Novel role of Egr-1 in nicotine-related neointimal formation

Abstract

Aims

The aim of this study was to investigate the mechanisms by which nicotine increases vascular smooth muscle cell (VSMC) proliferation and post-injury neointimal formation.

Methods and results

Vascular injury was inflicted in the right iliac artery of nicotine-treated and control rats. Nicotine increased post-injury VSMC proliferation (Ki67⁺ cells) and neointimal formation (neointima/media ratio, 0.42 ± 0.23 vs. 0.14 ± 0.07, P= 0.02). To determine the mechanisms by which nicotine exacerbates VSMC proliferation, cultured cells were exposed to nicotine, and signalling pathways leading to cell proliferation were studied. Nicotine activated extracellular signal-regulated kinase (ERK) 1/2 in a dose- and time-dependent manner. The blockade of this signalling axis abolished nicotine-mediated proliferation. Functional nicotinic acetylcholine receptors and Ca²⁺ influx were necessary for ERK1/2 activation and nicotine-induced mitogenesis in VSMCs. Downstream to ERK1/2, nicotine induced the phosphorylation of Ets-like gene 1 in a timely co-ordinated manner with the up-regulation of the atherogenic transcription factor, early growth response 1 (Egr-1). The treatment of balloon-injured arteries with a lentivirus vector carrying a short hairpin RNA against Egr-1 abolished the deleterious effect of nicotine on vascular remodelling.

Conclusion

Nicotine acts through its receptors in VSMC to activate the ERK–Egr-1 signaling cascade that induces cell proliferation and exacerbates post-injury neointimal development.

1. Introduction

Extensive epidemiological evidence indicates that cigarette smoking increases the risk of cardiovascular diseases.¹ Smokers are estimated to have a two- to three-fold higher risk of developing coronary disease than non-smokers.² Understanding the mechanisms by which cigarette smoking modifies the normal healing of the vasculature is essential to design effective therapeutic strategies to lessen the impact of smoking on cardiovascular diseases. Nicotine, the addictive substance of cigarettes, stimulates the sympathetic nervous system, increases cardiac output and causes endothelial injury.³ It also exaggerates post-injury neointimal hyperplasia in preclinical animal models of balloon angioplasty^4–6 and increases atherosclerosis burden in Apo null mice.^7,8 Despite compelling evidence of the harmful effect of nicotine on the vasculature, the mechanisms by which nicotine induces vascular smooth muscle cell (VSMC) proliferation and exaggerates neointimal development remain unknown.

Previous studies have suggested that nicotine modifies the biology of VSMCs to make them more atherogenic. Thyberg and associates demonstrated for first time that nicotine had a mitogenic effect on rat arterial VSMCs in vitro.⁹ They also showed that nicotine induced a phenotypic change in VSMCs, converting them from a contractile to a synthetic phenotype. These findings suggest the important role of nicotine in vascular disease, because recruitment and proliferation of synthetic VSMCs within the tunica intima of injured vessels are key events in the pathogenesis of vascular occlusive diseases.¹⁰ Subsequently, using human arterial VSMCs, Carty and associates confirmed the mitogenic effect of nicotine.¹¹ More recently, we and others have demonstrated that nicotine induces human aortic VSMC proliferation through its interaction with the non-neuronal nicotinic acetylcholine receptors (nAChRs) to increase the secretion of platelet-derived growth factor (PDGF) and to up-regulate the expression of the latter's receptors. The mechanisms by which nicotine induces mitogenesis in VSMCs are, however, not fully understood.^12,13

This study sought to elucidate the molecular mechanism(s) by which nicotine increases VSMC proliferation and neointimal formation after vascular injury. We have demonstrated that direct action of nicotine on VSMC nAChRs leads to the activation of the mitogen-activated kinase extracellular signal-regulated kinase 1 and 2 (ERK1/2) and the up-regulation of the atherogenic transcription factor early growth response 1 (Egr-1). We have also demonstrated that by targeting Egr-1 with specific short hairpin (sh) RNAs it is possible to prevent the harmful effects of nicotine on post-injury neointimal formation.

