—  SPECIALTY CONFERENCE  —

Cardiovascular Pathology

Case 4 - Sudden Cardiac Death


Richard N. Mitchell
Brigham and Women's Hospital
Boston, Massachusetts


Click on each slide thumbnail image for an enlarged view
Clinical History:
The patient was a 34 year old male with a history of congenital heart disease (D-transposition of the great arteries); a Mustard repair procedure at age 4 was complicated by right phrenic nerve damage and right diaphragmatic paralysis. He subsequently developed progressive failure of his systemic ventricle, and underwent cardiac transplantation in 1990 (at the age of 22); his post-transplant immunosuppression consisted of cyclosporine A, azothioprine, and corticosteroids. His course was notable for two episodes of acute rejection in the first year following transplantation, treated with solumedrol boluses. In addition, within 4 months of transplant, he developed a post-transplant lymphoma in a left submandibular node. This was resected, and he experienced no further recurrence after his immunosuppressive therapy was slightly tapered. Other complications included obesity (eventually developing Pickwickian symptomatology), hypertension, osteoporosis, and hyperlipidemia.


Slide 12 - Anterior half of heart sectioned in an echocardiographic 4-chamber view. The lateral free wall and mid-septal walls exhibit focal scarring, as well as depressed glassy-appearing areas indicating granulation tissue.
Slide 13 - Section from mid-left anterior descending coronary artery demonstrating moderate concentric intimal hyperplasia consisting of chronic inflammatory cells, smooth muscle cells, and extracellular matrix. This is representative of the appearance of all the epicardial arteries as well as many of the penetrating intramyocardial arterioles.



Slide 14 - Section of interventricular septum exhibiting diffuse, severe subendocardial myocyte vacuolization indicative of chronic sub-lethal ischemic injury. When seen on routine endomyocardial biopsy in transplant recipients, these changes are highly specific for allograft arteriopathy (See reference: Winters GL and Schoen FJ. J Heart Lung Transplant 1997;16:985-993).
Slide 15 - Section of left ventricular free wall showing well-circumscribed subendocardial microscopic infarction. Such watershed microscopic infarcts are also strongly associated with allograft arteriopathy (See reference: Winters GL and Schoen FJ. J Heart Lung Transplant 1997;16:985-993).

The patient had annual angiographic evaluation of his coronaries; beginning approximately 10 years after transplant, diffuse coronary artery disease was first seen, most apparent in the smaller coronary arteries. Intravascular ultrasound also documented moderate-severe diffuse epicardial vessel disease with up to 85% chronic stenoses. Beginning 11 years after transplant, the patient developed progressive, severe exertional dyspnea and biventricular failure (R>L), although ejection fractions were largely maintained; moderate pulmonary hypertension was documented. PFT's documented profound restrictive and obstructive lung disease; although he was found to be a carrier for cystic fibrosis, his findings were felt overall to be most consistent with central neurogenic hypoventilation.

The patient presented terminally with right lower abdomenal discomfort; CT evaluation showed only mild thickening of the distal ileum and he was discharged to be worked up electively. He subsequently suffered a cardiac arrest at home, and could not be resuscitated. Slides available for review are a representative coronary artery and myocardium from the failed allograft.

Differential Diagnosis

  • allograft arteriopathy with sudden cardiac death
  • pulmonary embolism
  • GI perforation/hemorrhage due to lymphoma or opportunistic infection
  • severe acute rejection
  • sepsis

Autopsy Findings
The lungs showed only minimal edema (combined weights of 920 g), and focal non-specific parenchymal hemorrhage; there was no evidence of acute or remote embolization, and no significant parenchymal fibrosis. The liver exhibited only mild central congestion. The kidneys were severely autolyzed and arteriolar hyalinization could not be evaluated. There was no morphologic cause apparent to explain the pre-terminal right lower quadrant pain; specifically there was no evidence of gastrointestinal tract ischemia, infarction, or inflammation. There was no mesenteric lymphadenopathy, and no evidence of lymphoma. Post-mortem cultures showed only typical post-mortem contaminants.

The heart exhibited moderate cardiomegaly (wt = 600 g), with well-healed atrial and vascular anastomoses. There was biventricular hypertrophy and moderate biventricular dilation, with mild-moderate myxomatous degeneration of the mitral valve and a bland, poorly organized thrombus on the anterior leaflet of the mitral valve. Grossly, there was evidence of focal remote myocardial scarring, as well as focal granulation tissue consistent with more recent infarction. There was moderate diffuse concentric intimal hyperplasia of all major epicardial vessels (allograft arteriopathy) causing 50-70% chronic stenoses without any acute thrombosis. Notably, the intramyocardial arterioles exhibited severe allograft arteriopathy with focal sub-total occlusions (see histology sections). The myocardium exhibited diffuse subendocardial myocyte vacuolization indicative of chronic low-grade ischemia; focal remote sub-endocardial microscopic infarcts were also present, consistent with allograft arteriopathy. In addition, focal scattered acute rejection with myocyte injury and Quilty A-type lesions were also present, without any significant myocardial edema.

