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Intracoronary Low-Dose Ionizing Irradiation (β or γ) for Prevention of Restenosis: Could It Succeed Where Pharmacotherapy Failed?
Intracoronary Low-Dose Ionizing Irradiation (b or g) for Prevention of Restenosis: Could It Succeed Where Pharmacotherapy Failed? Michael B. Gravanis, MD,* and Ron Waksman, MD† * Anatomical Pathology, Department of Pathology and Laboratory Medicine; and Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia
† Interventional
11 Although the precise pathogenesis of restenosis after percutaneous transluminal coronary angioplasty (PTCA) remains somewhat elusive, our understanding about the reparative phenomena at the site of dilatation has been significantly improved in recent years. Thus, restenosis appears to be the result of migration, proliferation, and excessive matrix formation by smooth muscle cells plus vascular wall remodeling leading to chronic recoil (constriction). Proposed pharmacotherapies to prevent restenosis have been ineffective in humans, in spite of a relative success in certain experimental animals. The rationale for low-dose irradiation (either b or g) in order to prevent restenosis is based on the known ability of ionizing irradiation to arrest cell division and, therefore, to reduce the number of clonal progenitors in situations like angioplasty. © 1997 by Elsevier Science Inc. Cardiovasc Pathol 1997;6:11–21
The major limitation of percutaneous transluminal coronary angioplasty (PTCA) is the occurrence of restenosis in 25% to 45% of patients within the first 6 months after the procedure. Experimental and clinical data indicate that restenosis results partly from recoil and vessel wall remodeling, but also from migration, proliferation, and excessive matrix deposition by smooth muscle cells after vascular injury (balloon dilation). In addition, changes in vessel redox state may have important consequences on remodeling by altering the nature of the matrix produced by smooth muscle cells and fibroblasts. Our recent study strongly supports the concept that vessel redox state is an important determinant of response to injury (1). Despite intensive research for almost 3 decades, the precise pathogenesis of the restenotic process remains elusive and proposed pharmacotherapies ineffective. The use of intracoronary stents for prevention of restenosis has been increasing in recent years. This increased popularity of stents is based on the assumption that these metallic
Manuscript received March 5, 1996; accepted May 31, 1996. Address for reprints: Dr. Michael B. Gravanis, Department of Pathology and Laboratory Medicine, Emory University Hospital, 1364 Clifton Road NE, Atlanta, GA 30322. Cardiovascular Pathology Vol. 6, No. 1, January/February 1997:11–21 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
scaffoldings will reduce restenosis after angioplasty by achieving a larger initial lumen size and reducing both the acute and chronic recoil phenomena. However, stents are known to be associated with more pronounced neointimal formation than balloon angioplasty. This has been demonstrated in animal models (2) and more recently in a clinical study using intravascular ultrasound (3). It is worth mentioning, though, that 1-year clinical follow-up of patients included in the Stress and Benestent randomized studies, comparing elective stent implantation with balloon angioplasty in patients with stable angina in a de novo coronary artery lesion, showed significant reduction in restenosis rate and requirement for repeat coronary intervention after stent placement (4,5). Attempts to improve the metallic stent surface by introducing polymer-coated devices in order to reduce thrombogenicity and provide local drug delivery have met with limited success because of problems with chronic tissue compatability (6). However, recent studies with heparincoated stents have reported reduction of the restenosis rate to 13% without subacute thrombosis (7). Restenosis after balloon angioplasty involves a complex interplay among endothelial cells, platelets, vascular smooth
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muscle cells, inflammatory cells, and blood proteins (8). Similar phenomena are also involved after stent placement, although the barotrauma to the arterial wall by the intravascular prosthesis is considered greater (3). According to some investigators, mural thrombosis may play a significant role in restenosis (9), although failure of aspirin, heparin, and the low molecular weight heparin to reduce restenosis in humans has, in most part, refuted this hypothesis. Furthermore, histologic evidence of preceding mural thrombosis in restenotic lesions is lacking (8).
Effects of Ionizing Radiation on Mammalian Cells Ionizing radiation may affect several cellular molecules, although its most damaging effects are seen on the nucleus, resulting in single- or double-strand breaks of the purine and pyrimidine bases of DNA (10). There is significant variation in regard to the effect of ionizing radiation on mammalian cells, depending on the amount of energy delivered, dose rate, and fractionation. Thus, radiation energy may produce no detectable results; may induce latent DNA changes which manifest in subsequent generations (oncogenesis); inflict DNA damage that may allow somatic life, but no cellular division (reproductive death); or instant somatic death (11). Factors pertaining to cell status may also determine the radiation effect. The response to radiation energy of hypoxic cells is less than that of oxygenated ones. The stage of the reproductive cycle of the cell is also an important determinant. Actively divided cells are more radioresponsive. Finally, the ability of the cell to repair radiation damage determines its survival (12).
Radiation Effects on Blood Vessels Similar to other mammalian tissues, the effects of radiation energy on blood vessels can be characterized as acute and chronic. Furthermore, the radiation effect depends on the size of the vessel, location, dose volume, dose rate factors, and preexisting vascular disease. Whereas all levels of the vascular system may be affected by ionizing radiation small vessels such as arterioles, capillaries and sinusoids are more radiosensitive.
Acute Phase Effects After clinically used doses of irradiation (for malignancies), there is increased capillary permeability manifested as interstitial edema, capillary and arteriolar dilation, and endothelial cell swelling and degeneration. In some tissues, such as the heart and skin, acute lesions may be associated with an inflammatory infiltrate (11).
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Delayed or Chronic Vascular Changes During this phase, the capillary wall basement membrane appears thickened, and the leakage of plasma elements is decreased. Some capillaries may eventually thrombose, whereas in others, the endothelium may recover and the patency of the vessel be maintained. In vessels of small and medium caliber, the morphologic changes are primarily seen in the intima. These include swelling and vacuolization of endothelial cells and myointimal proliferation with luminal narrowing and even occlusion. Lipid-laden macrophages may be seen in the intima a few days after irradiation, and they may persist for several years. Some reports have emphasized that in medium-sized arteries, radiation may induce multiplication of the internal or external elastic membranes, fragmentation or focal loss of medial elastic lamellae, medial necrosis with subsequent fibrosis, and obliteration of vasa vasorum. Although large artery damage is less frequent, coronary artery disease is a delayed manifestation that can produce ischemic myocardial lesions (12). One rather interesting feature of radiation vasculopathy is that it is not associated with leukocytic reaction.
Therapeutic Applications of Ionizing Irradiation Based on the inhibitory effect on cellular proliferation, ionizing radiation has been used widely in the treatment of neoplastic conditions. Furthermore, the use of low doses of ionizing radiation is utilized for benign conditions: superficial x-rays after surgery has been effective in the prevention of hypertrophic scarring, inhibits keloid formation, and heterotopic ossification after total hip arthroplasty. Experimental in vitro studies have shown that ionizing radiation inhibits thymidine uptake and collagen synthesis in cultured fibroblasts (13,14). 90Strontium, a pure b-emitter with a half-life of 28 years, is in clinical use for the treatment of recurrent pterygium.
Ionizing Irradiation: Experimental Models The rationale in all recent experimental studies to apply low-dose radiation in order to prevent restenosis is based on the fact that the latter is mediated at least in part by an uncontrolled proliferation of smooth muscle cells and abundant extracellular matrix synthesis, initiated by vascular injury (dilation). Thus, because radiation particularly affects proliferating tissues by arresting cell division, it could reduce the number of cells in situations such as angioplasty. Our experimental work with low-dose radiation was carried out on the pig model we developed in 1992 (2). In this model, an oversized balloon inflation in the coronary arter-
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ies of normolipemic juvenile pigs induces an acute injury to the tunica media, a disruption in the form of a crater. The subsequent healing process, analogous to hypertrophic scarring, has a close resemblance to the histopathologic responses of human coronary arteries after angioplasty as has been reported in animal experiments and autopsy studies (8,15). In our first experiment, after an overstretch injury to the pig coronary arteries (left anterior descending and circumflex) was performed, a 3-cm ribbon of 192Ir was positioned at the site of only one vessel at the angioplasty site and was left in place for a period sufficient to deliver the assigned dose to a depth of 2 mm (8 to 38 minutes, depending on dose and source activity), whereas the contralateral injured artery remained as the control (16). The prescribed doses in this experiment were 3, 7, or 14 Gy. The durability and the safety of the radiation treatment was studied in miniswines that underwent similar injury and radiation therapy but were sacrificed after 6 months. The objective of our next study was to determine whether the use of a b-emitting radioisotope would have similar effects with g-radiation on intimal proliferation and to establish the dose-response relations. The same normolipemic swine model was used in this study (17). A 2.5-cm length train with seeds of encapsulated 90Strontium/ 90Yttrium source (a pure b-emitter) was left in place to deliver one of four doses: 7, 14, 28, or 56 Gy to a depth of 2 mm in 90 to 720 seconds. Pure b-emitters such as 90Sr/Y have a distinct advantage over the use of g-emitters in that particles with high activity have a limited penetration in tissue and deliver significantly less energy beyond the desired point than do g-emitters
Figure 1. Control nonirradiated coronary artery 2 weeks after oversized balloon dilation. Note segmental medial disruption, neointimal proliferation (N) and destruction of the internal elastica (arrows). (Verhoeff’s van Gieson elastic stain, 390.)
