Learning from rejection: What transplantation teaches us about (other) vascular pathologies

Allograft vasculopathy is an accelerated intimal hyperplastic lesion leading to progressive vascular stenosis; it represents the major long-term limitation to successful solid organ transplant. Although allograft vasculopathy is not formally an autoimmune disease, nor does it constitute a major cause of cardiovascular disease on a purely numerical basis, its pathogenesis provides an important window on the mechanisms by which immune injury can drive more common vascular pathologic entities. Thus, insights gleaned from vascularized solid organ transplants can shed new mechanistic (and therapeutic) light on: 1) the intimal vascular responses accompanying typical atherosclerosis and other inflammatory vessel diseases (e.g., scleroderma); 2) the pathogenesis of vascular stenosis versus aneurysm formation; 3) the sources of intimal smooth muscle cells in the healing of any vascular injury; and 4) the mechanisms by which smooth muscle cells are recruited into intimal lesions. Indeed, research on allograft vasculopathy has led to the understanding that interferon-γ plays a similar pathogenic role in a host of vascular stenosing lesions—and that Th2 cytokines can drive vascular remodeling and aneurysm formation. Moreover, circulating precursors (and not just medial smooth muscle cells) contribute to the intimal hyperplasia seen in atherosclerosis and in-stent restenosis. That non-vessel smooth muscle cells can be recruited to sites of vessel injury further suggests that chemokine and adhesion molecule interactions may be viable targets to limit vascular stenosis in a wide range of vascular lesions. This review will describe the pathogenesis of allograft vasculopathy, and will relate how understanding the underlying pathways informs our understanding of both human transplant-associated disease, as well as other human vascular pathologies.     

Accelerated atheromatous lesions in mouse hearts transplanted to apolipoprotein-E-deficient recipients.

Arteriopathy, sometimes termed accelerated atherosclerosis, often impairs transplants. We employed apolipoprotein-E-deficient, hypercholesterolemic mice to determine how the hyperlipidemic environment affected transplanted hearts. Strain 129 hearts transplanted to C57BL/6 normal or C57BL/6 apolipoprotein-E-deficient recipients were evaluated by immunochemical and histological techniques. Analyses were possible both of differences in the coronary lesions that developed in a normolipidemic as compared with a hyperlipidemic environment and of the coronary atherosclerotic process in transplanted hearts compared with native hearts in the same hyperlipidemic environment. Aortas and coronary arteries of transplanted aortas in both recipient groups developed florid intimal thickening by 4 to 10 weeks, with marked lipid deposition, foamy macrophages, and infiltration of smooth muscle alpha-actin-positive cells in apolipoprotein-E-deficient mice. Lipid was layered against the internal elastic lamina as in human transplants. VCAM-1 was increased in various sites in both groups. Allotransplants to apolipoprotein-E-deficient recipients had more severe aortic and coronary lesions with characteristic T cell infiltration than native hearts. In this sense, transplants suffered from accelerated atherosclerosis. The character of coronary vascular changes in transplanted hearts was distinctly affected by their lipid environment, but their severity, in terms of luminal encroachment, was not markedly different.     

Studies on the pathogenesis of atheroarteriosclerosis induced in rabbit cardiac allografts by the synergy of graft rejection and hypercholesterolemia.

