| Summary: | 博士 === 國立臺灣大學 === 臨床醫學研究所 === 91 === 柒、論文英文簡述
Objective:
In this study, we sought to assess the incidence of transplant coronary artery disease in Chinese heart recipients, to find the possible contributing factors for different result, and to investigate whether plasma soluble markers in coronary sinus and peripheral blood is a good way to predict clinical status of transplant recipients. At last, a method of proteomic approach to discover a novel protein was briefly described.
Part I:
Materials and methods. From October 1987 to June 1998, we had performed 92 cases of heart transplantation at the National Taiwan University Hospital. Eighty-eight patients underwent orthotopic heart transplantation. All patients received triple-drug immunosuppressive therapy according to our heart transplantation protocol.
Endomyocardial biopsy was performed weekly in the first month, biweekly in the second month, monthly in the 6 months and yearly 6 months after transplantation.
All patients discharged received 100mg aspirin daily and 25mg dipyridamole 3 times per day for prevention of transplant coronary artery disease. Hyperlipidemia was treated with dietary modification and body weight control first. Hypertriglyceridemia was treated with bezafibrate. Hypercholesterolemia was treated with pravastatin and acipimox. Hyperuricemia was treated with probenecid and allopurinol. Hyperglycemia was treated with oral hypoglycemic agent and insulin. Hypertension was treated with angiotensin-converting enzyme inhibitors and calcium antagonists. All patients were followed at special cardiac transplantation clinic.
For those patients surviving for more than 6 months after transplantation, coronary angiography was performed annually for surveillance of transplant coronary artery disease. Side-by-side comparisons were done. The diagnosis of transplant coronary artery disease was made from the evidence of any coronary artery irregularity or diffuse narrowing in proximal and distal vessels. Cardiac events including myocardial infarction, left ventricular dysfunction, arrhythmia and sudden death were recorded. The recipient characteristics (age, sex, body weight, smoking, hypertension, diabetes, cholesterol level, triglyceride level and viral infection), donor characteristics (age, sex, preexisting coronary artery disease and allograft ischemic time), rejection episodes, medications and HLA match were recorded. Cytomegalovirus infection was defined as a 4-fold rise in antibody, cytomegalovirus inclusion body, or positive culture. Rejection was defined as a clinical event leading to specific immunosuppressive intervention.
Fifty patients survived for more than one year and were included in this study. Among these 50 patients, there were 42 men and 8 women. The clinical diagnosis of end-stage heart failure was idiopathic dilated cardiomyopathy in 31 (62%), ischemic heart disease in 13 (26%), valvular heart disease in 4 (8%), congenital heart disease and postpartum cardiomyopathy in one patient. The mean recipient age was 44.1+13.6 years at the time of transplantation. The mean donor age was 29.4+10.8 years (range 15 to 55). Positive cytomegalovirus antibody was present in 44 of 48 recipients (91.7%) and 27 of 30 donors (90%) at the time of transplantation. The allograft ischemic time was 45 to 223 minutes (mean 101.1). Complete HLA information on the A, B, and DR loci was available for both donor and recipient in 47 patients. Seven patients (14.9%) had 2 or fewer HLA mismatches, whereas 3 patients (6.4%) had mismatches of all 6 HLA loci.
Results. Among these 92 heart transplant recipients, 7 patients died within the first month after transplantation because of bleeding in 3, acute rejection in 2, arrhythmia and cerebral injury in one patient. Another 10 patients died within the first year after transplantation because of infection in 5, rejection in 3 (two for medical noncompliance), arrhythmia and gastrointestinal bleeding each in one patient. The operative mortality rate was 7.6%. The one-year survival rate was 81.5%.
Thirty-two patients had biopsy-proven episode of acute rejection during the first year after transplantation, with an incidence of rejection of 64 % within one year after transplantation. Among the 92 cases of heart transplantation, 2 patients had acute vascular rejection and died in spite of mechanical circulatory support, plasmapheresis, and immunoadsorption therapy. Rejection rarely occurred in patients more than one year after transplantation.
