Eur J Cardio-thorac Surg (1992)6:422-426
Effect of aspirin on intimal proliferation and tissue cholesterol in long-term experimental bypass grafts* R. W. Landymore, M. A. MacAnlay, and J. Fris Departments of Surgery and Pathology, Dalhousie University, Halifax, Nova Scotia, Canada
Abstract. A single daily dose of aspirin (ASA) reduces the incidence of early graft thrombosis after coronary bypass operations. Recent data indicate that aspirin may not prevent intimal proliferation and cholesterol uptake in experimental bypass grafts which suggests that aspirin may not improve long-term graft patency. To further clarify the effects of aspirin on intimal proliferation and cholesterol metabolism, we performed femoral interposition vein grafts in 12 dogs receiving a 2% cholesterol diet. Six controls (CON) received the diet alone while the remaining animals received the diet with 160 mg aspirin daily before and for 9 months following operation. A segment of each graft was removed at 3 months for measurement of intimal thickness and tissue cholesterol. The entire graft was then harvested at 9 months. Intimal thickness increased rapidly during the first 3 months. A slow and progressive increase in intimal thickness was observed between 3 and 9 months. There was, however, no difference in intimal thickness between the two groups. Tissue cholesterol increased similarly in both groups. Rapid cholesterol uptake occurred within the first 3 months and then decreased between 3 and 9 months. Conclusions: (1) ASA failed to reduce intimal proliferation and cholesterol uptake in experimental bypass grafts suggesting that ASA may not prevent late graft failure, (2) Accelerated intimal proliferation and cholesterol uptake occurred within the first 3 months emphasizing the importance of developing and instituting anti-proliferative therapy immediately after aortocoronary bypass. [Enr J Cardio-thorac Snrg (1992) 63422-4263 Key words: Experimental
vein grafts - Intimal hyperplasia - Aspirin - Cholesterol
Aspirin will reduce the incidence of early graft thrombosis after coronary bypass operations [6,8,16,30] but may fail to prevent the long-term attrition of bypass grafts. Thrombotic occlusion [6, 8, 16, 301 causes graft failure within the first few months following operation while late graft failure is secondary to atherosclerosis [7, 12,35,36]. Although Boerboom [2], Bonchek [4] and Landymore [22] have shown that a combination of aspirin and Dipyridamole or a single daily aspirin will decrease cholesterol uptake in experimental vein grafts within the first 6- 12 weeks following operation, aspirin and Dipyridamole appear to have relatively little influence on cholesterol metabolism in chronic experimental grafts [3]. Aspirin may also fail to modify the proliferative response. Landymore [21] demonstrated that aspirin in low, medium, and high doses failed to reduce intimal
proliferation in experimental grafts within the first 6 weeks after operation. Although aspirin decreases the incidence of early graft thrombosis and beneficially influences cholesterol metabolism within the first few weeks following operation, the role of aspirin in the prevention of late graft failure is not fully understood. To clarify the effects of aspirin for the prevention of late graft failure we measured intimal thickness and tissue cholesterol in experimental grafts during the first 9 months after operation. Material and methods External jugular vein grafts were interposed between bilaterally divided femoral arteries in 12 adult mongrel dogs weighing between 25-30 kg. The animals were housed in air-conditioned pens and were cared for in accordance with the guidelines for animal care outlined by the Canadian Council on Animal Care.
Presented at the Poster Session of the 5th Annual Meeting of the European Association for Cardio-thoracic September 23-25, 1991
Surgery, London,
UK,
* Supported by a Medical Research Council Grant and a Heart and Stroke Foundation of Canada Grant
Surgical preparation Anaesthesia was induced with Somnotol and maintained with halothane. The animals were ventilated with a Harvard volume
423 ventilator after endotracheal intubation. A long segment of undistended jugular vein was removed from the neck and both femoral arteries were exposed after preparation of the skin with Betadine. A small segment of each graft was saved for baseline measurements of intimal thickness and tissue cholesterol. The remaining vein was then divided into two equal grafts. Heparin was administered in a dose of 5 mg/kg and the femoral arteries were clamped and divided. The grafts were anastomosed to the ends of the divided arteries with 6-O Prolene and with the aid of optical magnification. The incisions were then closed and the animals were allowed to recover. The animals were returned to the operating room at 3 and 9 months. At 3 months, half of each graft was removed for measurement of intimal thickness and analysis of cholesterol content. The ends of the divided graft were then reanastomosed with 6-O Prolene and the incisions were closed. At 9 months, the animals were sacrificed and the remainder of each graft was removed.
Treatment groups The animals were fed a commercially prepared 2% cholesterol diet I. The lipid-supplemented diet has been employed in numerous earlier investigations [19]. The diet causes a two-fold increase in serum cholesterol which accelerates intimal proliferation in experimental bypass grafts. The animals were divided into two equal groups. Six animals (controls) received the diet alone, while the remaining animals (treatment group) received the diet and 160 mg of aspirin daily 2 days before operation and for 9 months following operation.
Histological preparation and measurement of intimal hyperplasia Serial cross-sections were prepared at 2 mm intervals from graft specimens obtained before implantation and at 3 and 9 months. The cross-sections were stained with hematoxylin-eosin and masson stains. Measurements of intimal thickness were made by a blinded observer with a Zeiss interactive image analysing system. The Zeiss computerized image analyzer has been described in detail in an earlier report [19].
