Fibrocellular tissue responses to endovascular and external beam irradiation in the porcine model of restenosis.

PURPOSE: Endovascular radiation has reduced postangioplasty restenosis in preclinical and early clinical studies. External radiation treatment may have advantages over endovascular therapy. We examined vascular and perivascular tissue responses to endovascular and external irradiation in pig coronary arteries. METHODS AND MATERIALS: Ninety-one animals received endovascular or external radiation following balloon injury and were sacrificed at 14, 30, or 180 days. Injured segments of coronary vessels including perivascular and myocardial tissues were evaluated with histochemistry. RESULTS: Endovascular radiation was associated with delayed arterial wound healing as late as 6 months, evidenced by paucity of smooth muscle alpha-actin in neointimal cells compared to control. External treatment was associated with increased collagen in neointima and adventitia, and focal interstitial necrosis in adjacent myocardium. CONCLUSIONS: These investigations showed whole-heart 14 Gy external radiation treatment following coronary injury exacerbated certain aspects of arterial healing. In addition focal myocardial necrosis and fibrosis was observed following external but not endovascular irradiation. Endovascular radiation has some advantages over external irradiation; however the persistence of a synthetic smooth muscle cell phenotype in the neointima at 6 months suggests ionizing radiation in general may have profound effects on vessel architecture over the long term.

Inhibition of platelet deposition with local delivery of heparin using a double balloon catheter.

Heparin is an effective agent in the treatment of unstable angina and myocardial infarction. The clinical utility of heparin is limited by bleeding complications. This study was performed to determine whether static delivery of heparin could effectively inhibit further platelet deposition. Thrombogenic graft segments were incorporated into chronic arteriovenous shunts in pigs. Autologous platelets were labeled with 111Indium. Platelet deposition was quantitated with gamma camera imaging. The grafts were exposed to blood flow for 15 min in order to induce platelet deposition on the thrombogenic surface. Heparin was delivered locally either by direct exposure or with a double balloon catheter. After a 15 minute exposure period, the heparin solution was removed and subsequent platelet deposition was monitored for 90 minutes. Heparin, administered with the double balloon catheter in doses as low as 12.5 U, effectively inhibited further platelet deposition. An intravenous injection of 100 U of heparin, the highest dose use for local delivery, did not perturb bleeding time or the activated partial thromboplastin time. In conclusion, platelet deposition can be inhibited with static local delivery of heparin at doses that are not associated with systemic bleeding.

Intracoronary radiation decreases the second phase of intimal hyperplasia in a repeat balloon angioplasty model of restenosis.

PURPOSE: Repeat balloon angioplasty is likely to induce intimal proliferation, which is associated with a higher restenosis rate. This study examined the effect of intracoronary ionizing radiation on restenotic lesions using repeat balloon injury in a normolipemic swine. METHODS AND MATERIALS: Eight domestic normolipemic pigs underwent overstretch balloon angioplasty with a 3.5 mm balloon in the LAD and LCX, followed by repeat balloon injury at the same sites 4 weeks after the initial injury. At that time a high activity 192Iridium source was introduced immediately after the angioplasty by random assignment to deliver 14 Gy at 2 mm in eight of the injured coronary arteries (LAD and LCX). One month later the animals were killed and the coronary arteries pressure perfusion fixed. Serial sections were stained with H&E and VVG, then evaluated by histopathologic and morphometric techniques. Maximal intimal thickness (MIT), intimal area (IA), and intimal area corrected for the extent of injury (IA/FL) were measured in the irradiated and control arteries and were compared to control arteries with single injuries from previous studies. RESULTS: Repeat balloon injury induced significant additional medial damage, which was associated with marked intimal hyperplasia in a concentric pattern. Intracoronary irradiation significantly decreased the total of neointima area formation (IA 93 + 0.35 mm2 compared to control 1.38 + 0.33 mm2 p < 0.01) and the MIT was also significantly reduced in the irradiated vessels (0.57 + 0.18 mm vs. 0.71 + 0.08 mm, p = 0.05). CONCLUSIONS: Intracoronary irradiation immediately after the second balloon dilatation inhibits the intimal hyperplasia due to that injury. However, there was no effect on the existing neointima from the initial injury.

