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Circulation. 1997;96:1944-1952

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(Circulation. 1997;96:1944-1952.)
© 1997 American Heart Association, Inc.

Articles

Effect of Intravascular Irradiation on Cell Proliferation, Apoptosis, and Vascular Remodeling After Balloon Overstretch Injury of Porcine Coronary Arteries

Ron Waksman, MD; José C. Rodriguez, MS; Keith A. Robinson, PhD; Gustavo D. Cipolla, DVM; Ian R. Crocker, MD; Neal A. Scott, MD, PhD; Spencer B. King, III, MD; ; Josiah N. Wilcox, PhD

From the Department of Medicine (R.W., J.C.R., K.A.R., G.D.C., N.A.S., S.B.K., J.N.W.) and the Department of Radiation Oncology (I.R.C.), Emory University School of Medicine, Atlanta, Ga. Dr Waksman is now at the Cardiology Research Foundation, Washington, DC.

Correspondence to Josiah N. Wilcox, PhD, Emory University, Department of Medicine, Division of Hematology/Oncology, 1639 Pierce Dr, Room 1115 WMRB, Atlanta, GA 30322. E-mail medjnw{at}emory.edu


*    Abstract
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 *Abstract
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 down arrowResults
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Background Ionizing radiation has been shown to reduce vascular lesion formation after balloon overstretch injury of pig coronary arteries. The present series of experiments examines the mechanism by which this occurs.

Methods and Results Balloon injury was performed on porcine coronary arteries, followed immediately by ionizing radiation using either a source train of 90Sr/Y or 192Ir seeds designed to deliver 14 or 28 Gy at a depth of 2 mm from the source. The animals were killed 3, 7, or 14 days after injury. Bromodeoxyuridine was administered 24 hours before euthanasia to label proliferating cells. Cell proliferation was significantly reduced on day 3 in the adventitia and media of the irradiated vessels compared with controls. Two weeks after injury, there were fewer {alpha}-actin–positive myofibroblasts in the adventitia of the irradiated vessels than in nonirradiated controls, and morphometric analysis indicated that the vessel perimeter of the irradiated vessels was significantly larger than in controls. Together, these results suggest a positive effect of intravascular irradiation on vascular remodeling. Apoptosis was estimated by terminal transferase dUTP-biotin nick-end labeling (TUNEL) 3 and 7 days after injury. TUNEL-labeled cells were found primarily in the adventitia at the medial tear, but no differences were detected between irradiated and control vessels.

Conclusions These studies suggest that intracoronary radiation primarily inhibits the first wave of cell proliferation in the vessel wall and demonstrates a favorable effect on late remodeling by preventing adventitial fibrosis at the injury site.


Key Words: restenosis • remodeling • proliferation • angioplasty • radiation


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
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A number of studies indicate that radiation treatment reduces vascular lesion formation after balloon overstretch injury of porcine coronary arteries. A significant reduction in the size of the neointima has been observed when balloon angioplasty is immediately preceded or followed by intracoronary ionizing radiation with both {gamma}- and ß-emitting radioactive sources, including 192Ir,1 2 3 90Sr/Y, and 90Y.4 5 6 Similar results have been reported on the prevention of lesion development in stented vessels either by intravascular irradiation at the time of stent placement7 or in studies using radioactive stents.8 9 The effect on lesion development appears to be stable and long-lasting, and at least two of the pig studies have been carried out to 6 months with good results.2 4 Currently, there are several clinical trials in progress that will assess the effect of intravascular irradiation on clinical restenosis.

The use of endovascular irradiation for reducing restenosis after angioplasty is derived from the concept that restenosis has many similarities to wound healing. The use of radiation to modify the wound-healing response has been well documented.1 2 10 Clinically, low-dose radiation is in use for a number of different proliferative conditions, including the prevention of keloids11 12 13 and heterotopic bone formation.14 15

Many different mechanisms have been proposed to explain the sequence of events leading to arterial narrowing or restenosis after angioplasty. Previously, the major focus had been on the medial smooth muscle cells, which were thought to give rise to a restenosis lesion. It is well documented in the rat and rabbit injury models that balloon injury stimulates the proliferation of medial smooth muscle cells, which migrate to the intima, where they continue to proliferate and produce matrix proteins, forming a neointima.16 17 18 19 However, such injuries rarely produced a narrowing of the arterial lumen that is associated with clinical restenosis. More recently, it has been suggested that geometric remodeling may be more important than neointima formation in the restenosis process. This is supported by intravascular ultrasound studies in patients20 21 22 23 and a number of animal studies,20 21 24 all of which indicate that there is a reduction in the diameter of the external elastic lamina after angioplasty and that this decrease in the overall vessel size is a better correlate with the degree of luminal narrowing than the size of the intimal mass.

