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(Circulation Research. 1997;81:904-910.)
© 1997 American Heart Association, Inc.

Articles

Activation of MAP Kinase In Vivo Follows Balloon Overstretch Injury of Porcine Coronary and Carotid Arteries

J. M. Pyles, K. L. March, M. Franklin, K. Mehdi, R. L. Wilensky, , L. P. Adam

From the Krannert Institute of Cardiology (J.M.P., K.L.M., M.F., K.M., R.L.W.) and the Richard L. Roudebush Veterans Administration Medical Center (K.L.M.), Indiana University School of Medicine, Indianapolis, and The Boston Biomedical Research Institute and Harvard Medical School (L.P.A.), Boston, Mass.

Correspondence to Keith L. March, MD, PhD, FACC, Department of Cardiology, Krannert Institute of Cardiology, 1111 West 10th St, Indianapolis, IN 46202. E-mail march{at}kimail.dmed.iupui.edu

	Abstract

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Abstract Vascular restenosis involves contraction, proliferation, and remodeling of the arterial wall in response to overstretch injury. Mitogen-activated protein kinases (MAPKs) are implicated in both contraction and proliferation of vascular smooth muscle (VSM), and studies of porcine carotid arterial muscle strips have shown that mechanical stretch leads to the activation of the extracellular signal–regulated kinase (ERK) family of MAPKs in vivo. We, therefore, analyzed the acute effect of mechanical overstretch injury on ERK-MAPK (herein referred to simply as MAPK) activity in porcine coronary and carotid arteries in vivo. Balloon angioplasty catheters were inflated to 6 atm three times over 5 minutes at a balloon-artery ratio of 1.2:1 in either porcine coronary or carotid arteries. The arteries were snap-frozen after angioplasty, and MAPK activity was measured. Angioplasty of the left anterior descending (LAD, n=5), left circumflex (LCx, n=5), and carotid (n=5) arteries effected an increase in MAPK activity compared with the activity in uninstrumented right coronary arteries (RCAs) or carotid arteries from the same animals used for controls. Balloon angioplasty of carotid arteries led to an increase in MAPK activity that was 7.7-fold over the activity in control arteries and comparable to the activity in stretched carotid arterial muscle strips in vivo. The increase in coronary artery kinase activity on angioplasty was variable from animal to animal. The increase in MAPK activity over that in control arteries ranged from 4.5- to 31.7-fold (mean±SEM, 10.7±5.3) in the LAD and 1.8- to 31.3-fold (mean±SEM, 9.7±5.7) in the LCx. There were no apparent inherent differences in the levels of MAPK activity in the three different types of coronary arteries (RCA, LAD, and LCx) without instrumentation. MAPK activation occurs rapidly during angioplasty, suggesting that this kinase may play an early role in initiating the injury response in both porcine coronary and carotid arteries. MAPKs may be key enzymes targeted to treat or prevent restenosis.

Key Words: stretch • mitogen-activated protein kinase • angioplasty • smooth muscle • restenosis

	Introduction

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Mitogen-activated protein kinase (MAPK) is a family of proline-directed protein kinases activated as part of a cascade by phosphorylation on both threonine and tyrosine residues. MAPKs are present in both the contractile and proliferative phenotypes of VSM and are activated by mechanical stress and growth factors including PDGF and EGF.^1,2 Members of the MAPK family include the ERKs (ERK-1 and ERK-2 [p44^MAPK and p42^MAPK]) and the SAPKs, of which JNK, implicated in smooth muscle hypertrophy,³ is a member. In proliferating cells, it has been postulated that activated ERK-MAPKs phosphorylate specific cytoplasmic and nuclear proteins needed for passage through certain checkpoints in the cell cycle (eg, G₁/S and G₂/M).^4–7 In highly differentiated and nonproliferating tissues such as muscle, the function of the MAPKs is poorly understood. In fully differentiated VSM, one role of MAPK may be to phosphorylate caldesmon,⁸ thereby altering either a mechanical property of the muscle or the organization of actin filaments. Alternatively, MAPK activation may lead to altered growth or proliferation of vascular muscle cells.

