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(Circulation. 1995;92:1230-1235.)
© 1995 American Heart Association, Inc.

Articles

Smooth Muscle Cell Proliferation Is Proportional to the Degree of Balloon Injury in a Rat Model of Angioplasty

Ciro Indolfi, MD; Giovanni Esposito, MD; Emilio Di Lorenzo, MD; Antonio Rapacciuolo, MD; Antonio Feliciello, MD; Antonio Porcellini, MD; Vittorio E. Avvedimento, MD; Mario Condorelli, MD; Massimo Chiariello, MD

From the Division of Cardiology, Department of Medicine & Molecular and Cellular Pathology, Federico II University, Naples, and the Department of Experimental and Clinical Medicine, Facoltà di Medicina di Catanzaro, Italy.

Correspondence to Ciro Indolfi, MD, FACC, Division of Cardiology, Federico II University, Via Pansini, 5 80131 Napoli, Italy.

	Abstract

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Background A variable degree of smooth muscle cell (SMC) proliferation after balloon injury has been reported in previous rat studies. It is unknown whether balloon injury induces c-fos expression and whether it is related to the degree of vascular injury in vivo. Therefore, we tested the hypothesis that proportional increases in neointimal formation and c-fos expression might be present after different degrees of balloon dilation.

Methods and Results Angioplasty of the carotid artery was performed with a balloon catheter. Vascular injury was evaluated at 0, 0.5, 1.0, 1.5, and 2 atm (n=6 for all). In 40 additional rats, total RNA dot blots were performed to assess the effect of various degrees of balloon injury on c-fos expression. SMC proliferation proportional to the increases of inflation pressure was found between 0 and 2 atm with neointimal areas of 0.002±0.002, 0.069±0.014, 0.128±0.043, 0.190±0.010, and 0.255±0.041 mm², respectively. When the degree of SMC proliferation (neointima and neointima/media ratio) was plotted against balloon inflation pressure, a linear relation was observed (r=.733, P<.001 and r=.755, P<.001, respectively). An increase in c-fos expression proportional to the degree of injury was found 30 minutes after injury.

Conclusions Neointimal proliferation produced by balloon injury is related to balloon inflation pressure, supporting the concept of an SMC proliferative response proportional to the degree of injury. The increase in SMC proliferation is associated with a proportional increase in the early expression of the c-fos nuclear proto-oncogene.

Key Words: muscle, smooth • stenosis • angioplasty

	Introduction

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The most common characteristic of the response of blood vessels to injury is the formation of a new structure called neointima; this occurs rapidly after angioplasty but is also observed in hypertension, renal and radiation injuries, or trauma to any layer of the vessel wall.¹ Neointimal formation after injury has been viewed as an important initial step in the progression of atherosclerotic lesions and the rapid restenotic changes observed after angioplasty.^{2 3}

The balloon catheter–injured artery has become the standard rat model for studying smooth muscle cell (SMC) proliferation in vivo.^{4 5 6} This model has been used extensively to study the factors involved in this process⁶ and to assess the potential inhibitory effects of various types of interventions on SMC proliferation.^{7 8}

Although most of the rat studies adopted the standard method described by Clowes and associates⁹ to produce balloon injury, a variable degree of SMC proliferation after balloon injury was reported in different studies.^{5 6 10 11 12 13 14 15} The standard technique produces injury of the carotid artery by use of a Fogarty catheter passed three times with the balloon distended sufficiently to generate slight resistance.⁵ The lack of a quantified balloon dilation might cause the variable neointimal proliferation observed in different studies. In fact, with the same animal model (rat) and the same balloon catheter (2F Fogarty catheter), a large variability of both neointima and neointima/media ratio is found by analysis of the published data (from 0.002 to 0.17 mm² and from 0.4 to 1.04, respectively).^{5 6 10 11 12 13 14 15} These differences make comparing or referring to previous studies difficult because baseline control data are different.

To explain the different degree of intimal proliferation, it might be hypothesized that the proliferative response of the injured vessel wall is related to the degree of injury. In this regard, a recent study demonstrated that pressure promotes DNA synthesis in rat cultured vascular SMC through an activation of phospholipase C and protein kinase C.¹⁶ In that study, a special device was used to separate effects caused by pure pressure from those caused by vessel stretch (or tension) induced by pressure.¹⁶

It has also been demonstrated that SMC proliferation is caused by the transduction of signals from the extracellular environment to the cell nucleus. Several genes become transiently activated during manipulation of SMCs.^{17 18 19 20} Studies in cultured SMCs have demonstrated that c-fos, c-myc, and c-myb proto-oncogenes are activated shortly after mitogenic stimuli.^{21 22} Similar results were obtained in in vivo studies.¹⁹ The activation of nuclear proto-oncogenes appears to be a final common pathway onto which mitogenic signals converge.²³ However, it has not been investigated whether the expression of these proto-oncogenes is related to the degree of injury.

