Cell Replication in the Arterial Wall

Activation of Signaling Pathway Following In Vivo Injury

From the Department of Pathology, University of Washington, Seattle.

Correspondence to Michael A. Reidy, PhD, Department of Pathology/Vascular Biology, Box 357335, University of Washington, Seattle, WA 98195-7335. E-mail mar1{at}u.washington.edu

	Abstract

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Abstract—This study examined intracellular signal events of arterial cells following balloon catheter injury to rat carotid artery. Within 30 minutes, a marked increase in extracellular signal–regulated kinase-1/2 (ERK1/2) activity was observed. This activity remained elevated for 12 hours but had decreased to control levels by day 1. No increase in ERK1/2 was detected at any later times. Injection of anti–fibroblast growth factor 2 antibody (60 mg IV) significantly inhibited the activation of ERK1/2 at 30 minutes after the injury. PD98059 (80 µmol/L), a selective inhibitor of mitogen-activated protein kinase/ERK kinase-1 (MEK1), decreased ERK1/2 activity in injured arteries and also reduced the medial cell replication. In contrast, PD98059 did not block the intimal cell replication at day 8. Mitogen-activated protein kinase phosphatase-1 (MKP-1) was expressed within hours after injury but only weakly at later times; MKP-1 was again expressed after 7 and 14 days. The expression of MKP-1 was associated with an activation of c-Jun amino-terminal kinase. Injury to the arterial wall also stimulated the activity of p70 S6 kinase from 30 minutes to 12 hours, suggesting an alternative pathway in mitogenic signaling of early cell replication. These findings demonstrate that fibroblast growth factor 2–induced ERK1/2 activation promotes medial cell replication after balloon injury; however, signaling of intimal cell replication may not be linked to the MEK1-dependent ERK pathway.

Key Words: balloon injury • cell replication • extracellular signal–regulated kinase-1/2 • mitogen-activated protein kinase phosphatase-1 • p70 S6 kinase

	Introduction

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Injury to the arterial wall invariably initiates a high rate of cell replication in the rat carotid artery that continues for 14 days and then slows down in a very predictable manner.¹ This prolonged replication is mainly restricted to the intimal SMC.¹ We know that early cell replication is stimulated by FGF2 released from damaged cells and that this is responsible for the early medial SMC replication.^{2 3 4} Very little is known about the replication of intimal SMC; despite many attempts, no candidates have thus far been identified as mitogenic stimuli or as inhibitors of this continued replication. Therefore, we decided to take an alternative approach and examine the intracellular signaling pathways that are activated in these arteries. This approach may not make it possible to identify those factors that are critical for this proliferation, but it should provide insight as to pathways that are necessary for SMC replication. Furthermore, such experiments might suggest any differences between the replicative process in medial and intimal SMCs.

Relatively little is known about which signaling pathways are necessary for proliferation of SMCs, although several recent studies have pointed to activation of specific signaling events in proliferating arteries.^{5 6 7 8 9} In the present study, we decided to examine the activation of the ERK1/2 pathway in injured arteries. The MAPK (now referred to as ERK, to distinguish it from other MAPKs) pathway is commonly associated with the interaction of growth factors with their receptors.¹⁰ Indeed, one reason for focusing on the ERK cascade in the present study is that activation of this pathway might suggest a role for a mitogen necessary for the chronic SMC replication in the intima. The main feature of the ERK cascade involves activation of Raf, which then initiates a series of phosphorylation steps, resulting in the activation of two substrates, namely ERK1 and ERK2.^{11 12} These phosphorylated kinases then act on several substrates, including p90 ribosomal S6 kinase, c-Fos, and c-Jun.^{11 12} PDGF,¹³ epidermal growth factor,¹⁴ nerve growth factor,¹⁵ and FGF,¹⁶ all activate this pathway. Furthermore, blockade of the ERK cascade prevents cell replication,^{17 18} and it is commonly believed that activation of this pathway is critical for cell replication.

One control point in the activation of the ERK cascade involves the action of MKP-1, which is a member of a dual-specificity tyrosine phosphatase family and dephosphorylates the phosphotyrosine and the phosphoserine/phosphothreonine of target proteins.¹⁹ Expression of MKPs has a negative effect on fibroblast proliferation through inactivation of ERK1/2,^{20 21} whereas phosphatase inhibitors increase the activation of MAPKs.²² A recent report has shown that expression of MKP-1 varies significantly after balloon injury of carotid arteries and that at times when replication is known to occur, MKP-1 expression is significantly reduced.⁷ MKP-1 could, therefore, act as a critical control molecule in injury-induced SMC replication, and one of the aims of the present study was to determine whether MKP-1 is expressed at times when SMC replication stops.