We have recently demonstrated that angioplasty of porcine coronary arteries stimulates adventitial myofibroblast proliferation, leading to a fibrotic response in the adventitia.25 These cells show increased synthesis of {alpha}-smooth muscle actin25 and nonmuscle myosin heavy chain26 and accumulate in the adventitia surrounding the injury site. In healing dermal wounds, similar cells are involved in the process of scar contraction.27 28 We hypothesize that the adventitial myofibroblasts may constrict the injured vessel in a similar fashion, thus contributing to the process of geometric remodeling and late lumen loss after angioplasty.25

In the present study, we have undertaken a series of experiments to determine the effect of ionizing radiation on cell proliferation in the media and adventitia, arterial remodeling, and apoptosis to understand the mechanisms by which ionizing radiation prevents vascular lesion formation after balloon injury in porcine coronary arteries.


*    Methods
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 up arrowIntroduction
 *Methods
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All experiments and animal care conformed to National Institutes of Health and American Heart Association guidelines for the care and use of animals and were approved by the Emory University Institutional Animal Care and Use Committee.

Balloon Overstretch Injury and Radiation Treatment
The model of overstretch injury has been described previously.29 30 Seventy-two female domestic pigs (Sus scrofa, 18 to 27 kg) were given aspirin (325 mg) 1 day before and on the day of the procedure. They were sedated with a combination of tiletamine HCl (10 mg/kg IM) and atropine (0.6 mg/kg IM), and general anesthesia was maintained with 1% to 2% Isothane with a Harvard respirator.

After placement of an 8F introducer sheath in the right femoral artery by surgical cutdown, each animal received a single dose of heparin (200 U/kg) and bretylium tosylate (2.5 mg/kg). Under fluoroscopic guidance, an 8F hockey stick guiding catheter was positioned in the left coronary ostium. After the intracoronary administration of nitroglycerin (200 µg), coronary angiography was performed in the 45° left anterior oblique and 45° right anterior oblique views and recorded by cineangiography (Phillips Cardiodiagnost).

Balloon overstretch injury was performed with a 3.5-mm clinical angioplasty balloon positioned in the proximal segments of the left anterior descending and left circumflex arteries and inflated to 10 atm three times for 30 seconds in each artery. Inflation periods were separated by 1-minute deflation periods to restore coronary perfusion. After the completion of the third inflation, the angioplasty balloon was withdrawn and additional nitroglycerin (200 µg) was administered to limit coronary spasm. Repeat angiography was then performed to assess vessel patency and degree of injury.

One of the injured coronary arteries in each swine was assigned randomly to receive radiation treatment with either a ß- or {gamma}-emitting source train. In 19 of the animals, a ß-source was positioned at the angioplasty site of the assigned artery within a 5F delivery catheter (Novoste Corp) introduced over a flexible 0.014-in wire. After the guidewire was withdrawn, a 2.5-cm-long train with five seeds of 90Sr/Y was positioned at the site of injury in the target vessel by cinefluoroscopic visualization within the delivery catheter. The seeds were left in place for a period sufficient to deliver the assigned dose (14 or 28 Gy) to a depth of 2 mm (180 to 360 seconds). In 4 pigs, the radiation treatment was administered with 192Ir as follows. A 4F perfusion-delivery catheter (USCI) was introduced over a flexible 0.014-in wire across the injury site of the assigned artery, the guidewire was withdrawn, and a 3.0-cm source train with nine seeds of 192Ir was positioned at the site of the injury and was left in place for a period sufficient to deliver a dose of 28 Gy to a depth of 2 mm (28 to 58 minutes, depending on the source activity). All animals receiving 192Ir were part of the 28-Gy dose group killed 3 days after angioplasty for BrdU analysis, such that 4 of 6 animals in that group received this form of radiation. Data from both ß- and {gamma}-irradiated vessels were combined, because previous work indicated that both forms of radiation produced similar results with respect to lesion formation if the dose was the same.5 31 After irradiation, the delivery and guiding catheters were removed and the femoral cutdown was repaired. Nitroglycerin ointment was administered topically, and the animals were returned to routine care.

