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Allograft aortopathy: An in vivo study of donor aorta involvement in cardiac allograft vasculopathy
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AIIograft aortopathy: An in vivo study of donor aorta involvement in cardiac allograft vasculopathy Mandeep R. Mehra, MD, Hector O. Ventura, MD, Dwight D. Stapleton, MD, Arun K. Karsan, MD, Frank W. Smart, MD, Stephen R. Ramee, MD, and Tyrone J. Collins, MD New Orleans, La. Limited histopathologic studies of failed cardiac allografts have demonstrated that cardiac allograft vasculopathy extends into the donor aorta; however, no study has examined the development of allograft aortic intimal proliferation in vivo in conjunction with coronary intimal hyperplasia. By using simultaneous intracoronary and intraaortic ultrasound, we studied 20 consecutive heart transplant recipients at 2.5 ± 2.1 years after transplantation. The degree of coronary intimal thickening was strongly correlated with the development of intraaortic intimal hyperplasia (r=0.90; p < 0.0001). Multivariate predictors of aortic intimal thickening included years after transplant (r = 0.47; p = 0.03), serum cholesterol level (r-- 0,65, p = 0.003), and serum triglyceride level (r= 0.51; p = 0.03). AIIograft aortopathy occurs in a similar manner to allograft coronary disease, thus providing support for the notion that an immunologic stimulus operating across the allograft vascular bed may be responsible for the development of cardiac allograft vasculopathy. Furthermore, this investigation provides insight into the putative role of hyperlipidemia in allograft vascular disease. (Am Heart J 1997;133:698-702.)
Cardiac allograft vasculopathy, a unique and accel: erated form of coronary arteriopathy manifested by diffuse and longitudinal myointimal proliferation, is currently the leading cause of cardiac allograft attrition after the first year following transplantation. 1 The exact cause of this disorder remains uncertain; however, several lines of clinical and experimental evidence suggest that this disorder is an immunologically initiated and metabolically propagated form of coronary arteriopathy. 2, 3 In fact, an early clinical investigation by Hess et al. 2 suggested that the From the Department of Internal Medicine, Section on Cardiology, Ochsner Medical Institutions. Received for publication Aug. 1, 1996; accepted March 7, 1997. Reprint requests: Mandeep R. Mehra, MD, Department of Internal Medicine, Section on Cardiology, Ochsner Medical Institutions, 1514 Jefferson Highway, New Orleans, LA 70121. Copyright © 1997 by Mosby-Year Book, Inc. 0002-8703/97/$5.00 + 0 4/1/81799
698
development of diffuse intimal proliferation depended on the concerted interaction of cytotoxic B-cell antibodies and hypercholesterolemia. If the immunologic hypothesis is to be endorsed, cardiac allograft vasculopathy must then extend to the allograft vascular structures beyond the coron a r y arteries. Indeed, limited histopathologic studies of failed cardiac allografts with terminally extensive coronary arteriopathy have suggested that cardiac allograft vasculopathy is characterized by extension into the donor aorta up to the suture lines and, in some instances, even into the allograft venous structures. 4-6 However, no study to date has assessed the early development of aortic intimal thickening in vivo in conjunction with the genesis of coronary intimal proliferation. Intravascular ultrasound has recently been validated as an effective clinical imaging device in the accurate assessment of cardiac allograft vasculopathy. 7,s Several studies have directed attention to the remarkable reproducibility and safety of intravascular ultrasound in serially characterizing the development of allograft coronary arteriopathy by delineating vessel wall morphologic characteristics in vivo. 9,1° Thus intravascular ultrasound can be extended to study the genesis of coronary arterial involvement in cardiac allograft vasculopathy and to investigate the simultaneous occurrence of donor aortic involvement in this diffuse, proliferative process by performing intraaortic ultrasonography. The purpose of this study was threefold: (1) to assess the early development ofintimal proliferation in the allograft aorta with intravascular ultrasound; (2) to compare the degree of development of aortic disease with coronary intimal proliferation; and (3) to define the risk factors associated with the development of allograft aortopathy in heart transplant recipients.
