This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Salame, M. Y. Articles by Robinson, K. A. Search for Related Content PubMed PubMed Citation Articles by Salame, M. Y. Articles by Robinson, K. A.

(Circulation. 2000;101:1087.)
© 2000 American Heart Association, Inc.

Brief Rapid Communications

The Effect of Endovascular Irradiation on Platelet Recruitment at Sites of Balloon Angioplasty in Pig Coronary Arteries

Mahomed Y. Salame, MRCP; Stefan Verheye, MD; Stephen P. Mulkey, BS; Nicolas A. F. Chronos, MD; Spencer B. King, III, MD; Ian R. Crocker, MD; Keith A. Robinson, PhD

From the Andreas Gruentzig Cardiovascular Center, Department of Medicine (Cardiology) (M.Y.S., S.V., S.B.K.), the Department of Radiation Oncology, Emory University School of Medicine (I.R.C.), and the Atlanta Cardiovascular Research Institute (S.P.M., N.A.F.C., K.A.R.), Atlanta, Ga.

Correspondence to Keith A. Robinson, PhD, Atlanta Cardiovascular Research Institute, c/o Cardiology of Georgia, 1996 Cliff Valley Way Suite 200, Atlanta, GA 30329. E-mail krobinson{at}acri.com

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Endovascular irradiation (EI) inhibits balloon-induced neointima formation in animals and is now in clinical trials for restenosis prevention. However, little is known of the effect of EI on vessel thrombogenicity due to delayed arterial healing. We investigated EI effects on platelet recruitment in pig coronary arteries.

Methods and Results—EI was performed using ⁹⁰Sr/Y at 0 Gray (Gy), 15Gy, or 30Gy at 2 mm after balloon overstretch injury. At 1 day, 1 week, and 1 month, platelet recruitment and thrombus formation were assessed using autologous ¹¹¹In-oxine-platelet labeling and light and scanning electron microscopy. In balloon-injured nonirradiated vessels, there was complete reendothelialization at 1 month, and platelet recruitment was similar to normal uninjured arteries. In irradiated vessels, scanning electron microscopy showed incomplete reendothelialization at 1 month, and these areas demonstrated attachment of activated platelets. Light microscopy of irradiated coronaries showed adherent partially organized thrombi and incomplete resolution of intramural hemorrhages. There was a significant increase in platelet recruitment at 1 month in arteries receiving EI at 15Gy (5.1±2.8x10⁶, P=0.02) or 30Gy (12.5±9.9x10⁶, P=0.005) compared with nonirradiated controls (2.7±1.5x10⁶); 30Gy was also higher than 15Gy (P=0.05). Platelet recruitment was also increased for 30Gy compared with control at 1 day.

Conclusions—Endovascular irradiation at 15Gy or 30Gy after balloon angioplasty results in incomplete endothelial recovery, impaired resolution of intramural hemorrhage, and a dose-dependent increase in platelet recruitment at 1 month.

Key Words: balloon angioplasty • restenosis • radiation • blood platelets • thrombosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Restenosis remains the major complication of percutaneous transluminal coronary angioplasty.^{1 2} Stent implantation initially appeared promising, with rates of around 20% to 30% in so-called "ideal" lesions.^{3 4} However, with improved stent technology, indications for stenting have broadened, and the restenosis rate has climbed.^{5 6 7} More recently, endovascular irradiation (EI) has emerged and progressed into clinical trials.^{8 9 10 11} Although radiation appears to delay postangioplasty arterial healing, little is known about the effects of EI on endothelial recovery or thrombus formation. We investigated the effect of EI on platelet recruitment at various time points after balloon injury in the coronaries of adult pigs.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
All experiments and animal care conformed to National Institutes of Health and American Heart Association guidelines and were approved by the Institutional Animal Care and Use Committee of Emory University. Female and castrated male adult minipigs (Yucatan strain, Lone Star Swine, Seguine, Texas) received balloon overstretch injury of all 3 coronary arteries followed by EI at doses of 15 Gray (Gy) or 30Gy or no EI (0Gy controls), according to constrained randomization. All animals received periprocedural aspirin and heparin but no chronic anticoagulant or antiplatelet agent.