Calculation of Radiation Dose
The treatment times for 192Ir were determined in standard fashion by entering the activity and length of the 192Ir ribbon (Best Industry Inc) into a commercial radiation treatment planning system (CMS Modulex) and calculating the dose rate at 2.0 mm from the center of the source train. The dose rate around the 2.5-cm 90Sr/Y source train was calculated with Monte Carlo electron transport code (ITS). The energy spectrum of 90Sr/Y was obtained from Cross et al.32 No in vivo dosimetry was performed. The delivery systems for the ß- and {gamma}-emitters were not centered, and therefore there was potential variability in the dose delivered to the arterial wall.

BrdU Injection and Tissue Preparation
The effect of radiation on cell proliferation was determined by BrdU injections and immunohistochemistry in 29 pigs killed either 3 (n=17) or 7 (n=12) days after injury. BrdU (Sigma Chemical Co) was administered via the ear vein of the pigs in three doses of 50 mg/kg at 24, 16, and 8 hours before tissue harvest to label the proliferating cells. The animals were killed with an overdose of barbiturate, the hearts removed, and the injured arteries perfused in situ with saline followed by 4% paraformaldehyde in NaPO4 buffer (pH 7.4) at 100 to 110 mm Hg pressure for 5 minutes. The arteries were then dissected from the heart and immersed overnight in 15% sucrose-PBS. The following day, the vessels were divided into serial 3.0-mm segments and frozen in liquid nitrogen embedded in optimal cutting temperature compound (O.C.T., Miles Laboratories) in a manner allowing the serial reconstruction of each vessel. Histological analysis was performed on 6-µm cryosections collected onto glass slides (Fisher SuperFrost Plus).

TUNEL Assay
Apoptosis was estimated by the TUNEL assay, which relies on the incorporation of labeled dUTP at sites of DNA breaks.33 Tissue sections were incubated in 4.0% paraformaldehyde for 20 minutes, followed by rinsing in PBS and treatment with 0.1% triton/0.1% sodium citrate for 2 minutes at 4°C before addition of the TUNEL reaction mixture containing TdT and fluorescein-labeled UTP as described by the manufacturer (In situ Cell Death Detection kit, Alkaline Phosphatase; Boehringer Mannheim) and incubation for 60 minutes at 37°C. The slides were then washed in PBS, and 100 µL of converter-AP (anti-fluorescein antibody linked to an alkaline phosphatase reporter; Boehringer Mannheim) was added to the sections, which were then incubated in a humid chamber for 30 minutes at 37°C. The slides were again washed in PBS, followed by a final wash in 100 mmol/L Tris (pH 8.2). The presence of alkaline phosphatase was detected with Vector Blue substrate as described by the manufacturer (Vector Laboratories). The slides were subsequently washed in distilled water and lightly counterstained with hematoxylin. Positive control slides, treated with 200 µg/mL DNase I (Promega) for 10 minutes at room temperature, and negative control slides in which the TdT was omitted from the reaction buffer were included in every experiment.

Immunohistochemistry
BrdU-containing cells were detected in the tissues by immunohistochemistry using a specific BrdU monoclonal antibody as described.25 Tissue sections were predigested with proteinase K (1 µg/mL) and 4N HCl, washed in PBS, and incubated with the anti-BrdU antibody (1/20 dilution; Dako) for 60 minutes at room temperature. The BrdU antibody was then detected with biotinylated horse anti-mouse IgG (1/400 dilution; Vector Laboratories) and the ABC-AP kit with Vector Red substrate as described by the manufacturer (Vector Laboratories), followed by counterstaining with hematoxylin. Immunohistochemistry using an {alpha}-smooth muscle actin–specific antibody was used to identify myofibroblasts in the adventitia as previously described25 (SM1, 1/800 dilution; Sigma Chemical) with biotinylated horse anti-mouse IgG and the ABC-AP kit with Vector Red.

Image Analysis
The counting of cells labeled by BrdU immunohistochemistry or the TUNEL assay was performed as previously described.25 Color video images of 280x360-µm fields were captured and digitized with a x25 objective with a Sony DXC-760MD video camera, a RasterOps 24XLTV video card, and Media Grabber software on a Macintosh Quadra 950 computer. The region of interest of each captured image was indicated by the operator and its area automatically determined by the computer after standardization with a microscale slide. The digital images were then analyzed by splitting the color images into their red and blue components for the determination of blue (hematoxylin-positive) and red (BrdU- or TUNEL-positive) cells, respectively, with the IP Lab Spectrum software package (Signal Analytics Corp). Positive and negative cells of each color were differentiated by setting threshold values and cell size discriminators that yielded the best identification of positive cells as judged by the operator. Each analysis was subjected to critical examination by a blinded operator, and cells were added or removed from the computer count to accurately reflect the number of red or blue cells in the microscope field. This analysis has been validated by comparison to manual counts alone and yields essentially the same results with a correlation of r=.892 (P=.0005). Consistency was determined throughout the computer analysis by repeated analysis of five control fields from a control BrdU-labeled vessel that showed a variation of <3%.