Hematological and biochemical analysis Hematocrit, prothrombin time, partial thromboplastin time, platelet counts, bleeding time, and serum cholesterol were measured before the diet and then at monthly intervals during the 9 months following operation. Graft cholesterol was measured from segments of vein obtained before implantation and from grafts removed at 3 and 9 months following operation. The analysis was carried out by an independent laboratory ’ in a blinded fashion. The methodology has been described in an earlier report [22].
Statistical analysis The results are reported as the arithmetic mean f standard error of the mean. The Student’s t-test and analysis of variance (ANOVA) were used for statistical comparisons.
control group and 4.0 f 0.4 mM/l in those animals receiving aspirin (PC 0.09). The diet caused a two-fold increase in the serum cholesterol in both groups. One month after institution of the diet, the serum cholesterol had increased to 9.7 + 0.9 mM/l (CON) and to 8.8 & 1.4 mM/l (ASA) group (P < 0.05). Serum cholesterol was measured monthly for 9 months following operation. The diet maintained a two-fold increase in serum cholesterol in both groups. (No significant difference between groups ANOVA). Prothrombin time, partial thromboplastin time and platelet counts were similar in both groups and were not affected by aspirin. However, the clotting time was prolonged in those animals receiving aspirin. The clotting time measured 101 Ifr5 s before operation and increased to 140-L-3 s following the administration of aspirin (P< 0.0001). Graft cholesterol concentration
Cholesterol concentration of the vein graft before implantation measured 1.69 + 0.1 mM/g. (The animals had been receiving the commercially prepared cholesterol diet for 1 week prior to operation and removal of the vein graft.) Cholesterol concentration increased to 2.5kO.2 mM/g at 3 months in the control group and 2.5 + 0.3 mM/g in the animals receiving aspirin (p < 0.1). At 9 months, the concentration of cholesterol in the grafts had decreased to 1.6fO.l mM/g in the control group and 1.3 ) 0.1 mM/g in those animals receiving aspirin (JJ< 0.2). Intimal thickness
The intimal thickness of the grafts before implantation measured 5 f 0.8 urn. Microscopically the intima represented a single layer of endothelial cells. By 3 months, the intima had increased to 31 f 6 urn in the control animals and to 25+ 3 urn in those animals receiving aspirin (P < 0.4). The intima measured 79 + 22 urn at 9 months in the control group and 67 + 12 urn in those animals receiving aspirin (P < 0.6). The greatest increase in intimal proliferation occurred within the first 3 months. The intima increased 520% in the control group and 400% in the ASA group. The accelerated intimal proliferation observed within the first 3 months gave way to a much more gradual and progressive increase in intimal thickness during the next 6 months. Between 3 and 9 months, the intima increased 155% in the control group and 168% in the animals taking aspirin.
Results Discussion
Serum cholesterol tended to be higher in the control group before the diet, measuring 4.9kO.3 mM/l in the 1 I.C.N. Nutritional Biochemicals, Cleveland, Ohio, USA ’ Efamol Research Incorporated, P.O. Box 818, Kentville, Scotia, Canada B4N 4H8
Nova
Earlier experimental investigations indicating that antiplatelet drug therapy would reduce intimal thickening in vein grafts [5, 28, 291 led to prospective randomized clinical trials which demonstrated that a combination of aspirin and Dipyridamole would reduce the incidence of
424
early graft failure after coronary bypass operations [6,8, 16,301. Aspirin and Dipyridamole were prescribed for all patients after coronary bypass until Brown [6] and Goldman [16] published data that suggested that a single daily aspirin was as effective as the combined drug regimen. The beneficial effects of aspirin on early graft patency, the efficacy of aspirin for the prevention of myocardial infarction [I, 331 and the cost effectiveness of aspirin have led to the prescription of aspirin for all cardiac patients. Thrombosis is the most common cause of graft failure within the first 3 months after coronary bypass while intima1 hyperplasia and atherosclerosis [7, 1I, 35, 361 are the leading causes of late graft failure. Early graft thrombosis may be reduced with antiplatelet drug therapy [6, 8, 16, 301or by the prescription of anticoagulants after myocardial revascularization [15]. The optimum drug regimen for the prevention of late graft failure is unknown. Drug therapy for the prevention of late graft failure must inhibit intimal proliferation and prevent the graft from accumulating cholesterol. There is increasing evidence to suggest that aspirin may not fulfill this role and that adjunctive therapy may be necessary to prevent the gradual attrition of vein bypass grafts. Boerboom [3] measured graft cholesterol concentration from 1 to 18 months after experimental vein bypass. Although cholesterol uptake was greater in the control group within the first 3 months in comparison with those animals in the treatment group, aspirin and Dipyridamole had no influence on cholesterol metabolism between 3 and 18 months. One group of animals discontinued aspirin and Dipyridamole 9 months following operation while a second group, on an identical drug regimen, continued antiplatelet drug therapy for a total of 18 months. Interestingly, the tissue cholesterol concentration after 18 months was identical in both groups which suggests that antiplatelet drug therapy may not influence cholesterol metabolism after the early accelerated phase. Our data supports the observations from Boerboom’s laboratory. In an earlier investigation [22], we demonstrated that aspirin reduced cholesterol uptake in experimental grafts within the first 6 weeks after veno-arterial bypass. Our current data, however, has shown that aspirin fails to influence cholesterol metabolism in long-term experimental grafts between 3 and 9 months after operation. Graft cholesterol was similar in both groups at 3 months and then decreased to baseline values by 9 months. This unexpected decrease in graft cholesterol between 3 and 9 months parallels the marked decrease in the rate of intimal proliferation occurring during the same time. We conclude from this data that cholesterol metabolism in a canine model is only accelerated during the early phase of accelerated intimal proliferation. The effects of antiplatelet drug regimens on intimal hyperplasia have been extensively investigated in a number of animal models [2, 19, 281. Independent investigators, more than a decade ago, reported that a combination of aspirin and Dipyridamole would reduce intimal proliferation in experimental bypass grafts [5, 28, 291. Although this drug combination is known to inhibit the early proliferative response, there has been a lack of data describing the individual effects of these drugs on intimal