2. Methods

2.1. Animal and surgical procedures

Sprague–Dawley rats were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN, USA). Nicotine was given in the drinking water (100 mg/L) for 3months before surgery until the animals were killed. Cotinine was measured in plasma by ELISA (Calbiotech, Spring Valley, CA, USA). Enzymatic determination of total serum cholesterol was performed according to Allain et al.¹⁴ Serum triglycerides were determined by quantitative enzymatic measurement of glycerol (Sigma-Aldrich, St Louis, MO, USA). Tail-cuff blood pressure was measured with a CODA non-invasive blood pressure system for rodents (Kent Scientific Corporation, Torrington, CT, USA). Plasma levels of interleukin (IL)-6, IL-1α and IL-1β were quantified using a LINCOplex bead assay kit (Millipore, Billerica, MA, USA).

All surgeries were performed under isoflurane anaesthesia. Vascular injury was inflicted in the right iliac artery.¹⁵ An aortotomy was made in the abdominal aorta to insert a 2 French Fogarty embolectomy catheter to the level of the right iliac artery. The balloon was inflated to 1.5 atmospheres and retracted to the aortotomy site. This was repeated three times to assure a good vascular injury. The aortic incision was repaired with 8–0 nylon sutures (0.4 metric, Ethicon, San Lorenzo, PR, USA). Rats were killed 3weeks after surgery using an overdose of isoflurane by inhalation, and the injured arteries were harvested and formalin fixed for histology.

Animal care was in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Institutes of Health (NIH publication No. 85-23, revised 1996). All animal procedures were revised and approved by the University of Miami Institutional Animal Care & Use Committee, protocols 06-078 and 09-074.

2.2. Histopathology and immunohistochemistry

Paraffin embedding and sectioning were performed by American Histolabs, SA (Bethestha, MD, USA). Morphometric analysis was performed on elastic Van Gieson or haematoxylin and eosin stained slides. The area of each vascular layer was measured to calculate the neointima-to-media ratio (N/M), where N/M = N/(M + N). Morphometric measurements and cell countings were performed on digital images using Image Pro Plus (Media Cybernetics, Inc., Bethesda, MD, USA).

For immunohistochemistry, sections were deparaffinized and rehydrated by serially immersing them in xylene, alcohol and water. After tissue rehydration, endogenous peroxidase was blocked with 3% hydrogen peroxide. Epitope retrieval was performed by boiling slides in citrate buffer (10 mM sodium citrate, pH 6.0) for 25 min. Non-specific binding was blocked with 0.5% blocking solution (DAKO, Carpinteria, CA, USA). Rabbit anti-Ki67 polyclonal antibodies (DAKO) were added for 1 h at room temperature. Bound primary antibodies were detected using the DAKO Universal link kit (DAKO). Colour was developed with a DAB chromogenic solution (DAKO). Nuclei were counterstained with Meyer's haematoxylin and mounted in Entellan mounting medium (EMD, Gibbstown, NJ, USA). Images were obtained with an Olympus 1X71 camera fitted to an Olympus BX 40 microscope (Olympus America Inc., Center Valley, PA, USA).

2.3. Cell culture and transfections

Rat VSMCs up to passage 22 were grown in serum-rich medium, Dulbecco's modified Eagle's medium–F12–fetal bovine serum (50:30:20).¹³ Cells were serum starved in 0.1% fetal bovine serum medium for 24 h to synchronize all cells at the G0 phase of the cell cycle. Nicotine hydrogen tartrate salt (Sigma-Aldrich, St Louis, MO, USA) was added to cells in 2% fetal bovine serum medium as indicated. Vascular smooth muscle cells were transfected by Amaxa's Nucleofector technology as described by the manufacturer (Lonza Cologne AG, Germany). Transfection efficiency was around 30% (Supplementary material online, Figure S1).