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(18). Thus, the use of b-irradiation for coronary application would appear to have distinct advantages in terms of both short treatment time and radiation exposure practicality for patients and healthcare workers.
Histopathologic and Morphometric Findings after g and b Irradiation The histologic and morphometric analyses of swine coronary arteries treated with the same doses of b- and g-radiation demonstrated overall similarity. Thus, one may surmise that the biological effect on neointimal formation depends on the absorbed dose and not on the specific type of isotope or the variation in treatment times seen with the different approaches. Control arteries revealed at about 2 weeks the filling of the medial defect created by the oversized balloon, by stellate and spindle-shaped cells in a loose extracellular matrix (Figure 1). The majority of these cells showed positive immunostaining for a-actin and had the structural characteristics of modified synthetic SMC (8). In contrast, the neointima formation at the site of the medial defect (crater) was minimal in irradiated arteries (Figure 2). Furthermore, in arteries that received high doses of irradiation (28 and 56 Gy), there was virtually no neointima formation. Whereas in treated arteries that received up to 14 Gy, the luminal surface was covered by a monolayer of endothelial cells by 14 days, in arteries that received high doses (28 and 56 Gy), the endothelial lining was discontinuous; nevertheless, none of these sections, except one (56 Gy) (Figure 3) showed evidence of thrombosis (16,17).
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Figure 2. (A) Irradiated coronary artery 2 weeks after treatment (14 Gy). The neointimal proliferation (arrows) at the site of the crater is very modest. (Verhoeff’s van Gieson elastic stain, 390.) (B) Irradiated coronary artery 2 weeks after treatment (28 Gy). Note that there is no intimal proliferation at the site of the crater (C). (Verhoeff’s van Gieson elastic stain, 390.)
We did not observe nuclear pyknosis or necrosis in the media or adventitia of the irradiated group; and the perivascular nerve fibers, ganglia, adipose tissue, and adjacent myocardium appeared normal. However, in animals killed 2 days after irradiation, there was periadventitial edema and a modest round cell infiltrate (Figure 4). In pigs killed 6 months after balloon injury and irradiation, there was no evidence of fibrosis of the media, perivascular tissues, or adjacent myocardium. Coronary angiography performed just before tissue was harvested showed no evidence of either significant stenosis or aneurysm formation.
Morphometric studies revealed that the ratio of intimal area (IA) to medial fracture length (FL 5 arch between fractured ends of media) is inversely correlated with increasing radiation dose [IA/FL ratio (m 5 0.0028, p , 0.0001, r 5 0.75)]. Analysis of long-term specimens showed reduction of IA/FL in the irradiated arteries with 7 Gy (0.3, p 5 0.009) and 14 Gy (0.31, p 5 0.001) compared with control arteries (0.50). Scanning electron microscopic studies demonstrated that radiation doses up to 14 Gy do not retard reendothelializa-
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Figure 3. Irradiated coronary artery (56 Gy) 2 days after treatment. Note the large intramural thrombus (T) and the dissection between media and adventitia (D). (Verhoeff’s van Gieson elastic stain, 390.)
tion. These studies also revealed that 2 weeks after irradiation (14 Gy), there were only rare small areas of incomplete reendothelialization, as well as occasional adherent leukocytes. However, similar findings were observed with approximately equal frequency in control arteries. In the study by Schwartz and associates, external x-ray radiation was administered to the site of overexpanded, percutaneously delivered tantalum wire coils in pigs. Their findings, however, revealed that g-irradiation (delivered variable doses and times) did not inhibit the proliferative intima response, but rather accentuated it (19).
Figure 4. Irradiated coronary artery (14 Gy) 2 days after treatment. Note the adventitial and periadventitial edema adjacent to the crater (C), myocardial edema (M) and a diffuse mononuclear infiltrate. (Hematoxylin-eosin stain, 390.)
The study by Wiedermann et al. (20) used the swine model of balloon overinflation and iridium-192, a g-radiation emitter. Their results indicate a 71.4% reduction in neointimal area and a 63.0% reduction in percent area stenosis in the irradiated arteries in comparison to controls. Although the authors did not observe any radiation-associated changes in the adventitia, epicardial fat, or myocardium, they detected a diffuse interstitial fibrosis in the media 30 days after the oversized balloon angioplasty. On the basis of these findings, they hypothesized that the inhibitory effect of radiation may be due to medial cell death, and
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therefore, a decrease in the proliferative potential of the remaining mature SMC precursors. Furthermore, they suggested that the media fibrosis may provide a diffusion barrier for mediators of chemokinesis, chemotaxis, and cellular proliferation. In order to study the short- and long-term effects of intracoronary irradiation on endothelial and smooth muscle function and to assess functional recovery and identify persistent vasomotor abnormalities acutely and at 32 days, Wiedermann et al. (21) used intravascular ultrasound. According to the report, irradiated sites acutely displayed vasoconstriction to acetylcholine, with loss of smooth muscle respond to nitroglycerin. A restudy at 32 days showed restoration of normal vasodilatory response to acetylcholine, but persistent loss of response to nitroglycerin. These findings may imply endothelial dysfunction due to acute radiation injury or perhaps inability of vascular smooth muscle to respond to NO shortly after irradiation. The fact, however, that there is no nitroglycerin responsiveness acutely after radiation favors the second mechanism. The authors concluded that whereas intracoronary irradiation does not produce lasting functional or morphologic impairment of the endothelium, the injury to the smooth muscle is lasting and thus responsible for decreased neointimal formation (fibrosis). However, it is possible that this decreased radiosensitivity may not be present in human coronary endothelial cells.
Long-Term Studies In our experimental studies, media fibrosis, either focal or diffuse, was not noted even in animals killed at 6 months. This finding of ours, plus the absence of any detectable histologic changes in the vasa vasorum, is rather reassuring in that the integrity of the media will not be compromised after low-dose radiation of the coronary arteries (l6,17). A recent report by Wiedermann et al. (22) in which the pigs were killed at 6 months after iridium-192 radiation reconfirmed the beneficial effect on neointimal proliferation by this technique and answered several questions raised in their previous report. Additional morphologic findings from this study included scattered foam cells and mononuclear cells in the neointimal proliferation and mild adventitial fibrosis in one-third of the irradiated animals. Our long-term (6-month) follow-up studies did not reveal any quantitative differences in the degree of adventitial fibrosis at 2 weeks and 6 months, between irradiated animals and controls. Furthermore, adventitial fibrosis observed in our experiments was not circumferential, but rather focal at the vicinity of the medial destruction (crater) from the oversized balloon injury. This rather encouraging finding in our studies may have important implications in regard to the late, chronic vascular recoil. However, the amount of morphometrically estimated neointima in the irradiated arteries at 6 months was greater than that observed at 2 weeks, although significantly less than the control arteries. This find-
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ing raises a very important question whether radiation indeed prevents neointima formation or simply delays the healing process.
Intracoronary Radiation Adjunct to Stent Implantation As we have alluded earlier, stents, in spite of the recent increased popularity, are known to induce more pronounced neointimal formation than balloon angioplasty. Our recent experimental study aimed to determine whether endovascular irradiation of the pig coronary artery before stent implantation would affect neointima formation (23). In a group of normolipemic pigs (#9), a high activity 192Ir source was used to deliver 14 Gy to a segment of either the LAD or circumflex coronary arteries. In another group of pigs (#3), the radiation source was a pure b-emitter (90S/Y). After the irradiation delivery, a 3.5-mm tantalum stent was implanted at the irradiated site of the coronary artery. In both groups, one artery, either the LAD or the circumflex, was used as a control (stented without irradiation). The results of this experiment demonstrated that the intimal area was significantly reduced in the irradiated stented arteries compared with control arteries treated with stent only (1.98 mm2 with 192Ir and 1.45 mm2 with 90Sr/Y versus 3.82 mm2 in the control-stented arteries, p , 0.005) (Figure 5). There were no significant morphological or morphometric differences between arteries treated with the b- or g-emitting isotopes. The luminal area was significantly larger in both irradiation groups compared to control, whereas the vessel perimeter and the vessel area were unchanged by the radiation treatment. Another finding was that the inflammatory infiltrates of mononuclear and giant cells often seen to surround the stent wires were significantly reduced in the irradiated arteries. Interestingly, using the same dose of b radiation, but reversing the order by stenting first and then irradiating, the beneficial effect of radiation compared to control was no longer apparent. There are two possible explanations for this unexpected finding. The radiation dose was either inadequate for the poststented increase in the diameter of the artery, or the stent struts prevented the radiation energy from reaching the arterial wall. The concept of combining low-dose endovascular radiation with stent placement led to the recent emergence of radioactive stents. Adding a radioisotope to a stent is relatively easy and the potential of such an approach was supported initially from in vitro studies on cultured human and animal smooth muscle cells and endothelial cells. Fischell et al. (24) designed a study to investigate the ability of local emission of b-particles from a 32P-impregnated titanium “stent” wire source to determine the doseresponse characteristics of the inhibition of smooth muscle cells. The authors’ data from their in vitro study suggest that a stent impregnated with a low concentration of 32P may
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Figure 5. (A) Control nonirradiated coronary artery 1 month after stent placement. Note the marked neointimal (N) proliferation and significant reduction of the luminal diameter. S 5 stent wire, M 5 media. (Toluidine blue stain, 330). (B) Irradiated coronary artery (14 Gy) immediately before stenting. Note significant reduction in neointima in comparison to the control artery. M 5 media, S 5 stent wire, N 5 neointima. (Toluidine blue stain, 330.) With permission [23] from Scientific Publications Department of the American Heart Association; © 1995.