Interventional Procedure
We performed coronary balloon overstretch injury in pigs (balloon-to-artery ratio of 1.2 to 1.3, resulting in medial rupture) followed by ß-radiation, as previously described.¹² In the case of control animals (no irradiation), sham treatment was performed using the source delivery catheter. Radiation treatment times were 3 minutes and 17 seconds for 15Gy and 6 minutes and 34 seconds for 30Gy based on the dose rate and dose distribution of the ⁹⁰Sr/Y source train measured by the National Institute of Standards and Technology.

Platelet Labeling and Tissue Processing
At serial time points (1, 7, and 28 days) after catheterization, animals were euthanized 2 hours after reinfusion of autologous ¹¹¹Indium-labeled platelets. The heart was rapidly excised and perfusion-fixed. Arterial segments (2.5 cm) were trimmed of excess tissue, and ¹¹¹In activity was measured; platelet recruitment was determined using methods previously described and expressed as number of platelets/vessel after determination of activity/platelet from simultaneously harvested blood samples.^{13 14} Sections of these segments were randomly allocated for light or scanning electron microscopy (SEM) and prepared appropriately. Complete data collection included samples of at least 6 arteries from at least 2 animals per treatment group at each time point.

Light and SEM
Paraffin-embedded sections (4 µm) taken at 2-mm intervals were stained with hematoxylin-eosin or elastin-trichrome stain to qualitatively assess the overall extent of arterial injury, presence of intramural hemorrhage, and healing response. Planimetry was used to determine vessel, neointimal, and intimal thrombus size, as well as extent of injury (medial fracture length). Samples for SEM were fixed in glutaraldehyde and processed using standard techniques¹² to qualitatively assess endothelialization and morphology, as well presence of adherent platelets, microthrombi, and leukocytes.

Statistical Analysis
Data are presented as mean±standard deviation. Two-way ANOVA using Sigma-Stat Software (Jandel Scientific) was performed to examine the effects of radiation and time on platelet recruitment. Follow-up comparisons were made using unpaired t tests, or when assumptions of normal distribution or equal variance were violated, Mann-Whitney rank-sum tests were performed. An alpha level of P<0.05 was considered to indicate a significant treatment effect or between-groups difference.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Light microscopy demonstrated that all vessels underwent medial rupture to an equivalent degree (FL in Table 1). Control arteries developed fibrous neointima; thrombus was present at 1 week but resorbed by 1 month (Table 1). In contrast, neointima of irradiated arteries (15Gy or 30Gy) at 1 month was almost exclusively inspissated thrombus with little cellular ingrowth (Table 1). Intimal thrombus area in controls, but not in 15- or 30-Gy treated vessels, decreased from 1 week to 1 month.

View this table: [in this window] [in a new window]   Table 1. Cross-Sectional Area of Vessels, Intima, and Intimal Thrombus, as Well as Medial Fracture Length at 1 Week and 1 Month

SEM of control vessels showed that injured areas were completely covered by a monolayer of endothelial-like cells by 1 month with only rare adherent platelets. However, irradiated arteries (15Gy or 30Gy) were not reendothelialized at 1 month and showed an abundance of adherent dendritic platelets, fibrin, and leukocytes (Figure 1). Approximately 25% of 15Gy-treated vessel and 50% of 30Gy-treated vessel surfaces were not endothelialized at 1 month.

View larger version (172K): [in this window] [in a new window]   Figure 1. SEM of pig coronary arteries fixed 1 month after balloon angioplasty with or without EI. Left-hand panels, low-magnification survey micrographs; right-hand panels, higher magnification images. a and b, control; c and d, 15Gy; e and f, 30Gy. Note recovery of luminal surface with confluent endothelial-like cells in control but lack of re-endothelialization and adherent activated platelets and leukocytes in irradiated vessels.

