Oncological miR-182-3p, a Novel Smooth Muscle Cell Phenotype Modulator, Evidences From Model Rats and PatientsHighlights
Objective—Vascular smooth muscle cell (VSMC) phenotype change is a hallmark of vascular remodeling, which contributes to atherosclerotic diseases and can be regulated via microRNA-dependent mechanisms. We recently identified that asymmetrical dimethylarginine positively correlates to vascular remodeling–based diseases. We hypothesized that asymmetrical dimethylarginine induces smooth muscle cell (SMC) phenotypic change via a microRNA-dependent mechanism.
Approach and Results—Microarray analysis enabled the identification of downregulation of miR-182-3p in asymmetrical dimethylarginine–treated human aortic artery SMCs. The myeloid-associated differentiation marker (MYADM) was identified as the downstream target of miR-182-3p and implicated to contribute to miR-182-3p knockdown–mediated SMC phenotype change, which was evidenced by the increased proliferation and migration and reduced expression levels of phenotype-related genes in human aortic artery SMCs through the ERK/MAP (extracellular signal-regulated kinase/mitogen-activated protein) kinase–dependent mechanism. When inhibiting MYADM in the presence of miR-182-3p inhibitor or overexpressing MYADM in the presence of pre-miR-182-3p, human aortic artery SMCs were reversed to the differentiation phenotype. In vivo, adeno-miR-182-3p markedly suppressed carotid neointimal formation by using balloon-injured rat carotid artery model, specifically via decreased MYADM expression, whereas adeno-miR-182-3p inhibitor significantly promoted neointimal formation. Atherosclerotic lesions from patients with high asymmetrical dimethylarginine plasma levels exhibited decreased miR-182-3p expression levels and elevated MYADM expression levels.
Conclusions—miR-182-3p is a novel SMC phenotypic modulator by targeting MYADM.
- asymmetrical dimethylarginine
- myeloid-associated differentiation marker
- smooth muscle cell
- vascular remodeling
Vascular remodeling is a common pathological process central to neointimal lesion formation that also contributes to atherosclerotic diseases, such as atherosclerosis, postangioplasty restenosis, and coronary heart disease. Although the signals for vascular remodeling are diverse in different pathological contexts, the switching of vascular smooth muscle cells (VSMCs) from the differentiated phenotype (also called the contractile phenotype) to the dedifferentiated phenotype (also called the synthetic phenotype) is consistently observed.1 This pathological change is called phenotype change, which mainly manifests as the increased proliferation, migration, and synthesis of extracellular matrix components and the reduced expression of differentiation markers (ie, smooth muscle α-actin [SMA] and calponin) in smooth muscle cells (SMCs).2
After vascular injury, biologically active components are released that trigger the SMC switch from the differentiated phenotype to the dedifferentiated phenotype and subsequently induce neointimal formation. Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, markedly increases in the plasma after endothelial injury, is positively associated with carotid neointimal formation,3 and mediates atherosclerotic plaque formation.4 Recently, ADMA was demonstrated to directly affect SMCs by increasing SMC migration, apoptosis, and proliferation.5Therefore, studies on the effects of ADMA on SMC phenotype change are critical to elucidate the key mechanisms central to vascular remodeling and atherosclerotic diseases.
MicroRNAs (miRNAs) are a class of highly conserved small noncoding RNAs that regulate target genes by binding to the 3′ untranslated region (3′-UTR) of messenger transcripts to repress their translation or regulate their degradation.6 Some critical roles of miRNAs in the SMC phenotype change have been documented.7However, whether an miRNA-dependent mechanism contributes to ADMA-induced changes in the SMC phenotype remains unknown.
In this study, we hypothesized that ADMA exerts effects on the miRNA transcriptome in SMCs, leading to phenotype change in SMCs. We aimed to identify miRNA-regulated targets and to inhibit the SMC phenotype change by modulating the miRNA-dependent mechanism. We also aimed to identify a correlation between plasma ADMA concentrations and miRNA (and its specific target) expression levels both in carotid plaques from patients who had undergone carotid endarterectomy and in plasma from patients with coronary heart disease.