2.4. Proliferation

Proliferation was assessed by direct counting of cells after nicotine stimulation. Cells were seeded into six-well plates at a concentration of 1×10⁵ cells per well in serum-rich medium for 24 h. After starvation, cells were stimulated with nicotine (1 µM) for an additional 24 h. Pharmacological agents were used as indicated and added to cultures 2h before nicotine stimulation. Cell counts were performed after trypsinization with a single threshold Coulter counter (Model ZF, Coulter Electronics, Miami, FL, USA).

2.5. Western blot analysis

Vascular smooth muscle cells (2 × 10⁵ cells per well) seeded in six-well plates were incubated with or without pharmacological agents for 2 h before nicotine stimulation. Cells were rinsed twice with ice-cold phosphate-buffered saline and lysed in RIPA buffer (Millipore) supplemented with leupeptin (10 µg/mL), aprotinin (20 mU/mL), phenylmethylsulphonyl fluoride (10 µM), NaF (10 µM), and NaVO₄ (10 µM). Cell lysates were loaded onto the QIAshredder homogenizer columns (Qiagen, Valencia, CA, USA) and centrifugated at 9 000 g for 3 min. Total proteins were quantified with the Bio-Rad Dc Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). The protein extracts were diluted 1:1 (vol/vol) in Laemmli buffer, and 15 µg of protein was loaded on each lane of a 4–12% Tris–glycine gel (Invitrogen, Carlsbad, CA, USA). The electrophoresed proteins were transferred to Amersham Hybond-ECL nitrocellulose membranes (GE Healthcare, Piscataway, NJ, USA). Blots were developed with the WesternBreeze Chemiluminescent kit (Invitrogen). The integrated optical density of each band and gel background was measured with the ImageJ NIH image software. The amount of each phosphorylated kinase was normalized with respect to the amount of the total kinase determined in a parallel blot.

Primary antibodies were: rabbit anti-MEK1/2 (no. 9122), rabbit anti-pMEK1/2 (Ser217/221, no. 9121), rabbit anti-p44/42 ERK1/2 (no. 9102), mouse anti-pp44/42 ERK1/2 (Thr202/Tyr204, no. 9106), rabbit anti-p38 MAPK (no. 9212), mouse anti-pp38 MAPK (Thr180/Tyr182, no. 9216), rabbit anti-stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) (no. 9252), mouse anti-pSAPK/JNK (Thr183/Tyr185, no. 9255), rabbit anti-ERK5 (no. 3372), rabbit anti-pERK5 (Thr218/Tyr220, no. 3371), rabbit anti-Ets-like gene 1 (Elk-1) (no. 9182), rabbit anti-pElk-1 (Ser 383, no. sc-135646), and rabbit anti-Egr-1 (no. 4152). All antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA) except for the one against p-Elk-1, which was from SantaCruz Biotechnology (SantaCruz, CA, USA).

2.6. Egr-1 promoter activity

Cells co-transfected with the pEgr-1 and pRL-TK (Promega, Madison, WI, USA) plasmids were seeded in six-well plates at a concentration of 2 × 10⁵ cells per well. pEgr-1 carries the firefly luciferase gene under the control of the rat Egr-1 promoter,¹⁶ while pRL-TK, the transfection control plasmid, carries the Renilla luciferase gene under the HSV TK promoter. After starvation and nicotine stimulation for 30 min, cells were lysed in 500 µL of passive lysed buffer (Promega) at room temperature for 15 min. Control cells were treated as described but omitting the nicotine. Luciferase activities were determined using the double luciferase reporter assay system (Promega) in a Tuner Biosystems Lumminometer model TD 20/20 (Mountain View, CA, USA). Luciferase activity was normalized based on the Renilla luciferase activity of the transient transfection control vector. Promoter activity was expressed as multiples of the control values.

