Amlodipine induces vasodilation via Akt2/Sp1‐activated miR‐21 in smooth muscle cells

Abstract

Background and Purpose

The calcium antagonist amlodipine exerts important cardioprotective effects by modulating smooth muscle and endothelial functions. However, the mechanisms underlying these effects are incompletely understood.

Experimental Approach

Western blotting was used to compare the expression of key genes involved in vascular smooth muscle cell (VSMC) phenotype conversion. Recombinant adeno‐associated virus system was used to regulate miRNA expression in rats via tail vein. Bioinformatics was used to predict the transcriptional regulation of miR‐21 upstream followed by biochemical validation using quantitative real‐time polymerase chain reaction, ChIP‐qPCR and EMSA assays.

Key Results

Only the calcium antagonist amlodipine, and no other type of anti‐hypertensive drug, induced miR‐21 overexpression in plasma and aortic vessels in the animal model. Real‐time PCR and luciferase assays showed that amlodipine induced miR‐21 overexpression in vascular smooth muscle cells. Western blot and immunofluorescence assays demonstrated that amlodipine activated Akt2, rather than Akt1, followed by activation of transcription factor Sp1, which regulated VSMC phenotype conversion via binding to the miR‐21 promoter. Furthermore, bioinformatic analyses and luciferase assays demonstrated that amlodipine activated miR‐21 transcription at the ‐2034/‐2027 Sp1‐binding site, which was further demonstrated by ChIP‐qPCR and EMSA assays. Consistently, small‐interfering RNA‐mediated knockdown of Akt2 and Sp1 significantly attenuated the effects of amlodipine on miR‐21 expression in smooth muscle cells.

Conclusion and Implications

These results indicate that amlodipine induces smooth muscle cell differentiation via miR‐21, which is regulated by p‐Akt2 and Sp1 nuclear translocation, thereby providing a novel target for cardiovascular diseases.

Abbreviations

ARBs

angiotensin II receptor blockers

CCBs

calcium channel blockers

CNN1

calponin 1

OPT

osteopontin

PDCD4

programmed cell death 4

PE

phenylephrine

PTEN

phosphatase and tensin homologue

SHRs

spontaneously hypertensive rats

VSMCs

vascular smooth muscle cells

What is already known

• The calcium antagonist amlodipine exerts important cardioprotective effects.

• Amlodipine is involved in smooth muscle and endothelial function modulating.

What this study adds

• Amlodipine regulates VSMC differentiation dependent on miR‐21 expression.

• Amlodipine promotion of Sp1 nuclear translocation requires Akt2 phosphorylation.

What is the clinical significance

• New mechanism of amlodipine protection of smooth muscle function is revealed.

• miR‐21 may be a target to treat high BP patients.

1. INTRODUCTION

It is well known that vascular tension‐associated cardiovascular diseases, such as hypertension and atherosclerosis, can be ameliorated by reducing arterial pressure (Gupta et al., 2018). Intensive BP lowering has been reported to provide more benefits from vascular protection than standard regimens, especially in high‐risk patients (Xie et al., 2016). Up to now, the classic antihypertensive drugs, including diuretics, β‐blockers, ACE inhibitors, calcium channel blockers (CCBs), and angiotensin II receptor blockers (ARBs), have been widely used in the treatment of hypertension. However, there is still a large number of resistant hypertensive patients whose BPs are uncontrolled even with more than three types of antihypertensive drugs (Dudenbostel et al., 2015). Therefore, it is important to elucidate the detailed mechanisms of these drugs and optimize therapeutic strategies.

CCBs are one of the key treatments for patients with hypertension due to their potent antihypertensive effects. CCBs are generally used in both monotherapy and combined therapies with other antihypertensive medicines, and they always serve as first‐line treatments. In the Antihypertensive and Lipid‐Lowering Treatment to Prevent Heart Attack Trial (ALLHAT), CCBs exhibited much more powerful effects in the control of arterial pressure compared with diuretics in hypertensive individuals (ALLHAT Officers and Coordinators for the ALLHAT Collaborative Research Group. The Antihypertensive and Lipid‐Lowering Treatment to Prevent Heart Attack Trial, 2002). Compared with the ACE inhibitor group, the incidences of stroke, combined cardiovascular diseases, gastrointestinal bleeding, and angioedema were lower in the CCB group in hypertensive patients according to ALLHAT (Ma, Lee, & Stafford, 2006). Hypertensive patients in the CCB group had a 4‐mmHg lower systolic BP than patients in the ARB group, and myocardial infarction occurrence in the CCB group was less than that in the ARB group (Epstein, Vogel, & Palmer, 2007). In the Anglo‐Scandinavian Cardiac Outcomes Trial Blood Pressure Lowering Arm, the risk of stroke and the BP variability in hypertensive patients taking CCBs were lower than those in patients taking β‐blockers (Poulter et al., 2009).

The first‐generation dihydropyridine L‐type CCBs, such as nifedipine, have been widely used in the treatment of vascular diseases due to their strong relaxation effect (De Leeuw & Birkenhager, 2002). To improve the selectivity of nifedipine, several generations of CCBs have been gradually developed (Elkayam, 1998). A third‐generation series of L‐type CCBs, such as amlodipine, show extra benefits in the treatment of heart and vascular diseases. Amlodipine not only lowers BP but also reduces cardiovascular events and displays several additional features such as anti‐atherosclerotic effects. For instance, amlodipine reduced ischaemia in patients with coronary artery disease in the Circadian Anti‐ischaemia Program in Europe trial, and it also reduced hospitalizations for unstable angina and revascularization in patients with angiographically documented coronary artery disease in the Prospective Randomized Evaluation of the Vascular Effects of Norvasc Trial (Pitt et al., 2000). In the Coronary Angioplasty Amlodipine Restenosis Study, amlodipine reduced the need of revascularization in patients with stable angina. In addition, amlodipine has been found to be the most effective antihypertensive agent in the management of BP variability in hypertensive patients with diabetes, and it has also been shown to prevent cardiovascular morbidity and mortality (Sethi, Baruah, & Kumar, 2017).

