MicroRNA-15b/16 Attenuates Vascular Neointima Formation by Promoting the Contractile Phenotype of Vascular Smooth Muscle Through Targeting YAP

Abstract

Objective: To investigate the functional role of the microRNA (miR)-15b/16 in vascular smooth muscle (SM) phenotypic modulation.

Approach and results: We found that miR-15b/16 is one of the most abundant mRs expressed in contractile vascular smooth muscle cells (VSMCs). However, when contractile VSMCs get converted to a synthetic phenotype, miR-15b/16 expression is significantly reduced. Knocking down endogenous miR-15b/16 in VSMCs attenuates SM-specific gene expression but promotes VSMC proliferation and migration. Conversely, overexpression of miR-15b/16 promotes SM contractile gene expression while attenuating VSMC migration and proliferation. Consistent with this, overexpression of miR-15b/16 in a rat carotid balloon injury model markedly attenuates injury-induced SM dedifferentiation and neointima formation. Mechanistically, we identified the potent oncoprotein yes-associated protein (YAP) as a downstream target of miR-15b/16 in VSMCs. Reporter assays validated that miR-15b/16 targets YAP's 3' untranslated region. Moreover, overexpression of miR-15b/16 significantly represses YAP expression, whereas conversely, depletion of endogenous miR-15b/16 results in upregulation of YAP expression.

Conclusions: These results indicate that miR-15b/16 plays a critical role in SM phenotypic modulation at least partly through targeting YAP. Restoring expression of miR-15b/16 would be a potential therapeutic approach for treatment of proliferative vascular diseases.

Keywords: YAP; microRNA; neointima formation; phenotypic modulation; smooth muscle.

Figure 1. Expression of miR-15b/16 is reduced during smooth muscle phenotypic switching from contractile to synthetic phenotype

A. qRT-PCR was performed to evaluate expression of miR15b/16 or smooth muscle contractile marker gene Hic-5 using the samples either from the freshly isolated rat thoracic aorta or rat thoracic aorta cultured in 20% FBS medium for 3 days. N=4. Culture vs Tissue, *p<0.05. B. Rat primary aortic SMCs were treated with PDGF-BB 50ng/ml for 48 hours then total RNA was harvested for qRT-PCR to measure microRNA expression as indicated. N=4. PDGF-BB vs Control, *p<0.05. C. 14 days post rat carotid artery balloon injury control or injured arterial vessels were harvested for qRT-PCR to assess miR-15b/16 expression as indicated. N=5. Control vs Injury, *p<0.05.

Figure 2. miR-15b/16 inhibits VSMC proliferation and migration

A. Adenovirus encoding control GFP or miR-15b/16 were transduced into rat primary aortic SMCs and then proliferation was measured using a cell proliferation WST-1 kit (Roche) in the culture media as indicated. *p<0.05. B. After transduction with miR-15b/16 or control GFP adenovirus, rat primary aortic SMCs were harvested for propidium iodide staining to analyze cell cycle by a flow cytometry. *p<0.05. C. After over-expression of miR-15b/16, rat primary aortic SMCs were seeded at equal density at day (D) 0 and counted at each time point as indicated. N=3. *p<0.05. D. HCASMCs were transduced by control GFP or miR-15b/16 virus and cell numbers were counted as described in “C”. E. WST-1 assay was performed in rat primary aortic SMCs cultured in the different culture media as indicated after transfection either with control or antisense oligonucleotides against miR-15b/16. *p<0.05. F. After silencing miR-15b/16, rat primary aortic SMCs were seeded at equal density at day (D) 0 and counted at each time point as indicated. N=3. *p<0.05. G. Quantification of Boyden chamber assay to assess cell migration after 4h plating of rat aortic SMCs cells that either GFP or miR-15b/16 adenovirus transduced. *p<0.05. H. Control or miR-15b/16 antisense oligonucleotides were transfected into rat aortic SMCs and the Boyden chamber assay was performed to examine cell migration. *p<0.05.

