During the first year after cardiac transplantation significant structural changes occur in the coronary artery that adversely affect long-term survival (1, 2). Reduction in vessel size (negative remodeling) and progression of intimal thickening, may both contribute to a loss of lumen diameter. Intravascular ultrasound (IVUS) is currently the most sensitive method for detecting these changes (3). Past studies using IVUS have demonstrated varying degrees of alteration in vessel size, both negative and positive remodeling, and varying amounts of intimal thickening early after transplantation (4–10). The discrepancies in these studies are likely explained by the use of two-dimensional IVUS analysis and/or the lack of sufficient serial data in the same patients. With automated catheter pullback devices and improved data integration and analysis software, it is now possible to construct three-dimensional IVUS images of the coronary arteries and perform a highly accurate assessment of volumetric changes (11).
The clinical determinants of lumen loss after transplantation are incompletely characterized. In particular, the role of cytomegalovirus (CMV) infection in the development of cardiac allograft vasculopathy continues to be debated (12). Determining the degree to which lumen loss occurs as a result of negative remodeling as opposed to intimal thickening and identifying the clinical factors associated with these changes remain central to identifying strategies to prevent and treat cardiac allograft vasculopathy.
Therefore, the first aim of this prospective, longitudinal study was to employ serial, volumetric IVUS analysis to clarify the type of vessel remodeling and the degree of intimal thickening and to define their impact on lumen loss early after cardiac transplantation. The second aim was to evaluate clinical correlates of these anatomic changes, particularly focusing on CMV infection and prophylaxis.
MATERIALS AND METHODS
Consecutive cardiac transplant recipients at Stanford University Medical Center between January 2002 and June 2005 were asked to participate in this prospective longitudinal NHLBI-funded study, intended to elucidate the role of cytomegalovirus in cardiac allograft vasculopathy. Those patients who were over the age of 18, provided informed, written consent, were clinically stable with a serum creatinine ≤2 mg/dL, and were not undergoing a repeat transplant are included in this analysis. All patients were scheduled for a baseline coronary angiogram and IVUS study within 8 weeks of transplantation and a follow-up angiogram and IVUS study 1 year after transplantation. Donor and recipient clinical characteristics, medications, and rejection episodes were prospectively entered into an electronic database developed and supervised by the Data Coordinating Center of the Biostatistics department of Stanford University. The protocol was approved by Stanford University’s Administrative Panel on Human Subjects.
All patients received standard immunosuppressive therapy, including induction therapy with daclizumab, corticosteroids, an antiproliferative agent (rapamycin or mycophenolate mofetil), and a calcineurin inhibitor (cyclosporine or tacrolimus). Recipients were monitored for rejection by performance of right ventricular endomyocardial biopsy on a weekly basis for the first 4 weeks, bimonthly basis for the next 8 weeks and then at 6, 9, and 12 months after transplantation. Rejection was graded using the standard ISHLT scoring system (13). A grade of ≥3A was chosen as a cutoff value based on previous work and on the fact that we generally treat this degree of rejection (14). All recipients received standard CMV prophylaxis consisting of 4 weeks of intravenous ganciclovir. Those recipients who were CMV antibody negative and received a heart from a CMV antibody positive donor received an additional 3 month course of CMV hyperimmune serum and up to 80 days of valganciclovir (aggressive CMV prophylaxis). CMV IgG antibody was tested for before transplantation and again at 1 year after transplantation. Only one patient developed evidence of active CMV infection requiring treatment in the first year after transplantation. All patients were treated with a calcium channel blocker and statin unless adverse side effects occurred. Angiotensin converting enzyme inhibitors or angiotensin receptor blockers were used if patients were intolerant to calcium blockers or required additional antihypertensive therapy.
After documentation of lack of significant coronary artery disease based on angiography, 3,000 to 5,000 units of heparin was administered intravenously and 200 ìg of intracoronary nitroglycerin was given via a 6F-guiding catheter in the left main coronary artery. A 0.014 guidewire was advanced under fluoroscopic guidance to the mid to distal left anterior descending artery (LAD). A 2.6F, 40 mH mechanical ultrasound catheter, connected to a Galaxy IVUS system (Boston Scientific Corporation) was advanced over the wire to the mid to distal LAD and an automated pullback of the catheter was performed at 0.5 mm/sec. Images were recorded for offline analysis of the first 50 mm of the LAD.