Materials and Methods
Materials and Methods are available in the online-only Data Supplement.
Downregulation of miR-182-3p in ADMA-Treated Human Aortic Artery SMCs
ADMA caused an miRNA transcriptome change in human aortic artery SMC (hASMC), of which miR-182-3p was most changed with the smallest P value. To corroborate these findings, the expression of miR-182-3p was further evaluated in the ADMA-induced hASMCs using quantitative real-time polymerase chain reaction (RT-PCR). As shown in Figure 1A and 1B, the treatment of human SMCs with ADMA significantly downregulated the expression of miR-182-3p in a time- and dose-dependent manner. VSMCs and endothelial cells are 2 major cell types in normal vascular walls. Here, the expression levels of miR-182-3p were validated to be reduced in human VSMCs from different tissues but not changed in endothelial cells (Figure I in the online-only Data Supplement) after ADMA treatment. The in situ hybridization of miR-182-3p (green color) showed that it was expressed primarily in the vessel media, where VSMCs were localized (Figure 1C).
Downregulation of miR-182-3p Mediates ADMA-Induced hASMCs Phenotype Change
To investigate the role of miR-182-3p knockdown in the ADMA-mediated SMC phenotype switch, we performed loss-of-function studies using the adenovirus-mediated delivery of a specific inhibitor of miR-182-3p into hASMCs. The transduction of SMCs with Ad-miR-182-3p inhibitor resulted in the reduced expression of miR-182-3p in a dose-dependent manner (Figure IIA in the online-only Data Supplement). Both ADMA and the miR-182-3p inhibitor reduced the expression of VSMC differentiation marker genes, such as SMA and calponin, as shown from the Western blotting, increased hASMC migration levels, and enhanced the hASMC proliferation rate, as measured from the BrdU (5-bromo-2′-deoxyuridine) incorporation rate (Figure 2A and 2B). These findings indicate that both ADMA and the reduction of miR-182-3p effectively induced the human SMC phenotype switch.
Then, we performed gain-of-function studies using the adenovirus-mediated delivery of miR-182-3p into hASMCs (Figure IIB in the online-only Data Supplement) to delineate whether ADMA induced the hASMC phenotype change via the inhibition of miR-182-3p. Increased miR-182-3p expression clearly restored the expression of SMA and calponin, to a normal level, suggesting that the ADMA-induced reduction in SMC differentiation marker proteins is miR-182-3p–knockdown associated. Accordingly, miR-182-3p overexpression significantly blunted ADMA-induced hASMC migration and proliferation (Figure 2C). Taken together, our results indicate that ADMA induces hASMCs to change from the differentiated phenotype to the dedifferentiated phenotype via an miR-182-3p–dependent mechanism.
Myeloid-Associated Differentiation Marker Is the Direct Target of miR-182-3p
A proteome-wide screening for miR-182-3p–specific target genes was performed using isobaric tags for relative and absolute quantitation–coupled 2D LC-MS/MS (liquid chromatograph-mass spectrometer) approaches to identify changes in the proteome of hASMCs. miR-182-3p overexpression led to the altered expression (P<0.05) of 65 proteins 48 hours after treatment (Table I in the online-only Data Supplement), of which the human myeloid–associated differentiation marker (gi|66932929, NM_001020818) was most reduced with the smallest P value (Figure III in the online-only Data Supplement). Using the online target gene prediction software program TargetScan (http://www.targetscan.org/mamm_31/), we identified a predicted miR-182-3p-binding site in the 3′-UTR region of human MYADM (Figure VIA in the online-only Data Supplement).
