- Email: [email protected]
Contribution of pre-existing vascular disease to allograft vasculopathy in a murine model
Transplant Immunology 22 (2009) 93–98
Contents lists available at ScienceDirect
Transplant Immunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t r i m
Contribution of pre-existing vascular disease to allograft vasculopathy in a murine model☆ Amr M. Zaki a, Gregory M. Hirsch a,b, Timothy D.G. Lee a,b,c,⁎ a b c
Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 Department of Surgery, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5
a r t i c l e
i n f o
Article history: Received 17 April 2009 Received in revised form 15 July 2009 Accepted 15 July 2009 Keywords: Allograft vasculopathy Graft vascular disease Cardiac allograft vasculopathy Graft arteriosclerosis
a b s t r a c t Allograft vasculopathy (AV) has emerged as a major obstacle for long-term graft survival after cardiac transplantation. The shortage of donor hearts has meant fewer restrictions have been placed on acceptable hearts over the past few years resulting in an increase in the number of older hearts in the donor pool. This increase has subsequently led to the increase of donor hearts containing pre-existing disease. The importance of this pre-existing donor vascular disease in AV outcomes remains controversial. In this study we address this by taking advantage of the fact that B6 Apolipoprotein-E knockout mice develop atherosclerotic lesions in their aortic tracts that closely model human naturally occurring vascular disease. By using these mice as donors, we transplant known levels of pre-existing disease into fully disparate (C3H) recipients. Cyclosporin A is used to prevent acute rejection and allow for allograft vasculopathy. We found that pre-existing lesions are retained in this model after transplantation and that they contribute to increase in lesion size and to increased lumenal narrowing. The de novo AV lesions overlay the pre-existing lesions and this leads to areas of eccentric lesion formation in the vessels with likely accompanying exacerbation of flow perturbation. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Allograft vasculopathy (AV) has emerged as a major obstacle for long-term graft survival after cardiac transplantation. Recent intravascular ultrasound (IVUS) studies have demonstrated that approximately 75% of cardiac transplant patients have identifiable AV 3 years post-transplant and 95% have marked AV by 10 years post-transplant [1]. Median graft survival rates are approximately 10 years [2]. IVUS data have also demonstrated that at least 30% of donor organs have significant pre-existing atherosclerosis at the time of transplant [3,4]. The importance of pre-existing donor vascular disease to AV outcomes remains controversial. The majority of studies have provided evidence of negative outcomes associated with donor organs with pre-existing vasculopathy [5–10] but other studies have suggested that these donor lesions do not affect long-term survival of cardiac grafts [3,4]. Current animal models have not incorporated preexisting donor vascular disease and, as such, fall short of addressing this important issue.
☆ This study was supported by a grant from the Heart and Stroke Foundation of Canada. ⁎ Corresponding author. Atlantic Centre for Transplantation Research, Dalhousie University, Tupper Medical Building Rm 10A-D, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5. Tel.: +1 902 494 3882; fax: +1 902 494 5125. E-mail address: [email protected] (T.D.G. Lee). 0966-3274/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2009.07.002
AV is characterized by a loss of smooth muscle cells from the media and a progressive concentric vascular lesion in the coronary arteries made up of α-actin positive myofibroblastic cells and infiltrating leukocytes [11]. We have provided significant data [12–15] to support a hypothesis that cardiac AV is a “response to injury” process, the purpose of which is to repair vessels weakened by immunological damage. In the tightly controlled rodent models this repair mechanism is characterized by the formation of a de novo lesion created by the influx of myofibroblastic cells into the sub-endothelial space and subsequent myofibroblastic expansion [16–18]. One of the most important discrepancies between the data arising from animal models and data from human studies relates to the origin of the myofibroblastic cells in the neointimal lesion. Evidence from a variety of rodent models has demonstrated that these are completely, or almost completely, of recipient origin [16–23]. This has held true for rodent studies using fully disparate transplants and therapeutic levels of calcineurin inhibitor (CNI) immunosuppression to ablate acute rejection [23]. In contrast, human neointimal lesions examined at autopsy are chimeric, containing both donor and recipient elements [24–28]. A retention, or possibly expansion, of pre-existing disease coupled with de novo allograft vasculopathy would explain the chimeric neointima seen in the human studies and could account for the reported poorer outcomes. In this study we address this issue by using the B6 Apolipoprotein-E knockout (B6 ApoE−/−) mouse as a donor (with a fully disparate C3H mouse recipient) to ascertain the contribution of pre-existing vasculopathy on the development of AV. B6 ApoE−/− mice have been well-
94
A.M. Zaki et al. / Transplant Immunology 22 (2009) 93–98
described as an appropriate model for human naturally occurring vascular disease [29,30]. Moreover, these studies are done in the presence of CNI immunosuppression (Cyclosporin A; CyA) at a dose we have previously [14] verified to be sufficient to control acute rejection (50 mg/kg/day). We demonstrate that pre-existing donor vascular lesions are retained in the allograft, under CNI immunosuppression, and that they contribute to the vascular lesion characteristic of allograft vasculopathy. 2. Materials and methods 2.1. Animals Male, 8–9-week-old and 30–32-week-old C57BL/6 (B6; H-2b) and 30–32-week-old B6.129P2-Apoetm1Unc/J (B6 ApoE−/−; H-2b) mice were used as donors. Male 8–9-week-old, C3H/HeJ (C3H; H-2k) and C57BL/6Tg(ACTB-EGFP)1Osb/J (B6 GFP; H-2b) mice were used as recipients. Aortas were removed from 8 to 9-week-old male B6 mice for preparation of denuded aortic segments for grafting. All mice were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained in the Carlton Animal Care Facility, at the Sir Charles Tupper Medical Building in a pathogen free environment. Mice were maintained on standard chow diet and water ad libitum. All animal experimentation was undertaken in compliance with the guidelines of the Canadian Council on Animal Care and conforms to NIH guidelines. 2.2. Aortic transplantation Abdominal aortic segments were transplanted as we have previously described [31]. Briefly, a section of the abdominal aorta approximately 1 mm in length was harvested from the donor mouse and flushed with cold saline solution for transfer into the recipient. B6 ApoE−/− aortic segments that were used for transplantation contained a single, focal, eccentric atherosclerotic lesion, identified at the time of harvest using a Zeiss operating microscope. Aortic segments were kept in cold saline until transplant with a cold ischemic time of less than 20 min. The recipient infrarenal abdominal aorta was isolated and, after proximal and distal clamping, the recipient aorta was transected. The donor aorta was then interposed with end to end micro-anastamoses using an 11–0 suture in an interrupted fashion. The clamps were removed and blood flow was confirmed by direct inspection. In one experiment, grafts taken from 8 to 9-week-old wild type B6 mice were stripped of endothelium with a short pulse of 1.3 × 10− 4 M EDTA treatment as we have described previously [12] and allowed to reendothelialize in similarly aged B6 GFP mice. After 21 days the reendothelialized grafts were removed for examination as described below.
For visualization of GFP tissue, C3H or B6 aortic grafts harvested from GFP mice were fixed in 4% paraformaldehyde in phosphate buffer (0.1 M sodium phosphate) overnight followed by incubation in 30% sucrose in phosphate buffer for 15 h. Segments were then embedded in 10% gelatin. Gelatin blocks were then incubated in 4% paraformaldehyde/PB overnight (to fix to the gelatin support) followed by incubation in 30% sucrose/phosphate buffer. Gelatin blocks were then frozen and sectioned (10 μm) on a Leica Cryostat (SM2000R). Sections were visualized by fluorescence microscopy on a Zeiss Fluorescent microscope. Excitation was achieved at a wavelength of 450–490 nm. Histological images were captured using a Zeiss AxioCam camera (Carl Zeiss, Thornwood, NY). The intimal areas were quantified using ImageJ software (National Institute of Health, Bethesda, MD). Mean intimal area and percent vessel occlusion were calculated using intimal area from 6 individual specimens. 2.5. Immunocytochemistry Briefly, aortic segments were placed in an OCT:20% sucrose (2:1) solution frozen in liquid nitrogen and stored at −80 °C until use. Cross sections (6–7 µm) of the aortic segments were cut on a cryostat and mounted on Fisherbrand SuperFrost/Plus positively-charged microscope slides. Sections were fixed in acetone for 2 min. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide in 0.2 M PBS. Non-specific sites were blocked with a universal serum-free protein block (DAKO). The sections were incubated with either anti-CD8a monoclonal antibody (mAB) (1:25; PharMingen, San Diego, CA), antiCD4 mAB (1:25; PharMingen, San Diego, CA), anti-macrophage/ monocyte mAB (1:25; Serotec, Raleigh, NC) or anti-α smooth muscle actin mAB (1:2000; clone 1A4; Sigma, St. Louis, Missouri). Primary antibody was detected with polyclonal biotinylated anti-rat IgG antibody (1:50; PharMingen, San Diego, CA). Polyclonal biotinylated antimouse IgG reagent (Vector Labs Inc, Burlingame, CA) was used as the secondary antibody for the primary anti-α smooth muscle actin mAB. Sections were washed in PBS and incubated with a peroxidase avidin/ biotin complex (Vector Labs, Inc. Burlingame, CA). Staining was visualized with 3,3′-diaminobenzidine (DAB; Dako; Mississauga, ON) and sections were counterstained with Mayer's hematoxylin (Sigma, St Louis, Missouri). 2.6. Statistical analysis Data are presented as the mean (±SEM) for each group. A nonparametric one-tailed student's t-test was used to determine if there were significant differences between two groups. p b 0.05 was considered significant.
