miR-145 Alleviates Smooth Muscle Cell Phenotype Transition via ADAM17-Mediated ACE2 Shedding

It has been shown that miR-145 is involved in the differentiation of vascular smooth muscle cells (VSMCs) and may regulate vascular remodeling. However, the molecular mechanisms behind these pathological processes in hypertension are not fully elucidated. The present study was to examine whether miR-145 modulates phenotypic transformation of VSMCs under normal state and synthetic state and to explore the possible role of ADAM17-mediated ACE2 shedding and ACE2-Ang-(1–7)-Mas receptor axis. Wistar rats were fed with high-sucrose/high-fat diet for 30 weeks to establish a metabolic hypertension animal model. VSMCs were cultured and treated with Ang II with or without miR-145 mimics or miR-145 inhibitor. Results showed the expression of contractile markers α-SMA and SM22α, miR-145, ACE2, and Mas receptor reduced in the thoracic aorta of metabolic hypertensive rats (MHRs), while that of synthetic marker OPN increased as compared to the control group. In in vitro study, miR-145 inhibitor inhibited the expression of α-SMA, SM22α, ACE2, Mas receptor, and the Ang-(1–7) excretion and induced the expression of synthetic markers OPN, EREG, and MMP2. However, miR-145 mimic produced opposite effects on the VSMCs. In addition, in the synthetic VSMC induced by Ang II, miR-145 inhibitor partially reversed the induced expression of OPN, EREG, and MMP2 by Ang II, while further decreasing the expression of α-SMA and SM22α and ACE2-Ang-(1–7)-Mas receptor. Cotreatment with ADAM17 siRNA partially reversed the inducible effect of miR-145 inhibitor on the EREG and MMP2, induced Ang-(1–7) excretion, and upregulated ACE2 and Mas receptor expression. In conclusion, miR-145 alleviates phenotype transition from contractile to synthetic type via ADAM17-mediated ACE2 shedding in VSMCs and retains the activation of ACE2-Ang-(1–7)-Mas axis, which may benefit the vascular structural remodeling in the metabolic hypertension.


Introduction
Hypertension is one of the most frequent cardiovascular disorders, and a leading global risk factor for death and disability worldwide [1]. It is reported that around 9.4 million deaths annually were attributed to hypertension [2], and the number of individuals diagnosed with hypertension is estimated to reach 1.5 billion globally by 2025 [1,3]. Although treatment in hypertension can prevent the onset of cardiovascular events, existing therapies are only partially efective. Te lack of defnitive data as to the mechanisms for the hypertension contributes to this sign. A key pathological hallmark of hypertension is increased peripheral vascular resistance due to structural and functional changes in large (conductive) and small (resistance) arteries. Vascular remodeling is an important step in the occurrence and development of hypertension [4]. Smooth muscle cells (SMCs) are involved in the physiological and pathological vascular remodeling due to their remarkable ability to dynamically modulate their phenotypes. Te phenotypic switching of SMCs refers to the shift between a diferentiated "contractile" phenotype and a dediferentiated "synthetic" phenotype in the SMCs. Synthetic phenotype is characterized by the loss of SMC markers, the increase in extracellular matrix (ECM) component synthesis, and the proliferation and migration of SMCs in response to various stimuli and environmental cues. Phenotypic evolution of SMC can be assessed by SMC markers, in vivo lineage-tracing and fate mapping [5,6]. It has been established that α-SMA and SM22α are classical contractile markers and OPN, epiregulin (EREG), and matrix metalloproteinase-2 (MMP2) are synthetic markers [7,8]. Adaptive phenotype alterations are essential for vascular development and repair, but dysregulation of this process plays a critical role in the pathological vascular remodeling [9]. Te elucidation of mechanisms underlying the regulation of phenotype transition is of utmost importance for better understanding of vascular diseases.
MicroRNAs (miRNAs or miRs) are endogenous small noncoding RNAs that can regulate translation and degradation of messenger RNAs (mRNAs) at the posttranscriptional level [10][11][12]. miR-145 is a conserved miR-NAs encoded by a bicistronic gene cluster and most frequently expressed miRNA in SMCs. It targets a network of transcription factors, including Kruppel-like factor 4 (Klf4), myocardin, and Elk-1 (ELK1, member of ETS oncogene family), to promote the diferentiation and repress the proliferation of SMCs [13]. A disintegrin and metalloprotease 17 (ADAM17) is responsible for the EGFR transactivation, which subsequently modulates vascular remodeling. Compared with control mice, vascular ADAM17-defcient mice and ADAM17 antibody treated mice show alleviated cardiac hypertrophy, vascular medial hypertrophy, and perivascular fbrosis induced by Ang II via activating EGFR, which is independent of blood pressure regulation [14,15]. ADAM17 governs angiotensin converting enzyme 2 (ACE2) shedding from cell membranes and promotes ACE/angiotensin II (Ang II) pressor and tissue remodeling action. It has been revealed that ADAM17 and miR-145 share the common binding domain, and ADAM17 is a direct substrate of miR-145 [16], which contributes to the migration and invasion of cancer [17][18][19][20]. However, the role of miR-145/ADAM17 pathway in the vascular remodeling in case of hypertension and the potential mechanisms is still poorly understood.
In the present study, the role of miR-145 pathway in the phenotype transition of VSMC induced by Ang II was investigated in vitro and in a rat model of metabolic hypertension, in which maladaptive vascular remodeling was obvious in our previous study [21]. Furthermore, the potential regulatory efects of miR-145/ADAM17 pathway on the vascular remodeling and RAAS activity were explored in vitro. Our fndings will help reveal the role and mechanism of microRNAs in vascular remodeling of metabolic hypertension and provide experimental evidence for the development of new therapeutic targets for vascular remodeling.