proliferation and almost no investigations designed specifically to examine the effects of antiplatelet drug regimens on the fate of long-term grafts. Our laboratory, however, became interested in examining the individual effects of these drugs on intimal proliferation when the prescription of Dipyridamole fell into disrepute. Interestingly, we discovered that aspirin in low, medium, and high doses failed to reduce intimal hyperplasia in experimental bypass grafts within the first 2 months [17]. On the other hand, Dipyridamole affected a marked reduction in intimal proliferation following operation [ 161.The effects of these drugs on long-term intimal proliferation has recently been reported by Boerboom’s laboratory and confirmed by this report. Boerboom [2] reported the effects of aspirin and Dipyridamole on intimal hyperplasia in a primate animal model. Intimal thickening was reported as the area of the intima in relationship to the total cross-sectional area of the graft. The intima represented 13% of the cross-sectional area of the graft before implantation and increased to 33% of the total cross-sectional area at 1 month. The increase in intimal thickness was less dramatic after the first month with the intima measuring 59% of the total cross-sectional area at 3 months. Our data have confirmed the presence of an accelerated proliferative phase within the first few months following arterial bypass. We observed a 400% -500% increase in intimal thickness during the first 3 months. This rapid increase in intimal thickness that occurred during the first 3 months, however, was followed by a slow and more gradual increase in intimal proliferation between 3 and 9 months. Independent laboratories have confirmed that antiplatelet drug therapy with aspirin and Dipyridamole [2] or aspirin alone [18] will decrease the uptake of cholesterol during the early accelerated phase of cholesterol uptake that occurs within the first 2 months following operation but that these drugs failed to influence longterm cholesterol metabolism. More importantly, however, these laboratories [3,18] have shown that aspirin therapy has very little influence on the long-term proliferative response which suggests that aspirin may not prevent late graft failure. Clinical observations support these experimental investigations. Chesebro [9] has reported that Dipyridamole and aspirin, although effective in preventing early graft occlusion after aortocoronary bypass operations, fail to prevent smooth muscle cell proliferation in chronic aortocoronary bypass grafts [9]. These findings are not unexpected in the light of more recent data that has demonstrated that platelets, monocytes and endothelial cells all release growth factors which stimulate smooth muscle cell proliferation [31]. Thus, therapy directed specifically against platelets will fail because monocyte released growth factor and endotheliai released growth factor will undoubtedly continue to stimulate smooth muscle cell proliferation.
Relevance
01 the animal model
The effects of drug interactions on intimal proliferation and atherosclerosis have been investigated in small and
425 large animal models. Small animal models have included mice [26], rats [17], rabbits [32] and pigeons [27]. These animals develop fatty streaks after catheter abrasion and have been used extensively in the investigation of atherosclerosis. Unfortunately, their size renders them unacceptably small for veno-arterial bypass. Large animals have been used extensively to investigate the effects of drug regimens on intimal hyperplasia. The intimal proliferative response is recognized as the pathological lesion that precedes the development of atherosclerosis [7, 35,361. Large animal models that lend themselves to veno-arterial bypass have included canine, swine and non-human primate models. The canine model has been used by Metke [29], McCann [28] and Landymore [19, 251 to investigate the effects of drug regimens on the proliferative response following arterial bypass. The dog develops accelerated intimal proliferation after veno-arterial bypass when fed a cholesterol supplemented diet [18], although this animal model does not develop typical atherosclerotic lesions within the first year after bypass. The advantage of the canine model is related to the fact that this animal is readily available and is easy to care for following operation. Swine [37] have been used as a veno-arterial bypass animal model but are difticult to intubate, operate upon and grow to excessive size and are thus hard to handle for a long-term experiment. The nonhuman primate model [2, 31 when fed a cholesterol-supplemented diet develops a lipid profile that is similar to humans, intimal hyperplasia and atherosclerotic coronary artery lesions, but unfortunately is extremely expensive and difficult to handle so that non-human primates are not allowed to be utilized in our Animal Care Centre. The canine animal model was used in earlier experiments to investigate the effects of aspirin and Dipyridamole on intimal proliferation and graft patency [28, 291. The results of these studies have led to randomized double-blind clinical trials that supported the use of antiplatelet drug regimens in the prevention of intimal proliferation in graft occlusion [8, 91. Other drug regimens that reduce the proliferative response in experimental canine grafts have also shown to be useful in man. Our laboratory demonstrated the efficacy of cod-liver oil in the prevention of intimal hyperplasia [23 -251 while later studies indicate that a fish oil supplement will reduce intimal proliferation in veno-arterial shunts [13] and following coronary angioplasty [l 11. Interestingly, results from Boerboom’s laboratory [2, 31 using a non-primate model that more closely simulates the effects of antiplatelet drug regimens on human coronary arteries are identical to the observations made in our laboratory using canine animal model. Thus, although the canine animal model may not be appropriate for the investigation of atherosclerosis, this model may be used to examine the effects of drug interventions on the proliferative response.
