Intracoronary Low-Dose β-Irradiation Inhibits Neointima Formation After Coronary Artery Balloon Injury in the Swine Restenosis Model

Restenosis after successful PTCA is the major limitation to long-term success of the procedure.¹ It is mediated largely by proliferation and extracellular matrix synthesis by modified SMC that have migrated in response to mechanical stretch and disruption at the site of the balloon angioplasty as well as overall vascular remodeling.² The development of the neointimal component of the restenosis lesion is the result of a healing process initiated by vascular injury, predominantly to the less-atheromatous aspect of the dilated artery.² Vascular smooth muscle does not normally display actively dividing cell populations. However, mechanical injury or other stimuli can induce a response of SMC characterized by migration, proliferation, and matrix synthesis.²³⁴ In this situation, ionizing radiation may effectively inhibit neointima formation presumably by killing the more rapidly dividing SMC.⁵⁶⁷⁸

The efficacy of low-dose γ-irradiation to inhibit neointima formation after injury has been demonstrated in several animal models: Shimotakahara and Mayberg⁹ have shown reduction of neointimal hyperplasia in injured rat carotid arteries using external radiation. Wiederman et al,¹⁰ Mazur et al,¹¹ and Waksman et al¹² have demonstrated the efficacy of endovascular low-dose γ-irradiation using ¹⁹²Ir to inhibit neointima formation after balloon overstretch injury in pig coronaries. With 6-months’ follow-up, the durability of the beneficial effect in the treated group without evidence of excess fibrosis was demonstrated in two studies.¹²¹³ The only work in humans using radiation after balloon angioplasty was carried out in peripheral arteries.¹⁴ These studies were all carried out using ¹⁹²Ir. The clinical study and one of the animal studies used a remote controlled high dose-rate afterloader (Nucleotron) to deliver the source to the treatment site; the other animal studies used a ¹⁹²Ir ribbon delivered manually to the treatment site. This isotope, although suitable for use in animal studies and peripheral arteries, has serious limitations for use in human coronaries because it is deeply penetrating and not effectively shielded by standard lead aprons. This would require the cardiology staff to remove themselves from the patient’s proximity during treatment.

In addition the highest activity (135 mCi), commercially available (Best Ind.) 3-cm ribbon, which is hand-delivered, requires a treatment time of approximately 30 minutes for 14 Gy at 2 mm, during which time the delivery catheter is continuously present in the coronary artery. One means of reducing the treatment time is to use a high-dose rate afterloader in which a very high activity (10 Ci) ¹⁹²Ir source is delivered under remote control to the treatment site. These treatment machines usually are only found in radiation oncology departments and require special shielding beyond what is present in most cardiac catheterization suites. Although the treatment time might be reduced, the potential need to add shielding to the catheterization suite or to transfer the patient to a radiation oncology facility for treatment presents significant problems.

As a result of the above concerns, we initiated development of a new treatment device for endovascular irradiation with the use of ⁹⁰Sr/Y, a pure β-emitter as the radioactive source. This isotope has favorable characteristics in terms of permitting delivery of dose to the required depth in tissue (2 to 3 mm), with little dose measured beyond 1 centimeter from the source. Novoste Corporation was able to supply a delivery system and source train with this isotope that met our specifications for profile (delivery within a 4.5F catheter) and treatment time (less than 4 minutes).

The purposes of this study were to (1) test whether the β-radiation from this isotope could inhibit neointima formation after balloon catheter injury, (2) examine the dose-response relation using this source, and (3) estimate patient whole-body and operator exposure from clinical use of this source.

The swine model of restenosis based on oversized balloon catheter inflation in the coronary arteries of normal juvenile pigs was used to test the primary hypothesis regarding neointima formation.^1516171819

Methods

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.

Experimental Protocol

The model of overstretch injury has been described previously.^1516171819 Forty-two female domestic pigs (Sus scrofa, 19.2 to 29.7 kg) were given aspirin (325 mg) 1 day before and the day of the procedure. They were sedated with a combination of tiletamine HCl (10 mg/kg) and atropine (0.6 mg/kg) by intramuscular injection. An intravenous line was established, and the animals were intubated. The pigs were ventilated with oxygen (2 L/min), nitrous oxide (2 L/min), and isoflurane 1% (1.5 L/min) with the use of a Harvard respirator. Adequate anesthesia was confirmed by the absence of a limb withdrawal reflex. Limb-lead ECG (Honeywell E for M) was monitored throughout the procedure.