It is well known that vascular smooth muscle cell (VSMC) remodelling is a key event in hypertension. VSMCs express a large number of voltage‐dependent calcium channels. Various agonists can regulate cell‐cycle initiation/progression of VSMCs by maintaining intracellular calcium homeostasis (Gollasch et al., 1998). Several studies have suggested that amlodipine is a potent inhibitor of VSMC proliferation (Lai et al., 2002). Amlodipine inhibits VSMC growth by affecting the G₁‐phase cell cycle and inhibiting DNA synthesis (Stepien et al., 1998). In addition, amlodipine also regulates the VSMC phenotype via MAPK signalling (Umemoto et al., 2006). However, the detailed mechanisms responsible for the critical signal transductions affecting the VSMC phenotype induced by amlodipine in hypertension are still poorly understood.

Normal VSMCs are differentiated, stationary, and contractile, while injured VSMCs show dedifferentiated, proliferated, and synthetic phenotypes. Smooth muscle myosin heavy chain (SM‐MHC), α‐smooth muscle actin (α‐SMA), and calponin 1 (CNN1) are markers of the contractile phenotype of VSMCs, while the synthetic phenotype may lose these markers and increase matrix protein expression (Owens, Kumar, & Wamhoff, 2004). It has been increasingly recognized that VSMCs change their phenotypes from a contractile phenotype to a synthetic phenotype under various environmental stimuli, which plays a pivotal role in neointimal hyperplasia during the pathogenesis of vascular proliferative diseases (Heusch et al., 2014). Knowing the mechanisms involved in VSMC phenotype switch is important for understanding the pathology of cardiovascular diseases as well as in designing therapeutic agents for their treatment and prevention (Liao et al., 2015).

MicroRNAs (miRNAs) are a novel class of regulatory non‐coding RNAs that regulate gene expression at the posttranscriptional level by binding to 3′‐untranslated regions of target mRNA in a negative fashion (Huntzinger & Izaurralde, 2011). miRNAs have been reported to play key roles in determining cell differentiation and tissue homeostasis (Davis, Hilyard, Lagna, & Hata, 2008). It is well known that the development of VSMCs is dependent on the functional expression of miRNAs (Nazari‐Jahantigh, Wei, & Schober, 2012). Late embryonic lethality and haemorrhage occurs when the miRNA‐processing enzyme, Dicer, is lacking in SMCs during development, which is most likely due to impaired proliferation and differentiation of SMCs (Albinsson et al., 2010). miR‐21 has been reported to act as a positive regulator of smooth muscle contractile gene expression by down‐regulating programmed cell death 4 (PDCD4; Davis et al., 2008), which protects VSMCs from H₂O₂‐induced death and apoptosis (Lin et al., 2009). miR‐21 is also activated by bone morphogenetic protein 4, which promotes vascular smooth muscle proliferation and inhibits apoptosis (Ji et al., 2007). Furthermore, our previous study indicated that miR‐21 translocates into mitochondria to counteract mt‐Cytb down‐regulation, and it significantly decreases BP in the spontaneously hypertensive rat (SHR) model (Li, Zhang, et al., 2016).

Since Minami et al. reported that miR‐221/miR‐222 is regulated by atorvastatin in 2009 (Minami et al., 2009), several studies have shown that the expression of certain miRNAs is regulated by statins (Li, Yin, et al., 2016). For instance, miR‐33b is up‐regulated by statins, which subsequently represses the expression and function of c‐Myc (Takwi et al., 2012). MiR‐133a is inhibited by statins in the vascular endothelium, which prevents endothelial dysfunction (Li, Yin, et al., 2016). Lovastatin‐induced miR‐29b reduces oxidative stress in rats with hyperglycaemia, dyslipidaemia, and hyperhomocysteinaemia (Wang et al., 2017). It remains unclear if miR‐21 is regulated by antihypertensive drugs.

The purpose of this study was to explore the effect of antihypertensive drugs on miR‐21 expression. The present study demonstrated that amlodipine increases the expression of miR‐21 by activating Akt2 and up‐regulating Sp1 expression in rat aorta artery SMC nuclei, which in turn targets PDCD4 to promote differentiation gene expression. The present study provided new insights into the mechanisms underlying phenotypic switch of VSMC by amlodipine, suggesting a potential therapeutic target for ameliorating VSMC‐related diseases.

2. METHODS

2.1. Animals

Six‐week‐old male Wistar–Kyoto (WKY) rats (WKY group) and SHRs were purchased from the Vital River Laboratory Animal Technology Company (Beijing, China). The weight of the animals was around 180 g. The animal experimental protocols complied with standards stated in the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the Chinese Academy of Sciences, and they were approved by the Tongji Hospital Ethics Committee. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010) and with the recommendations made by the British Journal of Pharmacology. Also, animal studies complied with the principles of replacement, refinement, and reduction (the 3Rs). All the animals used were raised in the specific pathogen‐free animal centre of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Animals were housed at a room temperature of 23 ± 1°C and 50% humidity with 12‐hr light/dark cycles and allowed free access to water and food. The food was sterilized by subjecting it to high pressure. The drinking water was supplied by a reverse osmosis water treatment system and sterilized by filtration and ozone. We changed the water bottles every 3 days. All the rats were housed in ventilated, polycarbonate cages and received irradiated corncob bedding. There were two rats in each cage. The cages were changed every week, while the food was changed every 3 days.

