Cardiac allograft vasculopathy (CAV), an accelerated form of coronary disease, is a leading cause of mortality after heart transplant (HTx) accounting for 30% of deaths after transplantation (1). Although there is a wealth of evidence indicating that cellular immune responses play a critical role in CAV development, current immunosuppressive agents, such as calcineurin inhibitors (CNIs), corticosteroids, and purine synthesis inhibitors, have, unfortunately, shown little efficacy in preventing CAV (2). Consequently, novel therapeutic agents targeting CAV are required, and there is considerable interest in everolimus and sirolimus, two proliferation signal inhibitors (PSIs) that have antiproliferative effects on vascular smooth muscle cells and fibroblasts and have been shown to prevent or even reverse intimal growth in experimental models of CAV (3), potentially providing an attractive option for use in clinical practice.
It has been previously demonstrated that treatment of de novo HTx recipients with PSIs is associated with a significant decrease in progression of CAV. Eisen et al. (4) compared the effect of everolimus versus azathioprine (AZA) on CAV progression evaluated by intravascular ultrasound (IVUS) examination in 211 de novo HTx recipients and demonstrated that everolimus significantly reduced the severity of CAV. However, there are no similar trials investigating the effect of PSIs on CAV progression among maintenance HTx recipients, and notably, there is evidence indicating that the underlying pathophysiologic mechanisms may be markedly different in the early versus late stages of CAV development (5, 6). Therefore, the results of PSI trials based on de novo HTx recipients may not be directly applicable to maintenance recipients with established CAV.
We have demonstrated in the recent Nordic Certican Trial in Heart and lung Transplantation (NOCTET) trial that the introduction of everolimus with CNI reduction achieves a significant improvement in renal function in maintenance HTx recipients (7). In this substudy of the NOCTET trial, we used IVUS to evaluate CAV progression among patients treated with everolimus and low-dose CNI therapy versus standard CNI immunosuppression. We also performed parallel assessment of a range of inflammatory markers to assess whether there was any significant interaction between the two treatment regimens, the development of CAV and markers of systemic inflammation.
In total, 190 HTx recipients were included in the NOCTET trial, and 111 patients completed the IVUS substudy with matching evaluable IVUS examinations at baseline and follow-up (Fig. 1). Mean time since HTx was 5.8±4.3 years, and there was no significant difference between the two trial arms (Table 1). Patients treated with everolimus were significantly older than the control group (60.0±9.5 vs. 55.7±11.3 years, P=0.03; Table 1). However, there was no significant difference in baseline characteristics of the 111 patients completing the IVUS substudy when compared with the 190 enrolled HTx recipients. Mean baseline maximal intimal thickness (MIT) for patients completing the IVUS study was 0.06±0.03 mm, and the majority of this study population (58 of 111 [52%]) demonstrated evidence of advanced CAV on inclusion (defined as MIT >0.5 mm ).
Of the 111 patients completing the IVUS substudy, 48 patients had been assigned to receive everolimus in combination with low-dose CNI therapy, whereas 63 patients received standard CNI therapy (controls). The apparent uneven distribution of patients in two treatment groups was attributable to a higher incidence of adverse events and discontinuation in the everolimus group as described previously (7). Further subgroup analysis was performed according to background usage of AZA and mycophenolate mofetil (MMF), and the four subgroups were (1) everolimus+low-dose CNI and AZA (n=16); (2) standard CNI+AZA (n=23); (3) everolimus+low-dose CNI and MMF (n=31); and (4) standard CNI+MMF (n=39). Two patients did not receive AZA or MMF.
Everolimus trough levels remained within target range (3–8 ng/mL) throughout the study, and the mean dose at 12 months was 1.2±0.5 mg/day. From baseline to month 12, mean cyclosporine A trough levels in the everolimus group decreased by 59% when compared with 23% in the control group (P<0.01). Similarly, the decline in mean tacrolimus levels in the everolimus group was 55% when compared with 15% in the control arm (P<0.01).
