Mol Cell Biochem (2014) 390:115–121 DOI 10.1007/s11010-014-1962-1
Silencing heat shock protein 27 (HSP27) inhibits the proliferation and migration of vascular smooth muscle cells in vitro Jie Huang • Liang-di Xie • Li Luo • Su-li Zheng Hua-jun Wang • Chang-sheng Xu
•
Received: 25 July 2013 / Accepted: 14 January 2014 / Published online: 28 January 2014 Ó Springer Science+Business Media New York 2014
Abstract The objective of this study was to examine the role of heat shock protein 27 (HSP27) in proliferation and migration of vascular smooth muscle cells (VSMCs). Three complementary DNA sequences targeting rat HSP27 gene were designed, synthesized, and subcloned into lentiviral vector. The interfering efficiency was detected by reverse transcriptase-polymerase chain reaction and Western blot. Methyl thiazolyl tetrazolium bromide assay was used for examining cell proliferation. F-actin polymerization was detected by FITC-Phalloidin staining using confocal microscopy. Modified Boyden chamber technique was used to assess VSMCs migration. The recombinant lentivirus containing RNAi targeting HSP27 gene significantly inhibited expression of HSP27 at both mRNA and protein levels. The interfering efficiencies of pNL-HSP27-EGFP-1, pNL-HSP27-EGFP-2, and pNL-HSP27-EGFP-3 were 71 %, 77 %, and 43 %, respectively. Reorganization of actin stimulated by PDGF-BB was markedly blocked by pretreatment with pNL-HSP27-EGFP-2. Proliferation and migration rates of VSMCs induced by PDGF-BB were inhibited by 30.8 % and 45.6 %, respectively, by pNLHSP27-EGFP-2 (all P \ 0.01). To conclude, these data
J. Huang (&) L. Xie (&) L. Luo S. Zheng H. Wang C. Xu Fujian Hypertension Research Institute, First Affiliated Hospital of Fujian Medical University, 350005 Fuzhou, China e-mail: [email protected] L. Xie e-mail: [email protected] J. Huang Department of Cardiology of the Zhengzhou, Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, Hean Province, China
indicate that HSP27 may regulate the proliferation, actin reorganization, and the migration of VSMCs. RNAi targeting at HSP27 may be a potential approach for inhibition of cell migration involved in pathogenesis of proliferative vascular diseases. Keywords Heat shock protein 27(HSP27) RNA interference (RNAi) Vascular smooth muscle cells (VSMCs) Cytoskeleton Migration
Introduction Vascular smooth muscle cells (VSMCs) are major constituents of the blood vessel wall. Unlike the majority of differentiated cells, VSMCs have ability of phenotypic plasticity and can switch between a differentiated (also termed contractile) phenotype and a dedifferentiated (also termed synthetic) state in response to cellular stimuli [1–3]. VSMCs phenotypic switching is modulated by various environmental stimuli, including cytokines, cell–cell contact, cell adhesions, extracellular matrix interactions, mechanical force, and many other stimuli [4, 5]. Phenotypic modulation is a prerequisite for VSMC proliferation and migration. Platelet-derived growth factor (PDGF), an important kind of growth factors, reduced expression of contractile genes and increased rate of proliferation and migration of VSMCs [6]. It is shown that the proliferation and migration of VSMCs contribute to atherosclerotic plaque formation and stability. Many of the intercellular proteins are involved in VSMCs migration including p38mitogen-activated protein kinases, cytoskeletal, and actin remodeling proteins such as heat shock protein 27(HSP27). HSP27, a molecular chaperone in cellular responses for a variety of stresses such as heat shock,
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toxicants, and oxidative stress [7], is an important member of the small heat shock protein family. HSP27 plays a role in regulation of cell growth and differentiation, and involves in F-actin assembly [8, 9]. In smooth muscle, HSP27 is constitutively expressed and, when phosphorylated, colocalizes with contractile proteins [10]. Our group previously reported that HSP27 played an important role in the migration of VSMCs derived from spontaneously hypertensive rats (SHR) [11, 12]. RNA interference (RNAi), an endogenous process to regulate gene expression to maintain the homeostasis, is a powerful genesilencing mechanism in most eukaryotic cells [13]. RNAi phenomenon can be triggered by chemically synthesized and enzymatically produced RNA duplexes containing 21–25 nucleotides in mammalian cells [14, 15]. The effects of RNAi are sequence specific with high efficiency [16, 17]. So far, there have been no studies showing the direct effects of HSP27 silencing on migration in VSMCs. In this study, we used recombinant lentiviral vector targeting HSP27 to test the role of HSP27 in the proliferation and migration of VSMCs.