Heterotopic cardiac allografts were placed in the necks of 48 rabbits. In rabbits that were not immunosuppressed, allografts beat as long as 12 days, while in immunosuppressed rabbits allografts beat as long as 101 days. Coronary arterial lesions in donor hearts of rabbits fed a lipid-poor diet were found in arteries of all sizes and were mainly proliferative without fatty change. In cholesterol-fed rabbits, arterial lesions were similarly distributed, but the majority of lesions in longer surviving transplants were fatty-proliferative and some bore close resemblance to chronic human coronary atheroselerosis. In contrast to findings in cardiac homotransplants, only occasional predominantly fatty lesions were induced in small intramyocardial arteries of cholesterol-fed recipients. By electron microscopy, early arterial lesions in allografts were characterized by platelet aggregates in widened junctions between endothelial cells, sloughing of endothelium without intimal thickening but with adherence of platelets to the denuded arterial wall, and platelets deep within essentially normal media. Platelets were also seen adhering to the lining cells overlying the thickened intima of more advanced arterial lesions. Results indicate that immunologic arterial injury due to allograft rejection acting in synergy with hypercholesterolemia resulting from a dietary supplement of cholesterol can lead to rapidly developing atherosclerosis. Observations of early and evolving lesions indicate that endothelial injury and platelet interaction with the arterial wall are early and continuing events and may be of primary importance in the pathogenesis of experimental graft-induced atheroarteriosclerosis. In man, similar mechanisms may be involved in the pathogenesis of graft-induced athererosclerosis and in other instances of atherosclerosis. (Am J Pathol 87:415-442, 1977).     

10-Year Experience with HLA-G in Heart Transplantation

The Human Leukocyte Antigen-G (HLA-G) is a MHC-class Ib molecule with robust immunomodulatory properties; in transplant, it inhibits cytotoxic activity of immune cells and thus has a pivotal role in protecting the allograft from immune attack. The present review details a 10-year experience investigating the influence of HLA-G on heart transplantation, allograft rejection and cardiac allograft vasculopathy development. Exploration of HLA-G in transplantation began with the initial findings of its increased expression in allograft hearts. Since then, HLA-G has been recognized as an important factor in transplant immunology. We discuss inducers of HLA-G expression, and the importance of HLA-G as a potential biomarker in allograft rejection and heart failure. We also highlight the importance of polymorphisms and how they may influence both HLA-G expression and clinical outcomes. There remains much to be done in this field, however we hope that findings from our group and other groups will ignite interest and facilitate further expansion of HLA-G research in transplantation.     

Allograft heart accelerated atherosclerosis: evidence for cell-mediated immunity in pathogenesis

The problem of accelerated atherosclerosis in the allograft heart continues to adversely effect the long term survival of transplant patients. Most traditional risk factors influencing atherogenesis do not appear to play an important role in accelerated atherosclerosis. Additional causative factors proposed in recent years are not without controversy. An immune-mediated endothelial damage by cytotoxic B-cell antibodies as the initial injury in the induction of accelerated atherosclerosis has not been confirmed on morphologic grounds. This study is based on six cases (four autopsies and two explanted hearts) from transplant patients whose hearts were examined at different post-transplantation intervals (24 h to 14 mo). Our morphologic findings of a segmental coronary vasculitis involving primarily the outer two-thirds of the media and adventitia suggest that the early vascular manifestations may reflect tissue rejection similar to that seen in the myocardium. Furthermore, our findings from the medium size coronary arteries are suggestive of a cell-mediated immune injury probably directed against the smooth muscle cells of the media. Although the end result (occlusive coronary lesion) may reveal some morphologic similarities in both naturally occurring atherosclerosis and accelerated atherosclerosis, the pathogenetic mechanisms involved in these two conditions may differ.     

ASSOCIATION OF CORONARY PLAQUE RUPTURE AND ATHEROSCLEROTIC INFLAMMATION

Coronary plaque inflammation may promote plaque rupture and thrombosis. To test this hypothesis, 351 coronary plaques from 83 patients were formalin-fixed and stained with haematoxylin and eosin. There were six groups: (1) ruptured plaques; (2) intact plaques from recently infarcted hearts; (3) plaques from hearts with severe coronary atherosclerosis without identifiable thrombosis; (4) native explanted hearts with severe coronary atherosclerosis; (5) cardiac transplant atherosclerosis; and (6) fatalities unrelated to coronary atherosclerosis. Selected arteries were immunostained for leukocyte markers and serially sectioned to identify plaque rupture. There were infiltrates of CD68-positive macrophages and CD3- and CD8-positive T cells adjacent to all plaque ruptures. Labelling with HLA-DR and CD30 indicated inflammatory cell activation. Plaque rupture was strongly statistically associated with the severity and frequency of superficial plaque inflammation but not that of deep plaque inflammation. Although atherosclerotic inflammation has been identified adjacent to rupture, this is its first comparison with control plaques. These results support the concept that inflammation in the fibrous cap is particularly associated with plaque rupture. © 1997 by John Wiley & Sons, Ltd.     