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 was recorded by cineangiography (Phillips Cardiodiagnost).

Coronary overstretch injury was performed with a 3.5-mm angioplasty balloon, which was positioned in the proximal segments of the LAD and LCx coronary arteries, 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 then was 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. Over a flexible 0.014-inch wire, a 4.5F delivery catheter (Novoste Corp) was introduced to the injury site of the assigned artery and positioned at the angioplasty site, the guide wire was withdrawn, and a 2.5-cm-length train with 5 seeds of ⁹⁰Sr/Y was positioned at the site of injury in the target vessel using cinefluoroscopic visualization within the delivery catheter. It was left in place for a period sufficient to deliver the assigned dose (7 Gy , 14 Gy, 28 Gy, or 56 Gy) to a depth of 2 mm (90 to 720 seconds). The delivery catheter without the radioactive source was placed in the control injured artery in the same manner as for the treated artery. After irradiation the delivery and guiding catheters were removed and the femoral cutdown was repaired. Nitroglycerin ointment (1 inch) was administered topically and the animals were returned to routine care.

The pigs were killed 14 days after the initial injury. The animals were heparinized, a lethal dose of barbiturate was given, the chest was opened, and the heart rapidly excised. The left coronary system was perfusion-fixed at 100 to 110 mm Hg driving pressure with buffered 10% formaldehyde for 15 minutes, the heart was stored overnight in the same fixative, and the injured coronary artery segments were prepared for histopathological analysis.

To determine the long-term consequences of β-radiation on the coronary arteries and to test for any evidence of arterial injury from the irradiation, an additional three pigs (mature female Hanford miniature swine, Charles River Laboratories) were treated in similar fashion and received 7 and 14 Gy in either the LAD or LCx, with the contralateral artery treated by angioplasty only and serving as a control. These pigs were killed 6 months after the procedure and the tissues processed as described above.

Radiation Dosimetry

The activity of each seed and the total source train was determined by the manufacturer with a National Institute of Standards and Technology traceable standard. The absorbed dose distribution and dose rate around the 2.5-cm ⁹⁰Sr/Y line source was calculated with the use of the Monte Carlo electron transport code ITS.²⁰ The β energy spectrum of ⁹⁰Sr/Y was obtained from Cross et al.²¹ As part of the verification of ITS, a determination of the dose distribution around the ¹⁹²Ir line source from our previous study was carried out and compared with the calculations done with the CMS Treatment Planning System. This was found to agree within 5% of the results obtained from this FDA-approved commercial system. The ITS results of the radial absorbed dose distributions at the center of the ⁹⁰Sr/Y and ¹⁹²Ir sources are shown in Fig 1. Because of the inherent problems associated with measuring the dose from β sources at a finite point in close proximity to the source, no confirmatory in vivo dosimetry was carried out. There was no self-centering of the catheter within the arterial lumen nor was there any attempt made to account for curvature of the artery and the radiation line source. Effective dose rates, shown in Table 1, were measured at the chest surface and at approximately 1-m distance while the pig was undergoing endovascular β-irradiation, with a thin-walled ionization chamber (Bicron RSO-5).

Tissue Harvest, Preparation, and Analysis

The injured segments of the LAD and LCx were located with the guidance of the coronary angiograms and then were dissected free from the heart. Serial 2- to 3-mm transverse segments were processed and embedded in paraffin. Cross sections (4 μm) were stained with HE or VVG. HE-stained sections were examined by an experienced observer blinded to the treatment group. Each specimen was evaluated for the presence of neointima formation, luminal encroachment, medial dissection, alteration of the internal and external elastic lamina, and morphological appearance of the cells within the media, adventitia, and neointima. Sections were also evaluated for the presence of intraluminal thrombus, intramural hemorrhage, and inflammatory cell infiltrate.

