purple is JunB staining; and blue is nuclear staining by DAPI. G, Quantification of JunB-positive cells showed that JunB was downregulated in Ad-miR-663-treated vessels compared with Ad-LacZ-treated vessels. *P<0.05 vs Ad-LacZ.