Ethics Statement.
Tis study was carried out with accordance to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Te protocol was approved by the Animal Ethics Committee of the 3rd Xiangya Hospital of Central South University. All eforts were made to minimize sufering.

Metabolic Hypertension Model.
Te metabolic hypertension rat model was established as previously reported [21]. Male Wistar rats weighing 250-290 g were purchased from Beijing Vital River Laboratory Animal Technology Co Ltd. After 7-day acclimatization, male Wistar rats were randomly assigned to 2 groups. In the control group (Ctrl, n � 10), animals were fed with standard chow diet (animal experiment center of the Tird Xiangya Hospital, Central South University, Changsha); in the metabolic hypertension group (MH, n � 12), animals were fed with a high-salt, highfat diet (HSHF, standard diet 58%, lard stearin 12%, yolk power 10%, sugar 5%, peanut 5%, milk power 5%, salt 4%, and sesame oil 1%) together with 20% fructose in the drinking water for 30 weeks. Te systolic blood pressure (SBP) was detected using a noninvasive computerized tailcuf system (NIBP, Shanghai Alcott Biotech). Before the measurement, rats were warmed for 10-20 min at 28°C to detect the steady arterial pulsations of the tail artery. Te SBP from 3 measurements was averaged for each animal. At the end of experiment, animals were euthanized with sodium pentobarbital (30 mg/kg, i.p), and the thoracic aorta was collected for histological examination as well as protein and RNA extraction.
2.9. ELISA. Te supernatant of cells was collected and centrifuged at 1000 g at 4°C. Ten, the supernatant was transferred to a sterilized tube. Ang-(1-7) ELISA kit (I190034618) was used to determine the level of Ang-(1-7) in the supernatant. All the procedures were carried out according to the manufacturer's instructions.

Luciferase Activity Assay.
A 286bp sequence containing the predicted miR-145 binding site at the 3′-untranslated regions (UTRs) of ADAM17 or a 286bp sequence containing a scrambled sequence was cloned into the XhoI/NotI site of the psiCHECK-2 vector (HonorGene, China) to generate PsiCHECK-2-ADAM17-WT and PsiCHECK-2-ADAM17mut vectors, respectively. For the luciferase assay, A7r5 cells were cultured in 12-well plates and cotransfected with 0.5 μg of PsiCHECK-2-ADAM17-WT or PsiCHECK-2-ADAM17mut and 50 nM of rno-miR-145-5p mimics or the negative control using the transfection reagent Lipofectamine 2000 (Invitrogen). 48 hours after transfection, the cells were harvested and luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Firefy luciferase was used for normalization. Experiments were repeated at least three times.
2.11. Statistical Analysis. All data are shown as mean-± standard error (SEM). One-way analysis of variance (ANOVA) following post hoc Student-Newman-Keuls test was used to determine the diferences among multiple groups. A value of P < 0.05 was considered statistically signifcant.