Three-dimensional IVUS analysis was performed by Stanford’s IVUS Core laboratory. Lumen, plaque, and vessel dimensions were manually traced in the first 50 mm of each LAD in 16 frame (0.5 mm) increments, resulting in 100 cross-sectional images from which volumetric reconstruction occurred automatically by interpolating the measurements of the remaining frames. Using Simpson’s method, vessel volume, lumen volume, and plaque (intimal) volume were quantified and the percent change in vessel, lumen, and plaque volumes from baseline to year 1 was calculated (echoPlaque, Indec Systems, Inc.) (15). The maximal intimal thickness was recorded at baseline and the intimal thickness was measured at the same spot at year 1. In addition, the maximal intimal thickness was measured at year 1.
The data were expressed as mean values±standard deviation (SD). Mean values were compared, in a paired fashion where appropriate, using a two-tailed, Student t test. Linear regression analysis was performed to determine the correlation between continuous variables. Multivariate regression analysis was applied to determine the independent predictors of changes in coronary artery anatomy. A P value <0.05 was considered significant. Statistical calculations were performed with Statview software (SAS Institute Incorporated).
There were 183 cardiac transplantations performed during the enrollment period, 158 of which met the inclusion criteria. Of these, 94 recipients consented to this study, but 35 did not have a baseline IVUS because of clinical reasons (e.g., elevated creatinine). Of the 59 patients who had baseline IVUS, 21 patients did not have a 1 year IVUS because of either clinical reasons or patient refusal. This report includes 36 patients who underwent IVUS evaluation of their LAD at baseline and 1 year follow-up and had acceptable recordings for volumetric analysis; two patients had baseline IVUS, but missed their 1 year follow-up and had follow-up IVUS at 2 years. A complete 50-mm pullback was available for analysis in all but two patients; in each of those two patients one of the two IVUS studies had a pullback of only approximately 30 mm. In these cases, the analysis of the other IVUS study was adjusted to the shorter pullback length. None of the patients had an angiographic stenosis greater than 50% in the LAD at baseline or 1 year follow-up.
The clinical characteristics of these patients are outlined in Table 1. The average time after transplantation for the first IVUS study was 27±20 days, and for the second study was 397±72 days. There were no patients actively using tobacco. The choice of antihypertensive was at the discretion of the treating physician. Twelve patients received aggressive 3-month CMV prophylaxis. There were no significant differences in prevalence of the clinical variables listed in Table 1 in the patients who received aggressive CMV prophylaxis compared to those who did not. Eleven patients received rapamycin instead of mycophenolate mofetil during the first 12 months after transplantation. Fifteen patients were CMV seronegative at the time of transplantation and three of these received hearts from CMV seronegative donors. Of the 23 CMV seropositive recipients, six received hearts from CMV seronegative donors.
Two-dimensional and volumetric analysis demonstrated significant changes in coronary anatomy during the first year after transplantation. On average, vessel size decreased significantly and plaque volume increased significantly, resulting in a highly significant decrease in lumen dimensions. These changes are outlined in Table 2. Two-dimensional maximum and minimum vessel, lumen, and plaque measurements also changed significantly in a similar fashion. The maximal intimal thickness at baseline did not change when compared with the matched cross-section at year 1 (0.87±0.58 vs. 0.86±0.63, P=0.92) but did increase significantly when compared with the maximal intimal thickness at year 1 (0.87±0.58 vs. 1.03±0.63, P=0.005).
There was a strong correlation between vessel volume and lumen volume at baseline (r=0.92, P<0.0001) and at 1 year (r=0.85, P<0.0001). Plaque volume did not correlate with lumen volume at baseline (r=0.26, P=0.11) or at year 1 (r=0.13, P=0.42) (Fig. 1). There was a strong correlation between the % change in vessel volume and the % change in lumen volume from baseline to year 1 (r=0.82, P<0.0001). The % change in plaque volume did not correlate with the % change in lumen volume (r=0.08, P=0.64), however there was a highly significant positive correlation between the % change in plaque volume and the % change in vessel volume (r=0.53, P=0.0006) (Fig. 2).