2.3. Immunosuppression 3. Results
Cyclosporine (Sandimmune™; CyA) immunosuppression was given subcutaneously at a dose of 50 mg/kg/day in sterile saline, for the duration of the experiment. This dose has been previously shown to be sufficient to ablate acute cardiac rejection but allow for allograft vasculopathy in cardiac and aortic grafts [14]. CyA was obtained from the Capital District Health Authority pharmacy. 2.4. Histology Transplanted sections of aorta were removed taking care not to damage the graft and frozen in an OCT:20% sucrose (2:1) solution immediately upon harvest. Sections (6–7 µm) were cut on a Leica Cryostat (CM1900 UV) and subsequently fixed for 10 min in 10% formalin to retain lipid moieties. Fixed slides were stained for 15 min with Oil Red O (0.003 g/ml in 60% isopropanol, Sigma-Aldrich), and counterstained for 2 min with Mayer's hematoxylin (Sigma-Aldrich) for qualitative observation of lipid accumulation.
3.1. The AV neointimal lesion is recipient in origin under CyA immunosuppression in mice Confirmation of the recipient origin of the neointimal lesion under CNI immunosuppression is fundamental to the understanding of the etiology of AV and the exploration of the role of pre-existing disease. Although we have demonstrated this using a rat model we had not yet provided evidence in the mouse model. In our first experiment in this study we confirmed the recipient origin of the mouse neointimal lesion in the presence of 50 mg/kg/ day CyA immunosuppression which is sufficient to ablate acute rejection [14]. Aortic segments from C3H donor mice were transferred into fully disparate B6 GFP mice. After 10 weeks the intimal lesions present in these C3H grafts were assessed for the presence of recipient B6 GFP cells. In all sections (10 sections per mouse) taken from the 3 mice transplanted, we found that the neointimal lesion was GFP positive (Fig. 1a), thus confirming the recipient origin of the lesion. In all these mice the media was GFP negative, as expected. As a control for this experiment we also visualized GFP in the endothelium and the media of cross sections of native aorta from GFP mice (Fig. 1b). As a further control we demonstrate the GFP positivity of the endothelium of aortic sections that had been harvested from B6 wild type (GFP negative) mice, stripped of their endothelium and reendothelialized in B6 GFP positive mice (Fig. 1c). As expected, the media in Fig. 1c was GFP negative, indicating the donor origin of the media in this syngeneic combination. These controls demonstrate the specificity of the GFP model.
A.M. Zaki et al. / Transplant Immunology 22 (2009) 93–98
95
actin. (Fig. 3a) In contrast, de novo neointimal lesion elements were Oil Red O negative (Fig. 3a) and α-actin positive (Fig. 3b). Both the Oil Red O and the α-actin staining suggest that the de novo lesion overlays the pre-existing lesion, although some mixing of the lesion elements cannot be discounted at this level of resolution. Immunocytochemistry for macrophages and CD4+ T cells in serial sections of the lesion above indicated that the pre-existing lesion was predominantly composed of lipid bearing foamy macrophages staining highly positive for the macrophage stain (Fig. 4a). Macrophage presence in the de novo lesion was also demonstrated but at a reduced level (Fig. 4a). CD4+ T cells were seen only in the de novo lesion and did not infiltrate the preexisting lesion (Fig. 4b). 3.4. The presence of pre-existing lesions increases both total intimal lesion area and percent lumen occlusion at 10 weeks post-transplant To ascertain whether pre-existing lesions contribute to overall lesion size in animals expressing AV, we compared the overall intimal area in aortic transplants from WT B6 donors to intimal areas in aortic transplants from B6 ApoE−/− donors (with preexisting lesions as above). The data shown in Fig. 5a demonstrate that, at 10 weeks post-transplant, there is a significant (p b 0.05, n = 6 per group) increase in overall lesion size in those grafts that had pre-existing lesions at the outset. In most cases this increase in intimal area was associated with a focal eccentric lesion corresponding to the pre-existing lesion (as shown in Fig. 3a). The data shown in Fig. 5b demonstrate that this increase in lesion size is mirrored by a significant increase in lumen occlusion in grafts with pre-existing lesions.
4. Discussion
Fig. 1. The AV neointimal lesion is recipient in origin in the presence of CNI immunosuppression. (a) WT C3H grafts into B6 GFP mice. The neointimal lesion is GFP (recipient) positive but the underlying media is GFP negative. (b) Native GFP mouse aorta. Media and intima are GFP positive. (c) WT B6 aorta re-endothelialized with GFP endothelium. Endothelium is GFP positive and media is GFP negative. M = Media. NI = Neointima.
3.2. B6 ApoE−/− mice develop abdominal aortic atherosclerosis by 30–32 weeks of age To develop a model of transplantation with pre-existing donor disease we used B6 ApoE−/− donor mice. We ascertained the development of atherosclerotic lesions in the abdominal aortas of these mice over time (12, 14 and 16 and 32 weeks of age). Younger mice only showed lesion progression in the aortic root and arch (data not shown). Only the 32-week-old mice expressed focal eccentric atherosclerotic lesions in their abdominal aorta. These were clearly visible under the operating microscope (Fig. 2a). Oil Red Ostained cross-sections confirmed the lipid rich nature of these lesions (Fig. 2b and c). 3.3. In recipients of B6 ApoE−/− aortic grafts the neointimal lesions are a combination of donor and recipient elements For this experiment we selected aortic segments from the B6 ApoE−/− mice that exhibited small-moderate sized lesions (approximately 150 µm in diameter) that would not, themselves, create obstruction. These were transplanted into fully disparate C3H mice along with CNI immunosuppression (with 50 mg/kg/day CyA). Histological examination of grafts at 10 weeks post-transplantation revealed the presence of a neointimal lesion along with clear evidence of the retention of elements of the preexisting atherosclerotic lesion. Fig. 3a and b shows the serial sections of the same lesion stained with Oil Red O (Fig. 3a) and for α-actin (Fig. 3b). There was clear evidence of retention of the pre-existing lesion as evidenced by staining of the pre-existing elements with the Oil Red O but not (or very weakly) with the antibody directed to α-
There have been many isolated reports of early vascular lesions in late adolescence and early adulthood including evidence of focal, early atheromatous lesions [32–38]. Since the majority of donor hearts come from within this demographic, questions have been raised as to exactly how much pre-existing disease is transmitted to transplant patients. In one study, 56% of the cardiac transplant patients studied had detectable donor atherosclerotic lesions when examined by IVUS [39]. The fact that the mean donor age in this study was 32 years displays the high prevalence of pre-existing disease in the general population. Due to the fact that there is a shortage of donor hearts, fewer restrictions have been placed on acceptable hearts over the past few years resulting in an increase in the number of older hearts in the donor pool [2]. This increase has subsequently led to the increase of donor hearts containing pre-existing disease. A number of studies have suggested that the neointimal AV lesions have a predilection for the proximal segments of the coronary arteries [3,40]. Available data suggest that the disease is progressive, starting at the proximal segments of the coronary arteries then advancing to medial and distal sites. Tuzcu and colleagues showed, by IVUS, a dichotomous pattern of AV [41]. Non-circumferential lesions were more common in the proximal segments of the coronary arteries than in the mid- and distal sites. Diffuse, circumferential lesions were more common in the mid- and distal sites. Pre-existing donor atherosclerosis is more frequently involved in the proximal segments of coronary arteries [3,42,43] and this may contribute to eccentric AV lesions in proximal segments. Donor atherosclerosis is rarely found at distal sites and thus the concentric nature of the lesions in these sites is probably solely due to the de novo AV lesion. The contribution of pre-existing lesions to the developing pathology of AV has been very difficult to ascertain given the tools available for human study. The animal models used to mimic human allograft vasculopathy do not account for the presence of pre-existing vasculopathy found in the coronary arteries of the donor hearts since the donor murine vessels are pristine at the time of transplantation. This study using aortic interposition grafts with pre-existing disease was designed to more appropriately model the human disease. The model chosen closely mimics the human situation in that acute rejection, which would normally be associated with fully disparate transplants, is controlled by CNI immunosuppression. This use of fully disparate transplants and CNI immunosuppression overcomes many of the weaknesses inherent in earlier AV models. Since the recipient origin of the de novo neointimal lesion is critical to our interpretation of the subsequent data we first confirmed, using
96
A.M. Zaki et al. / Transplant Immunology 22 (2009) 93–98
Fig. 2. 30–32-week-old B6 ApoE−/− mice develop atherosclerotic lesions in their abdominal aortas. (a) A representative 30–32-week-old B6 ApoE−/− abdominal aorta as seen under a dissecting microscope. White-bordered square demonstrates the pre-existing lesion. (b) Aortic segments with atherosclerosis were cross-sectioned and stained with Oil Red O. (c) Higher magnification of lesion evident in panel (b). Black arrow points to the internal elastic lamina.
