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Eur J Cardiothorac Surg 2001;20:177-186
© 2001 Elsevier Science NL
Transplant Unit, Wythenshawe Hospital, Manchester, UK
Received 10 October 2000; received in revised form 21 February 2001; accepted 30 March 2001.
Corresponding author. Transplant Unit, Freeman Hospital, Freeman Road, High Heaton, Newcastle upon Tyne NE7 7DN, UK. Tel.: +44-191-2843111; fax: +44-191-2912091
e-mail: tarekaziz55{at}hotmail.com
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results (study beginning...4. DiscussionReferences |
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Key Words: Transforming growth factor-beta • Heart transplantation • Echocardiography • Left ventricular dysfunction
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results (study beginning...4. DiscussionReferences |
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Cytokines and growth factors have many complex effects on all processes involved in fibrosis and collagen metabolism. Transforming growth factor-beta (TGF-ß) is one of a number of cytokines and growth factors, which may influence matrix accumulation. The contribution of TGF-ß to the development of impaired diastolic function in heart transplants is undefined. Increased expression of TGF-ß in human fibrotic lesions has been previously reported [3]. In solid organ transplantation TGF-ß has been implicated in the development of glomerulosclerosis mediated by its dual action of increasing deposition and decreasing degradation of extracellular matrix [5].
The involvement of TGF-ß was examined in the present study, in association with histological and clinical factors, in the generation of impaired left ventricular diastolic function following orthotopic heart transplantation.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results (study beginning...4. DiscussionReferences |
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One hundred and fifty-two patients survived over 2 years and were eligible to be included in our analysis. The age at transplantation was 45±11 years and 90% were males. The standard technique [6] was performed in 84 recipients and the bicaval [7] procedure in 68 recipients.
Follow-up was complete to October 1999 or to the time of death and ranged from 24 to 60 months.
2.2. Endomyocardial biopsy
Endomyocardial biopsy (EMB) was performed via a percutaneous modified Seldinger right internal jugular approach with a 9F sheath and a Stanford–Caves bioptome using strict aseptic technique. Usually four to five satisfactory specimens were retrieved. All biopsy specimens were evaluated for rejection using International Society of Heart and Lung Transplantation (ISHLT) criteria [8].
2.3. Immunosuppression
Triple-drug immunosuppression with cyclosporin (3.5–5.0 mg/kg per day), azathioprine (1.5–2.5 mg/kg per day) and steroid (0.75–1.0 mg/kg per day) therapy was used in all patients. Cytolytic induction therapy (Antithymocyte globulin 2 mg/kg) was used in every patient during the initial 3 days. Cyclosporin therapy was adjusted to maintain serum trough level of 180–250 ng/ml during the first 2 years following transplantation. Azathioprine dose was adjusted to maintain white blood cell count of >4000/µl. Steroid dose was tapered gradually after the peri-operative induction to 0.125 mg/kg within 2–3 weeks after the operation. Acute rejection was treated with bolus methyl-predenislone (500 mg daily for 3 days) and follow-up EMB was performed 1–2 weeks later to assess outcome of treatment.
2.4. Transthoracic echocardiography
Doppler echocardiographic studies were analyzed for every recipient. Echocardiographic studies are routinely performed at the same schedule of endomyocardial biopsy. An experienced echocardiographer performed conventional two-dimensional and Doppler echocardiography with patients in the left lateral decubitus position. All studies were performed with a Hewlett Packard Sonos 2000 or 2500 machine equipped with a 2.5 MHz transducer. M-mode and Doppler recordings were made at a sweep speed of 50 or 100 mm/s and studies were recorded on 0.75 ins SVHS videotape. The transmitral flow velocity profile was recorded from the apical four-chamber view with the pulsed wave sample volume located at the tips of the mitral valve leaflets in diastole. The maximum E and A wave velocity and the time velocity integral of E and A wave were recorded. The time velocity integrals were measured by planimetry of the area under the mitral flow velocity curve. Mitral deceleration time (MDT) was measured as the time from peak E velocity to the point of intercept of the deceleration slope with the baseline. The isovolumic relaxation time (IVRT) represents the interval between aortic valve closure and mitral valve opening was recorded by pulsed wave Doppler evaluation with the sample volume midway between the mitral leaflets and the aortic annulus. Using this technique the timing of aortic closure and mitral opening could be identified on the same Doppler trace.