Conclusions Our data indicate that the acceleration of intimal proliferation that occurs immediately after veno-arterial by-
pass is coupled with a phase of accelerated cholesterol uptake. Similarly, the decelerated phase of intimal proliferation that is observed between 3 and 9 months after operation is associated with reduced cholesterol uptake. Thus, it would appear that cholesterol metabolism closely follows the rate of smooth muscle cell proliferation. The failure of aspirin to modify the proliferative response and reduce cholesterol metabolism [3] suggests that renewed efforts are necessary to increase our understanding of the pathophysiology of the proliferative response. Furthermore, our observations suggest that we should develop targeted therapy to prevent the accelerated phase of intimal proliferation and cholesterol uptake that occurs within the first few weeks following operation.
References 1. Antiplatelet trialists’ collaboration (1988) Secondary prevention of vascular disease by prolonged antiplatelet treatment. Br Med J 296:320-331 2. Boerboom L, Olinger G, Liu T-Z, Rodriquez ER, Ferrans V, Kissebach A (1990) Histologic, morphometric, and biochemical evolution of vein bypass grafts in a non-human primate model: sequential changes within the first three months. J Thorac Cardiovasc Surg 99: 97- 106 3. Boerboom L, Olinger G, Liu T-Z, Rodriquez E, Ferrans V, Kissebach A (1990) A histologic, morphometric, and biochemical evolution of vein bypass grafts in a non-human primate model III: Long-term changes and their modification by platelet inhibition with aspirin and Dipyridamole. J Thorac Cardiovasc Surg 99: 426-432 4. Bonchek L, Boerboom L, Olinger G, Pepper J, Munns J, Hutchinson L, Kissebach A (1982) Prevention of lipid accumulation in experimental vein bypass grafts by antiplatelet therapy. Circulation 66: 338 -341 5. Brody W, Brown J, Reitz R, Fry D, Michaelis L (1977) Effects of Dipyridamole and methylprednisolone on intimal thickening in vein grafts. J Thorac Cardiovasc Surg 73: 602-604 6. Brown B, Cukingham R, DeRouen T, Goede L, Wong M, Fee H, Roth J, Carey J (1985) Improved graft patency in patients treated with platelet-inhibiting therapy after coronary bypass surgery. Circulation 72: 138 - 146 I. Campeau L, Enjalbert M, Lesperance J, Vaislic C, Grondin C, Bourassa M (1983) Atherosclerosis and late closure of aortocoronary saphenous vein grafts: sequential angiographic studies at 2 weeks, 1 year, 5 to 7 years, and IO to 12 years after surgery. Circulation 68 [Suppl II]: l-7 (PT 2) 8. Chesebro J, Clements I, Fuster V, Elveback L, Smith H, Bardsley W, Frye R, Holmes D, Vlietstra R, Pluth J, Wallace R, Puga F, Orszulak T, Piehler J, Schaffh-Danielson G (1982) A plateletinhibitor drug trial in coronary-artery bypass operations. N Engl J Med 307: 73-78 9. Chesebro J, Fuster V, Elveback L, Smith H, Jacobs J, Pendergast W, Puga F (1984) Does Dipyridamole plus aspirin affect aortocoronary vein graft narrowing over the first postoperative year? Circulation 70 [Suppl II]: 105 (Pt 2) IO. Decampli W, Kosek J, Mitchell R, Handen C, Miller C (1988) Effects of aspirin, Dipyridamole, and cod-liver oil on accelerated myointimal proliferation in canine veno-arterial allografts. Ann Surg 208: 746-754 11. Dehmer G, Popma J, Van Den Berg E, Eichorn E, Prewitt J, Campbell W, Jennings L, Willerson J, Schmitz J (1988) Reduction in the rate of early restenosis after coronary angioplasty by a diet supplemented with N-3 fatty acids. N Engl J Med 319: 733 -740
426 12. Dilley R, McGeachie J, Prendergast F (1988) A review of the histologic changes in vein-to-artery grafts, with particular reference to intimal hyperplasia. Arch Surg 123: 691-696 13. Diskin C, Thomas C, Zellner C, Lock S, Tanja J (1990) Fish oil to prevent intimal hyperplasia and access thrombosis. Nephron 55445-447 14. Ferraris V, Ferraris S, Lough F, Berry W (1988) Preoperative aspirin ingestion increases operative blood loss after coronary artery bypass grafting. Ann Thorac Surg 45: 71-74 15. Gohlke H, Gohlke-Barwolf C, Sturzenhofecker P, Gornandt L, Ritter B, Reichelt M, Buchwalsky R, Schmuziger M, Roskamm H (1981) Improved graft patency with anticoagulant therapy after aortocoronary bypass surgery: a prospective, randomized study. Circulation 64 [Suppl II]: 22-27 16. Goldman S, Copeland J, Moritz T, Henderson W, Zadina K, Ovitt T, Doherty J, Read R, Chesler E, Sako Y, Lancaster L, Emery R, Sharma G, Josa M, Pacold I, Montoya A, Parikh D, Sethi G, Holt J, Kirklin J, Shabetai R, Moores W, Alderidge J, Masud Z, DeMots H, Floten S, Haakenson C, Harker L (1988) Improvement in early saphenous vein graft patency after coronary artery bypass surgery with antiplatelet therapy: results of a veterans administration cooperative study. Circulation 77: 132441332 17. Joris I, Zand T, Nunnari J, Krolikowski J, Majno G (1983) Studies on the pathogenesis of atherosclerosis. Am J Path01 113:341-358 18. Karayannacos P, Hostetler J, Bond G, Kakos G, Williams R, Kilman J, Vasko J (1978) Late failure in vein grafts. Ann Surg 187: 183-188 19. Landymore R, Kinley C, Cameron C (1985) Intimal hyperplasia in autogenous vein grafts for arterial