Vascular Remodeling and Phenotypes of VSMCs in Aortic
Arteries of Hypertensive Rats. Rats were fed a high-sucrose/ high-fat diet for 30 weeks to establish a rat MH model, which shows an obvious vascular remodeling [21]. Immunofuorescence staining and Western blotting were used to International Journal of Hypertension examine the location and expression of contractile markers (α-SMA and SM22α) and synthetic marker (OPN) in thoracic aorta, respectively. Compared to the control rats, MH rats showed a marked increase in the SBP after 6 weeks, which remained until 30 weeks (Figures 1(a) and 1(b)). α-SMA, SM22α, and OPN were mainly expressed in the cytoplasm of VSMCs (Figures 1(c)-1(e)). Western blotting and immunofuorescence staining showed that the expression of α-SMA and SM22α was signifcantly downregulated in the thoracic aorta of MH rats after 30 weeks, which was the most obvious in the VSMCs of the media layer (Figures 1(c),1(d), 1(f ), 1(g), 1(j), and 1(k), P < 0.01). Interestingly, empty zone was observed in the medial wall of MHRs (Figures 1(c) and 1(d)). In addition, the expression of OPN signifcantly increased in the thoracic aorta of MH rats (Figures 1(e), 1(h), and 1(l), P < 0.01), which was observed in the VSMCs of medium and adventitia of aortic wall (Figure 1(e)).

Expression of miR-145, ADAM17, and ACE2-Ang-(1-7)-Mas Receptor Axis in Aortic Arteries of Hypertensive Rats.
To compare the expression of miR-145, ADAM17, ACE2, and MASR in the thoracic aorta between control rats and MH rats, Western blotting and real-time PCR were used to detect the protein and gene expression of these molecules, respectively. Te miR-145 expression was signifcantly lower in the MH rats than in the control rats ( Figure 2(d), P < 0.05). Te mRNA expression of ACE2 and Mas receptor signifcantly reduced in the MH rats as compared to the control rats (Figures 2(a) and 2(c), P < 0.01). However, ADAM17 expression increased markedly in the MH rats ( Figure 2(e), P < 0.01 vs control rats).

miR-145 Promoted VSMC Phenotype Transition from Synthetic to Contractile Phenotype and Augmented Activation of ACE2-Ang-(1-7)-Mas Receptor Axis.
To examine the efect of miR-145 on phenotype transition in VSMCs, Western blotting was used to detect the expression of synthetic and contractile markers. Results indicated that the gene expression of miR-145 was greatly promoted by miR-145 mimic and signifcantly inhibited by miR-145 inhibitor (P < 0.001, supplement fgure 1C). Te expression of α-SMA and SM22α in the miR-145 mimic-treated group increased signifcantly (Figures 3(a) and 3(b), P < 0.01), and the OPN and EREG expression decreased markedly as compared to the control group (Figure 3(c), P < 0.01). However, miR-145 inhibitor treatment signifcantly increased the expression of OPN, EREG, and MMP2, but decreased the expression of α-SMA and SM22α (Figures 3(a)-3(c), P < 0.01).

