Effects of cyclic stretch on the molecular regulation of myocardin in rat aortic vascular smooth muscle cells
© Chiu et al.; licensee BioMed Central Ltd. 2013
Received: 9 April 2013
Accepted: 10 July 2013
Published: 15 July 2013
The expression of myocardin, a cardiac-restricted gene, increases during environmental stress. How mechanical stretch affects the regulation of myocardin in vascular smooth muscle cells (VSMCs) is not fully understood. We identify the mechanisms and pathways through which mechanical stretch induces myocardin expression in VSMCs.
Rat VSMCs grown on a flexible membrane base were stretched to 20% of maximum elongation, at 60 cycles per min. An in vivo model of aorta-caval shunt in adult rats was also used to investigate myocardin expression. Cyclic stretch significantly increased myocardin and angiotensin II (AngII) expression after 18 and 6 h of stretch. Addition of extracellular signal-regulated kinases (ERK) pathway inhibitor (PD98059), ERK small interfering RNA (siRNA), and AngII receptor blocker (ARB; losartan) before stretch inhibited the expression of myocardin protein. Gel shift assay showed that myocardin-DNA binding activity increased after stretch. PD98059, ERK siRNA and ARB abolished the binding activity induced by stretch. Stretch increased while myocardin-mutant plasmid, PD98059, and ARB abolished the promoter activity. Protein synthesis by measuring [3H]proline incorporation into the cells increased after cyclic stretch, which represented hypertrophic change of VSMCs. An in vivo model of aorta-caval shunt also demonstrated increased myocardin protein expression in the aorta. Confocal microscopy showed increased VSMC size 24 h after cyclic stretch and VSMC hypertrophy after creation of aorta-caval shunt for 3 days.
Cyclic stretch enhanced myocardin expression mediated by AngII through the ERK pathway in cultured rat VSMCs. These findings suggest that myocardin plays a role in stretch-induced VSMC hypertrophy.
KeywordsMyocardin Stretch Vascular smooth muscle cells ERK pathway
In recent years, vascular smooth muscle cell (VSMC) hypertrophy has been increasingly associated with the development of atherosclerotic disease . Hypertrophy of VSMCs may result in plaque rupture and vulnerability in atherosclerosis . Hypertrophy of VSMCs may be induced by certain cardiac-restricted genes through specific pathways under different types of environmental stress [3–5].
Myocardin is a potent cardiovascular regulated gene and transcriptional cofactor, which functionally synergizes with serum response factor (SRF; a transcriptional factor) and has been documented to have measurable effects on cardiac embryo development and both VSMC and cardiomyocyte hypertrophy [6–10]. An earlier study showed myocardin knockout mice resulted in embryonic lethality at E10.5, and was associated with failed VSMC differentiation . Previous studies performed on rat carotid artery VSMCs following vascular injury have demonstrated that myocardin can selectively regulate SRF binding to the degenerate CArGs on VSMC actin to increase transcriptional activity. Loss of myocardin may contribute to the supression of actin expression in response to vascular injury. Vascular injury has been associated with induced expression of myocardin . In addition, transfection with dominant-negative forms and small interfering RNA (siRNA) of myocardin in cultured VSMC decreased transcription of VSMC maker genes. Previous reports also indicated that either exogenous addition or stress-induced expression of angiotensin II (AngII) secretions would result in myocardin expression and subsequent VSMC or cardiac myocyte hypertrophy [12–15]. So myocardin itself may act as a cardiac regulated gene and cooperate with AngII in regulating VSMC or/and cardiac myocyte hypertrophy through specific signal transduction pathways under conditions of stress or injury to the cardiovascular system.
The application of cyclic stretch to cultured VSMCs has been widely used as an in vitro experiment to study molecular events in response to mechanical overload [16–20]. It has previously been reported that cyclic mechanical stretch induced hypertrophy in VSMCs [21–24]. Cells in the cardiovascular system are permanently subjected to mechanical forces due to the pulsatile variation of blood flow and shear force, created by the beating heart. These hemodynamic forces play an important role in the regulation of vascular development, remodeling, repair and formation of atherosclerotic stenosis [25–28]. Mechanical stretch can modulate several different cellular functions in VSMCs. These functions may include cell proliferation and differentiation, migration, survival or apoptosis, vascular remodeling, as well as autocrine or paracrine functions [29, 30]. This study aimed to identify the cellular and molecular effects of mechanical stretch on VSMCs regulated by myocardin, and to identify its signal transduction pathway and relationship with AngII. Knowing the impact of mechanical stretch on the cardiovascular system is crucial to the understanding of the pathogenesis of cardiovascular diseases, and a key to providing new insight into the prevention and therapy of cardiovascular diseases.
