Allograft arteriopathy

Cardiovascular Pathology 13 (2004) 33 – 40

Allograft arteriopathy Pathogenesis update Richard N. Mitchell * Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, 77 Avenue Louis Pasteur / NRB 730D, Boston, MA 02115, USA Received 12 September 2003

Abstract Allograft arteriopathy is a vascular intimal proliferative process that occurs in all solid organ transplants and stands as the single most significant obstacle to successful long-term solid organ transplantation; it shares a number of pathologic features with restenosis lesions and atherosclerosis. This article will review some of the newer developments in our understanding of the immunological and vascular biology underpinnings of the disease, including the roles played by cytokine and chemokine mediators in recruiting and activating both inflammatory cells, as well as smooth muscle cell precursors. D 2004 Elsevier Inc. All rights reserved. Keywords: Transplantation; Arteriopathy; Rejection; Cytokine; Chemokine; Immunology; Atherosclerosis

1. Introduction Progressively better therapies now allow us to largely prevent or at least effectively treat acute allograft rejection; 1-year survival statistics for most solid organs exceeds 80 – 90%. Consequently, the long-term survival of solid organ transplants has increasingly become limited primarily by the development of allograft arteriopathy (AA). With a mean onset of detectable disease occurring at approximately 5 –7 years, AA manifests as severe, diffuse vascular intimal hyperplastic lesions leading eventually to lumenal stenoses and ischemic graft failure; interestingly, it is most prevalent in cardiac transplants [1,2]. Variously called chronic rejection, graft vascular sclerosis, graft arterial disease, or transplant associated arteriosclerosis, AA lesions consist primarily of smooth muscle cells (SMCs) and associated extracellular matrix, admixed with infiltrating mononuclear leukocytes (see below). In addition to progressive vascular stenoses, a presumably similar pathway in lung transplants results in SMC proliferation and matrix synthesis that gradually occludes airways (bronchiolitis obliterans). In the case of cardiac transplants, patients may develop con-

* Tel.: +1-617-278-0314. E-mail address: [email protected] (R.N. Mitchell). 1054-8807/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/S1054-8807(03)00108-X

gestive heart failure due to progressive loss of functioning myocardium, or they may have a sudden cardiac death due to a lethal arrhythmia or large infarct [3]. Clearly, such an outcome makes AA in the heart of greater immediate clinical import than in other organs where there may be a more insidious decline in graft function.

2. Histology and diagnosis AA is an intimal fibroproliferative process that differs from conventional atherosclerosis in that it forms concentric, often lipid-poor, longitudinally diffuse lesions, extending from epicardial vessels into intramyocardial arterioles (Fig. 1). The histologic appearance of AA differs from that seen in arteries undergoing acute rejection, the latter manifesting predominantly as an acute necrotizing vasculitis. AA lesions typically exhibit minimal acute necrosis with little to suggest a healed vasculitis, i.e., no fragmentation or reduplication of the internal elastic lamina [3,4]. Although there is minimal necrosis, the intimas of affected vessels show variable mononuclear cell infiltrates comprised of activated T lymphocytes and macrophages [3– 6]. The bulk of the cellularity in the expanded intimas derives from SMCs and associated ECM. Although the majority of the loss of lumenal diameter has been attributed to the intimal hyperplasia, advential scarring (also called constrictive

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R.N. Mitchell / Cardiovascular Pathology 13 (2004) 33–40

Fig. 1. Schematic comparing typical atherosclerosis and AA.

remodeling) can cause a fixed restrictive element around the vessel and contribute to the lumenal stenosis [7,8]. Since AA typically involves an extensive length of the coronary anatomy, affected hearts are not amenable to conventional bypass surgery or angioplasty, and the only therapeutic recourse in most cases is retransplant. Moreover, the diffuseness of the disease renders traditional angiographic diagnostic techniques relatively insensitive; intravascular ultrasound is increasingly being used as the preferred imaging modality, at least for the proximal vasculature accessible to the ultrasound probe [9 – 11]. Certain histologic findings on routine endomyocardial biopsies may also be helpful in suggesting a diagnosis of AA. Thus, subendocardial myocyte vacuolization indicative of chronic sublethal ischemic injury, and/or well-circumscribed subendocardial watershed microscopic infarcts are highly specific (if not necessarily sensitive) indicators of AA [12].