Wild-type apo A-I and apo A-IMilano gene transfer reduce native and transplant arteriosclerosis to a similar extent

Apolipoprotein (apo) A-I_Milano is an apo A-I mutant characterized by a cysteine for arginine substitution at position 173. Apo A-I_Milano carriers have much less atherosclerosis than expected from their low plasma high-density lipoprotein cholesterol levels, suggesting that this mutant may have superior atheroprotective properties. Here, we compare the effect of hepatocyte-directed gene transfer of wild-type human apo A-I and human apo A-I_Milano on endothelial progenitor cell (EPC) biology and on the progression of native atherosclerosis and allograft vasculopathy in C57BL/6 apo E^−/− mice. Human apo A-I and apo A-I_Milano transfer resulted in an equivalent increase of EPC number and function as well as EPC incorporation and endothelial regeneration in allografts and inhibited the progression of native atherosclerosis and allograft vasculopathy to a similar extent. In conclusion, the current head-to-head comparison indicates that human apo A-I_Milano transfer is not superior compared to wild-type human apo A-I transfer. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Pathologic findings in long-term cardiac transplants

Since the introduction of cardiac transplantation at our institution 15 years ago, major advances have occurred in the monitoring and treatment of these patients, resulting in many long-term survivors. We defined the pathologic features in 14 cardiac transplants with survival times longer than one year. Only one heart showed no evidence of rejection, while the remaining 13 hearts showed advanced chronic rejection, which was the main cause of death or of graft failure in 11 patients. One patient died of gastric carcinoma, one of Kaposi's sarcoma, and one of cerebral embolus. The most obtrusive change in the donor hearts was an obliterative arteritis, which in the epicardial coronary arteries mimicked atherosclerosis. Superadded thrombosis often resulted in myocardial infarction. These severe vascular lesions bore no constant relationship to survival time and took from 1.1 to 12.5 years to evolve.     

Human coronary transplantation-associated arteriosclerosis. Evidence for a chronic immune reaction to activated graft endothelial cells

Occlusive disease of coronary arteries of engrafted hearts is the major obstacle to long-term survival of human cardiac allografts. The pathogenesis of this process remains uncertain. The identity and localization of cells found in transplantation-associated arteriosclerosis lesions from human cardiac allografts were evaluated, and their expression of class II major histocompatibility complex (human leukocyte antigen-DR [HLA-DR]), surface molecules required for recognition of foreign cells by CD4+ T lymphocytes, was noted. Expanded intimas of transplanted coronary arteries contain T lymphocytes (both CD4+ and CD8+ in approximately equal number) and HLA-DR+ macrophages, both localized primarily in a ring immediately below the luminal endothelium, a distribution strikingly different from that in typical atherosclerosis. Coronary arterial endothelium from six of six transplanted hearts studied bore high levels of HLA-DR. Normal human arteries or usual atherosclerotic lesions have few if any HLA-DR+ endothelial cells. The significance of these findings was tested by evaluating the ability of HLA-DR+ arterial cells to interact with allogeneic T cells in vitro. Endothelial cells (but not smooth muscle cells) cultured from human arteries stimulated foreign CD4+ T cells to proliferate and augmented their secretion of interleukin-2. These findings suggest that ongoing stimulation of recipient T lymphocytes by HLA-DR+ endothelium of donor coronary arteries contributes to a sustained regional immune response. Consequent local release of cytokines may regulate smooth muscle cell proliferation and matrix accumulation within the coronary arteries of allografted hearts.     