The earliest biochemical events identified in response to balloon denudation of rat and rabbit aorta^9–12 include an increase in vascular mRNA and the protein content of several proto-oncogenes, including c-fos, c-myc, and c-jun. The increase in proto-oncogene expression is thought to result from mechanical stretch of the smooth muscle; stretch is a stimulus for many functions in VSM, including the generation of myogenic tone¹³ and hypertrophy. Stretch activates several types of surface membrane ion channels¹⁴ as well as MAPK¹⁵ in cardiomyocytes and may serve to signal the proliferation of smooth muscle cells. However, the nature of the molecular pathway specifically coupling mechanical stretch to the proliferation of smooth muscle during restenosis is unknown.

MAPK activity increases rapidly after mechanical stretch of porcine carotid arterial muscle strips in vivo.¹⁶ This increase in activity could possibly result in the expression of proto-oncogenes and the proliferation of smooth muscle cells after stretch. Because of the potential involvement of MAPK in the proliferation of VSM during restenosis, we evaluated MAPK content and activity in vascular tissue in vivo. Measurements of MAPK-specific phosphotransferase activity were made after angioplasty of porcine carotid and coronary arteries. Our data show that MAPK is activated on balloon stretch in both types of arteries.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Most chemicals and reagents used were purchased from Sigma. [³²P]ATP and [¹²⁵I]protein-A were from DuPont–New England Nuclear. Nitrocellulose was from Schleicher and Schuell, and the MAPK-specific polyclonal antibody was purchased from Biodesign (Ab-2). The female domestic swine were purchased from Hardin farm (Danville, Ind).

In Vitro Carotid Artery Handling
Porcine carotid arteries were dissected from animals and transported to the laboratory in an ice-cold physiological saline solution consisting of 140 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L Na₂HPO₄, 1.2 mmol/L MgSO₄, 1.6 mmol/L CaCl₂, 0.02 mmol/L EDTA, 5.6 mmol/L glucose, and 2 mmol/L MOPS, pH 7.4. Arterial muscle segments were cleaned, dissected, and attached to force transducers for the measurement of tension. Muscle strips were stretched by the application of a mechanical preload to the length optimal for development of contraction.¹⁶ MAPK activity in muscle strips treated in this manner was compared with the activity measured in arteries frozen immediately after dissection from anesthetized animals as described below.

Animal Preparation for Angioplasty
All procedures conformed to the Indiana University Animal Care and Use Committee guidelines. Female domestic swine (25 to 30 kg, n=5) were administered an intramuscular preanesthetic cocktail consisting of 800 mg ketamine, 30 mg acepromazine, and 0.6 mg atropine. Intravenous access was obtained, and 200 mg of sodium pentobarbital was given. The pigs were intubated and mechanically ventilated, and general anesthesia (2% isoflurane, 1.5 L/min nitrous oxide, and 1.5 L/min oxygen) was administered throughout the experiment. The right femoral artery was surgically exposed, an 8F or 9F femoral sheath was inserted into the artery, and a 5000 U bolus of heparin followed by 50 mg bretylium and 150 µg of nitroglycerin was administered intravenously. Cineangiography of the LAD and LCx coronary arteries was performed with a 1:1 mixture of ionic contrast and 0.9% saline in the anterior-posterior view. Spot angiograms of the contrasted left coronary arteries were obtained to measure the size of the LAD and LCx arteries with digital calipers. The same procedure was followed in sizing the carotid arteries in each pig.

Balloon Angioplasty
The right and left carotid arteries were surgically exposed to ensure immediate and careful harvesting of the vessels before balloon angioplasty of the coronary arteries. The left carotid artery was used as a control. Balloon dilation of the carotid arteries was performed at a 1.2:1 balloon-artery ratio, as described below for coronary arteries. While the balloon was inflated in the right carotid artery, india ink was used to mark the proximal and distal sites of injury. Immediately after injury, the segment between the india ink marks and a segment of the left carotid artery (control) were harvested. The artery segments were snap-frozen using liquid nitrogen–cooled tongs and stored at -80°C. The time between harvesting and snap-freezing was 2 to 5 minutes.

To obtain as much information as possible on the effects of angioplasty in coronary arteries and to maximize data acquisition for a given animal, we investigated the effects of overstretch injury on both the LAD and LCx arteries from the same animal. An appropriately sized angioplasty balloon catheter was passed over a 0.014-in guide wire to the selected segments of the left coronary arteries that achieved a balloon-artery ratio of 1.2:1. The balloon was then inflated 3 times to 6 atmospheres for 1 minute each separated by 1-minute intervals in the LCx artery followed within 20 minutes by a similar angioplasty of the LAD. A midline thoracotomy was performed before angioplasty of the LAD to visually define the area of angioplasty before harvesting the artery. The proximal and distal angioplasty sites of the LAD were carefully marked by 2–0 silk sutures. The heart was harvested within 5 minutes of the LAD angioplasty, and the relevant arterial segments were rapidly but carefully removed, along with a midregion of the uninstrumented RCA, which was used as a control. The arteries were then rapidly frozen using liquid nitrogen–cooled tongs and stored at -80°C.