Effect of intravascular irradiation on cell proliferation, apoptosis, and vascular remodeling after balloon overstretch injury of porcine coronary arteries.

BACKGROUND: Ionizing radiation has been shown to reduce vascular lesion formation after balloon overstretch injury of pig coronary arteries. The present series of experiments examines the mechanism by which this occurs. METHODS AND RESULTS: Balloon injury was performed on porcine coronary arteries, followed immediately by ionizing radiation using either a source train of 90Sr/Y or 192Ir seeds designed to deliver 14 or 28 Gy at a depth of 2 mm from the source. The animals were killed 3, 7, or 14 days after injury. Bromodeoxyuridine was administered 24 hours before euthanasia to label proliferating cells. Cell proliferation was significantly reduced on day 3 in the adventitia and media of the irradiated vessels compared with controls. Two weeks after injury, there were fewer alpha-actin-positive myofibroblasts in the adventitia of the irradiated vessels than in nonirradiated controls, and morphometric analysis indicated that the vessel perimeter of the irradiated vessels was significantly larger than in controls. Together, these results suggest a positive effect of intravascular irradiation on vascular remodeling. Apoptosis was estimated by terminal transferase dUTP-biotin nick-end labeling (TUNEL) 3 and 7 days after injury. TUNEL-labeled cells were found primarily in the adventitia at the medial tear, but no differences were detected between irradiated and control vessels. CONCLUSIONS: These studies suggest that intracoronary radiation primarily inhibits the first wave of cell proliferation in the vessel wall and demonstrates a favorable effect on late remodeling by preventing adventitial fibrosis at the injury site.

Endoluminal local delivery of PCNA/cdc2 antisense oligonucleotides by porous balloon catheter does not affect neointima formation or vessel size in the pig coronary artery model of postangioplasty restenosis.

Localized delivery of antisense oligonucleotides directed against cell cycle regulatory proteins has been proposed as a means to prevent restenosis after angioplasty. To test whether single endoluminal delivery of a combination of proliferating cell nuclear antigen (PCNA) and cell-division cycle 2 kinase (cdc2) antisense might affect restenosis, we delivered 2 ml of lipid-complexed PCNA/cdc2 antisense oligomers (1.35 mg) to the coronary arteries of pigs after balloon overstretch angioplasty (AS group) and performed planimetric histomorphometry on arterial sections of the tissue, harvested at 4 wk. Compared with controls receiving 3'-5' reversed sequence oligomers (REV group), there were no differences in absolute intimal area (AS 1.36 +/- 0.08 mm2, REV 1.23 +/- 0.10 mm2, P = NS), intimal area normalized to extent of injury (AS 0.67 +/- 0.03, REV 0.77 +/- 0.10, P = NS), or vessel perimeter (AS 7.72 +/- 0.19 mm, REV 7.36 +/- 0.22 mm, P = NS). We conclude that single endoluminal delivery of antisense against key cell cycle regulatory proteins does not affect neointima formation or vessel size in this model of restenosis.

Pharmacokinetics and tissue localization of antisense oligonucleotides in balloon-injured pig coronary arteries after local delivery with an iontophoretic balloon catheter.

When delivered locally to the arterial wall by passive fluid transfer systems such as perforated balloons, water-soluble compounds in aqueous solution are not readily taken up by tissue, show low levels of cellular localization, and are quickly lost by wash-out. One approach to improve delivery is addition of an "active" component to the catheter system to change the nature of the drug-to-tissue interaction. Using an iontophoretic balloon catheter to deliver antisense oligonucleotide (ODN) to pig coronary arteries after balloon angioplasty, we determined the quantity and localization of ODN in the tissue. By radiolabeling, 7.3 +/- 2.4 micrograms ODN was present at 30 min, 1.5 +/- 0.6 at 2 h, 0.52 +/- 0.35 at 24 h, and 0.26 +/- 0.11 at 7 d. By fluorescent labeling, circumferential medial uptake and adventitial delivery at the site of medial injury was observed, with primarily cellular localization. The iontophoretic catheter thus appears to be a useful device for ODN delivery to arterial tissue.