Role of anti-vimentin antibodies in allograft rejection

Production of anti-vimentin antibodies (AVA) after solid organ transplantation are common. Although classically thought to be expressed mainly within the cytosol, recent evidence demonstrates that extracellular or cell surface expression of vimentin is not unusual. This review examines the evidence to assess whether AVA contribute to allograft pathology. Clinical studies suggest that AVA are associated with cardiac allograft vasculopathy in heart transplant recipients. Studies in non-human primates confirm that production of AVA after renal and heart transplantation are not inhibited by Cyclosporine. Experimental studies have demonstrated that mice pre-immunised with vimentin undergo accelerated acute rejection and vascular intimal occlusion of cardiac allografts. Adoptive transfer of hyperimmune sera containing AVA into B-cell-knock-out mice caused accelerated rejection of allografted hearts, this is clear evidence that antibodies to vimentin accelerate rejection. AVA act in concert with the alloimmune response and AVA do not damage syngeneic or native heart allografts. Confocal microscopy of allografted organs in vimentin immunised mice shows extensive expression of vimentin on endothelial cells, apoptotic leukocytes and platelet/leukocyte conjugates, co-localising with C4d. One explanation for the ability of AVA to accelerate rejection would be fixation of complement within the graft and subsequent pro-inflammatory effects; there may also be interactions with platelets within the vasculature.     

Delineation of the extent of coronary atherosclerosis by high-frequency epicardial echocardiography

Postmortem studies suggest that coronary angiography does not always accurately delineate the extent of coronary-artery disease. We examined this problem in living human hearts by performing high-frequency epicardial echocardiography at the time of cardiac surgery. The ratio of the diameter of the lumen of the coronary artery to the thickness of its wall was used to quantify the severity of coronary lesions. In 11 patients with no angiographic evidence of coronary disease anywhere in the coronary tree, the mean (+/- SEM) ratio was 5.9 +/- 0.3. In 21 patients with angiographic disease at the site evaluated by echocardiography, the mean ratio was lower (2.3 +/- 0.2, P less than 0.05), reflecting encroachment into the arterial lumen by atherosclerotic plaque. In 15 patients with arterial segments that were angiographically normal but with arterial stenoses elsewhere in the coronary tree, the mean ratio was 4.1 +/- 0.3, with marked overlap with the values in the patients who had angiographic disease at the site of the echocardiographic evaluation. These results demonstrate, in living human hearts, that diffuse coronary atherosclerosis is often present when coronary angiography reveals only discrete stenoses. This finding suggests that coronary angiography may underestimate the severity and extent of coronary disease.     

Incidence of cardiovascular disease in the dissecting room: a valuable teaching asset

The purpose of this study was to determine the incidence of cardiovascular pathology in 50 cadavers in the dissecting room of the Department of Anatomy at Guy's Campus, King's College, London, and to demonstrate the importance of dissection in teaching the anatomy of normal and pathological hearts. After external evaluation of each heart the four chambers were dissected and studied. The features noted included evidence of coronary atherosclerosis, myocardial infarction, variations in coronary artery anatomy, valvular disease, variations in left ventricular wall thickness and atrial dimensions, and atrial anomalies. All the hearts studied had at least one pathology. The majority had severe coronary atherosclerosis (44) and aortic valve pathology (23). A large number had left ventricular hypertrophy (13) and left atrial enlargement (9). A small number showed evidence of myocardial infarction (4). Anatomical anomalies were also found, and included persistent foramen ovale (1), three coronary arterial ostia (3), and anatomical variations of the orientation of the main stem of the left coronary artery (2). This study demonstrates that dissection is not only an excellent way of studying normal cardiac anatomy, but also a valuable method for introducing common cardiac pathologies to the medical student.     

