1 INTRODUCTION

Asymmetric dimethylarginine (ADMA), a methylated amino acid derived from L-arginine, inhibits nitric oxide (NO) synthesis in vivo.1 It has been shown that plasma ADMA levels are significantly increased in patients with cardiovascular disorders associated with a reduction in NO synthesis.2-4 While much is known about the role of ADMA in endothelial function, few studies has focused on the role of ADMA in vascular smooth muscle cells (VSMCs) in hypertension. ADMA has been reported to contribute to the proliferation and migration of VSMCs and the enhancement of VSMC apoptosis.5, 6 However, the underlying mechanisms remain unclear.

Profilin-1 is a well-known highly conserved and ubiquitously expressed multifunctional actin-binding protein (12–15 kD) that plays an essential role in the regulation of cytoskeleton rearrangement and redistribution by promoting actin polymerization and vascular hypertrophy, and regulating cell signaling.7 One of its main functions is the sequestering function of the actin cytoskeleton, sequestering actin monomers in a 1:1 complex. It has been reported that ox-LDL and Ang II could significantly upregulate the expression of profilin-1 in cultured VSMCs, leading to VSMC proliferation.8, 9 Downregulation of profilin-1 expression attenuates the hypertrophy and apoptosis of VSMCs induced by advanced glycation end products (AGEs).10 Overexpression of profilin-1 dramatically promotes aortic remodeling and knockdown of profilin-1 inhibits vessel size, wall thickness, and collagen content in ouabain-induced hypertensive rats.11 In addition, the expression of profilin-1 was found to be dramatically elevated in the hypertrophic myocardium of SHRs, and interference of profilin-1 expression in SHRs significantly lowers hypertension-induced cardiac hypertrophy and fibrosis.12 Thus, these findings suggest that profilin-1 activates hypertrophic signaling cascades that contribute to vascular hypertrophy and remodeling in hypertension.

Based on these findings, we hypothesized that a close relationship exists between ADMA and profilin-1 in vascular injury and remodeling during hypertension. However, to date, there have been few studies on the role of profilin-1 in vascular injury and VSMC proliferation induced by ADMA. Therefore, this study aimed to better define the role of profilin-1 in vascular injury, especially regarding the proliferative effects of ADMA on VSMCs and to elucidate the underlying mechanisms.

2 MATERIALS AND METHODS 2.1 Chemicals and reagents

Rat aortic smooth muscle cells (RASMCs) A10 were purchased from ATCC (Manassas, VA, USA). ADMA standard, anti-β-actin antibody, and G418 were purchased from Sigma (St. Louis, MO, USA).

Fetal bovine serum (FBS) was supplied by Gibco BRL (Gaithersburg, MD, USA). Phenylmethylsulfonyl fluoride (PMSF) and bicinchoninic acid (BCA) protein kits were purchased from Beyotime. NO kits were purchased from Nanjing Juli Biological Limited Company (Jiangsu, China). Human Von Willebrand Factor (vWF) ELISA Kit was purchased from Shanghai Sun Biotech Co. Ltd. (Shanghai, China). TNF-α and IL-8 ELISA Kits were purchased from Shanghai Senxiong Biotech Co. Ltd. (Shanghai, China). Polyclonal antibodies against profilin-1 were purchased from Abcam (Cambridge, UK), and small hairpin RNAs (shRNAs) against profilin-1 (Pfn-1 shRNA) were obtained from Genechem, Inc. The primers were synthesized by Sangon Co. (Shanghai, China). RT reagent kits were obtained from Thermo Scientific (Waltham, MA, USA). Profilin-1 shRNA adenovirus vectors (Ad-profilin-1 shRNA) and blank control adenovirus vectors (Ad-GFP) were constructed by Hanbio Company (Shanghai, China). FuGENE HD was obtained from Roche (Wettsteinplatz, Basel, Switzerland).

2.2 Study populations

Forty healthy subjects and forty-two matched patients with newly diagnosed essential hypertension without antihypertensive drugs were enrolled in this study. The diagnosis of hypertension was based on the guidelines for the prevention, detection, evaluation, and management of high blood pressure in adults.13 The individuals were enrolled with blood pressure (BP) values ≥140/90 mmHg, with three measurements made on different dates. Subjects with congestive heart failure, secondary or malignant hypertension, coronary heart disease, severe cardiac arrhythmia, valvular heart disease, diabetes mellitus (type 1 or 2), autoimmune diseases, pregnant women, history of cerebrovascular accident or syncope, and severe hepatic and renal insufficiency were excluded. Subjects with coronary heart disease were excluded by clinical physical examination, electrocardiogram (ECG), treadmill exercise, B-ultrasound, SPECT, or coronary angiography. The protocol was approved by the Ethical Committee of Central South University, and all patients provided written consent to participate in this study.