have a salutory effect on the restenosis process. Their data also indicated that endothelial cells (bovine) are much more radioresistant than smooth muscle cells and therefore endothelial cell regrowth may occur before the antiproliferative effects of the b-emission on smooth muscle cells have been lost. The emergence of low-dose radioactive stents was rather swift, because ion implantation to make the stent radioactive does not require the use of a potentially biologically active substance such as a biodegradable polymer with its associated chronic tissue compatability problems. Hehrlein et al. (25) from Germany reported a study in which PalmazSchatz stents were made radioactive in a cyclotron. These stents had a very low activity and thus, manipulation did not require extensive radiation protection. The report indicates that whereas after 1 week, common iliac arteries with non-
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radioactive stents began to endothelialize, the endothelialization of arteries with radioactive stents was delayed in a dose-dependent fashion. However, in spite of the delayed endothelialization, radioactive stents did not reveal increased propensity to thrombus formation compared with nonradioactive stents. Two additional morphologic features were observed: i) the cellularity of the neointima covering radioactive stents was decreased compared with nonradioactive stents and ii) the presence of macrophages in the neointima, after radioactive stent implantation, a finding probably related to the delayed endothelialization. Although the study by Hehrlein et al. demonstrated that stents made radioactive in a cyclotron effectively inhibit SMC proliferation and neointimal hyperplasia in rabbits, their technique has some potential drawbacks, one of which is having g-irradiation and multiple radioisotopes (55,56,57Co, 52Mg, and 53Fe) including some with half-lives as long as 2.7 years. Recently, Hehrlein reported similar response on suppression of smooth muscle cell proliferation and neointima formation by using a b-particle-emitting radioisotope stent (32P) (26). Although radioactive stents have the potential advantage of application of the radioisotope close to the proliferating tissue, the practical issues of active half-life and the logistical problem of having available the necessary activity of a rapidly decaying source may be problematic. Therefore, we believe the ease and safety of a catheter-based delivery system of an isotope before stent implantation is an attractive alternative. There is, however, a valid argument in that the delivery catheter may not always be centered within the vessel lumen, resulting in no uniformity of dose delivery into the vessel wall. Studies using radioisotope stent (32P) were also reported recently by Laird et al. (27). The authors stressed the advantages of b-emitters over the g-irradiation such as the short half-life (14.3 days) and limited range of the b-particles in tissue (3 to 4 mm). Furthermore, they emphasized that by using b-emitting radioactive stents, they essentially eliminated the risk to catheterization laboratory personnel and have minimized the exposure of surrounding cardiac and pulmonary tissues to ionizing radiation. The reported results were a 37% reduction in neointimal area and a 32% reduction in percent area stenosis for b-particle-emitting stent compared with control stents at 28 days in the pig model (27). Scanning electron microscopy revealed endothelialization of the radioactive stents at 4 weeks after implantation and no evidence of thrombosis. This study, however, was terminated at 28 days and thus important information whether the beneficial effect was maintained at long-term follow-up is lacking. In addition, the stent placement, according to the authors, was not associated with any significant vessel injury. Understandably, several concerns have been voiced in regard to the implantation of radioactive stents. Some of those concerns include the potential leaching of the radioac-
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tive material from the metallic stent and the possible thrombosis on the stent wire due to delayed endothelialization of the stent struts. From a biologic point of view, there is a concern that the area at the terminal edges of the stent and beyond, which is more vulnerable to developing restenosis, will not be treated with enough dose of radiation if at all. The low dose rate in these watershed areas may not be sufficient to prevent the proliferation of smooth muscle cells and the migration that occurs primarily in the first 72 hours after the injury. Furthermore, a continuous delivery of radiation energy by a permanently implanted stent beyond the time required to inhibit restenosis might meet some resistance from both patients and physicians. However, in spite of the extremely low doses of radiation that such stents might emit, it will require careful and objective assessment of all the risks versus the benefits involved in order to overcome the reluctance of physicians and particularly that of the interventional cardiologists to permanently implanting a radioactive source (stent).
Clinical Trials with Endovascular Irradiation for Prevention of Restenosis The clinical use of endovascular irradiation for prevention of recurrent stenosis after stent implantation was reported by Liermann et al. (28). In this study, 29 patients with restenosis in stented femoropopliteal arteries were treated with 12 Gy using 192Ir delivered by high-dose rate afterloader “Nucleotron” to the angioplasty site. Long-term follow-up of these patients revealed no restenosis at the treated sites up to 5 years. Furthermore, the radiation did not affect the adjacent nerve; reported tolerance of peripheral nerves to a single radiation dose is considered to be about 15 Gy (29). Another potential application for using endovascular radiotherapy is in prevention of restenosis in patients after angioplasty for treatment of narrowed dialysis arteriovenous grafts. A feasibility study from our institution involved 10 patients with 15 lesions, all of which failed prior treatment with angioplasty. This study has demonstrated that radiation therapy postangioplasty using high activity 192Iridium administered by high rate afterloader is feasible, safe, and results in lower rate of restenosis (20%) at 6 months followup (30). Further clinical studies are required to determine whether this therapy can extend the survival of these grafts. There are several ongoing clinical trials of intracoronary radiation therapy post-PTCA. However, the only completed clinical trial of intracoronary radiation therapy at the time of balloon angioplasty was conducted in Caracas, Venezuela, by Condado et al. (31). The study involved 21 patients in which high activity 192Ir source was hand-delivered at the time of angioplasty. The intended treatment dose was 20 Gy and 25 Gy, but the mean actual treatment dose corrected to the measured luminal diameter by quantitative computer-
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ized angiography was 35.6 6 11 Gy (range 19.5 to 55.0 Gy). Angiographic follow-up was performed at 24 hours in 18 patients, and at 60 days in 12 patients. All patients had late clinical and angiographic >6 months follow-up. Delivery of the radioactive source wire was successful in all patients. Intracoronary radiation was free of procedural major complications. Angiographic study at 24 hours post-procedure demonstrated elastic recoil with reduction of luminal diameter from 1.92 6 0.55 mm immediately after the procedure to 1.40 6 0.27 mm at 24 hours, and one patient with acute closure. At 60 days, repeat angiograms demonstrated total occlusion in two arteries and false aneurysm in one artery, and at .6 months follow-up all arteries remained patent with mean luminal diameter of 1.65 6 0.8 mm. The overall angiographic patency rate at late follow-up in this series was 90.9%. Clinical events at follow-up were myocardial infarction in one patient, repeat angioplasty in four patients, and angina symptoms persisted in seven patients. These preliminary results demonstrate that intracoronary radiation following coronary intervention is feasible, safe, and associated with reduction of angiographic late loss at long-term follow-up (31). However, larger study populations will be required to determine whether this new therapy will influence the restenosis rate after angioplasty.
Analysis of Data from Experimental Studies Utilized Intravascular Irradiation to Prevent Restenosis In analyzing our results, as well as those of others in regard to the intravascular irradiation, one should take into consideration a number of important points, two of which have to do with the pig model. The first is that in this model one should expect variable healing results depending on the degree of the initial injury, which is created by the oversized balloon. Thus, minimal initial injury with modest subsequent smooth muscle proliferation may be erroneously interpreted as a favorable result (Figure 6). The second point is that one must appreciate that the pig model of oversized balloon injury does not by any means simulate the human atherosclerotic plaque either before or after angioplasty. Among the many differences between species, the presence of inflammatory cells and oxidized lipids in the atherosclerotic vessel makes the human coronary environment one in which a state of oxidation is likely to exist. There are additional important points as we analyze our finding. The issue of placing the irradiation source at equal distances from the arterial circumference is very important. Perhaps a centering device, such as a centering balloon, may prevent unequal distribution of the ionizing energy. Another point also needs clarification and understanding, particularly for those who interpret histopathologic findings in radiated human tissues. Although it has been stated by
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Figure 6. (A) Control coronary artery 2 weeks after oversized balloon dilatation. Note mildly injured media (M), disrupted internal elastica (arrow), and minimal neointimal proliferation (N). (Verhoeff’s van Gieson elastic stain, 390.) (B) Control coronary artery 2 weeks after oversized balloon dilation. The segmental injury to the media and adventitia is limited and the neointimal proliferation (N) modest. M 5 media. (Verhoeff’s van Gieson elastic stain 390.) (C) Control coronary artery 2 weeks after oversized balloon injury. There is marked medial destruction, adventitial damage (A), and pronounced neointimal (N) proliferation. M 5 media. (Verhoeff’s van Gieson elastic stain 390.)