Next, we determined whether the MYADM 3′-UTR was a functional target of miR-182-3p. As shown in Figure 3A, the overexpression of an miR-182-3p mimic, but not the nontargeting mimic control, substantially repressed the activity of luciferase fused with the wild-type-3′-UTR of MYADM but had no effect on luciferase activity when the miR-182-3p-binding sites in the 3′-UTR of MYADM were mutated, suggesting that miR-182-3p can directly bind to the 3′-UTR of MYADM and regulate its expression. Moreover, transfection with the miR-182-3p inhibitor, but not the nontargeting inhibitor control, significantly increased the activity of luciferase fused with the wild-type-3′-UTR of MYADM (Figure 3A).
Using Western blotting in cultured hASMCs, we confirmed that miR-182-3p overexpression by transduction with Ad-miR-182-3p reduces MYADM protein expression, whereas miR-182-3p knockdown by transduction with Ad-miR-182-3p inhibitor increases MYADM protein expression (Figure 3B). Similar to the Ad-miR-182-3p inhibitor, ADMA increased MYADM protein expression in hASMCs in a concentration-dependent manner (Figure 3C), which suggests that ADMA inhibits miR-182-3p expression and thus increases MYADM expression because ADMA has been verified to reduce miR-182-3p expression.
Role of miR-182-3p/MYADM in the VSMC Phenotype Change
To substantiate the functional significance of MYADM in SMC function, we performed a loss-of-function study using MYADM siRNA. As shown in Figure 4A, the transfection of VSMCs with MYADM siRNA significantly inhibited both basal and ADMA-induced MYADM expression. Accordingly, the knockdown of MYADM by specific siRNA markedly restored the expression of ADMA-reduced SMC contractile genes (SMA and calponin) to normal levels, as determined by a Western blot analysis. In addition, ADMA-induced SMC proliferation and migration was significantly blocked by the MYADM knockdown in hASMCs (Figure 3D).
To further determine the contribution of MYADM in miR-182-3p–mediated hASMC function, we examined the effect of Ad-miR-182-3p inhibitor/Ad-miR-182-3p on MYADM expression. First, in the absence of Ad-miR-182-3p, the transfection of hASMCs with an MYADM overexpression plasmid significantly increased the expression of MYADM and induced the hASMC phenotype change, whereas pretreatment with Ad-miR-182-3p in hASMCs markedly attenuated the effects of MYADM overexpression on hASMC phenotype switch and MYADM expression (Figure IVA in the online-only Data Supplement). Second, in the absence of the Ad-miR-182-3p inhibitor, the knockdown of MYADM by transfection with a specific siRNA significantly reduced the expression of MYADM and blunted the ADMA-induced hASMC phenotype change, whereas the knockdown of miR-182-3p with Ad-miR-182-3p inhibitor in hASMCs markedly attenuated the effect of MYADM siRNA on the ADMA-induced hASMC phenotype switch and MYADM expression (Figure IVB in the online-only Data Supplement). These results suggest that MYADM is a critical regulator for SMC differentiation, proliferation, and migration and is involved in the miR-182-3p knockdown–mediated effects on VSMC function.
Taken together, miR-182-3p/MYADM is critically implicated in the ADMA-induced VSMC phenotype change because ADMA has been verified to regulate miR-182-3p/MYADM expression.
MYADM Activates the Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase Signaling Pathway in hASMCs
The effects of miR-182-3p/MYADM on hASMCs, at least in part, associated with the activation of extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase signaling pathway. As shown in Figure 4A, ADMA treatment caused an increase in MYADM expression and a 0.45-fold decrease in the expression of the ERK/MAP kinase inhibitor Sprouty2 (SPRY2), leading to enhanced ERK1/2 phosphorylation, which was blunted by the overexpression of miR-182-3p in hASMCs, as shown by the return of MYADM, SPRY2 expression, and ERK1/2 phosphorylation to normal levels. It suggests that overexpression of MYADM via the reduction of miR-182-3p associates with the activation of ERK1/2 signaling way.