The aim of the present study was to induce a reproducible quantitative balloon injury of the carotid artery and to assess the effects of different degrees of balloon-induced vascular injury on neointimal formation in a rat model of angioplasty. Finally, the role of different degrees of vascular injury on c-fos expression was also assessed.

	Methods

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The rats in this study were handled according to the animal welfare regulation of the Federico II University of Naples, and the protocol was approved by the animal use committee of that institution. All rats received humane care in accordance with the animal use principles of the American Society of Physiology. All rats were maintained under identical conditions of temperature (21±1°C), humidity (60±5%), and light/dark cycle and had free access to normal rat chow.

Animal Preparation
Seventy-nine Wistar rats weighing 350 to 400 g (at 14 weeks of age) purchased from Morini were included in the present study. Rats were anesthetized with an intramuscular injection of ketamine (Ketalar, Parke-Davis) 100 mg/kg and xylazine (Rompun, Bayer AG) 5 mg/kg. Angioplasty of the carotid artery was performed with a balloon embolectomy catheter as previously described.^{5 9} In brief, the balloon catheter (2F Fogarty, Edwards Laboratories) was introduced through the right external carotid artery into the aorta, and the balloon was inflated at different atmospheres (see below). The vessel was damaged by passing an inflated balloon through the lumen three times. Preliminary pilot studies performed in our laboratory demonstrated that the time necessary to pass the inflated balloon catheter back and forth into the carotid artery three times was 18 seconds. Therefore, to keep the time of the injury constant (it might influence the SMC proliferation per se), we decided to maintain the time of balloon inflation constant at 18 seconds.

The balloon catheter was inflated with a calibrated, commercially available inflation device (Indeflator Plus 20, Advanced Cardiovascular System, Inc). The Indeflator is essentially a polypropylene syringe coupled to a pressure regulator. The syringe handle is a molded piston rod engineered to supply the maximum amount of force while dispensing pressure over the palm of the hand. A one-way stopcock is bonded to the distal Luer tip of the syringe. The accuracy and the reproducibility of the pressure measurements were assessed before the experiments. In fact, we checked the pressures measured by the Indeflator Plus 20 with another sophisticated electronic system (Intelliflator). A linear relation between the pressures measured with the two devices was found (coefficient of variation, 0.01±0.00).

Arterial pressure and heart rate were measured indirectly by a tail-cuff plethysmographic technique (Harvard Apparatus, model 50-0002).²⁴

Study Design
The effect of various degrees of injury was assessed in different groups of rats. To assess the relation between the inflation pressures and the degree of neointimal proliferation, vascular injury was evaluated at 0 (passing only the deflated balloon three times), 0.5, 1.0, 1.5, and 2.0 atm (n=6 for all). In three additional experiments, we inflated the balloon to 2.5 atm. At 2.5 atm, however, recovering the catheter in the external carotid artery was extremely difficult. Therefore, the maximal balloon inflation used in the present study was 2.0 atm. In six other rats, the effects of the anesthesia and the surgical procedure (without the balloon injury) on SMC proliferation were also assessed. The carotid arteries were removed 14 days after the injury and processed as described below.

Morphology
At the time of the final experiment (2 weeks later), the animals were anesthetized with an intramuscular injection of ketamine 100 mg/kg and xylazine 5 mg/kg, and the carotid arteries were fixed by perfusion at 120 mm Hg with 100 mL of PBS (pH 7.2) followed by 80 mL of prepared PBS containing 4% paraformaldehyde through a large cannula placed in the left ventricle.

The carotid arteries were removed, and six cross sections were cut (each 6 µm thick) from the approximate midportion of the artery, with three of the sections stained with hematoxylin and eosin to demarcate cell types. The remaining three sections were stained with aldehyde fuchsin and counterstained with van Gieson's solution to demarcate the internal elastic lamina (IEL). The sections were photographed under low power, blindly videodigitized, and stored in the image analysis system (Mipron, Kontron Electronics) in a 512x512 matrix with an 8-bit gray scale with a 12-cm field of view. The media, neointima, and vessel wall were traced carefully, and the ratios between neointima and media were calculated.