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many that radiation induces recognizable acute and delayed lesions, these morphologic changes in mammalian tissues are not specific and definitely not pathognomonic. During our experimental work with the pig model, we were concerned whether intravascular irradiation may encourage the formation of mural thrombus, because of its effect on the endothelium. We did not, however, observe any significant difference in the rate of mural thrombosis between control and irradiated animals. We did observe, however, mural thrombosis in one safety case in which 56 Gy were administered. We suspect that this high dose was responsible for the mural thrombus and also prevented the sealing back of the disrupted medial ends (Figure 3). It is possible though that irradiation may interfere with the fibrinolytic system by depressing tissue plasminogen activator. An additional concern of ours, which other investigators apparently share, has been whether intravascular irradiation may compromise the integrity of the arterial wall, leading for example to aneurysmal dilation. Contrary to others, we did not observe any morphologic alterations in the noninjured by the balloon media in either the 2 weeks or 6 months irradiated animals. The discrepancy with other reports indicating focal or diffuse media fibrosis 30 days after irradiation in the pig model may reflect differences in the technique of delivery of the radiation energy. In addition, we did not detect any morphologic changes in the vasa vasorum of the irradiated artery or adjacent nerves and ganglia. Lack of any detectable histologic change in the vasa vasorum is most reassuring in that the integrity of the media will not be compromised. Perhaps this is an important distinguishing feature between the endovascular brachytherapy and the external beam irradiation (19) in which the tunica adventitia shows the maximum response in the form of fibrosis that may affect and even obliterate the vasa vasorum. Our studies have adequately answered the question, raised by many investigators, whether intracoronary irradiation might delay the restenosis kinetics, such as smooth muscle proliferation. Our data indicate that a larger neointimal area was noted in animals killed at 6 months compared with those studied at 2 weeks. The importance of long-term follow-up is obvious. It has been reported that small amounts of irradiation energy might have a stimulatory effect on smooth muscle cells, resulting in their proliferation. Although we did not specifically design an experiment to test this hypothesis, our findings with very low-dose radiation (3 Gy) does not support the above contention. There is, however, ample evidence that large doses of irradiation in humans can induce intimal proliferation in medium-sized arteries, such as the coronary arteries, although large series of patients treated with irradiation for chest malignancies did not always reveal significant differences in death rate from coronary disease post-irradiation. We would like to emphasize, how-
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ever, that there is an obvious causative role of radiation on ischemic heart events in young individuals with no conventional risk factors. The issue whether there is late recoil in the swine oversized balloon model and whether intravascular irradiation has an inhibitory effect on such chronic recoil has not been adequately studied. Such a study will be needed, particularly now that reports suggest involvement of adventitia in the vascular repair process after medial injury. These reports have shown that after oversized balloon injury in the pig model, there is hypercellularity of the adventitial layer, proliferation of fibroblasts and modulation of their phenotype to myofibroblasts all contributing to a thickened adventitia. Furthermore, the proliferating activity in the adventitia exceeded that observed in the media at all times after injury. The hypercellular adventitia may be subsequently replaced by collagen-rich scar which could account for late, chronic recoil, a form of unfavorable geometric remodeling that prevents the artery from undergoing compensatory dilatation during neointimal formation (32,33). In our opinion, a future study should be designed to quantitatively calculate the degree and distribution of adventitial and periadventitial fibrosis in irradiated and control animals. Finally, we need to stress that although proliferative response to the arterial injury in the pig is quite similar to that observed after angioplasty in human coronary arteries, there are a number of differences in these two settings. There is no fibrous cap or atheromatous component in the normolipemic pig model. We do know though that in humans the stability of atherosclerotic plaque depends on the collagenous and elastic tissue synthesis by the smooth muscle cells to secure a thick, intact fibrous cap. Whether irradiation may inhibit proliferation and encourage apoptosis of smooth muscle cells in the fibrous cap of the human plaque and therefore compromise its integrity remains to be clarified by clinical trials. Such a clinical trial is underway in our institution at present.
References 1. Nunes GL, Sgoutas DS, Redden RA, et al. Combination of vitamins C and E alters the response to coronary balloon injury in the pig. Arteriosclerosis Thromb 1995;15:1–10. 2. Karas SP, Gravanis MB, Santoian EC, et al. Coronary intimal proliferation after balloon injury and stenting in swine: an animal model of restenosis. J Am Coll Cardiol 1992;20:467–474. 3. Mintz GS, Pichard AD, Kent K, et al. Endovascular stents reduce restenosis by eliminating geometric arterial remodeling: a serial intravascular ultrasound study. J Am Coll Cardiol 1995;35A:701-705. 4. Macaya C, Serruys PW, Raygrok P, et al. Continued benefit of coronary stenting versus balloon angioplasty: one-year clinical follow-up of Benestent trial. J Am Coll Cardiol 1996;27:255–261. 5. Fischman DL, Leon MB, Baim DS, et al. For the stent restenosis study investigators. A randomized comparison of coronary stent placement and balloon angioplasty in the treatment of coronary artery disease. N Engl J Med 1994;331:496–501.
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6. Murphy JG, Schwartz RS, Edwards WD, et al. Percutaneous polymeric stents in porcine arteries: initial experience with polyethylene terephthalate stents. Circulation 1992;86:1595–1604. 7. Serruys PW, Emanuelson H, Gissen W, et al. Heparin-coated PalmazSchatz stents in human coronary arteries: early outcome of the Benestent-II Pilot Study. Circulation 1996;93:412–422. 8. Gravanis MB, Robinson KA, Santoian EC, et al. The reparative phenomena at the site of balloon angioplasty in humans and experimental models. Cardovasc Pathol 1993;2:263–273. 9. Schwartz RS, Holmes DH, Topol BJ. The restenosis paradigm revisited: An alternative proposal for cellular mechanism. J Am Coll Cardiol 1992;20:1284–1293. 10. Kaplan HS. Biochemical basis of reproductive death in irradiated cells. Am J Roentgenol 1963;90:907–916. 11. Fajardo LF. Basic mechanisms and general morphology of radiation injury. Semin Roentgenol 1993;28:297–302. 12. Fajardo LF, Berthrong M. Vascular lesions following radiation. Pathol Ann 1988;23:297–330. 13. Fisher-Dzoga K. Mimitrievich GS, Griem ML. Differential radiosensitivity of aortic cells in vitro. Radiat Res 1984;99:536–546. 14. Hall EJ. Cell-Survival Curves. Radiobiology for the Radiologist (3rd ed). Philadelphia, PA: JB Lippincott Co, 1988;18–38. 15. Gravanis MB, Roubin GS. Histopathologic phenomena at the site of percutaneous transluminal coronary angioplasty: the problem of restenosis. Hum Pathol 1989;20:477–485. 16. Waksman R, Robinson KA, Crocker IR, et al. Endovascular low-dose irradiation inhibits neointima formation after coronary balloon injury in swine. A possible role for radiation therapy in restenosis prevention. Circulation 1995;91:1533–1539. 17. Waksman R, Robinson KA, Crocker IA, et al. Intracoronary low-dose b-irradiation inhibits neointima formation after coronary artery balloon injury in the swine restenosis model, Circulation 1995;92: 3024– 3031. 18. Gross WG, Ing H, Freedman N. A short atlas of beta-ray spectra. Phys Med Biol 1983;23(11):1251–1260. 19. Schwartz RS, Koval TM, Edwards WD, et al. Effect of external beam irradiation on neointimal hyperplasia after experimental coronary artery injury. J Am Coll Cardiol 1992;19:1106–1113. 20. Wiedermann JG, Marboe C, Amols H, et al. Intracoronary irradiation reduces restenosis after balloon angioplasty in a porcine model. J Am Coll Cardiol 1994;23:1491–1498.