Local intracoronary heparin delivery with a microporous balloon catheter.

Arterial thrombosis plays a major role in the pathogenesis of acute coronary syndromes such as unstable angina and acute myocardial infarction. Heparin is efficacious in treating both disorders; however, systemically administered heparin is associated with bleeding complications. Local intracoronary delivery of heparin may be a safer, more effective method of administration. This study was performed to determine the fate of heparin infused with a specially designed catheter for local intracoronary delivery. To quantitate heparin delivery, tritiated-labeled heparin was dissolved in a solution of unlabeled heparin (1,000 U/ml). A microporous balloon catheter was placed in the left anterior descending (LAD) and left circumflex arteries of anesthetized pigs (n = 15), and 1 ml of the heparin solution was infused. The animals were euthanized within 1 hour, and the treated arteries and controls were harvested, processed, and the tritiated activity was measured. To assess the distribution of the heparin in the arterial wall, 1 ml of fluorescein-isothiocyanate (FITC)-labeled heparin was locally delivered into the walls of the LAD and left circumflex arteries with the microporous balloon catheter. To visualize the dynamic fluid transfer of the device, a microporous balloon catheter was inflated in the LAD, and 1 ml of diluted contrast medium was infused under cinefluoroscopy. The arteries treated with tritiated-labeled heparin contained 0.6% +/- 0.2% of the infused heparin dose. Control arteries contained 0.01% of the administered heparin. Animals that were infused with FITC-labeled heparin displayed fluorescence throughout all layers of the artery, especially in the adventitia. In animals that were injected with 1 ml of diluted contrast medium through the microporous balloon, a relatively large amount of the infusate appeared in the arterial lumen proximal to the balloon. In conclusion, these results suggest that heparin can be delivered to coronary arteries with a microporous balloon catheter. However, <1% of the infused dose can be found in the artery 1 hour after delivery. Infused heparin is distributed throughout the arterial wall, but most of the infused solution appears in the arterial lumen proximal to the inflated balloon and is probably washed downstream after balloon deflation.

Identification of a potential role for the adventitia in vascular lesion formation after balloon overstretch injury of porcine coronary arteries.

BACKGROUND: In the present series of experiments, we examined the onset of cell proliferation and growth factor expression after balloon overstretch injury to porcine coronary arteries. METHODS AND RESULTS: Domestic juvenile swine underwent balloon overstretch injury to the left anterior descending and circumflex coronary arteries with standard percutaneous transluminal coronary angioplasty balloon catheters. To identify proliferating cells, 5-bromo-2-deoxyuridine (BrDU) was administered over a period of 24 hours before the animals were killed at either 1, 3, 7, or 14 days after injury. Immunohistochemistry was performed with monoclonal antibodies to BrDU and smooth muscle cell markers. Three days after injury, a large number of proliferating cells were located in the adventitia, with significantly fewer positive cells found in the media and lumen. Seven days after injury, proliferating cells were found primarily in the neointima, extending along the luminal surface. In situ hybridization for PDGF A-chain and beta-receptor mRNAs revealed that the expression of these two genes was closely correlated with the sites of proliferation at each time point. Studies in which BrDU was injected between days 2 and 3 and the animals were killed on day 14 suggested that the proliferating adventitial cells may migrate into the neointima. CONCLUSIONS: These data suggest that adventitial myofibroblasts contribute to the process of vascular lesion formation by proliferating, synthesizing growth factors, and possibly migrating into the neointima. Increased synthesis of alpha-smooth muscle actin observed in the adventitial cells after arterial injury may constrict the injured vessel and contribute to the process of arterial remodeling and late lumen loss after angioplasty.

Intracoronary low-dose beta-irradiation inhibits neointima formation after coronary artery balloon injury in the swine restenosis model.