Data obtained from both the left anterior descending and circumflex arteries were combined. Two cross sections from each vessel {approx}3 mm apart were stained for either BrdU or TUNEL and counted. Cell proliferation was analyzed at x250 magnification in five regions in each vessel as follows: region 1, in the media adjacent to the medial tear; region 2, in the media on the side opposite the medial tear; region 3, in the adventitia adjacent to the medial tear; region 4, in the adventitia on the side opposite the medial tear; and region 5, in the intima defined as the luminal side of the external elastic lamina between the torn ends of the media. All the cells in each region were counted; this meant that depending on the size of the vessel, two to four fields at x250 magnification were captured and analyzed in each region. The percentage of proliferating cells was calculated (total number of BrdU-labeled cells/total number of hematoxylin-labeled cellsx100) in each region and averaged over the number of fields and the number of cross sections examined for each vessel. The vessel means, determined for each region of each vessel at each time point, were then used as individual data points for statistical comparison.

Only those vessels with distinct medial tears corresponding to the injury classification II to III (a clear break in the internal elastic lamina and media with a 25% to 50% gap in the media without compromising the external elastic lamina) as previously described34 were analyzed. This eliminated from the study those vessels in which the balloon catheter failed to break the internal elastic lamina and media or in which multiple fractures of the media were found. Thrombus formation was not a significant feature in the vessels, although hemorrhage into the adventitial space localized to the region of injury was seen consistently in all vessels examined.

Determination of Vascular Remodeling
To assess the effect of intravascular brachytherapy on arterial remodeling after angioplasty, we measured changes in the artery size by computer-based morphometry. Forty-three animals underwent balloon injury as described, followed by either no treatment (n=20 arteries) or exposure to either 14 Gy (n=19 arteries) or 28 Gy (n=4 arteries) intravascular irradiation. The animals were killed 14 days after injury, and the vessels were harvested, pressure-perfused with 4.0% paraformaldehyde, and embedded in paraffin. Cross sections of these vessels were sectioned and stained for elastin, and the vessel perimeter and luminal area were measured by computerized morphometry with an IBM-based system (Bioscan 2, Thomas Optical Measurement System Inc) as previously described.2 5 The distribution of myofibroblasts in the adventitia surrounding the injury site was also examined in these vessels by immunohistochemistry with an {alpha}-smooth muscle actin–specific antibody (SM1). Previous studies from our laboratory indicated that myofibroblasts containing {alpha}-smooth muscle actin develop a fibrotic scar in the adventitia surrounding the injury site.25

Statistical Analysis
Data are expressed as mean±SEM. The numbers of proliferating cells as determined by BrdU immunohistochemistry and computer-aided image analysis were compared by one-way ANOVA and the Tukey-Kramer multiple comparison test with the InStat statistical package (GraphPad Software, version 2.01). The histomorphometric measurements were compared by Student's t tests with Bonferroni correction. Statistical significance was established at the 95% confidence level (P<.05).


*    Results
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 up arrowMethods
 *Results
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Effect on Cell Proliferation
Cell proliferation was examined in control and irradiated vessels 3 and 7 days after angioplasty by BrdU immunohistochemistry, and the number of proliferating cells relative to the total number of cells was determined by computer-based image analysis (Fig 1Down). In agreement with our previous observations,25 cell proliferation in the control vessels 3 days after angioplasty was greatest in the adventitia in region 3 (30.14±2.21%, mean±SEM) compared with the medial wall even at the site of the medial tear in region 1 (14.9±2.23%). Intravascular irradiation significantly reduced the number of proliferating cells compared with controls in both the adventitia in region 3 (14 Gy, 15.5±2.6%, P<.001; 28 Gy, 4.9±1.0%, P<.001) and the medial wall in region 1 (14 Gy, 5.0±1.4%, P<.05; 28 Gy, 3.8±1.6%, P<.01) at this time (Fig 2Down). The higher, 28-Gy dose resulted in a much greater inhibition of cell proliferation in the adventitia in region 3 compared with 14 Gy (P<.05). Although not significant, there was a tendency for the higher, 28-Gy dose to produce a greater inhibition of cell proliferation than 14 Gy in the other regions analyzed as well.