Excitation propagation in three-dimensional engineered hearts using decellularized extracellular matrix

Engineering of three-dimensional (3D) cardiac tissues using decellularized extracellular matrix could be a new technique to create an "organ-like" structure of the heart. To engineer artificial hearts functionally comparable to native hearts, however, much remain to be solved including stable excitation-propagation. To elucidate the points, we examined conduction properties of engineered tissues. We repopulated the decellularized hearts with neonatal rat cardiac cells and then, we observed excitation-propagation of spontaneous beatings using high resolution cameras. We also conducted immunofluorescence staining to examine morphological aspects. Live tissue imaging revealed that GFP-labeled-isolated cardiac cells were migrated into interstitial spaces through extravasation from coronary arteries. Engineered hearts repopulated with Ca²⁺-indicating protein (GCaMP2)-expressing cardiac cells were subjected to optical imaging experiments. Although the engineered hearts generally showed well-organized stable excitation-propagation, the hearts also demonstrated arrhythmogenic propensity such as disorganized propagation. Immunofluorescence study revealed randomly-mixed alignment of cardiomyocytes, endothelial cells and smooth muscle cells. The recellularized hearts also showed disarray of cardiomyocytes and markedly decreased expression of connexin43. In conclusion, we successfully demonstrated that the recellularized hearts showed dynamic excitation-propagation as a "whole organ". Our strategy could provide prerequisite information to construct a 3D-engineered heart, functionally comparable to the native heart.     

Prominence of coronary arterial wall lipids in human heart allografts. Implications for pathogenesis of allograft arteriopathy.

Transplant arteriopathy is a major late complication in human heart allograft recipients and the pathogenesis of such arteriopathy remains uncertain. The degree to which lipids and atheromata are involved in the arteriopathic lesions remains unsettled, and there is uncertainty regarding the significance of insudation or retention of lipids within the coronary artery walls of transplanted hearts. On current immunosuppressive regimens, most patients experience an increased serum total cholesterol and low-density lipoprotein cholesterol after transplant. Elevation of these blood lipids has an undetermined relationship to arteriopathy. We carried out morphological, morphometric, immunohistochemical, ultrastructural, and biochemical studies of particular coronary artery segments from 23 unselected explant or autopsy allografts and donor age-matched native coronary controls. Patients died of cardiac and non-cardiac reasons over a period of 4 to 1610 days after transplant. Atheromata were frequent, and diffuse intra- and extra-cellular accumulation of lipids in both intimal and medial walls was documented by oil red O positivity, immunohistochemical staining (muscle-specific alpha-actin), transmission and scanning electron microscopy, and biochemical analysis. Mean total cholesterol, esterified cholesterol, free cholesterol, and phospholipid content (microgram/cm2 intimal surface area) and concentration (microgram/mg dry defatted weight) in arteriopathic coronaries were > 10-fold higher than in comparable native coronary segments. Extent of lipids in the arterial walls was highly correlated with digitized percent luminal narrowing, mean daily and cumulative cyclosporin dose, and mean cumulative prednisone dose. Our data suggests strongly that lipid accumulation is an important early and persistent phenomenon in the development of transplant arteriopathy.     

Coronary ostial stenosis complicating coronary arteriography.

Coronary ostial stenosis is a cause of angina pectoris and sudden death. It is due mainly to atherosclerosis, syphilis, and iatrogenic disease. The last is of growing importance because of the frequency of coronary arteriography and surgical procedures on the aortic valve. Since both may cause stenosis of the coronary ostia, these procedures raise the topic from an obscure morphologic entity to an important consideration in the treatment of cardiac disease. We describe a case of ostial stenosis that was complicated by coronary arteriography.     

