Weis, Michael*; Wildhirt, Stephen M.†; Schulze, Costas†; Pehlivanli, Sinan*; Rieder, Gabriele‡; Wolf, Wolf P.*; Wilbert-Lampen, Ute*; Meiser, Bruno M.†; Enders, Georg‡; von Scheidt, Wolfgang*
Cardiac allograft vasculopathy remains a serious long-term complication of heart transplantation and is the major cause of death in patients surviving 1 year after transplantation (1). Several experimental models suggest that immunologic mechanisms operating in a milieu of non-immunologic risk factors constitute the principal stimuli that result in progressive myointimal hyperplasia (1,2). Repetitive alterations of the allograft endothelial barrier result in response to injury mechanisms leading to endothelial activation and dysfunction (2). Damage to the endothelium may alter various physiologic endothelial functions, predisposing the coronary artery to inflammation, thrombosis, vasoconstriction, and vascular smooth muscle cell growth. Recently, Davis et al. (3) demonstrated that early endothelial dysfunction (15 ± 3 days after transplantation) predicts the development of intravascular ultrasound-visible cardiac allograft vasculopathy at 1 year after heart transplantation. Using a predictive model for adverse cardiac events from cardiac allograft vasculopathy in a cohort of 107 consecutive heart transplant recipients Mehra et al. (4) detected a prognostic impact of immunosuppression and cellular rejection on cardiac allograft vasculopathy. A higher daily cyclosporine (CyA) dose was found to be protective against adverse cardiac events, whereas a greater prednisone consumption was substantially deleterious. Thus quality and quantity of immunosuppressive medication may have an impact on development of cardiac allograft vasculopathy (5). Recently, clinical trials with TKL and mycophenolate mofetil (MMF) have been started in clinical cardiac transplantation (6,7). TKL appears to exert its effects through a molecular mechanism of action similar to that of CyA but has been suggested to be more potent, presumably by inhibiting cytokine synthesis by T cells infiltrating the allograft (7). MMF, an antimetabolite derived from mycophenolic acid with antiproliferative properties, was shown to prolong cardiac allograft survival, induce donor-specific tolerance, and reverse ongoing acute cellular rejection in rodent and primate models (7). However, the effects of TKL and MMF on coronary endothelial function and subsequent development of human cardiac allograft vasculopathy are unknown.
The present study was designed to investigate functional (epicardial and microvascular endothelial function) and morphologic (epicardial intimal thickening) coronary alterations within 6 months after cardiac transplantation with respect to different immunosuppressive regimens. We hypothesized that coronary endothelial dysfunction in the transplanted heart is mediated by alterations in endothelium-derived and inducible vasoactive factors and cytokines. Thus gene expression of vasoactive mediators as well as transcardiac cytokine, endothelin-1, and nitrate release were determined.
This study was performed in the early, stable postoperative phase (1-6 months after transplantation). The 31 orthotopic heart transplant recipients were selected according to predefined exclusion criteria including acute infection or rejection episode (≥grade 1b according to the International Society of Heart and Lung Transplantation) and impaired renal function (serum creatinine > 1.8 mg/dl). University of Wisconsin or Celsior-solution was used for allograft preservation (Table 1). No induction therapy with antibody preparations was performed. In the first 17 patients the immunosuppressive protocol was randomized TKL, azathioprine, and prednisone versus CyA, azathioprine, and prednisone. Since August 1996 all patients in our institution received TKL, MMF, and prednisone as a standard immunosuppressive regimen. All TKL-treated patients received the drug i.v. for the first 2-3 postoperative days followed by oral dosing, targeting TKL trough levels of 13-15 ng/ml. MMF was administered orally after extubation at a dose of 2 g/day. The patients were divided in three groups corresponding to their individual immunosuppressive protocol. Group 1 consisted of eight transplant recipients treated with CyA, azathioprine, and prednisone. Group 2 consisted of nine transplant recipients treated with TKL, azathioprine, and prednisone. Group 3 consisted of 14 patients treated with TKL, MMF, and prednisone. Importantly, no significant differences were noted with respect to donor age, ischemic time, cytomegalovirus, and HLA-mismatch, whereas the number of treated rejection episodes was significantly reduced in group 3 (Table 1). The investigation was performed with approval by the institutional ethics committee and all patients gave written informed consent to the investigation performed during the routinely scheduled cardiac catheterization. The investigation conforms with the principles outlined in the Declaration of Helsinki. The patients were in the fasting state for at least 12 h and all vasoactive drugs were discontinued 12 h before the investigation.