The small interfering RNA–mediated reduction of SPRY2 and the overexpression of MYADM were both used to determine the correlation between MYADM induction and ERK1/2 activation. Similar to the effects of ADMA, both SPRY2 reduction and MYADM overexpression resulted in increased ERK1/2 phosphorylation (Figure 4B). The fact that the knockdown of SPRY2 did not change the MYADM expression level, whereas the overexpression of MYADM reduced the SPRY2 expression level, indicates that the overexpression of MYADM leads to the inhibition of SPRY2 expression and hence increases the activation of the ERK signaling pathway. Hence, the ADMA-induced reduction of miR-182-3p leads to the overexpression of MYADM and then the ERK/MAP kinase signaling pathway in hASMCs is activated.
miR-182-3p/MYADM Modules the hASMC Phenotype Change via the ERK1/2 Signaling-Dependent Mechanism
To further delineate the roles of ERK1/2 signaling pathway in the miR-182-3p/MYADM-modulated hASMC phenotype change, cells were transfected with human MYADM siRNA/overexpression plasmid or treated with the ERK1/2 ERK signaling pathway blocker PD9805.
As shown in Figure VA in the online-only Data Supplement, both MYADM overexpression–induced and ADMA-induced ERK1/2 phosphorylation were blunted by Ad-miR-182-3p or PD9805. Accordingly, the effects of MYADM overexpression and ADMA on the hASMC phenotype change were both attenuated by Ad-miR-182-3p or PD9805, which was shown in the normalized expression levels of SMC differentiation marker genes and proinflammation mediators, such as intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and osteopontin (OPN), as well as normal migration and proliferation capacities (Figure VA in the online-only Data Supplement).
Comparable to the effects of ADMA, Ad-miR-182-3p inhibitor also reduced the expression levels of SMC differentiation marker genes and increased the expression levels of proinflammation mediators, such as ICAM-1, VCAM-1, and OPN. Both MYADM knockdown and PD9805 treatment prevented ADMA-mediated and Ad-miR-182-3p inhibitor–mediated ERK1/2 phosphorylation and altered calponin, SMA, ICAM-1, VCAM-1, and OPN protein gene expression levels in the hASMCs. Additionally, hASMC proliferation and migration abilities were both reduced to the normal levels (Figure VB in the online-only Data Supplement).
This indicates that both ADMA- and miR-182-3p inhibition–induced hASMC phenotype changes occur via the MYADM overexpression–dependent and ERK1/2 signaling–dependent mechanism. In consideration of the activation of MYADM on the ERK1/2 signaling way and in view of the inhibition of ADMA on miR-182-3p, we deduced that ADMA reduced miR-182-3p expression level, which is followed by the MYADM-mediated activation of ERK1/2 signaling way and, hence, induced the phenotype change in hASMCs.
miR-182-3p Inhibits Balloon Injury–Induced Neointimal Formation in Rat Carotid Arteries via the Knockdown of MYADM
The binding site of miR-182-3p in the MYADM 3′-UTR is conserved among different species, such as humans, rats, and mice (Figure VIA in the online-only Data Supplement). Thus, to investigate whether the inhibition of miR-182-3p induces the SMC phenotype change in other species, we performed studies using rat SMCs. Similar to our findings in human cells, the inhibition of miR-182-3p increased the expression of MYADM in rat SMCs (Figure VIB in the online-only Data Supplement).
To determine whether miR-182-3p is involved in vascular lesion formation in vivo, a rat carotid artery balloon injury model was used and neointimal formation was examined 14 days after carotid injury. Rat carotid arteries were transduced with either Ad-miR-182-3p (1010 pfu/mL) or ctrl-Ad-miR (1010 pfu/mL). Compared with the ctrl-Ad-miR–treated group, the expression of miR-182-3p in the carotid arteries of the Ad-miR-182-3p–treated rats was markedly increased, and the target, MYADM, was significantly reduced (Figure VIIA and VIIB in the online-only Data Supplement), as determined by quantitative RT-PCR. Balloon injury led to a substantial increase in neointimal formation in carotid arteries transduced with ctrl-Ad-miR. The transduction of carotid arteries with Ad-miR-182-3p, however, substantially inhibited balloon injury–induced neointimal formation by decreasing the neointimal-to-media ratio and the intimal–media thickness (Figure 5A and 5B).