2.7. RT–PCR and TaqMan real time PCR

Total RNA was purified using the TRIzol reagent (Invitrogen). RT–PCRs for detection of 15 rat nAChR subunits were performed as previously described.^17,18

Rat egr-1, pdgf-B, and β-actin mRNAs were quantified using the TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed on an ABI Prism 7500 Fast Real-Time PCR System (96-well plate). Relative gene expression was determined using the ΔΔCT method.¹⁹

2.8. Gene knockdown of egr-1

SureSilencing™ shRNA Plasmids (SABiosciences, Frederick, MD, USA) were used to specifically knockdown the expression of the rat egr-1 gene by RNA interference in cultured VSMCs. This vector carries a puromycin-resistant gene and expresses the shRNA under control of the U1 promoter. Only one of the four tested plasmids effectively silenced egr-1 gene expression. This functional sequence was 5′-CTACTCCCAACACTGACATTT-3′. Vascular smooth muscle cells were selected in serum-rich medium supplemented with 6 µg/mL puromycin. Knockdown of egr-1 gene in VSMCs was confirmed by Western blot.

Post-injury egr-1 gene expression was also silenced via shRNA. A human micro RNA 30²⁰ containing the above functional sequence was chemically synthesized and inserted between the XhoI and EcoRI restriction sites into the pGIPZ lentiviral vector (Thermo Scientific Open Biosystems, Huntsville, AL, USA). The viral stocks were generated from 293T cells previously co-transfested with the shRNA lentiviral vector and the packaging and envelope plasmids psPAX2 and pMD2G (Addgene Inc., Cambridge, MA, USA) as previously described.²¹ Cell supernatants were passed through 0.45 µm filters and viruses concentrated using PEG-it precipitation solution (System Biosciences, Mountain View, CA, USA). Viral stocks contained at least 10⁸ transducing units per millilitre titrated on rat VSMCs. Lentiviruses were applied perivascularly at the time of surgery in 100 µL Pluronic-127 gel containing 30 µL of the viral stock.²² This formulation forms a gel after contact with the vessel and releases the viruses for approximately 2 days.

2.9. Statistics

Data were expressed as means ± SEM of at least three independent experiments. Two-group comparison was performed using Student's t-test for independent samples. Multiple group statistical analyses were performed by one-way ANOVA followed by Bonferroni's correction for multiple comparisons. Statistics were calculated with GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).

3. Results

3.1. Nicotine increases intimal thickness and VSMC proliferation after balloon injury in rats

To study the effect of nicotine on post-injury VSMC proliferation and neointimal formation, Sprague–Dawley rats were chronically exposed to nicotine for 3months before undergoing vascular injury. Nicotine intake was determined by measuring cotinine blood levels. The blood cotinine in treated rats was 578.27 ± 149.94 ng/mL, a level similar to the one found in heavy smokers.²³ There were no differences in blood cholesterol and triglycerides between treated and control rats. The inflammatory markers IL-6, IL-1α, and IL-1β were similar between the two groups. Rats drinking nicotine were slightly more hypotensive than control animals (mean arterial blood pressure = 99.68 ± 6.89 vs. 109.99 ± 4.42 mmHg, P = 0.02; Supplementary material online Table S1), though both treated and control rats had similar heart rates.

The effect of nicotine on neointimal formation was examined in animals that underwent balloon injury in the right iliac artery. The intima-to-media ratios in nicotine-treated and control animals were 0.22 ± 0.04 and 0.12 ± 0.03, respectively (P = 0.014, Figure 1A). Neointimas of treated animals had 1.8 times more Ki67⁺ cells than those of control animals (P = 0.043, Figure 1B). In agreement with previous studies,⁵ these data confirm the pro-stenotic property of nicotine in the injured vessels.

Figure 1.

Nicotine exacerbates neointimal formation and increases VSMC proliferation after vascular injury in rats. (A) Vascular injury was inflicted in the iliac artery of nicotine-treated (n = 12) and control rats (n = 13). Animals were killed 3weeks after injury, and the intima-to-media ratios (by area) were measured in elastic Van Gieson stained sections. Neointima is indicated between arrowheads. (B) Cell proliferation in the neointima was detected by immunohistochemistry with an anti-Ki67 monoclonal antibody. Representative Ki67⁺ cells (dark spots) are indicated with arrowheads. Each bar represents the mean ± SEM. Significance of the difference between the two groups was calculated by a Student's two tailed t-test with unequal variances.