JNK is a subfamily of the MAPK family, which parallels the pathway of the ERK cascade.¹² JNK is markedly activated by inflammatory cytokine, tumor necrosis factor-, and ultraviolet and various cellular stress and is generally thought to inhibit cell growth.^{23 24} A recent study has shown that activation of JNK upregulates MKP-1 expression, suggesting that the JNK pathway negatively affects the activation of ERK1/2.²⁵ Balloon injury is known to cause cell death, and we therefore wished to determine whether the stress of balloon injury is able to activate this pathway.

Another intracellular signaling known to significantly affect SMC replication involves p70^S6K.^{26 27} Lane et al²⁸ have shown that p70^S6K plays an important role in G₁ phase progression of the cell cycle.²⁸ Furthermore, recent studies using rapamycin, a potent inhibitor of p70^S6K, have suggested that activity of p27^Kip1 and cdk may be regulated by the activation of p70^S6K.^{29 30 31} The signaling pathway of p70^S6K is distinct from that of ERK1/2.³² Thus, the p70^S6K pathway might be an alternative candidate promoting SMC replication after balloon injury.

	Materials and Methods

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Balloon Catheter Injury
Male Sprague-Dawley rats (B&K Universal, Kent, Wash), aged 3 to 4 months, were used in all experiments, and all surgery was performed using general anesthesia with an intraperitoneal injection of xylazine (Xyla-ject, 4.6 mg/kg body wt, Phoenix Pharmaceutical Inc) and ketamine (Ketaject, 70 mg/kg body wt, Phoenix Pharmaceutical Inc). The common carotid arteries were injured by three rotating passes with a 2F Fogarty catheter (Baxter Healthcare Co) introduced through the external carotid artery.¹ Rats were killed at various times after the balloon injury with an overdose of pentobarbital (intravenous Nembutal, Abbott Laboratories), and ice-cold lactated Ringer's solution (Baxter Healthcare Co) was infused at physiological pressure in retrograde fashion from the abdominal aorta. Blood was drained from both sides of the jugular vein. After the solution cleared, the whole length of the carotids was excised, and the adventitia was stripped on an ice-cold dish. To assess a difference of each wall layer, some rats received an injection of Evans blue (200 µL of a 5% solution) 10 minutes before death at 14 days after the injury. The blue part of the carotid artery was isolated as described above and opened longitudinally, and the neointima was stripped gently at the internal elastic lamina. In this way, the carotid artery was divided into intima (14-day intima) and media containing little adventitia (14-day media). In one group of carotids, the artery was left intact (14-day whole), so that we could estimate whether the process of the intimal stripping, by itself, could influence kinase activity. All specimens were then snap-frozen in liquid nitrogen and stored at -80°C. The experimental design of these studies is shown in Fig 1.

View larger version (27K): [in this window] [in a new window]   Figure 1. Experimental design of blocking with anti-FGF2 antibody and inhibition experiments of both medial and intimal SMC replication with PD98059.

ERK1/2 In-Gel Kinase Assay
Tissues from at least 3 animals per each time were pooled and pulverized under liquid nitrogen and incubated in ice-cold 0.1% Triton lysis solution (10 mmol/L HEPES [pH 7.4], 50 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 5 mmol/L EDTA, 5 mmol/L EGTA, 100 µmol/L Na₃VO₄, 50 mmol/L NaCl, 0.1% Triton X-100, 500 µmol/L phenylmethylsulfonyl fluoride, and 10 µg/mL leupeptin) for 30 minutes. Insoluble matter was removed by centrifugation, and the protein concentration was measured by bicinchoninic acid assay (Pierce). Equal amounts (20 µg of total protein) of each lysate were boiled for 5 minutes with sample buffer (50 mmol/L Tris [pH 6.8], 10% glycerol, 0.01% bromophenol blue, 1% SDS, and 360 mmol/L 2-mercaptoethanol [final concentration]) and separated on 10% SDS-PAGE containing 0.4 mg/mL MBP (Sigma). Gel preparation, kinase reaction, and detection by autoradiograph were performed as described by Duff et al.³³