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Construction of lentiviral vectors encoding siRNA for HSP27 For knocking down HSP27 gene expression, three separate primers, 50 -GCCTCTTCGATCAAGCTTTCG-30 , 50 -GGT GCTTCACCCGGAA-ATACA-30 , and 50 -GCCCAAAGC AGTCACACAATC-30 , were designed and synthesized by Invitrogen from rat HSP27 (GenBank, accession number M86389). Oligonucleotides containing 21 nt of sense and 21 nt of complementary (antisense) sequences with an intervening loop were synthesized and inserted into the pSUPER vector. The H1 promoter and siRNA sequences were released from pSUPER vectors with EcoR I and Hind III, and inserted into lentiviral pNL-EGFP vector. The sequences of all three HSP27 siRNA constructs were verified by sequencing. The lentivirus was produced by cotransfection of packaging vector (pHelper) with vesicular stomatitis virus glycoprotein (VSVG) into 293T cells. Lentiviral particles were harvested 48 h after transfection, and purified with ultracentrifugation as described previously [18–20]. Efficacy of pNL-HSP27-EGFP
Materials and methods Xba I (R0145M, NEB, USA), HindIII (R104M, NEB, Ipswich, USA), Ale I (R0634L, NEB, Ipswich, USA), Hinc II(R0103L, NEB, Ipswich, USA), EcoR I (R0101L, NEB, Ipswich, USA), T4 DNA polymerase (R0203L, NEB, Ipswich, USA), Lipofectamine 2000 (11668-019, Invitrogen, USA), phalloidin-FITC (Sigma-Aldrich, Vienna, USA), Total HSP27 antibody(Santa Cruz Biotechnology, USA), immunoblotting chemiluminescence kit (Santa Cruz Biotechnology, USA), and Platelet-derived growth factorBB (Peprotech, USA) were commercially purchased. pNLEGFP lentivirus vector was kindly provided by Dr. Chen Yi-Pin (Tulane University, USA). Boyden chamber and polycarbonate membrane were from Molecular Probes (USA). All other reagents were of analytic grade.
Cell culture VSMCs were isolated from thoracic aortas of 8-week-old SHR by explant technique as described previously [11, 12] and were cultured in DMEM (PAA Laboratories GmbH, Haidmannweg, Pasching, Austria), supplemented with 15 % (v/v) fetal bovine serum (FBS), and 1 % (v/v) penicillin and streptomycin (100 lg/mL) in a humidified chamber at 37 °C in the presence of 5 % CO2. All the animal experimental procedures were approved by the Animal Care and Use Committee of Fujian Medical University.
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VSMCs were seeded at a density of 5.0 9 104 cells/mL in six-well plates and cultured overnight. When the cells reached 70 %–80 % confluence, 0.5 lg of pNL-HSP27EGFP-1, pNL-HSP27-EGFP-2, or pNL-HSP27- EGFP-3 was transfected into VSMCs using LipofectamineTM2000 based on manufacturer’s protocol. Same amount of pNLEGFP was used as an internal control. 24 h after transfection, the transfected cells were analyzed under a fluorescence microscope to determine the transfection efficiency. Total RNA was extracted from the cells according to the manufacturer’s instructions (Dalian Takara). The RNA concentration was determined byan ND1000 Spectrophotometer (Motogen, China), cDNA was synthesized according to the manufacturer’s protocol (M5101, Promega,USA). Levels of the mRNA transcripts encoding pNL-HSP27-EGFP-1, pNL-HSP27-EGFP-2, pNL-HSP27-EGFP-3, and b-actin genes were determined by reverse transcriptase polymerase chain reaction (RTPCR). Real-time PCR was performed with the AB17000 (ABI Biosystems). Each reaction was performed in triplicate, and the DCt method was used for the gene expression analysis [21]. To avoid technical error, each PCR experiment was repeated three times. Western blotting Western blotting analysis was performed as described previously [12]. In short, after extraction, proteins were separated by SDS-PAGE and immunoblot analysis was
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carried out with anti-total HSP27 (1:500) and anti-b-actin (1:2000). Specific antibody binding was detected using a chemiluminescence kit according to the manufacturer’s recommendations, and quantified by densitometer analysis. Analysis of actin cytoskeleton by confocal microscopy The effect of pNL-HSP27-EGFP-2 treatment on the reorganization of cytoskeleton of VSMCs was determined by confocal microscopy as described previously [11, 12]. In brief, VSMCs cultured on glass coverslips were treated with pNL-HSP27-EGFP-2 and pNL-EGFP for 24 h prior to stimulation with PDGF-BB for 2 h. VSMCs were then fixed with 4 % paraformaldehyde, permeabilized with 0.1 % Triton, and then incubated with Phalloidin-FITC for 60 min in dark for examination of organization of actin cytoskeleton. Images of actin filaments were captured and analyzed using a confocal laser scanning microscope (Leica, Tes-sp5, Germany)
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membrane were scraped away. The VSMCs that had migrated to the lower surface of the membrane were fixed and stained. The number of migrated cells on the lower side of the filter was quantitated by counting four highpowered fields per membrane. Assays were conducted in duplicate and repeated for six times.
Statistical analysis Data were expressed as mean ± SD, and One-way analysis of variance (ANOVA) was used to test statistical significance among different groups. Difference of data among groups was considered significant at P \ 0.05.