Morphometric analysis was performed on each segment with evidence of medial fracture, 2 to 6 in each artery (Table 2). Intramural deposits of fibrin as well as larger organized mural thrombi were included in area measurements of neointima. The histopathological features were measured with the use of a computerized IBM-based system (Bioscan 2, Thomas Optical Measurement System Inc). VVG-stained sections were magnified ×26, digitized, and stored in a frame-grabber board. The maximal intimal thickness (MIT) was determined by a radial line drawn from the lumen to the external lamina at the point of greatest tissue growth. The arc length of the medial fracture (FL), traced through the neointima from one dissected medial end to the other, was used as a measure of the extent of injury. Area measurements were obtained by tracing the lumen perimeter (luminal area, mm²), neointima perimeter (intimal area, IA; mm², defined by the borders of the internal elastic lamina, lumen, media, and external elastic lamina), and external elastic lamina (vessel area, mm²). The ratio of IA to FL (IA/FL) was calculated to correct for the extent of injury. The MIT and the absolute IA reflect the new tissue formation after vessel injury and serve as reliable indicators of the capacity for a potential therapy to inhibit neointima formation after injury. The IA/FL is somewhat more precise because it provides an adjustment for the extent of medial fracture, to which IA is directly correlated.¹⁹ Measurements were made by two experienced observers blinded to the treatment groups and were found to vary less than 10%.

In two arteries the effect of β-irradiation with 14 Gy on reendothelialization of the luminal surface at 14 days after injury was examined by scanning electron microscopy. These samples were compared with one control artery in the same pig; they were fixed by perfusion with buffered 2.5% glutaraldehyde at 100 mm Hg pressure and prepared for conventional secondary electron imaging using by postfixation in 1% OsO₄, dehydration in graded ethanol series to 100%, critical point drying from liquid CO₂, and sputter coating with 15-nm Au/Pd alloy. They were imaged in a Topcon DS-130 equipped with a LaB₆ emitter, and photographic documentations of the luminal surfaces were recorded.

Statistical Analysis

Data are expressed as mean±SD. A one-way ANOVA was used to test for an overall treatment effect, with follow-up t tests using the Bonferroni correction to analyze specific group differences. Linear regression analysis was used to test for a dose-response effect. Significance was established at the 95% confidence level (P<.05) except for Bonferroni-corrected t tests (P<{.05/4}=.0125).

Results

Group Characteristics

Twenty-two animals underwent interventions on 44 coronary arteries. There were no significant differences in body weight between groups at the time of balloon injury (Table 2). Angiographic arterial diameter was similar between groups (2.64±0.16 mm), and although balloon to artery ratio was therefore the same between groups, the extent of medial injury (FL) was significantly higher in the 14 and 28 Gy group compared with controls (3.45±1.77 and 4.57±1.5 mm versus 2.21±1.1 mm, P<.0001, Table 2).

Histological Analysis

HE- and VVG-stained sections of all arterial segments were examined. In injured segments of both control and β-irradiated arteries there was a variable degree of rupture of the tunica media resulting in a vessel wall defect. Control arteries showed replacement of the medial defect, with a substantial neointima consisting mostly of stellate and spindle-shaped cells in a loose extracellular matrix. The majority of neointimal cells in this model show positive immunostaining for α-actin and have characteristics of modified synthetic SMC or myofibroblasts by ultrastructural analysis.¹⁶¹⁷ Frequently, sections showed adventitial reaction; occasional perivascular and rare intramyocardial hemorrhages also were seen. A moderate number of sections showed hematomas in the media-adventitia dissection planes. In most sections there was perivascular edema and mild to moderate round cell infiltration.