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American Heart Journal
METHODS Patients. The study population consisted of a prospectively assembled cohort of 24 consecutive heart transplant recipients aged 53 -+ 9 years (18 men and six women) who underwent intravascular ultrasound examination at the time of routine annual examination at a mean duration of 2.5 -+ 1.8 years after transplantation. All recipients received identical triple immunosuppressive therapy (cyclosporine, predulsone, and azathioprine) and did not have acute rejection or infection at the time of the study. Informed consent was obtained from all patients, and the study was approved by the Institutional Review Board. Immunologic risk factors. Episodes of cellular or humeral rejection requiring treatment and the number of human leukocyte antigens (HLA) A, B, or DR matches between the donor and recipient were examined in all patients. A first-year mean biopsy rejection score was determined for all patients in an effort to assess the influence of the severity and frequency of rejection and to account for insidious episodes of cellular rejection. This score was adapted from the standardized criteria established by the International Society for Heart and Lung Transplantation. 11 Individual scores were assigned to each defined biopsy grade as follows: grade 0, 0; grade IA, 1; grade IB, 2; grade 2, 3; grade 3A, 4; grade 3B, 5; and grade 4, 6. The mean biopsy score was computed as the average of all biopsy scores during the first year after transplantation. Furthermore, detailed analysis of immunosuppressive regimens, including cyclosporine dose and levels, prednisone dose and cumulative consumption, azathioprine dose, and use of induction OKT3 was also performed. Nonimmunologic risk factors. Donor age and sex, obesity indexes (percentage weight gain, body mass index), hypertension, history of cytomegalovirus infection requiring therapeutic intervention with intravenous ganciclovir, fasting lipid profile (triglyceride, total cholesterol, lowdensity lipoprotein cholesterol and high-density lipoprotein cholesterol levels) at the time of ultrasound examination, history of diabetes mellitus, ischemic time, and time from transplantation were assessed in all heart transplant recipients. Intravascular ultrasound A o r t i c a n d c o r o n a r y u l t r a s o u n d procedure. Intracoronary ultrasound was performed with a commercially available 2.9F catheter with a central frequency of 30 MHz (Cardiovascular Imaging Systems, Sunnyvale, Calif.). The procedure for performing intravascular ultrasound has been previously described, s After performance of the intracoronary ultrasound, intraaortic ultrasonography was performed with an 8F ultrasound catheter with a central frequency of 20 MHz (Cardiovascular Imaging Systems, Sunnyvale, Calif.) over a 0.018-inch guide wire. The ultrasound catheter was advanced to the aortic valve and slowly withdrawn while imaging the allograft aorta up to the suture lines. Additionally, fluoroscopic pictures and audio annotations were used to ensure the correct localization of the artery segment for subsequent offiine analysis. A o r t i c a n d c o r o n a r y u l t r a s o u n d a s s e s s m e n t . All images
699
Table I. Clinical characteristics in cardiac transplant recipients without and with aortic intimal thickness Aortic intimal thickness
No (n = 10)
Yes (n = 14)
Age (yr) Time after transplant (yr) Ischemic time (min) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) % Weight gain after transplant Donor age (yr)
55 =~11 1.3 -+ 1.4 157 -+ 64 202 _+43 124 --+41 48 -+ 15 153 -+ 46 11 _+8 25 -+ 10
52 _+9 3.3 -+ 2.1" 192 -+ 47 261 -+ 47* 151 :~ 49 40 + 16 264 _+152" 14 _+6.8 31 _+11
LDL, Low-densitylipoprotein;HDL, high-densitylipopretein.
*p < 0.05. were analyzed twice by two independent observers who had no knowledge of the results from prior assessments of nonimmunologic risk factors. Three coronary sites per vessel (proximal, mid, and distal), for a total of 72 coronary artery segments, were evaluated. Maximal intimal thickness and intimal area were obtained by tracing the lumen vessel wall interface and the external border of the intimal layer. The gradation of severity of intimal thickness was based on an established classification scheme as previously described by St. Gear et al.,12 as follows: class 0, no measurable intimal layer; class I (minimal), an intimal layer <0.3 mm and <180 degrees of the vessel circumference; class II (mild), an intimal layer <0.3 mm but measurable in >180 degrees of the vessel circumference; class III (moderate), an intimal layer 0.3 mm to 0.5 mm thick and >180 degrees or an intimal layer >0.5 mm thick involving <180 degrees of the vessel circumference; and class IV (severe), >0.5 mm intimal thickening involving >180 degrees of the vessel circumference or any intimal layer >1.0 mm in any one area of the vessel circumference. Aortic image analysis was similarly perfo~zned and mean maximal intimal thickness was measured sector by sector to encompass all 360 degrees of the allograft aortic surface. Statistical analysis. Comparisons of aortic and coronary intimal proliferation as well as aortic intimal hyperplasia risk factor analysis were performed with univariate and multivariate regression analysis on Statview IV software (Abacus Concepts, Berkeley, Calif.) with a Macintosh computer (Apple Computers). Comparisons between groups were performed by Student's t test or chi-square tests as appropriate. All results were reported as mean -+ 1 SD. A p value <0.05 was considered significant.
RESULTS Patient characteristics. The total s t u d y population included 24 subjects (18 m e n a n d six women) with a m e a n age of 53 _+ 9 years. The clinical a n d i m m u n o logic characteristics of h e a r t t r a n s p l a n t recipients divided according to presence (n = 14) or absence (n = 10) of aortic intimal thickness are delineated in Tables I a n d II. Intraaortic and intracoronary ultrasound analysis. In-
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Table II. Immunologic characteristics in cardiac transplant recipients without and with aortic intimal thickness Aortic intimal thickness
No (n = 10)
Yes (n = 14)
Cyclosporine dose (mg/kg/day) Prednisone dose (mg/kg/day) Cumulative prednisone (gm) Azathioprine dose (mg/kg/day) Biopsy rejection score HLA matches
3.2 + 0.5 0.11 +- 0.04 7.9 -+ 0.4 1.3 +- 0.3 0.8 -+ 0.3 1.2 +_0.9
4.8 + 1.7 0.13_+0.05 10.4 -+ 6.7 1.6 _+0.6 0.95 -+ 0.30 0.8 -+ 0.99
Table III. Comparison of degree of intracoronary intimal
hyperplasia with intraaortic intimal thickening Intracoronary ultrasound degree of intimal thickening (ram) Minimal to mild Moderate Severe (0.10 +_0.03) (0.40 +_0.04)(0.75 +_0.05)
Allograft aorta intimal thickening (mm)
0.24-+ 0.10 0.80+- 0.10" 1.10 _+0.30*
*p < 0.01 (comparedwith other groups).