Previous reports have provided strong evidence that myocardin plays an important role in VSMC hypertrophy related to AngII secretion . However, no previous study has shown how cyclic mechanical stretch affects myocardin in the hypertrophy of VSMCs. Thus, in this study, we firstly investigated the mechanism of myocardin expression in cyclic mechanical stretch. Secondly, we investigated the effect and signal transduction pathway of myocardin expression induced by cyclic stretch.
Vascular smooth muscle cell culture
Primary cultures of VSMC were grown by the explant technique from the thoracic aorta of 200–250 g male Sprague–Dawley rats, as previously described [31, 32]. Cells were cultured in medium containing 20% fetal calf serum, 0.1 mmol/L non-essential amino acids, 1 mmol/L sodium pyruvate, 4 mmol/L L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C under 5% CO2/95% air in a humidified incubator. When confluent, monolayers of VSMCs were passaged every 6–7 days after trypsinization and were used for experiment from the 4th to 6th passages. These 4th to 6th passage cells were then cultured in Flexcell I flexible membrane dishes in medium containing 0.5% fetal calf serum, and the cells were incubated for a further 2 days to render them quiescent before initiating each experiment. The study was reviewed and approved by the Institutional Animal Care and Use Committee of Shin Kong Wu Ho-Su Memorial Hospital and conforms to Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 2011).
In vitro cyclic stretch on cultured vascular smooth muscle cells
The strain unit Flexcell FX-2000 (Flexcell International Co., NC, USA) consists of a vacuum unit linked to a valve controlled by a computer program. VSMCs cultured on the flexible membrane base were subjected to cyclic stretch produced by this computer-controlled application of sinusoidal negative pressure, as previously characterized and described in detail [33, 34]. A 10% or 20% cyclic stretch was performed with a frequency of 1 Hz (60 cycles/min).
Antibodies and reagents
Rabbit polyclonal antibodies against myocardin, mouse monoclonal antibodies (mAbs) against c-Jun N-terminal kinase (JNK) and anti-GAPDH antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse mAbs against p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and phospho-ERK were purchased from BD Bioscience Pharmingen (San Diego, CA). PD98059, SB203580, and SP600125 were purchased from Calbiochem (San Diego, CA). All other chemicals of reagent grade were obtained from Sigma (St Louis, MO). The roles of JNK, p38 MAPK, and ERK in stretch-induced myocardin expression were determined by pretreatment of the VSMCs with 25 μM SP600125, 3 μM SB203580, or 50 μM PD98059 for 30 min before cyclic stretch. SP600125 is a potent, cell-permeable, selective, and reversible inhibitor of JNK. SB203580 is a highly specific, cell-permeable inhibitor of p38 MAPK. PD98059 is a specific and potent inhibitor of the ERK pathway. The AngII and AngII antibodies came from Bachem AG (Torrance, CA). To examine the effect of ARB (AngII type 1 receptor blocker), VSMCs were treated with 100 nM losartan (Merck & Co, Inc).
Western blot analysis
Western blot was performed as previously described .
Total RNA was isolated from VSMCs using the single-step acid guanidinium hiocyanate/phenol/chloroform extraction method. The cDNA produced by reverse transcription (RT) was used to generate myocardin probes by polymerase chain reaction (PCR) as previously described . Primers were designed for detection of myocardin gene expression. The primers for myocardin were: 5′-GGACTGCTCTGGCAACCCAGTGC-3′; reverse: 5′-CATCTGCTGACTCCGGGTCATTTGC - 3′. GAPDH gene expression was used as internal controls. The primers for GAPDH were: 5′-GAGAGGCTCTCTGTCGACTAC-3′; reverse: 5′-TAGTGTAGGTTGGGCGCTCAA-3′.
VSMCs were transfected with 800 ng of ERK- and myocardin-annealed siRNA (Dharmacon, Lafayette, CO). ERK siRNAs are target- specific 20- to 25-nt siRNAs designed to knock down gene expression. The siRNA sequences were 5′-GACCGGAUGUUAACCUUUAUU (sense) and 5′-PUAAAGGUUAACA UCCGGUCUU (antisense) for ERK. The myocardin siRNA sequences were 5′-UGCAACUGCAGAAGCAGAAUU (sense) and 5′-UGCAACUGGUCUUGCAGAAUU (antisense). As a negative control, a non-targeting (control) siRNA (Dharmacon) was used. For transfection of rat VSMCs with siRNA oligonucleotides, we used Effectene transfection reagent according to the manufacturer’s instructions (Qiagen, Valencia, CA). After incubation at 37°C, cells were subjected to stretch and analyzed by Western blot.