Results Effects of pNL-HSP27-EGFP-2 on the expression of HSP27 mRNA and protein in VSMCs
Cell proliferative assay Methyl thiazolyl tetrazolium bromide (MTT) assay was used to determine the activity of cells proliferation at 24 h posttransfection as described before [20]. In brief, the VSMCs were treated by 1. PDGF-BB 10 ng/ml; 2. pNLHSP27-EGFP-2 ? PDGF-BB; 3. pNL-EGFP ? PDGFBB; 4. pNL-HSP27-EGFP-2; and 5. pNL-EGFP. Untreated cells served as control. PDGF-BB used in the above experiments was the final concentration. To determine the proliferative activity of cells, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay was performed at 24 h posttransfection. Cells were plated at a density of 5.0 9 104 cells/ml in 24-well plates. Then, 20 ll of MTT reagent (5 g/L) was added to each well, and the cells were incubated for 4 h at 37 °C. Subsequently, 100 ll of dimethylsulfoxide (DMSO) was added to each well. The amount of MTT formazan was quantitated at the absorbance values A560 using Universal Microplate Spectrophotometer. All the experiments were performed in triplicate. Cell migration assay Cell migration was performed using a modified Boyden chamber as described previously [11, 12]. The cells, treated with pNL-HSP27-EGFP-2 for 24 h, were fasted in serumfree DMEM for 24 h prior to migration experiments. Cell suspension at a density of 104 cells/well was prepared and loaded into the upper wells of the chamber with a gelatincoated, polycarbonate membrane separating two chambers. The lower wells were filled with DMEM–BSA containing 10 ng/ml PDGF-BB or vehicle. The cells were incubated for 4 h and then the cells on the upper side of the
HSP27 mRNA expression is significantly downregulated in VSMCs, which were transfected with the RNAi targeting HSP27 gene compared to the cells that were transfected with an empty vector. The interfering efficiencies of pNLHSP27-EGFP-1, pNL-HSP27-EGFP-2, and pNL-HSP27EGFP-3 were 71, 77, and 43 %, respectively. The lentivirus with the highest interfering efficiency was pNL-HSP27EGFP-2 (Fig. 1). Western blot showed that the silencing of the HSP27 gene considerably decreased the expression of HSP27 protein compared to the cells that were transfected with an empty vector. pNL-HSP27-EGFP-2 showed the highest interfering efficiency of HSP27 protein expression in VSMCs (Fig. 2). Effect of pNL-HSP27-EGFP-2 on proliferation induced by PDGF-BB in VSMCs The proliferation of VSMCs in response to 10 ng/ml PDGF-BB was significantly increased by 193.7 % compared with control (P \ 0.01); proliferation of VSMCs, stimulated by PDGF, was remarkably inhibited by pNLHSP27-EGFP-2 (P \ 0.05) with an inhibition rate of 30.8 %, compared with the PDGF-BB-treated cells. Vector control does not show any antiproliferating effect on VSMCs (Fig. 3). Effect of pNL-HSP27-EGFP-2 on F-actin organization induced by PDGF-BB in VSMCs Treatment with PDGF-BB (10 ng/ml) resulted in a substantial increase in the number of stress fibers and
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Fig. 1 Downregulation of HSP27 mRNA expression in VSMCs transfected with pNL-HSP27-EGFP-1, pNL-HSP27-EGFP-2, and pNL-HSP27-EGFP-3 detected with real-time RT-PCR. Data are expressed as the average of three repeated experiments. pNL-HSP27EGFP-2 showed significant inhibition effects of HSP27 mRNA expression in VSMCs, with an inhibition rate of about 77 %. *P \ 0.01 versus empty vector
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Fig. 3 Effect of pNL-HSP27-EGFP-2 on MTT assay in SHR VSMCs in response to PDGF-BB stimulation. The proliferation of VSMCs was assessed by MTT assays. An empty vector (pNL-EGFP) showed no effect on the MTT absorbance. PDGF-BB significantly increases the absorbance in MTT assay. Cotransfection with pNL-HSP27EGFP-2 decreased the absorbance in MTT assay in VSMCs compared to that in the PDGF-BB group. Data expressed as mean ± SD. # P \ 0.01 versus empty vector, ##P \ 0.05 versus PDGF-BB
Effect of pNL-HSP27-EGFP-2 on the migration of VSMCs induced by PDGF-BB It showed that PDGF-BB significantly increased the migration of VSMCs compared to untreated cells. Migration of VSMCs provoked by PDGF-BB was inhibited by pNL-HSP27-EGFP-2 with an inhibition rate of 45.6 %. There was no significant difference between the PDGF and the pNL-EGFP ? PDGF-treated cells (Fig. 5).
Discussion
Fig. 2 HSP27 protein expression in different clones of VSMCs by Western blot: Lane 1 empty vector, Lane 2 control, Lane 3 pNLHSP27-EGFP-1, Lane 4 pNL-HSP27-EGFP-2, and Lane 5 pNLHSP27-EGFP-3; Data expressed as mean ± SD. pNL-HSP27-EGFP2 showed significant inhibition effects of HSP27 protein expression in VSMCs, with an inhibition rate of about 77 %
rearrangement of these structures into ordered parallel arrays in cultured VSMCs. Reorganization of actin cytoskeleton stimulated by PDGF-BB was inhibited by pNLHSP27-EGFP-2 (Fig. 4).