To confirm that the effects of miR-182-3p on MYADM and downstream molecular events were localized to the VSMCs, we performed coimmunofluorescence with the smooth muscle marker SMA (red color) and MYADM, calponin, proliferating cell nuclear antigen (PCNA), ICAM-1, VCAM-1, OPN, or SPRY2 (green color) in the carotid artery. Interestingly, balloon injury led to an obvious increase in the ratio of MYADM-positive to SMA-positive cells, which was substantially reversed by Ad-miR-182-3p transduction in the carotid arteries compared with the Ctrl-Ad-miR–treated arteries. Moreover, the ratios of PCNA-, OPN-, ICAM-, and VCAM-positive cells were significantly decreased, whereas the number of calponin-positive and SRY2-positive cells was increased in Ad-miR-182-3p–transduced arteries compared with those of the Ctrl-Ad-miR–treated arteries (Figure 5C and 5D). In addition, these immunohistochemical changes could be quantitatively documented by a Western blot analysis (Figure VIII in the online-only Data Supplement). These results suggest that miR-182-3p inhibits balloon injury–induced neointimal formation by suppressing MYADM-mediated SMC migration, proliferation, and phenotype change. In addition, in the normal carotid arteries, MYADM distributes in nucleus and cytoplasm. The balloon injury significantly induced MYADM translocation into the nuclei in the neointima, which was blunted by the transduction of Ad-miR-182-3p in rat carotid arteries (Figure IX in the online-only Data Supplement). Nevertheless, the mechanism of MYADM translocation into the nuclei is still unknown.
Inhibition of Endogenous miR-182-3p Promotes Balloon Injury–Induced Neointimal Formation in Rat Carotid Arteries via MYADM Overexpression
Another rat carotid artery balloon injury model was used to determine whether the inhibition of endogenous miR-182-3p promotes balloon injury–induced neointimal formation. The carotid arteries of the rats were transduced with either the adenovirus-mediated delivery of a specific inhibitor of miR-182-3p (the Ad-miR-182-3p-inhibitor, 1010 pfu/mL) or a ctrl-Ad-miR-inhibitor (1010 pfu/mL).
Fourteen days after transduction, the expression of miR-182-3p in the rat carotid arteries was markedly decreased, and the target MYADM was significantly increased (Figure VIIC and VIID) in the Ad-miR-182-3p inhibitor–treated group compared with the Ctrl-miR inhibitor–treated group, as determined by quantitative RT-PCR. Notably, the transduction of carotid arteries with Ad-miR-182-3p inhibitor substantially promoted balloon injury–induced neointimal formation, as evidenced by the increased neointimal-to-media ratio and the intimal–media thickness compared with the ctrl-Ad-miR inhibitor–treated arteries (Figure XA–XC in the online-only Data Supplement). The ratios of MYADM-positive, PCNA-, ICAM-1-, VCAM-1-, and OPN-positive cells to SMA-positive cells were significantly increased, whereas the number of calponin- and SPRY2-positive cells was significantly decreased, in the Ad-miR-182-3p inhibitor–transduced arteries compared with the ctrl-Ad-miR inhibitor–treated arteries. Taken together, these results suggest that the inhibition of endogenous miR-182-3p promotes balloon injury–induced neointimal formation in vivo by increasing MYADM-mediated SMC proliferation, migration, and phenotype switching. Additionally, these immunohistochemical changes could be quantitatively analyzed by Western blotting (Figure XI in the online-only Data Supplement). Moreover, the balloon injury–induced MYADM translocation into the nuclei was significantly promoted by the transduction of Ad-miR-182-3p inhibitor in rat carotid arteries (Figure XII in the online-only Data Supplement), which indicates the miR-182-3p involved in the regulation of MYADM translocation into the nuclei.
miR-182-3p/MYADM Is Expressed in Human Atherosclerotic Plaques and Is Correlated With Plasma ADMA Levels
Next, to translate our in vitro and in vivo findings into a clinical scenario, plaques were collected from patients who had undergone carotid endarterectomy for extracranial high-grade internal carotid artery stenosis at the Chinese People’s Liberation Army General Hospital (n=12; Table II in the online-only Data Supplement). Based on the ADMA median concentration (0.5250 μM), the patients were divided into a high ADMA group (≥0.5250 μM) and a low ADMA group (<0.5250 μM).