All studies were analyzed without prior knowledge of their immunohistochemical staining. Echocardiographic studies performed during on going cellular rejection (any grade) or studies performed in recipient who got significant angiographically detected coronary artery disease (>50% stenosis of left main stem, left anterior descending, circumflex coronary arteries) were excluded.
Left ventricular end-systolic and end-diastolic dimensions were recorded in the parasternal long axis view immediately distal to the mitral valve tips from the two-dimensional-guided M-mode image. Left ventricular ejection fraction was estimated qualitatively according to the usual practice in our laboratory. Colour-flow Doppler imaging was used to quantify mitral and tricuspid regurgitation. The left ventricular ejection fraction was calculated from 2-dimensional M-mode measurement, in accordance with the standard (cub method) clinical formula used by transplant Centre, where LVEF=EDV-ESV)/EDV, and EDV=EDD3, ESV=ESD3. ESV: end systolic dimension, EDD: end diastolic dimension.
In all study subjects Doppler echocardiographic parameters were measured off line from videotape recordings by an independent observer who was unaware of the patient's clinical status. Ectopic and post-ectopic beats were disregarded. The mean of at least five cardiac cycles was calculated for each Doppler parameter and cycles preceded by recipient atrial contraction during systole were disregarded to minimize the effects of recipient atrial contraction.
2.5. Right heart catheterization
Right heart catheterization was performed annually with each biopsy using a multipurpose cordis 7F vascular catheter (Cordis, Miami, FL) connected to an AE 840 (Mikro Elektronik A/S) pressure transducer. Intracardiac pressures were recorded at the levels of the right atrium, right ventricular body and pulmonary artery. Pulmonary capillary wedge pressure was also recorded.
2.6. Coronary angiography and left heart catheterization
Each patient underwent annual surveillance coronary angiography during left heart catheterization performed from the femoral approach. The severity of coronary atherosclerosis was assessed in the left main stem coronary artery main coronary vessels (proximal 2/3 of the LAD, Circumflex, dominant right coronary arteries). Left intracardiac pressures were recorded with a fluid-filled pigtail catheter attached to micromanometer transducer.
2.7. Histological examination
A total of 3278 endomyocardial biopsies from the interventricular septum obtained during baseline follow-up and at the time of angiographic studies. For the purpose of this study, a rejection episode was defined as the presence in at least one biopsy of ISHLT grade >0 rejection. Subsequent positive biopsies were considered to be the same rejection episode if not separated by a rejection-free biopsy.
2.7.1. Immunohistochemistry
Only EMB specimens taken at least 2 years after transplantation were selected for immunohistochemical assessment. To avoid the immediate effects of current rejection, specimens taken from recipients in whom the previous biopsy result showed evidence of cellular rejection (any grade other than 0) were excluded from immunohistochemical staining (n=633 biopsies). Specimens taken from patients requiring additional immunosuppression for any reason were also excluded (n=163 biopsies).
Paraffin-embedded sections fixed in 4% formaldehyde were dewaxed and rehydrated with Citroclear, alcohol and water for 10 min then treated with 10% proteinase K (Dako) in Tris buffered saline (TBS). Non-specific binding was blocked with 10% normal swine serum (Chemicon International Ltd., Harrow, UK). Mouse anti-human TGF-ß antibody was diluted 1/10 in TBS and applied to three of the four sections on each slide whilst the remaining section received only TBS without antibodies. The slides were incubated for 1 h, then washed and stained with a peroxidase-conjugated anti-mouse IgG at 1/1000 dilution (Sigma Immunochemical, USA) for 2 h, after which slides were transferred to fresh diaminobenzidine. The slides were counterstained with Meyer's Haemalum, dehydrated and mounted with diputyl polystyrene xylene (DPX). To confirm the staining specificity, blocking studies were performed with recombinant TGF-ß (R+D Systems, UK) to inhibit binding of anti-TGF-ß antibodies. Macrophages were identified in sections using an indirect immunoperoxidase technique with a mouse monoclonal antibody, CD68 (reagent PGM-1, Dako, Bucks., UK).