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The goal of this study was to examine the effect of RNAi targeting at HSP27 on the proliferation and migration of VSMCs induced by the PDGF-BB. We successfully constructed lentivirus vector carrying RNAi targeting HSP27 gene and screened the efficient interfering segments. It was found that the transfection of siRNA targeting HSP27 resulted in a significant reduction in the expression of HSP27 gene at mRNA and protein levels. It was demonstrated that the proliferation and migration of VSMCs induced by PDGF-BB were significantly suppressed by pNL-HSP27-EGFP-2 in vitro. The proliferation and migration of VSMCs have been suggested to contribute to pathogenesis of atherosclerosis. They are also the key elements in the development of the blood vessels, as well as in the formation of neointima during vascular injury [22–24]. VSMCs modulate their phenotype from the contractile to the proliferative or synthetic state in response to vascular injury and cytokine, such as PDGF. As a potent mitogen, PDGF plays a key role in VSMC proliferation and migration. PDGF binds with its
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Fig. 5 The inhibitory effects of pNL-HSP27-EGFP-2 on VSMCs migration induced by PDGF-BB. Data expressed as mean ± SD (n = 6). #P \ 0.01 versus control group, *P \ 0.05 versus PDGF-BB
Fig. 4 Inhibitory effects of pNL-HSP27-EGFP-2 on the reorganization of actin cytoskeleton induced by PDGF-BB (10 ng/ml) under confocal microscopy. Cells were also infected with an empty vector (pNL-EGFP) without transgene insert. The cells were fixed, stained with FITC-labeled phalloidin, and examined by confocal microscopy. a Control, b PDGF 10 ng/ml, c PDGF 10 ng/ml ? pNL-EGFP, and d PDGF 10 ng/ml ? pNL-HSP27-EGFP-2
receptor leading to activation of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) cascades. Cell migration depends on the reorganization of cytoskeletal proteins such as actin and myosin, and it has been shown that blocking of actin reorganization inhibits cell motility [25]. It was showed in this study that the reorganization of actin cytoskeleton stimulated by PDGF-BB was inhibited by pNL-HSP27-EGFP-2. Elevated HSP27 expression has been found in a number of tumors. It is also expressed at high levels in a variety of normal tissues including cardiovascular tissues [26, 27]. Several experiments have indicated that HSP27 might be a sole effector of actin remodeling in the process of cell migration [28, 29]. This is consistent with the hypothesis that HSP27 promotes F-actin remodeling, which is necessary for migration of smooth muscle cell. Phosphorylation of HSP27 was shown to be necessary for F-actin formation, stabilization of focal adhesions, and cell migration induced by growth factors and other chemoattractant’s stimulation [30–32]. A large body of evidence has shown that HSP27 is a downstream molecule of the MAPK and PI3K cascades, and activation of MAPK and PI3K/Akt results in phosphorylation of HSP27. Significantly, HSP27 is well referenced to support cytoskeletal integrity, particularly by protecting F-actin disruption under stress condition [33, 34]. Our previous research works demonstrated that the reorganization of actin and the migration of VSMCs stimulated by PDGF-BB were significantly inhibited by the HSP27 inhibitor quercetin via P38MAPK pathway in a concentration-dependent manner [11, 12]. Hedges et al. [35] also demonstrated that HSP27 phosphorylation is increased in the smooth muscle cells treated with PDGF, suggesting that p38MAPK activation promotes cell migration possibly by regulating actin remodeling.
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In this study, the proliferation of VSMCs was quantified by MTT assay, which was an established well-recognized stable method of determining viable cell number in proliferation studies. Dalya et al.’s [36], Jiangbin Sun et al.’s [37], and our previous work [20] used this method to examine the effect of cell proliferation. Furthermore, we demonstrated that there was no statistically significant difference of OD values between the control and the empty vector group in MTT assay. This result demonstrated that viral transfection slightly hardly influenced the viability of cells. Our results showed that the decreased levels of HSP27 with RNAi technique significantly inhibited the proliferation and migration of VSMCs, which has been approved to be responsible for the formation of plaque and restenosis remodeling. This is the first study, to our knowledge, to report that siRNA targeting at HSP27 gene has inhibitory effects on the proliferation and activation of cytoskeletal, and the migration of VSMCs. RNAi holds substantial promise for efficient and tissue-specific tool to treat atherosclerosis, and may be superior to other known methods previously tested in gene therapy experiments. In conclusion, the findings of present study indicate that downregulation of HSP27 with RNAs inhibited the activation of cytoskeletal and the migration of VSMCs, suggesting that HSP27 may be a potential target for gene therapy. Acknowledgments This work was supported by Grants from the Clinical Key Program of the Fujian Medical University (XK201107) and the Fujian Natural Science Foundation (2011J01160).
