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Influence of cytomegalovirus infection in the development of cardiac allograft vasculopathy after heart transplantation
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Influence of cytomegalovirus infection in the development of cardiac allograft vasculopathy after heart transplantation Juan F. Delgado, MD, PhD,a Ana García Reyne, MD, PhD,b Santiago de Dios, MD,a Francisco López-Medrano, PhD,b Alfonso Jurado, MD,a Rafael San Juan, MD, PhD,b María José Ruiz-Cano, MD,a M. Dolores Folgueira, MD, PhD,c Miguel Ángel Gómez-Sánchez, MD, PhD,a José María Aguado, MD, PhD,b and Carlos Lumbreras, MD, PhDb From the aCardiology Department; bInfectious Disease Unit; and the cMicrobiology Department, University Hospital 12 de Octubre, Instituto de Investigación Hospital 12 de Octubre, Madrid, Spain.
KEYWORDS: cardiac allograft vasculopathy; heart transplantation; cytomegalovirus infection; calcium channel blocker; cardiac angiography
BACKGROUND: Cardiac allograft vasculopathy (CAV) is a major cause of long-term morbidity and mortality after heart transplantation (HTx), whose relationship with CMV infection is uncertain. This study evaluated the influence of CMV infection in the development of CAV. METHODS: We enrolled 166 consecutive HTx recipients who underwent their first transplant from January 1995 to July 2002. All patients received 14 days of intravenous ganciclovir and were prospectively monitored for CMV infection during the first year after HTx. CAV was diagnosed by coronary angiography performed at 1, 5, and 10 years after HTx, following the new criteria of the International Society for Heart and Lung Transplantation. We collected all variables potentially related with the development of CAV. Risk factors were studied using a complementary log-log model. RESULTS: After a median follow-up of 11 years (range, 1–17 years), 72 patients (43%) developed CAV (63.8% CAV1, 15.2% CAV2, 20.8% CAV3). Symptoms secondary to CAV were present in 32% of these patients, and 8% died because of it. In the regression multivariate analysis, independent variables associated with the development of CAV were donor age (hazard ratio [HR], 1.028; 95% confidence interval [CI], 1.002–1.053; p o 0.028), presence of cellular acute rejection Z 2R (HR, 1.764; 95% CI, 1.011–3.078; p o 0.0414), CMV infection (HR, 2.334; 95% CI, 1.043– 5.225; p o 0.0354), and not having been treated with a calcium channel blocker (HR, 0.472; 95% CI, 0.275–0.811; p o 0.0055). CONCLUSIONS: Standardized angiographic criteria show CMV infection is associated with the development of CAV. J Heart Lung Transplant 2015;34:1112–1119 r 2015 International Society for Heart and Lung Transplantation. All rights reserved.
Cardiac allograft vasculopathy (CAV) remains the Achilles heel of heart transplantation (HTx). Current prevalence of Reprint requests: Juan F. Delgado, MD, PhD, Heart Failure and Transplant Unit, Cardiology Department, University Hospital 12 de Octubre, Av. de Córdoba sn., 28041 Madrid, Spain. E-mail address: [email protected]
CAV among HTx patients is 20% at 3 years, 30% at 5 years, and 45% at 8 years after HTx.1 According to the International Society for Heart and Lung Transplantation (ISHLT) Registry, CAV is one of the most important causes of death after HTx, accounting for 10% to 13% of deaths occurring more than 1 year after HTx and may be present in patients who die after the first year due to “graft failure” (16% to 27%).1
1053-2498/$ - see front matter r 2015 International Society for Heart and Lung Transplantation. All rights reserved. http://dx.doi.org/10.1016/j.healun.2015.03.015
Delgado et al.