There was no significant difference in CAV progression according to treatment, with no differences in ΔMIT between the everolimus (n=48) and control (n=63) cohort (Fig. 2 and Table 2). Similarly, there was no significant difference in Δtotal atheroma volume (TAV) and Δpercent atheroma volume (PAV) between the two treatment arms. Given the observed imbalance in recipient age between the two treatment groups, change in IVUS parameters was also evaluated with adjustment for recipient age (included as a covariate in analysis of covariance model) but did not significantly influence the results.
Duration since HTx varied considerably among the study population, and the effect of everolimus on CAV among early versus late HTx recipients was also evaluated by categorization of patients into tertiles (time since HTx <3, 3–7, and >7 years). This revealed no significant effect of everolimus on CAV progression in the early, intermediate, or late stage after HTx as assessed by all three IVUS endpoints (MIT, PAV, or TAV—data not shown).
The Interaction of Everolimus With Concomitant Immunosuppression on CAV
When considering patients established on AZA (n=39), a significantly slower rate of CAV progression was observed among patients treated with everolimus+AZA when compared with standard CNI+AZA (ΔMIT 0.00±0.04 and 0.04±0.04 mm, ΔPAV 0.2%±3.0% and 2.6%±2.5%, and ΔTAV 0.25±14.1 and 19.8±20.4 mm3, respectively; all P<0.05; Fig. 3A). In contrast, when considering patients receiving MMF (n=70), there was a significant increase in CAV progression among patients treated with everolimus+MMF versus CNI+MMF as assessed by ΔMIT (0.06±0.12 vs. 0.02±0.06 mm, respectively, P=0.04) and ΔPAV (4.0%±6.3% vs. 1.4%±3.1%, respectively, P=0.02) but not by TAV measurements (Fig. 3B).
Given this difference in everolimus efficacy according to background usage of AZA or MMF, further comparison of baseline characteristics was performed for the four combinations: everolimus+AZA, CNI+AZA, everolimus+MMF, and CNI+MMF. We found that time since HTx was significantly greater for patients receiving concomitant treatment with AZA versus MMF. Similarly, donor age and high-density lipoprotein levels were significantly lower in patients treated with AZA versus MMF. However, none of these baseline variables were significantly different when comparing everolimus+AZA versus CNI+AZA patients or when comparing everolimus+MMF versus CNI+MMF patients. Hence, no confounding factors seemed to account for the attenuated rate of CAV progression among patients treated with everolimus+AZA when compared with CNI+AZA and the increased rate of CAV progression among patients treated with everolimus+MMF when compared with CNI+MMF.
Only a small number of patients received concomitant therapy with tacrolimus (n=13), and subgroup analysis of these patients revealed no significant difference in CAV progression between patients treated with everolimus versus controls (data not shown).
Immune Marker Profile
We observed no significant difference in change in levels of C-reactive protein (CRP; P=0.41), von Willebrand factor (vWf; P=0.76), vascular cell adhesion molecule (VCAM)-1 (P=0.46), soluble tumor necrosis factor receptor 1 6 (s-TNF-R1) (P=0.67), CCL16 (0.18), and interleukin-8 (P=0.43) when comparing patients treated with everolimus versus standard CNI therapy. However, when patients were stratified according to background usage of AZA versus MMF, we found that patients treated with everolimus+AZA demonstrated a significant decline in levels of CRP and VCAM-1 when compared with their counterparts treated with standard CNI+AZA (Fig. 4A). In contrast, a significant increase in levels of CRP, vWf, and VCAM-1 was noted among patients treated with everolimus+MMF when compared with CNI+MMF (Fig. 4B).
In this multicenter, randomized controlled trial, we have used matched IVUS analysis to demonstrate that conversion to everolimus and reduced CNI exposure does not significantly influence CAV progression during a 12-month follow-up period. However, a significantly reduced rate of CAV progression was evident among patients assigned to everolimus and low-dose CNI where background immunosuppression included AZA, and this was accompanied by a significant decline in CRP and VCAM-1. Conversely, an accelerated rate of CAV progression was observed among MMF patients treated with everolimus and low-dose CNI with a parallel increase in levels of CRP, vWf, and VCAM-1.