3. Role of alloimmune responses in AA AA lesions do not occur in syngeneic grafts in animal models and are not seen in the atrial remnants in human transplant recipients. Therefore, the arteriopathy is almost certainly the culmination of an initial alloimmune response to graft antigens [13 –15]. Experimental data suggest that indirect allorecognition of the graft — that is, processing of graft antigens by host cells for presentation to host lymphocytes rather than direct stimulation of host lymphocytes by graft cells — may be the most important route by which AA is induced [16,17]. Distressingly, AA lesions also typically develop in the setting of immunosuppression regimens sufficient to block most acute parenchymal rejection; indeed, AA incidence in most human studies does not strictly correlate with episodes or severity of allograft rejection [18]. Nevertheless, transient parenchymal rejection (involving injury to myocytes) has been demonstrated to potentiate the process of AA development [19 –21]. Moreover, as an integral part of an allograft, vascular wall cells (endothelial

cells [ECs] and SMCs) will obviously also experience rejection-induced injury. However, there is no consensus in ways to evaluate any such ‘‘vascular rejection’’ (at least in the heart) and vascular injury has not been traditionally assessed in human allografts [22]. Despite the lack of agreement and our inability to systematically assess vessel ‘‘rejection,’’ it is nevertheless reasonable to conclude that AA occurs secondary to immune-mediated injury of the graft vasculature. Mechanistically, it is most likely that the injury to the vessel wall cells is some form of delayed-type hypersensitivity (DTH) [13,23]. In fact, it has been postulated that the chronicity of the vascular injury may be due to a DTH response that is intrinsically ineffective in eliminating donor antigens (i.e., the ECs lining the vessels). In support of this, allogeneic ECs have been demonstrated to induce a unique subset of cytotoxic CD8+ T cells [24,25], that are less efficient at target cell killing. Moreover, significant numbers of donor arterial wall ECs and medial SMCs can be demonstrated in long-term murine cardiac allografts — even in those with well-developed AA — suggesting that persistence of alloantigen in the absence of efficient cell killing may also partly explain a chronic alloresponse [26]. Despite the fact that alloresponses undoubtedly underlie AA pathogenesis, the vascular lesions do not require ongoing allogeneic stimulation to develop. Thus, transient acute rejection with subsequent complete ablation of the alloresponse [20], or return of an allograft into an autologous recipient after an episode of rejection [27 – 29] still ultimately results in AA lesions. The implication is that initial allospecific activation may cause the recruitment and activation of secondary effector cells, including macrophages and natural killer (NK) cells. Through selected cellular recruitment (via chemokines) and subsequent development of a local milieu of inflammatory mediators (called cytokines), the effector cells may establish an environment sufficient for the subsequent alloantigen-independent development of AA. Perioperative ischemic injury [30], antibodymediated processes [31 –33], cytokine-induced endothelial dysfunction, and toxic or infectious insults (e.g., with CMVor other viruses [34 – 36] may also participate in alloantigenindependent activation of ECs, SMCs, or inflammatory cells and may serve to amplify the allospecific pathways.

4. Cellular vs. humoral immunity in AA Regardless of the initial cellular targets, some combination of humoral (antibody) and cellular immune effector mechanisms are brought to bear against the graft vasculature (or airways). In the last few years, particularly with the advent of C4d immunofluorescence staining to identify putative humoral rejection [37 –39], there has been substantial renewed interest in the role played by alloantibodies in AA [40,41]. Indeed, animal models indicate that alloantibodies can participate in the development of arteriopathy