Platelet recruitment measured by ¹¹¹In-labeling was significantly increased by radiation treatment (F=7.26, P=0.002). Follow-up comparisons showed 30Gy resulted in a significant increase in platelet recruitment compared with controls at both 1 day (52.2±35.9 versus 13.9± 14.5x10⁶, P=0.03) and 1 month (12.5±9.9 versus 2.7±1.5x 10⁶, P=0.005), respectively. There was increased platelet recruitment at 1 month for 15Gy compared with control (5.1±2.8 versus 2.7±1.5x10⁶, P=0.02) and for 30Gy compared with 15Gy (12.5±9.9 versus 5.1±2.8x10⁶, P=0.05; Table 1 and Figure 2).

View larger version (18K): [in this window] [in a new window]   Figure 2. Platelet recruitment in pig coronary arteries 1 month after balloon angioplasty with or without endovascular irradiation.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We evaluated the magnitude of platelet recruitment in pig coronary arteries treated with 3 doses of EI (0Gy, 15Gy, and 30Gy) and at 3 time points (1 day, 1 week, and 1 month) after balloon angioplasty. We also performed morphological assessment of these vessels. A dose of 15Gy at 2 mm from the source-center was used because it was within the range of doses used for clinical studies,^{8 9 10 11} whereas 30Gy was included to help define dose ranges for biological effects.

Light microscopy demonstrated endovascular irradiation at 15Gy or 30Gy resulted in delayed reendothelialization and incomplete resolution of intramural hemorrhages. This provides support for the concept that radiation delays healing after arterial injury. Mural thrombi were inspissated and showed minimal organization in sections from irradiated vessels with superimposed fresh platelet-rich thrombi. SEM confirmed irradiated arteries were not reendothelialized at 1 month in contrast to controls. The non-reendothelialized areas displayed abundant adherent dendritic platelets and leukocytes.

For quantitation of platelet recruitment, we used autologous ¹¹¹In-oxine platelet labeling, a method applied extensively for studies of arterial thrombosis,¹⁴ including pig coronaries.¹³ The labeled platelet population remains functionally normal after reinjection.¹⁵

Although variation between vessels existed within individual animals in the present study, ANOVA revealed that radiation increased platelet recruitment (P=0.002). A dose-response effect of irradiation on recruitment at 1 month was seen (0Gy<15Gy<30Gy). Importantly, the clinically relevant dose (15Gy) was associated with a doubling of platelet recruitment at this time point compared with controls. The level of recruitment in the control group was 2.7±1.5, similar to normal nonirradiated nonballoon-injured arteries (2.9±0.8).

These data, in concert with the correlative microscopic observations, demonstrate a profound influence of endovascular irradiation in delaying arterial healing and reendothelialization after angioplasty and thereby promote luminal surface thrombogenicity. Although these are interim results in an animal preparation without chronic antiplatelet therapy, they document the potential for thrombosis in this setting. These results suggest that aggressive and prolonged antiplatelet therapy may be helpful in endovascular irradiation for restenosis prevention. This is further substantiated by the appearance of late thrombotic occlusion in recent clinical trials of intracoronary brachytherapy.¹⁶

Limitations
This study was performed using an animal preparation that mimics some but not all features of coronary angioplasty in the clinical environment, so these findings cannot be used to directly predict responses in that setting.

View this table: [in this window] [in a new window]   Table 2. Platelet Recruitment (x106/Vessel) at Balloon-Injured Sites in Pig Coronary Arteries After Endovascular Irradiation

	Acknowledgments

 
The authors thank Richard A. Hillstead, Jon Lampkin, and Dr Jianhua Cui for assistance in performing animal experiments; Jill McCullers for help with the manuscript preparation; Dr Robert Apkarian for help with SEM; and Dr Stephen R. Hanson for advice and assistance with platelet labeling procedures. This study was supported by grant No. 1-RO1-HL60184-01 from the National Heart, Lung, and Blood Institute (K.A.R.) and by gifts from the Rich Foundation, Atlanta, GA.

