Introduction

Migration of vascular smooth muscle cells (VSMCs) has been suggested to be a key process in the early initiation of lesions of atherosclerosis and restenosis after percutaneous coronary intervention (PCI). Cell migration is believed to be promoted by growth factors and vascular bioactive peptides. However, complete inhibition of the mainstream pathways of intracellular signallings for a specific factor could not fundamentally prevent focus forming, and blockade of a mainstream intracellular signalling for some factors may also inhibit cell migration induced by other chemostimulants believed by another intracellular pathway. There may be a common final pathway key confluence, mediating different active stimulants, contributed to the migration of VSMCs and formation of atherosclerotic plaque. It has been a research orientation toward effectively preventing the VSMCs proliferation-associated diseases by regulating the key gene of such final common paths in cell migration.

Recent reports from several groups suggested that heat shock protein 27 (HSP27) might be a sole effector of actin remodeling [1–4]. Phosphorylation of HSP27 plays an important role in modulating its structural and functional coupling. Nonphosphorylated monomeric HSP27 binds to F-actin as a capping protein and thereby blocks actin polymerization, whereas phosphorylated HSP27 does not affect actin polymerization. Phosphorylation of HSP27 was shown to be necessary for F-actin formation, stabilization of focal adhesions, and promotion of cell migration induced by growth factor stimulation [5–7]. As a result, it is potential that regulating the activity of HSP27 be a therapeutic target for inhibition of cell migration.

The present study was designed to access the role of HSP27 phosphorylation in the migration of VSMCs induced by angiotensin II (AngII) and platelet derived growth factor-BB (PDGF-BB).

Methods

Materials

Platelet derived growth factor-BB was from PeproTech. AngII, Quercetin and phalloidin-FITC were obtained from Sigma-Aldrich Co. Phospho-HSP27 antibody was purchased from Alexis. Total-HSP27 antibody and immunoblotting chemiluminescence kit were provided from Santa Cruz Biotechnology. All other reagents were of analytic grade.

Cell culture

Vascular smooth muscle cells were isolated from thoracic aortas of 8-week-old spontaneously hypertensive rats (SHR) by explant technique and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum, 100 kU/l benzylpenicillin, 100 mg/l streptomycin. And the procedures of animal manipulation were approved by the Animal Care and Use Committee of Fujian Medical University. The cells showed a spindle-shaped appearance, and confluent VSMCs in culture showed the characteristic of “hill and valley” growth pattern. The identification of VSMCs was performed by smooth muscle α-actin staining. Cells were passaged by trypsinization and cells between passage three and five were used for experiments.

Western blotting

The cells were washed twice with ice-cold PBS, and lysed with SDS sample buffer (Tris–HCl 62.5 mmol/l, SDS 2%, glycerol 10%, DTT 50 mmol/l, bromophenol blue 0.1 %, pH 6.8). Solubilized protein samples were centrifuged (12,000g, 15 min, 4°C) and supernatant protein concentration was determined by Bradford protein assay.

Equal amounts of protein were then separated by SDS-PAGE and transferred to nitrocellulose membranes. Non-specific binding was blocked by 5% fat-free milk powder in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature. The membranes were incubated with primary antibodies of mouse anti-human for Phospho-HSP27 (1:70), and goat anti-human for total-HSP27 (1:500) overnight at 4°C. After washing with TBST, the membranes were incubated for 90 min with secondary antibody (goat anti-mouse for Phospho-HSP27, rabbit anti-goat for total-HSP27) at a dilution of 1:2,000 at room temperature. Membranes were then washed three times with TBST and once with TBS. The signals were detected using a chemiluminescence kit and quantified by densitometer analysis.

Analysis of actin cytoskeleton by confocal microscopy

Vascular smooth muscle cells cultured on glass coverslips were treated with Quercetin for 1 h prior to stimulation with AngII and PDGF-BB for 2 h. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton, and then incubated with Phalloidin-FITC for 60 min in dark. Images of actin filaments were captured and analyzed with a confocal microscopy.