CMV Infection and CAV After HTx
CAV is a long-term complication of HTx, but most studies of the pathogenesis of CAV are hampered by a very short follow-up and the lack of uniform criteria in its definition. CAV is detected mainly by coronary angiography,2 which is the best screening tool to detect the presence of CAV. However, the different angiographic definitions used make it impossible to draw solid conclusions regarding the influence of a specific factor in the development of CAV. The recently published standardized nomenclature for CAV by the ISHLT will be an important step forward to allow a better comparability between studies3 and a useful tool for prognostic stratification after HTx.4 Several factors have been linked with the development of CAV, including donor and recipient characteristics and post-HTx complications.1 In particular, cytomegalovirus (CMV) infection has been occasionally linked with CAV as a part of the so-called indirect effects in HTx recipients.5 However, a recent meta-analysis of risk factors associated with the development of CAV did not find a conclusive association between CMV infection and CAV.6 Thus, the main objective of our study was to evaluate the influence of CMV infection and other risk factors for the development of CAV.
Methods The Local Ethical Committee approved this investigation.
Study design A single-center, retrospective, observational study from prospectively collected data was designed to evaluate the relationship between CMV infection and CAV. We conducted this study by following the recommendations for observational studies of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement.7
Study population Consecutive patients who received their first HTx from January 1995 to July 2002 and survived at least 1 year were included in the study. Patients were excluded when they underwent repeat HTx, had an inadequate CMV sampling (o90% of the protocolized samples), or did not have an adequate coronary angiographic study. Follow-up continued until death, retransplantation, or until followup ended in December 31, 2011.
Immunosuppressive therapy Induction immunosuppressive therapy included OKT3 (5 mg/day for 14 days) or, after June 2002, 2 doses of basiliximab (20 mg) on Days 1 and 4 after surgery. Methylprednisolone was administered at 500 mg intravenously before surgery and at 125 mg intravenously every 8 hours for 3 doses after the operation, followed by prednisone at 1 mg/kg/day orally, tapered by 0.1 mg/kg on alternate days to 0.2 mg/kg/day and reduced to 0.1 mg/kg/day after 1 year. Azathioprine was administered at 2 mg/kg/day orally. After January 2002, azathioprine was substituted by mycophenolate mofetil (2–3 g/day). As a calcineurin inhibitor we used
1113 cyclosporine A (5 to 8 mg/kg/day) to maintain serum cyclosporine A levels within the range of 250 to 350 ng/ml during the first year and from 100 to 200 ng/ml for the second year and all following.
CMV diagnosis and CMV-related definitions Fourteen CMV anti-genemias were collected during the first 6 months after HTx (Monofluo kit CMV, BioRad, Marnes-la Coquette, France). It was measured in number of total leukocytes pp65 positive/200,000 leukocytes). CMV infection was defined as evidence of CMV replication regardless of symptoms, upon the sole presence of a positive antigenemia test. CMV disease was defined as evidence of CMV infection with attributable symptoms. CMV disease can be further categorized as a viral syndrome with fever, malaise, leukopenia, and thrombocytopenia or as a tissue-invasive disease.8,9 Asymptomatic viremia was diagnosed when a positive antigenemia test was not accompanied by any clinical symptom. Patients with asymptomatic viremia were compared only with patients without CMV infection, and therefore, patients with CMV disease were excluded from this analysis. We defined a “high CMV viremia” if 3 or more consecutive or non-consecutive anti-genemia tests showed more than 10 cells/ 200,000 leukocytes.
Antiviral therapy All patients, irrespective of their CMV serostatus, received universal CMV prophylaxis with intravenous ganciclovir (5 mg/kg/day) during the first 14 days after HTx as the only anti-CMV prophylactic therapy. Preemptive therapy was not performed during the follow-up, but patients with asymptomatic viremia were monitored closely for signs and symptoms of CMV disease, and when present, intravenous ganciclovir treatment for 14 to 21 days was administered.