bypass: a canine mode. Cardiovasc Res 19: 589-592 20. Landymore R, Karmazyn M, MacAulay M, Sheridan B, Cameron C (1988) Correlation between the effects of aspirin and Dipyridamole on platelet function and prevention of intima1 hyperplasia in autologous vein grafts. Can J Cardio14: 5659 21. Landymore R, MacAulay MA, Manku M (1990) The effects of low, medium, and high dose aspirin in intimal proliferation in autogenous veins used for arterial reconstruction. Eur J Cardiothorac Surg 4: 441-444 22. Landymore RW, MacAulay MA, Fris J (1991) Effect of aspirin on intimal hyperplasia and cholesterol uptake in experimental bypass grafts. Can J Cardiol 7: 87-90 23. Landymore RW, Cameron C, Sheridan B, MacAulay MA (1986) Reduction of intimal hyperplasia in canine autologous vein grafts with a combination of cod-liver oil and dipyridamole. Can J Surg 29: 357-358 24. Landymore RW, Kinley CE, MacAuley MA, Cooper J, Sheridan B, Cameron C (1985) Cod-liver oil in the prevention on intimal hyperplasia in autogenous vein grafts used for arterial bypass. J Thorac Cardiovasc Surg 89: 351-357
25. Landymore RW, MacAuley MA, Sheridan B, Cameron C (1987) Cumulative experience with eicosapaentoic acid and other platelet inhibitors for the prevention of intimal hyperplasia in autologous vein grafts. In: Lands WEM (ed) Proceedings of the AOCS Short Course of Polyunsaturated Fatty Acids and Eicosanoids, p 340 26. Leake D, Rankin S (1990) The oxidative modification of lowdensity lipoproteins by macrophages. Biochem J 270: 741-748 27. Lewis J, Taylor R, Jones N, St. Clair R, Cornhill J (1982) Endothelial surface characteristics in pigeon coronary artery atherosclerosis I. cellular alterations during the initial stages of dietary cholesterol challenge. Lab Invest 46: 123-138 28. McCann R, Hagen P-O Fuchs J (1980) Aspirin and Dipyridamole decrease intimal hyperplasia in experimental vein grafts. Ann Surg 191:238-243 29. Metke M, Lie J, Fuster V, Josa M, Kaye M (1979) Reduction of intimal thickening in canine coronary bypass vein grafts with Dipyridamole and aspirin. Am J Cardiol 43: 11441148 30. Mayer J, Lindsay W, Castaneda W, Nicoloff D (1981) Influence of aspirin and Dipyridamole on patency of coronary artery bypass grafts. Ann Thorac Surg 31:204-210 31. Ross R, Raines E, Bowen-Pope D (1982) Growth factors from platelets, monocytes, and endothelium: their role in cell proliferation. Ann NY Acad Sci 397: 18-24 32. Sarembock I, LaVeau P, Sigal S, Timms I, Sussman J, Handenschild C, Ezekowitz M (1989) Influence of inflation pressure and balloon size on the development of intimal hyperplasia after balloon angioplasty. Circulation 80: 1029-1040 33. Steering Committee of the Physician’s Health Study Research Group Preliminary Report (1988) Findings from the aspirin component of the ongoing physician’s health study. N Engl J Med 318:262-269 34. Tennant M, Dilley R, McGeachie J, Prendergast F (1990) Histogenesis of arterial intimal hyperplasia and atherosclerosis. Aust NZ J Surg 60: 79-85 35. Unni K, Kottke B, Titus J, Frye R, Wallace R, Brown A (1974) Pathologic changes in aortocoronary saphenous vein grafts. Am J Cardiol 34: 526-532 36. Vlodaver Z, Edwards J (1971) Pathologic changes in aorticcoronary arterial saphenous vein grafts. Circulation 44: 719728 37. Weiner B, Ockene I, Levine P, Cuenoud H, Fisher M, Johnson B, Daoud A, Jarmolych J, Hosmer D, Johnson M, Natale A, Vaudrevil C, Hoogasian J (1986) Inhibition of atherosclerosis by cod-liver oil in a hyperlipemic swine model. N Engl J Med 315:841-846 Dr. R. W. Landymore Room 3065, R. C. Dickson Centre Victoria General Hospital Halifax, Nova Scotia Canada B3H 2Y9
Effect of aspirin on intimal proliferation and tissue cholesterol in long-term experimental bypass grafts* R. W. Landymore, M. A. MacAnlay, and J. Fris Departments of Surgery and Pathology, Dalhousie University, Halifax, Nova Scotia, Canada
Abstract. A single daily dose of aspirin (ASA) reduces the incidence of early graft thrombosis after coronary bypass operations. Recent data indicate that aspirin may not prevent intimal proliferation and cholesterol uptake in experimental bypass grafts which suggests that aspirin may not improve long-term graft patency. To further clarify the effects of aspirin on intimal proliferation and cholesterol metabolism, we performed femoral interposition vein grafts in 12 dogs receiving a 2% cholesterol diet. Six controls (CON) received the diet alone while the remaining animals received the diet with 160 mg aspirin daily before and for 9 months following operation. A segment of each graft was removed at 3 months for measurement of intimal thickness and tissue cholesterol. The entire graft was then harvested at 9 months. Intimal thickness increased rapidly during the first 3 months. A slow and progressive increase in intimal thickness was observed between 3 and 9 months. There was, however, no difference in intimal thickness between the two groups. Tissue cholesterol increased similarly in both groups. Rapid cholesterol uptake occurred within the first 3 months and then decreased between 3 and 9 months. Conclusions: (1) ASA failed to reduce intimal proliferation and cholesterol uptake in experimental bypass grafts suggesting that ASA may not prevent late graft failure, (2) Accelerated intimal proliferation and cholesterol uptake occurred within the first 3 months emphasizing the importance of developing and instituting anti-proliferative therapy immediately after aortocoronary bypass. [Enr J Cardio-thorac Snrg (1992) 63422-4263 Key words: Experimental