R.N. Mitchell / Cardiovascular Pathology 13 (2004) 33–40

lesions [32,33]. However, it remains controversial whether such alloantibodies play any significant role in human AA, particularly in the heart. Indeed, AA in animal models can also occur in the absence of alloreactive antibodies, presumably driven by cytotoxic T cell and natural killer cell injury as well as by soluble mediators [42,43]. Perhaps in another few years, a more definitive analysis can be written on the topic of humoral rejection in AA. In lieu of that future manuscript, the current review will highlight some of the newer progress in our understanding of the specific cellular mechanisms that occur downstream of the inciting immunologic injury and that mediate AA development. Until very recently, the working hypothesis was that cytopathic mechanisms marshalled by the immune system (e.g., cytotoxic T cells, activated macrophages, alloantibodies and complement, etc.) promulgated persistent allograft vascular damage [13,44]. In turn, inflammatory cells, and/or activated, dysfunctional ECs and medial SMCs secreted chemoattractant molecules (chemokines) and growth factors (cytokines) that induced the migration of SMCs from the donor media into the arterial intima, as well as stimulated their proliferation and matrix synthesis. This model of the pathogenesis now requires some revision in light of the recent demonstration in aortic and heart experimental systems [45 – 48], as well as in human renal and cardiac transplants [49,50], that intimal SMCs are actually derived from circulating host precursors. While many of the newer insights reviewed here derive from work using animal experimental models, reference will be made to human data whenever possible.

5. Role of cytokines in AA Cytokines are protein mediators that are secreted by a variety of cell types, and in general have pleiotropic effects. Many cytokines are produced primarily by cells mediating antigen-specific immunity (e.g., T and B cells) and have roles in modulating immunologic responses. Some cytokines act in an autocrine or paracrine fashion, to influence the behavior of neighboring T and B cells. Other cytokines act primarily on nonlymphoid cells such as macrophages, SMCs, and ECs. For example, interferon-g (IFNg) activates macrophages, augments inflammatory cell recruitment by up-regulating adhesion molecules and chemokine expression, and amplifies immune responses by increasing major histocompatibility complex (MHC) molecule expression on antigen-presenting cells (APCs) and ECs [51]. Cytokines produced by antigen nonspecific cells (e.g., macrophages) generally play a broad role in stimulating or inhibiting inflammatory responses. In this category are interleukin (IL)-1 and tumor necrosis factor-a (TNFa), both of which have sweeping proinflammatory actions including EC activation (promoting inflammatory cell adhesion and procoagulant activity) and SMC proliferation and synthetic function. Relevant to the pathogenesis of AA, some cyto-

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kines (e.g., TGFh) may also induce a local fibrosing response [52,53]. The observations that ECs and SMCs can differentially activate T cells — potentially via specific cytokine and chemokine elaboration [54 –56] — suggest a complex set of interactions occurring in the cytokine milieu of the vessel wall. IFNg is a cytokine that is absolutely necessary for the development of AA. Thus, intimal lesions do not occur in heterotopic cardiac allografts in IFNg-deficient mice, despite ongoing parenchymal rejection [20,57]; comparable results were seen in wild type (WT) recipients receiving weekly injections of anti-IFNg antibodies [57]. Relative to grafts transplanted into WT recipients, allografts transplanted into IFNg-deficient hosts showed similar numbers of helper and cytotoxic T cells, as well as macrophages, despite the fact that vascular wall chemokine elaboration (see below) and adhesion molecule expression were attenuated. Perhaps more importantly, the various inflammatory cells in IFNg-deficient hosts were not as activated as their wild type counterparts, as inferred by diminished MHC and costimulator molecule expression [57]. Based on these observations in experimental models, it is now generally accepted that AA results from an initial alloresponse ( potentially in addition to other forms of vascular injury) that affects the recruitment and activation of macrophages in a process dependent on IFNg [57]. Antigen nonspecific mononuclear phagocytes subsequently elaborate fibrogenic mediators such as TGFh, as well as SMC growth stimulators such as IL-1 and TNF [13,14,23] that drive the development of the final intimal hyperplastic lesion. It is also noteworthy that IFNg (frequently in conjunction with TNFa) has been shown to regulate the expression of chemokines, chemokine receptors, and adhesion molecules that direct the recruitment and activation of SMCs in atherosclerotic plaque [58 –60]. Although IFNg inhibits medial SMC proliferation in vitro [61], the presence or absence of IFNg correlates with the extent of disease in murine models of atherosclerosis. It is therefore conceivable that intimal SMCs in AA may respond differently than medial SMCs to this cytokine (and others in the perivascular cytokine milieu), and that the efficacy of IFNg blockade may in part also derive from diminished SMC recruitment and/or activation. If the animal models are indeed correct, proximal blockade of IFNg, or some of its distal effects (e.g., macrophage recruitment and/or activation) during periods of parenchymal rejection could conceivably ameliorate the later development of AA in human transplants. Unfortunately, to date, no such therapeutic agent specific for IFNg has been identified for treating human disease. Sirolimus (rapamycin) coating of intravascular stents has shown promising efficacy in suppressing the histologically similar intimal hyperplasia seen in stent restenosis [62]. Similar efficacy may be seen with rapamycin or related agents in preventing AA, potentially via blockade of inflammatory cell proliferative responses and/or cellular migration [63,64].