Measurement of AngII concentration by enzyme-linked immunosorbent assay (ELISA)
Conditioned medium from VSMCs subjected to cyclic stretch and those from unstretched cells were collected for AngII measurement. The level of AngII was measured by a quantitative sandwich enzyme immunoassay technique (R&D Systems, Minneapolis, MN, USA). The lowest limit of AngII ELISA kit was 52 pg/mL.
Electrophoretic mobility shift assay (EMSA)
Nuclear protein concentrations from cultured VSMCs were determined by the Bradford method as commercialized by Santa Cruz Biotechnology and EMSA was performed as previously described . Consensus and control oligonucleotides were labeled by polynucleotide kinase incorporation of [γ-32P] ATP. In each case, mutant or cold oligonucleotide was used as a control to compete with the labeled sequences. The oligonucleotide sequence of consensus binding site for SRF was sense: 5′- GGA TGT CCA TAT TAG GAC ATC T-3′ and reverse: 5′-CCT ACA GGT ATA ATC CTG TAG A-3′. The mutant oligonucleotide sequence was 5′- GGA TGT CCA TAT TAT TAC ATC T-3′.
Promoter activity assay
A −968 to +44 bp rat myocardin promoter construct was generated as previously described . The myocardin promoter contains myocardin binding sites for SRF (sequences: CGGTTTAGGG) located at −514 to −505 bp of the promoter region. We used the binding sites to detect the transcriptional activity of myocardin. For construction of the mutant SRF binding region of myocardin, we changed the sequences located at −507 to −506 bp from CGGTTTAGG G to CGGTTTATT G by using a mutagenesis kit (Stratagene, La Jolla, CA). Site-specific mutations were confirmed by DNA sequencing. Plasmids were transfected into VSMCs using a low-pressure accelerated gene gun (Bioware, Taipei, Taiwan). Rat genomic DNA was amplified with forward (GGACTGCTCTGGCAAC CCAGT GC) and reverse (CATCTGCTGACTCCGGG TCATTTGC) primers. The amplified product was digested with Mlu I and Bgl II restriction enzymes and ligated into pGL3-basic luciferase plasmid vector (Promega, Madison, WI) digested with the same enzymes. In brief, 2 μg of plasmid DNA was suspended in 5 mL of PBS and was delivered to the cultured VSMCs at a helium pressure of 15 psi. The transfection efficiency using this method is 30%. Following 12 h of cyclic stretching, cell extracts were prepared using the Dual-Luciferase Reporter Assay System (Promega) and measured for dual luciferase activity by luminometer (Turner Designs, Sunnyvale, CA, USA).
The migration activity of VSMCs was determined using the growth-factor-reduced Matrigel invasion system (Becton Dickinson), following the protocol provided by the manufacturer. The migration assay was performed as previously described .
Determination of protein synthesis
Protein synthesis was examined by measuring [3H]proline incorporation into the cells. Cultured VSMCs were divided into the following groups: (1) control group: the cells were cultured in serum-free DMEM; and (2) mechanical stretch group (20% cyclic stretch) added to serum-free medium. Each experiment was repeated 6 times. VSMCs were first grown in DMEM with 10% FBS and 200 mg/L L-glutamine, and then seeded in 24-well plates at 1 × 105 cells/well in DMEM + 10% FBS. After synchronization of VSMCs, the medium was changed to DMEM without serum. VSMCs were treated with cyclic stretch and exposed to [3H]proline at the concentration of 1 μCi/well for the last 12 h of the 24 h incubation period. After the incubation, the cells were washed with ice-cold PBS and 10% trichloroacetic acid. Acid-insoluble [3H]proline was collected on glass fiber filters (Whatman, Kent, UK) and determined by a liquid scintillation counter (LS 6500, Beckman, Fullerton, CA , USA).
Rat model of aorta-caval shunt
The aorta-caval shunt was produced as previously described . The vena cava and aorta were exposed via abdominal midline incision. In brief, the aorta was punctured at the union of the segment two-thirds caudal to the left renal artery and one-third cephalic to the aortic bifurcation, with an 18-gauge disposable needle held with a plastic syringe. The needle was advanced into the aorta, perforating its adjacent wall and penetrating the vena cava. The induced aorta-caval shunt produced a ratio of 1.7 of pulmonary to systemic flow. Sham-operated control animals were prepared in a similar manner, except that the aorta was not punctured.