Everolimus is a potent immunosuppressive agent that also possesses antiproliferative action that is not limited to the immune system. In a landmark clinical trial, Eisen et al. (4) demonstrated that everolimus initiated with 72 hr after HTx instead of AZA reduced the incidence and severity of CAV at 1-year follow-up. Studies have demonstrated that the incidence and severity of CAV increases with time since HTx, but several lines of evidence indicate that different factors influence CAV development in the early versus late stage after HTx (5, 6). Hence, the results of everolimus therapy based on de novo HTx recipients may not be directly applicable per se to maintenance recipients. Our study included HTx recipients with a mean time of 5.8 years since HTx and demonstrated that conversion to everolimus in combination with reduced CNI exposure does not significantly influence CAV progression. Although no previous trial has investigated everolimus and CAV development among maintenance HTx recipients, there are two previous smaller studies evaluating the effect of a similar PSI agent (rapamycin/sirolimus) among patients with established CAV. Mancini et al. (9) conducted a single-center open label randomized trial where 22 patients with severe CAV were converted from MMF/AZA to rapamycin, and this was shown to attenuate CAV progression as evaluated by a subjective semiquantitative catheterization score. However, the study group also performed IVUS examination among a small subset of patients, and this demonstrated no significant difference in intimal thickening between the two groups (9). Raichlin et al. (10) performed a nonrandomized study in 29 HTx recipients and demonstrated that substituting CNI with sirolimus slowed the progression of CAV assessed by IVUS, but no difference was observed in the subgroup of patients with a transplant evolution time of more than 2 years. Although the latter study (nonrandomized, single-center, and use of sirolimus) may not be directly comparable, we believe that the results are in concordance with our present everolimus study indicating that CAV progression among maintenance HTx recipients is not influenced by therapy with PSIs.
There are several potential explanations for the neutral effect of everolimus on CAV progression in this study when compared with previous everolimus trials with de novo HTx recipients. First, CAV development may not necessarily be a time linear process, and it is possible that everolimus has more opportunity to exert its antiproliferative effects among de novo HTx recipients. Second, histopathologic studies (5) have shown that diffuse fibrous intimal thickening is often observed in early stages of CAV development, whereas focal atherosclerotic plaques and diffuse intimal thickening tend to predominate in later stages, potentially reflecting different triggers of endothelial injury in various stages post-HTx (6, 11). Indeed, the study by Eisen et al. (4) showed that the use of everolimus was associated with reduced intimal thickening together with a lower incidence of acute rejection and fewer episodes of CMV infection, two well-known risk factors for CAV that are not particularly relevant in the setting of maintenance HTx recipients. Third, there is evidence from other clinical trials suggesting that treating established CAV is distinctly different from preventing CAV by early medical intervention. For example, late switch to MMF does not affect the progression of CAV (12), and statin therapy instituted more than 4 years post-HTx does not affect immune activity (13). Our study demonstrates that the same “window of opportunity” may apply to everolimus. Finally, in previous landmark everolimus trials, AZA/MMF were discontinued, but such therapy was continued in this study. Previous trials with everolimus have also employed full-dose CNI therapy, but the increased risk of nephrotoxicity with this combination is an important clinical concern (14), and reduced CNI dosing with everolimus as used in this study is now recommended in consensus guidelines (15). Hence, concomitant immunosuppressive therapy in the current trial is significantly different from previous trials and may limit the direct comparison of our results with previously published findings.
The Interaction of Everolimus With Concomitant Immunosuppression on CAV
Although the design of the current trial only included two treatment arms, subgrouping of the study population according to background immunosuppressive therapy was performed as this was prespecified in the study protocol. Remarkably, an attenuated rate of CAV progression was seen in patients treated with everolimus/AZA versus their counterparts receiving CNI/AZA therapy. In contrast, we observed a significantly accelerated rate of CAV progression among patients treated with everolimus/MMF when compared with CNI/MMF. Moreover, the attenuating effect of everolimus/AZA and the enhancing effect of everolimus/MMF on CAV progression were accompanied by antiinflammatory (everolimus/AZA) and inflammatory (everolimus/MMF) effects of these treatment regimens. We have recently shown an association between CRP and VCAM-1 and parameters of CAV in cross-sectional testing (16), and our findings in this study further suggest a link between inflammation (i.e., CRP) and endothelial cell activation (i.e., VCAM-1 and vWf) and the development of CAV as shown also during longitudinal testing. Furthermore, during everolimus therapy, these inflammatory processes seem to be differently affected depending on background immunosuppression.