2.3 Blood sample measurements

The concentration of ADMA was measured using high-performance liquid chromatography. NO levels were detected using the Griess test kit according to the manufacturer's protocol. The levels of profilin-1, vWF, TNF-α, and IL-8 were detected using ELISA. The standard curve for each assay was plotted according to the manufacturer's instructions.

2.4 Cell culture and transfection

RASMCs were placed in an incubator at 37°C in a 90% humidity atmosphere containing 5% CO2 and cultured in dulbecco’s modification of Eagle’s medium dulbecco (DMEM) supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin, which were characterized by immunofluorescence staining of α-smooth muscle actin. At 90% confluence, the cells were subcultured and incubated with ADMA at different concentrations for different times at passages 2–3 or pretreated with AG490 (5 × 10−5 M, Janus kinase 2 [JAK2] inhibitor) or rapamycin (10−8 M, inhibition of signal transducer and activator of transcription 3 [STAT3] activation).

To silence the expression of profilin-1 gene, a custom and predesigned siRNA (sense strand: 5′-ACCTTCAATGTCACTGTCA-3′; antisense strand: 5′-TGACAGTGACATTGAAGGT-3′) was transfected into RASMCs using FuGENE HD transfection reagent according to the manufacturer's instructions when cells were grown to 70% confluency. The transfected cells were then maintained in a culture medium containing 400 μg/ml G418. Clonal cell lines were derived from stably transfected pools. Transfection efficiency and cell viability were measured by the percentage of cells using a fluorescence microscope, and real-time PCR and western blotting analysis were used to measure the expression of profilin-1.

2.5 Cell proliferation assays

Cell proliferation was assessed using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide (MTT) assay or flow cytometry analysis. Cell viability and proliferation of VSMCs were determined using the MTT assay according to the manufacturer's instructions. In brief, RASMCs were seeded into 96-well plates at a density of 6000 cells in DMEM containing 10% FBS. After 24 h, the DMEM supplemented with 1% FBS was changed to quiescent cells for 24 h with different concentrations of ADMA (1, 3, 10, and 30 mM) for different periods of time (0, 6, 12, 18, and 24 h). MTT solution (5 mg/ml) in culture medium (20 ml) was added to each well, and the mixture was incubated for 4 h. Formazan crystals in each well were dissolved in 150 μl DMSO, and the absorbance was measured at 595 nm using a microplate reader (PerkinElmer, Waltham, MA, USA) to assess cell proliferation.

Cell cycle was analyzed by flow cytometry, cells were grown into six-well culture plates at 1 × 105 cells/well for 24 h, and they were changed to DMEM containing 1% FBS and quiescented the cells for at least 24 h, and then preincubated RASMCs with AG490 (5 × 10−5 M), or rapamycin (10−8 M) for 1 h and treated with ADMA (30 mM) for 24 h. The cells were fixed in 70% ethanol at 4°C overnight, washed twice thoroughly with PBS, and stained with propidium iodide solution for 20 min in the dark. Then, the stained cells were subjected to flow cytometry to assess cell cycle analysis.

2.6 Real-time PCR analysis

Total RNA was extracted from cultured RASMCs using TRIzol reagent. The primer pairs were as follows: 5′-GAACGCCTACATCGACAGCC-3′ (sense) and 5′-CTTTGCCTACCAGGACACCA-3′ (anti-sense) for rat profilin-1 (157 bp); 5′-TGGCCTCCAAGGAGTAAGAAAC-3′ (sense) and 5’-GGCCTCTCTCTTGCTCTCAGTATC-3′ (anti-sense) for GAPDH (372 bp), and each reverse transcription reaction was conducted with 1 μg of total RNA, and cDNA was amplified by PCR using SYBR Green Master Mix. After an initial denaturation step (94°C, 30 s), the following amplification conditions were used: 40 cycles of 94°C for 30 s, 60°C for 60 s, and 72°C for 1 min. All assays for each sample were carried out in triplicate, and the results are expressed as the profilin-1/GAPDH mRNA ratio.