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6. Role of chemokines in AA Chemokines are small molecular weight (8 – 10 kDa) secreted proteins responsible for the recruitment and activation of a variety of inflammatory and noninflammatory cells. Chemokine receptors are G-protein-coupled seventransmembrane-spanning proteins with discrete cellular distributions and binding specificities [55,65]; there are over 20 so far described. Greater than 50 chemokines have been identified and are broadly classified into four subfamilies based on the position of internal cysteine residues (e.g., adjacent cysteines in the CC chemokines, or cysteines separated by some other amino acid in CXC chemokines). For example, mediators responsible for the recruitment of neutrophils and monocyte – macrophages involved in innate immunity include the so-called CXC chemokines such as macrophage inflammatory protein-2a (MIP-2a), Groa, and interleukin-8, as well as CC chemokines such as macrophage chemoattractant protein-1 (MCP-1). Mediators that contribute to the recruitment of antigen-specific T cells include CXC chemokines such as monokine induced by IFNg (Mig), IFNg-inducible protein 10 (IP-10), and IFNginducible T cell alpha chemoattractant (I-TAC), as well as CC chemokines like regulated on activation, normal T cell expressed and secreted (RANTES). Of the CXC chemokines, IP-10, Mig, and I-TAC are all strongly induced by IFNg and may therefore be extremely relevant in the development of AA. The role of chemokines in acute allograft rejection has been the subject of a number of recent excellent reviews [66 – 69]; potentially unique roles for chemokines in the development of AA have only recently been proposed [70]. The roles of specific chemokines in the recruitment of inflammatory cell subsets and SMCs and in the develop-

ment of AA have been summarized in schematic form in Fig. 2. Graft ECs represent the initial site where host inflammatory cells encounter and recognize foreign donor MHC. Recipient inflammatory cells must adhere to ECs and subsequently pass through the vessel wall into the donor parenchyma. The process begins with antigen nonspecific neutrophils and monocyte/macrophages, cells of innate immunity recruited into allografts as a consequence of allograft ischemic injury [67,71]. This initial wave promotes the recruitment and activation of alloreactive T cells, which in turn drive the recruitment of additional macrophages, and eventually SMC precursors (see below) leading to AA. Interrupting chemokine cellular conscription pathways at any of these stages — innate immunity, allospecific immune responses, secondary macrophage recruitment, or intimal SMC recruitment — can potentially yield beneficial effects in attenuating the development of AA lesions. The plethora of chemokines and receptors, as well as the apparent promiscuity of chemokine – receptor interactions, raises the relevant issue whether any single chemokine (e.g., MCP-1) or receptor can have a central nonredundant role in a given inflammatory process such as AA. It appears that selected MCP-1 or CCR2 ablations in genetically deficient mice do materially impact monocyte recruitment and activation in DTH lesions [72,73] and attenuate the development of atherosclerosis [74,75]. Relevant to transplantation, monoclonal antibody blockade of the IFNg-induced chemokine Mig diminished allograft rejection [76]. Targeted deletion of the CCR1 chemokine receptor reduced both acute parenchymal rejection as well as the development of AA [77]. Moreover, a selective nonpeptide CCR1 receptor antagonist (BX471) developed by Berlex Biosciences (Richmond, CA) has efficacy in acute cardiac rejection models in rats and rabbits [78,79].

7. Source of intimal cells in AA

Fig. 2. Chemokines involved in the development of AA lesions. The three major phases of the process are temporally dominated by subsets of chemokines that in turn drive the recruitment of cells of innate immunity, allospecific immune T cells, and SMCs. The major chemokines are indicated at each stage (notably IL-8, MIP-2, Mig, IP-10, and I-TAC are all regulated by IFNg); the major sources of the chemokines are indicated in parentheses below each set of chemokines.