	Footnotes

 
Drs Crocker, King, and Robinson have shareholding and/or consulting relationships with Novoste Corporation, which supplied the radiation delivery system used in this study.

Received October 20, 1999; revision received December 22, 1999; accepted January 24, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Hillegass WB, Ohman EM, Califf RM. Restenosis: the clinical issues. In: Topol E, ed. Textbook of Interventional Cardiology. Philadelphia, PA: WB Saunders Inc, 1994:15–435.
   
2. Klein LW, Rosenblum J. Restenosis after successful percutaneous transluminal coronary angioplasty. Prog Cardiovasc Dis. 1990;32:365–382.[Medline] [Order article via Infotrieve]
   
3. Fischman DL, Leon MB, Baim DS, Schatz RA, Savage MP, Penn I, Detre K, Veltri L, Ricci D, Nobuyoshi M. A randomized comparison of coronary-stent placement and balloon angioplasty in the treatment of coronary artery disease. Stent Restenosis Study Investigators. N Engl J Med. 1994;331:496–501.[Abstract/Free Full Text]
   
4. Serruys PW, de Jaegere P, Kiemeneij F, Macaya C, Rutsch W, Heyndrickx G, Emanuelsson H, Marco J, Legrand V, Materne P. A comparison of balloon-expandable-stent implantation with balloon angioplasty in patients with coronary artery disease. Benestent Study Group. N Engl J Med. 1994;331:489–495.[Abstract/Free Full Text]
   
5. Dussaillant GR, Mintz GS, Pichard AD, Kent KM, Satler LF, Popma JJ, Wong SC, Leon MB. Small stent size and intimal hyperplasia contribute to restenosis: a volumetric intravascular ultrasound analysis. J Am Coll Cardiol. 1995;26:720–724.[Abstract]
   
6. Elezi S, Kastrati A, Neumann FJ, Hadamitzky M, Dirschinger J, Schomig A. Vessel size and long-term outcome after coronary stent placement. Circulation. 1998;98:1875–1880.[Abstract/Free Full Text]
   
7. Mintz GS, Hoffmann R, Mehran R, Pichard AD, Kent KM, Satler LF, Popma JJ, Leon MB. In-stent restenosis: the Washington Hospital Center experience. Am J Cardiol. 1998;81:7E–13E.[Medline] [Order article via Infotrieve]
   
8. King SB, Williams DO, Chougule P, Klein JL, Waksman R, Hilstead R, Macdonald J, Anderberg K, Crocker IR. Endovascular beta-radiation to reduce restenosis after coronary balloon angioplasty: results of the beta energy restenosis trial (BERT). Circulation. 1998;97:2025–2030.[Abstract/Free Full Text]
   
9. Teirstein PS, Massullo V, Jani S, Popma JJ, Mintz GS, Russo RJ, Schatz RA, Guarneri EM, Steuterman S, Morris NB, Leon MB, Tripuraneni P. Catheter-based radiotherapy to inhibit restenosis after coronary stenting. N Engl J Med. 1997;336:1697–1703.[Abstract/Free Full Text]
   
10. Condado JA, Waksman R, Gurdiel O, Espinosa R, Gonzalez J, Burger B, Villoria G, Acquatella H, Crocker IR, Seung KB, Liprie SF. Long-term angiographic and clinical outcome after percutaneous transluminal coronary angioplasty and intracoronary radiation therapy in humans. Circulation. 1997;96:727–732.[Abstract/Free Full Text]
    
11. Verin V, Urban P, Popowski Y, Schwager M, Nouet P, Dorsaz PA, Chatelain P, Kurtz JM, Rutishauser W. Feasibility of intracoronary beta-irradiation to reduce restenosis after balloon angioplasty: a clinical pilot study. Circulation. 1997;95:1138–1144.[Abstract/Free Full Text]
    
12. Waksman R, Robinson KA, Crocker IR, Wang C, Gravanis MB, Cipolla GD, Hillstead RA, King SB. Intracoronary low-dose beta-irradiation inhibits neointima formation after coronary artery balloon injury in the swine restenosis model. Circulation. 1995;92:3025–3031.[Abstract/Free Full Text]
    
13. Scott NA, Robinson KA, Nunes GL, Thomas CN, Viel K, King SB, Harker LA, Rowland SM, Juman I, Cipolla GD. Comparison of the thrombogenicity of stainless steel and tantalum coronary stents. Am Heart J. 1995;129:866–872.[Medline] [Order article via Infotrieve]
    
14. Harker LA, Kelly AB, Hanson SR. Experimental arterial thrombosis in nonhuman primates. Circulation. 1991;83:IV41–IV55.
    