2.7.2. Immunohistochemistry quantification
We employed the TGF-ß scoring system, which was developed, by our laboratory and has been used to assess the immunohistochemical staining in heart and lung transplant recipients [9,10]. The system consists of evaluation of both cellular and fibrous scoring.
2.7.2.1. TGF-ß staining assessment
The total scoring for TGF-ß and CD+68 scoring took into account two factors:
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By the end of the second post transplant year, each patient had of five to seven biopsies. Each single biopsy stained for:
TGF-ß staining assessment was calculated for each separate pieces and then added together to calculate the mean TGF-ß score for each single biopsy. Slides were reviewed blindly twice. Similar TGF-ß staining score was achieved in 87% of the biopsies. Slides with TGF-ß staining or fibrosis scoring differences of more than 1 point were reviewed to reach an agreement regarding the final score of the slides.
2.7.3. Development of TGF-ß scoring system and echocardiographic parameters for each patient
Mean TGF-ß score and Doppler echocardiographic studies were calculated for each patient starting at the study beginning point at 24 months after the operation. In total 2685 endomyocardial biopsy specimens were stained for the entire study population. Patients were classified according to their degree TGF-ß staining score. Echo-cardiographic assessment was repeated annually to assess the long-term progress of diastolic left ventricular function. In total 587 Doppler echo-cardiographic assessment during the time target (Patient death or 5 years after transplantation).
2.8. Statistical analysis
All statistical analysis were completed with SPSS software (window 7.5; SPSS, Inc, Chicago, IL). Results are expressed as mean±standard of deviation. Data between the two groups were compared with unpaired t-test for parametric data and the Mann–Whitney test, or Fisher's exact test where applicable. Parameters within the same group were compared using Student's t-test. Some variables required log transformation to achieve approximate normality or constancy or additivity of scale. The regression and correlation analysis was used to compare the value of immunohistochemistry TGF score, and echocardiographic studies. Coronary artery disease was assessed using the Kaplan–Meier method and compared by log-rank test. A P value of less than 0.05 was defined as statistically significant.
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3. Results (study beginning point: 2 years after transplantation) |
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TopAbstract1. Introduction2. Methods3. Results (study beginning...4. DiscussionReferences |
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7).
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3.3. Haemodynamic parameters
The mean arterial blood pressure was not significantly different between the two groups (Table 4). Twenty seven percent of recipients in group A (n=12 patients) were on anti-hypertensive treatment medication (Calcium channel blocker) compared to 33% of recipients in group 2 (n=32 patients) P=0.6. Three patients from group A and four patients in group B required more than single antihyperenstive agent (P=0.4). Right ventricular systolic and diastolic pressure, mean pulmonary arterial pressure and pulmonary capillary wedge pressure were statistically higher in group A (Table 4). Mean right atrial pressure was also significantly different between the two groups. Left ventricular end-diastolic pressure was higher for group A. Haemodynamic and echocardiographic variables were significantly different in recipients with the bicaval technique according to their TGF-ß staining (Table 5).
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3.6. Clinical status at 2 years after transplantation
Mean NYHA status for patients in group A was 2.7±1.3 compared to 1.17±0.4 for patients in group B (P=0.002). Of the 56 patients in group A, 33/56 were in NYHA class III or IV or required anti-failure treatment compared with only 17/ 96 in group B (Table 6).
3.7. Follow-up (2–5 years after transplantation)
Restrictive filling pattern was persistent in recipients at group A and statistically significant compared to those at group B up to 5 years follow-up (Fig. 2)
. At the end of the study period, MDT was 118±11 ms for recipients in group A compared to 156±12 ms for those in group B (P=0.006). Similar difference was noticed in measuring of the IVRT was 62±7 ms for recipients in group A compared to 80±5 ms for those in group B. The actuarial development of subsequent coronary artery disease (>50% stenosis) in one or more of the major coronary artery vessel was 17 and 29% for recipients in group A compared to 4 and 6% for recipients in group B at 3 and 5 years follow-up, respectively (P=0.01 and P=0.005, respectively).