Western Blot Analysis
For analysis of phosphorylated ERK1/2 and ERK2, equal amounts (10 µg of total protein) of sample lysed in 0.1% Triton lysis solution were separated on 10% SDS-PAGE, and equal amounts (20 µg of total protein) of the lysate were electrophoresed on 8% SDS-PAGE for p70^S6K. To detect MKP-1, samples were lysed in SDS lysis solution (50 mmol/L Tris [pH 7.6], 100 mmol/L NaCl, 1% SDS, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 mg/mL leupeptin), and equal amounts (10 µg of total protein) of the lysate were separated on 12% SDS-PAGE. Transfer to nitrocellulose membrane (PROTRAN, Shleicher & Schuell), blocking, incubation with antibody, washing, and detection by enhanced chemiluminescence (Amersham) were performed according to the protocol described previously.³⁴ The following were used as primary antibodies: rabbit polyclonal antibodies against phosphorylated ERK1/2 (1:1000, No. 9101S, New England Biolabs Inc), ERK2 (1:1000, No. sc-154, Santa Cruz Biotechnology Inc), MKP-1 (1:500, No. sc-1199, Santa Cruz), and p70^S6K (1:500, No. sc-230, Santa Cruz).

Blocking of ERK1/2 Activity With Antibody Against FGF2
To evaluate the role of FGF2 in ERK1/2 activation after balloon injury, a neutralizing goat antibody against FGF2 (60 mg) was injected intravenously 10 minutes before the balloon injury: nonimmune goat IgG (60 mg) was injected in control rats in the same manner (Fig 1). The specificity of the anti-FGF2 antibody has been documented in a previous study.³⁴ Rats were killed at 30 minutes after the balloon injury, and ERK1/2 activity was detected using the in-gel kinase assay described above. The sample of normal carotid was also applied to the same gel, and the activity of ERK1/2 was quantified by autoradiographic densitometry using NIH Image 1.55. This experiment was repeated three times.

Inhibition of Medial Cell Replication With MEK1 Inhibitor
The MEK1 inhibitor PD98059 (New England Biolab Inc) is a selective inhibitor of MEK1 activation and the ERK1/2 cascade.^{18 35} To assess the significance of ERK1/2 in medial cell replication after balloon injury, PD98059 was used in rats subjected to the balloon injury (Fig 1). After anesthesia, proximal and distal parts of the common carotid and the external carotid were exposed. The proximal common carotid was clamped with a vessel clip, and a PE-10 tube (Clay Adams) was introduced into the common carotid through the external carotid. After flashing with Ringer's solution, 200 µL of PD solution (80 µmol/L PD98059 and 0.8% DMSO in Ringer's solution) was gently infused into the common carotid through the tube. The distal common carotid was then clamped, and the common carotid was left for 1 hour. The vessel clips of the carotid artery were then removed, and the carotid was subjected to balloon injury in the same manner as described above. Immediately after injury, 200 µL of PD solution containing 25% Pluronic gel (Pluronic F-127, Sigma) was applied around the vessel, and the wound was closed. In control rats, vehicle solution (0.8% DMSO in Ringer's solution) was applied instead of PD solution.

To evaluate the effect of the inhibitor, rats were killed at 30 minutes after the balloon injury, and ERK1/2 activity was measured by the in-gel kinase assay described. Other rats were killed at 2 days after balloon injury to analyze cell replication in media. At 1, 9, and 17 hours before death, each rat was injected subcutaneously with BrdU (25 mg/kg body wt, Boehringer-Mannheim). After infusion of Ringer's solution, rats were fixed by perfusion of 4% phosphate-buffered paraformaldehyde (0.1 mol/L PO₄ buffer, pH 7.3) at 120 mm Hg. Three segments of the common carotid (5 to 7 mm, 7 to 9 mm, and 9 to 11 mm from carotid bifurcation) were cut out and embedded in paraffin. A section was cut from each segment, replicating cells were identified by monoclonal antibody against BrdU (Bu 20a, DAKO Co), and counterstaining was performed with hematoxylin, as described.⁴ BrdU labeling index (cell replication index) was calculated as the number of labeled nuclei÷total nucleix100 in media, and the average of each value from three sections per animal was used for statistical analysis.³⁴

Inhibition of Intimal Cell Replication With MEK1 Inhibitor
To evaluate the role of ERK1/2 in the intimal cell replication, PD98059 was administered to the rats subjected to balloon injury 6 days before the experiment (Fig 1). PD98059 or vehicle was infused into the injured carotid using the technique described above, and the same solution containing Pluronic gel was applied around the vessel. Rats were killed at 2 days after the drug application (8 days after the balloon injury), and BrdU labeling index of both intima and media was quantified.