green fluorescent protein expressing recipient mice, that the cells constituting the neointimal lesion in this fully disparate mouse strain combination with therapeutic levels of CyA immunosuppression are of recipient, not donor, origin. We have previously shown this in a rat model [23] but this had yet to be proven in a mouse model. Having confirmed this, we characterized the atherosclerotic disease occurring in B6 ApoE−/− mice over time on a normal chow diet, with specific reference to the appearance of lesions in the abdominal aorta. Recent imaging studies have shown that, on a normal chow diet, vascular disease in B6 ApoE−/− mice eventually occurs in both proximal and distal vessels in the aortic tree [44,45]. In this study we demonstrate that lesions are present in the abdominal aorta at 32 weeks of age but not at 16 weeks of age or earlier. Similar to other studies [44,46,47], atherosclerotic lesions were seen in the aortic root and proximal aorta much earlier. One unexpected but substantial advantage was that the atherosclerotic lesion could be visualized from the external surface of the aorta. This made it possible to select lesions of a specific size and location for transplant, thus dramatically increasing the accuracy of the resulting data. As such, small to moderate sized focal lesions (approximately 150 µm diameter) were selected for transplant to most closely approximate the human situation. Moreover, as part of the model development, a significant number of lesions were assessed macroscopically and subsequently removed for histological examination. This provided information regarding the relationship between macroscopic
and histological appearance at donor graft harvest that was invaluable for our interpretation of the 10 weeks harvest data. Our study results demonstrate a number of important points that are relevant to the human disease. First, the use of calcineurin inhibitor immunosuppression allows for the retention of the donor atherosclerotic lesion. This is of significant importance since it might be postulated that allo-immune rejection responses could have destroyed the pre-existing lesion rendering any contribution of this lesion to AV insignificant. Second, the pre-existing lipid rich lesion does not appear to form a mixed lesion with the AV lesion in that the two lesions appear to remain relatively discreet, with the AV lesion growing over the pre-existing lesion. This suggests that the two lesions do not share a common etiology. Third, the de novo neointimal lesion appears to overlay the preexisting lesion. Since the pre-existing lesions were eccentric, in areas of pre-existing lesion luminal narrowing is accentuated. This has important implications to flow dynamics and thrombosis in these large vessels. Fourth, the contribution of the pre-existing lesions resulted in an increase in overall intimal area, and associated decrease in overall lumen size, in the affected transplants. Vessels containing pre-existing lesions demonstrated lumenal occlusion of 65% (±7.2%) while vessels without pre-existing lesions demonstrated lumenal occlusion of 49% (±4.5%), a difference of 16%. Since pre-existing, pre-transplant, lesions occluded the vessels by 19% (±9.6%, data not shown) it would appear that the
A.M. Zaki et al. / Transplant Immunology 22 (2009) 93–98
97
Fig. 3. The de novo AV lesion appears to overlay the pre-existing atherosclerotic lesion. (a) Oil Red O staining of cross section of B6 ApoE−/− aortic allograft in C3H recipients 10 weeks after transplant. (b) Immunocytochemistry for α-actin in a similar (serial) section. IEL = Internal Elastic Lamina.
contribution of the pre-existing lesion is additive rather than synergistic and that the pre-existing lesions do not grow or diminish significantly after transplant in the presence of CyA immunosuppression.
Fig. 5. The presence of pre-existing lesions contributes to overall intimal lesion area and increases lumen occlusion at 10-week post-transplant. Digital image analysis of 10 weeks total intimal lesion areas and percent lumen occlusion of wild type B6 to C3H transplants and B6 ApoE−/− to C3H transplants. * = p b 0.05, n = 6 per group.
In summary, donor pre-existing lesions survive the allo-immune response in CNI immunosuppressed mice and they contribute to increased lesion size and lumenal narrowing. The increased lesion size and the eccentric nature of the chimeric lesions magnifies the potential for exacerbation of the effects of luminal narrowing and may explain the data suggesting poor outcomes associated with transplantation of cardiac grafts with mild to moderate pre-existing atherosclerosis.
References
Fig. 4. Immunocytochemistry for macrophages and CD4 T cells in the lesion. Immunocytochemistry for macrophages (a) and CD4+ T cells (b) in serial cross sections of B6 ApoE−/− aortic allografts in C3H recipients were stained with 10 weeks after transplant. IEL = Internal Elastic lamina. Small arrows indicate location of CD4+ T cells in the de novo lesion.
[1] Ramzy D, Rao V, Brahm J, Miriuka S, Delgado D, Ross HJ. Cardiac allograft vasculopathy: a review. Can J Surg 2005;48:319–27. [2] Taylor DO, Edwards LB, Aurora P, Christie JD, Dobbels F, Kirk R, et al. Registry of the International Society for Heart and Lung Transplantation: twenty-fifth official adult heart transplant report—2008. J Heart Lung Transplant 2008;27:943–56. [3] Kapadia SR, Nissen SE, Ziada KM, Guetta V, Crowe TD, Hobbs RE, et al. Development of transplantation vasculopathy and progression of donor-transmitted atherosclerosis: comparison by serial intravascular ultrasound imaging. Circulation 1998;98:2672–8. [4] Li H, Tanaka K, Anzai H, Oeser B, Lai D, Kobashigawa JA, et al. Influence of pre-existing donor atherosclerosis on the development of cardiac allograft vasculopathy and outcomes in heart transplant recipients. J Am Coll Cardiol 2006;47:2470–6. [5] Sandler D, McKenzie FN, Menkis AH, Novick RJ, Pflugfelder PW, Kostuk WJ. Early death after cardiac transplantation—the role of unsuspected donor coronary artery disease. J Heart Lung Transplant 1991;10:172. [6] Grauhan O, Hetzer R. Impact of donor-transmitted coronary atherosclerosis. J Heart Lung Transplant 2004;23:S260–2. [7] Grauhan O, Patzurek J, Hummel M, Lehmkuhl H, Dandel M, Pasic M, et al. Donortransmitted coronary atherosclerosis. J Heart Lung Transplant 2003;22:568–73. [8] Grauhan O, Siniawski H, Dandel M, Lehmkuhl H, Knosalla C, Pasic M, et al. Coronary atherosclerosis of the donor heart—impact on early graft failure. Eur J Cardiothorac Surg 2007;32:634–8. [9] Wong CK, Yeung AC. The topography of intimal thickening and associated remodeling pattern of early transplant coronary disease: influence of pre-existent donor atherosclerosis. J Heart Lung Transplant 2001;20:858–64.