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Silencing heat shock protein 27 (HSP27) inhibits the proliferation and migration of vascular smooth muscle cells in vitro Jie Huang • Liang-di Xie • Li Luo • Su-li Zheng Hua-jun Wang • Chang-sheng Xu
•
Received: 25 July 2013 / Accepted: 14 January 2014 / Published online: 28 January 2014 Ó Springer Science+Business Media New York 2014
Abstract The objective of this study was to examine the role of heat shock protein 27 (HSP27) in proliferation and migration of vascular smooth muscle cells (VSMCs). Three complementary DNA sequences targeting rat HSP27 gene were designed, synthesized, and subcloned into lentiviral vector. The interfering efficiency was detected by reverse transcriptase-polymerase chain reaction and Western blot. Methyl thiazolyl tetrazolium bromide assay was used for examining cell proliferation. F-actin polymerization was detected by FITC-Phalloidin staining using confocal microscopy. Modified Boyden chamber technique was used to assess VSMCs migration. The recombinant lentivirus containing RNAi targeting HSP27 gene significantly inhibited expression of HSP27 at both mRNA and protein levels. The interfering efficiencies of pNL-HSP27-EGFP-1, pNL-HSP27-EGFP-2, and pNL-HSP27-EGFP-3 were 71 %, 77 %, and 43 %, respectively. Reorganization of actin stimulated by PDGF-BB was markedly blocked by pretreatment with pNL-HSP27-EGFP-2. Proliferation and migration rates of VSMCs induced by PDGF-BB were inhibited by 30.8 % and 45.6 %, respectively, by pNLHSP27-EGFP-2 (all P \ 0.01). To conclude, these data
J. Huang (&) L. Xie (&) L. Luo S. Zheng H. Wang C. Xu Fujian Hypertension Research Institute, First Affiliated Hospital of Fujian Medical University, 350005 Fuzhou, China e-mail: [email protected] L. Xie e-mail: [email protected] J. Huang Department of Cardiology of the Zhengzhou, Central Hospital Affiliated to Zhengzhou University, Zhengzhou 450007, Hean Province, China
indicate that HSP27 may regulate the proliferation, actin reorganization, and the migration of VSMCs. RNAi targeting at HSP27 may be a potential approach for inhibition of cell migration involved in pathogenesis of proliferative vascular diseases. Keywords Heat shock protein 27(HSP27) RNA interference (RNAi) Vascular smooth muscle cells (VSMCs) Cytoskeleton Migration
Introduction Vascular smooth muscle cells (VSMCs) are major constituents of the blood vessel wall. Unlike the majority of differentiated cells, VSMCs have ability of phenotypic plasticity and can switch between a differentiated (also termed contractile) phenotype and a dedifferentiated (also termed synthetic) state in response to cellular stimuli [1–3]. VSMCs phenotypic switching is modulated by various environmental stimuli, including cytokines, cell–cell contact, cell adhesions, extracellular matrix interactions, mechanical force, and many other stimuli [4, 5]. Phenotypic modulation is a prerequisite for VSMC proliferation and migration. Platelet-derived growth factor (PDGF), an important kind of growth factors, reduced expression of contractile genes and increased rate of proliferation and migration of VSMCs [6]. It is shown that the proliferation and migration of VSMCs contribute to atherosclerotic plaque formation and stability. Many of the intercellular proteins are involved in VSMCs migration including p38mitogen-activated protein kinases, cytoskeletal, and actin remodeling proteins such as heat shock protein 27(HSP27). HSP27, a molecular chaperone in cellular responses for a variety of stresses such as heat shock,
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toxicants, and oxidative stress [7], is an important member of the small heat shock protein family. HSP27 plays a role in regulation of cell growth and differentiation, and involves in F-actin assembly [8, 9]. In smooth muscle, HSP27 is constitutively expressed and, when phosphorylated, colocalizes with contractile proteins [10]. Our group previously reported that HSP27 played an important role in the migration of VSMCs derived from spontaneously hypertensive rats (SHR) [11, 12]. RNA interference (RNAi), an endogenous process to regulate gene expression to maintain the homeostasis, is a powerful genesilencing mechanism in most eukaryotic cells [13]. RNAi phenomenon can be triggered by chemically synthesized and enzymatically produced RNA duplexes containing 21–25 nucleotides in mammalian cells [14, 15]. The effects of RNAi are sequence specific with high efficiency [16, 17]. So far, there have been no studies showing the direct effects of HSP27 silencing on migration in VSMCs. In this study, we used recombinant lentiviral vector targeting HSP27 to test the role of HSP27 in the proliferation and migration of VSMCs.