MAPK Peptide Phosphotransferase Activity
Frozen arteries were ground to a fine powder under liquid nitrogen, and MAPK was extracted into 500 µL of extraction buffer containing 20 mmol/L Tris, pH 7.5, 5 mmol/L EGTA, 1 mmol/L DTT, phosphatase inhibitors (1 mmol/L Na₃VO₄, 20 mmol/L ß-glycerophosphate, and 10 mmol/L NaF), and protease inhibitors (1 µg/mL aprotinin and 0.1 mmol/L each of phenylmethylsulfonyl fluoride, TPCK, and TLCK). After extraction for 30 minutes at 4°C, the samples were clarified by centrifugation at 100 000g for 10 minutes. MAPK phosphotransferase activity was assayed in the supernatant fraction immediately after extraction and clarification using the peptide substrate APRTPGGRR.¹⁷ Briefly, 10 µL of the tissue extract was incubated with peptide (500 µmol/L) for 30 minutes at room temperature in 50 µL of a buffer that consisted of 12.5 mmol/L MOPS, pH 7.2, 12.5 mmol/L ß-glycerophosphate, 7.5 mmol/L MgCl₂, 0.5 mmol/L EGTA, 0.05 mmol/L NaF, 0.5 mmol/L Na₃VO₄, 2 mmol/L DTT, and 0.25 mmol/L [-³²P]ATP. Reactions were terminated by the addition of trichloroacetic acid (10% final volume) and centrifuged for 5 minutes at 14{ths}000g. The supernatant was spotted onto phosphocellulose paper (P81, Whatman) and washed four times in 500 mL of 50 mmol/L H₄PO₃ at 4°C. Filters were washed with 100 mL of 95% ethanol, and the amount of radioactivity was determined by liquid scintillation counting. This procedure has been shown previously to measure only p44^MAPK (ERK-1) and p42^MAPK (ERK-2) activity in arteries.¹⁶

MAPK Activity Normalization
In previous work measuring MAPK activity in smooth muscle tissue (carotid arteries in vitro),¹⁶ protein in the supernatant fraction was quantified by the method of Lowry et al,¹⁸ and kinase activity was reported in terms of the amount of phosphate incorporated into the peptide substrate per minute per milligram protein. In the present work, kinase activity was normalized to the content of MAPK quantified using radioimmunoblotting techniques (and, specifically, to total ERK1 content). This analysis was performed in lieu of normalization to total protein to avoid bias caused by protein originating from contaminating blood that was variable in the different tissue samples. Small amounts of blood contain a large amount of albumin that would interfere with normalization if performed by protein content. Proteins in the 100 000g supernatant fraction were separated by SDS-PAGE and transferred to nitrocellulose. Nitrocellulose strips were incubated with a polyclonal antibody specific for ERK-MAPKs (1:200) followed by [¹²⁵I]protein-A (0.25 µCi/mL). The p44^MAPK (ERK1) band was identified by autoradiography, and the amount of radioactivity was quantified by gamma counting. The antibody used recognizes both ERK1 and ERK2; however, the signal strength to ERK1 was much stronger than that for ERK2. Conversely, the signal to ERK2 was variable, weak, and at times not present. For this reason we chose to normalize the values to total ERK1 content. MAPK-specific activity was calculated as the amount of phosphate incorporated into the peptide substrate per minute per amount of MAPK determined by immunoblot analysis. For coronary arteries, these values were normalized to the MAPK activity in the right coronary artery of each animal used as a control; the activity in instrumented right carotid arteries was normalized to the value in noninstrumented left carotid arteries.