Right heart catheterization. Endomyocardial biopsy was performed through the percutaneous jugular venous approach for the detection of acute rejection. Two additional myocardial biopsies were immediately frozen in liquid nitrogen and stored at −80°C for molecular investigations. A 7-Fr multipurpose catheter was then placed in the coronary sinus and 20-ml blood samples were withdrawn for determination of cardiac cytokines, endothelin-1, and nitrate release.
Left heart catheterization. After the diagnostic procedure including left ventriculography and coronary angiography, a Doppler flow-wire (FloWire, Cardiometrics Inc., Mountain View, CA, U.S.A.), was placed in the proximal left anterior descending or circumflex artery, permitting measurement of coronary blood flow velocities. Five thousand internation units of heparin were given intravenously. During baseline conditions, the position of the Doppler flow-wire was adjusted to optimize the audio flow-velocity signal and the phasic flow-velocity waveform. Doppler flow-wire positions were not changed thereafter. Intracoronary blood flow velocity and arterial pressure were recorded continuously at baseline and during administration of the study agents. First, the endothelium independent agent adenosine was infused into the left coronary system at different dose levels (80 and 160 μg/min over 5 min each) to achieve maximal (endothelium-independent) coronary flow. Second, the endothelium-dependent agent acetylcholine (Acetylcholine, Dispersa, Germering, Germany), was infused into the left coronary system (1 and 30 μg/min over 5 min each) to investigate epicardial and microvascular endothelial function. At the end of the protocol, nifedipine (Bayer AG, Leverkusen, Germany) was administered (0.2 mg i.c.) to achieve maximal epicardial coronary artery vasodilatation. At the end of each infusion, coronary arteriography was performed with a biplane imaging system in a right and left oblique position with adequate cranial or caudal angulation for optimal analysis of the left coronary tree on end-diastolic frames. Position was kept constant during the protocol. Throughout each infusion, heart rate, arterial pressure, coronary flow velocity, and electrocardiogram were monitored continuously and documented on videotape for additional off-line analysis. All measurements were recorded in steady-state conditions. For determination of transcardiac cytokines, endothelin-1, and nitrate-release, aortic blood samples (20 ml) were withdrawn.
Coronary flow reserve. The coronary flow velocity reserve as a marker of microvascular integrity was determined by the ratio of the maximal coronary flow velocity (cm/s) after pharmacologic stimulation to the basal flow velocity. Coronary microvascular smooth muscle dysfunction was defined as coronary flow velocity reserve <2 in response to adenosine and nifedipine to exclude an isolated adenosine-defect (8). Coronary microvascular endothelial dysfunction was defined as coronary flow velocity reserve <2 in response to acetylcholine.
Quantitative coronary angiography. Quantitative coronary angiography was performed to investigate epicardial vasomotor response using a computerized automatic-analysis system (HICOR, Siemens, Erlangen, Germany). Nonstenotic proximal and distal coronary arterial segments identified between easily visualized branch points were selected for analysis in the anterior descending or circumflex artery. Epicardial endothelial dysfunction was defined as >10% diameter reduction in response to acetylcholine compared with baseline. Epicardial smooth muscle cell dysfunction was defined as <10% diameter increase in response to nifedipine. The prevalence of pathologic findings in the proximal and distal coronary parts was determined and mean values were calculated.