BACKGROUND: Neointima formation contributing to recurrent stenosis remains a major limitation of percutaneous transluminal angioplasty. Endovascular low-dose gamma-irradiation has been shown to reduce intimal thickening (hyperplasia) after balloon overstretch injury in pig coronary arteries, a model of restenosis. The objective of this study was to determine whether the use of a beta-emitting radioisotope for this application would have similar effects and to examine the dose-response relations with this approach. METHODS AND RESULTS: Normal domestic pigs underwent balloon overstretch injury in the left anterior descending and left circumflex and coronary arteries. A flexible catheter was introduced by random assignment into one of these arteries and was afterloaded with a 2.5-cm ribbon of encapsulated 90Strontium/90Yttrium sources (90Sr/Y, a pure beta-emitter). It was left in place for a period of time sufficient to deliver one of four doses: 7, 14, 28, or 56 Gy, to a depth of 2 mm. Animals were killed 14 days after balloon injury, the coronary vasculature was pressure-perfusion fixed, and histomorphometric analysis of arterial cross sections was performed. All arteries treated with radiation demonstrated significantly decreased neointima formation compared with control arteries. The ratio of intimal area to medial fracture length was inversely correlated with increasing radiation dose: control (no radiation), 0.47; 7 Gy, 0.34; 14 Gy, 0.20; 28 Gy, 0.08; and 56 Gy, 0.02 (r = -.78, P < .000001). Scanning electron microscopy demonstrated a confluent layer of endothelium-like cells both in control and in 14 Gy-irradiated arteries. There was neither evidence of significant necrosis nor excess fibrosis in the media, adventitia, or perivascular space of the coronary arteries or adjacent myocardium in the irradiated groups. Furthermore, the exposure to the staff and the total body exposure to the pig with the beta source was a small fraction of the dose previously measured and calculated with 192Ir, a gamma-emitting radioisotope. CONCLUSIONS: Administration of endovascular beta-radiation to the site of coronary arterial overstretch balloon injury in pigs with 90Sr/Y is technically feasible and safe. Radiation doses between 7 and 56 Gy showed evidence of inhibition of neointima formation. A dose-response relation was demonstrated, but no further inhibitory effect was seen beyond 28 Gy. These data suggest that intracoronary beta-irradiation is practical and feasible and may aid in preventing clinical restenosis.

Intracoronary radiation before stent implantation inhibits neointima formation in stented porcine coronary arteries.

BACKGROUND: Stent implantation has been shown to reduce restenosis by establishing a larger lumen but not by reducing neointima formation. We have previously shown that ionizing radiation reduced neointima formation after balloon injury in a swine model of restenosis. The purpose of this study was to determine whether endovascular irradiation of the coronary artery before stent implantation would affect neointima formation. METHODS AND RESULTS: Nine normolipemic pigs underwent coronary angiography, and segments of the left anterior descending and left circumflex arteries were chosen as targets for stenting. A high-activity 192Ir source was used to deliver 14 Gy by random assignment to one of the vessels. After this, 3.5-mm tantalum stents were implanted in both arteries. Three additional pigs were treated with a 90Sr/Y source (a pure beta-emitter) delivering 14 Gy to five segments of coronary vessels that were stented immediately after irradiation. Stent-to-artery ratio was similar in the radiated and the control arteries. Animals received aspirin 325 mg daily and were killed at 28 days. 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 2.53 mm2 with 90Sr/Y versus 3.82 mm2 in the control stented arteries, P < .005). CONCLUSIONS: Endovascular radiation before coronary stenting reduces neointima formation and may further reduce the restenosis rate after stent implantation.

Comparison of the thrombogenicity of stainless steel and tantalum coronary stents.