98
A.M. Zaki et al. / Transplant Immunology 22 (2009) 93–98
[10] Gao HZ, Hunt SA, Alderman EL, Liang D, Yeung AC, Schroeder JS. Relation of donor age and preexisting coronary artery disease on angiography and intracoronary ultrasound to later development of accelerated allograft coronary artery disease. J Am Coll Cardiol 1997;29:623–9. [11] Libby P, Pober JS. Chronic rejection. Immunity 2001;14:387–97. [12] Currie M, Zaki AM, Nejat S, Hirsch GM, Lee TD. Immunologic targets in the etiology of allograft vasculopathy: endothelium versus media. Transpl Immunol 2008;19:120–6. [13] Skaro AI, Liwski RS, Zhou J, Vessie EL, Lee TD, Hirsch GM. CD8+ T cells mediate aortic allograft vasculopathy by direct killing and an interferon-gamma-dependent indirect pathway. Cardiovasc Res 2005;65:283–91. [14] Vessie EL, Hirsch GM, Lee TD. Aortic allograft vasculopathy is mediated by CD8(+) T cells in Cyclosporin A immunosuppressed mice. Transpl Immunol 2005;15:35–44. [15] Nejat S, Zaki A, Hirsch GM, Lee TD. CD8(+) T cells mediate aortic allograft vasculopathy under conditions of calcineurin immunosuppression: role of IFN-gamma and CTL mediators. Transpl Immunol 2008;19:103–11. [16] Shimizu K, Sugiyama S, Aikawa M, Fukumoto Y, Rabkin E, Libby P, et al. Host bonemarrow cells are a source of donor intimal smooth- muscle-like cells in murine aortic transplant arteriopathy. Nat Med 2001;7:738–41. [17] Sata M, Hirata Y, Nagai R. Circulating recipient cells contribute to graft coronary arteriosclerosis. J Cardiol 2002;39:48–9. [18] Hillebrands JL, Klatter FA, Rozing J. Origin of vascular smooth muscle cells and the role of circulating stem cells in transplant arteriosclerosis. Arterioscler Thromb Vasc Biol 2003;23:380–7. [19] Hu Y, Davison F, Ludewig B, Erdel M, Mayr M, Url M, et al. Smooth muscle cells in transplant atherosclerotic lesions are originated from recipients, but not bone marrow progenitor cells. Circulation 2002;106:1834–9. [20] Saiura A, Sata M, Hirata Y, Nagai R, Makuuchi M. Circulating smooth muscle progenitor cells contribute to atherosclerosis. Nat Med 2001;7:382–3. [21] Li J, Han X, Jiang J, Zhong R, Williams GM, Pickering JG, et al. Vascular smooth muscle cells of recipient origin mediate intimal expansion after aortic allotransplantation in mice. Am J Pathol 2001;158:1943–7. [22] Hillebrands JL, Klatter FA, van den Hurk BM, Popa ER, Nieuwenhuis P, Rozing J. Origin of neointimal endothelium and alpha-actin-positive smooth muscle cells in transplant arteriosclerosis. J Clin Invest 2001;107:1411–22. [23] Johnson P, Carpenter M, Hirsch G, Lee T. Recipient cells form the intimal proliferative lesion in the rat aortic model of allograft arteriosclerosis. Am J Transplant 2002;2:207–14. [24] Glaser R, Lu MM, Narula N, Epstein JA. Smooth muscle cells, but not myocytes, of host origin in transplanted human hearts. Circulation 2002;106:17–9. [25] Hruban RH, Long PP, Perlman EJ, Hutchins GM, Baumgartner WA, Baughman KL, et al. Fluorescence in situ hybridization for the Y-chromosome can be used to detect cells of recipient origin in allografted hearts following cardiac transplantation. Am J Pathol 1993;142:975–80. [26] Grimm PC, Nickerson P, Jeffery J, Savani RC, Gough J, McKenna RM, et al. Neointimal and tubulointerstitial infiltration by recipient mesenchymal cells in chronic renalallograft rejection. N Engl J Med 2001;345:93–7. [27] Quaini F, Urbanek K, Beltrami AP, Finato N, Beltrami CA, Nadal-Ginard B, et al. Chimerism of the transplanted heart. N Engl J Med 2002;346:5–15. [28] Atkinson C, Horsley J, Rhind-Tutt S, Charman S, Phillpotts CJ, Wallwork J, et al. Neointimal smooth muscle cells in human cardiac allograft coronary artery vasculopathy are of donor origin. J Heart Lung Transplant 2004;23:427–35. [29] Russell JC, Proctor SD. Small animal models of cardiovascular disease: tools for the study of the roles of metabolic syndrome, dyslipidemia, and atherosclerosis. Cardiovasc Pathol 2006;15:318–30. [30] Zadelaar S, Kleemann R, Verschuren L, de Vries-Van der Weij J, van der Hoorn J, Princen HM, et al. Mouse models for atherosclerosis and pharmaceutical modifiers. Arterioscler Thromb Vasc Biol 2007;27:1706–21.
[31] Koulack J, McAlister VC, Giacomantonio CA, Bitter-Suermann H, MacDonald AS, Lee TD. Development of a mouse aortic transplant model of chronic rejection. Microsurgery 1995;16:110–3. [32] McGill Jr HC, McMahan CA. Determinants of atherosclerosis in the young. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group. Am J Cardiol 1998;82:30T–6T. [33] Velican D, Velican C. Atherosclerotic involvement of the coronary arteries of adolescents and young adults. Atherosclerosis 1980;36:449–60. [34] Enos WF, Holmes RH, Beyer J. Coronary disease among United States soldiers killed in action in Korea; preliminary report. J Am Med Assoc 1953;152:1090–3. [35] McNamara JJ, Molot MA, Stremple JF, Cutting RT. Coronary artery disease in combat casualties in Vietnam. JAMA 1971;216:1185–7. [36] Berenson GS, Wattigney WA, Tracy RE, Newman III WP, Srinivasan SR, Webber LS, et al. Atherosclerosis of the aorta and coronary arteries and cardiovascular risk factors in persons aged 6 to 30 years and studied at necropsy (The Bogalusa Heart Study). Am J Cardiol 1992;70:851–8. [37] St Goar FG, Pinto FJ, Alderman EL, Fitzgerald PJ, Stinson EB, Billingham ME, et al. Detection of coronary atherosclerosis in young adult hearts using intravascular ultrasound. Circulation 1992;86:756–63. [38] Strong JP, Malcom GT, McMahan CA, Tracy RE, Newman III WP, Herderick EE, et al. Prevalence and extent of atherosclerosis in adolescents and young adults: implications for prevention from the Pathobiological Determinants of Atherosclerosis in Youth Study. JAMA 1999;281:727–35. [39] Tuzcu EM, Hobbs RE, Rincon G, Bott-Silverman C, De Franco AC, Robinson K, et al. Occult and frequent transmission of atherosclerotic coronary disease with cardiac transplantation. Insights from intravascular ultrasound. Circulation 1995;91:1706–13. [40] Hiemann NE, Wellnhofer E, Knosalla C, Lehmkuhl HB, Stein J, Hetzer R, et al. Prognostic impact of microvasculopathy on survival after heart transplantation: evidence from 9713 endomyocardial biopsies. Circulation 2007;116:1274–82. [41] Tuzcu EM, De Franco AC, Goormastic M, Hobbs RE, Rincon G, Bott-Silverman C, et al. Dichotomous pattern of coronary atherosclerosis 1 to 9 years after transplantation: insights from systematic intravascular ultrasound imaging. J Am Coll Cardiol 1996;27:839–46. [42] Rahmani M, Cruz RP, Granville DJ, McManus BM. Allograft vasculopathy versus atherosclerosis. Circ Res 2006;99:801–15. [43] Nissen SE, Yock P. Intravascular ultrasound: novel pathophysiological insights and current clinical applications. Circulation 2001;103:604–16. [44] Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb 1994;14:133–40. [45] Isobe S, Tsimikas S, Zhou J, Fujimoto S, Sarai M, Branks MJ, et al. Noninvasive imaging of atherosclerotic lesions in apolipoprotein E-deficient and low-density-lipoprotein receptor-deficient mice with annexin A5. J Nucl Med 2006;47:1497–505. [46] Choudhury RP, Fayad ZA, Aguinaldo JG, Itskovich VV, Rong JX, Fallon JT, et al. Serial, noninvasive, in vivo magnetic resonance microscopy detects the development of atherosclerosis in apolipoprotein E-deficient mice and its progression by arterial wall remodeling. J Magn Reson Imaging 2003;17:184–9. [47] Itskovich VV, Choudhury RP, Aguinaldo JG, Fallon JT, Omerhodzic S, Fisher EA, et al. Characterization of aortic root atherosclerosis in ApoE knockout mice: highresolution in vivo and ex vivo MRM with histological correlation. Magn Reson Med 2003;49:381–5.