2.7 Western blotting analyses

The cells were lysed in RIPA lysis buffer (containing 0.01% PMSF); then, precipitated RNA was collected by centrifugation at 12,000 × g for 20 min, and the BCA protein assay was used to measure the protein concentrations. Each sample was mixed with sample buffer containing sodium dodecyl sulfate (SDS), incubated for 5 min at 100°C, and equal amounts of proteins were loaded onto a 15% SDS-polyacrylamide gel electrophoresis (PAGE) gel, and then transferred onto a polyvinylidene fluoride (PVDF) membrane using standard procedures. The membranes were blocked for 1 h at 37°C in 5% non-fat dry milk in tris-buffered saline with Tween 20 (TBST) buffer and probed with primary antibodies at a suitable dilution (profilin-1, 1:1000; β-actin, 1:3000) overnight at 4°C. The membrane was rinsed three times using TBST, and then incubated for 1 h at 37°C with horseradish peroxidase(HRP)-conjugated secondary antibodies (Millipore, 1:5000). The secondary antibody was removed by 3 min washes, the targeted protein bands were visualized and quantified using Image analysis software, and the intensities of the target bands were normalized with β-actin as an internal standard.

2.8 Statistical analysis

Data are presented as means ± SEM, and differences between groups were analyzed using Student's t-test or ANOVA followed by a Student–Newman–Keuls post hoc test for multiple groups. Statistical significance was set at p < 0.05.

3 RESULTS

There were no significant differences in age, sex, blood lipids, blood glucose, body mass index, and smoking status between hypertensive and normotensive subjects. Compared to normotensive subjects, the levels of SBP and DBP, plasma ADMA, vWF, TNF-α, and IL-8 were significantly elevated, whereas levels of NO were lower in hypertensive subjects (p < 0.05, p < 0.01; Table 1).

TABLE 1. The features in normotensive subjects and essential hypertensive patients Normotensive subjects (40) Hypertensive subjects (42) Age (years) 45.25 ± 7.49 46.76 ± 6.77 Sex (male/female) 23/17 20/22 BMI (kg/m2) 22.69 ± 2.50 23.49 ± 2.69 Smoking (%) 47.5% 45.5% TC (mmol/L) 4.77 ± 0.66 4.56 ± 0.62 TG (mmol/L) 1.12 ± 0.35 1.22 ± 0.34 LDL-C (mmol/L) 2.74 ± 0.54 2.58 ± 0.55 HDL-C (mmol/L) 1.51 ± 0.34 1.42 ± 0.41 FBS (mmol/L) 5.07 ± 0.62 5.27 ± 0.56 SBP (mmHg) 118.3 ± 9.8 157.2 ± 12.5** DBP (mmHg) 74.9 ± 8.0 99.5 ± 9.9** ADMA (mmol/L) 0.72 ± 0.20 0.84 ± 0.26* NO (mmol/L) 24.34 ± 3.35 16.91 ± 1.49* Profilin-1 (pg/ml) 8.97 ± 3.46 25.40 ± 12.04** vWF (ng/L) 90.27 ± 38.38 122.02 ± 63.53** IL-8 (ng/L) 1.78 ± 0.22 2.61 ± 0.33* TNF-α (ng/L) 66.22 ± 5.27 99.48 ± 13.23* * p < 0.05 versus normotensive subjects. ** p < 0.01 versus normotensive subjects. 3.1 Upregulation of profilin-1 expression induced by ADMA

As shown in Figure 1A,B, compared with the control group, different concentrations of ADMA (1, 3, 10, and 30 mM) for different periods of time (0, 6, 12, 18, and 24 h) upregulated the mRNA and protein expression of profilin-1 in cultured RASMCs (Figure 1A,B, p < 0.05, p < 0.01). Moreover, incubation with ADMA induced the proliferation of RASCMs in a concentration- and time-dependent manner (Figure 1C,D, p < 0.01). ADMA at a concentration of 30 μM for 24 h had the most robust effect on the expression of profilin-1 and proliferation of RASMCs, concomitantly with a remarkable increase according to the formazan absorbance and the percentage of cells in the S + G2/M phase by flow cytometry analysis (Figures 2B and 3C, p < 0.01).

The effect of exogenous ADMA on the expression of profilin-1 and proliferation ability in cultured RASCMs. (A) The effect of ADMA (1, 3, 10, and 30 μM) for 24 h on the protein expression of profilin-1 in RASMCs, **p < 0.01 versus control. (B) The effect of ADMA (30 μM) for different times (6, 12, 18, and 24 h) on the protein expression of profilin-1 in RASMCs, *p < 0.05 versus control and **p < 0.01 versus control. Data are from three independent experiments (n = 3). (C) The effect of ADMA (1, 3, 10, and 30 μM) for 24 h on the proliferation of RASMCs by MTT, **p < 0.01 versus control. (D) The effect of ADMA (30 μM) for different times (6, 12, 18, and 24 h) on the proliferation of RASMCs by MTT, **p < 0.01 versus control. Data were obtained from six independent experiments (n = 6)