vein grafts - Intimal hyperplasia - Aspirin - Cholesterol
Aspirin will reduce the incidence of early graft thrombosis after coronary bypass operations [6,8,16,30] but may fail to prevent the long-term attrition of bypass grafts. Thrombotic occlusion [6, 8, 16, 301 causes graft failure within the first few months following operation while late graft failure is secondary to atherosclerosis [7, 12,35,36]. Although Boerboom [2], Bonchek [4] and Landymore [22] have shown that a combination of aspirin and Dipyridamole or a single daily aspirin will decrease cholesterol uptake in experimental vein grafts within the first 6- 12 weeks following operation, aspirin and Dipyridamole appear to have relatively little influence on cholesterol metabolism in chronic experimental grafts [3]. Aspirin may also fail to modify the proliferative response. Landymore [21] demonstrated that aspirin in low, medium, and high doses failed to reduce intimal
proliferation in experimental grafts within the first 6 weeks after operation. Although aspirin decreases the incidence of early graft thrombosis and beneficially influences cholesterol metabolism within the first few weeks following operation, the role of aspirin in the prevention of late graft failure is not fully understood. To clarify the effects of aspirin for the prevention of late graft failure we measured intimal thickness and tissue cholesterol in experimental grafts during the first 9 months after operation. Material and methods External jugular vein grafts were interposed between bilaterally divided femoral arteries in 12 adult mongrel dogs weighing between 25-30 kg. The animals were housed in air-conditioned pens and were cared for in accordance with the guidelines for animal care outlined by the Canadian Council on Animal Care.
Presented at the Poster Session of the 5th Annual Meeting of the European Association for Cardio-thoracic September 23-25, 1991
Surgery, London,
UK,
* Supported by a Medical Research Council Grant and a Heart and Stroke Foundation of Canada Grant
Surgical preparation Anaesthesia was induced with Somnotol and maintained with halothane. The animals were ventilated with a Harvard volume
423 ventilator after endotracheal intubation. A long segment of undistended jugular vein was removed from the neck and both femoral arteries were exposed after preparation of the skin with Betadine. A small segment of each graft was saved for baseline measurements of intimal thickness and tissue cholesterol. The remaining vein was then divided into two equal grafts. Heparin was administered in a dose of 5 mg/kg and the femoral arteries were clamped and divided. The grafts were anastomosed to the ends of the divided arteries with 6-O Prolene and with the aid of optical magnification. The incisions were then closed and the animals were allowed to recover. The animals were returned to the operating room at 3 and 9 months. At 3 months, half of each graft was removed for measurement of intimal thickness and analysis of cholesterol content. The ends of the divided graft were then reanastomosed with 6-O Prolene and the incisions were closed. At 9 months, the animals were sacrificed and the remainder of each graft was removed.
Treatment groups The animals were fed a commercially prepared 2% cholesterol diet I. The lipid-supplemented diet has been employed in numerous earlier investigations [19]. The diet causes a two-fold increase in serum cholesterol which accelerates intimal proliferation in experimental bypass grafts. The animals were divided into two equal groups. Six animals (controls) received the diet alone, while the remaining animals (treatment group) received the diet and 160 mg of aspirin daily 2 days before operation and for 9 months following operation.
Histological preparation and measurement of intimal hyperplasia Serial cross-sections were prepared at 2 mm intervals from graft specimens obtained before implantation and at 3 and 9 months. The cross-sections were stained with hematoxylin-eosin and masson stains. Measurements of intimal thickness were made by a blinded observer with a Zeiss interactive image analysing system. The Zeiss computerized image analyzer has been described in detail in an earlier report [19].
Hematological and biochemical analysis Hematocrit, prothrombin time, partial thromboplastin time, platelet counts, bleeding time, and serum cholesterol were measured before the diet and then at monthly intervals during the 9 months following operation. Graft cholesterol was measured from segments of vein obtained before implantation and from grafts removed at 3 and 9 months following operation. The analysis was carried out by an independent laboratory ’ in a blinded fashion. The methodology has been described in an earlier report [22].