Knockdown of ADAM17 Reversed Phenotype Transition
Induced by miR-145 In Vitro. ADAM17 is a direct substrate of miR-145, and whether ADAM17 mediates miRNA-145induced efect on the phenotypic transition is still unknown. In our experiment, results showed miR-145 mimic inhibited ADAM17 expression and Ang II-induced ADAM17 expression (Figure 4(d), both P < 0.01), while miR-145 inhibitor further augmented Ang II-induced effect on ADAM17 (Figure 4(d), P < 0.01), which indicated that ADAM17 mediated the efect of miR-145 on the phenotype transition of VSMCs. Compared with the control group, ADAM17 knockdown promoted the expression of α-SMA ( Figure 5(a), P < 0.01) and SM22α ( Figure 5(b), P < 0.01) and inhibited the expression of OPN ( Figure 5(c), P < 0.01). Of interest, ADAM17 itself had no efect on the expression of EREG and MMP2, while ADAM17 siRNA at least partially alleviated the induced efect of miR-145 inhibitor on these two synthetic markers (Figures 5(d) and 5(e), P < 0.01). To experimentally confrm that ADAM17 is a direct substrate of miR-145 by targeting the putative binding site, we performed luciferase reporters by cloning the wild-type 3′-UTR of ADAM17 or its mutant versions downstream of the frefy luciferase open reading frame. Te result showed that miR-145 reduced the ADAM17 3′-UTR luciferase activity compared with the negative control, and no efects was observed on the mutated ADAM17 3′-UTR luciferase reporter ( Figure 5(g)), indicating that ADAM17 is a direct target of miR-145. Terefore, miR-145 is involved in the phenotype transition likely via ADAM17 in the VSMCs.  Figure 1: SMC phenotypes in aortic arteries of hypertensive rats. 22 male Wistar rats were randomly divided into control group (con group, n � 10) or metabolic hypertension group (MHR group, n � 12). Standard rat chow diet was administered to the control group and a highsucrose/high-fat diet was administered to the MHR group. Immunoblot analysis: (c-e) the thoracic aorta was collected to detect α-SMA-, SM22α-, and OPN-positive cells by immunofuorescence staining (green), and nuclei were stained with DAPI (blue). (f-h) Te quantifcation of α-SMA, SM22α, and OPN expression from panel (c-e). Western blotting: (j-l) Te quantifcation of α-SMA, SM22α, and OPN expression in the aorta from panel (i) and the data were expressed after normalization to β-actin. (i) Representative fgures from Western blotting of α-SMA, SM22α, OPN, and β-actin. (a, b) SBP and DBP were measured with the tail-cuf method every 6 weeks in groups. * * P < 0.01 between groups, and * P < 0.05 between groups. # P < 0.01 vs. baseline SBP. All the data are expressed as mean ± SEM. MHR, metabolic hypertension rats; SBP, systolic blood pressure; and DBP, diastolic blood pressure.