2.1.1. Experimental procedures

Experiment 1

The WKY group was administered a vehicle p.o. (10‐mM PBS, pH = 7.4), and the SHRs were randomly treated, p.o., with a vehicle (10‐mM PBS, pH = 7.4), amlodipine (2 mg·kg⁻¹·day⁻¹), valsartan (20 mg·kg⁻¹·day⁻¹), indapamide (1.5 mg·kg⁻¹·day⁻¹), hydrochlorothiazide (10 mg·kg⁻¹·day⁻¹), captopril (100 mg·kg⁻¹·day⁻¹), or metoprolol (50 mg·kg⁻¹·day⁻¹; n = 8 in each group). The antihypertensive drugs were dissolved in 10‐mM PBS and mixed well to make a suspension. Each rat received a suspension of 2 ml, by p.o. gavage, for 10 weeks.

Experiment 2

The WKY group was treated with a vehicle, and the SHRs were randomly treated with a vehicle (10‐mM PBS, pH = 7.4), amlodipine (2 mg·kg⁻¹·day⁻¹), amlodipine + recombinant adeno‐associated virus (rAAV)–GFP or amlodipine + rAAV–miR‐21–TUD (n = 8 in each group). rAAV was prepared by triple plasmid cotransfection in HEK293T cells, as previously described (Li, Zhang, et al., 2016). Three weeks after injection of the rAAV delivery system (1 × 10¹¹ genome copies in 100 ml of saline solution per rat), animals we administered amlodipine p.o. for 4 weeks.

At the end of treatments, rats were anaesthetized using isoflurane and then killed by decapitation. Tissue samples were snap frozen in liquid nitrogen or collected for paraffin embedding.

2.1.2. Randomization and blinding

Animals were randomized for intervention group. Data collection and evaluation of all experiments were performed blindly of the group identity. The design in this study complies with the recommendations on experimental design described previously (Curtis et al., 2018).

2.1.3. Validity of animal species or model selection

In the present study, SHRs were used to simulate the state of high BP, because they have been confirmed as a classical hypertensive animal model.

2.2. Cell culture and chemicals

Rat aortic artery‐derived VSMCs (rat VSMCs) and human aortic artery‐derived VSMCs (human VSMCs) were obtained from the American Type Culture Collection and were routinely cultured in DMEM supplemented with 10% FBS and penicillin–streptomycin (100 IU·ml⁻¹) in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. Twenty‐four hours prior to drug treatment, cells were transferred to media containing 2.5% FBS for the duration of the experiment as indicated. VSMCs cultured in 2.5% FBS proliferate slowly and do not differentiate spontaneously (Fetalvero et al., 2006).

2.3. Western blotting

Western blotting was performed as described previously (Inoue & Node, 2009). Briefly, the protein concentration was measured using a bicinchoninic acid protein assay reagent kit (Boster, Wuhan, China). Blots were developed using enhanced chemiluminescence reagents according to the manufacturer's recommendations. Antibodies used in the present study are listed in Table S1.

2.4. Cell transfection and treatments

Cells were transfected with miRNA mimic/inhibitor or small‐interfering RNA (siRNA; 100 nM, similarly hereinafter) using MegaTran (OriGene, MA) according to the manufacturer's protocol. Twenty‐four hours after transfection, cells were treated with 5‐μM amlodipine for an additional 24 hr and then collected.

2.5. RNA isolation, detection, and quantitative RT‐PCR assays

RNA isolation, detection, and quantitative RT‐PCR assays were performed as previously described (Yin et al., 2016). The PCR primers are listed in Table S2.

2.6. Cloning of miR‐21 promoter

Various miR‐21 promoter fragments containing different binding sites were cloned into the SacI/BglII sites of the pGL3 promoterless vector (Promega, Madison, WI) by PCR using human genomic DNA templates. The PCR primers are listed in Table S3.

2.7. Expression vector construction

To express human Sp1, human Akt2, rat Sp1, and rat Akt2, the full‐length sequence of their protein‐coding sequences (CDS) was amplified by PCR using the primers listed in Table S4. The amplicons were then ligated into the pcDNA3.1 expression vector (Invitrogen, Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. DNA sequencing analysis was performed to confirm the nucleotide sequences of the plasmids constructed. Plasmid DNA was prepared with the E.Z.N.A. Endo‐free Plasmid Maxi Kit (Omega BioTek, Norcross, GA) before transfection following the manufacturers' protocol as described previously.

2.8. Immunoprecipitation assay

The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology. VSMCs and HEK293T cells were washed with ice‐cold PBS and lysed in immunoprecipitation buffer (Beyotime Institute of Biotechnology, Nanjing, China). After centrifugation at 4°C, the supernatant was collected and pre‐cleared with protein A/G agarose beads (Beyotime Institute of Biotechnology, Nanjing, China) for 1 hr at 4°C. The supernatant was collected after centrifugation and incubated with specific antibodies or normal IgG (as control) followed by incubation with protein A/G beads overnight at 4°C (Li et al., 2013). The immunoprecipitated proteins were then identified by Western blotting as described above using specific antibodies.

2.9. Immunofluorescence staining

Immunofluorescence staining was performed as previously described (Joyal et al., 2014). Evaluations were performed in a blinded fashion by two investigators.

2.10. Chromatin immunoprecipitation assay

Human VSMCs, rat VSMC and HEK293T cells were transfected with expression vector‐coding Sp1 and flag, and 48 hr after transfection, the cells were harvested and cross‐linked with 4% paraformaldehyde at 37°C for 10 min followed by quenching with glycine and flash freezing. Then, chromatin immunoprecipitation assays were performed as previously described (Musunuru et al., 2010). The presence of immunoprecipitated DNA sequence around −2,034/−2,027 was detected by quantitative PCR, and primers are listed in Table S5. The values of the immunoprecipitated target were normalized to values of IgG chromatin.

2.11. Electrophoretic mobility shift assay

Nuclear extracts were prepared from human VSMCs using NE‐PER nuclear and cytoplasmic extraction reagents (Beyotime Institute of Biotechnology, Nanjing, China) and stored in aliquots at ‐80°C until further use. DNA probes containing the miR‐21 promoter fragment (from −2,034 to −2,027) were synthesized, biotinylated, and annealed. The probe sequences and mutation probe sequences are listed in Table S6. EMSA was performed according to a method described previously (Suzuki et al., 2010).