Fig. 1. Intracoronary and intraaortic ultrasound images
of normal cardiac allograft (upper panel) compared with abnormal allograft (lower panel) demonstrating severe inritual proliferation in both allograft coronary artery and allograft aorta. timal t h i c k e n i n g was identified in all allograft aortas of subjects whose coronary arteries revealed presence of significant intimal thickening. Conversely, w h e n the coronary vessels were devoid of intimal hyperplasia, c o n c u r r e n t intraaortic u l t r a s o u n d demo n s t r a t e d no evidence of intimal proliferation. In h e a r t t r a n s p l a n t recipients with a n y detectable intireal proliferation, the m e a n m a x i m a l i n t r a c o r o n a r y intimal thickness was 0.41 _+ 0.18 ram, w h e r e a s intraaortic intimal thickness was 0.90 _+ 0.28 mm. F u r t h e r m o r e , intraaortic intimal proliferation was strongly correlated with the degree of coronary intimal t h i c k e n i n g (r = 0.90; p < 0.0001) (Fig. 1 a n d Table III). Ultrasound analysis reproducibility. I n t e r o b s e r v e r and i n t r a o b s e r v e r variability for this analysis were 2.1% a n d 3.4%, respectively. I n t r a o b s e r v e r variability was d e t e r m i n e d b y comparing the first a n d second m e a s u r e m e n t s of each observer. I n t e r o b s e r v e r variability was d e t e r m i n e d by comparing the first m e a s u r e m e n t s of the two different observers. Univariate and multivariate predictors of aortic intimal proliferation. Significant u n i v a r i a t e predictors of aor-
tic intimal t h i c k e n i n g included y e a r s after t r a n s plant, total cholesterol level, s e r u m triglyceride level,
Table IV. Significant predictors of aortic intimal hy-
perplasia Variable
r Value
p Value
Coronary intimal thickening Years after transplant Total cholesterol Triglycerides
0.90 0.47 0.65 0.51
<0.0001 0.03 0.003 0.03
and daily cyclosporine dose (Table IV). W h e n the interaction of daily cyclosporine dose with lipid abnormalities was assessed, a significant t r e n d of a direct association o f h y p e r t r i g l y c e r i d e m i a a n d h i g h e r daily cyclosporine dose e m e r g e d (r = 0.55; p = 0.06). On m u l t i v a r i a t e analysis, only years after t r a n s p l a n t , hypercholesterolemia, a n d h y p e r t r i g l y c e r i d e m i a rem a i n e d as significant correlates of aortic intimal thickening. DISCUSSION
The results of this in vivo i n t r a v a s c u l a r u l t r a s o u n d s t u d y indicate t h a t cardiac allograft v a s c u l o p a t h y a t ~ c t s the allograft a o r t a in a similar m a n n e r to its involvement of the c o r o n a r y arteries of h e a r t t r a n s p l a n t recipients. Indeed, the degree o f i n t r a a o r t i c intimal proliferation was strongly correlated with coro n a r y intimal t h i c k e n i n g (r = 0.90; p < 0.0001), suggesting the simultaneous genesis of allograft v a s c u l o p a t h y across both vascular beds. F u r t h e r more, h y p e r l i p i d e m i a and y e a r s after t r a n s p l a n t were found to be significant predictors of "allograft
Volume 133, Number 6 American Heart Journal
aortopathy," a term we have used to denote the involvement of the donor aorta in the process of cardiac allograft vasculopathy. The participation of the great vessels in the process of cardiac allograft vasculopathy has only been sparsely studied in pathologic examinations of failed cardiac allografts. The earliest clinical report that assessed the histologic status of the allograft aorta appeared almost 2 decades ago by Kennedy et al. 13In their study of nine failed allografts, the investigators found evidence of cellular intimal thickening in the allograft aorta strikingly early after transplantation. These initial observations have since been expanded in a necropsy study by Russell et al., 4 who studied 19 cardiac allografts that failed as a result of cardiac allograft vascul0pathy and who demonstrated the universal development of intimal proliferation in the allograft aorta in conjunction with coronary arteriopathy. For the first time, our intraaortic ultrasound findings further delineate in vivo the close association of intracoronary and aortic intimal hyperplasia that develops in a time-dependent manner and the genesis of coronary arterial intimal proliferation. More recent evidence suggests that an alloimmunologic response to the allograft vasculature may be primarily responsible for the development of cardiac allograft vasculopathy. This school of thought has been strengthened by the demonstration of an association among acute cellular rejection,14 presence of cytotoxic B cell antibodies, 2 and the presence of donor-specific HLA antibodies 15 with the subsequent development of cardiac allograft vasculopathy. Because alloimmune stimuli would necessarily operate throughout the vascular bed, the finding of allograft aortopathy lends further credibility to this thesis. On the other hand, as our investigation demonstrates, systemic metabolic factors might also be important in the genesis of this disease because we have demonstrated a significant relation between hypercholesterolemia and hypertriglyceridemia with the development of allograft aortopathy. One intriguing finding in our study was the association of a higher dose of cyclosporine with the severity of aortic intimal proliferation. This observation is speculative but deserves mention. The relation of cyclosporine and the development of cardiac allograft vasculopathy has been a matter of previous investigation. Sedmak et al. 16 alluded to the occurrence of a cyclosporine-inducedendothelial injury as a result of its cytotoxicity. Unfortunately this theory has not been validated in clinical heart transplantation. An angiographic study by Gao et al.17 demonstrated a 41% incidence of cardiac allograft vascul-