Contents lists available at ScienceDirect
Transplant Immunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / t r i m
Contribution of pre-existing vascular disease to allograft vasculopathy in a murine model☆ Amr M. Zaki a, Gregory M. Hirsch a,b, Timothy D.G. Lee a,b,c,⁎ a b c
Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 Department of Surgery, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5 Department of Microbiology & Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5
a r t i c l e
i n f o
Article history: Received 17 April 2009 Received in revised form 15 July 2009 Accepted 15 July 2009 Keywords: Allograft vasculopathy Graft vascular disease Cardiac allograft vasculopathy Graft arteriosclerosis
a b s t r a c t Allograft vasculopathy (AV) has emerged as a major obstacle for long-term graft survival after cardiac transplantation. The shortage of donor hearts has meant fewer restrictions have been placed on acceptable hearts over the past few years resulting in an increase in the number of older hearts in the donor pool. This increase has subsequently led to the increase of donor hearts containing pre-existing disease. The importance of this pre-existing donor vascular disease in AV outcomes remains controversial. In this study we address this by taking advantage of the fact that B6 Apolipoprotein-E knockout mice develop atherosclerotic lesions in their aortic tracts that closely model human naturally occurring vascular disease. By using these mice as donors, we transplant known levels of pre-existing disease into fully disparate (C3H) recipients. Cyclosporin A is used to prevent acute rejection and allow for allograft vasculopathy. We found that pre-existing lesions are retained in this model after transplantation and that they contribute to increase in lesion size and to increased lumenal narrowing. The de novo AV lesions overlay the pre-existing lesions and this leads to areas of eccentric lesion formation in the vessels with likely accompanying exacerbation of flow perturbation. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Allograft vasculopathy (AV) has emerged as a major obstacle for long-term graft survival after cardiac transplantation. Recent intravascular ultrasound (IVUS) studies have demonstrated that approximately 75% of cardiac transplant patients have identifiable AV 3 years post-transplant and 95% have marked AV by 10 years post-transplant [1]. Median graft survival rates are approximately 10 years [2]. IVUS data have also demonstrated that at least 30% of donor organs have significant pre-existing atherosclerosis at the time of transplant [3,4]. The importance of pre-existing donor vascular disease to AV outcomes remains controversial. The majority of studies have provided evidence of negative outcomes associated with donor organs with pre-existing vasculopathy [5–10] but other studies have suggested that these donor lesions do not affect long-term survival of cardiac grafts [3,4]. Current animal models have not incorporated preexisting donor vascular disease and, as such, fall short of addressing this important issue.
☆ This study was supported by a grant from the Heart and Stroke Foundation of Canada. ⁎ Corresponding author. Atlantic Centre for Transplantation Research, Dalhousie University, Tupper Medical Building Rm 10A-D, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5. Tel.: +1 902 494 3882; fax: +1 902 494 5125. E-mail address: [email protected] (T.D.G. Lee). 0966-3274/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2009.07.002
AV is characterized by a loss of smooth muscle cells from the media and a progressive concentric vascular lesion in the coronary arteries made up of α-actin positive myofibroblastic cells and infiltrating leukocytes [11]. We have provided significant data [12–15] to support a hypothesis that cardiac AV is a “response to injury” process, the purpose of which is to repair vessels weakened by immunological damage. In the tightly controlled rodent models this repair mechanism is characterized by the formation of a de novo lesion created by the influx of myofibroblastic cells into the sub-endothelial space and subsequent myofibroblastic expansion [16–18]. One of the most important discrepancies between the data arising from animal models and data from human studies relates to the origin of the myofibroblastic cells in the neointimal lesion. Evidence from a variety of rodent models has demonstrated that these are completely, or almost completely, of recipient origin [16–23]. This has held true for rodent studies using fully disparate transplants and therapeutic levels of calcineurin inhibitor (CNI) immunosuppression to ablate acute rejection [23]. In contrast, human neointimal lesions examined at autopsy are chimeric, containing both donor and recipient elements [24–28]. A retention, or possibly expansion, of pre-existing disease coupled with de novo allograft vasculopathy would explain the chimeric neointima seen in the human studies and could account for the reported poorer outcomes. In this study we address this issue by using the B6 Apolipoprotein-E knockout (B6 ApoE−/−) mouse as a donor (with a fully disparate C3H mouse recipient) to ascertain the contribution of pre-existing vasculopathy on the development of AV. B6 ApoE−/− mice have been well-
94
A.M. Zaki et al. / Transplant Immunology 22 (2009) 93–98
described as an appropriate model for human naturally occurring vascular disease [29,30]. Moreover, these studies are done in the presence of CNI immunosuppression (Cyclosporin A; CyA) at a dose we have previously [14] verified to be sufficient to control acute rejection (50 mg/kg/day). We demonstrate that pre-existing donor vascular lesions are retained in the allograft, under CNI immunosuppression, and that they contribute to the vascular lesion characteristic of allograft vasculopathy. 2. Materials and methods 2.1. Animals Male, 8–9-week-old and 30–32-week-old C57BL/6 (B6; H-2b) and 30–32-week-old B6.129P2-Apoetm1Unc/J (B6 ApoE−/−; H-2b) mice were used as donors. Male 8–9-week-old, C3H/HeJ (C3H; H-2k) and C57BL/6Tg(ACTB-EGFP)1Osb/J (B6 GFP; H-2b) mice were used as recipients. Aortas were removed from 8 to 9-week-old male B6 mice for preparation of denuded aortic segments for grafting. All mice were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained in the Carlton Animal Care Facility, at the Sir Charles Tupper Medical Building in a pathogen free environment. Mice were maintained on standard chow diet and water ad libitum. All animal experimentation was undertaken in compliance with the guidelines of the Canadian Council on Animal Care and conforms to NIH guidelines. 2.2. Aortic transplantation Abdominal aortic segments were transplanted as we have previously described [31]. Briefly, a section of the abdominal aorta approximately 1 mm in length was harvested from the donor mouse and flushed with cold saline solution for transfer into the recipient. B6 ApoE−/− aortic segments that were used for transplantation contained a single, focal, eccentric atherosclerotic lesion, identified at the time of harvest using a Zeiss operating microscope. Aortic segments were kept in cold saline until transplant with a cold ischemic time of less than 20 min. The recipient infrarenal abdominal aorta was isolated and, after proximal and distal clamping, the recipient aorta was transected. The donor aorta was then interposed with end to end micro-anastamoses using an 11–0 suture in an interrupted fashion. The clamps were removed and blood flow was confirmed by direct inspection. In one experiment, grafts taken from 8 to 9-week-old wild type B6 mice were stripped of endothelium with a short pulse of 1.3 × 10− 4 M EDTA treatment as we have described previously [12] and allowed to reendothelialize in similarly aged B6 GFP mice. After 21 days the reendothelialized grafts were removed for examination as described below.
For visualization of GFP tissue, C3H or B6 aortic grafts harvested from GFP mice were fixed in 4% paraformaldehyde in phosphate buffer (0.1 M sodium phosphate) overnight followed by incubation in 30% sucrose in phosphate buffer for 15 h. Segments were then embedded in 10% gelatin. Gelatin blocks were then incubated in 4% paraformaldehyde/PB overnight (to fix to the gelatin support) followed by incubation in 30% sucrose/phosphate buffer. Gelatin blocks were then frozen and sectioned (10 μm) on a Leica Cryostat (SM2000R). Sections were visualized by fluorescence microscopy on a Zeiss Fluorescent microscope. Excitation was achieved at a wavelength of 450–490 nm. Histological images were captured using a Zeiss AxioCam camera (Carl Zeiss, Thornwood, NY). The intimal areas were quantified using ImageJ software (National Institute of Health, Bethesda, MD). Mean intimal area and percent vessel occlusion were calculated using intimal area from 6 individual specimens. 2.5. Immunocytochemistry Briefly, aortic segments were placed in an OCT:20% sucrose (2:1) solution frozen in liquid nitrogen and stored at −80 °C until use. Cross sections (6–7 µm) of the aortic segments were cut on a cryostat and mounted on Fisherbrand SuperFrost/Plus positively-charged microscope slides. Sections were fixed in acetone for 2 min. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide in 0.2 M PBS. Non-specific sites were blocked with a universal serum-free protein block (DAKO). The sections were incubated with either anti-CD8a monoclonal antibody (mAB) (1:25; PharMingen, San Diego, CA), antiCD4 mAB (1:25; PharMingen, San Diego, CA), anti-macrophage/ monocyte mAB (1:25; Serotec, Raleigh, NC) or anti-α smooth muscle actin mAB (1:2000; clone 1A4; Sigma, St. Louis, Missouri). Primary antibody was detected with polyclonal biotinylated anti-rat IgG antibody (1:50; PharMingen, San Diego, CA). Polyclonal biotinylated antimouse IgG reagent (Vector Labs Inc, Burlingame, CA) was used as the secondary antibody for the primary anti-α smooth muscle actin mAB. Sections were washed in PBS and incubated with a peroxidase avidin/ biotin complex (Vector Labs, Inc. Burlingame, CA). Staining was visualized with 3,3′-diaminobenzidine (DAB; Dako; Mississauga, ON) and sections were counterstained with Mayer's hematoxylin (Sigma, St Louis, Missouri). 2.6. Statistical analysis Data are presented as the mean (±SEM) for each group. A nonparametric one-tailed student's t-test was used to determine if there were significant differences between two groups. p b 0.05 was considered significant.