RNA Extraction and Northern Blot Analysis
Arterial tissues from at least 6 animals per each time were mixed and ground to a fine powder under liquid nitrogen, and total cellular RNA was isolated by acid thiocyanate extraction.³⁶ The RNA concentration was measured by absorption at 260 nm. Equal amounts (15 µg of total RNA) of each RNA were separated on formaldehyde agarose gel (1.2%) and transferred to nylon membranes (Zeta-Probe, Bio-Rad Laboratories). After cross-linking by shortwave UV light, exposure to methylene blue staining was used to verify loading in each lane. Prehybridization, hybridization, washing, and autoradiography were carried out as previously described.³⁷ A cDNA probe of rat MKP-1 (a 336-bp fragment; a gift from Dr A. Misra-Press, Molecumetics Ltd, Bellevue, Wash) was labeled with ³² P by random primer extension (Multi-Prime, Amersham).

JNK Kinase Activity Assay
Kinase activity of JNK was measured via solid-phase kinase assay as described by Hibi et al.³⁸ Equal amounts (250 µg of total protein) of each extract, lysed in 0.1% Triton lysis solution, were mixed with 3 µg of GST-c-Jun (No. 1-169) bound to glutathione-agarose beads (30 µL, No. 14–178, Upstate Biotechnology) and rotated at 4°C for 3 hours. After three washes with 1 mL of washing buffer (12.5 mmol/L MOPS [pH 7.2], 12.5 mmol/L ß-glycerophosphate, 0.5 mmol/L EGTA, 0.25 mol/L NaCl, 7.5 mmol/L MgCl₂, 1 mmol/L dithiothreitol, and 1% NP-40), the pelleted beads were resuspended in 45 µL of kinase buffer A (12.5 mmol/L MOPS [pH 7.2], 12.5 mmol/L ß-glycerophosphate, 0.5 mmol/L EGTA, 17.5 mmol/L MgCl₂, 1 mmol/L MnCl₂, 1 mmol/L dithiothreitol, and 1% NP-40) containing 50 µmol/L cold ATP and 20 µCi [-³² P]ATP and incubated at 30°C for 30 minutes. Phosphorylated fusion proteins were eluted by boiling with sample buffer and separated in 10% SDS-PAGE. The gel was dried and autoradiographed.

p70^S6K Kinase Activity Assay
p70^S6K activity was measured by an immune complex kinase assay using S6 peptide (RRRLSSLRA, No. 12–124, Upstate Biotechnology Inc). Tissues from two carotids per each time were lysed in 1% Triton lysis solution (20 mmol/L Tris [pH 7.5], 137 mmol/L NaCl, 2 mmol/L EDTA, 148 µmol/L Na₃VO₄, 10% glycerol, 1% Triton X-100, 250 µmol/L phenylmethylsulfonyl fluoride, and 5 µg/mL leupeptin), and the protein concentration was measured, as described. Lysates containing equal amounts of protein (200 µg of total protein) were precleared with protein G–Sepharose (Pharmacia Biotech Inc) and incubated with 1 µg of polyclonal antibody against p70^S6K for 3 hours at 4°C. The immune complex was precipitated with 50 µL of protein G–Sepharose (50% slurry) for 1 hour at 4°C, and the immunoprecipitates were washed twice with 1% Triton lysis solution and twice with PBS. The precipitates were resuspended in 40 µL of kinase buffer B (30 mmol/L HEPES [pH 7.3], 15 mmol/L ß-glycerophosphate, 2 mmol/L EGTA, 15 mmol/L MgCl₂, 750 µmol/L dithiothreitol, 230 µmol/L Na₃VO₄, and 150 µg/mL BSA) containing 75 µmol/L cold ATP, 2 µCi [-³² P]ATP, and 375 µmol/L S6 peptide and incubated at 30°C for 20 minutes. The reaction was stopped by adding 20 µL of 100 mmol/L EDTA. After centrifugation, 40 µL of the supernatant was spotted onto phosphocellulose filter paper (P81, Whatman Inc) and washed five times (10 minutes each) with 1% phosphoric acid and twice with ethanol. After the filter paper was dried, radioactivity was quantified by scintillation counting. The experience was repeated at least three times for each time point.

Statistics
The difference in ERK1/2 activity, BrdU index, and number of nuclei on a section of each group was analyzed by unpaired Student's t test, and analysis of p70^S6K activity was performed by ANOVA followed by the method of Bonferroni. Data were considered significant at P<.05.