During the follow-up period, 8 patients had major infection. Among these 50 recipients, seven patients (14%) were hepatitis B carriers. Two of 7 carriers had liver failure due to hepatitis B reactivation and were treated successfully with lamivudine. Two patients (4%) had cytomegalovirus infection and were treated with ganciclovir therapy. One patient had aspergillus infection and was cured by amphotericin infusion and wedge resection of infected lung. There was one patient with malignant lymphoma of small intestine. No patient had solid organ tumor or Kaposi’s sarcoma after transplantation.
Cardiac events occurred in 3 patients. One patient had atrial arrhythmia. One patient had biopsy-negative left ventricular dysfunction and the coronary angiogram was patent. Another patient with heterotopic heart transplantation had sudden arrest of donor heart. The coronary artery was patent and the pathology was massive coagulation necrosis. The patient was doing well for improved native heart function due to prolonged unloading of native heart after heterotopic heart transplantation.
At the time of this study, obesity was present in 17, hypertension in 6, hyperuricemia in 27, diabetes in 17 and hyperlipidemia in 34 of 50 patients. No patients used diltiazem. Pravastatin was used in 12 patients.
Coronary angiography was reviewed by at least 2 cardiologists at the time of study. Among 74 angiograms of 50 patients reviewed, only one patient had discrete stenosis less than 50% in the middle part of left anterior descending artery at one year after transplantation. No coronary angioplasty was performed then, because he was free of angina and left ventricular dysfunction. No patient had diffuse narrowing of coronary artery. The incidence of transplant coronary artery disease was 2%(1/50) at one year and none at 2 and 4 years after transplantation.
Part II:
Previous studies have reported the levels of nitric oxide metabolites and cytokines of coronary sinus in heart transplant recipients, but their clinical correlation were not appropriately assessed. In our second study, we investigated whether plasma soluble markers in coronary sinus is better to predict clinical status of transplant recipients than in peripheral blood.
Materials and methods. The patients in this cross-sectional study were parts of the transplant program in our hospital. Between February 1998 and January 2001, 51 patients admitted for endomyocardial biopsy were included in this study. The median age at transplantation of this group was 52 years (range, 18 to 69), and 80 % were males. All patients received triple-drug immunosuppressive therapy according to our heart transplantation protocol.
Endomyocardial biopsy was performed weekly in the first month, biweekly in the second month, monthly in the 6 months and yearly six months after transplantation. Rejection was defined as a clinical event leading to specific immunosuppressive intervention. The scale of the International Society for Heart Transplantation is used for grading of rejection. This scale ranges from grade 0, which denotes no sign of acute rejection through 1A, 1B, 2, 3A, 3B to grade 4 which denotes diffuse aggressive inflammation, myocyte necrosis and damage, edema, hemorrhage, and vasculitis. Humoral rejection was diagnosed by the immunofluorescent staining of immunoglobulins and complement.
For those patients surviving for more than 6 months after transplantation, coronary angiography was performed annually for surveillance of transplant coronary artery disease. Side-by-side comparisons were done. The diagnosis of transplant coronary artery disease was made from the evidence of any coronary artery irregularity or diffuse narrowing in proximal and distal vessels.
Clinical events including acute rejection, infection, transplant coronary artery disease and death were recorded. The recipient characteristics including age, sex, body weight, smoking, hypertension, diabetes, cholesterol level, triglyceride and medications were recorded. The clinical events of the transplant recipient were recorded as: 1) early post-transplant; 2) infection; 3) long-term uneventful status; 4) acute rejection and 5) transplant coronary artery disease. Early post-transplant was defined as within one month after transplantation. Infection was defined as clinical evidence of local or systemic infection with or without leukocytosis. Acute rejection was defined as a clinical event leading to specific immunosuppressive intervention and the rejection grade was > 2 by endomyocardial biopsy. Long-term uneventful status was defined as no clinical significant complications, including rejection and infection, after transplantation..