Immediately after Doppler flow measurement, intracoronary ultrasound (ICUS) was performed to detect intimal hyperplasia not visible with angiography. Before intravascular positioning, 200 μg of nitroglycerin were injected into the left coronary artery. The imaging system consisted of a 30-MHz ultrasound transducer enclosed within an acoustic housing on the tip of a 2.9-Fr flexible, rapid-exchange catheter (CVIS Inc., Sunnyvale, CA, U.S.A.). The catheter was advanced to the distal left anterior descending or circumflex artery, with careful observance of a lumen-ICUS catheter diameter ratio of >1.5. During the subsequent standardized pullback maneuver, images were documented on SVHS videotape for further off-line-analysis. The three sites with the most severe intimal proliferation were evaluated quantitatively concerning the radial and circumferential extent of intimal hyperplasia, and the averaged maximal intimal thickness was calculated. Averaged mean intimal thickness was determined using a minimum of 5 randomized proximal to distal coronary sites (9).
Myocardial molecular investigations
RNA extraction and cDNA preparation. Samples were homogenized with OMNI 2000 (SLB, Gauting, Germany) in 600 μl of lysis buffer (Quiagen, Hilden, Germany). Insoluble material was separated from the lysate by centrifugation at 10,000g for 3 min. Total RNA was extracted from the supernatant using spin columns with a selective binding silica-based membrane (RNeasy Kit, Qiagen). Total RNA was quantified by measuring the optical density at 260 nm and was confirmed by gel electrophoresis. Complementary DNA (cDNA) was prepared from 2 μg of total RNA in 30 μl reverse transcription buffer (Gibco BRL, Paisley, U.K.) supplemented with 0.6 mM each of dATP, dGTP, dCTP, and dTTP (New England Biolabs, Frankfurt-am-Main, Germany), 32 U RNase inhibitor (Boehringer, Mannheim, Germany), 400 U of Moloney murine leukemia virus reverse transcriptase (MMLV-RT; Gibco BRL), 10 mM dithiothreitol, and 1.5 μM p(dt)15 primer (Boehringer) at 37°C for 60 min. Subsequently, the reaction mixture was heat-inactivated for 10 min at 95°C.
Polymerase chain reaction procedure. An aliquot (3 μl) of cDNA was amplified by polymerase chain reaction (PCR) with a DNA thermal cycler (Perkin-Elmer 480, Cetus Corp., Norwalk, CT, U.S.A.). The amplification reaction was carried out in a total volume of 50 μl of PCR buffer containing 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl, 50 mM KCl, 200 μM each of dATP, dGTP, dCTP, and dTTP, 400 nM each of 3′ and 5′ primers, and 1 U Taq DNA polymerase (Boehringer). PCR amplification of cDNA was performed with specific primers for endothelin-1 (MWG, Ebersberg, Germany), inducible and endothelial nitric oxide synthases (iNOS and eNOS) (MWG), prostacyclinsynthase, and thromboxansynthase (kindly provided by Professor V. Ulrich, Konstanz, Germany). The primers (Table 2) were designed such that the expected products were only obtained from cDNA, but not from genomic DNA.
The PCR reaction mixture was covered with 25 μl of light mineral oil (Sigma Chemie, Deisenhofen, Germany). After 1 min predenaturation at 94°C, the PCR conditions were as follows: denaturation at 94°C for 45 s, annealing at 62°C for 45 s, and extension at 72°C for 1 min. To ensure detection of low-abundance mRNA, 35 amplification cycles were performed. A sample (10 μl) of each amplified product was subjected to electrophoresis in a 1% agarose gel (Promega, Madison, WI, U.S.A.), stained with ethidium bromide and visualized by ultraviolet illumination. To determine the amount of PCR product, we used a semiquantitative scoring scheme that was based on visual assessment of photographic negatives of ethidium bromide agarose gels. This standardized method, previously described and validated (10) has been optimized for the semiquantitative measurement of inducible mRNA transcripts from multiple samples and can be used to discern at least fourfold differences in the transcription or quantity of a given mRNA. The semiquantitative grades were scored by two independent; blinded observers. The reaction cycles of PCR were performed in the range that demonstrated a linear correlation between the amount of cDNA and the yield of PCR products. Each signal was normalized for the DNA standard of the same gel to account for variances between gels. Normalization for the house-keeping gene GAPDH was performed to account for the variability of sample quality.