2.3. Immunosuppression 3. Results
Cyclosporine (Sandimmune™; CyA) immunosuppression was given subcutaneously at a dose of 50 mg/kg/day in sterile saline, for the duration of the experiment. This dose has been previously shown to be sufficient to ablate acute cardiac rejection but allow for allograft vasculopathy in cardiac and aortic grafts [14]. CyA was obtained from the Capital District Health Authority pharmacy. 2.4. Histology Transplanted sections of aorta were removed taking care not to damage the graft and frozen in an OCT:20% sucrose (2:1) solution immediately upon harvest. Sections (6–7 µm) were cut on a Leica Cryostat (CM1900 UV) and subsequently fixed for 10 min in 10% formalin to retain lipid moieties. Fixed slides were stained for 15 min with Oil Red O (0.003 g/ml in 60% isopropanol, Sigma-Aldrich), and counterstained for 2 min with Mayer's hematoxylin (Sigma-Aldrich) for qualitative observation of lipid accumulation.
3.1. The AV neointimal lesion is recipient in origin under CyA immunosuppression in mice Confirmation of the recipient origin of the neointimal lesion under CNI immunosuppression is fundamental to the understanding of the etiology of AV and the exploration of the role of pre-existing disease. Although we have demonstrated this using a rat model we had not yet provided evidence in the mouse model. In our first experiment in this study we confirmed the recipient origin of the mouse neointimal lesion in the presence of 50 mg/kg/ day CyA immunosuppression which is sufficient to ablate acute rejection [14]. Aortic segments from C3H donor mice were transferred into fully disparate B6 GFP mice. After 10 weeks the intimal lesions present in these C3H grafts were assessed for the presence of recipient B6 GFP cells. In all sections (10 sections per mouse) taken from the 3 mice transplanted, we found that the neointimal lesion was GFP positive (Fig. 1a), thus confirming the recipient origin of the lesion. In all these mice the media was GFP negative, as expected. As a control for this experiment we also visualized GFP in the endothelium and the media of cross sections of native aorta from GFP mice (Fig. 1b). As a further control we demonstrate the GFP positivity of the endothelium of aortic sections that had been harvested from B6 wild type (GFP negative) mice, stripped of their endothelium and reendothelialized in B6 GFP positive mice (Fig. 1c). As expected, the media in Fig. 1c was GFP negative, indicating the donor origin of the media in this syngeneic combination. These controls demonstrate the specificity of the GFP model.
A.M. Zaki et al. / Transplant Immunology 22 (2009) 93–98
95
actin. (Fig. 3a) In contrast, de novo neointimal lesion elements were Oil Red O negative (Fig. 3a) and α-actin positive (Fig. 3b). Both the Oil Red O and the α-actin staining suggest that the de novo lesion overlays the pre-existing lesion, although some mixing of the lesion elements cannot be discounted at this level of resolution. Immunocytochemistry for macrophages and CD4+ T cells in serial sections of the lesion above indicated that the pre-existing lesion was predominantly composed of lipid bearing foamy macrophages staining highly positive for the macrophage stain (Fig. 4a). Macrophage presence in the de novo lesion was also demonstrated but at a reduced level (Fig. 4a). CD4+ T cells were seen only in the de novo lesion and did not infiltrate the preexisting lesion (Fig. 4b). 3.4. The presence of pre-existing lesions increases both total intimal lesion area and percent lumen occlusion at 10 weeks post-transplant To ascertain whether pre-existing lesions contribute to overall lesion size in animals expressing AV, we compared the overall intimal area in aortic transplants from WT B6 donors to intimal areas in aortic transplants from B6 ApoE−/− donors (with preexisting lesions as above). The data shown in Fig. 5a demonstrate that, at 10 weeks post-transplant, there is a significant (p b 0.05, n = 6 per group) increase in overall lesion size in those grafts that had pre-existing lesions at the outset. In most cases this increase in intimal area was associated with a focal eccentric lesion corresponding to the pre-existing lesion (as shown in Fig. 3a). The data shown in Fig. 5b demonstrate that this increase in lesion size is mirrored by a significant increase in lumen occlusion in grafts with pre-existing lesions.
4. Discussion
Fig. 1. The AV neointimal lesion is recipient in origin in the presence of CNI immunosuppression. (a) WT C3H grafts into B6 GFP mice. The neointimal lesion is GFP (recipient) positive but the underlying media is GFP negative. (b) Native GFP mouse aorta. Media and intima are GFP positive. (c) WT B6 aorta re-endothelialized with GFP endothelium. Endothelium is GFP positive and media is GFP negative. M = Media. NI = Neointima.
3.2. B6 ApoE−/− mice develop abdominal aortic atherosclerosis by 30–32 weeks of age To develop a model of transplantation with pre-existing donor disease we used B6 ApoE−/− donor mice. We ascertained the development of atherosclerotic lesions in the abdominal aortas of these mice over time (12, 14 and 16 and 32 weeks of age). Younger mice only showed lesion progression in the aortic root and arch (data not shown). Only the 32-week-old mice expressed focal eccentric atherosclerotic lesions in their abdominal aorta. These were clearly visible under the operating microscope (Fig. 2a). Oil Red Ostained cross-sections confirmed the lipid rich nature of these lesions (Fig. 2b and c). 3.3. In recipients of B6 ApoE−/− aortic grafts the neointimal lesions are a combination of donor and recipient elements For this experiment we selected aortic segments from the B6 ApoE−/− mice that exhibited small-moderate sized lesions (approximately 150 µm in diameter) that would not, themselves, create obstruction. These were transplanted into fully disparate C3H mice along with CNI immunosuppression (with 50 mg/kg/day CyA). Histological examination of grafts at 10 weeks post-transplantation revealed the presence of a neointimal lesion along with clear evidence of the retention of elements of the preexisting atherosclerotic lesion. Fig. 3a and b shows the serial sections of the same lesion stained with Oil Red O (Fig. 3a) and for α-actin (Fig. 3b). There was clear evidence of retention of the pre-existing lesion as evidenced by staining of the pre-existing elements with the Oil Red O but not (or very weakly) with the antibody directed to α-
There have been many isolated reports of early vascular lesions in late adolescence and early adulthood including evidence of focal, early atheromatous lesions [32–38]. Since the majority of donor hearts come from within this demographic, questions have been raised as to exactly how much pre-existing disease is transmitted to transplant patients. In one study, 56% of the cardiac transplant patients studied had detectable donor atherosclerotic lesions when examined by IVUS [39]. The fact that the mean donor age in this study was 32 years displays the high prevalence of pre-existing disease in the general population. Due to the fact that there is a shortage of donor hearts, fewer restrictions have been placed on acceptable hearts over the past few years resulting in an increase in the number of older hearts in the donor pool [2]. This increase has subsequently led to the increase of donor hearts containing pre-existing disease. A number of studies have suggested that the neointimal AV lesions have a predilection for the proximal segments of the coronary arteries [3,40]. Available data suggest that the disease is progressive, starting at the proximal segments of the coronary arteries then advancing to medial and distal sites. Tuzcu and colleagues showed, by IVUS, a dichotomous pattern of AV [41]. Non-circumferential lesions were more common in the proximal segments of the coronary arteries than in the mid- and distal sites. Diffuse, circumferential lesions were more common in the mid- and distal sites. Pre-existing donor atherosclerosis is more frequently involved in the proximal segments of coronary arteries [3,42,43] and this may contribute to eccentric AV lesions in proximal segments. Donor atherosclerosis is rarely found at distal sites and thus the concentric nature of the lesions in these sites is probably solely due to the de novo AV lesion. The contribution of pre-existing lesions to the developing pathology of AV has been very difficult to ascertain given the tools available for human study. The animal models used to mimic human allograft vasculopathy do not account for the presence of pre-existing vasculopathy found in the coronary arteries of the donor hearts since the donor murine vessels are pristine at the time of transplantation. This study using aortic interposition grafts with pre-existing disease was designed to more appropriately model the human disease. The model chosen closely mimics the human situation in that acute rejection, which would normally be associated with fully disparate transplants, is controlled by CNI immunosuppression. This use of fully disparate transplants and CNI immunosuppression overcomes many of the weaknesses inherent in earlier AV models. Since the recipient origin of the de novo neointimal lesion is critical to our interpretation of the subsequent data we first confirmed, using
96
A.M. Zaki et al. / Transplant Immunology 22 (2009) 93–98
Fig. 2. 30–32-week-old B6 ApoE−/− mice develop atherosclerotic lesions in their abdominal aortas. (a) A representative 30–32-week-old B6 ApoE−/− abdominal aorta as seen under a dissecting microscope. White-bordered square demonstrates the pre-existing lesion. (b) Aortic segments with atherosclerosis were cross-sectioned and stained with Oil Red O. (c) Higher magnification of lesion evident in panel (b). Black arrow points to the internal elastic lamina.