AZA and MMF are both purine synthesis inhibitors, but the latter agent is associated with a more favorable side-effect profile because of its selective effect on lymphocytes (17), and several trials have established that CNI-based immunosuppression regimens with MMF instead of AZA is associated with lower rates of acute rejection (18) and improved survival (19). Furthermore, the inhibitory effect of MMF on smooth muscle cells and fibroblast proliferation in the vascular wall is relevant for CAV progression and confers important advantages on MMF in comparison with AZA (20, 21). Nevertheless, our data reveal a beneficial role of AZA+everolimus supported by evidence of decreased inflammatory activity when compared with an opposite pattern with MMF+everolimus. Most previous everolimus trials have replaced MMF or AZA with everolimus, and to the best of our knowledge, there are no previous large randomized trials investigating the effect of everolimus as an add-on antiproliferative agent. Although speculative, our surprising results could, therefore, potentially reflect hitherto unknown interactions between AZA+everolimus and everolimus+MMF and warrants further investigation. Furthermore, although multivariate analysis was performed to correct for differences in baseline characteristics, the confounding influence by other less established risk factors for CAV cannot be excluded. For example, it has been demonstrated that cyclosporine A-based maintenance immunosuppression containing MMF instead of AZA seems to be associated with a higher incidence of nonlethal viral infections (20, 22), which again potentially could affect proliferation of smooth muscle cells and endothelial cell function. Such less-studied factors may also be relevant in the setting of combined usage of the two antiproliferative agents MMF and everolimus. Nonetheless, although the mechanisms are not clear, our results should encourage further studies on the interactions between the different immunosuppressive drugs that are used in HTx recipients.
This study had a 12-month follow-up period, and the observed increase in MIT was of a smaller magnitude than the established cutoff level (MIT increase >0.5 mm) associated with a significantly increased risk of all-cause death and myocardial infarction (23). The clinical significance of the level of intimal thickening found in this study is not fully clear and requires cautious interpretation pending studies with a longer follow-up duration. Another potential limitation of this study is the higher discontinuation rate observed among patients treated with everolimus because of the occurrence of serious adverse events (details previously published in Ref. 7).
We only used IVUS data from the patients who had undergone immune marker profiling when correlating CAV development with immune marker changes. However, lack of immune marker data for the entire study population is a potential limitation. Furthermore, in the NOCTET study, we hypothesized that the combined use of everolimus and AZA/MMF may allow effective CNI minimization in maintenance HTx recipients with parallel improvement in renal function and allograft vasculopathy. Although this strategy has shown promising results in trials exploring CNI elimination protocols (24), most clinicians considering CNI minimization strategies in HTx recipients will generally substitute everolimus for AZA or MMF, not continue both. Hence, the design of this study may confound the true effect of everolimus and limits comparison with previous trials.
This study demonstrates that conversion to everolimus and reduced CNI exposure does not significantly influence CAV development and inflammation among maintenance HTx recipients. However, background immunosuppressive therapy seems to be of importance as AZA+everolimus patients demonstrated attenuated CAV progression and a significant decline in levels of CRP and VCAM-1. Conversely, everolimus+MMF therapy was associated with accelerated CAV progression and a parallel increase in levels of CRP, vWf, and VCAM-1. The different effect of everolimus when combined with AZA versus MMF could potentially reflect hitherto unknown interactions. However, these findings require validation in a prospective trial of everolimus where patients are also randomized to AZA or MMF as concomitant background immunosuppressive medication.