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4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results (study beginning...4. DiscussionReferences |
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Interstitial myocardial fibrosis and stiffness of the left ventricle have been claimed to be the main pathophysiological features in chronic left ventricular diastolic dysfunction [12]. The mechanism of chronic left ventricular diastolic dysfunction in the non-rejecting allograft has not been fully studied.
Left ventricular diastolic dysfunction has been demonstrated in patients with coronary artery disease with or without systolic dysfunction [1], valvular disease [2], cardiomypathies [2] and a variety of systemic diseases [3]. However, the causes and implications of chronic diastolic left ventricular dysfunction of the human cardiac allograft are not clear. There is an evolving body of evidence that abnormalities of diastolic filling play an important role in the clinical status and prognosis of patients with heart disease [10–12]. Ross et al. [4] reported that impaired left ventricular diastolic function within 6 months of transplant was associated with an increased late mortality.
4.2. Pathophysiology of left ventricular diastolic dysfunction in heart transplant recipient
Stinson et al. [13] found a persistent elevation left ventricular end-diastolic pressure on exercise in patients studied 1 year after heart transplantation. Theoretically, particularly in view of the fact that myocardial dysfunction during rejection is at least in part due to leukocyte cytokines which are released acutely [14], recovery of function may be expected in these circumstances. Valentine et al. [15], however, have shown that this recovery may often be incomplete with the development of a chronic pattern of restrictive physiology characterized by an elevation of left ventricular end-diastolic pressure. This pattern is characterised by myocytes loss and fibrous replacement. The healing response may bring about an irreversible progressive decline in compliance and leading to chronically deranged diastolic function. Skowronski et al. [16] noticed that increased chamber stiffness following acute rejection remained abnormal despite resolution of histological abnormalities. Diastolic function abnormalities in the cardiac allograft need not relate solely to those caused directly by rejection. Very significant abnormalities may occur at various stages following transplantation and are attributable to factors as disparate as perioperative ischaemia, reperfusion injury and hypertension. Drug therapy with cyclosporin may contribute in some patients and, in-fact, it has been suggested that the mechanism of diastolic dysfunction caused by cyclosporin [17] may be mediated directly by myocardial fibrosis. Because some patients with chronic left ventricular dysfunction appear to develop clinically significant heart failure, it is important that the aetiology and pathology of diastolic abnormalities be explored. An early report [17] of myocardial fibrosis occurring in association with cyclosporin treatment prompted speculation that cyclosporin may contribute to diastolic abnormalities of the cardiac allograft. More recently, however, Greenberg [18] failed to confirm an association between myocardial fibrosis and the use of cyclosporin. In our study, we were unable to confirm a direct correlation between cyclosporin and impaired left ventricular function as the patients in both groups were on long term treatment of cyclosporin. However, as group A recipients have been suffering from more frequent rejection episodes, it is logical to conclude that the level of cyclosporin was needed to be higher than average during these rejection episodes.
4.3. The potential role of TGF-ß
TGF-ß modulates a number of crucial events potentially central to the genesis and maintenance of chronic graft injury. These include macrophage chemotaxis, suppression of lymphocyte function, fibroblast chemotaxis and proliferation in addition to the modulation of collagen synthesis [19]. TGF-ß is also a strong stimulator of extracellular matrix synthesis [20]. Many different cells can synthesise and release TGF-ß, including activated macrophages, lymphocytes and platelets [19,20]. Previous reports have confirmed increased expression of TGF-ß in fibrotic lesions of human allografts [5].