3.2. Nicotine induces a rapid phosphorylation of ERK1/2 in VSMCs

To elucidate the molecular mechanism by which nicotine induces mitogenesis in VSMCs four major signalling pathways leading to cell proliferation were studied. Nicotine induced a rapid activation of ERK1/2 (p44/p42) in a dose-dependent manner (Figure 2A). It occurred within 5 min after exposure and returned to basal levels 20 min later (Figure 2B). The nuclear translocation of ERK in treated VSMCs was further confirmed by immunofluorescence microscopy (Supplementary material online, FigureS2). The effect of nicotine on ERK phosphorylation was faster and less lasting than that produced by PDGF. Nicotine caused only little activation of p38MAPK and had no effect on SAPK/JNK and ERK5 activation (Supplementary material online, FigureS3).

Figure 2.

Nicotine induces a rapid activation of ERK1/2 in VSMCs. (A) Nicotine induces ERK1/2 (p44/p42) phosphorylation in VSMCs. Treated cells were harvested 3 min after exposure to the indicated nicotine concentration. (B) Activation of ERK1/2 by nicotine (1 µM) was rapid and reversible after 10 min. Total (ERK) and phosphorylated ERK1/2 (pERK) were detected by Western blot. (C) Nicotine rapidly activates MEK1/2, the main ERK upstream kinase. (D) Inhibition of MEK1 with PD98059 blocks nicotine activation of ERK1/2. (E) Inhibition of MEK1/2 inhibits nicotine-induced mitogenesis in VSMCs. Each bar represents the mean ± SEM, n = 5–7.

3.3. MAP kinase (MEK1/2) inhibitors block nicotine-induced ERK phosphorylation in VSMCs

We also investigated whether inhibition of MEK1/2 kinases, the upstream components of the ERK signalling axis, would prevent nicotine-mediated VSMC proliferation. Nicotine induced a rapid phosphorylation of MEK1/2 as shown by Western blot with phospho-specific antibodies (Figure 2C). The incubation of VSMCs with MEK inhibitors U0126 and PD98059 prior to nicotine exposure completely abolished the activation of ERK1/2 and cell proliferation (Figures 2D and E).

3.4. nAChRs are essential for nicotine-mediated activation of the ERK1/2 pathway in VSMCs

The expression of nAChR subunits in rat VSMCs was analysed by RT–PCR. This VSMC line was free of any endothelial or fibroblast contamination as previously shown.²⁴ VSMCs contained abundant mRNAs for α2, α5, α7, β1, and β2 subunits (Figure 3A). The role of these receptors on nicotine-mediated proliferation was further confirmed with pharmacological agents. The nAChR agonist epibatidine induced a rapid activation of ERK1/2 in doses ranging from 100 to 0.1 nM (data not shown). Nicotinic AChRs on VSMCs were blocked with two different antagonists, mecamylamine, and hexamethonium dichloride. These two blockers inhibited activation by nicotine of ERK at concentrations ranging from 0.05 to 50 µM (Figure 3B). These same antagonists blocked nicotine-induced proliferation of VSMCs (Figure 3C). These data demonstrate the presence of operational non-neuronal nAChRs on rat VSMCs that mediate nicotine-induced proliferation.

Figure 3.

Multiple nAChR subunits are expressed on VSMCs. (A) The expression of 15 nAChR subunits in VSMCs was assessed by RT–PCR. (B and C) Blockade of non-neuronal nAChRs with 0.1 µM of either mecamylamine (Mec) or hexamethonium dichloride (Hex) inhibits nicotine-mediated phosphorylation of ERK1/2 (B) and proliferation (C). (D) Calcium mediates nicotine signalling in VSMCs. Pretreatment of VSMCs with BAPTA, an intracellular Ca²⁺ chelator, prevented the activation of ERK1/2 by nicotine. Each bar represents the average of six independent experiments.