Cell migration assay

Cell migration was carried out using a modified Boyden chamber. Cells were fasted in serum-free DMEM for 24 h prior to migration experiments. Cells were suspended and counted, and then 0.4 ml was added on the upper chamber with a gelatin-coated, polycarbonate membrane (8.0 μm pore) separating two chambers. After 4 h, cells on the upper side of the membrane were scraped using a cotton swab. Cells that migrated to the lower side of the membrane were fixed with 95% alcohol and stained with 0.5% toluidine. The number of migrated cells on the lower side of the filter was counted in five fields under 10× magnification. Assays were conducted in duplicate and repeated six times using cultured cells from different animals (n = 6).

Statistical analysis

Values were expressed as mean ± SD, and assessed by one-way ANOVA test.

Results

Effect of AngII on activity of HSP27 in VSMCs

The basal level of HSP27 phosphorylation was low in quiescent VSMCs. AngII (10−7 mol/l) induced a rapid, transient increase of HSP27 phosphorylation, with a peak at 30 min. The phosphorylation of HSP27 induced by AngII was increased in a concentration-dependent manner in VSMCs. The phosphorylation of HSP27 in response to 10−5 mol/l AngII stimulation was increased by 338.9% compared with control (P < 0.01) (Fig. 1).

Fig. 1
figure 1

Effect of AngII on the phosphorylation of HSP27 in VSMCs. a Time-dependent phosphorylation of HSP27 induced by AngII (10−7 mol/l) in VSMCs. b Concentration-dependent phosphorylation of HSP27 induced by AngII (30 min). ** P < 0.05, * P < 0.01 versus control

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Effect of PDGF-BB on activity of HSP27 in VSMCs

The peak of HSP27 phosphorylation stimulated by PDGF-BB (30 ng/ml) was at 30 min. PDGF-BB induced HSP27 phosphorylation in a concentration-dependent manner in VSMCs. The phosphorylation of HSP27 in response to 100 ng/ml PDGF-BB was increased by 159.1% compared with control (P < 0.01) (Fig. 2).

Fig. 2
figure 2

Effect of PDGF-BB on the phosphorylation of HSP27 in VSMCs. a Time-dependent HSP27 phosphorylation of cultured VSMCs induced by PDGF-BB (30 ng/ml). b Concentration-dependent phosphorylation of HSP27 induced by PDGF-BB (30 min) in VSMCs. ** P < 0.05, * P < 0.01 versus control

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Effect of Quercetin on activity of HSP27 induced by AngII and PDGF-BB in VSMCs

The phosphorylation of HSP27 induced by AngII and PDGF-BB was blocked by the selective HSP inhibitor Quercetin in a concentration-dependent manner, with maximal inhibition rate at 71.7% and 66.7%, respectively (P < 0.01) (Fig. 3).

Fig. 3
figure 3

Concentration-dependent inhibitory effects of Quercetin on the activity of HSP27 induced by AngII (10−7 mol/l, 30 min) and PDGF-BB (30 ng/ml, 30 min) in VSMCs. a and b autoradiogram; c quantitation of immunoreactive bands. # P < 0.01 versus control. ** P < 0.05, * P < 0.01 versus positive

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Effect of Quercetin on F-actin induced by AngII and PDGF-BB in VSMCs

Treatment with AngII and PDGF-BB resulted in a substantial increase in the number of stress fibers and rearrangement of these structures into ordered parallel arrays in cultured SMCs. Reorganization of actin cytoskeleton stimulated by AngII and PDGF-BB was inhibited by 100 μmol/l Quercetin pretreatment (Fig. 4).

Fig. 4
figure 4

Inhibitory effects of Quercetin (100 μmol/l) on reorganization of actin cytoskeleton induced by AngII (10−6 mol/l) and PDGF-BB (10 ng/ml) for 2 h. The cells were fixed, stained with FITC-labeled phalloidin and examined by confocal microscopy. a control, b AngII, c AngII + Quercetin, d PDGF-BB, e PDGF-BB + Quercetin

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Effect of Quercetin on the migration of VSMCs induced by AngII and PDGF-BB

The migration of VSMCs was promoted by AngII and PDGF-BB. Migration of VSMCs was significantly inhibited by Quercetin. The inhibition rates of 100 μmol/l Quercetin on migration of VSMCs induced by AngII and PDGF-BB were at 55.3% and 53.6% (P < 0.01) (Fig. 5).