Coronary angiographic studies CAV diagnosis was done through a retrospective review of all coronary angiographic and echocardiographic studies performed in every patient by protocol at 1, 5 and 10 years after HTx, following standardized ISHLT nomenclature.3 Coronary angiography was performed using standard techniques after pre-treatment with nitroglycerine. Two expert cardiologists, blinded to the clinical course of patients, examined all angiograms. We defined the presence of CAV as a status Z CAV1.
Risk factor analysis Risk factor data were collected by a retrospective review. More than 100 variables were collected as potential risk factors for CAV, including recipient and donor characteristics, immunosuppression, specific treatments, and complications (acute rejection episodes, severe infections different than CMV, and malignancies). We defined severe infections other than CMV as all bacterial, viral, and fungal infections that needed hospitalization or intravenous anti-biotic treatment and also infections caused by varicella-zoster virus. In addition, we collected classical cardiovascular risk factors before HTx and at 1, 5 and 10 years after HTx, including diabetes, hypertension, smoking status, hypercholesterolemia (defined as cholesterol 4 220 mg/dl) and hypertriglyceridemia (triglyceride
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value 4 200 mg/dl). The real effect of hypertension was difficult to measure after HTx because most patients were receiving antihypertensive drugs, so we measured the effect of anti-hypertensive drugs including angiotensin-converting enzyme inhibitors or calcium channel blockers (CCBs). The same occurred with hypercholesterolemia analysis, so we also analyzed the use of statins. Acute cellular rejection (ACR) surveillance was standardized and performed in all patients. Endomyocardial biopsy specimens were graded according to the 2005 ISHLT classification as 1R, 2R, and 3R.10 We included in the analysis all ACR episodes during the first year after transplantation. Patient survival, CAV-related death, and non-fatal major adverse cardiovascular events (NF-MACE) were analyzed. NFMACE included acute myocardial infarction, congestive heart failure, need for percutaneous cardiac intervention, coronary artery bypass grafting, cardiac defibrillator placement, cerebral strokes, and peripheral vascular disease.11
Statistical analysis The patients’ data were prospectively sampled and stored in a computerized database. They were summarized by descriptive statistics that characterized continuous variables by mean ⫾ standard deviation and categoric variables by proportions. As a first step, a univariate analysis was performed of the association between single putative risk factors and the presences of CAV during follow-up. The chi-square test or Fisher’s exact test for qualitative variables and Student’s t-test or Wilcoxon’s rank test for quantitative variables were used. Because CAV is assessed at discrete time points (1, 5, and 10 years), risk factor analysis (univariate and multivariate) was done using complementary log-log model for interval-censored survival times. This model is analogous to the Cox proportional hazards model with continuous time and is often used to estimate incidence rates at discrete time points while taking into account differential patient follow-up. All continuous and categoric variables that were significant in the univariate analysis with a p o 0.1 were subsequently entered into multivariate analysis. Predicted survival distribution of CAV for CMV was also done using complementary log-log model and was plotted on a graph. We also estimated by logistic regression the predicted probability of developing CAV during the 10 years of follow-up in relation with the intensity of CMV viremia (“high degree viremia”) and we also plotted it on a graph. Statistical significance was defined as a p-value of o0.05. We used SAS/STAT 9 software.
Results Overall, 213 patients received their first HTx at our institution during the study period. We excluded 4 patients who underwent re-HTx, 31 who died during the first year after HTx, 5 whose CMV sampling was inadequate, and 7 without angiographic studies due to vascular access problems. Thus, we finally included 166 HTx recipients. Table 1 describes the main baseline characteristics of the cohort. Most patients received induction therapy with OKT3 and triple therapy with cyclosporine, azathioprine, and prednisone. Median follow-up was 11 years, with an overall mortality of 37%. A NF-MACE event developed in 16% of patients during follow-up.