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  		Figure 1. Determination of percent of BrdU-positive cells in control (open bars), 14-Gy (hatched bars), and 28-Gy (dotted bars) irradiated vessels 3 (A) and 7 (B) days after balloon overstretch injury of porcine coronary arteries. Number of BrdU-positive proliferating cells was determined by computer-assisted image analysis ("Methods") in five regions of vessel wall after angioplasty as follows: region 1, media adjacent to medial tear; region 2, media on side opposite medial tear; region 3, adventitia adjacent to medial tear; region 4, adventitia on side opposite medial tear; and region 5, intima defined as luminal side of external elastic lamina between torn ends of media (counted only on day 7 when intima was formed). Data are mean percent of BrdU-positive cells±SEM (*P<.05, **P<.01, ***P<.001 vs controls; #P<.05 vs 14 Gy).



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  		Figure 2. Distribution of BrdU-positive cells in control (A and D), 14-Gy (B and E), and 28-Gy (C and F) irradiated vessels 3 days (A through C) or 7 days (D through F) after balloon overstretch injury of porcine coronary arteries. Three days after injury, BrdU-positive proliferating cells were localized primarily in adventitia (adv) and broken end of media (m) in control vessels, whereas fewer proliferating cells were seen in irradiated vessels. Seven days after injury, intimal proliferation dominated in control vessels, whereas little proliferation was seen in vessels treated with 14 or 28 Gy. Less intimal formation is apparent in irradiated vessels, which tended to artificially increase percent of proliferating cells in region 5 of these tissues (Fig 1Up). Magnification x50.

Careful examination of individual ß-irradiated vessels on a segment-by-segment basis through the injury and irradiation site demonstrated some variability in the number of proliferating cells in adjacent vessel segments as close as 3 mm apart (Fig 3Down). For example, in one section, 38.1% of the adventitial cells were BrdU positive, whereas in a vessel segment in the next block only 3 mm away, 8.5% of the adventitial cells were proliferating. Although the total mean percentage of proliferating cells analyzed statistically across all of the segments indicated that there was a significant reduction in proliferation by radiation, it should be realized that this represents an average of irradiated segments from the same animal showing both a very high and a very low rate of proliferation. It is likely that this represents a problem with the early prototype versions of the radiation sources delivering the radiation evenly across the injured arterial segment rather than a heterogeneity of the effect of radiation on cell proliferation in these tissues. This is supported by our evaluation of the distribution of radiation emanating from the source train in these prototype catheters. The 90Sr/Y sources were left in place in a Lucite storage device after these experiments were completed. Radiation released from these sources interacted with the Lucite, producing a brown discoloration that is seen visually and shows the radiation effect to be segmental rather than continuous (Fig 3CDown).



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  		Figure 3. Variation in adventitial cell proliferation in adjacent segments of vessel treated with 14-Gy intravascular irradiation. Two adjacent segments, 3 mm apart, of a single irradiated vessel were analyzed by BrdU immunohistochemistry 3 days after injury and showed large variation in distribution of proliferating cells in region 3 (A and B, adventitia just beneath external elastic lamina and between broken ends of media). Similar variation in cell proliferation in medial wall in these segments was noted (not shown). Segmental variation in cell proliferation may be due to uneven release of radiation from device used to irradiate vessels. This is indicated by segmented appearance of discoloration of Lucite block in which catheter containing radioactive source was stored (C). Magnification, A and B, x100.

Seven days after angioplasty, no differences in cell proliferation in the media or adventitia between the irradiated and control arteries were detected, although there was an apparent reduction in the degree of intimal development at this time point (Fig 2Up). Morphometric analysis indicated that the average intimal area per cross section was significantly smaller in the 14-Gy (0.073±0.016 mm2) and 28-Gy (0.057±0.007 mm2) groups than in controls (0.144±0.014 mm2) (P<.05 and P<.01, respectively), although there was no difference in the density of intimal cells (number of hematoxylin-stained nuclei/mm2) between the three groups (Table 1Down).