Discussion
Our results revealed a novel role of miR-145 in regulating vascular remodeling. miR-145, which preserved the contractile phenotype of VSMC, was a robust inducer of ACE2-Ang-(1-7)-MAS axis, and ADAM17 modulated this efect.
Vascular remodeling is a characteristic pathological feature of hypertension, and VSMCs dysfunction is the important foundation of vascular remodeling. Phenotype transition of VSMCs is one key factor of vascular remodeling. miR-145 is a member of noncoding single stranded RNA, which has been proved to be highly expressed in VSMC and necessary for the diferentiation and function of these cells, as well as an important determinant for phenotypic transition of VSMCs in response to vascular injury, such as subarachnoid hemorrhage [22]. In our study, the expression of miR-145 signifcantly decreased in rats with metabolic hypertension (Figure 2), which was consistent with fndings from patients with essential hypertension [23]. miR-145 is able to regulate the proliferation and migration of VSMCs in spontaneous hypertension rats (SHRs) [24], while its function in phenotype transition of VSMCs is still unknown. Our fndings for the frst time indicated that miR-145 promoted the phenotype switch of VSMCs from synthetic to contractile phenotype ( Figure 3). As Ang II is an  from panel (f ) and the data were normalized to that of GAPDH. * * P < 0.01 vs. control group; * P < 0.05 vs. control group; ## P < 0.01 vs. Ang II group; and # P < 0.05 vs. Ang II group. All the data are expressed as mean ± SEM from three independent experiments. miRNA NC, negative control miRNA. 8 International Journal of Hypertension    International Journal of Hypertension important inducible factor of vascular remodeling in case of hypertension, Ang II was used to establish a cell model in which phenotype transition from contractile and synthetic phenotype was observed. Tese results showed that miR-145 attenuated Ang II-induced phenotype transition, which implies that miR-145 may be an important factor maintaining contractile phenotype in both normal VSMCs and pathological VSMCs. ADAM17, as the most important sheddase, is also involved in the vascular remodeling in the case of hypertension. In our animal model, the ADAM17 expression signifcantly increased in the thoracic aorta of rats with metabolic hypertension as compared to the control group ( Figure 2). Tere is evidence showing that vascular ADAM17-defcient mice and ADAM17 antibody treated mice have alleviated Ang II-induced cardiac hypertrophy, vascular medial hypertrophy, and perivascular fbrosis via activating EGFR, which is independent of blood pressure regulation [14,25]. Our results further revealed that knockdown of ADAM17 with siRNA signifcantly increased the expression of contractile phenotype markers α-SMA and SM22α and decreased the expression of synthetic phenotype markers OPN (Figure 5), which indicate that ADAM17 is involved in the regulation of vascular remodeling.
Interestingly, in our experiment, miR-145 inhibited ADAM17 expression both in normal and pathological state (Figure 4(d)), which indicates that ADAM17 mediates the efect of miR-145 on the phenotype transition. Our study and other studies have revealed that ADAM17 and miR-145 share the common binding domain, and miR-145 can directly target the ADAM17 3′-UTR and suppress ADAM17 expression [26]. Tis ADAM17/EGFR/miR-145 feedback loop contributes to the migration and invasion of cancers such as nasopharyngeal cancer, colon cancer, hepatocellular cancer, renal cancer, and so on [16,19,27,28]. However, the role of miR-145/ADAM17 feedback loop in the vascular remodeling in case of hypertension is poorly understood. In the present study, ADAM17 siRNA partially alleviated the induced efect of miR-145 inhibitor on the phenotype transition in VSMCs ( Figure 5). Of interest, ADAM17 itself had no efect on the expression of EREG and MMP2, while cotreatment of ADAM17 siRNA reversed the induced efect of miR-145 inhibitor on these two synthetic markers, which indicates a regulatory role of ADAM17 in the miR-145induced phenotypic switch. Tus, the miR-145/ADAM17 feedback loop may provide a new target for the treatment of vascular remodeling.
Te potential downstream molecules involved in the miR-145/ADAM17 pathway were also explored in phenotype transition of VSMC. It has been confrmed that RAAS is involved in the regulation of vascular remodeling in case of hypertension [29], which involves two axes: ACE-Ang II-AT1 receptor axis and ACE2-Ang-(1-7)-Mas receptor axis. ACE2-Ang-(1-7)-Mas receptor axis is thought to be a nonclassical pathway and plays a protective role in the pathogenesis and development of hypertension [30,31]. ACE2 cleaves the octapeptide Ang II to Ang-(1-7), a vasodilator peptide. Tis cleavage results in nitric oxide (NO) release and reduced sympathetic output, while restoring barorefex sensitivity [32]. Similarly, our results showed that the expression of ACE2 and Mas receptor was signifcantly lower in the thoracic aorta of MH rats than in the control group ( Figure 2). ADAM17, the most important sheddase, was also found to mediate the proteolysis and ectodomain shedding of ACE2 [33]. Our results suggest that knockdown of ADAM17 with siRNA can enhance the expression of ACE2 and Mas receptor and Ang-(1-7) concentration in both normal and pathological VSMCs. Quantifcation of ACE2 (c) and MASR (d) expression determined by Western blotting, and the data were normalized to that of GAPDH. * * P < 0.01 vs. control group; * P < 0.05 vs. control group; and ## P < 0.01 vs. miR-145 inhibitor group. All the data are expressed as mean ± SEM of three independent experiments. NC siRNA, negative control siRNA.

Conclusions
In conclusion, our study for the frst time indicates that miRNA-145 induces aortic SMC phenotype transition via activating ADAM17, which may lead to vascular remodeling in the metabolic hypertension. Furthermore, ACE2-Ang-(1-7)-Mas receptor axis is involved in the regulation of miR-145/ADAM17 on the VSMCs. However, there were limitations in the present study. More studies are required to better understand the role of miR-145/ADAM17 in the pathophysiology of vascular remodeling in vivo and in the development of treatments for vascular remodeling due to hypertension.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.