Statistical analysis The results are reported as the arithmetic mean f standard error of the mean. The Student’s t-test and analysis of variance (ANOVA) were used for statistical comparisons.
control group and 4.0 f 0.4 mM/l in those animals receiving aspirin (PC 0.09). The diet caused a two-fold increase in the serum cholesterol in both groups. One month after institution of the diet, the serum cholesterol had increased to 9.7 + 0.9 mM/l (CON) and to 8.8 & 1.4 mM/l (ASA) group (P < 0.05). Serum cholesterol was measured monthly for 9 months following operation. The diet maintained a two-fold increase in serum cholesterol in both groups. (No significant difference between groups ANOVA). Prothrombin time, partial thromboplastin time and platelet counts were similar in both groups and were not affected by aspirin. However, the clotting time was prolonged in those animals receiving aspirin. The clotting time measured 101 Ifr5 s before operation and increased to 140-L-3 s following the administration of aspirin (P< 0.0001). Graft cholesterol concentration
Cholesterol concentration of the vein graft before implantation measured 1.69 + 0.1 mM/g. (The animals had been receiving the commercially prepared cholesterol diet for 1 week prior to operation and removal of the vein graft.) Cholesterol concentration increased to 2.5kO.2 mM/g at 3 months in the control group and 2.5 + 0.3 mM/g in the animals receiving aspirin (p < 0.1). At 9 months, the concentration of cholesterol in the grafts had decreased to 1.6fO.l mM/g in the control group and 1.3 ) 0.1 mM/g in those animals receiving aspirin (JJ< 0.2). Intimal thickness
The intimal thickness of the grafts before implantation measured 5 f 0.8 urn. Microscopically the intima represented a single layer of endothelial cells. By 3 months, the intima had increased to 31 f 6 urn in the control animals and to 25+ 3 urn in those animals receiving aspirin (P < 0.4). The intima measured 79 + 22 urn at 9 months in the control group and 67 + 12 urn in those animals receiving aspirin (P < 0.6). The greatest increase in intimal proliferation occurred within the first 3 months. The intima increased 520% in the control group and 400% in the ASA group. The accelerated intimal proliferation observed within the first 3 months gave way to a much more gradual and progressive increase in intimal thickness during the next 6 months. Between 3 and 9 months, the intima increased 155% in the control group and 168% in the animals taking aspirin.
Results Discussion
Serum cholesterol tended to be higher in the control group before the diet, measuring 4.9kO.3 mM/l in the 1 I.C.N. Nutritional Biochemicals, Cleveland, Ohio, USA ’ Efamol Research Incorporated, P.O. Box 818, Kentville, Scotia, Canada B4N 4H8
Nova
Earlier experimental investigations indicating that antiplatelet drug therapy would reduce intimal thickening in vein grafts [5, 28, 291 led to prospective randomized clinical trials which demonstrated that a combination of aspirin and Dipyridamole would reduce the incidence of
424
early graft failure after coronary bypass operations [6,8, 16,301. Aspirin and Dipyridamole were prescribed for all patients after coronary bypass until Brown [6] and Goldman [16] published data that suggested that a single daily aspirin was as effective as the combined drug regimen. The beneficial effects of aspirin on early graft patency, the efficacy of aspirin for the prevention of myocardial infarction [I, 331 and the cost effectiveness of aspirin have led to the prescription of aspirin for all cardiac patients. Thrombosis is the most common cause of graft failure within the first 3 months after coronary bypass while intima1 hyperplasia and atherosclerosis [7, 1I, 35, 361 are the leading causes of late graft failure. Early graft thrombosis may be reduced with antiplatelet drug therapy [6, 8, 16, 301or by the prescription of anticoagulants after myocardial revascularization [15]. The optimum drug regimen for the prevention of late graft failure is unknown. Drug therapy for the prevention of late graft failure must inhibit intimal proliferation and prevent the graft from accumulating cholesterol. There is increasing evidence to suggest that aspirin may not fulfill this role and that adjunctive therapy may be necessary to prevent the gradual attrition of vein bypass grafts. Boerboom [3] measured graft cholesterol concentration from 1 to 18 months after experimental vein bypass. Although cholesterol uptake was greater in the control group within the first 3 months in comparison with those animals in the treatment group, aspirin and Dipyridamole had no influence on cholesterol metabolism between 3 and 18 months. One group of animals discontinued aspirin and Dipyridamole 9 months following operation while a second group, on an identical drug regimen, continued antiplatelet drug therapy for a total of 18 months. Interestingly, the tissue cholesterol concentration after 18 months was identical in both groups which suggests that antiplatelet drug therapy may not influence cholesterol metabolism after the early accelerated phase. Our data supports the observations from Boerboom’s laboratory. In an earlier investigation [22], we demonstrated that aspirin reduced cholesterol uptake in experimental grafts within the first 6 weeks after veno-arterial bypass. Our current data, however, has shown that aspirin fails to influence cholesterol metabolism in long-term experimental grafts between 3 and 9 months after operation. Graft cholesterol was similar in both groups at 3 months and then decreased to baseline values by 9 months. This unexpected decrease in graft cholesterol between 3 and 9 months parallels the marked decrease in the rate of intimal proliferation occurring during the same time. We conclude from this data that cholesterol metabolism in a canine model is only accelerated during the early phase of accelerated intimal proliferation. The effects of antiplatelet drug regimens on intimal hyperplasia have been extensively investigated in a number of animal models [2, 19, 281. Independent investigators, more than a decade ago, reported that a combination of aspirin and Dipyridamole would reduce intimal proliferation in experimental bypass grafts [5, 28, 291. Although this drug combination is known to inhibit the early proliferative response, there has been a lack of data describing the individual effects of these drugs on intimal