The reproducibility of the measurements of neointima (repeated analysis by the same "blinded" investigator) revealed a coefficient of variation of 0.02±0.01.

Total RNA Preparation and Dot-Blot Analysis of c-fos Expression
To assess the effect of various degrees of balloon injury on c-fos expression, a proto-oncogene codifying a nuclear protein involved in cell proliferation, experiments were performed at 0.5,⁷ 1.0,⁷ and 2.0 atm.⁸ The arteries were removed 30 minutes after balloon injury. Additional experiments were performed to assess the effects of injury 4 hours after balloon inflation (0.5 atm, n=6; 1.0 atm, n=6; 2 atm, n=6) on c-fos expression.

Total RNA was isolated by guanidinium thiocyanate–phenol–chloroform extraction.²⁵ We used dot-blot hybridization because it also allows detection of partially degraded mRNA molecules.²⁶

Total RNA dot blots were performed by use of a Schleider & Schuell Minifold II apparatus following the manufacturer's recommendations. RNA samples were mixed with three volumes of denaturing solutions (50% formamide, 2.2 mol/L formaldehyde, 20 mmol/L MOPS, 5 mmol/L sodium acetate, and 0.5 mmol/L EDTA), heated for 10 minutes at 65°C, and diluted twofold in 19x SSC (where SSC is 150 mmol/L NaCl and 15 mmol/L sodium citrate). The samples were then applied to a Hybond-N membrane (Amersham) equilibrated in 10x SSC.

The RNA was cross-linked to membrane by UV exposure (5 minutes). Prehybridization (1 hour at 65°C) and hybridization (15 hours at 65°C) were performed in sodium phosphate buffer (0.5 mol/L Na₂HPO₄ [pH 7.4], 7% SDS, and 1 mmol/L EDTA [pH 8]). The filters were washed (three times for 15 minutes at 65°C) in high-stringency buffer (40 mmol/L Na₂HPO₄ [pH 7.4], 1% SDS).

Filters were exposed to Kodak XAR-5 film (Eastman), sensitized by preflashing, with Du Pont Cronex intensifying screens (Du Pont) at -70°C. The probe used for hybridization was a fragment EcoRI–Xba I derived from murine c-fos.²⁷

Quantitative analysis of the autoradiograms was performed by laser densitometry on an LKB 2202 instrument.

Statistical Analysis
All data are shown as mean±SEM. Statistical analysis between groups was performed by ANOVA with a SYSTAT program.²⁸ When a significant overall effect was detected, Tukey's test was applied to compare single mean values.²⁹ Linear regression analysis was also performed to compare the degree of neointimal formation (and the neointima/media ratio) and the inflation pressure. A value of P<.05 was considered significant. The coefficient of variation was calculated by dividing the SD of the mean by the mean.

	Results

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No differences in arterial pressure and heart rate were found between the sham-operated rats and the experimental groups. In addition, blood pressure and heart rate were comparable in the different experimental groups.

Effects of Various Degrees of Vascular Injury on Neointimal Proliferation and c-fos Proto-oncogene Expression
In the sham-operated rats not subjected to vascular injury, no neointimal formation was detected, and the endothelium was found intact 14 days after surgery in both carotid arteries of all rats. Fig 1 shows representative cross sections of the carotid arteries from each group of rats studied. The passing of the deflated balloon catheter into the carotid artery did not produce neointimal formation in any of the rats except one, which showed very mild neointimal formation (0.010 mm², Fig 2).

View larger version (0K): [in this window] [in a new window]   Figure 1. Photomicrographs showing the effect of different inflation pressures on neointimal formation in rat carotid arteries subjected to balloon injury. Representative cross sections are from the carotid artery of a control rat (control) and after progressive vascular injury induced at 0 (passing only the deflated balloon three times), 0.5, 1.0, 1.5, and 2.0 atm. The proliferative response of smooth muscle cells was proportional to the degree of vascular injury.

View larger version (14K): [in this window] [in a new window]   Figure 2. Bar graph showing the media (open bars) and neointima (solid bars) of injured rat carotid arteries. Data are expressed as mean±SEM. *P<.05 vs 0 atm; **P<.05 vs 0 and 0.5 atm; ***P<.05 vs 0, 0.5, and 1.0 atm.

In contrast, in the experimental group neointimal formation was already present at 0.5 atm (0.069±0.014 mm²). This degree of pressure of the inflating balloon was also effective in increasing the neointima/media ratio (see the Table).