Fig. 5
figure 5

Inhibitory effects of Quercetin (100 μmol/l) on migration of VSMCs in response to AngII (10−6 mol/l) and PDGF-BB (10 ng/ml) for 4 h. The migrated cells were fixed in 95% alcohol, stained with 0.5% toludine and counted with a light microscopy at 200× magnification. # P < 0.01 versus control, * P < 0.01 versus PDGF, △ P < 0.01 versus AII

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Discussion

In this study, we showed that there was basic expression of HSP27 phosphorylation in VSMCs of SHR in vitro. AngII and PDGF-BB induced increasing expression of HSP27 phosphorylation in VSMCs in a time- and dose-dependent manner. Pretreatment of Quercetin may inhibit this inducible effect and migration of VSMCs.

Cell migration involves numerous spatially and temporally coordinated cellular processes and occurs in four steps: the formation of actin-rich protrusions such as lamellipodia, cell adhesion, the translocation of the cell body, and rear detachment. There is growing evidence that actin polymerization and the dynamic reorganization of the actin cytoskeleton play important roles in the regulation of cell migration.

HSP27, with a molecular weight of approximately 27 kDa, has been shown to form large aggregates of up to 800 kDa in the cytosol. Expression of HSP27 is up-regulated during the stress response, and correlated with increased survival ability in cells exposed to cytotoxic stimulus. It is a molecular chaperone with an affinity to interact with a large number of different proteins. It has been shown to prevent cell death caused by a variety of toxic agents that promote apoptosis. In addition to enhancing stress tolerance, HSP27 plays a key role in the arrangement of microstructure of actin, one or the most important cytoskeleton component, that is regulated by means of phosphorylation and dephosphorylation [8–10]. Purified unphosphorylated HSP27 homologs from mouse and chicken prevent actin from polymerization in vitro. Phosphorylation of HSP27 reverses the inhibition, presumably favoring formation of F-actin in vivo. Thus, HSP27 phosphorylation would be expected to increase the dynamics of actin assembly, which is necessary for VSMCs migration [11–13].

Previous reports have shown that the p38 mitogen-activated protein kinase (p38MAPK) pathway, through the activation of downstream kinases MAPK activated protein (MAPKAP) and p38-regulated/activated protein kinase, mediates the phosphorylation of HSP27 [14–20]. Takenaka et al. [19] reported that PDGF-BB phosphorylates HSP27 at Ser-15 and -85 via p38MAPK in cardiac myocytes. Meloche et al. [20] suggested that AngII, working through the AT1 receptor subtype, stimulated the enzymatic activity of p38, which then promoted the activation of MAPKAP kinase-2 and the phosphorylation of HSP27. Most importantly, incubation with the p38MAPK inhibitor SB203580 reduced the contractile effect of AngII on fresh rat aortic rings. The main finding of the present study is that AngII and PDGF-BB stimulate the phosphorylation of HSP27 in a time- and dose-dependent manner in VSMCs.

To determine whether HSP27 phosphorylation plays roles in the process of the actin reorganization and migration of VSMCs, we examined the effect of Quercetin on the phosphorylation of HSP27 and cell migration. Quercetin, a bioflavonoid extracted from gingko biloba, known as an inhibitor of heat shock factor protein 1 (HSF1), can block Hsps at the transcriptional levels. Our study demonstrated that the phosphorylation of HSP27 induced by AngII and PDGF-BB was partially blocked by Quercetin in a concentration-dependent manner. Reorganization of actin cytoskeleton stimulated by AngII and PDGF-BB was inhibited in 100 μmol/l Quercetin pretreatment. So we propose that the phosphorylation of HSP27 is an important pathway that contribute to the rearrangement of F-actin and migration of VSMCs in response to AngII and PDGF-BB.

In summary, our study clearly demonstrated that HSP27 phosphorylation plays a key role in actin filament remodeling and migration of VSMCs in response to AngII and PDGF-BB. HSP27 may be a potential target for the treatment of pathological process related to cell migration.