	Results

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ERK1/2 Is Activated Immediately After Balloon Injury
The activity of ERK1/2 was detected in balloon-injured rat arteries using an in-gel kinase assay with MBP as the substrate. No activity was detected in normal arteries, but within 30 minutes and 1 hour, a marked increase in activity of two bands at molecular masses of 44 kD (ERK1) and 42 kD (ERK2), was observed (Fig 2A). ERK1/2 activity was still detected at 12 hours, although by 1 day, the activity was nearly equal to that in the normal carotid. At all remaining times after injury, no increase in ERK1/2 was observed. An ERK1/2 activity assay was also carried out at day 14 on the whole artery and on the intima and media separately. The ERK1/2 activity of all three samples was almost equal to the level in the normal carotid with no difference in any sample (data not shown).

View larger version (49K): [in this window] [in a new window]   Figure 2. A, ERK1/2 activity in rat carotid after balloon injury. Cell lysate from injured rat carotids was separated on SDS-PAGE gel containing MBP and incubated with [-32 P]ATP. Phosphorylated MBP was observed at molecular masses of 44 kD (ERK1) and 42 kD (ERK2). B, Western blot for phosphorylated ERK1/2. The antibody is specific to phosphorylated ERK1/2. C, Western blot for ERK2. ERK2 was expressed equally through the time course. N indicates normal carotid; m, minutes after injury; h, hour(s) after injury; and d, day(s) after injury.

A Western blot of phosphorylated ERK1/2 showed two bands of phosphorylated proteins (42 and 44 kD) at 30 minutes and 1 hour after injury. The lower-sized protein, ERK2, showed an increase in phosphorylation at 12 hours and 1 and 2 days later. At other times, no increase in phosphorylation was detected in ERK1/2 (Fig 2B). Protein loading is shown in Fig 2C in which the arterial extracts were stained with an antibody to ERK2.

Anti-FGF2 Antibody Blocks Activation of ERK1/2
Our previous data have shown that FGF2 is the major mitogen for the early SMC replication observed after balloon injury.^{2 3 4} Since nearly all mitogens are thought to activate ERK1/2, we asked if an anti-FGF2 antibody (60 mg) would prevent activation of this signaling pathway. The antibody was given (intravenously) 10 minutes before balloon injury, and the activation of ERK1/2 was evaluated using the in-gel kinase assay 30 minutes after the balloon injury. The ERK1/2 activity of rats injected with anti-FGF2 antibody was significantly lower than in control animals that received the nonimmune IgG (Fig 3).

View larger version (51K): [in this window] [in a new window]   Figure 3. Anti-FGF2 antibody (60 mg) blocked ERK1/2 activation at 30 minutes after the balloon injury in kinase activity assay (A). The experiment was repeated three times, and kinase activity was quantified (B). The value of normal carotid was set at 1.0, and all data are presented as ratios of the test value to the normal carotid value. The ERK1/2 activity in injured carotid with anti-FGF2 antibody (60 mg) added was significantly lower than that with nonimmune IgG (60 mg). Values are shown as mean±SD. *P<.05; **P<.01.

Inhibition of ERK1/2 Suppresses Injury-Induced Medial Cell Replication
Our next experiment was to document whether the activation of ERK1/2 was related to the ability of medial cells to replicate. PD98059 has been shown to block the activation of MEK1, which lies upstream from ERK1/2.^{18 35} The carotid arteries were therefore preincubated with PD98059 for 1 hour, and the drug was added to the adventitia in a Pluronic gel immediately after balloon injury. This treatment caused a significant decrease in ERK1/2 activity at 30 minutes (Fig 4A) and a significant decrease in medial cell replication (Fig 4B). The drug appeared not to be toxic to arterial cells, since no difference in cell number was detected between groups (Fig 4C).

View larger version (41K): [in this window] [in a new window]   Figure 4. PD98059 (80 µmol/L, MEK1 inhibitor), compared with vehicle (0.8% DMSO), inhibited the ERK1/2 activation induced by balloon injury in kinase activity assay (A). Medial cell replication at 2 days after the injury was also blocked by PD98059 (B). No significant difference in medial nuclei number was detected in PD98059-treated animals (C). Values are shown as mean±SD.