The neointima from β-irradiated arteries was markedly smaller in size than the controls, with some sections showing a virtual absence of neointima formation (especially 28 and 56 Gy); when present, the cells of the neointima were morphologically similar to controls. In a moderate number of samples there were mural fibrin deposits. In the majority of the samples there was complete coverage of the luminal surface by a monolayer of endothelium-like cells. However, in the arteries treated with 28 Gy and 56 Gy some regions revealed no luminal cell lining; nevertheless, none of these sections showed evidence of thrombosis. Furthermore, we did not observe any significant necrosis or nuclear pyknosis in the media or adventitia in either the control or the radiation treatment groups. The perivascular nerve fibers, adipose tissue, and adjacent myocardium appeared normal. The overall histological findings in arteries treated with 56 Gy and 28 Gy were similar. Low magnification micrographs of VVG-stained sections from injured coronary arteries of pigs in five of treatment groups (control, 7, 14, 28, and 56 Gy) are shown in Fig 2, and higher-magnification images of HE-stained sections in Fig 3.

Coronary arteries from miniature pigs killed 6 months after balloon injury and irradiation were examined by the histopathologist (M.B.G.). There was no evidence of fibrosis of the arterial wall, perivascular tissue, or adjacent myocardium in the irradiated segments that differed from the changes seen in arteries treated by balloon injury only. In addition, coronary angiography performed just before tissue harvest showed no evidence of either significant stenosis or aneurysm formation.

Morphometric Analysis

The effect of β-radiation dose on four descriptors of the vessel response to injury is shown in Table 3. By ANOVA, a significant treatment effect of irradiation on MIT (F=48.2, P<.0001), IA (F=982.67, P<.0001), and IA/FL (F=26.85, P<.0001) was observed (Table 2 and Fig 3). Post hoc analysis by t test showed significant reductions comparing each treatment group with the control group for all three dependent variables. In addition, there was an inverse relation between the dose (control, 7, 14, 28, and 56 Gy) and IA/FL ratio (m=−0.0028, P<.0001, r=−.75).

Scanning Electron Microscopy

Representative low-magnification images of control and 14 Gy–irradiated, balloon-injured arteries are shown in Fig 4. Arteries from both groups displayed a largely confluent lining of endothelium-like cells showing occasional leukocyte adherence with apparent spreading and endothelial diapedesis as well as rare small (approximately 200 to 500 μm²) nonreendothelialized areas. No regions of significant mural thrombosis were seen in either control or irradiated vessels.

Discussion

Radiation Treatment System

The use of endovascular ionizing radiation to inhibit the neointima formation response to arterial injury is relatively recent. Intracoronary irradiation in the swine model of restenosis has been reported previously, only with ¹⁹²Ir either by high dose-rate afterloader technique or by a lower-activity, hand delivered ribbon.¹⁰¹¹¹²

This study demonstrates, to our knowledge, the first successful use of an endovascular β-emitter and shows the efficacy of this treatment for inhibiting neointima formation. Similar to our previous study using the γ-emitter ¹⁹²Ir, the present findings demonstrate that both 7 and 14 Gy at 2-mm depth had a significant effect in decreasing neointima formation compared with control at 2 weeks after injury. There appeared to be evidence of further reduction of neointima formation with 28 Gy but no additional benefit at 56 Gy.

Pure β-emitters such as ⁹⁰Sr/Y have a distinct advantage over the use of γ-emitters in that β particles have a limited penetration in tissue and deliver significantly less dose beyond the prescription point than do γ-emitters.¹⁴²¹ Effective skin doses to the patient of between 69 and 124 Sv, from cinefluoroscopy during an average PTCA procedure, have been reported in the medical literature. This translates to an effective total body exposure of 13 to 24 Sv.²²²³ We calculated an effective total body dose of 0.19 mSv from the brachytherapy intervention with the ⁹⁰Sr/Y source to treat one artery with 14 Gy at 2-mm depth in tissue. In contrast to this, one would expect an effective total body dose of approximately 1000 mSv with the use of ¹⁹²Ir. Therefore, the total body dose from the ⁹⁰Sr/Y is significantly less than that seen with ¹⁹²Ir and would constitute only 0.001% of the patient’s total body radiation dose from the PTCA procedure.