The effect of shRNA profilin-1 on the proliferation ability in cultured RASCMs. (A) Western blot analysis of the transfection efficiency of profilin-1 shRNA. **p < 0.01 versus Neg-CON. Data are from three independent experiments (n = 3). (B) Flow cytometry analysis detected the effect of shRNA profilin-1 on the proliferation of RASCMs incubated with ADAM. ++p < 0.01 versus Neg-CON. *p < 0.05 versus ADMA (30 μM). (C) MTT assay detected the effect of shRNA profilin-1 on the proliferation of RASCMs incubated with ADAM. ++p < 0.01 versus Neg-CON. *p < 0.05 versus ADMA (30 μM). Data are from six independent experiments (n = 3–6). CON, wild-type cells; ADMA, wild-type cells were treated with ADMA (30 μM) for 24 h; +Pfn-1 shRNA, profilin-1 shRNA transfected cells (4 mg) were treated with ADMA (30 μM) for 24 h; +Neg-CON, negative shRNA transfected cells were treated with ADMA (30 μM) for 24 h. Pfn-1 shRNA, cells were transfected with profilin-1 shRNA; Neg-CON, shRNA negative control

The effect of JAK2/STAT3 pathway in ADMA-mediated profilin-1 expression and proliferation of RASCMs. (A) Western blot analysis of profilin-1 expression ##p < 0.01 versus Neg-CON. (B,C) MTT and flow cytometry analyses revealed that the expression of profilin-1 and cell proliferation of RASCMs incubated with ADMA were partly inhibited in the presence of the JAK2 inhibitor AG490 (5 × 10−5 M) and STAT3 inhibitor rapamycin (10−8 M). #p < 0.05, ##p < 0.01 versus CON; *p < 0.05, **p < 0.01 versus ADMA (30 μM). Data are from three independent experiments (n = 3). Control, wild-type cells; ADMA, wild-type cells were treated with ADMA (30 μM) for 24 h; +AG490, cells were pretreated with AG490 (5 × 10−5 M) for 1 h before treatment with ADMA (30 μM) for 24 h. +Rapamycin, cells were pretreated with rapamycin (10−8 M) for 1 h before treatment with ADMA (30 μM) for 24 h. AG490, wild-type cells were treated with AG490 (5 × 10−5 M) for 24 h. Rapamycin, wild-type cells were treated with rapamycin (10−8 M) for 24 h; alcohol, wild-type cells were treated with 0.1% alcohol (AG490 solvent) for 24 h. DMSO, wild-type cells were treated with 0.1% DMSO (rapamycin solvent) for 24 h

3.2 Knockdown of profilin-1 inhibits ADMA-induced cell proliferation

As shown in Figure 2A, targeting profilin-1 using shRNA successfully achieved knockdown of expression in RASMCs at the mRNA and protein levels (p < 0.01). Furthermore, compared with the negative control, the stimulation of cell proliferation and the elevated percentage of cells in the S + G2/M phase induced by ADMA (30 M, 24 h) was blunted by profilin-1 silencing as observed in the MTT assays and flow cytometric analyses (p < 0.05; Figure 2B,C).

3.3 The role of JAK2/STAT3 pathway in ADMA-mediated cell proliferation and expression of profilin-1

To explore whether the JAK2/STAT3 pathway is involved in ADMA-induced vascular injury in RASMCs, the JAK2 inhibitor AG490 and inhibitor of STAT3 activation rapamycin were used. The results showed that upregulated expression of profilin-1 by ADMA (30 mM, 24 h) was markedly inhibited by pretreatment with AG490 (5 × 10−5 M) or rapamycin (10−8 M) for 1 h (p < 0.05; Figure 3A). In addition, pretreatment with AG490 (5 × 10−5 M) or rapamycin (10−8 M) also suppressed ADMA-induced proliferation of RASMCs by MTT and flow cytometry (p < 0.05, p < 0.01). Rapamycin inhibited the proliferation of RASMCs AG490 or rapamycin alone did not affect the profilin-1 expression of RASMCs, and AG490 alone had no effect on the proliferation of RASMCs. However, rapamycin alone markedly inhibited the proliferation of RASMCs (p < 0.05; Figure 3B,C).

4 DISCUSSION

The main findings of this study are as follows: (1) endothelial dysfunction exists in hypertensive patients, especially those with elevated levels of ADMA and profilin-1, (2) ADMA increases the expression of profilin-1 in cultured RASMCs, (3) knockdown of profilin-1 attenuates ADMA-mediated proliferative effects of RASMCs, and (4) the JAK2/STAT3 pathway may be involved in ADMA-induced cell proliferation and profilin-1 expression in RASMCs.