MEK1 Inhibitor Does Not Block Injury-Induced Intimal Cell Replication
The absence of any obvious activation of the ERK cascade in the carotid artery after 4 days suggests that SMC replication at these times is not dependent on this pathway. Since PD98059 was effective in inhibiting medial cells at 2 days, we wished to determine whether this MEK1 inhibitor affected replication of intimal cells. We therefore subjected rat carotid arteries to balloon injury, and after 6 days, when there is a high rate of intimal SMC replication, PD98059 was added in the same manner as described above. Intimal and medial cell replication was measured 2 days later, and no significant difference in intimal cell replication was detected in arteries treated with PD98059 (Fig 5A), although a significant difference in medial cell replication of the same arteries was observed (Fig 5B). No change in either intimal or medial nuclei number was noted (data not shown).

View larger version (37K): [in this window] [in a new window]   Figure 5. PD98059 or vehicle was applied to carotid arteries subjected to balloon injury 6 days earlier. PD98059 (80 µmol/L) did not block intimal cell replication (A), but medial cell replication was significantly reduced (B). Values are shown as mean±SD.

MKP-1 Expression After Balloon Injury
The activity of ERK1/2 is in part regulated by MKP-1, which acts to dephosphorylate these kinases. A Northern blot of MKP-1 showed that it was strongly expressed in normal arteries and in injured arteries up to 12 hours after injury (Fig 6A and 6B). By 1 day after balloon injury, there was a significant reduction in expression, and levels remained low until day 4. At day 7, and in the intimal cells after 14 days, expression of MKP-1 was significantly increased, though not as strongly as in the control arteries.

View larger version (74K): [in this window] [in a new window]   Figure 6. A, Northern blot analysis of MKP-1 after balloon injury. Expression of MKP-1 was abundant by 12 hours after the injury. Expression was downregulated at day 1 and upregulated again from day 7. B, Blot stained with methylene blue to verify loading. C, Western blot for MKP-1. MKP-1 protein was present by 6 hours after the injury and was expressed abundantly in the 14-day intima sample. N indicates normal carotid; h, hours after injury; d, day(s) after injury; I, 14-day intima; and M, 14-day media.

A Western blot showed that MKP-1 protein was present in normal carotids and injured carotids after 6 hours but was not detected at 1 or 2 days after the injury (Fig 6C). At day 7, a faint band of MKP-1 was detected, and there was strong expression in the intima 14 days after the injury.

JNK Is Activated After Balloon Injury
By use of the solid-phase kinase assay, JNK activity was detected in normal arteries, and a strong activation of JNK was observed at 30 minutes and 1 hour after balloon injury (Fig 7A). Maximal activity was at 30 minutes. The activity of JNK decreased after 12 hours, and after 2 days a gradual increase was observed. To estimate a difference between intima and media, JNK activity was assessed on the whole artery and on the intima and media separately. No difference between intima and media was observed, and it appeared that stripping of the intima did not increase JNK activity, since the activity in three samples was nearly equal (Fig 7B).

View larger version (39K): [in this window] [in a new window]   Figure 7. A, JNK activity in rat carotid after balloon injury. Cell lysate was mixed with GST-c-Jun bound to glutathione-agarose beads and rotated for 3 hours. Activation was detected at 30 minutes and 1 hour after the injury. B, JNK activity of 14-day whole, 14-day intima, and 14-day media samples. JNK activity in the intima was nearly equal to that in the media. N indicates normal carotid; m, minutes after injury; h, hour(s) after injury; d, day(s) after injury; W, 14-day whole; I, 14-day intima; and M, 14-day media.

The JNK kinase assay was also performed on the samples used in the blocking study with anti-FGF2 antibody; however, the antibody did not affect JNK activation (data not shown).

p70^S6K Is Activated After Balloon Injury
Immune complex kinase assay using S6 peptide showed a significant activation of p70^S6K at 30 minutes, 1 hour, and 12 hours after the injury, with a maximum at 30 minutes (Fig 8A). After 1 day, no significant change in p70^S6K activity was detected.

View larger version (33K): [in this window] [in a new window]   Figure 8. A, p70S6K activity in rat carotid after balloon injury. Cell lysate was treated with anti-p70S6K antibody, and specific kinase activity was measured by S6 peptide phosphorylation. Data were presented as the ratio of the test value to the value of the normal artery (set at 1.0). Significant activation was detected at 30 minutes, 1 hour, and 12 hours after the injury. B, Western blot of p70S6K. Slower migrating bands represent phosphorylated forms of p70S6K. N indicates normal carotid; m, minutes after injury; h, hour(s) after injury; and d, day(s) after injury. Values are shown as mean±SD. *P<.05; **P<.01.