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Construction of lentiviral vectors encoding siRNA for HSP27 For knocking down HSP27 gene expression, three separate primers, 50 -GCCTCTTCGATCAAGCTTTCG-30 , 50 -GGT GCTTCACCCGGAA-ATACA-30 , and 50 -GCCCAAAGC AGTCACACAATC-30 , were designed and synthesized by Invitrogen from rat HSP27 (GenBank, accession number M86389). Oligonucleotides containing 21 nt of sense and 21 nt of complementary (antisense) sequences with an intervening loop were synthesized and inserted into the pSUPER vector. The H1 promoter and siRNA sequences were released from pSUPER vectors with EcoR I and Hind III, and inserted into lentiviral pNL-EGFP vector. The sequences of all three HSP27 siRNA constructs were verified by sequencing. The lentivirus was produced by cotransfection of packaging vector (pHelper) with vesicular stomatitis virus glycoprotein (VSVG) into 293T cells. Lentiviral particles were harvested 48 h after transfection, and purified with ultracentrifugation as described previously [18–20]. Efficacy of pNL-HSP27-EGFP
Materials and methods Xba I (R0145M, NEB, USA), HindIII (R104M, NEB, Ipswich, USA), Ale I (R0634L, NEB, Ipswich, USA), Hinc II(R0103L, NEB, Ipswich, USA), EcoR I (R0101L, NEB, Ipswich, USA), T4 DNA polymerase (R0203L, NEB, Ipswich, USA), Lipofectamine 2000 (11668-019, Invitrogen, USA), phalloidin-FITC (Sigma-Aldrich, Vienna, USA), Total HSP27 antibody(Santa Cruz Biotechnology, USA), immunoblotting chemiluminescence kit (Santa Cruz Biotechnology, USA), and Platelet-derived growth factorBB (Peprotech, USA) were commercially purchased. pNLEGFP lentivirus vector was kindly provided by Dr. Chen Yi-Pin (Tulane University, USA). Boyden chamber and polycarbonate membrane were from Molecular Probes (USA). All other reagents were of analytic grade.
Cell culture VSMCs were isolated from thoracic aortas of 8-week-old SHR by explant technique as described previously [11, 12] and were cultured in DMEM (PAA Laboratories GmbH, Haidmannweg, Pasching, Austria), supplemented with 15 % (v/v) fetal bovine serum (FBS), and 1 % (v/v) penicillin and streptomycin (100 lg/mL) in a humidified chamber at 37 °C in the presence of 5 % CO2. All the animal experimental procedures were approved by the Animal Care and Use Committee of Fujian Medical University.
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VSMCs were seeded at a density of 5.0 9 104 cells/mL in six-well plates and cultured overnight. When the cells reached 70 %–80 % confluence, 0.5 lg of pNL-HSP27EGFP-1, pNL-HSP27-EGFP-2, or pNL-HSP27- EGFP-3 was transfected into VSMCs using LipofectamineTM2000 based on manufacturer’s protocol. Same amount of pNLEGFP was used as an internal control. 24 h after transfection, the transfected cells were analyzed under a fluorescence microscope to determine the transfection efficiency. Total RNA was extracted from the cells according to the manufacturer’s instructions (Dalian Takara). The RNA concentration was determined byan ND1000 Spectrophotometer (Motogen, China), cDNA was synthesized according to the manufacturer’s protocol (M5101, Promega,USA). Levels of the mRNA transcripts encoding pNL-HSP27-EGFP-1, pNL-HSP27-EGFP-2, pNL-HSP27-EGFP-3, and b-actin genes were determined by reverse transcriptase polymerase chain reaction (RTPCR). Real-time PCR was performed with the AB17000 (ABI Biosystems). Each reaction was performed in triplicate, and the DCt method was used for the gene expression analysis [21]. To avoid technical error, each PCR experiment was repeated three times. Western blotting Western blotting analysis was performed as described previously [12]. In short, after extraction, proteins were separated by SDS-PAGE and immunoblot analysis was
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carried out with anti-total HSP27 (1:500) and anti-b-actin (1:2000). Specific antibody binding was detected using a chemiluminescence kit according to the manufacturer’s recommendations, and quantified by densitometer analysis. Analysis of actin cytoskeleton by confocal microscopy The effect of pNL-HSP27-EGFP-2 treatment on the reorganization of cytoskeleton of VSMCs was determined by confocal microscopy as described previously [11, 12]. In brief, VSMCs cultured on glass coverslips were treated with pNL-HSP27-EGFP-2 and pNL-EGFP for 24 h prior to stimulation with PDGF-BB for 2 h. VSMCs were then fixed with 4 % paraformaldehyde, permeabilized with 0.1 % Triton, and then incubated with Phalloidin-FITC for 60 min in dark for examination of organization of actin cytoskeleton. Images of actin filaments were captured and analyzed using a confocal laser scanning microscope (Leica, Tes-sp5, Germany)
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membrane were scraped away. The VSMCs that had migrated to the lower surface of the membrane were fixed and stained. The number of migrated cells on the lower side of the filter was quantitated by counting four highpowered fields per membrane. Assays were conducted in duplicate and repeated for six times.
Statistical analysis Data were expressed as mean ± SD, and One-way analysis of variance (ANOVA) was used to test statistical significance among different groups. Difference of data among groups was considered significant at P \ 0.05.