MATERIALS AND METHODS
The NOCTET trial was a 12-month, open-label, multicenter, randomized, controlled study undertaken at transplant centers in Scandinavia, in which maintenance heart and lung transplant patients (n=282) were randomized to continue their current immunosuppressive regimen or start everolimus therapy with a predefined reduction in CNI exposure. Detailed accounts of the design, enrollment, and patient characteristics of NOCTET trial have been reported previously (7). This study was a prospectively designed substudy of the main NOCTET trial with CAV progression evaluated by IVUS as the primary endpoint and markers of inflammation as the secondary endpoint.
Of the 190 HTx recipients enrolled in the NOCTET study, 164 patients consented to IVUS examination (Fig. 1). Adverse events or consent withdrawal led to study discontinuation in 23 everolimus patients and 7 control patients with pneumonia and pulmonary embolism being the most common serious adverse event. A detailed description of all adverse events leading to discontinuation in the NOCTET trial has been reported previously (7). IVUS recordings for 23 patients could not be analyzed because of technical error or nonmatching segments. Hence, 111 patients completed the IVUS substudy with matching IVUS recordings at baseline and follow-up.
Written informed consent was obtained from all patients following institutional review board approval, and the study was carried out in accordance with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Harmonized Tripartite Guidelines for Good Clinical Practice, applicable local regulations and the Declaration of Helsinki.
IVUS examination of the same major coronary epicardial artery (preferentially the left-anterior descending coronary artery) was performed at baseline and after 12 months. After intracoronary administration of 200 μg nitroglycerin, imaging was performed using a 20 MHz, 2.9F, monorail electronic Eagle Eye Gold IVUS catheter (Volcano Corporation Inc, CA). IVUS images were acquired at a rate of 30 frames/sec and a pullback speed of 0.5 mm/sec resulting in 1-mm intervals between every 60 frames.
All IVUS analysis was conducted after trial closure by an external core laboratory (Cardialysis, Rotterdam, The Netherlands) blinded to patient treatment. Precise matching of the baseline and 12-month IVUS recordings was performed, and contour detection of both the lumen and external elastic membrane (EEM) was performed at 1-mm intervals using validated software (Curad, Wijk bij Duurstede, The Netherlands). Borders were reviewed by two independent operators according to the guidelines for acquisition and analysis of IVUS images by the American College of Cardiology and European Society of Cardiology (8).
The primary objective of IVUS analysis was to evaluate the mean change in MIT. This endpoint has been shown to be a powerful predictor of all-cause mortality, myocardial infarction, and angiographic abnormalities among HTx recipients (23, 25). In accordance to established guidelines, the largest distance from the intimal leading edge to the EEM was defined as MIT (8). The secondary efficacy variable was specified as TAV and PAV, which expresses the summation of atheroma areas in proportion to the EEM area using the equation: PAV=∑(EEMarea−Lumenarea)/∑EEMarea×100. The intraobserver variability for all IVUS measurements was less than 3%, whereas the interobserver variability was less than 5%.
Inflammatory Marker Analysis
In total, 58 (all enrolled at Oslo University Hospital) of the 111 patients underwent plasma sampling by standard venepuncture before IVUS examination. Plasma levels of sTNF-R1, interleukin-8, CXCL16, and VCAM-1 were measured by enzyme immunoassays obtained from R&D Systems (Minneapolis, MN). Plasma levels of CRP and vWf were measured by enzyme immunoassays as described previously (26, 27). All intraassay and interassay coefficients of variance were less than 10%.
Analyses were performed with the SPSS version 15.0 statistical software (SPSS Inc. Chicago, IL). Data are expressed as mean±standard deviation or as median (interquartile range) as appropriate, and a two-tailed P value less than 0.05 was considered statistically significant. Baseline characteristics were compared between the two treatment groups using Student's t test if normally distributed variables, Mann-Whitney U test if not normally distributed variables, and Fisher's exact test on categorical variables. Within group comparison of change in IVUS endpoints from baseline to 12-month follow-up were compared using a paired t test. When three or more independent groups were compared at one time point, a one-way analysis of variance was used to test for trend. Analysis of covariance was used to compare changes in IVUS endpoints between treatment groups with the baseline IVUS value, time since HTx, and recipient age included as covariates and treatment group as a fixed factor. The changes in immune markers between treatment groups were compared using the same statistical method.
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