The in situ hybridization of miR-182-3p (green color) showed that miR-182-3p was expressed in the plaques (Figure 6A). Plaques from high ADMA group patients exhibited significantly decreased miR-182-3p expression levels compared with those from low ADMA group patients (Figure 6A), which was verified by miR-182-3p expression levels detected using an RT-PCR method (Figure 6B). Moreover, the miR-182-3p expression in plaques was inversely correlated with ADMA plasma levels, as determined by a Spearman correlation model (r=−0.666; P=0.018; Figure 6D). Plaques from high ADMA group patients exhibited significantly increased MYADM expression compared with those from low ADMA group patients (Figure 6C). The expression levels of MYADM and miR-182-3p in the plaques are summarized in Table III in the online-only Data Supplement.
In the present study, we identified miR-182-3p as a novel modulator implicated in human SMC differentiation, proliferation, and migration. We uncovered 3 major findings. First, we showed that the expression of miR-182-3p is markedly downregulated in ADMA-treated hASMCs. Second, both loss-of-function and gain-of-function studies suggest that miR-182-3p plays a key role in the SMC phenotype switch in vitro and in vivo via the miR-182-3p–specific target gene MYADM, which activates the ERK1/2 signaling pathway. Third, we also observed that the ADMA-induced SMC phenotype change via the inhibition of miR-182-3p expression is associated with atherosclerotic lesion formation in clinical scenarios.
miR-182 has been designated an oncology-related miRNA8 whose level positively correlates with the initiation and development of a variety of tumors9 because of its role in the regulation of cell migration and proliferation. However, the involvement of miR-182 in cell differentiation, migration, and proliferation is controversial. For example, miR-182 mediates the inhibition of proliferation and migration by atorvastatin in prostate cancer cells10 but increases tumor cell proliferation and migration in human ovarian carcinomas,11 which suggests that miR-182-modulated proliferation and migration is cell-specific and dependent on the culture environment. The endogenous nitric oxide synthase inhibitor ADMA, as used in the culture environment in our present study, has been shown to induce the proliferation, migration, and apoptosis of rat SMCs. Here, we present evidence that ADMA reduces miR-182-3p expression in hASMCs, leading to the SMC phenotype change (manifested by the reduced expression of differentiation genes and increased proliferation and migration). In view of the pathological effects of ADMA on the cardiovascular system, it would be interesting to investigate whether miR-182-3p is involved in ADMA-associated cardiovascular events. In addition, it is worth noting that ADMA has been previously shown to upregulate the expression of miR-21 in endothelial cells12; miR-21 is another important miRNA involved in the SMC phenotype change, as shown by the fact that miR-21 overexpression induces the SMC phenotype switch, whereas miR-21 silencing inhibits cell proliferation in rat aortic SMCs and in injured rat carotid arteries.13 Although ADMA does not change miR-21 expression in hASMCs, our present study indicates that the reduction of miR-182-3p markedly contributes to the ADMA-induced VSMC phenotype change. Moreover, we validated the primary distribution of miR-182-3p in the vessel media by performing an in situ hybridization, and ADMA did not change the miR-182-3p expression levels in endothelial cell, as measured by miR-182-3p–specific RT-PCR. Hence, the action of miR-182-3p in response to ADMA is SMC-specific, and miR-182-3p is another SMC phenotype change modulator. Nevertheless, at this time, little is known about the role of miR-182-3p in SMC biology.