A subpopulation of peripheral circulating cells and bone marrow cells can differentiate into ECs [80,81], and host ECs have been shown to seed synthetic vascular grafts implanted in vivo [82]. However, the majority of ECs — as well as the medial SMCs — lining AA lesions in longterm cardiac allografts remain of donor origin [26,45,83]. This persistence of donor arteriolar ECs in cardiac allografts is significant in that they may well continue to drive an alloresponse that culminates in AA lesions. With regard to the intimal SMCs in AA, the story is more complex — and more interesting. In experimental models host SMCs were identified in synthetic grafts as well as in some arterial allografts [82,84]. Despite these earlier findings, it has nevertheless been generally assumed that the majority of intimal SMCs in AA lesions originated by ingrowth of donor SMCs from the media of engrafted vessels [85]. Indeed, the paradigm for all vascular pathologic lesions (i.e., atherosclerosis, restenosis after balloon injury, AA, etc.)

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has been that the intimal SMCs migrate from the underlying media. Although it has long been appreciated that intimal and medial SMCs have distinct phenotypes [86], such changes have been variously attributed to activation or dedifferentiation as the cells emigrated from one tunica to the other. Since the primary function of differentiated medial SMCs is to regulate vascular tone, the majority of SMCs in normal adult arteries are in a contractile state, and exhibit a substantially lesser synthetic function [87]. In contrast, neointimal SMCs recruited to effect a ‘‘repair’’ exhibit a distinct phenotype [86], variously described as ‘‘embryonic’’ growth phenotype [88], adult ‘‘platelet-derived growth-factor-independent’’ phenotype [89], or an embryonic/fetal/neonatal growth phenotype [88,90 – 92]. These intimal SMCs are synthetically active and share morphologic features with the cells of prenatal arteries [87]. Besides the phenotypic distinctions, there is also substantial evidence that intimal SMCs are clonal and potentially have a unique derivation [93]. Nevertheless, the paradigm view remained that the intimal cells derived from the underlying media. These assumptions led to unsuccessful experimental molecular interventions (e.g., gene therapy) targeting donor medial SMC proliferation to ameliorate AA [94,95]. In the last 2 years, we’ve been forced to rethink this paradigm. It has been now shown that intimal SMCs in vascular grafts are virtually all host-derived [45 –48]; in addition, it has been demonstrated that a proportion of these originate from host bone-marrow-derived cells [48]. This latter result is consistent with the observation that bone marrow stem cells are capable of developing into multiple mesenchymal lineages [96], and that peripheral blood contains CD34+, flt1+, and flk1+ precursors capable of SMC differentiation [97]. Since there are numerous important functional differences between medial SMCs and the neointimal cells [98 – 101], it is therefore perhaps more accurate to refer to the intimal population as smooth muscle-like cells (SMLCs) to distinguish them from the anatomically and phenotypically distinct medial SMCs [48]. These intimal SMLCs are elongated cells with oval nuclei and cytoplasmic filaments similar to those seen in medial SMCs [85]; they express characteristic SMC markers such as smooth muscle a-actin, calponin, and SM1 [48]. Interestingly, a similar circulating origin of SMLC-like cells in restenosis lesions and in atherosclerotic plaques in both mice [102] and humans [103] has now been demonstrated. These findings suggest that the intimal hyperplasia seen in all manner of vascular pathology may be more attributable to the recruitment of circulating precursors than to the recruitment of underlying medial SMCs! Even more recently, it has been demonstrated that vascular SMCs (both intimal and medial varieties) can produce IFNg directly [104,105]; the implication is that after appropriate stimulation, SMCs or SMLCs can potentially be an ongoing source of the cytokine critical to AA development — even after inflammatory cells have long vacated the perivascular area.

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In summary, while understanding the molecular mechanisms that regulate intimal SMLC replication and migration will be important to develop therapies to ameliorate AA, it is also likely that similar strategies may prove extremely relevant to preventing the intimal lesions in arterial restenosis and atherosclerosis.

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