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  		Table 1. Effect of Radiation on Cell Density in the Intima, Media, and Adventitia After Balloon Overstretch Injury of Porcine Coronary Arteries

Apoptosis
Apoptosis was estimated by TUNEL labeling at 3 and 7 days after balloon injury, and the number of apoptotic cells was counted by computer-based image analysis (Fig 4Down). These studies suggested that there was histochemical evidence of apoptosis in all of the injured vessels. However, there were no quantitative differences in the amount of labeling among irradiated and control vessels in any region examined on either day 3 or day 7. Three days after angioplasty, the number of TUNEL-labeled cells was greatest in region 5 along the luminal surface of the external elastic lamina, which was exposed by the tearing of the medial wall at the time of angioplasty. Morphological examination of these cells suggests that these were neutrophils that had accumulated at the injury site. Many TUNEL-labeled cells were also detected in the adventitia beneath the external elastic lamina between the broken ends of the media and in the torn ends of the medial wall (Fig 5Down). These are all sites of the greatest amount of cell proliferation at this time as determined by BrdU immunohistochemistry.



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  		Figure 4. Comparison of TUNEL labeling in control (open bars), 14-Gy (hatched bars), and 28-Gy (dotted bars) irradiated vessels 3 (A) and 7 (B) days after angioplasty determined by computer-assisted image analysis. TUNEL labeling was performed on irradiated and control vessels to estimate apoptosis in these tissues. TUNEL-positive cells in five regions (see Fig 1Up legend) of injured vessels were counted by computer-assisted image analysis (see "Methods"). There were no significant differences in percent of TUNEL-positive cells in any region examined. Data are mean percent of TUNEL-positive cells±SEM.



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  		Figure 5. Distribution of TUNEL-positive cells in control (A and B) or 14-Gy (C and D) irradiated vessels 3 days after angioplasty at site of medial break (A and C) or in media on opposite side of vessel away from break site (B and D). TUNEL-positive cells are blue and can be found distributed at broken end of media and in adventitia beneath external elastic lamina at break site (regions 1 and 3, respectively). Many TUNEL-positive cells were detected in external elastic lamina or on its luminal surface. Such cells were included in region 5 counts presented in Fig 4Up. Relatively few TUNEL-positive cells were detected in medial wall away from break site (region 2) (B and D). Magnification x50.

The cell density, calculated as the mean number of hematoxylin-stained nuclei per square millimeter, was examined in control and irradiated vessels. There were no significant differences in cell density between the control, 14-Gy, or 28-Gy arteries 3 days after injury in any portion of the media or adventitia (Table 1Up). Seven days after angioplasty, there appeared to be significantly more cells per square millimeter in region 2 (normal media on the side opposite the break) of the 28-Gy–treated vessels compared with either control or 14-Gy treatment (P<.01), but no other significant differences in cell number in the intima, media at the break site, or adventitia were found.

Effect on Remodeling
The recruitment of adventitial myofibroblasts to the injury site was assessed by immunohistochemistry for {alpha}-smooth muscle actin on days 3, 7, and 14 after injury. There was a clear difference in the extent of adventitial {alpha}-actin staining in the irradiated vessels compared with control vessels at all time points (Fig 6Down), suggesting an inhibition of adventitial fibrosis by the radiation treatment. Morphometric analysis among 18 specimens 2 weeks after balloon injury with and without radiation confirmed that there was a larger vessel perimeter in the irradiated vessels (Table 2Down).



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  		Figure 6. Effect of intravascular irradiation on recruitment of {alpha}-actin–positive myofibroblasts in adventitia 14 days after balloon overstretch injury of porcine coronary arteries. {alpha}-Actin staining (red) was performed on pressure-perfusion-fixed paraffin-embedded segments of coronary arteries from control animals (A) and animals receiving 14-Gy (B) or 28-Gy (C) intravascular irradiation at time of injury. Note reduction in {alpha}-actin staining in 14- and 28-Gy irradiated vessels as well as larger lumen of irradiated vessels vs controls. Arrows indicate breaks in medial wall; arrowheads indicate border of external elastic lamina. Magnification x50.