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opathy in the precyclosporine era compared with a 33% incidence in cyclosporine-treated subjects 3 years after transplantation. A more plausible explanation of the association of cyclosporine with cardiac allograft vasculopathy relates to the induction of adverse lipid metabolic parameters because cyclosporine has been demonstrated to decrease lipoprotein lipase activity and inhibit prednisone clearance by its interaction with the hepatic cytochrome P-450 system.iS, 19This interaction results in impaired very-low-density lipoprotein and low-density lipoprotein clearance and leads to hypercholesterolemia and hypertriglyceridemia.2° Indeed, a close relation between hypertriglyceridemia and cyclosporine dose was evident in our study cohort (r = 0.55; p = 0.06). Furthermore, the association of cyclosporine with allograft aortopathy was not evident in multivariate testing, whereas hyperlipidemia remained a significant correlate of aortic intimal proliferation. It is also important to emphasize that the association between cyclosporine and intimal proliferation might not be a causal relation but may underlie an attempt at enhanced immunosuppression against alloimmune responses that are the primary culprit responsible for the development ofintimal proliferation. Limitations. Certain limitations of this study deserve emphasis. First, the ultrasound device that we used provided sectional images in most cases, thus requiring rotation of the catheter to examine the entire 360-degree cross-sectional surface of the aortic intimal surface. This requirement might have been avoided by the passage of a larger 10F catheter system; however, this system requires placement of a larger vascular access and thus raises possibilities for a higher risk of antecedent vascular complications. Second, no histopathologic correlation of the study findings was possible. Lastly, serial investigations were not performed; therefore a correlation of the rate of increase in aortic intimal proliferation in conjunction with coronary intimal thickening cannot be made in this study. Conclusions. Allograft aortopathy, or donor aorta involvement in the transplanted heart, occurs in a similar manner to allograft coronary disease. Our findings provide support for the notion that an immunologic stimulus operating across the allograft vascular bed, coupled with the adverse influence of hyperlipidemia over time, may be responsible for the development of cardiac allograft vasculopathy. REFERENCES
1. Uretsky BF, Murali S, Reddy PS, Rabin B, Lee A, Griffith BP, et al. Development of coronary artery disease in cardiac transplant patients
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3. 4.
5.
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receiving immunosnppressive therapy with cyclosporine and prednisone. Circulation 1987;76:827-33. Hess ML, Hastillo A, Mohankumar DVM, Cowley MJ, Vetrovac G, Szentpetery S, et al. Accelerated atherosclerosis in cardiac transplantation: role of cytotoxic B-cell antibodies and hyperlipidemia. Circulation 1983;68(suppl II):94-101. Johnson MR. Transplant coronary disease: nonimmunologic risk factors. J Heart Lung Transplant 1992;11:$124-32. Russell ME, Fujita M, Masek MA, Rowan RA, Billingham ME. Cardiac graft vascular disease. Nonselective involvment of large and small vessels. Transplantation 1993;56:1599-601. Fujita M, Russell ME, Masek MA, Rowan RA, Nagashima t~ Billingh a m ME. Graft vascular disease in the great vessels and vasa vasorum. Hum Pathol 1993;24:1067-72. Oni AA, Ray J, Hosenpud JD. Coronary venous intimal thickening in explanted cardiac allograi~s. Evidence demonstrating that transplant coronary artery disease is a manifestation of a diffuse allograft vasculopathy. Transplantation 1992;53:1247-51. Ventura H e , White CJ, Jain SP, Smart FW, Jain A, Stapleton DD, et al. Assessment of intracoronary morphology in cardiac transplant recipients by angioscopy and intravascular ultrasound. Am J Cardiol 1993;72:805-9. Mehra MR, Ventura He, Chambers RB, Collins TJ, Ramee SR, Kates MA, et al. Predictive model to assess risk for Cardiac allograft vasculopathy: an intravascular ultrasound study. J Am Cell Cardiol 1995; 26:1537-44. Hausmann D, Lundkvist AS, Freidrich GJ, Mullen WL, Fitzgerald PJ, Yock PG. Intracorouary ultrasound imaging: intraobserver and interobserver variability of morphometric measurements. Am Heart J 1994; 128:674-80. Pinto FJ, St. Gear FG, Gao S-Z, ChenzbraunA, Fischell TA, Alderman EL, et al. Immediate and one-year safety of intracoronary ultrasonic imaging. Evaluation with serial quantitative augiography. Circulation 1993;88:1709-14.