In the 23 recipients who were CMV antibody positive at the time of transplantation the % decrease in vessel volume (−15.0±10.4 vs. −3.8±14.3%, P=0.008) and lumen volume (−21.6±12.1 vs. −12.7±11.7%, P=0.03) was significantly greater compared to the 15 who were not CMV seropositive. In the 12 patients who received aggressive CMV prophylaxis, there was significantly less loss of lumen volume (−11.7%±12.8% vs. −21.0%±11.5%, P=0.03) and vessel volume (−3.6%±15.5% vs. −13.8%±10.8%, P=0.02) compared to those who did not receive aggressive prophylaxis.
Univariate regression analysis using the clinical variables in Table 1 as independent variables revealed that recipient CMV antibody positivity (P=0.008) and lack of use of a calcium channel blocker at year 1 (P=0.01) were the only significant predictors of a greater % reduction in vessel volume (i.e., greater negative remodeling). On multivariate analysis both independent variables remained significant predictors of a greater % change in vessel volume, (P<0.01, coefficient=−10.2 for recipient CMV antibody positivity, and P=0.01, coefficient=9.9 for calcium blocker use at year 1). Of note, cellular and humoral rejection did not correlate with negative remodeling.
Univariate regression analysis using the clinical variables in Table 1 as independent variables revealed that lack of rapamycin therapy at year 1 (P=0.02), a history of tobacco use (P=0.02), and male donor gender (P<0.05) were the only significant predictors of a greater % change in plaque volume (i.e., more plaque volume). On multivariate analysis using these three independent variables, none remained significant.
Univariate regression analysis using the clinical variables in Table 1 as independent variables revealed that lack of calcium channel blocker use at year 1 (P=0.02), male donor gender (P=0.03), and recipient CMV antibody positivity (P=0.03) were the only significant predictors of % change in lumen volume (i.e., greater lumen loss). On multivariate analysis using these three independent variables, recipient CMV antibody positivity (P=0.02, coefficient=−8.6), male donor gender (P=0.02, coefficient=−11.8), and lack of calcium channel blocker use (P=0.03, coefficient=8.2) remained significant independent predictors of % change in lumen volume.
A correlation matrix including the variables in Table 1 revealed that recipient CMV antibody positivity at the time of transplantation and aggressive CMV prophylaxis were highly correlated (R=0.78). For this reason these two variables were not included together in the multivariate analysis. After substituting aggressive CMV prophylaxis into the multivariate regression equation for recipient CMV antibody positivity, aggressive CMV prophylaxis was a strong independent predictor of less change in vessel volume (P=0.03, coefficient=9.3) and less change in lumen volume (P=0.03, coefficient=8.4) (Table 3).
In this study we found that negative remodeling is primarily responsible for coronary artery lumen loss during the first year after cardiac transplantation. This change occurs more dramatically in CMV seropositive patients who are given standard prophylaxis compared to seronegative patients who receive more aggressive anti-CMV prophylaxis. Plaque volume does increase significantly during the first year after transplantation however, it occurs most in vessels that undergo the least degree of negative remodeling. The changes in plaque volume do not correlate with changes in lumen volume, but correlate positively with changes in vessel volume.
Anatomic Changes Early After Transplantation
Past IVUS studies early after cardiac transplantation demonstrate conflicting data with respect to changes in coronary artery vessel size and plaque burden and their respective contributions to lumen loss. Studies have consistently revealed increases in plaque burden early after transplantation, but some describe associated positive remodeling with little change in lumen dimensions, whereas others reveal negative remodeling in the most diseased segments with significant lumen narrowing (4–10).
In this study, we found that on average plaque volume increased significantly during the first year after transplantation, whereas vessel volume and lumen volume decreased significantly. This implies that on average lumen loss occurred because of a combination of negative remodeling and intimal thickening. However, because the changes in vessel volume correlated strongly with changes in lumen volume, while changes in plaque volume did not, it appears that the early lumen loss was primarily a result of negative remodeling. Changes in plaque volume correlate positively with changes in vessel volume. This relationship means that positive (compensatory) remodeling occurs in the vessels with the greatest increase in plaque volume, likely as a result of the phenomenon first described by Glagov et al. (16). Thus, intimal thickening contributes less to lumen loss early after transplantation. Very few patients had actual vessel enlargement as a result of positive remodeling, but the degree of negative remodeling was less. Presumably the effect on coronary flow by plaque development results in increased release of nitric oxide and other mediators of positive remodeling, which counteract the propensity for negative remodeling (17).