View this table: [in this window] [in a new window]   Table 1. Neointima and Neointima/Media Ratio Measured 14 Days After Injury in the Different Animal Groups

A proportional SMC proliferation was found after the inflation pressure was increased to 1, 1.5, and 2 atm with neointimal areas of 0.128±0.043, 0.190±0.010, and 0.255±0.041 mm², respectively. A similar trend was observed for the neointima/media ratio that reached 1.898±0.324 at 2 atm (Fig 3). The IEL was ruptured in only one experiment (at 2.0 atm).

View larger version (10K): [in this window] [in a new window]   Figure 3. Bar graph showing the effects of different balloon pressures on neointima/media ratio. There was a significant increase in the neointima/media ratio proportional to the degree of injury. Data are expressed as mean±SEM. *P<.05 vs 0 atm; ***P<.05 vs 0, 0.5, and 1.0 atm.

When the degree of SMC proliferation (neointima and neointima/media ratio) was plotted against the balloon inflation pressure, a significant linear relation was observed (r=.733, P<.001 and r=.755, P<.001, respectively).

Total RNAs were analyzed for the expression of c-fos proto-oncogene by dot-blot hybridization. The amount of c-fos expression 30 minutes after balloon dilation was related to the degree of injury. The values were computed as integrated density.

The actual numbers are 0.63±0.05, 1.13±0.1, and 1.20±0.09 for GAPDH at 0.5, 1.0, and 2.0 atm, respectively. The corresponding values for fos hybridization are 0.92±0.04, 1.28±0.12, and 2.3±0.3 (Fig 4). In uninjured control arteries, c-fos was undetectable.

View larger version (49K): [in this window] [in a new window]   Figure 4. Increasing inflation pressure stimulates c-fos expression. The RNA was extracted 30 minutes after the balloon injury. Top, Representative autoradiogram of hybridization of total RNA (3 mg) with c-fos–specific probe or with GAPDH probe. Bottom, Quantitative analysis of the dot blot by laser densitometry. The reported values represent the fold induction relative to the control. The values were calculated in arbitrary units relative to the GAPDH mRNA. The actual values were as follows: control, 0.92±0.04 (GAPDH, 0.63±0.05); 1 atm, 1.28±0.12 (GAPDH, 1.13±0.10); and 2 atm, 2.3±0.30 (GAPDH, 1.20±0.09). These data indicate a persistent c-fos stimulation induced by the inflation pressure.

We performed the same analysis 4 hours after balloon injury. The actual numbers are 0.43±0.05, 0.40±0.06, and 0.38±0.04 for GAPDH at 0.5, 1.0, and 2.0 atm, respectively. The corresponding values for fos hybridization are 0.36±0.04, 0.72±0.06, and 0.99±0.08.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The principal finding of the present study is that neointimal proliferation produced by balloon injury is related to the balloon inflation pressure, supporting the concept of an SMC proliferative response proportional to the degree of injury. In addition, the increase in SMC proliferation 14 days after injury is associated with a proportional increase of the early expression of the c-fos nuclear proto-oncogene.

Previous Studies
Conflicting data exist on whether the SMC proliferation is proportional to the degree of injury.^{30 31 32} In a previous study by Sarembock and associates,³⁰ the influence of two different inflation pressures on intimal hyperplasia was assessed in the rabbit. When the balloon inflation pressure was doubled, a significant increase in intimal hyperplasia was observed (from 0.9±0.5 to 1.7±0.9 mm²).³⁰ In contrast, two other studies were unable to demonstrate a relation between the degree of injury and the extent of SMC proliferation.^{31 32} The reasons for these different results are unclear and might be related to the different study protocols (ie, different balloon dilation procedures, use of nitrogen gas to desiccate the artery, diet, additional drugs, and species differences).

Our data demonstrated a clear linear relation between the degree of injury and the extent of neointimal formation. This might be due to the fact that we carefully monitored two major determinants of vessel injury: balloon inflation pressure (with a calibrated inflation device) and the total time of balloon inflation.

The transient increase of nuclear proto-oncogene mRNA after mitogenic stimulation has been shown as the cell enters the G₁ phase and appears to be necessary for the transition from the G₁ to the S phase.^{33 34} The importance of these oncogenes in SMC proliferation was demonstrated with antisense oligonucleotides directed against the mRNAs of these proteins that inhibit the process.^{7 35} The signal transduction pathway that links receptor activation with SMC proliferation includes induction of c-myc and c-fos. Induction of c-fos is maximal 30 minutes after the growth stimulus; after that, the specific mRNA decays at levels lower than the peak but above the basal. Our data demonstrated that, 30 minutes after balloon injury, c-fos is expressed proportionally to the degree of injury.