NO, known as the “endothelium-derived relaxing factor,” is a bioactive product of endothelial NO synthase (eNOS) and biosynthesized from L-arginine, oxygen, and NADPH, which regulates many endothelial cell functions. It contributes to vessel regulation of the cardiovascular system by signaling the surrounding smooth muscle to relax, inhibiting the proliferation of VSMCs, and preventing platelet aggregation.14-16 Growing evidence has shown that ADMA, an endogenous competitive inhibitor of NO synthase, plays a critical role during the process of endothelial dysfunction. It can reduce the synthesis of NO and result in endothelial dysfunction, platelet aggregation, leukocyte adhesion, and VSMC proliferation. Plasma levels of ADMA are significantly elevated in patients with endothelial dysfunction and VSMC proliferation disorders.17 In our study, we found that the levels of plasma ADMA, vWF, TNF-α, and IL-8 in essential hypertensive patients were significantly elevated, compared to normotensive subjects. It has been reported that patients with hypertension have higher ADMA concentrations than controls with normotension, and the L-arginine and ADMA are inversely related to endothelial function.18 Moreover, there is an association between high plasma ADMA levels and the presence of cardiovascular risk factors in hypertensive patients.19

Previously, it has been revealed that ADMA plasma levels are correlated with myointimal proliferation in the aorta of hypercholesterolemic rabbits, in which one-third of the proliferating cells are VSMCs.20 Furthermore, ADMA was shown to induce VSMC migration via phenotype change resulting from the activation of the Rho/ROCK signaling pathway.21 In this study, we confirmed that exogenous ADMA could stimulate the proliferation of VSMCs in a concentration- and time-dependent manner.

Profilin-1 plays a vital role in endothelial dysfunction-related and VSMC proliferation disorder-related cardiovascular diseases.7 It has been found that profilin-1 expression increases in endothelial cells, macrophages of atherosclerotic lesions, and diabetic endothelium.22, 23 It has been reported that the expression levels of profilin-1 were significantly increased in SHR rats that were injected with profilin-1 overexpression of adenoviral vector of pAd-Profilin-1-RES-EGFP, moreover, and changes in profilin-1 protein are considered to affect the occurrence and development of hypertensive vascular remodeling.24 Overexpression of profilin-1 is sufficient to induce cardiomyocyte hypertrophy and promotes the development of myocardial hypertrophy, which can be reversed by profilin-1 silencing, suggesting that profilin-1 is an important mediator of cellular hypertrophy.25-27 In this study, the effect of exogenous ADMA on profilin-1 protein expression in RASMCs was detected. The results found that ADMA stimulated the expression of profilin-1 in a concentration- and time-dependent manner. To further determine the contribution of profilin-1 in RASMC proliferation, we silenced the profilin-1 gene in RASMCs and discovered that knockdown of the expression of profilin-1 led to a significant decline in ADMA-mediated RASMC proliferation. It has been reported that overexpression of profilin-1 reduces a marked increase in vascular wall thickness and promotes aortic remodeling in SHRs.28 This study showed that profilin-1 plays a vital role in the proliferation of ADMA-induced VSMCs.

The JAK2/STAT3 pathway serves as a crucial downstream mediator for the signal transduction of a variety of hormones, cytokines, and growth factors.29 It has been shown that JAK activation is involved in the proliferation of VSMCs， and Ang II through the JAK2/STAT3 pathway increases nitroxidative stress, which contributes to the hyperproliferation of VSMC.30, 31 The study revealed that 7-ketocholesterol upregulated profilin-1 expression in aortic endothelial cells by activating the OSBP1/STAT3 pathway.32 In this study, JAK2 and STAT3 inhibitors weakened the expression and proliferation of profilin-1 in ADMA-mediated RASMCs. The results showed that ADMA upregulated profilin-1 expression and reduced VSMC proliferation through activation of the JAK2/STAT3 signaling pathway. Nevertheless, further investigation has indicated that Rho kinase is related to the proliferation of ADMA-mediated VSMCs (Figure 4).

The technical roadmap

In summary, our study demonstrates that profilin-1 mediates the ADMA-mediated proliferation of VSMCs, and that the JAK/STAT3 signaling pathway is activated through profilin-1. Disruption of profilin-1 gene expression attenuated the extent of VSMC proliferation, suggesting that profilin-1 may be involved in the vascular remodeling events that underpin endothelial abnormalities, particularly VSMC proliferation during atherosclerosis development. These new findings provide the basis for further understanding of the pathophysiological significance of ADMA and profilin-1 in cardiovascular diseases.

CONFLICT OF INTEREST

All authors declare no conflict of interest.