2.12. Data and statistical analysis

All data are presented as the means ± SEM. Statistical significance of differences among the groups was analysed by Student's t test or ANOVA for multiple comparisons with Tukey's test. Tukey's tests were run only when F achieved P < .05 and there was no significant variance inhomogeneity. Each experiment was performed at least five times independently. All statistical calculations were performed using SPSS 19.0 software, and P < .05 was considered a statistically significant difference. The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology.

2.13. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017).

3. RESULTS

3.1. Amlodipine induces miR‐21 expression and VSMC differentiation

Quantitative RT‐PCR assays showed that among the six antihypertensive drugs, amlodipine was the only one that induced miR‐21 overexpression in both plasma and aortic vessels in SHRs (Figure 1a,b). The animal characteristics are summarized in Table S7. In addition, amlodipine induced a dose‐dependent positive regulation on the expression of miR‐21 in rat VSMCs (Figure S1a). As shown in Figure S1b, amlodipine induced miR‐21 expression at 6 hr and then maintained a relatively high level of miR‐21 thereafter. Moreover, amlodipine treatment enhanced miR‐21–Luc promoter activity in human VSMCs and HEK293T cells (Figure S1c,d). Western blot assays showed that amlodipine increased the expression of α‐SMA, CNN1, and SM‐MHC but decreased osteopontin (OPN) expression in aortic vessels from SHRs (Figure 1c,d). Importantly, the inhibited expression of α‐SMA, CNN1, and SM‐MHC and enhanced expression of OPN by phenylephrine were attenuated by amlodipine in a dose‐dependent manner in VSMCs (Figure 1e,f). Moreover, amlodipine induced these gene expression alterations in VSMCs under normal conditions (Figure 1g,h). Consistently, the expression of differentiation genes induced by amlodipine increased in a time‐dependent manner in VSMCs (Figure 1i–l).

Figure 1.

Amlodipine (AM) induces vascular smooth muscle cell (VSMC) differentiation markers and inhibits dedifferentiation marker expression. (a) Expression of miR‐21 in plasma (n = 8). (b) Expression of miR‐21 in aorta vessels (n = 8). (c, d) Western blots of VSMC differentiation genes in aorta vessels in different groups. (e, f) Western blots of VSMC differentiation genes stimulated by PE combined with AM at different concentrations. (g, h) Western blots of VSMC differentiation genes stimulated by different concentrations of AM. (i, j) Western blots of VSMC differentiation genes stimulated by PE and 5‐μM AM at different time points. (k, l) Western blots of VSMC differentiation genes stimulated by 5‐μM AM at different time points. Data are presented as the mean ± SEM (n = 6). *P < .05 versus spontaneously hypertensive rat (SHR) + vehicle or control or 0 hr; ^% P < .05 versus Wistar–Kyoto (WKY) + vehicle; and ^& P < .05 versus PE or PE + 0 hr. α‐SMA, α‐smooth muscle actin; CNN1, calponin 1; OPN, osteopontin; PE, phenylephrine; SM‐MHC, smooth muscle myosin heavy chain

3.2. Amlodipine induces VSMC differentiation via miR‐21‐targeting PDCD4

The involvement of miR‐21 in the amlodipine‐induced phenotypic switch of VSMCs was investigated. Quantitative RT‐PCR assays showed that the expression of miR‐21 in aorta was significantly down‐regulated by rAAV–miR‐21–TUD (Figure S2a). Moreover, immunofluorescence showed that the target cell type of our delivery system is VSMCs in SHRs (Figure S2b). The animal characteristics were summarized in Table S8. Western blots showed that the rAAV–miR‐21–TUD significantly attenuated the amlodipine‐induced increase in α‐SMA, CNN1, and SM‐MHC protein levels as well as the amlodipine‐induced decrease in OPN protein levels in aortic vessels from SHRs (Figure 2a,b). We obtained similar results in VSMCs using miR‐21 inhibitor, while miR‐21 mimics displayed the opposite effects (Figure 2c–f). Further, the effects of miR‐21 on VSMC phenotypic switch were evidenced by immunofluorescence staining (Figure 2g). Among the reported targets of miR‐21, PDCD4 and phosphatase and tensin homologue (PTEN) were chosen to identify the downstream signals in VSMC phenotypic switch, because of their important roles in VSMC function. Results showed that PDCD4, a representative differentiation related target, but not PTEN, a representative proliferation differentiation related target, was inhibited by amlodipine in a dose‐ and time‐dependent manner, which was consistent with miR‐21 up‐regulation (Figure 2h–j).

Figure 2.

Amlodipine (AM)‐induced vascular smooth muscle cell (VSMC) differentiation requires miR‐21. (a, b) Western blots of VSMC differentiation genes in aorta vessels in different groups. (c–f) Protein levels of smooth muscle phenotype transition‐related genes were examined. (g) Immunofluorescence staining of VSMCs treated with or without AM after transfection with miR‐21 mimics, inhibitor, or negative control (con) for 24 hr. (h) Western blot analysis of programmed cell death 4 (PDCD4) and phosphatase and tensin homologue (PTEN) in VSMCs treated with miR‐21 mimics, inhibitor, or negative con. (i, j) PDCD4 and PTEN expression was determined by Western blot after AM treatment at different doses and times. Data are presented as the mean ± SEM (n = 6). *P < .05 versus Wistar–Kyoto (WKY) + vehicle or con or 0 hr; ^& P < .05 versus inhibitor–con + AM or inhibitor random; and ^# P < .05 versus miR – con + AM. α‐SMA, α‐smooth muscle actin; CNN1, calponin 1; OPN, osteopontin; SM‐MHC, smooth muscle myosin heavy chain