15. Savage B, McFadden PR, Hanson SR, Harker LA. The relation of platelet density to platelet age: survival of low- and high-density 111indium-labeled platelets in baboons. Blood. 1986;68:386–393.[Abstract/Free Full Text]
    
16. Costa MA, Sabaté M, van der Giessen WJ, Kay IP, Cervinka P, Lighthart JMR, Serrano P, Coen VLMA, Levendag PC, Serruys PW. Late coronary occlusion after intracoronary brachytherapy. Circulation. 1999;100:789–792.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				C. Deiner, E. Shagdarsuren, P. L. Schwimmbeck, P. Rosenthal, C. Loddenkemper, U. Rauch, M. Pauschinger, R. Dietz, H.-P. Schultheiss, R. Dechend, et al. Nf-{kappa}b and AP-1 activation is associated with late lumen loss after porcine coronary angioplasty and antiproliferative beta-irradiation Cardiovasc Res, July 1, 2007; 75(1): 195 - 204. [Abstract] [Full Text] [PDF]

				R Waksman, I M Leitch, J Roessler, H Yazdi, R Seabron, F Tio, R W Scott, R I Grove, S Rychnovsky, B Robinson, et al. Intracoronary photodynamic therapy reduces neointimal growth without suppressing re-endothelialisation in a porcine model Heart, August 1, 2006; 92(8): 1138 - 1144. [Abstract] [Full Text] [PDF]

				G. W. Stone, S. G. Ellis, C. D. O'Shaughnessy, S. L. Martin, L. Satler, T. McGarry, M. A. Turco, D. J. Kereiakes, L. Kelley, J. J. Popma, et al. Paclitaxel-Eluting Stents vs Vascular Brachytherapy for In-Stent Restenosis Within Bare-Metal Stents: The TAXUS V ISR Randomized Trial JAMA, March 15, 2006; 295(11): 1253 - 1263. [Abstract] [Full Text] [PDF]

				M. Togni, S. Windecker, P. Wenaweser, D. Tueller, A. Kaisaier, W. Maier, B. Meier, and O. M. Hess Deleterious Effect of Coronary Brachytherapy on Vasomotor Response to Exercise Circulation, July 13, 2004; 110(2): 135 - 140. [Abstract] [Full Text] [PDF]

				J. R Sindermann, V. Verin, J. W Hopewell, H. P. Rodemann, and J. H Hendry Biological aspects of radiation and drug-eluting stents for the prevention of restenosis Cardiovasc Res, July 1, 2004; 63(1): 22 - 30. [Abstract] [Full Text] [PDF]

				H.-J. Cho, H.-S. Kim, M.-M. Lee, D.-H. Kim, H.-J. Yang, J. Hur, K.-K. Hwang, S. Oh, Y.-J. Choi, I.-H. Chae, et al. Mobilized Endothelial Progenitor Cells by Granulocyte-Macrophage Colony-Stimulating Factor Accelerate Reendothelialization and Reduce Vascular Inflammation After Intravascular Radiation Circulation, December 9, 2003; 108(23): 2918 - 2925. [Abstract] [Full Text] [PDF]

				E. Cheneau, M. C. John, J. Fournadjiev, R. C. Chan, H.-S. Kim, L. Leborgne, R. Pakala, H. Yazdi, A. E. Ajani, R. Virmani, et al. Time Course of Stent Endothelialization After Intravascular Radiation Therapy in Rabbit Iliac Arteries Circulation, April 29, 2003; 107(16): 2153 - 2158. [Abstract] [Full Text] [PDF]