All results were expressed as means ± SEM. Statistical significance was evaluated using variance (GraphPad Software Inc., San Diego, CA, USA). Dunnett’s test was used to compare multiple groups to a single control group. Tukey-Kramer comparison was used for pairwise comparisons between multiple groups after ANOVA. A value of P < 0.05 was considered to denote statistical significance.
Cyclic stretch enhances myocardin protein and mRNA expression in vascular smooth muscle cells
Stretch-induced myocardin protein expression in vascular smooth muscle cells is mediated by the ERK pathway
Cyclic stretch increases the phosphorylation of ERK protein in rat vascular smooth muscle cells
We also found that ERK protein phosphorylation increased to its maximal level 24 h after 20% cyclic stretch and declined gradually. The ERK pathway inhibitor (PD98059) could effectively block the phosphorylation of ERK protein (Figure 2C and D).
Cyclic stretch stimulates secretion of angiotensin II from vascular smooth muscle cells
Exogenous addition of angiotensin II increases myocardin protein expression
To investigate the direct effect of AngII on myocardin expression in VSMCs, AngII at different concentrations was administered to the cultured medium for 24 h. As shown in Figure 3B and C, the effect of AngII on myocardin protein expression was dose-dependent. These findings suggested that exogenous addition of AngII also enhances myocardin expression without cyclic stretch. Addition of losartan 30 min before stretch significantly blocked the expression of myocardin induced by cyclic stretch for 24 h.
Cyclic stretch increases myocardin binding activity
Cyclic stretch increases myocardin promoter activity through the ERK pathway
To study whether the myocardin expression induced by stretch is regulated at the transcriptional level, we cloned the promoter region of rat myocardin (−968 to +44) and constructed a luciferase reporter plasmid (pGL3-Luc). The myocardin promoter construct contains myocardin binding sites. As shown in Figure 4B and C, transient transfection experiments on VSMCs using this reporter gene revealed that stretch for 6 h significantly induced myocardin promoter activity. This result indicated that myocardin expression in VSMCs is induced at transcriptional level during cyclic stretch. When the myocardin binding sites were mutated, the increased promoter activity induced by stretch was abolished. Moreover, addition of ERK pathway inhibitor (PD98059) and ARB (losartan) caused an inhibition of transcription. These results suggested that the binding site in the myocardin promoter is essential for transcriptional regulation by cyclic stretch. In addition, we also found that exogenous addition of AngII without stretch increases the transcriptional activity in VSMCs (Figure 4C).
Myocardin increases the migration of VSMCs
Cyclic stretch induces protein synthesis in VSMCs and VSMC hypertrophy
In vivo aorta-caval shunt increases aortic myocardin protein expression
In this study, we demonstrated several significant or novel findings. Firstly, cyclic stretch upregulates myocardin expression in rat VSMCs; secondly, cyclic stretch induces AngII expression in VSMCs; thirdly, AngII acts as an autocrine factor to mediate the increased myocardin expression induced by cyclic stretch; fourthly, ERK MAP kinase and SRF transcriptional factor are involved in the signaling pathway of myocardin induction; and fifthly, in vivo acute hemodynamic overload increases aortic myocardin expression. Myocardin was upregulated in both a time- and load- dependent manner by cyclic stretch. Cyclic stretch of VSMCs increased both myocardin protein and mRNA expression.
In our study, exogenous addition of AngII to non-stretched VSMCs was also sufficient to induce similar myocardin protein expression as that observed in stretched VSMCs. These results provide the first evidence that AngII mediates cyclic stretch-induced expression of myocardin in VSMCs. Our study revealed that AngII acts as an autocrine mediator in response to cyclic stretch in VSMCs. Previously, another study identified that AngII enhanced myocardin expression through AngII type 1 (AT1) receptor results in VSMC hypertrophy . We also have previously demonstrated that hypoxia in cardiomyocytes increased AngII secretion and myocardin expression and finally resulted in cardiac myocyte hypertrophy through the ERK pathway . In this study, we found that cyclic stretch also enhanced myocardin expression by AngII secretion and the ERK pathway, which had not been identified by previous studies.