Results Effects of pNL-HSP27-EGFP-2 on the expression of HSP27 mRNA and protein in VSMCs
Cell proliferative assay Methyl thiazolyl tetrazolium bromide (MTT) assay was used to determine the activity of cells proliferation at 24 h posttransfection as described before [20]. In brief, the VSMCs were treated by 1. PDGF-BB 10 ng/ml; 2. pNLHSP27-EGFP-2 ? PDGF-BB; 3. pNL-EGFP ? PDGFBB; 4. pNL-HSP27-EGFP-2; and 5. pNL-EGFP. Untreated cells served as control. PDGF-BB used in the above experiments was the final concentration. To determine the proliferative activity of cells, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay was performed at 24 h posttransfection. Cells were plated at a density of 5.0 9 104 cells/ml in 24-well plates. Then, 20 ll of MTT reagent (5 g/L) was added to each well, and the cells were incubated for 4 h at 37 °C. Subsequently, 100 ll of dimethylsulfoxide (DMSO) was added to each well. The amount of MTT formazan was quantitated at the absorbance values A560 using Universal Microplate Spectrophotometer. All the experiments were performed in triplicate. Cell migration assay Cell migration was performed using a modified Boyden chamber as described previously [11, 12]. The cells, treated with pNL-HSP27-EGFP-2 for 24 h, were fasted in serumfree DMEM for 24 h prior to migration experiments. Cell suspension at a density of 104 cells/well was prepared and loaded into the upper wells of the chamber with a gelatincoated, polycarbonate membrane separating two chambers. The lower wells were filled with DMEM–BSA containing 10 ng/ml PDGF-BB or vehicle. The cells were incubated for 4 h and then the cells on the upper side of the
HSP27 mRNA expression is significantly downregulated in VSMCs, which were transfected with the RNAi targeting HSP27 gene compared to the cells that were transfected with an empty vector. The interfering efficiencies of pNLHSP27-EGFP-1, pNL-HSP27-EGFP-2, and pNL-HSP27EGFP-3 were 71, 77, and 43 %, respectively. The lentivirus with the highest interfering efficiency was pNL-HSP27EGFP-2 (Fig. 1). Western blot showed that the silencing of the HSP27 gene considerably decreased the expression of HSP27 protein compared to the cells that were transfected with an empty vector. pNL-HSP27-EGFP-2 showed the highest interfering efficiency of HSP27 protein expression in VSMCs (Fig. 2). Effect of pNL-HSP27-EGFP-2 on proliferation induced by PDGF-BB in VSMCs The proliferation of VSMCs in response to 10 ng/ml PDGF-BB was significantly increased by 193.7 % compared with control (P \ 0.01); proliferation of VSMCs, stimulated by PDGF, was remarkably inhibited by pNLHSP27-EGFP-2 (P \ 0.05) with an inhibition rate of 30.8 %, compared with the PDGF-BB-treated cells. Vector control does not show any antiproliferating effect on VSMCs (Fig. 3). Effect of pNL-HSP27-EGFP-2 on F-actin organization induced by PDGF-BB in VSMCs Treatment with PDGF-BB (10 ng/ml) resulted in a substantial increase in the number of stress fibers and
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Fig. 1 Downregulation of HSP27 mRNA expression in VSMCs transfected with pNL-HSP27-EGFP-1, pNL-HSP27-EGFP-2, and pNL-HSP27-EGFP-3 detected with real-time RT-PCR. Data are expressed as the average of three repeated experiments. pNL-HSP27EGFP-2 showed significant inhibition effects of HSP27 mRNA expression in VSMCs, with an inhibition rate of about 77 %. *P \ 0.01 versus empty vector
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Fig. 3 Effect of pNL-HSP27-EGFP-2 on MTT assay in SHR VSMCs in response to PDGF-BB stimulation. The proliferation of VSMCs was assessed by MTT assays. An empty vector (pNL-EGFP) showed no effect on the MTT absorbance. PDGF-BB significantly increases the absorbance in MTT assay. Cotransfection with pNL-HSP27EGFP-2 decreased the absorbance in MTT assay in VSMCs compared to that in the PDGF-BB group. Data expressed as mean ± SD. # P \ 0.01 versus empty vector, ##P \ 0.05 versus PDGF-BB
Effect of pNL-HSP27-EGFP-2 on the migration of VSMCs induced by PDGF-BB It showed that PDGF-BB significantly increased the migration of VSMCs compared to untreated cells. Migration of VSMCs provoked by PDGF-BB was inhibited by pNL-HSP27-EGFP-2 with an inhibition rate of 45.6 %. There was no significant difference between the PDGF and the pNL-EGFP ? PDGF-treated cells (Fig. 5).
Discussion
Fig. 2 HSP27 protein expression in different clones of VSMCs by Western blot: Lane 1 empty vector, Lane 2 control, Lane 3 pNLHSP27-EGFP-1, Lane 4 pNL-HSP27-EGFP-2, and Lane 5 pNLHSP27-EGFP-3; Data expressed as mean ± SD. pNL-HSP27-EGFP2 showed significant inhibition effects of HSP27 protein expression in VSMCs, with an inhibition rate of about 77 %
rearrangement of these structures into ordered parallel arrays in cultured VSMCs. Reorganization of actin cytoskeleton stimulated by PDGF-BB was inhibited by pNLHSP27-EGFP-2 (Fig. 4).