Although c-fos induction is maximal 30 minutes after the growth stimulus, we have also measured the expression of this proto-oncogene 4 hours after injury. In fact, inefficient stimuli induce a labile c-fos induction that does not produce mitogenic effects. The demonstration of c-fos induction 4 hours after injury indicates a persistent and consistent entry in the cycle of the majority of the cells.

Therefore, overexpression of the c-fos proto-oncogene may play a role in the determination of the amount of SMC proliferation 14 days after balloon injury. Better understanding of the significance of this oncogene in SMC proliferation requires further studies. A previous study demonstrated that antisense c-myb oligonucleotides inhibit intimal SMC proliferation in vivo.⁷ Therefore, the antisense c-fos should also be tested in this model in future studies to assess its possibility of inhibiting SMC proliferation after balloon injury.

A previous study performed in a swine model of restenosis demonstrated that the neointimal lesions formed after balloon injury in the pig were reparative because they primarily filled the void left in the vessel wall at the point of IEL and medial disruption.^{36 37 38} Therefore, in the pig, neointimal hyperplasia occurs only when the IEL is ruptured. To focus on the role of the IEL, in our study, sections were stained with aldehyde fuchsin and counterstained with van Gieson's solution. In contrast with the swine model, our experiments demonstrate that the response of the rupture of the IEL is not the "conditio sine qua non"; the SMCs proliferate in a rat model of restenosis. In addition, we did not observe any relation between IEL damage and neointimal formation. It should be pointed out that, in human angioplasty, IEL rupture is not a prerequisite for the clinical occurrence of restenosis.^{39 40}

Finally, Fingerle and associates⁶ studied the role of the endothelium on SMC proliferation. They found a gentle denudation of the endothelium from rat carotid arteries by use of a rotating loop of 5/0 monofilament suture induced a mild SMC proliferative response (compared with a standard balloon technique) at 1, 4, and 12 weeks.⁶ Therefore, a denuding injury with no medial trauma induces minimal neointimal formation. The significantly greater proliferation observed in ballooned vessels might reflect a response of the medial cells to the trauma that occurs during denudation, as demonstrated in a previous in vitro study.¹⁶ A preliminary study by Vignale and associates⁴¹ also reported significantly greater thymidine uptake after balloon angioplasty compared with animals in which only the deflated balloon catheter or the angioplasty guide wire was used. Animal studies also showed that, when superficial denudation is carefully carried out in a large area without damaging the media, no intimal proliferation occurs despite platelet adherence on the surface and late recovery of the endothelium.⁴² Thus, simple denudation and exposure to platelets do not represent a sufficient stimulus to initiate marked intimal proliferation; direct injury to SMCs, either mechanical or inflammatory, is essential for this process.

Possible Clinical Implications
Over the past decade, percutaneous transluminal coronary angioplasty has gained wide acceptance as the procedure of choice in many patients with atherosclerotic coronary arteries. As experience with the procedure has been gained, its success rate has risen to approximately 95%, and the incidence of acute complication has fallen.⁴³ Despite these improvements, restenosis in the days, weeks, or months after successful angioplasty of a narrowed coronary artery occurs in 25% to 55% of patients.^{44 45 46 47 48 49 50} Use of an oversized balloon may increase the chance of both acute complications⁵¹ and restenosis.⁵²

Although extreme caution should be used in extrapolating animal studies to human clinical settings, our current data suggest that a high inflation pressure might increase the incidence of angiographic restenosis. If this is true, reduced injury should decrease proliferation and attenuate clinical restenosis.

However, this possibility and the role of the inflation pressure on restenosis rate are difficult to test clinically. In fact, such a study might be only a retrospective evaluation of studies based on the use of the same type of balloon catheter (ie, compliant or noncompliant balloons). In addition, the diameter of the balloon catheter used should be carefully matched to the normal vessel size through a quantitative method. Finally, an inverse relation between intimal thickness and the degree of wall shear stress⁵³ and between blood flow rate and the degree of intimal proliferation has been documented.⁵⁴ Therefore, because wall shear stress may influence SMC proliferation after injury, the regional coronary blood flow should also be measured.

	Acknowledgments

 
This work was partially supported by grants from the Associazione Italiana Ricerca Cancro, Progetti Finalizzati CNR Ingegneria Genetica, and Applicazioni Cliniche Ricerca Oncologica. We thank Armando Coppola for excellent technical assistance.

Received November 7, 1994; revision received February 27, 1995; accepted February 28, 1995.

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