				R. Bonvini, I. Baumgartner, D. D. Do, M. Alerci, J.-M. Segatto, P. Tutta, K. Jager, M. Aschwanden, E. Schneider, B. Amann-Vesti, et al. Late acute thrombotic occlusion after endovascular brachytherapy and stenting of femoropopliteal arteries J. Am. Coll. Cardiol., February 5, 2003; 41(3): 409 - 412. [Abstract] [Full Text] [PDF]

				E. Regar, K. Kozuma, G. Sianos, V.L.M.A. Coen, W.J. van der Giessen, D. Foley, P. de Feyter, B. Rensing, P. Smits, J. Vos, et al. Routine intracoronary beta-irradiation. Acute and one year outcome in patients at high risk for recurrence of stenosis Eur. Heart J., July 1, 2002; 23(13): 1038 - 1044. [Abstract] [Full Text] [PDF]

				K. Kozuma, M.A. Costa, W.J. van der Giessen, M. Sabate, J.M.R. Ligthart, V.L.M.A. Coen, I.P. Kay, A.J. Wardeh, A.H.M. Knook, P.J de Feyter, et al. Initial observation regarding changes in vessel dimensions after balloon angioplasty and stenting followed by catheter-based {beta}-radiation. Is stenting necessary in the setting of catheter-based radiotherapy? Eur. Heart J., April 2, 2002; 23(8): 641 - 649. [Abstract] [Full Text] [PDF]

				D. Scheinert, V. Strnad, R. Muller, R. Burckhard, S. Ropers, R. Sauer, W.G. Daniel, R. Bonan, and J. Ludwig High-Dose Intravascular {beta}-Radiation After De Novo Stent Implantation Induces Coronary Artery Spasm Circulation, March 26, 2002; 105(12): 1420 - 1423. [Abstract] [Full Text] [PDF]

				T. A. Fischell and R. Virmani Intracoronary Brachytherapy in the Porcine Model: A Different Animal Circulation, November 13, 2001; 104(20): 2388 - 2390. [Full Text] [PDF]

				P. K. Coussement, H. de Leon, T. Ueno, M. Y. Salame, S. B. King III, N. A.F. Chronos, and K. A. Robinson Intracoronary {beta}-Radiation Exacerbates Long-Term Neointima Formation in Balloon-Injured Pig Coronary Arteries Circulation, November 13, 2001; 104(20): 2459 - 2464. [Abstract] [Full Text] [PDF]

				R. Waksman, A. E. Ajani, R. L. White, E. Pinnow, R. Dieble, A. B. Bui, M. Taaffe, L. Gruberg, G. S. Mintz, L. F. Satler, et al. Prolonged Antiplatelet Therapy to Prevent Late Thrombosis After Intracoronary {{gamma}}-Radiation in Patients With In-Stent Restenosis : Washington Radiation for In-Stent Restenosis Trial Plus 6 Months of Clopidogrel (WRIST PLUS) Circulation, May 15, 2001; 103(19): 2332 - 2335. [Abstract] [Full Text] [PDF]

				G. L. Kaluza, A. E. Raizner, W. Mazur, D. G. Schulz, J. M. Buergler, L. F. Fajardo, F. O. Tio, and N. M. Ali Long-Term Effects of Intracoronary {beta}-Radiation in Balloon- and Stent-Injured Porcine Coronary Arteries Circulation, April 24, 2001; 103(16): 2108 - 2113. [Abstract] [Full Text] [PDF]

				A. Farb, S. Shroff, M. John, W. Sweet, and R. Virmani Late Arterial Responses (6 and 12 Months) After 32P {beta}-Emitting Stent Placement : Sustained Intimal Suppression With Incomplete Healing Circulation, April 10, 2001; 103(14): 1912 - 1919. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Salame, M. Y. Articles by Robinson, K. A. Search for Related Content PubMed PubMed Citation Articles by Salame, M. Y. Articles by Robinson, K. A.