Table 1
Heart Transplant Baseline Characteristics
Characteristics Age, mean ⫾ SD years Male sex, No. (%) Cytomegalovirus serology, No. (%) Donor positive/recipient negative Donor positive/recipient positive Donor negative/recipient positive Donor negative/recipient negative Immunosuppression, No. (%) Induction therapy OKT3 Basiliximab Maintenance therapy Cyclosporine and steroids Azathioprine Mycophenolate mofetil Follow-up, median (min–max) years NF-MACE, No. (%) Mortality, No. (%) General CAV-associated
Value (N ¼ 166) 53.03 ⫾ 12.47 99 (71) 16 (11.6) 83 (60.1) 34 (24.6) 5 (3.5) 166 (100) 161 (96.9) 5 (3.01) 166 (100) 130 (78.3) 36 (21.6) 11 (1–17) 27 (16.3) 61 (36.7) 6 (3.6)
CAV, cardiac allograft vasculopathy; NF-MACE, non-fatal major adverse cardiovascular events; SD, standard deviation.
We analyzed 394 coronary angiographic studies: 166 in the first year after HTx, 126 in the fifth year, and 102 in the tenth year. The reasons for the absence of a coronary angiography in patients who were alive in each study period were medical or surgical conditions that discouraged the procedure (n ¼ 14), severe peripheral vascular disease (n ¼ 8), and patient refusal (n ¼ 8). In 8 patients we did not perform angiography at the fifth year but it was performed in the tenth year and was included in the analysis. As expected, CAV presence increased during the followup. Some degree of CAV was present in 6.6% of studies in the first year, in 25% in the fifth year, and in 57% in the tenth year after HTx (Figure 1). At the end of the follow-up, CAV developed in 72 patients (43%): CAV1 (mild) in 46 patients (63.8%), CAV2 (moderate) in 11 patients (15.2%), and CAV3 (severe) in 15 patients (20.8%). The comparison between the 72 patients who developed CAV during follow-up and the 94 who did not is reported in Table 2. Pre-HTx CMV serostatus did not correlate with the development of CAV. CMV infection and disease were significantly more frequent in patients with CAV and in patients who had the previously defined “high-degree CMV viremia.” The mean value of the area under the curve of CMV viremia was also higher in those patients who developed CAV. The univariate analysis (Table 3) showed that several factors were statistically associated with the development of CAV, including recipient body mass index, donor age, CMV infection, CMV disease, high degree of CMV viremia, ACR Z 2R, hypertriglyceridemia, with the administration of CCB during follow-up a protective factor.
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CMV Infection and CAV After HTx
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Figure 1 Result of coronary angiographies in the first, fifth, and tenth year after heart transplantation. CAV, cardiac allograft vasculopathy; ISHLT, International Society of Heart and Lung Transplantation.
For the multivariate analysis we made different models in which we included 1 CMV-related variable and up to 6 other variables that were statistically significant in the univariate analysis. As reported in Table 4, CMV infection, Table 2
CMV disease, and CMV asymptomatic viremia were significantly associated with the development of CAV. Another risk factor associated with the development of CAV in all of the models was the administration of a CCB. Donor
Comparisons Between Patients With and Without Cardiac Allograft Vasculopathy No CAV
CAV
Variables
(n ¼ 94)
(n ¼ 72)