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  		Table 2. Effect of Intravascular Irradiation at the Time of Angioplasty on Vascular Remodeling in Porcine Coronary Arteries


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present series of experiments, we have shown that intravascular irradiation administered at the time of angioplasty effectively reduces cell proliferation 3 days after balloon injury in both the medial wall and adventitia. Radiation did not affect cell proliferation in the intima or media 7 days after angioplasty. These findings are correlated with the effect of radiation on the extent of lesion development assessed 2 weeks or 6 months after angioplasty.2 4 There were no significant differences in the percentage of TUNEL-positive cells in irradiated and control vessels in any region examined either 3 or 7 days after injury, suggesting that radiation did not increase apoptosis in the media or adventitia at these times. A positive effect of radiation treatment was seen on vascular remodeling, because the irradiated vessels had a larger vessel perimeter when measured 2 weeks after angioplasty. In addition, there was a reduction in adventitial {alpha}-smooth muscle actin staining in the irradiated vessels at 3, 7, and 14 days after angioplasty. We hypothesize that this represents a reduction in the distribution of myofibroblasts in the adventitia surrounding the injury site. Together, these results suggest that radiation treatment inhibits vascular lesion formation by reducing the first wave of cell proliferation in the media and adventitia and reducing the adventitial fibrosis, which may be the underlying cause of geometric remodeling associated with clinical restenosis.

A number of studies support the hypothesis that cell proliferation is the key contributor to vascular narrowing after angioplasty,35 36 37 38 but agents that inhibit cell proliferation do not necessarily reduce the size of the ultimate vascular lesion that develops. Proliferation in the arterial wall after angioplasty can be broken down into two components: early or first-wave proliferation occurs in the medial wall within 24 to 72 hours after injury; late or second-wave cell proliferation occurs in the intima and may continue for as long as 2 months after injury. Previous studies have shown successful reduction in first-wave proliferation (ie, by treatment with FGF antibodies), but such treatment did not reduce the size of the neointima.39 It has been hypothesized that there are multiple pathways that stimulate the growth of the neointima, such that inhibition of one pathway for growth stimulation may be replaced by another. Thus, the few cells that proliferated in the media in the presence of the FGF antibody migrated to the intima and continued to grow there, independent of FGF, giving rise to the lesion. Presumably, this ensures that proper repair mechanisms remain in place after vascular injury. In contrast, intravascular irradiation at the time of angioplasty effectively reduced first-wave proliferation 3 days after injury but had no effect on the second wave of growth, measured on day 7, in the intima. Yet this treatment successfully reduced lesion size measured not only at an early time point 2 weeks after angioplasty2 5 but 6 months later as well.2 4 This could mean one of two things: either intimal proliferation is not important in generating the final lesion or radiation may have caused an earlier termination of intimal proliferation sometime after day 7, the time point examined in this study. Given the present knowledge about the effect of radiation on cell growth in other systems, the latter hypothesis seems more tenable. Additional work will have to be done to determine whether intimal proliferation is suppressed at an earlier time point in irradiated versus nonirradiated vessels.

There was some variability in cell proliferation within the adjacent segments of an individual irradiated artery. These occurred primarily when radiation with the pure ß-emitter 90Sr/Y was applied and may be related to the lower penetration properties of the ß-radiation compared with the {gamma}-radiation combined with heterogeneous distribution of radiation emanating from the source train. It is possible that inhomogeneous packing of the radioactivity in the seeds caused some of this variability. Alternatively, the thick end caps placed on the seeds used in the first prototype version of the device resulted in spaces between the radioactive sources in the train, such that a lower dose was delivered to the tissues positioned at these junctions. Consequently, some segments of the vessel did not receive a sufficient radiation dose to inhibit cell proliferation. Alternatively, malpositioning of the source train at the injury site may also have spared a portion of the vessel from exposure to radiation. An examination of the Lucite block in which the source train was stored for several weeks suggests that the former hypothesis is correct. This has been corrected in latter versions of the radiation sources. These findings stress the importance of accuracy in positioning the source at the angioplasty site and the need to design the delivery device in such a way as to ensure the even distribution of radiation into the surrounding tissues. These results also indicate that intravascular radiation therapy using 90Sr/Y produces highly localized effects in the surrounding vessel. This may be important in that in clinical use, the radiation should not damage deep adventitial structures. Thus, a large population of resident adventitial cells will not have been exposed to high doses of radiation and should be capable of responding to maintain vessel integrity in the setting of a subsequent injury or cellular loss at that site. We hypothesize that this may reduce the potential for aneurysmal dilation of the vessel over long periods of time.