In-Gel Kinase Assay
Additional experiments were performed to further document MAPK activation in the arteries. We measured MAPK activity with an in-gel assay in which MBP was used as a substrate.^19,20 Arterial segments were rapidly frozen with liquid nitrogen–cooled pliers and then ground to a fine powder under liquid nitrogen. Proteins were then extracted into electrophoresis sample buffer and separated on a denaturing 7.5% polyacrylamide gel that was polymerized with 0.5 mg/mL MBP. The in-gel kinase assay was then performed according to published procedures.^19,20 Briefly, the gel was washed with 20% 2-propanol, 50 mmol/L Tris, pH 8.0, and then 50 mmol/L Tris, pH 8.0, and 5 mmol/L ß-mercaptoethanol (twice for 30 minutes). After these washes, the gel was denatured and then renatured by subsequently washing in 6 mol/L guanidine, 50 mmol/L Tris, pH 8.0, and 5 mmol/L ß-mercaptoethanol (twice for 30 minutes), followed by 50 mmol/L Tris, pH 8.0, 5 mmol/L ß-mercaptoethanol, and 0.04% Tween 40 (twice for 30 minutes and then overnight at 4°C). The gel was then washed with 40 mmol/L HEPES, pH 8.0, 2 mmol/L DTT, 0.1 mmol/L EGTA, and 5 mmol/L MgCl₂ (once for 30 minutes) and then incubated for 60 minutes in the same buffer containing 3 to 4 µCi/mL [-³²P]ATP. The reaction was terminated by washing the gel 10 times in 5% TCA and 1% sodium pyrophosphate, and phosphorylated MBP was visualized by autoradiography of the dried gels.

Statistical Analysis
Results are expressed as mean±SEM. To evaluate differences between MAPK activity in arteries undergoing angioplasty and the activity in paired control (uninstrumented) arteries within the same animal, a Student's t test was conducted to determine whether the log transformation of the ratio differed from zero. This analysis was performed in consultation with members of the Division of Biostatistics (Indiana University).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
MAPK Activity in Carotid Arteries
Previous work from our laboratory showed that ERK-MAPK activity in porcine carotid arteries increased on muscle stretch in vivo.¹⁶ To evaluate whether arterial stretch could lead to an increase in MAPK activity in vivo, we measured kinase activity in carotid arteries stretched by balloon angioplasty. However, in initial studies we found that we could not adequately wash the arterial muscle samples free of contaminating blood. Because the blood contamination was variable in the different tissue samples and contributed a significant amount of protein (predominantly albumin) to the extracts analyzed for kinase activity, the normalization of MAPK activity to protein content could not be made. We, therefore, normalized MAPK activity to the amount of p44^MAPK in the sample as determined by immunoblot. In control experiments we detected no p44^MAPK or MAPK phosphotransferase activity in porcine blood and conclude, therefore, that the kinase activity we measured was from the artery and not from any contaminating blood.

As shown in Fig 1, angioplasty of right carotid arteries led to a 7.7-fold increase in tissue MAPK activity when compared with the levels in uninstrumented left carotid arteries used as controls. In addition, MAPK activity in the arteries undergoing angioplasty was similar to the activity found in arteries placed under tension in vitro. The low level of kinase activity in unmanipulated arteries, in vivo, was somewhat surprising considering the in vitro muscle experiments. Arterial strips studied in vitro are typically positioned for study at their optimal length for force development. This positioning of the muscle has been thought to place the muscle at a length that is physiologically appropriate and comparable to what occurs in vivo. It might have been expected, therefore, that kinase activity in uninstrumented arteries would be nearer to that in stretched muscle strips in vitro and that angioplasty would increase the activity from that baseline. However, MAPK activity in instrumented carotid arteries in vivo was not different from the level of MAPK activity in maximally stretched carotid arteries in vitro, suggesting that the degree of stretch in either of these two animal models actually results in a similar activation of MAPK. Interestingly, the increase in MAPK activity on balloon angioplasty of carotid arteries was reversible after a 2-hour time interval (data not shown). In two animals, MAPK activities in right carotid arteries harvested 2 hours after angioplasty were not different from the activities in left carotid arteries taken from the same animals. These data are in agreement with the results of in vitro studies where it was observed that the increase in MAPK activity due to stretch of porcine carotid arterial muscle strips was reversed within an hour after unloading the muscle.²¹

View larger version (43K): [in this window] [in a new window]   Figure 1. MAPK activity in porcine carotid arteries in vivo. MAPK activity was quantified in extracts from carotid arteries that were dissected from animals, freeze-clamped, and ground to a fine powder within 2 minutes after experimental manipulation. The left carotid arteries served as controls for the RCAs undergoing PTA. MAPK activity in stretched porcine carotid arterial muscle strips in vitro and in arteries undergoing PTA were normalized to the activity in uninstrumented left carotid arteries. Results are expressed as the mean±SEM for four to eight experiments. Both the PTA and stretch (in vitro) points are statistically significantly different from control (P<.0005).