3.3. Amlodipine mediates VSMC differentiation by Sp1‐induced miR‐21

Next, we explored the transcriptional regulation of miR‐21 upstream to find out potential transcription factors, which may not only activate miR‐21 expression but also involved in the smooth muscle cell phenotype transition. Bioinformatics analyses (http://gene‐regulation.com/pub/databases.html) were used to search potential transcription factors that might regulate miR‐21 expression, which obtained 62 possible transcription factors. Referring to the existing researches, 15 transcription factors associated with smooth muscle phenotype switching were found. By comparing intersection, the number of candidate transcription factors was narrowed down to 7, including NFAT5, Klf4, Klf5, CREB3, TBP, Sp1, and TRB3 (Figure S3a). Among them, only the expression of Sp1 was increased significantly by amlodipine treatment (Figure S3b). Similar to miR‐21, amlodipine induced Sp1 expression in a dose‐ and time‐dependent manner (Figures 3a and S3c,d), and the same effects were observed in vivo and under phenylephrine treatment (Figure S3e–g). The involvement of Sp1 in amlodipine‐induced changes in VSMC differentiation genes was next tested. Western blots showed that the Sp1‐specific siRNA significantly attenuated the amlodipine‐induced increases in α‐SMA, CNN1, and SM‐MHC protein levels as well as the amlodipine‐induced decrease in OPN protein levels in VSMCs, while pcDNA3.1–Sp1 displayed the opposite effects (Figure 3b–e). Quantitative RT‐PCR and dual‐luciferase assays showed that Sp1 siRNA significantly attenuated the amlodipine‐induced increase in miR‐21 expression in VSMCs and HEK293T cells, while pcDNA3.1–Sp1 showed the opposite effects (Figures 3f–i and S4a–d). To validate the binding sites between Sp1 and miR‐21, four fragments of the miR‐21 promoter region (−2,173/+66, −1,398/+66, −781/+66, and −383/+66) were cloned into the pGL3 luciferase reporter vector. Amlodipine increased the fluorescence intensity of the −2,173/+66‐bp region fragment, while the other three fragments had no change (Figures 3j and S4e). Subsequently, two potential binding sites of Sp1, namely, −2,034/−2,027 and −1,986/−1,978, were identified (Figure 3k). A series of deletion mutants showed that the most powerful effects were induced by the −2,034/−2,027 region (Figures 3l and S4f), while the −1,986/−1,978 mutation caused no change. Consistently, the regulatory effect of −2,034/−2,027 region was further evidenced by chromatin immunoprecipitation–real‐time PCR and EMSA assays (Figures 3m,n and S4g).

Figure 3.

Amlodipine (AM)‐mediated changes in vascular smooth muscle cell (VSMC) differentiation genes and miR‐21 expression depend on Sp1. (a) Western blots detected the protein level of Sp1 induced by AM. (b, c) VSMC differentiation genes were detected in VSMCs treated with AM after cotransfection with Sp1 siRNA. (d, e) VSMC differentiation genes were detected after cotransfection with AM and pcDNA3.1–Sp1. (f) The expression of miR‐21 in rat VSMCs treated with AM after cotransfection with Sp1 siRNA. (g) Luciferase activity of pGL3‐f reporter plasmids stimulated by AM and Sp1 siRNA in human VSMCs. (h) miR‐21 expression in rat VSMCs treated with AM after cotransfection with pcDNA3.1–Sp1. (i) Luciferase activity of pGL3‐f reporter plasmids stimulated with AM or pcDNA3.1–Sp1 in human VSMCs. (j) Luciferase activity of pGL3 (−2,173/+66), pGL3 (−1,398/+66), pGL3 (−781/+66), and pGL3 (−383/+66) reporter plasmids stimulated by AM in human VSMCs. (k) Mutation of the binding sites of Sp1 and miR‐21 in the −2,173 to −1,398 region. (l) Luciferase activity of mutated binding sites of Sp1 and miR‐21 stimulated by AM in human VSMCs. (m) Interaction of the Sp1 and the promoter region of miR‐21 was examined by chromatin immunoprecipitation in human VSMCs (n = 8). (n) EMSA was performed with nuclear extracts from human VSMCs in the presence of the biotin‐labelled DNA oligonucleotides −2,034/−2,027—wild‐type (WT) or −2,034/−2,027—mutant (Mut). pGL3‐f represents the −2,173/+66 miR‐21 promoter region. Data are presented as the mean ± SEM (n = 6). *P < .05 versus control (con); ^& P < .05 versus siRNA (si)–con + AM; and ^% P < .05 versus AM. α‐SMA, α‐smooth muscle actin; CNN1, calponin 1; OPN, osteopontin; SM‐MHC, smooth muscle myosin heavy chain

3.4. Amlodipine mediates VSMC differentiation and miR‐21 expression via Akt2

It has been reported that amlodipine significantly increases p‐Akt (S473) expression in SHR aorta compared with vehicle (Umemoto et al., 2006). Thus, the effects of amlodipine on p‐Akt (S473) expression in VSMCs were investigated. Western blots showed that amlodipine induced p‐Akt (S473) expression in a concentration‐ and time‐dependent manner (Figure 4a,b). The similar results were observed in a high‐pressure environment in vitro (Figure S5a). Knockdown of Akt2, but not Akt1 or Akt3, using siRNA inhibited the amlodipine‐induced up‐regulation of α‐SMA, CNN1 and SM‐MHC up‐regulation, and the amlodipine‐induced down‐regulation of OPN, while pcDNA3.1–Akt2 expression showed the opposite effects (Figures 4c–f and S6a,b). Furthermore, amlodipine increased Akt2 phosphorylation (Ser474) and promoted its nuclear translocation in a time‐dependent manner while have no effect on Akt3 phosphorylation (Ser472; Figures 4g and S6c). Quantitative RT‐PCR and dual‐luciferase assays demonstrated that Akt2‐specific siRNA significantly attenuated the increased expression of miR‐21 in VSMCs and HEK293T cells induced by amlodipine, while pcDNA3.1–Akt2 displayed the opposite effects (Figures 4h–k, S6d,e, and S7a–d).