Diagnosis
Sudden cardiac death in the setting of remote and recent myocardial infarctions, secondary to allograft arteriopathy 12 years status post cardiac transplantation.

Discussion
Despite excellent therapies to prevent and treat acute allograft rejection, successful long-term solid organ transplant survival continues to be limited by allograft arteriopathy. With a mean onset of detectable pathology at approximately 5-7 years, transplanted organs typically develop severe, diffuse intimal hyperplastic lesions leading eventually to lumenal stenoses and to ischemic graft failure. The intimal lesions, variously called chronic rejection, allograft arteriopathy, graft vascular sclerosis, graft arterial disease, or transplant associated arteriosclerosis, result from an initial alloimmune response, although the entire set of effector mechanisms remain to be elucidated.1, 2  The lesions consist primarily of smooth muscle cells (SMC) and associated extracellular matrix, admixed with infiltrating mononuclear leukocytes. Until recently, the working hypothesis was that low-level endothelial or perivascular immune responses induced persistent allograft vascular damage.1, 3  In turn, inflammatory cells, and/or activated, dysfunctional endothelial cells (EC) and medial SMC would secrete growth factors that induced the migration of SMC from the donor media into the arterial intima, as well as stimulated their proliferation and matrix synthesis. This hypothesis has now been modified by the recent demonstration in aortic and heart experimental models,4-8  as well as in human renal transplantation,9  that intimal SMC are actually derived from circulating host precursors.

Allograft arteriopathy (AA)
In the current era of immunosuppression, graft failure due to acute rejection occurs relatively infrequently, and one-year survival statistics for most solid organs exceeds 90%. Thus, AA has emerged as the principal barrier to long-term solid organ allograft survival; interestingly, it is most prevalent in cardiac transplants.10, 11  In the case of cardiac allografts, patients may develop congestive 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.12  Distressingly, AA lesions occur even in the setting of immunosuppression regimens adequate to block most acute parenchymal rejection and does not strictly correlate with episodes or severity of allograft rejection.13 

As an integral part of an allograft, vessels will experience acute rejection prior to the development of AA. A number of workers have shown that although acute rejection episodes do not strictly correlate with the development of AA, transient acute rejection may potentiate the process.14-16  Consequently, AA likely occurs predominantly as a consequence of immune-mediated allospecific vascular injury in grafts,1  most likely a form of delayed-type hypersensitivity (DTH). The chronicity of the disease may be due a DTH response which is intrinsically ineffective in eliminate donor antigen (i.e., the EC). There is evidence that allogeneic EC induce a unique subset of cytolytic CD8+ T cells,17, 18  which are less efficient at target cell killing. As a source of persistent alloantigen, donor arterial vascular wall EC and medial SMC are still present in long-term murine cardiac allografts that had already developed extensive AA.19 

Moreover, AA does not require on-going allogeneic stimulation. Thus, transient acute rejection with subsequent complete ablation of the alloantigen response,15  or return of an allograft into an autologous recipient after an episode of rejection20-22  still culminates 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 specific cellular recruitment (via chemokines) and subsequent development of a local milieu of inflammatory mediators (called cytokines), these cells may establish an environment sufficient for the subsequent alloantigen-independent development of AA. Peri-operative ischemic injury,23  antibody-mediated processes,24-26  cytokine-induced endothelial dysfunction, and toxic or infectious insults (e.g. with CMV or other viruses27-29  may also contribute to alloantigen-independent activation of EC, SMC, or inflammatory cells and may serve to amplify the allospecific pathways.

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 of these act in an autocrine or paracrine fashion,to influence the behavior of neighboring T and B cells. Other cytokines act primarily on non-lymphoid cells such as macrophages, SMC, and EC. For example, interferon- g (IFN- g ) activates macrophages, augments inflammatory cell recruitment by up-regulating adhesion molecules and chemokine expression, and amplifies immune responses by increasing MHC I and II expression on antigen presenting cells (APC) and.30  Cytokines produced by antigen non-specific 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 (TNF- a ),both of which have sweeping pro-inflammatory actions including EC activation (promoting inflammatory cell adhesion and pro-coagulant activity) and SMC proliferation and synthetic function. Relevant to the pathogenesis of AA, some cytokines (e.g., TGF b )may also induce a local fibrosing response.31, 32  The observations that EC and SMC can differentially activate T cells--potentially via specific cytokine and chemokine elaboration-33-35  -suggest a complex set of interactions occurring in the cytokine milieu of the vessel wall.

IFNg is a cytokine central to the development of AA. Intimal lesions do not occur in heterotopic cardiac allografts in IFN g -deficient mice despite ongoing parenchymal rejection;15, 36  comparable results were seen in wild-type (WT) recipients receiving weekly injections of anti-IFN g antibodies.36  Relative to grafts transplanted into WT recipients, allografts transplanted into IFN g -deficient hosts showed similar (or even increased) numbers of helper and cytotoxic T cells, as well as macrophages. Nevertheless, cardiac allografts in IFN g -deficient recipients exhibited diminished co-stimulator and adhesion molecule expression relative to control recipients.36  IFNg can also potently affect the development of AA by inducing the vascular production of chemokines (see below) that regulate mononuclear inflammatory cell recruitment.