4.4. Current study
We have demonstrated that TGF-ß expression in the cardiac allograft is associated with impaired graft function implicating this cytokine as an important substrate for the development of diastolic dysfunction. In accordance with previous studies [21,22], we have documented an association between impaired left ventricular diastolic dysfunction of the human cardiac allograft and rejection incidence. These data suggest that cumulative immune mediated injury could be responsible for triggering and pathogenesis of left ventricular dysfunction.
The haemodynamic data in the present study are consistent with the Doppler echocardiographic findings. The relatively short isovolumic relaxation time in group A was paralleled by higher pulmonary capillary wedge pressures than in group B indicating a reduction in myocardial compliance characteristic of fibrosis. This fibrosis is a recognized consequence of several possible insults to the heart, of which recurrent inflammation is one. Thus increased fibrosis scores in patients with impaired left ventricular diastolic function may be reasonably attributed to higher rejection incidence in this group. Acute cardiac rejection is characterized by myocardial mononuclear cell infiltration and oedema in the perivascular and interstitial tissues [7,16]. These lead to temporary increased stiffness of the myocardium and changes in left ventricular filling properties. With more frequent rejection episodes, the amount and activity of mononuclear cell in the myocardium increases and is associated with an increasing cytokine production by these cells. Both cytokines and markers of activated cytotoxic T cells have been associated with diastolic dysfunction assessed by Doppler echocardiography.
The level of TGF-ß expression in the EMB was the most potent predictor of diastolic dysfunction. The number of ISHLT grade rejection episodes correlated significantly with higher myocardial TGF-ß deposition and the development of diastolic dysfunction. These findings suggest that frequent cellular rejection episodes during the first 2 post-transplant years predisposed to higher TGF-ß production initiate a series of inflammatory and immunological responses characterized by over-expression of TGF-ß. This culminates in myocardial fibrosis and subsequent impairment of left ventricular diastolic function.
4.5. Limitations
Our study was limited by the fact that the study population was analyzed retrospectively and restricted to recipients surviving for at least 2 years after transplantation who had undergone coronary angiography. We were unable, therefore, to provide information about the very early development of early diastolic dysfunction. The rationale for this approach was based on the requirement to avoid the temporary influence of acute allograft rejection on the echocardiographic assessment, particularly given the poor reliability of immunohistochemical staining during rejection. By including no recipient in whom echocardiographic evaluation was performed during or within 6 months of an acute rejection episode and excluding all recipients with significant coronary artery disease our study has not suffered from their potential confounding effects on ventricular filling. The study is also limited by the fact that two different techniques have been used for heart transplantation, which may influence the accuracy of the haemodynamic and echocardiographic variables. However, the difference in the percentage of the bicaval technique recipients in each group was statistically insignificant. The haemodynamic variables of the left ventricular filling pressures and echo-cardiographic assessment was different between the bicaval technique recipients according to their TGF staining score. In addition, previous studies from our institution [23] and others [24] have not suggested any differences in the diastolic left ventricular function between the bicaval and standard technique heart transplant recipients.
4.6. Conclusion
This study is the first to highlight the potential role of TGF-ß in the development of left ventricular diastolic dysfunction for long term survivors after heart transplantation. Increased understanding of the pathophysiology of diastolic dysfunction and the role of cytokines in its development will allow a clearer definition of recipients at high risk. We suggest that dealing with impaired left ventricular function in transplanted heart can be considered in stepwise fashion. First, individual identified by their echocardiographic pattern. Second, this high-risk group to be targeted for intra-coronary vascular ultra-sound even in the absence of angiographic evidence of coronary artery disease. Third, modification of the biopsy regime and immunosuppression may be important for these patients. Finally, we suggest that immunological strategies to manipulate cytokine expression in cardiac allografts may improve their function and survival.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstract1. Introduction2. Methods3. Results (study beginning...4. DiscussionReferences |
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This article has been cited by other articles:
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  M. Wilhelmi, K. Pethig, M. Wilhelmi, H. Nguyen, M. Struber, and A. Haverich Heart transplantation: echocardiographic assessment of morphology and function after more than 10 years of follow-up Ann. Thorac. Surg., October 1, 2002; 74(4): 1075 - 1079. [Abstract] [Full Text] [PDF]
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