Our second finding is that MYADM is the specific target of miR-182-3p and contributes to miR-182-3p–mediated hASMC function. MYADM is a novel hematopoietic-associated marker composed of 322 amino acids that are encoded by the MYADM gene in humans. MYADM is thought to mediate Rac1 targeting to ordered membranes, which is required for cell spreading and migration.14 Here, we further extend our knowledge regarding the role of MYADM in increased SMC phenotype change via direct regulation by an miR-182-3p–dependent mechanism both in vitro and in vivo using the gain-of-function and loss-of-function of miR-182-3p strategies. It is worth noting that in the neointima, MYADM expression is increased, which suggests that MYADM is a potential target of vascular remodeling. Moreover, our present study indicates that the effects of ADMA and miR-182-3p knockdown on MYADM expression are at least partially associated with the ERK/MAP kinase signaling pathway. These results are in agreement with those of previous studies demonstrating that ADMA induces oxidative stress via the ERK/MAP kinase activation induced by the inhibition of SPRY2.12 Furthermore, the ERK1/2 pathway is also downregulated by miR-182 in the posterior uveal melanoma,15 confirming our finding that the inhibition of miR-182-3p activates the ERK1/2 pathway. Thus, the inhibition of miR-182-3p expression and the subsequent increase in MYADM expression induced by ADMA is associated with ERK activation.
The SMC phenotype change contributes to atherosclerotic lesion formation.16 Although the cellular and molecular mechanisms involved remain elusive, it has been ascertained that the majority of intimal SMCs within atherosclerotic lesions are derived from resident medial SMCs that undergo phenotypic modulation and migration into the intima where they proliferate, produce extracellular matrix, and participate in fibrous cap formation.16,17 In fact, SMCs are the main cell type in early arterial intimal thickenings and a major component of most stages of human atherosclerosis.18 Recently, SMCs were observed to contribute >50% of the total foam cells in human coronary intimas, which suggests that SMCs are a major source of macrophage-like cells in human atherosclerotic plaques.19
In the present study, although the effects of miR-182-3p on SMCs in atherosclerotic plaques were not evaluated, it can be inferred that the ADMA-mediated deregulation of miR-182-3p/MYADM may be another cause of SMC phenotype switching in atherosclerotic lesions because ADMA has been confirmed to induce atherosclerotic plaque formation in ApoE−/− mice.20 In agreement with this result, an ApoE−/−/hDDAH1+/−transgenic mouse that overexpressed human isoform 1 of the ADMA-degrading enzyme dimethylarginine dimethylaminohydrolase was generated to reduce plaque formation in ApoE−/− mice by lowering ADMA, which constituted direct evidence of the role of ADMA in atherosclerotic plaque formation.17 Hence, our findings provide proof-of-principle that the ADMA-induced SMC phenotype change plays a causal role in atherosclerosis via an miR-182-3p knockdown–dependent mechanism, which raises the possibility that miR-182-3p is a potential therapeutic treatment for cardiovascular diseases, such as atherosclerosis.
Although the functions of various miRNAs have been identified in various cultured cells or animal models, the involvement of miRNAs in human atherosclerotic plaques has received little attention. In the present study, the role of the ADMA-induced SMC phenotype change via miR-182-3p inhibition was validated in clinical scenarios, as evidenced by the negative correlation between plasma ADMA concentrations and miR-182-3p expression levels in atherosclerotic plaques collected from patients with carotid artery stenosis. ADMA is a novel alarm signal and potent factor in vascular remodeling; hence, we suggests that miR-182-3p reduction might be an indicator of high system vascular remodeling. Nevertheless, one unavoidable limitation of our study is that we did not show direct evidence that the reduction in circulating miR-182-3p was derived from the ADMA-induced SMC phenotype change.