proliferation and almost no investigations designed specifically to examine the effects of antiplatelet drug regimens on the fate of long-term grafts. Our laboratory, however, became interested in examining the individual effects of these drugs on intimal proliferation when the prescription of Dipyridamole fell into disrepute. Interestingly, we discovered that aspirin in low, medium, and high doses failed to reduce intimal hyperplasia in experimental bypass grafts within the first 2 months [17]. On the other hand, Dipyridamole affected a marked reduction in intimal proliferation following operation [ 161.The effects of these drugs on long-term intimal proliferation has recently been reported by Boerboom’s laboratory and confirmed by this report. Boerboom [2] reported the effects of aspirin and Dipyridamole on intimal hyperplasia in a primate animal model. Intimal thickening was reported as the area of the intima in relationship to the total cross-sectional area of the graft. The intima represented 13% of the cross-sectional area of the graft before implantation and increased to 33% of the total cross-sectional area at 1 month. The increase in intimal thickness was less dramatic after the first month with the intima measuring 59% of the total cross-sectional area at 3 months. Our data have confirmed the presence of an accelerated proliferative phase within the first few months following arterial bypass. We observed a 400% -500% increase in intimal thickness during the first 3 months. This rapid increase in intimal thickness that occurred during the first 3 months, however, was followed by a slow and more gradual increase in intimal proliferation between 3 and 9 months. Independent laboratories have confirmed that antiplatelet drug therapy with aspirin and Dipyridamole [2] or aspirin alone [18] will decrease the uptake of cholesterol during the early accelerated phase of cholesterol uptake that occurs within the first 2 months following operation but that these drugs failed to influence longterm cholesterol metabolism. More importantly, however, these laboratories [3,18] have shown that aspirin therapy has very little influence on the long-term proliferative response which suggests that aspirin may not prevent late graft failure. Clinical observations support these experimental investigations. Chesebro [9] has reported that Dipyridamole and aspirin, although effective in preventing early graft occlusion after aortocoronary bypass operations, fail to prevent smooth muscle cell proliferation in chronic aortocoronary bypass grafts [9]. These findings are not unexpected in the light of more recent data that has demonstrated that platelets, monocytes and endothelial cells all release growth factors which stimulate smooth muscle cell proliferation [31]. Thus, therapy directed specifically against platelets will fail because monocyte released growth factor and endotheliai released growth factor will undoubtedly continue to stimulate smooth muscle cell proliferation.
Relevance
01 the animal model
The effects of drug interactions on intimal proliferation and atherosclerosis have been investigated in small and
425 large animal models. Small animal models have included mice [26], rats [17], rabbits [32] and pigeons [27]. These animals develop fatty streaks after catheter abrasion and have been used extensively in the investigation of atherosclerosis. Unfortunately, their size renders them unacceptably small for veno-arterial bypass. Large animals have been used extensively to investigate the effects of drug regimens on intimal hyperplasia. The intimal proliferative response is recognized as the pathological lesion that precedes the development of atherosclerosis [7, 35,361. Large animal models that lend themselves to veno-arterial bypass have included canine, swine and non-human primate models. The canine model has been used by Metke [29], McCann [28] and Landymore [19, 251 to investigate the effects of drug regimens on the proliferative response following arterial bypass. The dog develops accelerated intimal proliferation after veno-arterial bypass when fed a cholesterol supplemented diet [18], although this animal model does not develop typical atherosclerotic lesions within the first year after bypass. The advantage of the canine model is related to the fact that this animal is readily available and is easy to care for following operation. Swine [37] have been used as a veno-arterial bypass animal model but are difticult to intubate, operate upon and grow to excessive size and are thus hard to handle for a long-term experiment. The nonhuman primate model [2, 31 when fed a cholesterol-supplemented diet develops a lipid profile that is similar to humans, intimal hyperplasia and atherosclerotic coronary artery lesions, but unfortunately is extremely expensive and difficult to handle so that non-human primates are not allowed to be utilized in our Animal Care Centre. The canine animal model was used in earlier experiments to investigate the effects of aspirin and Dipyridamole on intimal proliferation and graft patency [28, 291. The results of these studies have led to randomized double-blind clinical trials that supported the use of antiplatelet drug regimens in the prevention of intimal proliferation in graft occlusion [8, 91. Other drug regimens that reduce the proliferative response in experimental canine grafts have also shown to be useful in man. Our laboratory demonstrated the efficacy of cod-liver oil in the prevention of intimal hyperplasia [23 -251 while later studies indicate that a fish oil supplement will reduce intimal proliferation in veno-arterial shunts [13] and following coronary angioplasty [l 11. Interestingly, results from Boerboom’s laboratory [2, 31 using a non-primate model that more closely simulates the effects of antiplatelet drug regimens on human coronary arteries are identical to the observations made in our laboratory using canine animal model. Thus, although the canine animal model may not be appropriate for the investigation of atherosclerosis, this model may be used to examine the effects of drug interventions on the proliferative response.
Conclusions Our data indicate that the acceleration of intimal proliferation that occurs immediately after veno-arterial by-
pass is coupled with a phase of accelerated cholesterol uptake. Similarly, the decelerated phase of intimal proliferation that is observed between 3 and 9 months after operation is associated with reduced cholesterol uptake. Thus, it would appear that cholesterol metabolism closely follows the rate of smooth muscle cell proliferation. The failure of aspirin to modify the proliferative response and reduce cholesterol metabolism [3] suggests that renewed efforts are necessary to increase our understanding of the pathophysiology of the proliferative response. Furthermore, our observations suggest that we should develop targeted therapy to prevent the accelerated phase of intimal proliferation and cholesterol uptake that occurs within the first few weeks following operation.
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