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The goal of this study was to examine the effect of RNAi targeting at HSP27 on the proliferation and migration of VSMCs induced by the PDGF-BB. We successfully constructed lentivirus vector carrying RNAi targeting HSP27 gene and screened the efficient interfering segments. It was found that the transfection of siRNA targeting HSP27 resulted in a significant reduction in the expression of HSP27 gene at mRNA and protein levels. It was demonstrated that the proliferation and migration of VSMCs induced by PDGF-BB were significantly suppressed by pNL-HSP27-EGFP-2 in vitro. The proliferation and migration of VSMCs have been suggested to contribute to pathogenesis of atherosclerosis. They are also the key elements in the development of the blood vessels, as well as in the formation of neointima during vascular injury [22–24]. VSMCs modulate their phenotype from the contractile to the proliferative or synthetic state in response to vascular injury and cytokine, such as PDGF. As a potent mitogen, PDGF plays a key role in VSMC proliferation and migration. PDGF binds with its
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Fig. 5 The inhibitory effects of pNL-HSP27-EGFP-2 on VSMCs migration induced by PDGF-BB. Data expressed as mean ± SD (n = 6). #P \ 0.01 versus control group, *P \ 0.05 versus PDGF-BB
Fig. 4 Inhibitory effects of pNL-HSP27-EGFP-2 on the reorganization of actin cytoskeleton induced by PDGF-BB (10 ng/ml) under confocal microscopy. Cells were also infected with an empty vector (pNL-EGFP) without transgene insert. The cells were fixed, stained with FITC-labeled phalloidin, and examined by confocal microscopy. a Control, b PDGF 10 ng/ml, c PDGF 10 ng/ml ? pNL-EGFP, and d PDGF 10 ng/ml ? pNL-HSP27-EGFP-2
receptor leading to activation of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) cascades. Cell migration depends on the reorganization of cytoskeletal proteins such as actin and myosin, and it has been shown that blocking of actin reorganization inhibits cell motility [25]. It was showed in this study that the reorganization of actin cytoskeleton stimulated by PDGF-BB was inhibited by pNL-HSP27-EGFP-2. Elevated HSP27 expression has been found in a number of tumors. It is also expressed at high levels in a variety of normal tissues including cardiovascular tissues [26, 27]. Several experiments have indicated that HSP27 might be a sole effector of actin remodeling in the process of cell migration [28, 29]. This is consistent with the hypothesis that HSP27 promotes F-actin remodeling, which is necessary for migration of smooth muscle cell. Phosphorylation of HSP27 was shown to be necessary for F-actin formation, stabilization of focal adhesions, and cell migration induced by growth factors and other chemoattractant’s stimulation [30–32]. A large body of evidence has shown that HSP27 is a downstream molecule of the MAPK and PI3K cascades, and activation of MAPK and PI3K/Akt results in phosphorylation of HSP27. Significantly, HSP27 is well referenced to support cytoskeletal integrity, particularly by protecting F-actin disruption under stress condition [33, 34]. Our previous research works demonstrated that the reorganization of actin and the migration of VSMCs stimulated by PDGF-BB were significantly inhibited by the HSP27 inhibitor quercetin via P38MAPK pathway in a concentration-dependent manner [11, 12]. Hedges et al. [35] also demonstrated that HSP27 phosphorylation is increased in the smooth muscle cells treated with PDGF, suggesting that p38MAPK activation promotes cell migration possibly by regulating actin remodeling.
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In this study, the proliferation of VSMCs was quantified by MTT assay, which was an established well-recognized stable method of determining viable cell number in proliferation studies. Dalya et al.’s [36], Jiangbin Sun et al.’s [37], and our previous work [20] used this method to examine the effect of cell proliferation. Furthermore, we demonstrated that there was no statistically significant difference of OD values between the control and the empty vector group in MTT assay. This result demonstrated that viral transfection slightly hardly influenced the viability of cells. Our results showed that the decreased levels of HSP27 with RNAi technique significantly inhibited the proliferation and migration of VSMCs, which has been approved to be responsible for the formation of plaque and restenosis remodeling. This is the first study, to our knowledge, to report that siRNA targeting at HSP27 gene has inhibitory effects on the proliferation and activation of cytoskeletal, and the migration of VSMCs. RNAi holds substantial promise for efficient and tissue-specific tool to treat atherosclerosis, and may be superior to other known methods previously tested in gene therapy experiments. In conclusion, the findings of present study indicate that downregulation of HSP27 with RNAs inhibited the activation of cytoskeletal and the migration of VSMCs, suggesting that HSP27 may be a potential target for gene therapy. Acknowledgments This work was supported by Grants from the Clinical Key Program of the Fujian Medical University (XK201107) and the Fujian Natural Science Foundation (2011J01160).
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