This study was designed to compare the thrombogenicity of stainless steel and tantalum coronary stents of the same design. Stainless steel and tantalum coronary stents are being evaluated for their utility in treating acute closure and restenosis. A major disadvantage of stainless steel stents is radiolucency. To determine whether radioopaque tantalum stents may be safely substituted for stainless steel stents, we compared the relative thrombogenicity of these materials in stents of identical design. Total platelet and fibrin deposition on the stents were determined from measurements of indium 111-labeled platelet and iodine 125-labeled fibrinogen accumulation after deployment into exteriorized chronic arteriovenous shunts in seven untreated baboons. In another series of experiments, 111In-platelet deposition was compared 2 hours after stent implantation in coronary arteries of pigs. In baboons, platelet thrombus formation on stainless steel and tantalum stents was equivalent and plateaued at approximately 2.5 x 10(9) platelets after 1 hour (p > 0.05). Fibrin deposition averaged approximately 1 mg/stent and did not differ between the stainless steel and tantalum stents (p > 0.05). In the porcine coronary model there was no significant difference in 111In-labeled platelet deposition between the stainless steel and tantalum stents (p > 0.05). This result was confirmed by scanning electron microscopic analysis of the coronary stents. Based on these two models, we conclude that there is no significant difference in the thrombogenicity of stainless steel and tantalum wire coil stents.

Endovascular low-dose irradiation inhibits neointima formation after coronary artery balloon injury in swine. A possible role for radiation therapy in restenosis prevention.

BACKGROUND: Restenosis after percutaneous transluminal coronary angioplasty remains a major limitation of the long-term success of this procedure. Restenosis is a form of wound healing. Low-dose ionizing radiation has been effective in inhibiting exuberant wound healing responses in a variety of clinical situations. METHODS AND RESULTS: Vascular neointimal lesions resembling human restenosis were created in the coronary arteries of normal pigs by overstretch balloon angioplasty injury. To test the effect of low-dose endovascular gamma radiation on lesion formation, a high-activity 192Ir source was introduced into one of the injured arteries in each animal and left in place for a period sufficient to deliver one of three doses: 350, 700, or 1400 cGy. To test potential benefits of delayed irradiation, 700 cGy was given in another group 2 days after injury. Animals were killed 14 days after balloon injury and the coronary vasculature was pressure-perfusion fixed. To test the late effect and safety of endovascular low-dose irradiation, 700 or 1400 cGy was given in miniswine coronary arteries after injury as well as in noninjured carotid arteries; this group was followed up for 6 months. Tissue sections were measured by computer-assisted planimetry. All arteries treated with radiation demonstrated significantly decreased neointima formation compared with control arteries. The ratio of intimal area-to-medial fracture length (IA/FL) was inversely correlated with the different radiation doses: control, 0.59; 350 cGy, 0.38; 700 cGy, 0.42; and 1400 cGy, 0.17 (r = -0.75, P < .0001). Delay of 700-cGy irradiation for 2 days after injury significantly decreased neointima formation compared with the same dose given immediately after injury. Analysis of long-term specimens showed reduction of IA/FL in the arteries irradiated with 700 cGy (0.3, P = .009) and 1400 cGy (0.31, P = .001) compared with control arteries (0.50). There was no excess fibrosis in the media, adventitia, or perivascular space of the coronary arteries or adjacent myocardium in pigs that received radiation compared with control animals. CONCLUSIONS: Low-dose intracoronary irradiation delivered to the site of coronary arterial overstretch balloon injury in pigs inhibited subsequent intimal thickening (hyperplasia). A dose-response relationship was demonstrated, and delay of treatment for 48 hours appeared to augment the inhibitory effect. Six months of follow-up without fibrosis or arteriosclerosis demonstrated the durability of the beneficial effect in the treated group. These data suggest that intracoronary irradiation therapy may aid in preventing clinical restenosis.

Use of the porous balloon in porcine coronary arteries: rationale for low pressure and volume delivery.