Based on these observations, it has been previously proposed that AA results from an initial alloresponse (and potentially from other forms of vascular injury) leading to secondary recruitment and activation of macrophages in a process largely driven by IFN g.36  In this model, the antigen non-specific mononuclear phagocytes subsequently elaborate fibrogenic mediators such as TGF b , as well as SMC growth stimulators such as IL-1 and TNFa.1  Consequently, proximal blockade of IFN g , 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. To date, no such IFN g -blocking therapeutic agent has been identified for treating human disease.

It is also noteworthy that IFN g (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 SMC in atherosclerotic plaque.37-39  Although IFNg inhibits medial SMC proliferation in vitro,40  the presence or absence of IFN g correlates with the extent of disease in murine models of atherosclerosis. It is therefore conceivable that intimal SMC in AA may respond differently that medial SMC to this cytokine (and others in the perivascular cytokine milieu), and that the efficacy of IFN g blockade may in part derive from diminished SMC recruitment and/or activation.

Role of chemokines in AA
Chemokines are small molecular weight (8-10 kD) secreted proteins responsible for the recruitment and activation of a variety of inflammatory and non-inflammatory cells. Greater than 50 chemokines have been identified and are broadly classified into four sub-families 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-2 a (MIP-2 a ), Gro a , 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), IFN g -inducible protein 10 (IP-10), and IFN g -inducible T cell alpha chemoattractant (I-TAC), as well as CC chemokines like RANTES (regulated upon activation, normal T cell expressed and secreted). Of the CXC chemokines, IP-10, Mig, and I-TAC are all strongly expressed by IFNg ; this is again noteworthy since the absence of IFN g abrogates the development of AA, as described above.15, 36  The role of these molecules in acute allograft rejection has been the subject of a number of recent excellent reviews,41-44  although potentially unique roles for chemokines in the development of AA have not been detailed.

Chemokine receptors are G-protein coupled seven transmembrane-spanning proteins with discrete cellular distributions and binding specificities;34, 45  there are over 20 so far described.

Graft EC represent the initial site where host inflammatory cells encounter and recognize foreign donor MHC. Recipient inflammatory cells must adhere to EC and subsequently pass through the vessel wall into the donor parenchyma. The process begins with antigen non-specific neutrophils and monocyte/macrophages, cells of innate immunity recruited into allografts as a consequence of allograft ischemic injury.42, 46  This initial wave promotes the recruitment and activation of alloreactive T cells, which in turn drive the recruitment of additional macrophages, and eventually host-derived SMC precursors leading to AA. Interrupting chemokine cellular conscription pathways at any of these stages-innate immunity, allospecific immune responses, secondary macrophage recruitment, or intimal SMC-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 non-redundant 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 delayed-type hypersensitivity lesions47, 48  and attenuate the development of atherosclerosis.49,50  Relevant to transplantation, monoclonal antibody blockade of the IFN g -induced chemokine Mig diminished allograft rejection.51  Targeted deletion of the CCR1 chemokine receptor reduced both acute parenchymal rejection, as well as the development of AA.52  Moreover, a selective non-peptide CCR1 receptor antagonist (BX471) developed by Berlex Biosciences (Richmond, CA) has efficacy in acute cardiac rejection models in rats and rabbits.53, 54 

Source of intimal cells in AA
A subpopulation of peripheral circulating cells and bone marrow cells can differentiate into EC.55, 56  However, the majority of EC (as well as the medial SMC) lining AA lesions in long-term cardiac allografts are donor-derived.4, 19, 57  The observation concerning the identity of arteriolar EC in cardiac allografts is significant in that persistence of donor cells may well continue to drive an alloresponse that culminates in AA lesions.

In some experimental models, host EC can seed synthetic vascular grafts implanted in vivo,58  and host SMC are identified in synthetic grafts and some arterial allograft models.58, 59  Despite these findings, it was generally assumed that the majority of intimal SMC in AA lesions originated by ingrowth of donor SMC from the media of engrafted vessels.60  These assumptions led to unsuccessful experimental molecular interventions (e.g., gene therapy) targeting donor medial SMC proliferation to ameliorate AA.61, 62 

It was subsequently shown that intimal SMC in vascular grafts are virtually all host-derived;4-6,8  in particular, it has been demonstrated that a proportion of these could originate from host bone marrow-derived cells.8  This result is consistent with the recent finding that bone marrow stem cells are capable of developing into multiple mesenchymal lineages.63  It also suggests that intimal SMC may have a functional phenotype distinct from the underlying vascular wall medial SMC, and may also have unique chemokine and cytokine responsiveness.

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