Under the guidance of fluoroscopy, we cannulated coronary sinus through right internal jugular approach. The correct position in coronary sinus was verified by analysis of the blood oxygen saturation. Study blood samples were collected directly from coronary sinus and peripheral blood. Ten millimeters of blood were collected. The samples are collected in vacuum tubes, kept in ice slush, and centrifugated. The plasma samples are then immediately frozen at —700C, in 500-ul aliquots, and stored until analysis. The plasma levels of interleukin-2 (IL-2), tumor necrosis factor-a (TNF-a), intercellular adhesion molecule-1 (ICAM-1) and P-selectin were determined by the commercially available enzyme-linked immunosorbent assays (ELISAs) (R&D Systems USA, Minneapolis, MN). Tropinin-I was also determined by ELISAs. High sensitive C-reactive protein (CRP) was determined by immunoturbidometry.
The measured plasma levels of troponin-I, CRP, IL-2, TNF-a, ICAM-1 and P-selectin of coronary sinus and peripheral blood were compared between patients with and without each clinical event of the transplant recipient. The plasma levels of these soluble markers were compared between coronary sinus and peripheral blood among patients without early post-transplant and infection. Whitney-Mann test and Spearman correlation were used for independent two-sample comparison. A p-value < 0.05 was considered statistically significant.
Blood were obtained in patients 1-2 years after the first sampling to assess the time course of each soluble marker in variable clinical events.
Results. There were 71 blood samples from 51 patients of heart transplantation. The post-transplantation period ranged from one week to 5 years.
Among the 71 clinical events, there were 19 early post-transplant, 16 acute rejection, 9 transplant coronary artery disease, 10 infection, and 32 long-term uneventful status.
Our data shoed that, in early post-transplant patients, there were higher levels of P-selectin, ICAM-1, CRP and troponin-I in both coronary sinus and peripheral blood. In patients with infection, there were higher levels of all soluble markers except IL-2 in both coronary sinus and peripheral blood.
In patients with long-term uneventful status, there was a lower level of CRP in peripheral blood but not in coronary sinus. In patients with acute rejection, there was a higher level of IL-2 in peripheral blood but not in coronary sinus. In patients with transplant coronary artery disease, there was a higher level of TNF-a in peripheral blood but not in coronary sinus. Among the 48 specimens in patients without early post-transplant and infection, patients with acute rejection had a higher level of IL-2, TNF-a, and CRP in peripheral blood but not in coronary sinus.
Among the 48 specimens in patients without early post-transplant and infection, the plasma levels of P-selectin, IL-2, and TNF-a were higher in coronary sinus than in peripheral blood, but the level of ICAM-1 was higher in peripheral blood than in coronary sinus.
Blood samples were available 1-2 years after the first test in 4 patients with acute rejection. The plasma levels of IL-2 and TNF-a declined after the episode of acute rejection. Blood samples were available 1-2 years after the first test in 5 patients with transplant coronary artery disease. The plasma levels of TNF-a increased 1-2 years after the diagnosis of transplant coronary artery disease.
Part III: Concluding remarks
Transplant coronary artery disease in Chinese. Numerous studies have investigate the possible risk factors for the development of transplant coronary artery disease. Controversy still exists regarding donor and recipient factors which promote the development and progression of coronary artery disease after transplantation. Both immunologic and non-immunologic factors could contribute to the occurrence of transplant coronary artery disease. Older recipient age, older donor age, male recipient, female donor, recipient with ischemic heart disease, allograft ischemic time, cytomegalovirus infection, hyperlipidemia, obesity, immunosuppression, acute rejection, prophylactic diltiazem use, HLA mismatches and racial disparity have been recognized as the risk factors.
We compared the donor and recipient characteristics between our patients and western patients. The donor age, recipient age, and the percentage of male gender were the same. Pre-transplant diagnosis of recipients in our study was less due to ischemic heart disease (26% vs 50%). The prevalence of ischemic heart disease is low in oriental countries possibly due to low fat and high vegetable diet.
Human cytomegalovirus infection has been recognized as a significant risk factor of transplant coronary artery disease and prophylactic ganciclovir therapy may protect against the development of transplant coronary artery disease. In this study, more than 90% of donors and recipients had positive cytomegalovirus antibody before transplantation. The incidence of cytomegalovirus mismatches between donors and recipients was also low. The percentage of cytomegalovirus seronegative recipients was only 8.3% in this study. Thus the chance of active cytomegalovirus infection after transplantation, especially primary infection, was reduced (4% vs 50%). In fact, people in Taiwan usually had cytomegalovirus infection by airborne transmission before adulthood. This accounts for the high incidence of past cytomegalovirus infection before transplantation.