green fluorescent protein expressing recipient mice, that the cells constituting the neointimal lesion in this fully disparate mouse strain combination with therapeutic levels of CyA immunosuppression are of recipient, not donor, origin. We have previously shown this in a rat model [23] but this had yet to be proven in a mouse model. Having confirmed this, we characterized the atherosclerotic disease occurring in B6 ApoE−/− mice over time on a normal chow diet, with specific reference to the appearance of lesions in the abdominal aorta. Recent imaging studies have shown that, on a normal chow diet, vascular disease in B6 ApoE−/− mice eventually occurs in both proximal and distal vessels in the aortic tree [44,45]. In this study we demonstrate that lesions are present in the abdominal aorta at 32 weeks of age but not at 16 weeks of age or earlier. Similar to other studies [44,46,47], atherosclerotic lesions were seen in the aortic root and proximal aorta much earlier. One unexpected but substantial advantage was that the atherosclerotic lesion could be visualized from the external surface of the aorta. This made it possible to select lesions of a specific size and location for transplant, thus dramatically increasing the accuracy of the resulting data. As such, small to moderate sized focal lesions (approximately 150 µm diameter) were selected for transplant to most closely approximate the human situation. Moreover, as part of the model development, a significant number of lesions were assessed macroscopically and subsequently removed for histological examination. This provided information regarding the relationship between macroscopic
and histological appearance at donor graft harvest that was invaluable for our interpretation of the 10 weeks harvest data. Our study results demonstrate a number of important points that are relevant to the human disease. First, the use of calcineurin inhibitor immunosuppression allows for the retention of the donor atherosclerotic lesion. This is of significant importance since it might be postulated that allo-immune rejection responses could have destroyed the pre-existing lesion rendering any contribution of this lesion to AV insignificant. Second, the pre-existing lipid rich lesion does not appear to form a mixed lesion with the AV lesion in that the two lesions appear to remain relatively discreet, with the AV lesion growing over the pre-existing lesion. This suggests that the two lesions do not share a common etiology. Third, the de novo neointimal lesion appears to overlay the preexisting lesion. Since the pre-existing lesions were eccentric, in areas of pre-existing lesion luminal narrowing is accentuated. This has important implications to flow dynamics and thrombosis in these large vessels. Fourth, the contribution of the pre-existing lesions resulted in an increase in overall intimal area, and associated decrease in overall lumen size, in the affected transplants. Vessels containing pre-existing lesions demonstrated lumenal occlusion of 65% (±7.2%) while vessels without pre-existing lesions demonstrated lumenal occlusion of 49% (±4.5%), a difference of 16%. Since pre-existing, pre-transplant, lesions occluded the vessels by 19% (±9.6%, data not shown) it would appear that the
A.M. Zaki et al. / Transplant Immunology 22 (2009) 93–98
97
Fig. 3. The de novo AV lesion appears to overlay the pre-existing atherosclerotic lesion. (a) Oil Red O staining of cross section of B6 ApoE−/− aortic allograft in C3H recipients 10 weeks after transplant. (b) Immunocytochemistry for α-actin in a similar (serial) section. IEL = Internal Elastic Lamina.
contribution of the pre-existing lesion is additive rather than synergistic and that the pre-existing lesions do not grow or diminish significantly after transplant in the presence of CyA immunosuppression.
Fig. 5. The presence of pre-existing lesions contributes to overall intimal lesion area and increases lumen occlusion at 10-week post-transplant. Digital image analysis of 10 weeks total intimal lesion areas and percent lumen occlusion of wild type B6 to C3H transplants and B6 ApoE−/− to C3H transplants. * = p b 0.05, n = 6 per group.
In summary, donor pre-existing lesions survive the allo-immune response in CNI immunosuppressed mice and they contribute to increased lesion size and lumenal narrowing. The increased lesion size and the eccentric nature of the chimeric lesions magnifies the potential for exacerbation of the effects of luminal narrowing and may explain the data suggesting poor outcomes associated with transplantation of cardiac grafts with mild to moderate pre-existing atherosclerosis.
References
Fig. 4. Immunocytochemistry for macrophages and CD4 T cells in the lesion. Immunocytochemistry for macrophages (a) and CD4+ T cells (b) in serial cross sections of B6 ApoE−/− aortic allografts in C3H recipients were stained with 10 weeks after transplant. IEL = Internal Elastic lamina. Small arrows indicate location of CD4+ T cells in the de novo lesion.
[1] Ramzy D, Rao V, Brahm J, Miriuka S, Delgado D, Ross HJ. Cardiac allograft vasculopathy: a review. Can J Surg 2005;48:319–27. [2] Taylor DO, Edwards LB, Aurora P, Christie JD, Dobbels F, Kirk R, et al. Registry of the International Society for Heart and Lung Transplantation: twenty-fifth official adult heart transplant report—2008. J Heart Lung Transplant 2008;27:943–56. [3] Kapadia SR, Nissen SE, Ziada KM, Guetta V, Crowe TD, Hobbs RE, et al. Development of transplantation vasculopathy and progression of donor-transmitted atherosclerosis: comparison by serial intravascular ultrasound imaging. Circulation 1998;98:2672–8. [4] Li H, Tanaka K, Anzai H, Oeser B, Lai D, Kobashigawa JA, et al. Influence of pre-existing donor atherosclerosis on the development of cardiac allograft vasculopathy and outcomes in heart transplant recipients. J Am Coll Cardiol 2006;47:2470–6. [5] Sandler D, McKenzie FN, Menkis AH, Novick RJ, Pflugfelder PW, Kostuk WJ. Early death after cardiac transplantation—the role of unsuspected donor coronary artery disease. J Heart Lung Transplant 1991;10:172. [6] Grauhan O, Hetzer R. Impact of donor-transmitted coronary atherosclerosis. J Heart Lung Transplant 2004;23:S260–2. [7] Grauhan O, Patzurek J, Hummel M, Lehmkuhl H, Dandel M, Pasic M, et al. Donortransmitted coronary atherosclerosis. J Heart Lung Transplant 2003;22:568–73. [8] Grauhan O, Siniawski H, Dandel M, Lehmkuhl H, Knosalla C, Pasic M, et al. Coronary atherosclerosis of the donor heart—impact on early graft failure. Eur J Cardiothorac Surg 2007;32:634–8. [9] Wong CK, Yeung AC. The topography of intimal thickening and associated remodeling pattern of early transplant coronary disease: influence of pre-existent donor atherosclerosis. J Heart Lung Transplant 2001;20:858–64.