A Western blot of p70^S6K showed multiple bands due to sequential phosphorylation of serine and threonine residues (Fig 8B).³⁹ From 30 minutes to 1 day after the injury, a shift in the mobility of the bands suggested an increase of phosphorylation at these times. After day 2, this shift in mobility of the bands was not detected.

	Discussion

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Balloon catheter injury of an artery stimulates a series of events that culminates in a significantly thickened intima, composed of SMCs and matrix.¹ Critical to the growth of this lesion is the replication of medial and, more important, intimal SMCs.¹ The kinetics of this SMC proliferation were first documented over 15 years ago,¹ and yet we still know comparatively little about the factors that control their growth. FGF2 was shown to be critical for the early replication of medial SMCs^{2 3 4} but not for the replication of the intimal cells.⁴⁰ Indeed, despite the fact that intimal SMCs are capable of significant growth within the first few weeks after injury, these cells are only weakly stimulated by FGF, PDGF, and angiotensin II.^{2 41 42} One other interesting feature is that intimal SMCs cease replication in a very predicable manner, regardless of the severity of injury and the early replication rate.^{1 43} Our data show that once SMCs have migrated into the intima, their replication rate is initially very high, in excess of 80%, but by 14 days this is reduced to <5%.¹ Thereafter, only minimal replication is detected in the intima. We have no explanation for this rapid and sustained phase of SMC growth nor for the predictable reduction in replication.

One aim of the present study was to determine whether the ERK1/2 pathway is activated throughout the replicative phase of SMCs after arterial injury. Our data show that ERK1/2 is activated within minutes after injury and that increased activity can still be detected up to 12 hours later. Many mitogens activate ERK1/2,^{13 14 15 16} and since FGF is known to be liberated by balloon catheter injury,³ it is reasonable to assume that the activation of ERK1/2 is a result of FGF receptor activation. This fact was confirmed by the use of a neutralizing anti-FGF2 antibody that was found to significantly reduce the activity of ERK1/2 30 minutes after injury. The importance of the ERK signaling pathway for SMC replication is illustrated by the significant decrease in medial cell replication that occurred after treatment with the MEK1 inhibitor PD98059. This drug has been shown to act on the upstream activator of ERK1/2, namely, MEK1, without significantly inhibiting the activity of other MAPKs.^{18 35} PD98059 was not able to totally block cell replication, which may be linked to the fact that not all ERK activity was suppressed by PD98059. This may be because we were unable to achieve an optimum concentration of the inhibitor or because MEK1-independent pathways may play a role in cell replication at this time. We believe that these data link FGF2 with the activation of the ERK pathway in arterial SMCs and that activation of this signaling pathway is critical for the early SMC replication detected after arterial injury at this time.

One surprising finding of the present study was that activation of the ERK cascade in the carotid artery was detected only at the onset of SMC replication and not at other times when replication is high, ie, between 4 and 7 days. This would suggest that the ERK signaling cascade is not necessary for cell replication of the arterial wall at these times. In contrast to this finding, Lai et al⁷ have shown activation of ERK1/2 at 3 days after injury with continued activation up to 14 days. It is possible, though unlikely, that there were differences in the severity of injury in these two experiments that might affect the rates of replication. There are few data on the signaling pathways that are activated in vivo, but in a model of hypertension known to induce cell replication, others have shown that the activation of the ERK pathway is only transitory despite the persistence of the high blood pressure.⁶ In addition, the MEK1 inhibitor PD98059 had no effect on intimal cell replication at 8 days but was able to significantly reduce the replication of the medial cell in the same artery. This clearly shows a marked difference in the pathways activated during replication of these cells and suggests that intimal cell replication is not dependent on the MEK1-ERK1/2 pathway. Since many growth factors are known to activate the ERK pathway,^{13 14 15 16} one possible conclusion from this finding is that the chronic intimal cell replication is not controlled via this classic growth regulatory pathway. If this conclusion is true, these data would support the mainly negative findings that have failed to link the expression of any mitogen to the replication of intimal cells.^{2 41 42}