June 1997 American Heart Journal
11. Billingham ME, Cary NR, Hammond ME, Kemnitz J, Marboe C, McCallister HA. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection. Heart Rejection Study Group. The International Society for Heart Transplantation. J Heart Lung Transplant 1990;9:587-93. 12. St. Gear FG, Pinto FJ, Alderman EL, Fitzgerald PJ, Stadius ML, Popp RL. Intravascular ultrasound imaging of angiographically normal coronary arteries: an in rive comparision with quantitative angiography. J Am Cell Cardiol 1991;18:952-8. 13. Kennedy LJ, Beiber CP, Mitchinson MJ. Aortic histopathology in hum a n cardiac allograi~s. J Thorac Cardiovasc Surg 1971;62:42-50. 14. Costanzo-Nordin MR. Cardiac a]lografi vasculopathy: relationship with acute cellular rejection and histocompatibihty. J Heart Lung Transplant 1992;11:$90-103. 15. Rose EA, Smith CR, Petrossian GA, Barr ML, Reemtsma K. Humeral immune responses after cardiac transplantation: correlation with fatal rejection and grai~ atherosclerosis. Surgery 1989;106:203-8. 16. Sedmak DD, Orosz CG. The role of vascular endothelial cells in transplantation. Arch Pathol Lab Med 1991;115:260-5. 17. Gao SZ, Alderman EL, Schroeder JS, Silverman JF, Hunt SA. Accelerated coronary vascular disease in the heart transplant patient: coronary arteriographic findings. J Am CoU Cardiol 1988;12:334-40. 18. Ost L. Effects of cyclosporine on prednisolone metabolism [letter]. Lancet 1984;1:451. 19. Chancerelle Y, Logeril MD, Viret R, Chiron B, Dureau G, Renaud S, et al. Increased lipid peroxidation in cyclosporin-treated heart transplant recipients. Am J Cardiol 1991i68:813-6. 20. Hess ML, Hastillo A, Thompson JA, Sansonetti DJ, Szentpetery S, Barnhart G, et al. Lipid mediators in organ transplantation: does cyclosporine accelerate coronary atherosclerosis? Transplant Proc 1987; 19:71-73.
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III
Surgery
AIIograft aortopathy: An in vivo study of donor aorta involvement in cardiac allograft vasculopathy Mandeep R. Mehra, MD, Hector O. Ventura, MD, Dwight D. Stapleton, MD, Arun K. Karsan, MD, Frank W. Smart, MD, Stephen R. Ramee, MD, and Tyrone J. Collins, MD New Orleans, La. Limited histopathologic studies of failed cardiac allografts have demonstrated that cardiac allograft vasculopathy extends into the donor aorta; however, no study has examined the development of allograft aortic intimal proliferation in vivo in conjunction with coronary intimal hyperplasia. By using simultaneous intracoronary and intraaortic ultrasound, we studied 20 consecutive heart transplant recipients at 2.5 ± 2.1 years after transplantation. The degree of coronary intimal thickening was strongly correlated with the development of intraaortic intimal hyperplasia (r=0.90; p < 0.0001). Multivariate predictors of aortic intimal thickening included years after transplant (r = 0.47; p = 0.03), serum cholesterol level (r-- 0,65, p = 0.003), and serum triglyceride level (r= 0.51; p = 0.03). AIIograft aortopathy occurs in a similar manner to allograft coronary disease, thus providing support for the notion that an immunologic stimulus operating across the allograft vascular bed may be responsible for the development of cardiac allograft vasculopathy. Furthermore, this investigation provides insight into the putative role of hyperlipidemia in allograft vascular disease. (Am Heart J 1997;133:698-702.)
Cardiac allograft vasculopathy, a unique and accel: erated form of coronary arteriopathy manifested by diffuse and longitudinal myointimal proliferation, is currently the leading cause of cardiac allograft attrition after the first year following transplantation. 1 The exact cause of this disorder remains uncertain; however, several lines of clinical and experimental evidence suggest that this disorder is an immunologically initiated and metabolically propagated form of coronary arteriopathy. 2, 3 In fact, an early clinical investigation by Hess et al. 2 suggested that the From the Department of Internal Medicine, Section on Cardiology, Ochsner Medical Institutions. Received for publication Aug. 1, 1996; accepted March 7, 1997. Reprint requests: Mandeep R. Mehra, MD, Department of Internal Medicine, Section on Cardiology, Ochsner Medical Institutions, 1514 Jefferson Highway, New Orleans, LA 70121. Copyright © 1997 by Mosby-Year Book, Inc. 0002-8703/97/$5.00 + 0 4/1/81799
698
development of diffuse intimal proliferation depended on the concerted interaction of cytotoxic B-cell antibodies and hypercholesterolemia. If the immunologic hypothesis is to be endorsed, cardiac allograft vasculopathy must then extend to the allograft vascular structures beyond the coron a r y arteries. Indeed, limited histopathologic studies of failed cardiac allografts with terminally extensive coronary arteriopathy have suggested that cardiac allograft vasculopathy is characterized by extension into the donor aorta up to the suture lines and, in some instances, even into the allograft venous structures. 4-6 However, no study to date has assessed the early development of aortic intimal thickening in vivo in conjunction with the genesis of coronary intimal proliferation. Intravascular ultrasound has recently been validated as an effective clinical imaging device in the accurate assessment of cardiac allograft vasculopathy. 7,s Several studies have directed attention to the remarkable reproducibility and safety of intravascular ultrasound in serially characterizing the development of allograft coronary arteriopathy by delineating vessel wall morphologic characteristics in vivo. 9,1° Thus intravascular ultrasound can be extended to study the genesis of coronary arterial involvement in cardiac allograft vasculopathy and to investigate the simultaneous occurrence of donor aortic involvement in this diffuse, proliferative process by performing intraaortic ultrasonography. The purpose of this study was threefold: (1) to assess the early development ofintimal proliferation in the allograft aorta with intravascular ultrasound; (2) to compare the degree of development of aortic disease with coronary intimal proliferation; and (3) to define the risk factors associated with the development of allograft aortopathy in heart transplant recipients.