Figure 4.

Akt2 is involved in vascular smooth muscle cell (VSMC) differentiation gene regulation and miR‐21 expression induced by amlodipine (AM). (a, b) Western blots detected the phosphorylation of Akt induced by AM. (c, d) VSMC differentiation genes were detected in VSMCs treated with AM after cotransfection with Akt1 and Akt2 siRNA. (e, f) VSMC differentiation genes were detected after cotransfection with AM and pcDNA3.1–Akt2. (g) Immunofluorescence staining using a p‐Akt2 (S474) antibody of rat VSMCs treated with AM at different time points. (h) Expression of miR‐21 in rat VSMCs treated with AM after cotransfection with Akt2 siRNA. (i) Luciferase activity of pGL3‐f reporter plasmids stimulated by AM and Akt2 siRNA in human VSMCs. (j) Expression of miR‐21 in rat VSMCs treated with AM after cotransfection with pcDNA3.1–Akt2. (k) Luciferase activity of pGL3‐f reporter plasmids stimulated by AM or pcDNA3.1–Akt2 in human VSMCs. pGL3‐f represents the −2,173/+66 miR‐21 promoter region. Data are presented as the mean ± SEM (n = 6). *P < .05 versus control (con) or 0 hr or si–con; ^& P < .05 versus si–con + AM; and ^% P < .05 versus AM. α‐SMA, α‐smooth muscle actin; CNN1, calponin 1; OPN, osteopontin; SM‐MHC, smooth muscle myosin heavy chain

3.5. Amlodipine triggers p‐Akt2‐dependent nuclear translocation of Sp1

In VSMCs, amlodipine‐induced Sp1 expression was reduced by si–Akt2 and not by si–Akt1 or si–Akt3 (Figures 5a and S8a). Akt2 and Akt2 phosphorylation levels were not regulated by Sp1 (Figure 5b,c), but exogenous expression of Akt2 increased Sp1 protein levels (Figure 5d). Further, Sp1 and Akt2 were tagged with flag tags and used in immunoprecipitation assays, which showed that the flag antibody simultaneously pulled Sp1, Akt2, and p‐Akt (S474) down (Figures 5e,f and S9a–d). In addition, p‐Akt2 (S474), Akt2, and Sp1 expression was increased in the nucleus but decreased in the cytoplasm after amlodipine treatment, and these changes occurred in a time‐dependent manner (Figure 5g). Moreover, Sp1 was reduced or increased in the nucleus accompanied with Akt2 expression (Figure 5h,i). The ability of LY294002, a PI3K inhibitor, to influence Sp1 expression was next tested. Western blot analysis showed that LY294002 (50 μM) intervention reduced Sp1 expression in the nucleus (Figures 5j and S10a). Overall, these data revealed that amlodipine addition increases phosphorylated Akt2 in the nucleus, thereby inducing Sp1 translocation from the cytoplasm to the nucleus.

Figure 5.

p‐Akt2 promotes Sp1 translocation into the nucleus. (a) Sp1 protein expression in rat vascular smooth muscle cells (VSMCs) after cotransfection with Akt2 siRNA. (b) Expression of phosphorylated Akt2 and Akt2 in rat VSMCs treated with amlodipine (AM) after cotransfection with Sp1 siRNA. (c, d) Western blots of Akt2 and Sp1 protein levels after overexpression of Sp1 and Akt2. (e, f) The interaction among Akt2, p‐Akt2 (S474), and Sp1 in a complex was examined by immunoprecipitation assays. (g) Western blots detected the protein levels of Akt2, Sp1, and p‐Akt2 (S474) in the nucleus and cytoplasm. (h–j) AM‐induced nuclear aggregation of Sp1 was dependent on p‐Akt2 (S474). Data are presented as the mean ± SEM (n = 6). *P < .05

4. DISCUSSION

The present study showed that amlodipine up‐regulates differentiation gene expression via miR‐21‐targeting PDCD4, which is regulated by p‐Akt2 and Sp1 nuclear translocation (Figure 6). These findings suggested that the maintenance of VSMC phenotype might be an additional cardiovascular protective effect of amlodipine. Furthermore, these observations revealed a novel pathway in which amlodipine might affect VSMC phenotypic modulation, thereby indicating a new therapeutic strategy against cardiovascular diseases.

Figure 6.

Signalling pathways showing mechanisms involved in the protective effects of amlodipine on vascular smooth muscle cells (VSMCs) subjected to phenotype conversion. Amlodipine promoted Akt2 activation, leading to the translocation of the Sp1 transcription factor into the nucleus, which increased binding activity to the miR‐21 promoter and regulated the expression of VSMC differentiation genes

Previous studies have indicated that amlodipine induces contractile‐type SM‐MHC isoform SM2 expression and reduces NMHC‐B/SMemb expression (Umemoto et al., 2006). Meanwhile, the roles of miRNAs in VSMCs proliferation, migration, and differentiation were well investigated recently, especially miR‐21. Early in 2008, high expression of miR‐21 was observed in the vascular wall of balloon‐injured rat carotid arteries, and posttranscriptional induction of miR‐21 by BMP4 inhibited VSMC migration and promoted the differentiated contractile phenotype of VSMCs via PDCD4 (Davis et al., 2008), which was consistent with our current study. Our previous study showed that miR‐21 is up‐regulated in hypertensive patients and hypertensive SHRs compared with controls (Li, Zhang, et al., 2016). The present study reported that amlodipine promoted VSMC differentiation via miR‐21‐mediated PDCD4 inhibition. PDCD4 and PTEN are direct targets of miR‐21, which is involved in the regulation of VSMCs function (Maegdefessel et al., 2012). In particular, PDCD4 is generally involved in phenotype transformation, while PTEN is related to proliferation. The present data showed that amlodipine reduced PDCD4 expression, but not PTEN expression, in a concentration‐ and time‐dependent manner. Interestingly, amlodipine‐induced contractile protein expression was repressed with miR‐21 inhibitor transfection.