One mechanism by which the ERK pathway can be inactivated is by dephosphorylation of the activated kinases. MKP-1 is known to dephosphorylate members of the ERK cascade and so inactivate the ERK pathway.¹⁹ Indeed, stimulation of MKP-1 expression coincides with a reduction in ERK activity in SMCs,⁷ whereas inhibition of MKP-1 with antisense oligonucleotides increases ERK1/2 activity of SMCs.³³ Thus, one possibility is that MKP-1 is responsible for the decrease in ERK activation that we observed in the injured artery. Our data, however, show that MKP-1 is expressed at 2 to 12 hours after injury, which is when ERK1/2 is activated. Also, when there was a significant decrease in MKP-1, between 12 hours and day 3, a concomitant increase in activation of the ERK pathway was not observed. A recent study showed that MKP-1 expression was diminished after arterial injury, and the authors suggested that this was linked to ERK-activated SMC replication.⁷ The same study, however, did not examine the early times after injury, and as mentioned above, we did not detect any significant activation of the ERK pathway when cell replication was high, ie, 3 to 7 days. Of interest is that MKP-1 was reexpressed starting at day 7 and was still expressed at day 14, and this coincided with a reduction in intimal SMC replication. Recently, we have noted that MKP-1 expression is abundant 6 weeks after injury (data not shown). Thus, apart from the first round of SMC replication, our data would suggest no role for activation of the ERK cascade in SMC replication in vivo. Furthermore, the expression of MKP-1 is not linked to the early decrease in ERK activity (1 to 7 days), but at 7 and 14 days after injury, an increase in MKP-1 expression is associated with a decrease in cell replication.

We were interested to know what factors might be regulating MKP-1 expression in the injured artery. One suggestion has been that the activation of JNK increases MKP-1 expression.²⁵ Our data showed that at times when JNK is activated, namely, at 30 and 60 minutes after injury, this coincides with MKP-1 expression. Furthermore, after 14 days, when there was a second increase in JNK activity, a similar increase in MKP-1 expression was found. Thus, these data would suggest a potential link between JNK and MKP-1 expression, although more work is required to confirm this. We are limited in our ability to discern the role of JNK activation in the injured artery, since it is not possible to block this pathway in vivo. It has been suggested that activation of the ERK pathway increases MKP-1 expression.⁴⁴ Our data would not support this observation, since at 7 and 14 days after injury, there was an increase in MKP-1 expression with no detectable ERK activity.

Activation of p70^S6K is associated with a mitogenic signaling that is distinct from the ERK pathway.^{28 32} Microinjection of p70^S6K antibodies into rat fibroblasts prevented serum-induced cell replication.²⁸ Our results show that p70^S6K is activated at early times after injury in a pattern similar to that of ERK1/2. p70^S6K phosphorylation is not mediated by the signaling pathway as is ERK1/2,³² but the timing of p70^S6K activation would support a role for its requirement as cells enter S phase. Although we have no supportive data, the inability of the MEK1 inhibitor to totally block early arterial cell replication may be explained by this finding. p70^S6K is also thought to regulate cell replication via its action on the cyclin/cdk inhibitor p27^Kip1,³¹ and Koyama et al²⁷ have suggested that this pathway plays a role in SMC replication. In preliminary studies, we have noted a reduction in p27^Kip1 levels within hours after injury, when p70^S6K is activated. It is difficult to draw any meaningful conclusions from these data, and further work on the association of p27^Kip1 with p70^S6K activity is required. Thus, p70^S6K activation does occur at early times after injury, when cells are starting to enter the cell cycle, but the lack of p70^S6K activation at late times would suggest that it plays no role in intimal cell replication.

In summary, the present study shows that the early cell replication that occurs after balloon catheter injury is linked to activation of the ERK signaling pathway, since the addition of the MEK1 inhibitor PD98059 caused a significant reduction in cell replication. Between 4 and 7 days after injury, when there is a high rate of cell replication, little or no activation of ERK1/2 could be detected, and the high intimal cell replication was not blocked by the MEK1 inhibitor. The expression of MKP-1 was associated with the activation of JNK and did not appear to coincide with decreases in ERK1/2. These data would suggest that apart from the initial replication, the MEK1-dependent ERK signaling pathway is not closely linked to intimal cell replication in vivo.

	Selected Abbreviations and Acronyms

 

BrdU = 5-bromo-2'-deoxyuridine cdk = cyclin-dependent kinase DMSO = dimethyl sulfoxide ERK = extracellular signal–regulated kinase FGF = fibroblast growth factor GST = glutathione S-transferase JNK = c-Jun amino-terminal kinase MAPK = mitogen-activated protein kinase MBP = myelin basic protein MEK = MAPK/ERK kinase MKP = MAPK phosphatase p70S6K = p70 S6 kinase PDGF = platelet-derived growth factor SMC = smooth muscle cell

	Acknowledgments

 
This study was supported by National Institute of Health grants HL-03174 and HL-41103.

Received September 12, 1997; accepted January 20, 1998.

	References

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