MAPK Activity in Uninstrumented Coronary Arteries
Since the activation of MAPK in carotid arteries is dependent on stretch and since the pathological process of restenosis may involve MAPK activation, we measured MAPK activity in coronary arteries subjected to PTCA. To determine if right coronary arteries could be used in subsequent studies as controls to identify the effects of balloon angioplasty in left coronary arteries, basal MAPK activity was measured in two pairs of uninstrumented RCAs and LADs (Fig 2). As with the carotid arteries, MAPK was extracted from the coronary arteries, and its phosphotransferase activity measured. The total content of p44^MAPK (ERK1) was quantified, by immunoblot, as described in "Materials and Methods" and shown in Fig 2. Peptide phosphotransferase activity was then normalized to the amount of p44^MAPK in the artery. We found that MAPK activities in the RCA and LAD for a given animal were similar, although there was variability in the activity for a given artery between the two animals. Because there were no apparent differences in basal levels of MAPK activity in the different types of coronary arteries, these data provided a basis for further studies in which noninstrumented right coronary vessels served as controls for instrumented left coronary vessels in individual experimental animals. Also, whenright versus left coronary arteries from a given animal assayed at the same time were compared, differences in the normalization process due to differential protein transfer to nitrocellulose or antibody binding were minimized.

View larger version (46K): [in this window] [in a new window]   Figure 2. MAPK activity in porcine coronary arteries from two control animals. RCAs and LADs from two animals that did not undergo angioplasty were harvested and assayed for MAPK activity as described in "Materials and Methods." Data in the upper panel show the phosphotransferase activity in the tissue extracts in units of radioactivity incorporated into the MAPK-peptide substrate over a 30-minute assay time. The bottom panel is an immunoblot showing the relative content of p44MAPK (ERK1) in the different tissue extracts. No inherent differences in activity between the RCA and LAD were detected on normalization of the phosphotransferase activity for MAPK content.

Effects of PTCA on MAPK Activity
Although MAPK activities in uninstrumented LADs and RCAs were not different within given animals, an increase in MAPK activity was found in all cases after PTCA of either the LAD or the LCx artery compared with the uninstrumented RCA in the same animal used as a control (Fig 3). As described in "Materials and Methods," the procedure we followed was to inflate the balloon in the LCx artery, then to inflate the balloon in the LAD (within 20 minutes), and then to harvest and freeze the tissue for subsequent biochemical analyses within an additional 5 minutes. The increase in MAPK activity that we measured on angioplasty was variable from animal to animal and tended to be greater in the LAD. This may be a result of our experimental protocol, since the LCx arteries underwent angioplasty before the LAD and, therefore, had a greater time (although less than 20 minutes in each case) to recover before harvesting and analysis of MAPK activity. Increases in MAPK activity ranged from 4.5- to 31.7-fold for the LAD, with a mean increment of 10.7-fold (±5.3, SEM, P=.0049) compared with the level in the RCA (n=5), and 1.8- to 31.3-fold for the LCx artery, with a mean increment of 9.7-fold (±5.7, SEM P=.048) versus that in the RCA (n=5). The variability in the increase that we observed in MAPK activity, in response to angioplasty, is perhaps not surprising given the variability observed in the severity of the restenosis response to angioplasty in various animal models and human studies.

View larger version (22K): [in this window] [in a new window]   Figure 3. Effects of balloon angioplasty on MAPK activity in porcine coronary arteries. MAPK activity was measured in coronary arteries from a series of animals. The arteries were either unmanipulated (RCA) or stretched using a balloon angioplasty catheter (LCx and LAD), as described in "Materials and Methods." Specimens were isolated and freeze-clamped 5 minutes (LAD) or less than 25 minutes (LCx) after angioplasty. MAPK phosphotransferase activity was normalized to MAPK content by immunoblot just as depicted in Fig 2. The values obtained for the LCx artery and LAD were compared with the values measured in the RCA of the same animal used as a control. PTCA resulted in an increase in arterial MAPK activity in comparison to the activity in control (RCA) arteries (P=.0003).