Using a high dose-rate afterloader with ¹⁹²Ir in patients would require the removal of the healthcare workers from the room during treatment and the installation of special shielding in the walls of the catheterization laboratory. The use of a manually delivered source might not require the same amount of additional shielding, but the treatment times are too long to be practical (approximately 30 minutes). In contrast to this, using ⁹⁰Sr/Y, we measured a dose of only 0.009 mSv, which would be received by the cardiologist if he remained at the patient’s bedside during the delivery of 14 Gy to the arterial wall at 2-mm depth (Table 3). If the cardiologist did five procedures per day, 50 weeks per year with this device, he would only receive 12.5 mSv, which is well below the 5000 mSv allowed to radiation workers per year and much less than the exposure from routine fluoroscopy.²⁴

The use of radioactive stents, or stents coated with a radioactive isotope, has been proposed by two groups.²⁵²⁶ Coating the stent with a β-emitting isotope would seem to be the most desirable approach of the two, but concerns regarding potential leaching of the radioactive material from the metallic stent and possible thrombosis on the stent wire caused by delayed reendothelialization should be addressed.

Radiobiological Effect

Substantial effects of irradiation on all morphometric descriptors of neointima formation were observed. Our data indicate that there was a linear dose-response effect from single doses of 7 to 28 Gy using the ⁹⁰Sr/Y source. There was virtual eradication of neointima formation at 2 weeks with 28 Gy. Consequently, there was no further inhibition of neointima formation at 56 Gy.

The absence of a necrotizing effect on the arterial tissue and the preservation of normal morphology in perivascular tissues and adjacent myocardium in this study suggest that these tissues are not damaged, at least acutely, by even very high doses of β-radiation. We know from our γ studies that doses up to 14 Gy are unlikely to cause any injury to the vessel from the radiation treatment. Our present results from scanning electron microscopic analysis demonstrate that the inhibitory effect of 14 Gy on neointima formation does not retard reendothelialization. At 28 and 56 Gy in paraffin-embedded tissue sections there was no evidence of recent mural thrombosis, and for the most part there was recovery of a periluminal cell layer in the medial defect. Furthermore, the results in three miniature pigs at 6 months provide evidence that there is no chronic injury to the arterial wall or surrounding tissues including adjacent myocardium, which is induced by β-radiation at doses of 7 and 14 Gy. It remains to be seen in ongoing chronic β studies whether higher doses (28 and 56 Gy) also will be free of any adverse effect from radiation treatment.

The histological and histomorphometric analyses of coronary arteries treated with the same doses of β- and γ-radiation demonstrate overall similarity.¹⁰ It can be surmised therefore that the biological effect on neointima formation is dependent on the absorbed dose and not on the type of isotope or the variation in treatment times seen with the different approaches.

Study Limitations

The pig restenosis model has several limitations. It is primarily a neointima formation model, and although the normal pig coronary resembles the normal human coronary, there is no atherosclerotic disease as in the human PTCA setting. Furthermore, there is considerable variability in the amount of injury that influences the proliferative response. Nonetheless, in the present study we demonstrated marked inhibition of neointima formation even in severely injured vessels with the use of β-radiation treatment.

This study does not provide long-term histomorphometric data. However, since the results of the present β study were similar to the γ study at 2 weeks, and the 6-month study with γ-radiation demonstrated the durability of the effect, we anticipate that similar morphometric results will be found with complete long term follow-up using ⁹⁰Sr/Y in the 7 and 14 Gy groups.

Conclusions

Intracoronary irradiation with ⁹⁰Sr/Y is feasible and compatible with use in a standard catheterization laboratory after coronary intervention. Radiation doses between 7 and 56 Gy effectively inhibited neointima formation at 2 weeks after coronary balloon injury. No dose response was seen beyond 28 Gy. Histological and histomorphometric results are similar between γ-irradiated (¹⁹²Ir) and β-irradiated (⁹⁰Sr/Y) arteries. However, the use of β-irradiation for this application would appear to have distinct advantages in terms of both safety and practicality for patients and healthcare workers.

Selected Abbreviations and Acronyms

From Andreas Gruentzig Cardiovascular Center, Division of Cardiology, Department of Medicine (R.W., K.A.R., G.D.C., S.B.K.); Department of Radiation Oncology (I.R.C.); Department of Pathology, Emory University School of Medicine (M.B.G.); Health Physics Program, Georgia Institute of Technology (C.W.); and Novoste Corporation, (R.A.H.), Atlanta, Ga.

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Footnotes