Using agents administered systemically, attempts to control the restenotic myoproliferative response associated with angioplasty have been unsuccessful. The porous balloon has the advantage of achieving high local concentrations by directly infusing agents into the arterial wall. The purpose of this study is to identify any acute and chronic morphological changes in swine coronary arteries infused with normal saline through the porous balloon at different driving pressures. In order to establish the safety of local arterial wall infusion through the porous balloon, swine underwent porous balloon infusion of 3, 6, or 10 ml of saline at 5 atmospheres, or infusion of 3 ml of normal saline delivered at either 2, 5, or 10 atmospheres of pressure into the normal left anterior descending and left circumflex arteries. To assess the histopathologic alterations induced by the porous balloon, sized 1.1 to 1 with respect to the artery, animals were sacrificed either immediately after porous balloon infusion or 14 ays later. Acute vessels were evaluated for the presence of medial injury, disruption and/or dissection, whereas chronic vessels underwent morphometric analysis measuring the residual luminal area (Lumen area/Intimal area+Lumen area) and the maximal intimal thickness. Adequate adventitial penetration was confirmed by infusing as little as 2-3 ml of methylene blue at 2 atmospheres of pressure. Infusion of 3 ml of normal saline at 2 atmospheres resulted in minor focal medial edema and disorganization, detected both acutely and 14 days after porous balloon infusion. At delivery pressures of 5 or 10 atmospheres, proportionally more acute injury was noted and measurable neointimal lesions were observed 2 weeks after infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Probucol decreases neointimal formation in a swine model of coronary artery balloon injury. A possible role for antioxidants in restenosis.

BACKGROUND: Restenosis after percutaneous transluminal coronary angioplasty is the major limitation of the long-term success of this procedure. The process of restenosis is similar to an accelerated form of atherosclerosis. Thus, therapeutic interventions that limit the progression and initiation of atherosclerosis may be beneficial in the treatment of restenosis. One such intervention is the antioxidant drug probucol, which has demonstrated benefit in animal models of atherosclerosis. METHODS AND RESULTS: Twenty-six female domestic swine were divided into three study groups (control, n = 9; low-dose probucol, n = 9; high-dose probucol, n = 8) before oversized balloon injury of the left anterior descending and left circumflex coronary arteries. Probucol (1 g/d, low-dose group; 2 g/d, high-dose group) was administered 2 days before balloon injury and was continued until the swine were killed 2 weeks after balloon injury. Morphometric analysis of the injured arteries included the intimal area (square millimeters), maximal intimal thickness (millimeters), and residual lumen (ratio of luminal to intimal plus luminal area). Treatment with high-dose probucol significantly reduced neointimal formation compared with control animals (decreases of 36% in intimal area, P = .007; 20% in maximal intimal thickness, P = NS; and an increase of 15% in residual lumen, P = .02). CONCLUSIONS: The major finding of this study is that the antioxidant drug probucol reduces neointimal formation after oversized balloon injury in a swine model of restenosis. This suggests that active oxygen species may play a role in restenosis.

Angiopeptin inhibits intimal hyperplasia after angioplasty in porcine coronary arteries.

BACKGROUND: Restenosis is mediated by uncontrolled neointimal growth at the site of coronary angioplasty. Angiopeptin is an octapeptide analogue of somatostatin that has been shown to decrease the experimental intimal hyperplasia associated with vascular injury in rats and rabbits. The study purpose was to determine if angiopeptin inhibits the development of intimal hyperplasia in normolipemic swine coronary arteries after overstretch-balloon injury. METHODS AND RESULTS: Overstretch-balloon injury was performed in normolipemic swine coronary arteries using a 3.5-mm angioplasty balloon. Treated animals received angiopeptin (50 micrograms/kg) 1 hour before and at the time of balloon injury. Angiopeptin was administered at 100 (micrograms/kg)/day SQ in two divided doses for 14 days. Animals were killed at 14 and 28 days (2 weeks after cessation of angiopeptin) after balloon injury. Treatment animals were compared with control animals receiving balloon injury alone. Angiopeptin significantly limited the experimental intimal hyperplasia estimated by the maximal intimal thickness and residual lumen (lumen area/lumen area+intimal area) compared with controls. CONCLUSIONS: Angiopeptin inhibits the development of intimal hyperplasia in swine coronary arteries after balloon injury. The beneficial effect was detectable 2 weeks after cessation of angiopeptin therapy.