Volume 133, Number 6
M e h r a et al.
American Heart Journal
METHODS Patients. The study population consisted of a prospectively assembled cohort of 24 consecutive heart transplant recipients aged 53 -+ 9 years (18 men and six women) who underwent intravascular ultrasound examination at the time of routine annual examination at a mean duration of 2.5 -+ 1.8 years after transplantation. All recipients received identical triple immunosuppressive therapy (cyclosporine, predulsone, and azathioprine) and did not have acute rejection or infection at the time of the study. Informed consent was obtained from all patients, and the study was approved by the Institutional Review Board. Immunologic risk factors. Episodes of cellular or humeral rejection requiring treatment and the number of human leukocyte antigens (HLA) A, B, or DR matches between the donor and recipient were examined in all patients. A first-year mean biopsy rejection score was determined for all patients in an effort to assess the influence of the severity and frequency of rejection and to account for insidious episodes of cellular rejection. This score was adapted from the standardized criteria established by the International Society for Heart and Lung Transplantation. 11 Individual scores were assigned to each defined biopsy grade as follows: grade 0, 0; grade IA, 1; grade IB, 2; grade 2, 3; grade 3A, 4; grade 3B, 5; and grade 4, 6. The mean biopsy score was computed as the average of all biopsy scores during the first year after transplantation. Furthermore, detailed analysis of immunosuppressive regimens, including cyclosporine dose and levels, prednisone dose and cumulative consumption, azathioprine dose, and use of induction OKT3 was also performed. Nonimmunologic risk factors. Donor age and sex, obesity indexes (percentage weight gain, body mass index), hypertension, history of cytomegalovirus infection requiring therapeutic intervention with intravenous ganciclovir, fasting lipid profile (triglyceride, total cholesterol, lowdensity lipoprotein cholesterol and high-density lipoprotein cholesterol levels) at the time of ultrasound examination, history of diabetes mellitus, ischemic time, and time from transplantation were assessed in all heart transplant recipients. Intravascular ultrasound A o r t i c a n d c o r o n a r y u l t r a s o u n d procedure. Intracoronary ultrasound was performed with a commercially available 2.9F catheter with a central frequency of 30 MHz (Cardiovascular Imaging Systems, Sunnyvale, Calif.). The procedure for performing intravascular ultrasound has been previously described, s After performance of the intracoronary ultrasound, intraaortic ultrasonography was performed with an 8F ultrasound catheter with a central frequency of 20 MHz (Cardiovascular Imaging Systems, Sunnyvale, Calif.) over a 0.018-inch guide wire. The ultrasound catheter was advanced to the aortic valve and slowly withdrawn while imaging the allograft aorta up to the suture lines. Additionally, fluoroscopic pictures and audio annotations were used to ensure the correct localization of the artery segment for subsequent offiine analysis. A o r t i c a n d c o r o n a r y u l t r a s o u n d a s s e s s m e n t . All images
699
Table I. Clinical characteristics in cardiac transplant recipients without and with aortic intimal thickness Aortic intimal thickness
No (n = 10)
Yes (n = 14)
Age (yr) Time after transplant (yr) Ischemic time (min) Total cholesterol (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Triglycerides (mg/dl) % Weight gain after transplant Donor age (yr)
55 =~11 1.3 -+ 1.4 157 -+ 64 202 _+43 124 --+41 48 -+ 15 153 -+ 46 11 _+8 25 -+ 10
52 _+9 3.3 -+ 2.1" 192 -+ 47 261 -+ 47* 151 :~ 49 40 + 16 264 _+152" 14 _+6.8 31 _+11
LDL, Low-densitylipoprotein;HDL, high-densitylipopretein.