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21. Wiedermann JG, Leavy J, Amols H, et al. Effects of high-dose intracoronary irradiation on vasomotor functions and smooth muscle histopathology. Am J Physiol 1994;267:H125–H132. 22. Wiedermann JG, Marboe C, Amols H, et al. Intracoronary irradiation markedly reduces neointimal proliferation after balloon angioplasty in swine: Persistent benefit at six month follow-up. J Am Coll Cardiol 1995;25:1451–1456. 23. Waksman R, Robinson KA, Crocker IR, et al. Intracoronary radiation before stent implantation inhibits neointima formation in stented porcine coronary arteries. Circulation 1995;92:1383–1386. 24. Fischell TA, Kharma BK, Fischell DR, et al. Low-dose, b-particle emission from “stent” wire results in complete, localized inhibition of smooth muscle cell proliferation. Circulation 1994;90:2956–2963. 25. Hehrlein C, Gollan C, Donges K, et al. Low-dose radioactive endovascular stents prevent smooth muscle cell proliferation and neointimal hyperplasia in rabbits. Circulation 1995;92:1570–1575. 26. Hehrlein C, Stinz RB, Kinchref R, et al. Pure b emitting stents inhibit neointima formation in rabbits. Circulation 1996;93:641–645. 27. Laird JR, Carter AJ, Kufs WM, et al. Inhibition of neointimal proliferation with low-dose irradiation from a b-particle-emitting stent. Circulation 1996;93:529–536. 28. Liermann D, Bottcher HD, Kollah J, et al. Prophylactic endovascular radiotherapy to prevent intimal hyperplasia after stent implantation in femoropopliteal arteries. Cardiovasc Interventional Radiol 1994;17: 12–16. 29. Kinsella TJ, Sinclair WF, DeLuca AM. Threshold dose for peripheral nerve injury following intraoperative radiotherapy (IDRT) in a large animal model. Int J Radiat Oncol Biol Phys 1988;I:205–211. 30. Waksman R, Crocker IR, Kikeri D, et al. Endovascular low dose radiation for prevention of restenosis following angioplasty for treatment of narrowed dialysis arteriovenous grafts. First International Radiation for Restenosis Meeting, Atlanta, GA, 1996. 31. Condado JA, Gurdiel O, Espinosa R, et al. Late follow-up after percutaneous transluminal coronary angioplasty (PTCA) and intracoronary radiation therapy (ICRT). First International Radiation for Restenosis Meeting, Atlanta, GA, 1996. 32. Shi Y, Pieniek M, Ford A, et al. Adventitial remodeling after coronary arterial injury. Circulation 1996;93:340–348. 33. Scott NA, Ross CE, Dunn B, et al. Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries. Circulation 1996 (in press).
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† Interventional
11 Although the precise pathogenesis of restenosis after percutaneous transluminal coronary angioplasty (PTCA) remains somewhat elusive, our understanding about the reparative phenomena at the site of dilatation has been significantly improved in recent years. Thus, restenosis appears to be the result of migration, proliferation, and excessive matrix formation by smooth muscle cells plus vascular wall remodeling leading to chronic recoil (constriction). Proposed pharmacotherapies to prevent restenosis have been ineffective in humans, in spite of a relative success in certain experimental animals. The rationale for low-dose irradiation (either b or g) in order to prevent restenosis is based on the known ability of ionizing irradiation to arrest cell division and, therefore, to reduce the number of clonal progenitors in situations like angioplasty. © 1997 by Elsevier Science Inc. Cardiovasc Pathol 1997;6:11–21
The major limitation of percutaneous transluminal coronary angioplasty (PTCA) is the occurrence of restenosis in 25% to 45% of patients within the first 6 months after the procedure. Experimental and clinical data indicate that restenosis results partly from recoil and vessel wall remodeling, but also from migration, proliferation, and excessive matrix deposition by smooth muscle cells after vascular injury (balloon dilation). In addition, changes in vessel redox state may have important consequences on remodeling by altering the nature of the matrix produced by smooth muscle cells and fibroblasts. Our recent study strongly supports the concept that vessel redox state is an important determinant of response to injury (1). Despite intensive research for almost 3 decades, the precise pathogenesis of the restenotic process remains elusive and proposed pharmacotherapies ineffective. The use of intracoronary stents for prevention of restenosis has been increasing in recent years. This increased popularity of stents is based on the assumption that these metallic
Manuscript received March 5, 1996; accepted May 31, 1996. Address for reprints: Dr. Michael B. Gravanis, Department of Pathology and Laboratory Medicine, Emory University Hospital, 1364 Clifton Road NE, Atlanta, GA 30322. Cardiovascular Pathology Vol. 6, No. 1, January/February 1997:11–21 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
scaffoldings will reduce restenosis after angioplasty by achieving a larger initial lumen size and reducing both the acute and chronic recoil phenomena. However, stents are known to be associated with more pronounced neointimal formation than balloon angioplasty. This has been demonstrated in animal models (2) and more recently in a clinical study using intravascular ultrasound (3). It is worth mentioning, though, that 1-year clinical follow-up of patients included in the Stress and Benestent randomized studies, comparing elective stent implantation with balloon angioplasty in patients with stable angina in a de novo coronary artery lesion, showed significant reduction in restenosis rate and requirement for repeat coronary intervention after stent placement (4,5). Attempts to improve the metallic stent surface by introducing polymer-coated devices in order to reduce thrombogenicity and provide local drug delivery have met with limited success because of problems with chronic tissue compatability (6). However, recent studies with heparincoated stents have reported reduction of the restenosis rate to 13% without subacute thrombosis (7). Restenosis after balloon angioplasty involves a complex interplay among endothelial cells, platelets, vascular smooth
1054-8807/97/$17.00 PII S1054-8807(96)00066-X
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muscle cells, inflammatory cells, and blood proteins (8). Similar phenomena are also involved after stent placement, although the barotrauma to the arterial wall by the intravascular prosthesis is considered greater (3). According to some investigators, mural thrombosis may play a significant role in restenosis (9), although failure of aspirin, heparin, and the low molecular weight heparin to reduce restenosis in humans has, in most part, refuted this hypothesis. Furthermore, histologic evidence of preceding mural thrombosis in restenotic lesions is lacking (8).
Effects of Ionizing Radiation on Mammalian Cells Ionizing radiation may affect several cellular molecules, although its most damaging effects are seen on the nucleus, resulting in single- or double-strand breaks of the purine and pyrimidine bases of DNA (10). There is significant variation in regard to the effect of ionizing radiation on mammalian cells, depending on the amount of energy delivered, dose rate, and fractionation. Thus, radiation energy may produce no detectable results; may induce latent DNA changes which manifest in subsequent generations (oncogenesis); inflict DNA damage that may allow somatic life, but no cellular division (reproductive death); or instant somatic death (11). Factors pertaining to cell status may also determine the radiation effect. The response to radiation energy of hypoxic cells is less than that of oxygenated ones. The stage of the reproductive cycle of the cell is also an important determinant. Actively divided cells are more radioresponsive. Finally, the ability of the cell to repair radiation damage determines its survival (12).
Radiation Effects on Blood Vessels Similar to other mammalian tissues, the effects of radiation energy on blood vessels can be characterized as acute and chronic. Furthermore, the radiation effect depends on the size of the vessel, location, dose volume, dose rate factors, and preexisting vascular disease. Whereas all levels of the vascular system may be affected by ionizing radiation small vessels such as arterioles, capillaries and sinusoids are more radiosensitive.
Acute Phase Effects After clinically used doses of irradiation (for malignancies), there is increased capillary permeability manifested as interstitial edema, capillary and arteriolar dilation, and endothelial cell swelling and degeneration. In some tissues, such as the heart and skin, acute lesions may be associated with an inflammatory infiltrate (11).
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Delayed or Chronic Vascular Changes During this phase, the capillary wall basement membrane appears thickened, and the leakage of plasma elements is decreased. Some capillaries may eventually thrombose, whereas in others, the endothelium may recover and the patency of the vessel be maintained. In vessels of small and medium caliber, the morphologic changes are primarily seen in the intima. These include swelling and vacuolization of endothelial cells and myointimal proliferation with luminal narrowing and even occlusion. Lipid-laden macrophages may be seen in the intima a few days after irradiation, and they may persist for several years. Some reports have emphasized that in medium-sized arteries, radiation may induce multiplication of the internal or external elastic membranes, fragmentation or focal loss of medial elastic lamellae, medial necrosis with subsequent fibrosis, and obliteration of vasa vasorum. Although large artery damage is less frequent, coronary artery disease is a delayed manifestation that can produce ischemic myocardial lesions (12). One rather interesting feature of radiation vasculopathy is that it is not associated with leukocytic reaction.
Therapeutic Applications of Ionizing Irradiation Based on the inhibitory effect on cellular proliferation, ionizing radiation has been used widely in the treatment of neoplastic conditions. Furthermore, the use of low doses of ionizing radiation is utilized for benign conditions: superficial x-rays after surgery has been effective in the prevention of hypertrophic scarring, inhibits keloid formation, and heterotopic ossification after total hip arthroplasty. Experimental in vitro studies have shown that ionizing radiation inhibits thymidine uptake and collagen synthesis in cultured fibroblasts (13,14). 90Strontium, a pure b-emitter with a half-life of 28 years, is in clinical use for the treatment of recurrent pterygium.