Cytokine assay technique. Serum levels (coronary sinus and aortic blood samples) of tumor necrosis factor (TNF) and interleukin (IL)-6 were measured by enzyme-linked immunoabsorbent assay (ELISA) (Medgenix GmbH, Ratingen, Germany). Soluble IL-2R, soluble TNF-R-p55 and -p75 were measured by Cobas Core (Hoffmann La Roche, Basel, Switzerland) with enzyme-linked immunologic assay (ELISA).
Endothelin-1 radioimmunoassay. Measurement of endothelin-1 has been described in detail elsewhere (11). In brief, blood samples were immediately placed on ice, centrifuged, and stored at −80°C until they were assayed. Endothelin-1 was extracted through Sep-Pak C18 cartridges washed with 10 mL 0.1% trifluoroacetic acid (TFA), and eluted with 5 mL 60% acetonitrile/0.1% TFA. For radioimmunoassay, eluates were deep-frozen, dried and redissolved in RIA buffer. Endothelin-1 antibody 2428 (Medor, Herrsching, Germany) was added to samples and standards (binding characteristics: endothelin-1, 100%; endothelin-2, 73%; endothelin-3, 31%; Big endothelin-1, 13%) for radioimmunoassay.
Griess reaction. Total nitrate-nitrite levels were measured by Griess reaction. The stable end products of nitric oxide generation were assayed by spectrophotometric analysis at 540 nm after reduction of nitrite to nitrate with nitrate reductase as described in detail elsewhere (12).
Differences in group means were compared by ANOVA followed by the post hoc Tukey's test. The Kolmogorov-Smirnov Z test was used for nonparametric analysis. Significant differences in the prevalence of pathologic findings were examined using χ2 test. A probability value of less than 0.05 was considered significant. All data are expressed as mean ± SD.
Coronary functional alterations
Table 3 summarizes the epicardial and microvascular endothelium-dependent (acetylcholine) and endothelium-independent vasomotor responses (adenosine). Epicardial endothelium-dependent vasomotor response was significantly reduced in patients treated with TKL, azathioprine, and prednisone (Fig. 1). In line, the prevalence of epicardial endothelial vasomotor dysfunction (>10% vasoconstriction to acetylcholine) was pronounced in the patient group treated with TKL, azathioprine, and prednisone as shown in Figure 2A (prevalence of epicardial dysfunction 78% in group 2 versus 44% [χ2 4,2; p < 0.05] and 46% [χ2 4,4; p < 0.05] in group 1 and 3, respectively). No significant differences in epicardial endothelium-independent response (Fig. 2B) were observed between the groups.
Microvascular endothelium-independent vasomotor response (but not endothelial response) was diminished in patients treated with TKL, azathioprine, and prednisone (Fig. 3). Prevalence of microvascular endothelial dysfunction was comparable between the groups (Fig. 4A), whereas the prevalence of pathologic microvascular endothelium-independent vasomotion (flow increase to adenosine <2.0) was markedly enhanced in the TKL- and azathioprine-treated group (56% vs 13% [χ2 5,7; p < 0.02] and 7% [χ2 6,7; p < 0.01] in group 1 and 3, respectively) as indicated in Figure 4B.
Coronary morphologic alterations
No differences could be observed concerning extent of coronary intimal thickening (Table 4), suggesting a comparable extent and distribution pattern of donor-transmitted disease among the groups in the early phase after transplantation.
mRNA gene expression of vasoactive mediators
Intramyocardial mRNA transcript levels for vasoactive enzymes are shown in Figure 5. Highest signals for iNOS mRNA were detected in recipients immunosuppressed with TKL and azathioprine (expression grade 2.7 ± 0.7 vs. 1.0 ± 0.6 and 1.6 ± 1 in recipients treated with CyA-based and TKL+MMF-based immunosuppression, respectively; p < 0.05), whereas eNOS mRNA expression was significantly decreased in the TKL and azathioprine-treated group (1.2 ± 1 vs. 2.3 ± 0.5 and 2.5 ± 0.9 in CyA-based and TKL+MMF-based group, respectively; p < 0.05). No differences were found in prostacyclinsynthase or thromboxansynthase mRNA signal intensity, whereas endothelin-1 mRNA expression tended to be elevated in the TKL + azathioprine group (Fig. 5).