3.5. Calcium is the early intracellular mediator of nicotine signal in VSMCs

Next, we looked for the early intracellular mediators of nicotine signalling in VSMCs. It has been reported that nAChRs on VSMCs have high Ca²⁺ permeability.²⁵ To test whether calcium mediated nicotine signalling in VSMCs, we pretreated cultured VSMCs with the intracellular Ca²⁺ chelator BAPTA (between 10  and 0.1 µM) before pulsing them with nicotine for 3min. BAPTA totally inhibited the nicotine-induced activation of ERK, demonstrating the role of calcium influx in nicotine-mediated mitogenesis in VSMCs (Figure 3D).

3.6. Egr-1 is an essential component in nicotine-induced VSMC proliferation and neointimal formation

As part of this study, we also searched for the components that are downstream of ERK1/2 in the transduction of nicotine signalling in VSMCs. Elk-1 is directly phosphorylated by ERK1/2 at multiple sites. Elk-1 is a strong transactivator of serum-responsive element, an element that resides within the egr-1 gene promoter.²⁶ We found that nicotine induced a tightly co-ordinated phosphorylation of Elk-1 in the Ser383 as shown by Western blot with phosphor-specific antibodies (Figure 4A). Phosphorylated ELK-1 was detected within 5min after ERK1/2 activation and 12 min after exposure to nicotine (Figure 4B).

Figure 4.

Nicotine activates the transcription factor Elk-1. (A) Phosphorylated Elk (pElk) was detected by Western blot in VSMCs after nicotine exposure (1 µM) at different time points. (B) Time course of nicotine-mediated activation of MEK1, ERK1/2 and Elk-1 in VSMCs. Each point represents the average of five independent experiments

The ELK-1 transcriptional activity was further confirmed by measuring the Egr-1 promoter activity. For this, VSMCs were transfected with pEgr-1, a plasmid that carries the firefly luciferase gene under the control of the rat Egr-1 promoter.¹⁶ Nicotine increased Egr-1 promoter activity by 50% with respect to untreated cells (Figure 5A). In agreement with these data, nicotine up-regulated egr-1 gene expression in VSMCs by 15-fold, and Egr-1 protein content by more than six-fold (Figure 5B). The egr-1 gene expression increased 1 h after nicotine exposure and returned to baseline levels after 4 h (Figure 5B). The activity of the Egr-1 transcription factor was indirectly measured through the PDGF-β mRNA levels. It is known that the binding of Egr-1 to this gene promoter is sufficient to initiate transcription.²⁷ The PDGF gene expression was temporally co-ordinated with the Egr-1 protein production. The PDGF-β mRNA level in rat VSMCs reached its zenith 2 h after nicotine exposure, which was 1 h later than the peak expression of the egr-1 gene (Figure 5B). The nicotine-mediated up-regulation of Egr-1 was significantly blocked by hexamethonium dichloride, a nAChR antagonist, and by the MEK inhibitors U0126 and PD98059 (Figure 5C).

Figure 5.

Egr-1 transcription factor is essential for nicotine-induced mitogenesis in VSMCs. (A) Nicotine activates Egr-1 promoter in VSMCs. Luciferase activity was determined after nicotine exposure in VSMCs carrying the luciferase gene driven by the rat Egr-1 promoter. Promoter activity is expressed as muliples of activity in control cells. (B) Nicotine up-regulates, in a timely, co-ordinated manner, the egr-1 and the PDGF-β gene expression in VSMCs. The mRNAs were quantified by TaqMan quantitative RT–PCR. The Western blot (top right) shows Egr-1 protein levels at different time points after exposure to nicotine. (C) The nAChR antagonist hexametonium (Hex) and MEK blockers (U0126 and PD 98059) inhibited nicotine (Nic)-mediated up-regulation of the egr-1 gene in VSMCs. (D) Egr-1 gene knockdown in VSMCs using a SureSilent shRNA. Egr-1 production in knockdown VSMCs was measured by Western blot in the presence and absence (vehicle) of nicotine. (E) Nicotine-mediated proliferation is blunted in Egr-1-deficient VSMCs. Values are expressed as multiples of the values for control, untreated cells. Each bar represents the mean ± SEM of n = 5–7. Significance of the difference between the two groups was calculated by Student's two-tailed t-test with unequal variances.