Ionizing Irradiation: Experimental Models The rationale in all recent experimental studies to apply low-dose radiation in order to prevent restenosis is based on the fact that the latter is mediated at least in part by an uncontrolled proliferation of smooth muscle cells and abundant extracellular matrix synthesis, initiated by vascular injury (dilation). Thus, because radiation particularly affects proliferating tissues by arresting cell division, it could reduce the number of cells in situations such as angioplasty. Our experimental work with low-dose radiation was carried out on the pig model we developed in 1992 (2). In this model, an oversized balloon inflation in the coronary arter-
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ies of normolipemic juvenile pigs induces an acute injury to the tunica media, a disruption in the form of a crater. The subsequent healing process, analogous to hypertrophic scarring, has a close resemblance to the histopathologic responses of human coronary arteries after angioplasty as has been reported in animal experiments and autopsy studies (8,15). In our first experiment, after an overstretch injury to the pig coronary arteries (left anterior descending and circumflex) was performed, a 3-cm ribbon of 192Ir was positioned at the site of only one vessel at the angioplasty site and was left in place for a period sufficient to deliver the assigned dose to a depth of 2 mm (8 to 38 minutes, depending on dose and source activity), whereas the contralateral injured artery remained as the control (16). The prescribed doses in this experiment were 3, 7, or 14 Gy. The durability and the safety of the radiation treatment was studied in miniswines that underwent similar injury and radiation therapy but were sacrificed after 6 months. The objective of our next study was to determine whether the use of a b-emitting radioisotope would have similar effects with g-radiation on intimal proliferation and to establish the dose-response relations. The same normolipemic swine model was used in this study (17). A 2.5-cm length train with seeds of encapsulated 90Strontium/ 90Yttrium source (a pure b-emitter) was left in place to deliver one of four doses: 7, 14, 28, or 56 Gy to a depth of 2 mm in 90 to 720 seconds. Pure b-emitters such as 90Sr/Y have a distinct advantage over the use of g-emitters in that particles with high activity have a limited penetration in tissue and deliver significantly less energy beyond the desired point than do g-emitters
Figure 1. Control nonirradiated coronary artery 2 weeks after oversized balloon dilation. Note segmental medial disruption, neointimal proliferation (N) and destruction of the internal elastica (arrows). (Verhoeff’s van Gieson elastic stain, 390.)
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(18). Thus, the use of b-irradiation for coronary application would appear to have distinct advantages in terms of both short treatment time and radiation exposure practicality for patients and healthcare workers.
Histopathologic and Morphometric Findings after g and b Irradiation The histologic and morphometric analyses of swine coronary arteries treated with the same doses of b- and g-radiation demonstrated overall similarity. Thus, one may surmise that the biological effect on neointimal formation depends on the absorbed dose and not on the specific type of isotope or the variation in treatment times seen with the different approaches. Control arteries revealed at about 2 weeks the filling of the medial defect created by the oversized balloon, by stellate and spindle-shaped cells in a loose extracellular matrix (Figure 1). The majority of these cells showed positive immunostaining for a-actin and had the structural characteristics of modified synthetic SMC (8). In contrast, the neointima formation at the site of the medial defect (crater) was minimal in irradiated arteries (Figure 2). Furthermore, in arteries that received high doses of irradiation (28 and 56 Gy), there was virtually no neointima formation. Whereas in treated arteries that received up to 14 Gy, the luminal surface was covered by a monolayer of endothelial cells by 14 days, in arteries that received high doses (28 and 56 Gy), the endothelial lining was discontinuous; nevertheless, none of these sections, except one (56 Gy) (Figure 3) showed evidence of thrombosis (16,17).
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Figure 2. (A) Irradiated coronary artery 2 weeks after treatment (14 Gy). The neointimal proliferation (arrows) at the site of the crater is very modest. (Verhoeff’s van Gieson elastic stain, 390.) (B) Irradiated coronary artery 2 weeks after treatment (28 Gy). Note that there is no intimal proliferation at the site of the crater (C). (Verhoeff’s van Gieson elastic stain, 390.)
We did not observe nuclear pyknosis or necrosis in the media or adventitia of the irradiated group; and the perivascular nerve fibers, ganglia, adipose tissue, and adjacent myocardium appeared normal. However, in animals killed 2 days after irradiation, there was periadventitial edema and a modest round cell infiltrate (Figure 4). In pigs killed 6 months after balloon injury and irradiation, there was no evidence of fibrosis of the media, perivascular tissues, or adjacent myocardium. Coronary angiography performed just before tissue was harvested showed no evidence of either significant stenosis or aneurysm formation.
Morphometric studies revealed that the ratio of intimal area (IA) to medial fracture length (FL 5 arch between fractured ends of media) is inversely correlated with increasing radiation dose [IA/FL ratio (m 5 0.0028, p , 0.0001, r 5 0.75)]. Analysis of long-term specimens showed reduction of IA/FL in the irradiated arteries with 7 Gy (0.3, p 5 0.009) and 14 Gy (0.31, p 5 0.001) compared with control arteries (0.50). Scanning electron microscopic studies demonstrated that radiation doses up to 14 Gy do not retard reendothelializa-
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Figure 3. Irradiated coronary artery (56 Gy) 2 days after treatment. Note the large intramural thrombus (T) and the dissection between media and adventitia (D). (Verhoeff’s van Gieson elastic stain, 390.)
tion. These studies also revealed that 2 weeks after irradiation (14 Gy), there were only rare small areas of incomplete reendothelialization, as well as occasional adherent leukocytes. However, similar findings were observed with approximately equal frequency in control arteries. In the study by Schwartz and associates, external x-ray radiation was administered to the site of overexpanded, percutaneously delivered tantalum wire coils in pigs. Their findings, however, revealed that g-irradiation (delivered variable doses and times) did not inhibit the proliferative intima response, but rather accentuated it (19).
Figure 4. Irradiated coronary artery (14 Gy) 2 days after treatment. Note the adventitial and periadventitial edema adjacent to the crater (C), myocardial edema (M) and a diffuse mononuclear infiltrate. (Hematoxylin-eosin stain, 390.)
The study by Wiedermann et al. (20) used the swine model of balloon overinflation and iridium-192, a g-radiation emitter. Their results indicate a 71.4% reduction in neointimal area and a 63.0% reduction in percent area stenosis in the irradiated arteries in comparison to controls. Although the authors did not observe any radiation-associated changes in the adventitia, epicardial fat, or myocardium, they detected a diffuse interstitial fibrosis in the media 30 days after the oversized balloon angioplasty. On the basis of these findings, they hypothesized that the inhibitory effect of radiation may be due to medial cell death, and
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therefore, a decrease in the proliferative potential of the remaining mature SMC precursors. Furthermore, they suggested that the media fibrosis may provide a diffusion barrier for mediators of chemokinesis, chemotaxis, and cellular proliferation. In order to study the short- and long-term effects of intracoronary irradiation on endothelial and smooth muscle function and to assess functional recovery and identify persistent vasomotor abnormalities acutely and at 32 days, Wiedermann et al. (21) used intravascular ultrasound. According to the report, irradiated sites acutely displayed vasoconstriction to acetylcholine, with loss of smooth muscle respond to nitroglycerin. A restudy at 32 days showed restoration of normal vasodilatory response to acetylcholine, but persistent loss of response to nitroglycerin. These findings may imply endothelial dysfunction due to acute radiation injury or perhaps inability of vascular smooth muscle to respond to NO shortly after irradiation. The fact, however, that there is no nitroglycerin responsiveness acutely after radiation favors the second mechanism. The authors concluded that whereas intracoronary irradiation does not produce lasting functional or morphologic impairment of the endothelium, the injury to the smooth muscle is lasting and thus responsible for decreased neointimal formation (fibrosis). However, it is possible that this decreased radiosensitivity may not be present in human coronary endothelial cells.
Long-Term Studies In our experimental studies, media fibrosis, either focal or diffuse, was not noted even in animals killed at 6 months. This finding of ours, plus the absence of any detectable histologic changes in the vasa vasorum, is rather reassuring in that the integrity of the media will not be compromised after low-dose radiation of the coronary arteries (l6,17). A recent report by Wiedermann et al. (22) in which the pigs were killed at 6 months after iridium-192 radiation reconfirmed the beneficial effect on neointimal proliferation by this technique and answered several questions raised in their previous report. Additional morphologic findings from this study included scattered foam cells and mononuclear cells in the neointimal proliferation and mild adventitial fibrosis in one-third of the irradiated animals. Our long-term (6-month) follow-up studies did not reveal any quantitative differences in the degree of adventitial fibrosis at 2 weeks and 6 months, between irradiated animals and controls. Furthermore, adventitial fibrosis observed in our experiments was not circumferential, but rather focal at the vicinity of the medial destruction (crater) from the oversized balloon injury. This rather encouraging finding in our studies may have important implications in regard to the late, chronic vascular recoil. However, the amount of morphometrically estimated neointima in the irradiated arteries at 6 months was greater than that observed at 2 weeks, although significantly less than the control arteries. This find-
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ing raises a very important question whether radiation indeed prevents neointima formation or simply delays the healing process.