Cardiac cytokine pattern
No difference was measurable in cardiac cytokine production between the groups despite cardiac IL-6 production, which was elevated in TKL and azathioprine-treated recipients compared with CyA and azathioprine-treated recipients (6 ± 6 pg/ml vs. −5 ± 4 pg/ml; p <0.01) (Table 5). Transcardiac IL-6 release was detectable in 100% of patients treated with TKL, azathioprine, and prednisone, whereas IL-6 release was detectable in only 20% and 50% from groups 1 and 3, respectively (p < 0.025). The prevalence of cardiac net release of IL-2R, TNF-α, and TNF-αR was similar between the groups.
Transcardiac endothelin-1 and nitrate gradient
Cardiac endothelin-1 release was not measurable in any patient, suggesting endothelin-1 net extraction by the heart. Cardiac nitrate release was similar between groups (Table 6).
The major finding of the present study is a significantly higher prevalence of epicardial endothelial and microvascular smooth muscle cell dysfunction in heart transplant recipients immunosuppressed with TKL, azathioprine, and prednisone, associated with increased iNOS expression, decreased eNOS expression, and increased cardiac IL-6 release, compared with both other immunosuppressive regimens.
Epicardial endothelial dysfunction contributes to ischemic manifestations of coronary artery disease. Moreover, endothelial dysfunction may extend to the coronary microcirculation, where no overt atheroma develop but where coronary blood flow, and hence myocardial perfusion, is regulated. Microvascular dysfunction may therefore contribute importantly to ischemic consequences of coronary artery disease, even in the absence of significant epicardial stenoses (13,14). Indeed, the endothelium is not only a target but also a mediator of atherosclerosis, and coronary endothelial dysfunction is considered a pivotal initial step in the pathogenesis of atherosclerosis (13).
Cyclosporin A has been shown to impair endothelial and smooth muscle cell function in animal experiments (15), but conflicting results have been reported in humans (16,17). Noteworthy, Stroes et al. (18) demonstrated that acute administration of CyA enhances both basal- and receptor-stimulated endothelial nitric oxide activity. Thus endothelium-derived nitric oxide constitutes an important counterregulating mechanism that protects against CyA-associated (likely endothelin-mediated) vasoconstriction in vivo. This potential endothelium-protective mechanism has not yet been described for TKL. It is unclear whether a direct (dose-dependent) pharmacologically induced endothelial/smooth muscle cell effect or a mechanism concerning the NOS pathway is responsible for the TKL-induced changes in coronary function as detected in the present study. Importantly, an increased suppression of NOS induction in smooth muscle cells by CyA when compared with TKL has been demonstrated (19,20). In the present study highest amounts of iNOS mRNA (with potential nitric oxide-mediated cytotoxic side effects) and lowest amounts of eNOS gene expression were detectable in the TKL and azathioprine-treated group, suggesting a possible link between enhanced iNOS expression, diminished eNOS expression, and endothelial dysfunction. Among possible explanations for the iNOS-mediated destructive influences on coronary endothelium are enhanced capacity of immune cells to injure (21) or the promotion of endothelial apoptotic cell loss (22). Although eNOS is constitutively expressed, both in vivo and in vitro studies have shown that its expression is subject to modest (but likely important) degrees of regulation (23). Several pathophysiologic factors have been shown to lower eNOS expression, such as exposure of cultured endothelial cells to TNF-α and hypoxia (23). Thus under proinflammatory conditions, a decrease in eNOS expression might have a significant impact on endothelial vasomotor regulation. In addition, other factors such as differences in cardiac cytokine expression (suppressed in a variable manner by different immunosuppressive agents) may also be of importance concerning changes in coronary endothelial function (24,25). In the present study we could demonstrate a significant cardiac IL-6 release only in patients treated with TKL and azathioprine, suggesting a sufficient inhibition on IL-6 activity by both other immunosuppressive regimens. It is known that TKL exerts only weak inhibitory effects on IL-6 activity in vitro and in vivo (26,27). Importantly, IL-6, as a predominantly T-cell-independent (macrophage-derived) cytokine, has been shown to induce specific changes in endothelial cell function and morphology and has been implicated in the development of sclerosis and fibrosis after renal transplantation (28-30).