Recipient age, mean ⫾ SD years Recipient BMI, mean ⫾ SD kg/m2 CAD before HTx, No (%) Donor age, mean ⫾ SD years CMV serology Dþ/R–, No. (%) ACR at first year, No. (%) ACR Z 2R at first year, No. (%) Severe infections different than CMV, No. (%) Severe infections, mean ⫾ SD No. CMV infection, No. (%) Asymptomatic viremia CMV disease High-degree CMV viremia Other risk factors, No. (%) At first year Calcium channel blocker Statin treatment Hypertriglyceridemia At fifth year Calcium channel blocker Statin treatment Hypertriglyceridemia At tenth year Calcium channel blocker Statin treatment Hypertriglyceridemia
54.73 ⫾12.5 23.68 ⫾ 3.5 50 (52.6) 25.36 ⫾ 8.48 8 (12.9) 65 (69.1) 12 (12.98) 54 (57.4) 1.42 ⫾ 1.53 71 (75.5) 60 (72.3) 11 (11.7) 15 (16)
50.80 ⫾ 12.08 24.85 ⫾ 3.54 39 (54.9) 29.89 ⫾ 10.49 6 (13) 59 (81.9) 22 (30.6) 36 (50) 1.3 ⫾ 1.768 64 (88.8) 41 (83.7) 23 (31.9) 22 (30.6)
0.044 0.037 0.769 0.002 0.983 0.06 0.05 0.253 0.633 0.029 0.136 0.001 0.025
85 (90.4) 51 (54.3) 5 (5.3)
52(72.2) 41 (56.9) 8 (11.1)
0.002 0.71 0.166
76 (80.9) 55 (58.5) 5 (5.3)
51 (70.8) 54 (75) 9 (12.5)
o0.0001 0.191 0.06
50 (53.2) 54 (57.4) 5 (5.3)
35 (48.6) 49 (68.1) 18 (25)
0.013 0.47 0.005
p-value
ACR, acute cellular rejection; BMI, body mass index; CAD, coronary artery disease; CAV, cardiac allograft vasculopathy; CMV, cytomegalovirus; D, donor; HTx, heart transplant; R, recipient; SD, standard deviation.
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Table 3 Univariate Analysis for Risk Factors Associated With Cardiac Allograft Vasculopathy Risk factor
HR
95% CI
p-value
Recipient age Recipient BMI Coronary artery disease before HTx Donor age ACR at first year ACR Z 2R at first year CMV infection CMV asymptomatic viremia CMV disease High-degree CMV viremia Diabetes mellitus after HTx Hypertriglyceridemia Treatment with Calcium channel blocker ACE inhibitor Statin
1 1.081 1.180
0.978–1.014 1.009–1.157 0.732–1.901
0.6524 0.0240 0.4885
1.035 1.57 1.730 2.352 1.92 2.258 1.718 1.322 2.070
1.010–1.060 0.845–2.895 1.021–2.932 1.108–4.991 0.885–4.171 1.343–3.795 1.023–2.886 0.783–2.259 1.088–3.924
0.0043 0.1458 0.0375 0.0230 0.092 0.0017 0.0367 0.2824 0.0237
0.390 1 0.910
0.235–0.656 0.604–1.646 0.527–1.587
0.0003 0.9910 0.7451
ACE, angiotensin-converting-enzyme; ACR is acute cellular rejection; BMI, body mass index; CI, confidence interval; CMV, cytomegalovirus; HR, hazard ratio; HTx, heart transplant.
age and cellular acute rejection during first year after HTx were not always associated with CAV in the different multivariate models. If we analyzed the probability of developing CAV during follow-up, those patients with CMV infection had a statistically higher probability of developing CAV than patients without CMV infections. Thus, 10 years after transplantation, only 35% of patients with CMV infection were CAV-free compared with 64% of patients without CMV infection (Figure 2). We also analyzed the effect of CMV on the severity of CAV. As can been seen in Table 5, CAV moderate to severe (CAV2 or higher), following the ISHLT score, was more frequent in patients with CMV disease and with a higher degree of anti-genemia, but this difference did not reach statistical significance. High-degree CMV viremia was not seen as statistically significant in the multivariate analysis. However, regarding the probability of developing CAV, it exhibited a lineal relationship with the development of CAV, suggesting that a high CMV viremia may increase the probability of CAV development (Figure 3).
Discussion Our study shows that CMV infection, including asymptomatic replication, is an independent risk factor associated with the development of CAV. We were able to demonstrate it through a special scenario that occurred during the interval we chose for the study: a homogeneous cohort who received intense immunosuppressive therapy, a short course of intravenous ganciclovir prophylaxis, standardized blood sampling to search for CMV replication, and a long-term angiographic follow-up.