Intracoronary Radiation Adjunct to Stent Implantation As we have alluded earlier, stents, in spite of the recent increased popularity, are known to induce more pronounced neointimal formation than balloon angioplasty. Our recent experimental study aimed to determine whether endovascular irradiation of the pig coronary artery before stent implantation would affect neointima formation (23). In a group of normolipemic pigs (#9), a high activity 192Ir source was used to deliver 14 Gy to a segment of either the LAD or circumflex coronary arteries. In another group of pigs (#3), the radiation source was a pure b-emitter (90S/Y). After the irradiation delivery, a 3.5-mm tantalum stent was implanted at the irradiated site of the coronary artery. In both groups, one artery, either the LAD or the circumflex, was used as a control (stented without irradiation). The results of this experiment demonstrated that the intimal area was significantly reduced in the irradiated stented arteries compared with control arteries treated with stent only (1.98 mm2 with 192Ir and 1.45 mm2 with 90Sr/Y versus 3.82 mm2 in the control-stented arteries, p , 0.005) (Figure 5). There were no significant morphological or morphometric differences between arteries treated with the b- or g-emitting isotopes. The luminal area was significantly larger in both irradiation groups compared to control, whereas the vessel perimeter and the vessel area were unchanged by the radiation treatment. Another finding was that the inflammatory infiltrates of mononuclear and giant cells often seen to surround the stent wires were significantly reduced in the irradiated arteries. Interestingly, using the same dose of b radiation, but reversing the order by stenting first and then irradiating, the beneficial effect of radiation compared to control was no longer apparent. There are two possible explanations for this unexpected finding. The radiation dose was either inadequate for the poststented increase in the diameter of the artery, or the stent struts prevented the radiation energy from reaching the arterial wall. The concept of combining low-dose endovascular radiation with stent placement led to the recent emergence of radioactive stents. Adding a radioisotope to a stent is relatively easy and the potential of such an approach was supported initially from in vitro studies on cultured human and animal smooth muscle cells and endothelial cells. Fischell et al. (24) designed a study to investigate the ability of local emission of b-particles from a 32P-impregnated titanium “stent” wire source to determine the doseresponse characteristics of the inhibition of smooth muscle cells. The authors’ data from their in vitro study suggest that a stent impregnated with a low concentration of 32P may
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Figure 5. (A) Control nonirradiated coronary artery 1 month after stent placement. Note the marked neointimal (N) proliferation and significant reduction of the luminal diameter. S 5 stent wire, M 5 media. (Toluidine blue stain, 330). (B) Irradiated coronary artery (14 Gy) immediately before stenting. Note significant reduction in neointima in comparison to the control artery. M 5 media, S 5 stent wire, N 5 neointima. (Toluidine blue stain, 330.) With permission [23] from Scientific Publications Department of the American Heart Association; © 1995.
have a salutory effect on the restenosis process. Their data also indicated that endothelial cells (bovine) are much more radioresistant than smooth muscle cells and therefore endothelial cell regrowth may occur before the antiproliferative effects of the b-emission on smooth muscle cells have been lost. The emergence of low-dose radioactive stents was rather swift, because ion implantation to make the stent radioactive does not require the use of a potentially biologically active substance such as a biodegradable polymer with its associated chronic tissue compatability problems. Hehrlein et al. (25) from Germany reported a study in which PalmazSchatz stents were made radioactive in a cyclotron. These stents had a very low activity and thus, manipulation did not require extensive radiation protection. The report indicates that whereas after 1 week, common iliac arteries with non-
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radioactive stents began to endothelialize, the endothelialization of arteries with radioactive stents was delayed in a dose-dependent fashion. However, in spite of the delayed endothelialization, radioactive stents did not reveal increased propensity to thrombus formation compared with nonradioactive stents. Two additional morphologic features were observed: i) the cellularity of the neointima covering radioactive stents was decreased compared with nonradioactive stents and ii) the presence of macrophages in the neointima, after radioactive stent implantation, a finding probably related to the delayed endothelialization. Although the study by Hehrlein et al. demonstrated that stents made radioactive in a cyclotron effectively inhibit SMC proliferation and neointimal hyperplasia in rabbits, their technique has some potential drawbacks, one of which is having g-irradiation and multiple radioisotopes (55,56,57Co, 52Mg, and 53Fe) including some with half-lives as long as 2.7 years. Recently, Hehrlein reported similar response on suppression of smooth muscle cell proliferation and neointima formation by using a b-particle-emitting radioisotope stent (32P) (26). Although radioactive stents have the potential advantage of application of the radioisotope close to the proliferating tissue, the practical issues of active half-life and the logistical problem of having available the necessary activity of a rapidly decaying source may be problematic. Therefore, we believe the ease and safety of a catheter-based delivery system of an isotope before stent implantation is an attractive alternative. There is, however, a valid argument in that the delivery catheter may not always be centered within the vessel lumen, resulting in no uniformity of dose delivery into the vessel wall. Studies using radioisotope stent (32P) were also reported recently by Laird et al. (27). The authors stressed the advantages of b-emitters over the g-irradiation such as the short half-life (14.3 days) and limited range of the b-particles in tissue (3 to 4 mm). Furthermore, they emphasized that by using b-emitting radioactive stents, they essentially eliminated the risk to catheterization laboratory personnel and have minimized the exposure of surrounding cardiac and pulmonary tissues to ionizing radiation. The reported results were a 37% reduction in neointimal area and a 32% reduction in percent area stenosis for b-particle-emitting stent compared with control stents at 28 days in the pig model (27). Scanning electron microscopy revealed endothelialization of the radioactive stents at 4 weeks after implantation and no evidence of thrombosis. This study, however, was terminated at 28 days and thus important information whether the beneficial effect was maintained at long-term follow-up is lacking. In addition, the stent placement, according to the authors, was not associated with any significant vessel injury. Understandably, several concerns have been voiced in regard to the implantation of radioactive stents. Some of those concerns include the potential leaching of the radioac-
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tive material from the metallic stent and the possible thrombosis on the stent wire due to delayed endothelialization of the stent struts. From a biologic point of view, there is a concern that the area at the terminal edges of the stent and beyond, which is more vulnerable to developing restenosis, will not be treated with enough dose of radiation if at all. The low dose rate in these watershed areas may not be sufficient to prevent the proliferation of smooth muscle cells and the migration that occurs primarily in the first 72 hours after the injury. Furthermore, a continuous delivery of radiation energy by a permanently implanted stent beyond the time required to inhibit restenosis might meet some resistance from both patients and physicians. However, in spite of the extremely low doses of radiation that such stents might emit, it will require careful and objective assessment of all the risks versus the benefits involved in order to overcome the reluctance of physicians and particularly that of the interventional cardiologists to permanently implanting a radioactive source (stent).
Clinical Trials with Endovascular Irradiation for Prevention of Restenosis The clinical use of endovascular irradiation for prevention of recurrent stenosis after stent implantation was reported by Liermann et al. (28). In this study, 29 patients with restenosis in stented femoropopliteal arteries were treated with 12 Gy using 192Ir delivered by high-dose rate afterloader “Nucleotron” to the angioplasty site. Long-term follow-up of these patients revealed no restenosis at the treated sites up to 5 years. Furthermore, the radiation did not affect the adjacent nerve; reported tolerance of peripheral nerves to a single radiation dose is considered to be about 15 Gy (29). Another potential application for using endovascular radiotherapy is in prevention of restenosis in patients after angioplasty for treatment of narrowed dialysis arteriovenous grafts. A feasibility study from our institution involved 10 patients with 15 lesions, all of which failed prior treatment with angioplasty. This study has demonstrated that radiation therapy postangioplasty using high activity 192Iridium administered by high rate afterloader is feasible, safe, and results in lower rate of restenosis (20%) at 6 months followup (30). Further clinical studies are required to determine whether this therapy can extend the survival of these grafts. There are several ongoing clinical trials of intracoronary radiation therapy post-PTCA. However, the only completed clinical trial of intracoronary radiation therapy at the time of balloon angioplasty was conducted in Caracas, Venezuela, by Condado et al. (31). The study involved 21 patients in which high activity 192Ir source was hand-delivered at the time of angioplasty. The intended treatment dose was 20 Gy and 25 Gy, but the mean actual treatment dose corrected to the measured luminal diameter by quantitative computer-
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ized angiography was 35.6 6 11 Gy (range 19.5 to 55.0 Gy). Angiographic follow-up was performed at 24 hours in 18 patients, and at 60 days in 12 patients. All patients had late clinical and angiographic >6 months follow-up. Delivery of the radioactive source wire was successful in all patients. Intracoronary radiation was free of procedural major complications. Angiographic study at 24 hours post-procedure demonstrated elastic recoil with reduction of luminal diameter from 1.92 6 0.55 mm immediately after the procedure to 1.40 6 0.27 mm at 24 hours, and one patient with acute closure. At 60 days, repeat angiograms demonstrated total occlusion in two arteries and false aneurysm in one artery, and at .6 months follow-up all arteries remained patent with mean luminal diameter of 1.65 6 0.8 mm. The overall angiographic patency rate at late follow-up in this series was 90.9%. Clinical events at follow-up were myocardial infarction in one patient, repeat angioplasty in four patients, and angina symptoms persisted in seven patients. These preliminary results demonstrate that intracoronary radiation following coronary intervention is feasible, safe, and associated with reduction of angiographic late loss at long-term follow-up (31). However, larger study populations will be required to determine whether this new therapy will influence the restenosis rate after angioplasty.