*p < 0.05. were analyzed twice by two independent observers who had no knowledge of the results from prior assessments of nonimmunologic risk factors. Three coronary sites per vessel (proximal, mid, and distal), for a total of 72 coronary artery segments, were evaluated. Maximal intimal thickness and intimal area were obtained by tracing the lumen vessel wall interface and the external border of the intimal layer. The gradation of severity of intimal thickness was based on an established classification scheme as previously described by St. Gear et al.,12 as follows: class 0, no measurable intimal layer; class I (minimal), an intimal layer <0.3 mm and <180 degrees of the vessel circumference; class II (mild), an intimal layer <0.3 mm but measurable in >180 degrees of the vessel circumference; class III (moderate), an intimal layer 0.3 mm to 0.5 mm thick and >180 degrees or an intimal layer >0.5 mm thick involving <180 degrees of the vessel circumference; and class IV (severe), >0.5 mm intimal thickening involving >180 degrees of the vessel circumference or any intimal layer >1.0 mm in any one area of the vessel circumference. Aortic image analysis was similarly perfo~zned and mean maximal intimal thickness was measured sector by sector to encompass all 360 degrees of the allograft aortic surface. Statistical analysis. Comparisons of aortic and coronary intimal proliferation as well as aortic intimal hyperplasia risk factor analysis were performed with univariate and multivariate regression analysis on Statview IV software (Abacus Concepts, Berkeley, Calif.) with a Macintosh computer (Apple Computers). Comparisons between groups were performed by Student's t test or chi-square tests as appropriate. All results were reported as mean -+ 1 SD. A p value <0.05 was considered significant.
RESULTS Patient characteristics. The total s t u d y population included 24 subjects (18 m e n a n d six women) with a m e a n age of 53 _+ 9 years. The clinical a n d i m m u n o logic characteristics of h e a r t t r a n s p l a n t recipients divided according to presence (n = 14) or absence (n = 10) of aortic intimal thickness are delineated in Tables I a n d II. Intraaortic and intracoronary ultrasound analysis. In-
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American Heart Journal
Table II. Immunologic characteristics in cardiac transplant recipients without and with aortic intimal thickness Aortic intimal thickness
No (n = 10)
Yes (n = 14)
Cyclosporine dose (mg/kg/day) Prednisone dose (mg/kg/day) Cumulative prednisone (gm) Azathioprine dose (mg/kg/day) Biopsy rejection score HLA matches
3.2 + 0.5 0.11 +- 0.04 7.9 -+ 0.4 1.3 +- 0.3 0.8 -+ 0.3 1.2 +_0.9
4.8 + 1.7 0.13_+0.05 10.4 -+ 6.7 1.6 _+0.6 0.95 -+ 0.30 0.8 -+ 0.99
Table III. Comparison of degree of intracoronary intimal
hyperplasia with intraaortic intimal thickening Intracoronary ultrasound degree of intimal thickening (ram) Minimal to mild Moderate Severe (0.10 +_0.03) (0.40 +_0.04)(0.75 +_0.05)
Allograft aorta intimal thickening (mm)
0.24-+ 0.10 0.80+- 0.10" 1.10 _+0.30*
*p < 0.01 (comparedwith other groups).
Fig. 1. Intracoronary and intraaortic ultrasound images
of normal cardiac allograft (upper panel) compared with abnormal allograft (lower panel) demonstrating severe inritual proliferation in both allograft coronary artery and allograft aorta. timal t h i c k e n i n g was identified in all allograft aortas of subjects whose coronary arteries revealed presence of significant intimal thickening. Conversely, w h e n the coronary vessels were devoid of intimal hyperplasia, c o n c u r r e n t intraaortic u l t r a s o u n d demo n s t r a t e d no evidence of intimal proliferation. In h e a r t t r a n s p l a n t recipients with a n y detectable intireal proliferation, the m e a n m a x i m a l i n t r a c o r o n a r y intimal thickness was 0.41 _+ 0.18 ram, w h e r e a s intraaortic intimal thickness was 0.90 _+ 0.28 mm. F u r t h e r m o r e , intraaortic intimal proliferation was strongly correlated with the degree of coronary intimal t h i c k e n i n g (r = 0.90; p < 0.0001) (Fig. 1 a n d Table III). Ultrasound analysis reproducibility. I n t e r o b s e r v e r and i n t r a o b s e r v e r variability for this analysis were 2.1% a n d 3.4%, respectively. I n t r a o b s e r v e r variability was d e t e r m i n e d b y comparing the first a n d second m e a s u r e m e n t s of each observer. I n t e r o b s e r v e r variability was d e t e r m i n e d by comparing the first m e a s u r e m e n t s of the two different observers. Univariate and multivariate predictors of aortic intimal proliferation. Significant u n i v a r i a t e predictors of aor-
tic intimal t h i c k e n i n g included y e a r s after t r a n s plant, total cholesterol level, s e r u m triglyceride level,
Table IV. Significant predictors of aortic intimal hy-
perplasia Variable
r Value
p Value
Coronary intimal thickening Years after transplant Total cholesterol Triglycerides
0.90 0.47 0.65 0.51
<0.0001 0.03 0.003 0.03
and daily cyclosporine dose (Table IV). W h e n the interaction of daily cyclosporine dose with lipid abnormalities was assessed, a significant t r e n d of a direct association o f h y p e r t r i g l y c e r i d e m i a a n d h i g h e r daily cyclosporine dose e m e r g e d (r = 0.55; p = 0.06). On m u l t i v a r i a t e analysis, only years after t r a n s p l a n t , hypercholesterolemia, a n d h y p e r t r i g l y c e r i d e m i a rem a i n e d as significant correlates of aortic intimal thickening. DISCUSSION