These studies suggest that it is important that a sufficient dose of radiation be delivered to the adventitial structures at the injury site to prevent arterial remodeling and restenosis. We have previously presented data suggesting that adventitial myofibroblasts may migrate into the developing neointima across the external elastic lamina and contribute to the mass of the lesion that develops after balloon overstretch injury of porcine coronary arteries.25 In addition, the proliferation of adventitial cells produces a fibrotic response around the injured vessel and probably contributes to the geometric remodeling associated with balloon injury. In the present study, we directed our dose not to the intimal surface (ideally calculated 1.5 mm from the center of the source train) but rather to a depth sufficient to ensure an adequate dose to the adventitia surrounding the injury site (2.0 mm). This reduced the proliferation of the adventitial myofibroblasts and the recruitment of {alpha}-smooth muscle actin–positive cells around the vessel. We hypothesize that the reduction of adventitial fibrosis prevented negative arterial remodeling, resulting in a larger vessel perimeter of the irradiated vessels. This may also have had the favorable secondary effect of inhibiting migration of adventitial myofibroblasts across the external elastic lamina into the neointima.

Apoptosis is also known as programmed cell death and is distinct from necrosis or other forms of cell death.40 Radiation therapy of tumors has been reported to cause signs of apoptotic death within 3 hours. If a tumor responds rapidly to a relatively low dose of radiation, it generally means that apoptosis is involved, because the process peaks at 3 to 5 hours after irradiation. Susceptibility to the induction of apoptosis may also be an important factor determining radiosensitivity, because apoptosis appears to be prominent early in radiosensitive mouse tumors and essentially absent in radioresistant tumors. It has been suggested that apoptosis is the dominant form of cell death in lymphoma cells treated with photodynamic therapy and that this process occurs more rapidly than after x-irradiation.41 42 43

Apoptosis is also a feature of human vascular pathology, including restenotic lesions.44 Coronary arterial specimens of patients with restenotic lesions retrieved via atherectomy demonstrated a high level of apoptosis, and it was suggested that apoptosis may modulate the cellularity of lesions that produce vascular obstruction. Balloon injury of rat carotid arteries also stimulates apoptosis in the medial wall and neointima in regions of greatest cell proliferation.45 These observations are similar to our finding of TUNEL-positive cells at the sites of greatest cell proliferation in the media and adventitia of the injured porcine coronary arteries. However, radiation did not increase TUNEL labeling either 3 or 7 days after injury compared with control vessels. Although this suggests that radiation may not work through an induction of apoptosis, these studies do not eliminate the possibility that radiation may induce an increase in apoptosis much earlier, within hours of treatment. Furthermore, it should be pointed out that TUNEL labeling alone is not a perfect measure of apoptosis and tends to overestimate actual rates of apoptosis in normal and atherosclerotic vessels.46 Additional studies will have to be done with additional markers of apoptosis at earlier time points to determine what proportion of the TUNEL-positive cells is beginning programmed cell death as a result of the radiation therapy.

Arterial remodeling has been described as a major contributor to the restenosis process. Several authors have suggested that vascular remodeling after angioplasty is more important than neointima formation in late luminal narrowing.20 21 Others relate vascular remodeling to the device used for the arterial dilation.47 48 In the present study, we observed an increase of the vessel perimeter and the luminal area of irradiated vessels compared with controls, with a positive relationship between the dose and vessel size. This morphometric observation is supported by the reduction in {alpha}-smooth muscle actin staining of adventitial myofibroblasts in the irradiated vessels. Therefore, we hypothesize that intravascular irradiation had a positive effect on vascular remodeling because of the reduction in the recruitment of adventitial myofibroblasts in the adventitia at the injury site that prevented constriction. Intravascular irradiation may be a substitute for intracoronary stenting if chronic vascular constriction after angioplasty is diminished by radiation.

These preliminary findings suggest that intracoronary radiation prevents vascular lesion formation after coronary intervention by reducing cell proliferation in the media and adventitia 3 days after injury. In addition, intravascular radiation may contribute to positive remodeling and reduction of vessel constriction by reduction of {alpha}-smooth muscle actin–containing cells in the adventitia. Additional work is needed to determine the effect of radiation on cell migration from the media or adventitia to the neointima and to determine the effects of radiation on normal and atherosclerotic vessels.


*    Selected Abbreviations and Acronyms
 
BrdU = bromodeoxyuridine
FGF = fibroblast growth factor
TdT = terminal deoxynucleotidyl transferase
TUNEL = TdT-mediated dUTP nick-end labeling


*    Acknowledgments
 
This work was supported by NIH grant HL-47838-04 (Dr Wilcox) and a grant from the Gruentzig Center for Interventional Cardiology. The authors would like to thank Cheryl Ross for her expert technical assistance in performing the immunohistochemistry experiments.

Received February 12, 1997; revision received April 2, 1997; accepted April 13, 1997.


*    References
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