Several groups investigated the influence of TKL on smooth muscle proliferation and development of cardiac allograft vasculopathy. Allografted hearts treated with TKL showed more severe grade of cardiac allograft vasculopathy compared with grafted hearts treated with CyA (31,32). In contrast, a prospective clinical trial in heart transplantation demonstrated no significant difference in freedom from angiographically visible cardiac allograft vasculopathy between patients treated with TKL (n = 80) or CyA (n = 80) (6). However, in the latter study coronary functional measurements and intravascular ultrasound were not performed.
Besides TKL, the use of MMF has recently been introduced in clinical heart transplantation (33). In a randomized, prospective multicenter study, the combination of CyA and MMF was associated with a 35% reduction in 3-year mortality, mostly because of a decrease in cardiovascular mortality, compared with CyA, and azathioprine (34). MMF inhibits both T-cell-mediated and T-cell-independent pathways, decreases adhesion molecule activity, and diminishes cytokine synthesis (30,35), and hence, may protect endothelial function in specific immunologic settings. Here, we demonstrated that MMF in combination with TKL and prednisone diminished the extent of epicardial endothelial and microvascular smooth muscle dysfunction and suppressed the cardiac iNOS and IL-6 expression seen in transplant recipients immunosuppressed with TKL and azathioprine. Importantly, MMF-therapy has been associated with reduction in smooth muscle cell and endothelium cell proliferation (36). Thus a potential direct anti-atherosclerotic action by MMF, in vivo, may delay the development of cardiac allograft vasculopathy.
The most important limitation of the study is the absent randomization of group 3 patients and the relatively small number of patients in each group. However, the patients were selected carefully according to predefined exclusion criteria and the groups did not differ significantly in donor or recipient characteristics or serum lipid and lipoprotein concentrations. Importantly, Celsior and University of Wisconsin preservation solutions are comparable concerning coronary vasomotor function early after transplantation (37). Thus the alterations in vasomotor function and the different expression of vasoactive factors are most likely attributable to the different immunosuppressive regimens. In addition, in the present investigation we have detected specific functional and molecular correlates of endothelial dysfunction early after transplantation. These possible markers of early functional coronary changes should be studied in future prospective randomized trials with respect to their impact on atherosclerotic coronary manifestations.
In summary, the immunosuppressive combinations of CyA + azathioprine and TKL + MMF appear to be superior to TKL + azathioprine regarding preservation of early coronary vasomotor function, eNOS expression, iNOS suppression, as well as cardiac IL-6 release. These functional and molecular graft alterations in the early period after transplantation may have an important impact on subsequent development of transplant coronary arteriosclerosis. In this regard, preliminary follow-up data from our patient population suggest that patient treated with TKL + azathioprine developed enhanced maximal epicardial intimal thickening compared with patients treated with CyA + azathioprine or TKL + MMF during the first year after transplantation (unpublished personal data). In line, in a recent prospective trial using volumetric intravascular ultrasound analysis for the prevention of cardiac allograft vasculopathy, a trend toward a more pronounced progression of intimal thickening was noted in the TKL versus the CyA group (38).
Acknowledgment: This study was in part supported by the Friedrich-Baur Foundation, Munich Germany. We would like to thank Peter Fraunberger for collaborative work (cytokine analysis).
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