On the other hand, it was reported that the miR‐21 promoted neointima SMC hyperplasia (Ji et al., 2007). Further, local anti‐miR‐21‐eluting stent effectively prevented experimental in‐stent restenosis (Wang et al., 2015). The complicated findings may be due to the following: (a) Phenotypes of VSMCs are not completely different. Contractile and proliferating/migrating “phenotypes” were not necessarily stable, irreversible, or mutually exclusive (Joshi, Comer, McLendon, & Gerthoffer, 2012). Some even assumed that the phenotype of smooth muscle cells was more graded than a binary phenomenon with cells in a particular tissue having a mosaic pattern of contractile protein gene expression (Larsson, McLean, Mecham, Lindahl, & Nelander, 2008; Owens et al., 2004). (b) The in vivo and in vivo models are different. The signal pathway involved in various models may be different. By bioinformatic analysis, it is predicted that one certain miRNA may have more than 100 potential targets. Moreover, the main functional targets, which are related to the key signals, are usually different among different biological processes. This maybe also the reason that the expression of PTEN, a proliferation‐related target, was regulated by miR‐21 in other models but not affected by miR‐21 in our current model. (c) The complicated effects of miR‐21. It was reported that miR‐21 played different roles in different cell types even in the same pathological process. In cardiac hypertrophy, direct effects of miR‐21 on cardiomyocytes protected against myocardial remodelling, while direct effects on fibroblasts promoted fibrosis (Bang et al., 2014; Yan et al., 2015). Using miR‐21 smooth muscle cell‐specific knockout or transgenic animals under different pathophysiological conditions may be better to confirm the effects of miR‐21 on the vascular phenotype switch.

Sp1 is one of four zinc finger transcription factors, and it plays an important role in the transcriptional activation of a wide range of genes. Previous studies have shown that posttranslational induction of Sp1 in the injured vessel wall may contribute to transcriptional activation of p27 and the cessation of VSMC proliferation at later phases during arterial remodelling (Andres, Urena, Poch, Chen, & Goukassian, 2001). A recent study has shown that solamargine inhibition of Sp1 is reversed by overexpression of Akt in H1299 and A549 cells (Chen et al., 2015). Another study has indicated that the effects of rosiglitazone on VSMC phenotype transformation regulation are induced by the increase of Sp1‐binding activity on the PKG promoter (Yang et al., 2013). Quantitative RT‐PCR and bioinformatics analyses showed that among the transcription factors (NFAT5, Klf4, Klf5, CREB3, TBP, Sp1, and TRB3) predicted to bind with the miR‐21 promoter region, only Sp1 was changed in amlodipine‐treated VSMCs. These results suggested that Sp1 may be involved in the regulation of miR‐21 expression. In addition, Sp1 played a pivotal role in amlodipine‐induced VSMC phenotype transformation. Furthermore, the present data also revealed that amlodipine‐regulated miR‐21 expression depended on Sp1. Dual‐luciferase assays showed that amlodipine dramatically increased the binding activity of Sp1 on the miR‐21 promoter. However, this promoter‐binding activity was decreased when cells were transfected with Sp1‐specific siRNA. Furthermore, when the Sp1‐binding site in the miR‐21 promoter (−2,034 to −2,027) was mutated, the binding activity was destroyed.

Akt, also known as PKB, is part of a family of PH domain‐containing serine threonine kinases. Akt has three isoforms, namely, Akt1, Akt2, and Akt3, and all of which are regulated by phosphorylation via a PI3K‐dependent process (Polytarchou et al., 2011). Similar to adiponectin and rapamycin, the present study showed that amlodipine required Akt2 activation to promote VSMC differentiation. In contrast to adiponectin but consistent with rapamycin, the present data suggested that amlodipine only activated Akt2 and not Akt1 or Akt3. The effect of amlodipine on Akt3 expression was also measured. Although Akt3 has been reported to promote VSMC proliferation (Sandirasegarane & Kester, 2001), no change induced by amlodipine was observed in the present study. Polytarchou et al. (2011) showed that the induction of miR‐21 is both necessary and sufficient for the growth advantage of Akt2‐expressing cells upon exposure to hypoxia. In the present study, dual‐luciferase assays and quantitative RT‐PCR showed that Akt2 participated in amlodipine‐mediated miR‐21 expression.

Several investigations have revealed that Sp1 expression is regulated by Akt, but the specific roles of each Akt isoform are poorly understood (Pore et al., 2004). The present study indicated that Sp1 expression was regulated by amlodipine via Akt2. Further, an immunoprecipitation assay showed that Sp1, Akt2, and p‐Akt2 were pulled down by a flag antibody, indicating that they may combined with each other directly. It has been revealed that TSSC3 induces Sp1 translocation from the cytoplasm to the nucleus through the PI3K/Akt pathway (Takao, Asanoma, Tsunematsu, Kato, & Wake, 2012). The present experiments revealed that amlodipine treatment induced the transfer of p‐Akt2 and Sp1 from the cytoplasm to the nucleus.

In summary, amlodipine promoted Akt2 activation, which led to nuclear translocation of the Sp1 transcription factor, thereby increasing the binding activity to the miR‐21 promoter to regulate the expression of VSMC differentiation genes. The present study revealed for the first time that miR‐21 expression is regulated by antihypertensive drugs. These findings provided a theoretical basis for miRNA‐based therapeutics against hypertension and other associated diseases.

4.1. Perspectives

Amlodipine induces smooth muscle cell differentiation via miR‐21, which is regulated by p‐Akt2 and Sp1 nuclear translocation, thereby providing a novel target for cardiovascular diseases.