Previous studies have shown that HLA mismatch is associated with a higher incidence and severity of transplant coronary artery disease. The pathogenic mechanism of transplant coronary artery disease is thought to be primarily immune-mediated. Both cellular and humoral immunities are involved. Besides, race and donor/recipient race mismatch may influence the development of transplant coronary artery disease. African-Americans have increased early mortality due to transplant coronary artery disease. In this study, the donors and recipients were all Chinese except one Caucasian donor from Ireland. Thus the race mismatch was only 1 of 50 recipients. The HLA mismatch was also low in this study. The incidence of HLA mismatches two or less in A, B, and DR loci was 14.9%, compared to 6% in western series.
Previous studies have shown that the risk of transplant coronary artery disease may be increased by acute graft rejection. In this study, we used antithymocyte globulin for induction and higher doses of cyclosporine for maintenance immunosuppression. However, the incidence of rejection within one year after transplantation was the same between this study (63%) and western series (64%). Rejection may not the causative factor of low incidence of transplant coronary artery disease in this study.
According to the angiographic findings, transplant coronary artery disease had 2 different morphological features: diffuse concentrated narrowing of distal vessels and eccentric stenosis of proximal vessels. The former is more frequent in heart transplant recipients. The major limitation of this study was that coronary angiography underestimates the presence and severity of transplant coronary artery disease. Intravascular ultrasonography provides more sensitive and accurate method for detecting transplant coronary artery disease. However, intravascular ultrasonography is an invasive and costly method in our hospital. Dobutamine stress echocardiography may be a sensitive and noninvasive method to diagnose transplant coronary artery disease.
Conclusion. Our first study confirms the low incidence of transplant coronary artery disease in Chinese heart transplant recipients in comparison with western transplant patients. Low percentage of ischemic heart disease in recipients, low occurrence of active cytomegalovirus infection after transplantation, less racial disparity, and lower HLA mismatches may be the important factors.
Soluble markers and heart transplantation. Inflammatory processes play a pivotal role in the pathogenesis of transplantation rejection and mediate many of the stages of rejection from acute cellular rejection to chronic rejection (transplant coronary artery disease). Noninvasive methods to assess immune activation would be helpful in optimizing therapy after heart transplantation to reduce rejection and complications caused by excessive immunosuppression. Elevated plasma levels of several soluble markers of the inflammatory cascade and tissue injury have been shown to predict future risk of rejection and graft loss. These markers included troponin-I, CRP, cytokines (TNF-a and ILs), and adhesion molecules. But the results were conflicting with limited clinical utility. The plasma levels of these soluble markers released from the transplant hearts were mixed with the blood from peripheral circulation. Therefore, the measured plasma levels represented the sums of coronary sinus and peripheral blood. Rejection is a focal inflammation and soluble markers are released into coronary sinus. It prompted us to investigate whether plasma soluble markers in coronary sinus are better to predict the clinical event of transplant recipients than in peripheral blood. Wildhirt et al. reported a series of studies of coronary sinus expression of nitric oxide metabolites, TNF-a, IL-2 and IL-6 in transplant recipients. Patients with acute rejection or infection were excluded. The primary end point of these studies was microvascular endothelial dysfunction, but not clinical event. Fyfe et al. in 1993 reported that elevated coronary sinus levels of TNF-a and IL-6 in transplant recipients. But they found no relation between the coronary sinus or vena cava cytokine concentration or profile and severity of rejection. Since then, there was no extensive study of coronary sinus expression of soluble markers in transplant recipients.
Previous studies have yielded conflicting data regarding whether a relationship exists between elevated cardiac troponin-I levels and acute allograft rejection in patients who have received heart transplants. In a large prospective study, all heart transplant recipients had elevated troponin-I levels during the first month after transplantation. Troponin-I levels remained persistently elevated during the first 12 months in one-half of patients. Patients with persistent elevation of serum troponin-I had significantly increased risk for subsequent development of chronic rejection. However, markers of tissue injury were inadequate predictors of acute rejection in cardiac allografts. We had a similar result. Patients in early post-transplant period had an elevated titer of troponin-I both in coronary sinus and peripheral blood. But the levels of tropinin-I, either in coronary sinus or in peripheral blood, was not high in patients with rejetcion.