Taken together, the results of the present study demonstrate that ADMA induces SMC dedifferentiation, proliferation, and migration via an miR-182-3p knockdown/MYADM–dependent mechanism that is associated with atherosclerotic plaque formation. Our findings raise the possibility that miR-182-3p is a new SMC phenotype change regulator and plays a critical role in regulating atherosclerotic plaque formation because the SMC phenotype change is critical in atherosclerotic plaque formation.
We thank the native English-speaking experts of Nature publishing group language editing for their text editing.
Sources of Funding
This work was supported by National Science Foundation (Grant number: 81102445 and 81100878), Beijing Natural Science Foundation (Grant number: 7162132), the PUMC Youth Fund and the Fundamental Research Funds for the Central Universities (Grant number: 33320140069), and the grant from the Key National Basic Research Program of China (2012CB517503, 2013CB530804).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.115.307412/-/DC1.
- Nonstandard Abbreviations and Acronyms
- asymmetrical dimethylarginine
- human aorta artery smooth muscle cell
- intercellular adhesion molecule 1
- myeloid-associated differentiation marker
- smooth muscle α-actin
- smooth muscle cell
- vascular cell adhesion molecule 1
- smooth muscle cell
- Received February 17, 2016.
- Accepted April 21, 2016.
- © 2016 American Heart Association, Inc.
- Kyuragi R,
- Matsumoto T,
- Harada Y,
- Saito S,
- Onimaru M,
- Nakatsu Y,
- Tsuzuki T,
- Nomura M,
- Yonemitsu Y,
- Maehara Y
- Li P,
- Zhu N,
- Yi B,
- Wang N,
- Chen M,
- You X,
- Zhao X,
- Solomides CC,
- Qin Y,
- Sun J
- Hasegawa K,
- Wakino S,
- Tatematsu S,
- Yoshioka K,
- Homma K,
- Sugano N,
- Kimoto M,
- Hayashi K,
- Itoh H
- Sun L,
- Zhang T,
- Yu X,
- Xin W,
- Lan X,
- Zhang D,
- Huang C,
- Du G
- Griffiths-Jones S,
- Grocock RJ,
- van Dongen S,
- Bateman A,
- Enright AJ
- Peng X,
- Li W,
- Yuan L,
- Mehta RG,
- Kopelovich L,
- McCormick DL
- Fleissner F,
- Jazbutyte V,
- Fiedler J,
- Gupta SK,
- Yin X,
- Xu Q,
- Galuppo P,
- Kneitz S,
- Mayr M,
- Ertl G,
- Bauersachs J,
- Thum T
- Ji R,
- Cheng Y,
- Yue J,
- Yang J,
- Liu X,
- Chen H,
- Dean DB,
- Zhang C
- Aranda JF,
- Reglero-Real N,
- Kremer L,
- Marcos-Ramiro B,
- Ruiz-Sáenz A,
- Calvo M,
- Enrich C,
- Correas I,
- Millán J,
- Alonso MA
- Gomez D,
- Owens GK
- Jacobi J,
- Maas R,
- Cardounel AJ,
- Arend M,
- Pope AJ,
- Cordasic N,
- Heusinger-Ribeiro J,
- Atzler D,
- Strobel J,
- Schwedhelm E,
- Böger RH,
- Hilgers KF
- Allahverdian S,
- Chehroudi AC,
- McManus BM,
- Abraham T,
- Francis GA
- Cherepanova OA,
- Pidkovka NA,
- Sarmento OF,
- Yoshida T,
- Gan Q,
- Adiguzel E,
- Bendeck MP,
- Berliner J,
- Leitinger N,
- Owens GK
miR-182-3p and its target myeloid-associated differentiation marker are potential therapeutic targets for combating vascular remodeling–associated diseases, such as atherosclerosis.
We found a deregulation of miR-182-3p and myeloid-associated differentiation marker expression levels in atherosclerotic plaque.
miR-182-3p expression levels in plasma and in plaque are negative correlation to plasma asymmetrical dimethylarginine (identified as an alarm signal and potent factor in vascular remodeling) level, which suggested that miR-182-3p reduction can be another plasma marker of high vascular remodeling risk.