To confirm this observation using an alternative method of measuring MAPK activity, we assayed for the ability of coronary arterial MAPK to phosphorylate MBP using an in-gel kinase assay (Fig 4). Panel A of Fig 4 is a Coomassie blue–stained gel of the proteins extracted from a control RCA, from regions of a LAD proximal to and at the site of balloon dilation, and from the dilation site of an LCx artery (lanes A, B, C, and D, respectively). Panel B depicts the results of the in-gel kinase assay, demonstrating enhanced levels of MAPK activity both in the region of artery traversed by the uninflated balloon (lane B), and the site of stretch due to dilation (lane C). Although MAPK activity is elevated in both of these arterial regions, the site of balloon angioplasty has a much greater increase in activity. The dilated region of the LCx (lane D) also had an increase in MAPK activity comparable to that of the LAD. Although not shown, we found that MAPK activity was also increased (in an amount comparable to that at the site proximal to balloon inflation) in two arteries distal to the site of injury that was only instrumented by the balloon guide wire. As mentioned previously, total protein content in the tissue extracts was somewhat variable because of differences in blood contamination. This variability in protein due to the presence of blood can be observed in the results shown in Fig 4, where the amounts of actin (45 kD) and ERK1 were similar in the four samples (depicted in panels A and C, respectively), whereas the amount of albumin (visible at approximately 66 kD in panel A) varied in the four samples. Interestingly, there was more MAPK activity associated with the 44-kD band (ERK1) than with the 42-kD band (ERK2), in agreement with the data of Lai et al,²² in rat carotid arteries.

View larger version (39K): [in this window] [in a new window]   Figure 4. MBP-kinase activity in mechanically manipulated and control porcine coronary arteries in vivo. Extracts from an unmanipulated RCA (lane A), regions of an LAD proximal to (lane B) and at the site of balloon angioplasty (lane C), and a region of an LCx artery at the site of angioplasty (lane D) were assayed for MAPK activity with an in-gel kinase assay using gels polymerized with MBP. Panel A is a Coomassie blue–stained gel of the tissue extracts, panels B and C are autoradiograms. Panel B shows incorporation of phosphate into MBP within the gel, and panel C is an immunoblot depicting relative p44MAPK(ERK1) content in the tissue extracts used in panels A and B.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we demonstrate that balloon angioplasty of porcine coronary and carotid arteries is sufficient for the activation of MAPK in vivo. The extent of MAPK activation after PTA of carotid arteries is similar to the activity in muscle strips stretched in vitro, further suggesting that in vitro studies on stretch-dependent increases in MAPK activity provide a relevant model for studying the effects of angioplasty on MAPK in vivo. However, the biochemical mechanisms regulating the increase in MAPK activity in the arterial wall after PTA are unknown. The biological response to mechanical stretch involves the opening or closing of a number of specific sarcolemmal ion channels, apparently depending on the cell type, and the activation of the protein kinase C family of serine/threonine protein kinases.¹³ Protein kinase C activation leads to the subsequent activation of Raf, MAPK kinase (MAPKK, also referred to as MEK for MAPK and ERK kinase), MAPK, and possibly at least two proteins involved in protein translation, S6 kinase and the eukaryotic initiation factor (eIF) binding protein PHAS-I.^14,23–26 These events can culminate in the phosphorylation of the 40S ribosomal protein S6 or the dissociation of PHAS-I from one of the subunits of eIF (eIF-4E), resulting in increased global cellular protein synthesis. Whether these or other events occur in vasculature during angioplasty remains to be elucidated.

The fact that MAPK activity increased immediately after angioplasty corroborates the results of muscle stretch in vitro, where it has been shown that stretch of carotid arteries, resulting from the application of mechanical loads to individual muscle strips, increases MAPK activity within 30 seconds.¹⁶ MAPK activity is also rapidly elevated, although transiently so, when cultured myocytes are grown on flexible tissue culture plates and stretched.^14,15,23,27 These data complement the results of Lai et al,²² who showed that MAPK activity was increased in rat carotid arteries stretched by balloon injury at a 2-day time point after balloon angioplasty. MAPK activity in the rat carotid artery model peaked 5 to 8 days after angioplasty and then decreased slightly. Consideration of this study in concert with our data suggests that there may be two phases to the activation of MAPK in response to mechanical manipulation of arteries. In the first phase, there is a rapid activation of the kinase in response to the stretch that accommodates within the time frame of the 2-day period observed by Lai et al. The time for this decrease in activity is likely to be on the scale of 1 to 2 hours, since in two pigs carotid arterial MAPK activity 2 hours after angioplasty was not different from the activity in control arteries. In the second phase, there is a more slowly developing increase in MAPK activity that occurs over the time scale of days. This protracted increase in activity may reflect the transformation of smooth muscle cells from a contractile to a proliferative, or secretory, phenotype thought to occur during the process of postangioplasty restenosis.