98
A.M. Zaki et al. / Transplant Immunology 22 (2009) 93–98
[10] Gao HZ, Hunt SA, Alderman EL, Liang D, Yeung AC, Schroeder JS. Relation of donor age and preexisting coronary artery disease on angiography and intracoronary ultrasound to later development of accelerated allograft coronary artery disease. J Am Coll Cardiol 1997;29:623–9. [11] Libby P, Pober JS. Chronic rejection. Immunity 2001;14:387–97. [12] Currie M, Zaki AM, Nejat S, Hirsch GM, Lee TD. Immunologic targets in the etiology of allograft vasculopathy: endothelium versus media. Transpl Immunol 2008;19:120–6. [13] Skaro AI, Liwski RS, Zhou J, Vessie EL, Lee TD, Hirsch GM. CD8+ T cells mediate aortic allograft vasculopathy by direct killing and an interferon-gamma-dependent indirect pathway. Cardiovasc Res 2005;65:283–91. [14] Vessie EL, Hirsch GM, Lee TD. Aortic allograft vasculopathy is mediated by CD8(+) T cells in Cyclosporin A immunosuppressed mice. Transpl Immunol 2005;15:35–44. [15] Nejat S, Zaki A, Hirsch GM, Lee TD. CD8(+) T cells mediate aortic allograft vasculopathy under conditions of calcineurin immunosuppression: role of IFN-gamma and CTL mediators. Transpl Immunol 2008;19:103–11. [16] Shimizu K, Sugiyama S, Aikawa M, Fukumoto Y, Rabkin E, Libby P, et al. Host bonemarrow cells are a source of donor intimal smooth- muscle-like cells in murine aortic transplant arteriopathy. Nat Med 2001;7:738–41. [17] Sata M, Hirata Y, Nagai R. Circulating recipient cells contribute to graft coronary arteriosclerosis. J Cardiol 2002;39:48–9. [18] Hillebrands JL, Klatter FA, Rozing J. Origin of vascular smooth muscle cells and the role of circulating stem cells in transplant arteriosclerosis. Arterioscler Thromb Vasc Biol 2003;23:380–7. [19] Hu Y, Davison F, Ludewig B, Erdel M, Mayr M, Url M, et al. Smooth muscle cells in transplant atherosclerotic lesions are originated from recipients, but not bone marrow progenitor cells. Circulation 2002;106:1834–9. [20] Saiura A, Sata M, Hirata Y, Nagai R, Makuuchi M. Circulating smooth muscle progenitor cells contribute to atherosclerosis. Nat Med 2001;7:382–3. [21] Li J, Han X, Jiang J, Zhong R, Williams GM, Pickering JG, et al. Vascular smooth muscle cells of recipient origin mediate intimal expansion after aortic allotransplantation in mice. Am J Pathol 2001;158:1943–7. [22] Hillebrands JL, Klatter FA, van den Hurk BM, Popa ER, Nieuwenhuis P, Rozing J. Origin of neointimal endothelium and alpha-actin-positive smooth muscle cells in transplant arteriosclerosis. J Clin Invest 2001;107:1411–22. [23] Johnson P, Carpenter M, Hirsch G, Lee T. Recipient cells form the intimal proliferative lesion in the rat aortic model of allograft arteriosclerosis. Am J Transplant 2002;2:207–14. [24] Glaser R, Lu MM, Narula N, Epstein JA. Smooth muscle cells, but not myocytes, of host origin in transplanted human hearts. Circulation 2002;106:17–9. [25] Hruban RH, Long PP, Perlman EJ, Hutchins GM, Baumgartner WA, Baughman KL, et al. Fluorescence in situ hybridization for the Y-chromosome can be used to detect cells of recipient origin in allografted hearts following cardiac transplantation. Am J Pathol 1993;142:975–80. [26] Grimm PC, Nickerson P, Jeffery J, Savani RC, Gough J, McKenna RM, et al. Neointimal and tubulointerstitial infiltration by recipient mesenchymal cells in chronic renalallograft rejection. N Engl J Med 2001;345:93–7. [27] Quaini F, Urbanek K, Beltrami AP, Finato N, Beltrami CA, Nadal-Ginard B, et al. Chimerism of the transplanted heart. N Engl J Med 2002;346:5–15. [28] Atkinson C, Horsley J, Rhind-Tutt S, Charman S, Phillpotts CJ, Wallwork J, et al. Neointimal smooth muscle cells in human cardiac allograft coronary artery vasculopathy are of donor origin. J Heart Lung Transplant 2004;23:427–35. [29] Russell JC, Proctor SD. Small animal models of cardiovascular disease: tools for the study of the roles of metabolic syndrome, dyslipidemia, and atherosclerosis. Cardiovasc Pathol 2006;15:318–30. [30] Zadelaar S, Kleemann R, Verschuren L, de Vries-Van der Weij J, van der Hoorn J, Princen HM, et al. Mouse models for atherosclerosis and pharmaceutical modifiers. Arterioscler Thromb Vasc Biol 2007;27:1706–21.
[31] Koulack J, McAlister VC, Giacomantonio CA, Bitter-Suermann H, MacDonald AS, Lee TD. Development of a mouse aortic transplant model of chronic rejection. Microsurgery 1995;16:110–3. [32] McGill Jr HC, McMahan CA. Determinants of atherosclerosis in the young. Pathobiological Determinants of Atherosclerosis in Youth (PDAY) Research Group. Am J Cardiol 1998;82:30T–6T. [33] Velican D, Velican C. Atherosclerotic involvement of the coronary arteries of adolescents and young adults. Atherosclerosis 1980;36:449–60. [34] Enos WF, Holmes RH, Beyer J. Coronary disease among United States soldiers killed in action in Korea; preliminary report. J Am Med Assoc 1953;152:1090–3. [35] McNamara JJ, Molot MA, Stremple JF, Cutting RT. Coronary artery disease in combat casualties in Vietnam. JAMA 1971;216:1185–7. [36] Berenson GS, Wattigney WA, Tracy RE, Newman III WP, Srinivasan SR, Webber LS, et al. Atherosclerosis of the aorta and coronary arteries and cardiovascular risk factors in persons aged 6 to 30 years and studied at necropsy (The Bogalusa Heart Study). Am J Cardiol 1992;70:851–8. [37] St Goar FG, Pinto FJ, Alderman EL, Fitzgerald PJ, Stinson EB, Billingham ME, et al. Detection of coronary atherosclerosis in young adult hearts using intravascular ultrasound. Circulation 1992;86:756–63. [38] Strong JP, Malcom GT, McMahan CA, Tracy RE, Newman III WP, Herderick EE, et al. Prevalence and extent of atherosclerosis in adolescents and young adults: implications for prevention from the Pathobiological Determinants of Atherosclerosis in Youth Study. JAMA 1999;281:727–35. [39] Tuzcu EM, Hobbs RE, Rincon G, Bott-Silverman C, De Franco AC, Robinson K, et al. Occult and frequent transmission of atherosclerotic coronary disease with cardiac transplantation. Insights from intravascular ultrasound. Circulation 1995;91:1706–13. [40] Hiemann NE, Wellnhofer E, Knosalla C, Lehmkuhl HB, Stein J, Hetzer R, et al. Prognostic impact of microvasculopathy on survival after heart transplantation: evidence from 9713 endomyocardial biopsies. Circulation 2007;116:1274–82. [41] Tuzcu EM, De Franco AC, Goormastic M, Hobbs RE, Rincon G, Bott-Silverman C, et al. Dichotomous pattern of coronary atherosclerosis 1 to 9 years after transplantation: insights from systematic intravascular ultrasound imaging. J Am Coll Cardiol 1996;27:839–46. [42] Rahmani M, Cruz RP, Granville DJ, McManus BM. Allograft vasculopathy versus atherosclerosis. Circ Res 2006;99:801–15. [43] Nissen SE, Yock P. Intravascular ultrasound: novel pathophysiological insights and current clinical applications. Circulation 2001;103:604–16. [44] Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb 1994;14:133–40. [45] Isobe S, Tsimikas S, Zhou J, Fujimoto S, Sarai M, Branks MJ, et al. Noninvasive imaging of atherosclerotic lesions in apolipoprotein E-deficient and low-density-lipoprotein receptor-deficient mice with annexin A5. J Nucl Med 2006;47:1497–505. [46] Choudhury RP, Fayad ZA, Aguinaldo JG, Itskovich VV, Rong JX, Fallon JT, et al. Serial, noninvasive, in vivo magnetic resonance microscopy detects the development of atherosclerosis in apolipoprotein E-deficient mice and its progression by arterial wall remodeling. J Magn Reson Imaging 2003;17:184–9. [47] Itskovich VV, Choudhury RP, Aguinaldo JG, Fallon JT, Omerhodzic S, Fisher EA, et al. Characterization of aortic root atherosclerosis in ApoE knockout mice: highresolution in vivo and ex vivo MRM with histological correlation. Magn Reson Med 2003;49:381–5.