The results of this in vivo i n t r a v a s c u l a r u l t r a s o u n d s t u d y indicate t h a t cardiac allograft v a s c u l o p a t h y a t ~ c t s the allograft a o r t a in a similar m a n n e r to its involvement of the c o r o n a r y arteries of h e a r t t r a n s p l a n t recipients. Indeed, the degree o f i n t r a a o r t i c intimal proliferation was strongly correlated with coro n a r y intimal t h i c k e n i n g (r = 0.90; p < 0.0001), suggesting the simultaneous genesis of allograft v a s c u l o p a t h y across both vascular beds. F u r t h e r more, h y p e r l i p i d e m i a and y e a r s after t r a n s p l a n t were found to be significant predictors of "allograft
Volume 133, Number 6 American Heart Journal
aortopathy," a term we have used to denote the involvement of the donor aorta in the process of cardiac allograft vasculopathy. The participation of the great vessels in the process of cardiac allograft vasculopathy has only been sparsely studied in pathologic examinations of failed cardiac allografts. The earliest clinical report that assessed the histologic status of the allograft aorta appeared almost 2 decades ago by Kennedy et al. 13In their study of nine failed allografts, the investigators found evidence of cellular intimal thickening in the allograft aorta strikingly early after transplantation. These initial observations have since been expanded in a necropsy study by Russell et al., 4 who studied 19 cardiac allografts that failed as a result of cardiac allograft vascul0pathy and who demonstrated the universal development of intimal proliferation in the allograft aorta in conjunction with coronary arteriopathy. For the first time, our intraaortic ultrasound findings further delineate in vivo the close association of intracoronary and aortic intimal hyperplasia that develops in a time-dependent manner and the genesis of coronary arterial intimal proliferation. More recent evidence suggests that an alloimmunologic response to the allograft vasculature may be primarily responsible for the development of cardiac allograft vasculopathy. This school of thought has been strengthened by the demonstration of an association among acute cellular rejection,14 presence of cytotoxic B cell antibodies, 2 and the presence of donor-specific HLA antibodies 15 with the subsequent development of cardiac allograft vasculopathy. Because alloimmune stimuli would necessarily operate throughout the vascular bed, the finding of allograft aortopathy lends further credibility to this thesis. On the other hand, as our investigation demonstrates, systemic metabolic factors might also be important in the genesis of this disease because we have demonstrated a significant relation between hypercholesterolemia and hypertriglyceridemia with the development of allograft aortopathy. One intriguing finding in our study was the association of a higher dose of cyclosporine with the severity of aortic intimal proliferation. This observation is speculative but deserves mention. The relation of cyclosporine and the development of cardiac allograft vasculopathy has been a matter of previous investigation. Sedmak et al. 16 alluded to the occurrence of a cyclosporine-inducedendothelial injury as a result of its cytotoxicity. Unfortunately this theory has not been validated in clinical heart transplantation. An angiographic study by Gao et al.17 demonstrated a 41% incidence of cardiac allograft vascul-
Mehra et al.
701
opathy in the precyclosporine era compared with a 33% incidence in cyclosporine-treated subjects 3 years after transplantation. A more plausible explanation of the association of cyclosporine with cardiac allograft vasculopathy relates to the induction of adverse lipid metabolic parameters because cyclosporine has been demonstrated to decrease lipoprotein lipase activity and inhibit prednisone clearance by its interaction with the hepatic cytochrome P-450 system.iS, 19This interaction results in impaired very-low-density lipoprotein and low-density lipoprotein clearance and leads to hypercholesterolemia and hypertriglyceridemia.2° Indeed, a close relation between hypertriglyceridemia and cyclosporine dose was evident in our study cohort (r = 0.55; p = 0.06). Furthermore, the association of cyclosporine with allograft aortopathy was not evident in multivariate testing, whereas hyperlipidemia remained a significant correlate of aortic intimal proliferation. It is also important to emphasize that the association between cyclosporine and intimal proliferation might not be a causal relation but may underlie an attempt at enhanced immunosuppression against alloimmune responses that are the primary culprit responsible for the development ofintimal proliferation. Limitations. Certain limitations of this study deserve emphasis. First, the ultrasound device that we used provided sectional images in most cases, thus requiring rotation of the catheter to examine the entire 360-degree cross-sectional surface of the aortic intimal surface. This requirement might have been avoided by the passage of a larger 10F catheter system; however, this system requires placement of a larger vascular access and thus raises possibilities for a higher risk of antecedent vascular complications. Second, no histopathologic correlation of the study findings was possible. Lastly, serial investigations were not performed; therefore a correlation of the rate of increase in aortic intimal proliferation in conjunction with coronary intimal thickening cannot be made in this study. Conclusions. Allograft aortopathy, or donor aorta involvement in the transplanted heart, occurs in a similar manner to allograft coronary disease. Our findings provide support for the notion that an immunologic stimulus operating across the allograft vascular bed, coupled with the adverse influence of hyperlipidemia over time, may be responsible for the development of cardiac allograft vasculopathy. REFERENCES
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