Analysis of Data from Experimental Studies Utilized Intravascular Irradiation to Prevent Restenosis In analyzing our results, as well as those of others in regard to the intravascular irradiation, one should take into consideration a number of important points, two of which have to do with the pig model. The first is that in this model one should expect variable healing results depending on the degree of the initial injury, which is created by the oversized balloon. Thus, minimal initial injury with modest subsequent smooth muscle proliferation may be erroneously interpreted as a favorable result (Figure 6). The second point is that one must appreciate that the pig model of oversized balloon injury does not by any means simulate the human atherosclerotic plaque either before or after angioplasty. Among the many differences between species, the presence of inflammatory cells and oxidized lipids in the atherosclerotic vessel makes the human coronary environment one in which a state of oxidation is likely to exist. There are additional important points as we analyze our finding. The issue of placing the irradiation source at equal distances from the arterial circumference is very important. Perhaps a centering device, such as a centering balloon, may prevent unequal distribution of the ionizing energy. Another point also needs clarification and understanding, particularly for those who interpret histopathologic findings in radiated human tissues. Although it has been stated by
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Figure 6. (A) Control coronary artery 2 weeks after oversized balloon dilatation. Note mildly injured media (M), disrupted internal elastica (arrow), and minimal neointimal proliferation (N). (Verhoeff’s van Gieson elastic stain, 390.) (B) Control coronary artery 2 weeks after oversized balloon dilation. The segmental injury to the media and adventitia is limited and the neointimal proliferation (N) modest. M 5 media. (Verhoeff’s van Gieson elastic stain 390.) (C) Control coronary artery 2 weeks after oversized balloon injury. There is marked medial destruction, adventitial damage (A), and pronounced neointimal (N) proliferation. M 5 media. (Verhoeff’s van Gieson elastic stain 390.)
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many that radiation induces recognizable acute and delayed lesions, these morphologic changes in mammalian tissues are not specific and definitely not pathognomonic. During our experimental work with the pig model, we were concerned whether intravascular irradiation may encourage the formation of mural thrombus, because of its effect on the endothelium. We did not, however, observe any significant difference in the rate of mural thrombosis between control and irradiated animals. We did observe, however, mural thrombosis in one safety case in which 56 Gy were administered. We suspect that this high dose was responsible for the mural thrombus and also prevented the sealing back of the disrupted medial ends (Figure 3). It is possible though that irradiation may interfere with the fibrinolytic system by depressing tissue plasminogen activator. An additional concern of ours, which other investigators apparently share, has been whether intravascular irradiation may compromise the integrity of the arterial wall, leading for example to aneurysmal dilation. Contrary to others, we did not observe any morphologic alterations in the noninjured by the balloon media in either the 2 weeks or 6 months irradiated animals. The discrepancy with other reports indicating focal or diffuse media fibrosis 30 days after irradiation in the pig model may reflect differences in the technique of delivery of the radiation energy. In addition, we did not detect any morphologic changes in the vasa vasorum of the irradiated artery or adjacent nerves and ganglia. Lack of any detectable histologic change in the vasa vasorum is most reassuring in that the integrity of the media will not be compromised. Perhaps this is an important distinguishing feature between the endovascular brachytherapy and the external beam irradiation (19) in which the tunica adventitia shows the maximum response in the form of fibrosis that may affect and even obliterate the vasa vasorum. Our studies have adequately answered the question, raised by many investigators, whether intracoronary irradiation might delay the restenosis kinetics, such as smooth muscle proliferation. Our data indicate that a larger neointimal area was noted in animals killed at 6 months compared with those studied at 2 weeks. The importance of long-term follow-up is obvious. It has been reported that small amounts of irradiation energy might have a stimulatory effect on smooth muscle cells, resulting in their proliferation. Although we did not specifically design an experiment to test this hypothesis, our findings with very low-dose radiation (3 Gy) does not support the above contention. There is, however, ample evidence that large doses of irradiation in humans can induce intimal proliferation in medium-sized arteries, such as the coronary arteries, although large series of patients treated with irradiation for chest malignancies did not always reveal significant differences in death rate from coronary disease post-irradiation. We would like to emphasize, how-
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ever, that there is an obvious causative role of radiation on ischemic heart events in young individuals with no conventional risk factors. The issue whether there is late recoil in the swine oversized balloon model and whether intravascular irradiation has an inhibitory effect on such chronic recoil has not been adequately studied. Such a study will be needed, particularly now that reports suggest involvement of adventitia in the vascular repair process after medial injury. These reports have shown that after oversized balloon injury in the pig model, there is hypercellularity of the adventitial layer, proliferation of fibroblasts and modulation of their phenotype to myofibroblasts all contributing to a thickened adventitia. Furthermore, the proliferating activity in the adventitia exceeded that observed in the media at all times after injury. The hypercellular adventitia may be subsequently replaced by collagen-rich scar which could account for late, chronic recoil, a form of unfavorable geometric remodeling that prevents the artery from undergoing compensatory dilatation during neointimal formation (32,33). In our opinion, a future study should be designed to quantitatively calculate the degree and distribution of adventitial and periadventitial fibrosis in irradiated and control animals. Finally, we need to stress that although proliferative response to the arterial injury in the pig is quite similar to that observed after angioplasty in human coronary arteries, there are a number of differences in these two settings. There is no fibrous cap or atheromatous component in the normolipemic pig model. We do know though that in humans the stability of atherosclerotic plaque depends on the collagenous and elastic tissue synthesis by the smooth muscle cells to secure a thick, intact fibrous cap. Whether irradiation may inhibit proliferation and encourage apoptosis of smooth muscle cells in the fibrous cap of the human plaque and therefore compromise its integrity remains to be clarified by clinical trials. Such a clinical trial is underway in our institution at present.
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21. Wiedermann JG, Leavy J, Amols H, et al. Effects of high-dose intracoronary irradiation on vasomotor functions and smooth muscle histopathology. Am J Physiol 1994;267:H125–H132. 22. Wiedermann JG, Marboe C, Amols H, et al. Intracoronary irradiation markedly reduces neointimal proliferation after balloon angioplasty in swine: Persistent benefit at six month follow-up. J Am Coll Cardiol 1995;25:1451–1456. 23. Waksman R, Robinson KA, Crocker IR, et al. Intracoronary radiation before stent implantation inhibits neointima formation in stented porcine coronary arteries. Circulation 1995;92:1383–1386. 24. Fischell TA, Kharma BK, Fischell DR, et al. Low-dose, b-particle emission from “stent” wire results in complete, localized inhibition of smooth muscle cell proliferation. Circulation 1994;90:2956–2963. 25. Hehrlein C, Gollan C, Donges K, et al. Low-dose radioactive endovascular stents prevent smooth muscle cell proliferation and neointimal hyperplasia in rabbits. Circulation 1995;92:1570–1575. 26. Hehrlein C, Stinz RB, Kinchref R, et al. Pure b emitting stents inhibit neointima formation in rabbits. Circulation 1996;93:641–645. 27. Laird JR, Carter AJ, Kufs WM, et al. Inhibition of neointimal proliferation with low-dose irradiation from a b-particle-emitting stent. Circulation 1996;93:529–536. 28. Liermann D, Bottcher HD, Kollah J, et al. Prophylactic endovascular radiotherapy to prevent intimal hyperplasia after stent implantation in femoropopliteal arteries. Cardiovasc Interventional Radiol 1994;17: 12–16. 29. Kinsella TJ, Sinclair WF, DeLuca AM. Threshold dose for peripheral nerve injury following intraoperative radiotherapy (IDRT) in a large animal model. Int J Radiat Oncol Biol Phys 1988;I:205–211. 30. Waksman R, Crocker IR, Kikeri D, et al. Endovascular low dose radiation for prevention of restenosis following angioplasty for treatment of narrowed dialysis arteriovenous grafts. First International Radiation for Restenosis Meeting, Atlanta, GA, 1996. 31. Condado JA, Gurdiel O, Espinosa R, et al. Late follow-up after percutaneous transluminal coronary angioplasty (PTCA) and intracoronary radiation therapy (ICRT). First International Radiation for Restenosis Meeting, Atlanta, GA, 1996. 32. Shi Y, Pieniek M, Ford A, et al. Adventitial remodeling after coronary arterial injury. Circulation 1996;93:340–348. 33. Scott NA, Ross CE, Dunn B, et al. Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries. Circulation 1996 (in press).
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