MAPK phosphorylates transcription factors required for the expression of genes involved in cell growth and proliferation,²⁸ as well as the high molecular weight isoform of caldesmon, found specifically in fully differentiated smooth muscle.¹⁶ MAPK activation in smooth muscle in vitro occurs in response to such diverse external signals as growth factors, oxidative stress, and mechanical stretch.²⁹ Shear stresses and mechanical stretch are evidently positive regulators of MAPK activity in several cell types, including bovine aortic endothelial cells subjected to fluid shear stress³⁰ and cardiac myocytes.^14,23,27 Mechanical stretch has also been demonstrated to induce a variety of immediate-early genes in cultured myocytes, including c-fos, c-myc, and c-jun. In addition, these proteins are induced in vivo after balloon injury of the artery wall, with peak levels between 30 minutes and 2 hours after injury in rat and rabbit models.^10–12 Fos and jun positively regulate transcription by binding to AP-1 sites in the promoter region of a number of genes involved in vascular responses to injury.²⁷ In our study, MAPK activity in porcine arteries was elevated within 5 minutes after angioplasty, suggesting a possible role for MAPK in the acute response to vascular injury. Although these data cannot formally exclude a contribution by adventitial tissue to the increase in observed kinase activity, the in vitro experiments using carotid arterial muscle strips devoid of adventitia support the interpretation that the observed kinase activation resides primarily in the media. Pharmacologically induced hypertension also results in MAPK and enhanced DNA-binding activity of AP-1.³ Therefore, MAPK is presumably activated by direct mechanical stretch of the arterial wall during balloon angioplasty and also by increased wall stress associated with the elevated intra-arterial pressure of hypertension.

Several lines of evidence suggest that proto-oncogene expression plays a pivotal role in VSM cell entry into the cell cycle and the proliferation of VSM cells in the arterial wall and in cell culture.³¹ Bennett et al³² showed that c-myc expression peaked at 2 hours after PTA and was significantly reduced after the administration of c-myc antisense oligodeoxynucleotides to the adventitial surface of the arterial wall. In addition, neointimal proliferation was reduced at 2 weeks with c-myc antisense oligodeoxynucleotides.³² Furthermore, angiotensin II, a potent vasoconstrictor that stimulates VSM proliferation and hypertrophy, also stimulates MAPK activity^33,34 and c-fos expression³⁵ in rat VSM cells; angiotensin-converting enzyme inhibitors suppress the early expression of c-fos and c-myc after balloon denudation, at doses leading to a reduction of neointimal hyperplasia.³⁶ Therefore, MAPK is a key enzyme activated during structural deformation and angiotensin II and growth factor stimulation of VSM and may be an important regulator of the increase in proto-oncogene expression and cell proliferation that occurs after angioplasty.

The data in the present study provide a link to show that a key regulator of vascular cell proliferation, MAPK, is activated in response to balloon overstretch injury. Alterations in the expression of various oncogenes after vascular stretch presumably result from the activation of MAPK. These observations suggest that modulation of the upstream activators of MAPK in the context of acute vascular injury may provide an effective approach to controlling the proliferation of VSM cells during the process of restenosis.

	Selected Abbreviations and Acronyms

 

DTT = dithiothreitol EGF = epidermal growth factor ERK = extracellular signal–regulated kinase JNK = c-Jun NH2-terminal kinases LAD = left anterior descending artery LCx = left circumflex MAPK = mitogen-activated protein kinase MBP = myelin basic protein PDGF = platelet-derived growth factor PTA = percutaneous transluminal angioplasty PTCA = percutaneous transluminal coronary angioplasty RCA = right coronary artery SAPK = stress-activated protein kinase VSM = vascular smooth muscle

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-56035 (Dr Adam) and grants from Cordis Corp (Drs March and Wilensky) as well as the Cryptic Masons Medical Research Foundation (Dr Mehdi). We thank David Mendel for his excellent technical expertise in helping to perform these experiments.

Received June 27, 1997; accepted September 17, 1997.

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