Figure 3. miR-15b/16 promotes smooth muscle contractile phenotype by targeting YAP

A. Adenovirus expressing GFP or miR-15b/16 were transduced into rat primary aortic SMCs for 3 days. Subsequently protein was collected for western blot. B. HCASMCs were transduced with GFP or miR-15b/16 adenovirus and western blot was performed to examine the protein expression as described in “A”. C. Total RNA were harvested from the rat primary aortic SMCs that infected with GFP or miR-15b/16 adenovirus and qRT-PCR was performed to measure mRNA expression as indicated. *p<0.05. D. Control or miR-15b/16 antisense oligonucleotides were transfected into rat primary aortic SMCs for 3 days and protein was harvested for Western blot as indicated. E. Schematic diagram of the luciferase reporters harboring wild-type (WT) or mutation of the miR-16 binding site in the 3′-UTR of human YAP. The shaded region indicates an evolutionarily conserved “seed” binding sequence among vertebrate species for miR-15b/16 recognition. The mutated nucleotides in YAP 3′-UTR reporter were underscored. F. The luciferase reporter constructs harboring WT or mutation of predicted miR-16 binding site of YAP 3′-UTR were co-transfected with the increasing amount of miR-15b/16 plasmid into HEK293 cells. Post transfection 36 hours, cells were harvested for dual luciferase assays. Reporter activity is normalized to a renilla luciferase internal control and expressed relative to the transfection with empty plasmid (set to 1). Data were presented as mean ± SE of 6 samples. *p<0.05. G. Rat primary aortic SMCs were transduced with the combination of GFP, miR-15b/16 or YAP as indicated for 2 days and then WST1 assays were performed to assess the cell proliferation and cell numbers were counted at the time points as indicated (H). miR-15b/16 or YAP vs GFP, *p<0.05; YAP+miR-15b/16 vs miR-15b/16, ^#p<0.05.

Figure 4. Restoring expression of miR-15b/16 attenuates arterial injury-induced neointima formation

A. Post injury 14 days, rat control RCA or balloon injured LCA arteries that infused with either GFP or miR-15b/16 adenovirus on the day of injury were harvested for qRT-PCR to evaluate miR-15b and miR-16 expression, respectively. The expression of miRs in control RCA in each group was set to 1. N=7 each group. B. After injury 14 days, carotid artery sections from either control or balloon-injured vessels that were transduced with GFP, or miR-15b/16 adenovirus were prepared for H&E staining. NI: neointima; M: media; AD: adventitial layer; C. Statistical analysis of neointima/media layer ratio and neointima area (D) of sections shown in the panel “B” using ImageJ software. N=7. *p<0.05.

Figure 5. Over-expression of miR-15b/16 promotes VSMC contractile phenotype in vivo

A. 14 days after injury, corresponding contralateral control RCA or balloon injured LCA (GFP or miR-15b/16 adenovirus transduced at time of injury) were harvested for western blot as indicated. Data presented are a representative blot from 7 rats in each group. B. Quantification of protein expression in panel “A” from 7 rats in each group. Expression was normalized to loading control vinculin then expressed relative to its corresponding uninjured control RCA (set to 1, red line). *p<0.05. C. Co-staining of proliferative marker Ki67 (green) and smooth muscle-specific marker SM a-actin (red) was performed in the rat control RCA or balloon injured LCA (14 days post injury) that were infused with either GFP or miR-15b/16 adenovirus at the time of injury. Sections that were incubated with 2^nd antibody alone served as a negative control. Arrows point to the representative Ki67 positive cells in the injured vessels. Arrow-head points to the internal elastic lamina. NI: neointima, M: media. AD: adventitial layer. D. Quantification of the percentage of Ki67 positive cells within the neointima area in the GFP or miR-15b/16 adenovirus infected LCA as shown in panel “C”. N=7. *P< 0.05. E. A simplified schematic diagram summarizing the findings of this study.