However, one study found no increased concentration of AngII in the medium collected from porcine VSMCs at 24 and 48 h after 25% stretch . Sotoudeh et al. used pulmonary VSMCs, whereas our study used rat aortic VSMCs. Different species, stretch intension, and stretch time may explain the discrepancy. Our results suggest that AngII is responsible for myocardin-DNA binding in VSMCs. In this study, we demonstrated that cyclic stretch stimulation of myocardin-DNA binding activity required at least phosphorylation of ERK since ERK pathway inhibitor (PD98059) and ERK siRNA abolished the myocardin/SRF binding activity. PD98059, a potent and specific inhibitor of ERK MAP kinase, also inhibited the myocardin expression induced by stretch, whereas inhibitors of p42/p44, p38, and c-JUN MAP kinase did not have this inhibitory effect. Thus, ERK MAP kinase is an important intracellular signaling pathway that regulates myocardin expression. We also demonstrated that ERK siRNA significantly inhibited myocardin expression induced by stretch. ARB likewise had an inhibitory effect on the stretch-induced myocardin expression. Since ARB is an AngII inhibitor, and mechanical stretch is known to affect the production of AngII, , our findings potentially indicate that AngII has a role in the induction of myocardin by mechanical stretch. In this study, we demonstrated via promoter activity assay that increased transcriptional activity of myocardin promoter by cyclic stretch was SRF dependent. These data imply that the ERK MAP kinase pathway, but not the other MAP kinase pathway, is the major pathway involved in the induction of myocardin by stretch and that it mediates the increased binding activity of myocardin and transcription to VSMCs.
Mechanical stretch can modulate several different cellular functions in VSMCs. These functions include cell alignment and differentiation, migration, survival or apoptosis, vascular remodeling, and autocrine or paracrine functions . However, use of different kinds of VSMCs (venous or arterial) and various species of animals used in different studies (mouse, rat, rabbit, swine and others), have resulted in sometimes controversial findings . Most of these studies used in vitro models. However, the cellular functions induced by in vitro mechanical stretch may not accurately represent cellular function in vivo. So, more studies are necessary to identify the real effects of mechanical stretch on VSMC functions and the mechanisms by which they occurr. Our study further confirmed the increased aortic myocardin expression in acute hemodynamic overload as that occurring with aorta-caval shunts. It has been previously reported that myocardin protein expression increased in the carotid artery balloon injury model in rats , suggesting myocardin may be enhanced during acute hemodynamic overload in vivo. The increased myocardin protein expression following acute hemodynamic overload may contribute to the regulation of vascular repair and remodeling, which involves VSMC proliferation .
With regard to the clinical application of cyclic stretch on VSMCs, mechanical stretch activates multiple intracellular signaling networks and regulates gene expressions and functional responses in VSMCs. The cellular and molecular effects of mechanical stretch on vascular cells may provide new insights in the pathogenesis of vascular diseases and therapeutic potentials. Mechanical stretch can modulate several different cellular functions in VSMCs, including cell alignment and differentiation, migration, survival or apoptosis, vascular remodeling, and autocrine and paracrine functions. Arterial VSMCs are aligned primarily in the circumferential direction in the media of the artery. Mechanical stretch from pulsatile blood flow is one of the key factors in regulating vascular remodeling. VSMC migration is important in the development of vascular diseases, including atherosclerosis and post-angioplasty restenosis. VSMC migration is found more frequently in curved and bifurcating blood vessels, which are exposed to non-laminar blood flow, than in straight arterial segments exposed to laminar blood flow. In this, we have also demonstrated that mechanical stretch increased the migration of VSMCs. The gene expression induced by mechanical stretch may be relevant to pathological complications in the cardiovascular system, including atherosclerosis, plaque instability and hypertension. The induction of genes by mechanical stretch may play a role in vascular remodeling. Understanding the molecular mechanisms regulating VSMC remodeling, migration, and proliferation under mechanical stretch supports the clinical application of ACEI (angiotensin-converting enzyme inhibitors), ARB, and statin in cardiac protection and in the prevention of vascular diseases. Therefore knowledge of the impact of mechanical stretch on VSMCs is vital in the understanding of the pathogenesis of cardiovascular diseases and is crucial in providing new insights into the prevention and therapy of cardiovascular diseases.
In summary, our study reports for the first time that cyclic mechanical stretch enhances myocardin expression in cultured rat VSMCs. The stretch-induced myocardin is mediated by AngII through the ERK pathway.
Vascular smooth muscle cells
Serum response factor
Small interfering RNA
C-Jun N-terminal kinase
Mitogen-activated protein kinase
Extracellular signal-regulated kinase
AngII type 1 receptor blockers
Polymerase chain reaction
Enzyme-linked immunosorbent assay
Electrophoretic motility shift assay
Dulbecco’s modified Eagle’s/F12 medium
Receptor: AngII type 1 receptor.
This study was sponsored in part by Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan.
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