The role of Egr-1 in nicotine-induced proliferation in VSMCs was further confirmed by knocking down the egr-1 gene expression with a specific shRNA. Figure 5D shows a significant reduction (by 75%) of the level of the Egr-1 protein in nicotine-treated VSMCs whose egr-1 gene expression had been blocked with a specific shRNA (Figure 5D). The nicotine-induced proliferative response of Egr-1-deficient VSMCs was 80% less than that of cells transfected with the mock shRNA (Figure 5E).

Finally, we examined the effect of egr-1 gene silencing on nicotine-mediated neointimal formation. The egr-1 gene expression was targeted using a lentiviral vector carrying a specific shRNA. Lentiviruses were embedded in pluronic gels and delivered perivascularly around the iliac artery at the time of surgery. The knockdown of egr-1 gene expression inhibited the neointimal formation after balloon injury (Figure 6). The neointimas of injured arteries treated with Egr-1 shRNA were significantly thinner than those of control arteries (0.44 ± 0.12 vs. 0.84 ± 0.06, P < 0.01).

Figure 6.

Silencing of the egr-1 gene prevents nicotine-enhanced neointimal formation in the rat balloon injury model. (A–C) Vascular injury was inflicted in the iliac artery of nicotine-treated and untreated rats. Animals received a lentiviral vector carrying either the Egr-1 specific (n = 7, C) or the scrambled shRNA (n = 6, A and B). Lentiviruses were embedded in pluronic gels and delivered perivascularly around the iliac artery at the time of surgery. This formulation forms a gel after contact with the vessel and releases the viruses for approximately 2days. Animals were killed 3weeks after injury, and the intima-to-media ratios (by area) were quantified. Neointima is indicated between arrowheads. (D) Neointima-to-media ratios (by area). Each bar represents the mean ± SEM of n = 5–7. Significance of the difference among groups was calculated by a one-way ANOVA, and comparisons were made using Boferroni's correction. *P < 0.05 and **P < 0.01.

4. Discussion

Nicotine, the main addictive component of cigarette smoke, is a pro-restenotic^4–6 and pro-atherosclerotic⁷ substance. Herein, we describe a comprehensive mechanism of action for nicotine-induced mitogenesis in VSMCs. We identify the atherogenic transcription factor Egr-1 as the main molecular player that translates nicotine extracellular signal into a proliferative response. Most importantly, we provide a mechanism for previous descriptive studies indicating that nicotine exposure increases the severity of neointimal formation after vascular injury.^4–6

The mitogenic property of nicotine on vascular cells is well documented.^12,13,28 Our results reported herein demonstrate that nicotine acts as an agonist of the nAChR on VSMCs to trigger an intracellular Ca²⁺ influx, which in turn activates the ERK proliferative pathway. The activated ERK1/2 in nicotine-treated cells phosphorylates Elk-1, which in turn binds to the Egr-1 promoter to up-regulate this gene expression. Egr-1 is a known atherogenic transcription factor that controls the production of VSMC mitogenic cytokines, such as PDGF-BB.²⁷

In agreement with other studies,^17,18 we found multiple nAChR subunits in rat VSMCs, suggesting that the effects of nicotine on VSMCs may depend upon the action of more than one nAChR receptor type. Nicotinic AChRs are pentameric ligand-gated ion channels made up of various subunits.^29,30 The subunit composition of nAChRs determines ligand specificity, ligand affinity, cation permeability, and channel kinetics. Among the nAChR subtypes present in VSMCs is the homomeric α7-nAChR that possesses high Ca²⁺ permeability and rapid onset of desensitization.³¹ It has been shown that the α7-nAChR mediates nicotine-induced angiogenesis⁸ and is an essential regulator of inflammation.³² The presence of the α7subunit in VSMCs reported herein (Figure 3) suggests that the α7-nAChR also participates in VSMC proliferation and post-injury vascular remodelling.