AUTHOR CONTRIBUTIONS

All authors have approved this manuscript and its contents, and they are aware of the responsibilities connected with authorship. Q.F. designed and performed the study and analysed the data; M.T., F.W., Z.Z., T.D., W.W., Y.Y., X.L., G.C., L.X., and H.W. participated in performing the study; Y.W. and C.C. designed and organized the study; and D.W.W. organized the study.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation and as recommended by funding agencies, publishers, and other organizations engaged with supporting research.

Supporting information

Figure S1. Amlodipine induces miR‐21 expression. (A) Multiple anti‐hypertensive drugs induced miR‐21 expression at different concentrations. (B) The miR‐21 level induced by amlodipine at different time points. (C and D) Luciferase activity of pGL3 reporter plasmids stimulated by amlodipine in human VSMCs and HEK293T cells. pGL3‐f represents the −2173/+66 miR‐21 promoter region. Data are presented as the mean ± SEM (n = 6). *P < 0.05 vs. Control or 0 h.

Figure S2. Effects of rAAV‐miR‐21‐TUD on miR‐21 expression in SHRs aorta. (A) MiR‐21 expression in SHRs aorta. (B) Immunofluorescence staining in SHRs aorta treated with amlodipine and rAAV‐GFP. Data are presented as the mean ± SEM (n = 8). *P < 0.05 vs. SHR + Vehicle; ^& P < 0.05 vs. SHR + Amlodipine.

Figure S3. Amlodipine induces the expression of Sp1. (A) The schematic for transcription factors prediction. (B) Expression of multiple transcription factors associated with VSMC phenotype in VSMCs treated with amlodipine. (C) Western blot assays of amlodipine‐induced Sp1 expression. (D) Western blot assays of PE‐induced Sp1 expression with or without amlodipine. (E) Immunofluorescence staining in rat VSMCs treated with amlodipine at different time points. (F and G) Western blot assays of amlodipine‐induced Sp1 expression in SHR aortas. Data are presented as the mean ± SEM (n = 6). *P < 0.05 vs. Con or 0 h or SHR + Vehicle.

Figure S4. Effects of siRNA and pcDNA3.1‐Sp1 on Sp1 and miR‐21 expression. (A and B) Sp1 protein expression in HEK293T cells after co‐transfection with Sp1 siRNA and pcDNA3.1‐Sp1. (C and D) Luciferase activity of pGL3‐f reporter plasmids stimulated by amlodipine and Sp1 siRNA or pcDNA3.1‐Sp1 in HEK293T cells. (E) Luciferase activity of pGL3 (−2173/+66), pGL3 (−1398/+66), pGL3 (−781/+66) and pGL3 (−383/+66) reporter plasmids stimulated by amlodipine in HEK293T cells. (F) Luciferase activity of mutated binding sites of Sp1 and miR‐21 stimulated by amlodipine in HEK293T cells. (G) Interaction of the Sp1 and the promoter region of miR‐21 was measured by ChIP in HEK293T cells (n = 8). pGL3‐f represents the −2173/+66 miR‐21 promoter region. Data are presented as the mean ± SEM (n = 6). *P < 0.05 vs. Control.

Figure S5. Amlodipine induces the expression of Akt2. (A) Amlodipine induced Akt2 activation in a high‐pressure environment. Data are presented as the mean ± SEM (n = 6). *P < 0.05 vs. PE+0 h.

Figure S6. The role of Akt3 in amlodipine mediated VSMC phenotype switch and miR‐21 expression. (A and B) Western blots of VSMC differentiation genes in VSMCs in different groups. (C) Western blots detected the phosphorylation of Akt3 induced by amlodipine. (D) MiR‐21 expression in rat VSMCs treated with amlodipine after co‐transfection with si‐Akt3. (E) Luciferase activity of pGL3‐f reporter plasmids stimulated by amlodipine or si‐Akt3 in human VSMCs. Data are presented as the mean ± SEM (n = 6). *P < 0.05 vs. si‐con

Figure S7. Effects of siRNA and pcDNA3.1‐Akt2 on miR‐21 expression. (A and B) Akt2 protein expression in HEK293T cells after co‐transfection with Akt2 siRNA and pcDNA3.1‐Akt2. (C and D) Luciferase activity of pGL3‐f reporter plasmids stimulated by amlodipine and Akt2 siRNA or pcDNA3.1‐Akt2 in HEK293T cell. pGL3‐f represents the −2173/+66 miR‐21 promoter region. Data are presented as the mean ± SEM (n = 6). *P < 0.05.

Figure S8. Effect of siRNA of Akt3 on Sp1 expression. (A) Sp1 protein expression in rat VSMCs after transfection with Akt3 siRNA. Data are presented as the mean ± SEM (n = 6). *P < 0.05.

Figure S9. The interaction among Akt2, p‐Akt2 (S474) and Sp1. (A and B) The interaction among Akt2, p‐Akt2 (S474) and Sp1 in a complex was examined in human VSMCs. (C and D) The interaction among Akt2, p‐Akt2 and Sp1 in a complex was examined in HEK293T cells.

Figure S10. Effects of LY294002 on p‐Akt2 (S474) and Akt2 expression. (A) The expression of p‐Akt2 (S474) and Akt2 protein in rat VSMCs after treatment with the PI3K inhibitor, LY294002, at different concentrations.

Table S1. List of Antibodies.

Table S2. The primers for qRT‐PCR assays.

Table S3. The primers for miR‐21 promoter cloning.

Table S4. The primers for expression vectors.

Table S5. The primers for chromatin immunoprecipitation assays.

Table S6. The primers for electrophoretic mobility shift assay.

Table S7. The characteristics of animals treated with different anti‐hypertensive drugs.

Table S8. The characteristics of animals treated with different rAAV vectors.

Fang Q, Tian M, Wang F, et al. Amlodipine induces vasodilation via Akt2/Sp1‐activated miR‐21 in smooth muscle cells. Br J Pharmacol. 2019;176:2306–2320. 10.1111/bph.14679