Elevated levels of plasma CRP were associated with decreased graft survival in cardiac transplant recipients. Although CRP was elevated more often in the presence of acute rejection, its sensitivity didn’t allow CRP to replace routine endomyocardial biopsy for monitoring rejection. In our study, the level of CRP was high in coronary sinus and peripheral blood in patients with early post-transplant and infection, but it failed to predict the occurrence of acute rejection and transplant coronary artery disease. But CRP seemed to have a predicting role in long-term uneventful status.
The cardiac allograft is a major source of cytokines and adhesion molecules after heart transplantation. Pathological studies suggested that immune events resulting in surface changes of coronary endothelial cells and production of cytokines play a role in the pathogenesis of acute rejection and may contribute to the long-term complication of transplant coronary artery disease. Cytokines are of great importance in the mechanisms of transplant rejection and this, in some cases, is reflected in the serum. However, this is not sufficiently consistent to be of diagnostic value. Measurement of serum cytokine levels in heart transplant recipients does not appear to correlate with findings on endomyocardial biopsy. We had a similar result. In patients with early post-transplant and infection, there was a higher level of all inflammatory markers except IL-2 in coronary sinus and peripheral blood. The depressed level of IL-2 was due to immunosuppression modification because most of immunosuppressive agents (cyclosporine and tacrolimus) were aimed to suppress IL-2 expression.
Positivity for circulating ICAM-1 in heart transplant recipients has been claimed to predict the development of coronary artery disease and risk of graft failure. However, more recent studies showed that increased circulating ICAM-1 levels did not correlate with endomyocardial biopsy scores. When the measured serum levels were correlated with the clinical status of the transplant recipient, soluble adhesion molecules only weakly discriminated between rejection and infection and the clinical utility of ICAM-1 in non-invasive monitoring is, therefore, limited. P-selectin level increased progressively with increasing rejection grade. In heart transplant recipients, P-selectin levels are highly predictive of organ rejection. Significant prolongation of graft survival was observed in mice treated with anti-P-selectin monoclonal antibodies. The enhanced endothelial mRNA expression of P-selectin was observed in the rejecting cardiac allografts. These results suggest that P-selectin is critically involved in the early development of acute rejection. In our study, patients with acute rejection had a higher level of IL-2, TNF-a, and CRP in peripheral blood but not in coronary sinus. Soluble markers in coronary sinus failed to predict the occurrence of acute or chronic rejections.
Conclusions. In patients with heart transplantation, soluble markers in coronary sinus is not better to predict the occurrence of acute or chronic rejections than in peripheral blood.
Part IV: Future perspectives.
One emerging approach to the study of cardiac disease is to identify proteins that have been specifically altered or modified in the diseased state. The identification of modified proteins will provide new diagnostic markers of myocyte injury. Disease-induced modification will substantively affect molecular function and contribute directly to cardiac dysfunction.
An approach to the issue of protein modification is to use a broad-scope screening method to observe as many proteins as possible. This approach, currently termed “proteomics”, 2-dimensional gel electrophoresis is used to resolve proteins and their modified proteins, which can then be characterized by mass spectrometry. The proteomic approach will prove powerful for documentation of the subtle and complex changes that occur along the continuum of cardiac diseases. Accordingly, we propose to identify novel serum biomarkers that can help us detect transplant coronary artery disease at earlier stages.
Specifically, we would like to investigate what protein or protein modifications in serum proteome are differentially expressed during progression of transplant coronary artery disease. These molecules will be used as indicator markers instrumental in rapid diagnosis as well as therapeutic management of patients with transplant coronary artery disease.
Our preliminary result is encouraging and it showed that our method using 2-D gel electrophoresis and silver staining provided a good tool to discover a novel marker of transplant coronary artery disease. Several spots were identified and further study was in progress.
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