Our results also extended previous findings that demonstrated the role of ERK signalling in the nicotine-mediated proliferation of VSMCs and fibroblasts.^18,33,34 The activation of ERKs, also called mitogen-activated protein kinases (MAPK), plays a critical role in signal transduction cascades from the cell surface to the nucleus, promoting gene expression, cell proliferation, and cell survival.³⁵ We demonstrated that nicotine activates ERKs in VSMCs via a Ca²⁺-dependent mechanism that may involve a variety of upstream effectors, including MEK1/2 kinases. The increase in ERK activity after exposure to nicotine confers a proliferative advantage to VSMCs and may therefore exacerbate neointimal formation after an acute vascular injury. We also sought to identify the molecules responsible for transduction of nicotine signalling into the VSMC nucleus. We found that ERK1/2 is translocated to nucleus of VSMCs after nicotine exposure to target the transcription factor Elk-1, which in turn up-regulates the expression of Egr-1. Egr-1 is a key player in atherosclerosis progression³⁶ and post-injury neointimal formation.³⁷ We demonstrated that the silencing of egr-1 gene expression with specific shRNA prevented the proliferative effects of nicotine on VSMCs, and thus inhibited the exaggerated neointimal formation after vascular injury. The latter data agree with previous results showing that the inhibition of Egr-1 activity with a DNA enzyme prevents the neointimal formation in response to balloon injury in the rat.³⁷ Egr-1 seems to be a potential target for the development of new therapies to prevent the deleterious effects of nicotine on the blood vessels.

Finally, we would like to point out a difference in the mechanisms of nicotine-induced proliferation in VSMCs and tumour cells. In both types of cells, nicotine-mediated proliferation depends on the activation of ERK signalling. In tumours, however, the action of nicotine relies on the physical interaction between nAChRs and β-arrestin to initiate ERK signalling.³⁸ In contrast, nicotine-mediated activation of ERK signalling in VSMCs depends more on nAChR permeability. Results from the present study do not rule out the existence of other mechanisms that involve β-arrestin in nicotine-induced mitogenesis in VSMCs.

4.1. Limitations of the study

Using nicotine alone vs. cigarette smoking had the advantage of isolating the biological effects of nicotine from those of other noxious components, such as ammonia, tar, and carbon monoxide, that may also have effects on post-injury vascular remodelling. Therefore, the mechanisms by which nicotine modifies vascular repair may be modified by the presence of other active components of tobacco. The rat balloon injury model also carries important limitations that need to be taken into account when interpreting the data. In this model, injury is induced in normal elastic artery with rare intimal cells, and little vasa vasorum. In contrast, arterial injury leading to restenosis in humans is mainly produced during percutaneus coronary interventions in atherosclerotic muscular coronary arteries. However, the rat balloon injury model is perhaps the most commonly used and has the advantages of availability, low cost, and the ability to develop a rapid, reproducible response to balloon injury.

4.2. Clinical significance

Nicotine may contribute to cardiovascular disease either by activating the sympathetic nervous system³ or by accelerating atherosclerosis⁷ and restenosis.^4–6 This study reveals new insights into the pathobiology of smoking-related cardiovascular diseases, specifically the mechanisms by which nicotine exacerbates post-injury VSMC proliferation. Most importantly, this study suggests that the atherogenic transcription factor Egr-1 is a plausible therapeutic target to ameliorate the deleterious effects of nicotine on vascular occlusive diseases.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.

Funding

This work was supported in part by Florida Department of Health Grants 11NIR06 (R.I.V.-P.), 07KT-02 (S.M.P. and K.A.W.), American Heart Association grant 0755568B (S.M.P.) and the NHLBI grant 1K01HL096413-01 (R.I.V.-P.).