Myocardial injury in patients with aortic stenosis

Clinical studies of patients with aortic stenosis suggest that left ventricular myocardial injury is frequent. To examine the morphologic basis of this observation, we studied 32 patients with isolated aortic stenosis, no cardiac surgery, and hearts studied at autopsy after postmortem arteriography and fixation in distension. The patients were 46-87 years old (average 69), and 21 (66%) were male. Calcific aortic stenosis was present in 19 hearts, 12 had congenital bicuspid aortic valve, and 1 had rheumatic aortic stenosis. In 19 hearts there was moderate or marked coronary atherosclerosis, and 12 hearts had 17 myocardial infarcts. However, among 13 hearts with no or mild coronary atherosclerosis, 9 had either subendocardial myocardial contraction band necrosis, a lesion occurring when periods of no perfusion are followed by reflow, or focal replacement fibrosis. In 13 hearts there was subendocardial vacuolization of myocytes, an alteration produced by ischemia, that was not accounted for by coronary artery disease. Our results are consistent with clinical observations and show that ischemic myocardial injury may be associated with isolated aortic stenosis in the absence of coronary artery obstruction. The contraction band necrosis and vacuolated myocytes suggest that both episodic and sustained reductions of subendocardial blood flow occur in the presence of aortic stenosis.     

Coronary atherosclerosis in Mangalore--a random post mortem study

A study was undertaken in Mangalore, South Kanara District of Karnataka, India to evaluate the rising prevalence of coronary artery diseases in our country. Seventy hearts were analysed, at post-mortem, between December 1996 and December 1997. The coronary arteries and the myocardium were examined, both grossly and histologically, to relate them with regard to the age and gender of the deceased. Fifty five hearts were from males and 15 were from females. Forty eight (68.59%) hearts were proven to be afflicted with coronary atherosclerosis. Twenty seven of these had three or four vessel disease. A total of 192 sections from the four major epicardial arteries of the 48 hearts were examined. Of these, 124 showed atherosclerosis (with 37 in advanced stages), of which 103 were occlusive lesions while the remaining 21 were fatty streaks. Severe stenosis (grade III or IV) was commonest in the left anterior descending artery. Twenty one (30%) of the 70 hearts showed histological evidence of myocardial ischaemia. Of these 6 were from females and 15 were males. Myocardial ischaemia was found to have no correlation to the severity of stenosis.     

Indwelling cardiac catheters. An autopsy study of associated endocardial lesions

An autopsy study was carried out to assess the relationship between indwelling intracardiac catheters and hemorrhagic, thrombotic, and infective lesions of the right heart endocardium and valve. Intracardiac catheters cause such lesions frequently, with a spectrum from uncomplicated valvular hemorrhage through nonbacterial thrombotic endocarditis to infective endocarditis. Pulmonary emboli were associated with the thrombotic, infective lesions. Endocardial lesions are more commonly seen with Swan-Ganz (SG) catheters; with these, lesions are more common and severe, with longer periods of catheterization. Pulmonic-valve lesions were only seen with SG catheters. However, not all hemorrhagic lesions in the right heart endocardium were associated with catheters, because a few inpatients without catheters had small subendocardial valvular hemorrhages; the cause of these hemorrhages is obscure because they were not seen in the hearts of patients who died outside the hospital.     

Renal pathology in the pediatric transplant patient

Renal transplantation is a therapeutic goal for children with advanced chronic kidney disease. There are many causes of renal dysfunction in children with allografts--the transplanted kidney can develop a variety of morphologic alterations leading to dysfunction. Evaluation of the kidney biopsy is one of the best methods of determining the cause of graft dysfunction. Rejection is a major cause of renal allograft failure in children. The morphologic hallmarks of acute antibody-mediated and cell-mediated rejection and chronic allograft nephropathy have been codified in classification strategies that are useful in adults and children. Viral infection and Epstein-Barr virus-driven posttransplant lymphoproliferative disease also occur in the pediatric transplanted kidney. Drug toxicity from immunosuppressive agents also causes characteristic morphologic alterations in the renal allograft. As the survival of pediatric heart and liver transplant patients improves, the incidence of immunosuppression therapy-related disease in the native kidney in these patients will likely become more important clinically. In addition to renal lesions related to the allograft state, glomerular disease can recur or occur de novo in renal allografts. Here, we describe the pathology of the more common morphologic lesions in kidneys of children with a renal allograft.