The discrepancy between these data and other studies may be related to the fact that previous work has used primarily two-dimensional IVUS analysis, which is likely less representative of global vascular change. In addition, in the present study a longer portion of the LAD (50 mm) was analyzed and the three-dimensional reconstruction was obtained from narrowly spaced cross-sectional images (100 images per vessel, or every 0.5 mm).
Studies by Kobashigawa et al. and Tuzcu et al. have shown that maximal intimal thickness is predictive of major cardiovascular events and patient mortality (1, 2). This study did not document a significant increase in maximal intimal thickness when comparing the maximal intimal thickness calculated in one cross-section at baseline and compared to the same cross-section at year 1, but did show a significant increase when comparing the cross section with the maximal intimal thickness at baseline to any cross section with the maximal intimal thickness at year 1. In both of these previous studies, multiple cross-sections were analyzed and the greatest change in maximal intimal thickness was recorded and used to classify patients with or without vasculopathy. It will be important to determine if the changes detected by volumetric analysis in this study are as powerful predictors as change in maximal intimal thickness in prior studies.
Clinical Correlates to Anatomic Changes
CMV infection has been implicated in the development of cardiac allograft vasculopathy, however its exact role remains unclear. Early studies demonstrated that CMV infection was associated with greater degrees of angiographic coronary disease (18). More recently, it has been shown that CMV infection requiring treatment is associated with less positive remodeling early after transplantation (19).
In this study, we extend these findings by showing that CMV seropositive recipients exhibit a significantly greater degree of negative remodeling. Active infection or clinically evident reactivation of CMV only occurred in one patient making it impossible to comment on its relationship to vascular changes. However, patients who received aggressive prophylaxis against CMV infection/reactivation did have significantly less lumen loss and less negative remodeling. Moreover, aggressive CMV prophylaxis was an independent predictor of less lumen loss and less negative remodeling. This finding may partially explain the association between CMV antibody positivity and greater negative remodeling, because seropositive recipients did not receive aggressive prophylaxis. This is in stark contrast to CMV seronegative patients, most of whom did received aggressive prophylaxis, (those who did not, received hearts from CMV antibody negative donors, rendering them at no risk for seroconversion). We did not observe any correlation between CMV DNA levels in peripheral blood leukocytes and negative remodeling in this series of patients, which was suggested in an earlier analysis (14).
CMV infection has been shown to impair endothelial function and lead to worse outcomes (20). Weis et al. demonstrated that CMV infection in cardiac transplant recipients resulted in higher levels of asymmetric dimethylarginine (ADMA), the endogenous inhibitor of nitric oxide synthase (21). Thus, CMV infection might predispose to negative remodeling based on its effects on the nitric oxide synthase pathway.
In this study we also found that treatment with calcium channel blockers was associated with lesser degrees of negative remodeling and lumen loss, supporting earlier work done at Stanford (22). Lastly, male donor gender was associated with greater degrees of lumen loss. Previous reports have suggested that recipients of female donor hearts are at greater risk for adverse events, presumably in part a result of greater cardiac allograft vasculopathy (23). The findings in this study with respect to donor gender should be interpreted with caution as there were only six recipients of female hearts and only two of these recipients were male (i.e., gender mismatched).
Significant coronary structural changes occur during the first year after cardiac transplantation. The unique findings in this study support the notion that progression of intimal thickening or atherosclerotic plaque may not be the most important change during this period. Conversely, we find that lumen volume is affected more by negative remodeling than by intimal thickening. Transplant recipients who are CMV seronegative and/or who have received aggressive CMV prophylaxis have significantly less negative remodeling and lumen loss. When evaluating new therapies to prevent and treat cardiac allograft vasculopathy, it will be important to consider their effects on arterial remodeling as well as plaque progression. It also will be necessary to determine the prognostic importance of negative remodeling, in comparison to increases in plaque volume.
This study involved a small number of patients, typical of studies of this nature. However, the prospective cohort design, extensive clinical characterization, and serial three-dimensional IVUS evaluation are distinct advantages over prior retrospective studies. The lack of randomization to CMV prophylaxis is a limitation. The results of this study may not apply to cardiac transplant recipients who do not receive induction therapy and hence may have a lower likelihood of CMV reactivation/infection. More universal use of angiotensin converting enzyme inhibitor or angiotensin receptor blocker therapy could also affect these results, as these medications have been shown to decrease the incidence of cardiac allograft vasculopathy (24). Larger, randomized studies will be important to confirm these findings.
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