Of course, our intention was not to generalize these data with what is currently occurring in HTx programs with a different immunosuppressive therapy and more potent antiviral prevention strategies. However, our data show that CMV infection and disease is definitely associated with CAV when the new, more sensitive ISHLT3 score is used that takes into account milder grades of CAV. This finding is useful for our current transplantation practices because it emphasizes the importance of detecting and avoiding CMV replication to improve the long-term outcome of HTx recipients. The current paradigm for the development of CAV includes the exposure of coronary graft vessels to immune and non-immune pathogenic factors that induce endothelial cell injury, leading to intimal hyperplasia and vascular remodeling.2,12 There are abundant data suggesting that infections may play a role in atherosclerosis, and CMV is one of the pathogens that has been most convincingly implicated.13–16 Several mechanisms have been evoked to explain this association. CMV has been shown to directly infect human vessels,17,18 leading to smooth muscle cell proliferation and migration, uptake of oxidized low-density lipoprotein, release of cytokines and chemokines, and increased procoagulant activity by endothelial cells.19–22 Alternatively, CMV may induce vascular lesions without direct invasion. Viral antigens can trigger an immune response that crossreacts with self-peptides expressed on the vascular wall.19 Finally, CMV is a driver of age-associated immune changes, which lead to a reduction of naive T cells, an expansion of terminally differentiated T cells, and an increase in proinflammatory cytokines.23 The data supporting a role for CMV infection in the pathophysiology of CAV is based on epidemiologic and observational data,24 experimental models,25–27 and therapeutic trials.15,28,29 However, available evidence has not been consistent for different reasons: a limited number of patients, studies handicapped by important heterogeneity, inadequate (usually too-short) follow-up, and heterogeneous definitions of risk factors, CAV, and CMV-related events. Actually, a recent systematic review of observational studies concluded that there is no evidence of a relationship between CMV and CAV.6 More recently, however, the association between CMV replication and atherosclerotic events was proven in kidney transplant patients,14 and previous studies in HTx patients have suggested that the suppression of viral replication could prevent vascular damage.30 Our study shows that not only CMV disease but also asymptomatic viral replication is likewise an independent risk factor. We were not able to probe in the multivariate analysis an independent association between elevated viremia and the development of CAV. However, as is shown in Figure 3, there was a lineal relationship between the degree of CMV viremia and the development of CAV. Our data may have important implications in the clinical management of CMV infection in HTx patients because it strongly suggests that avoiding CMV viremia after HTx might reduce the incidence of CAV. So, in this context,
Delgado et al.
CMV Infection and CAV After HTx
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Table 4 Models of Multivariate Analysis That Include One Way of Measuring Cytomegalovirus Infection and Other Risk Factors Associated With Cardiac Allograft Vasculopathy Multivariate analysis Model Model 1 CMV infection Acute rejection Z 2R in the first year Recipient BMI Donor age Calcium channel blocker treatment Model 2 CMV disease Acute rejection Z 2R in the first year Recipient BMI Donor age Calcium channel blocker treatment Model 3 CMV asymptomatic viremia Acute rejection Z 2R in the first year Hypertriglyceridemia Donor age Calcium channel blocker treatment
HR
95% CI
p-value
2.334 1.764 1.066 1.028 0.472
1.043–5.225 1.011–3.078 0.991–1.148 1.002–1.053 0.275–0.811
0.0354 0.0414 0.0816 0.0287 0.0055
1.755 1.337 1.069 1.024 0.457
1.011–3.046 0.695–2.572 0.993–1.152 0.998–1.050 0.267–0.784
0.0414 0.3741 0.0706 0.0606 0.0037
2.428 1.431 2.008 1.033 0.416
1.013–5.819 0.682–3.002 0.0865–4.665 1.002–1.064 0.220–0.786
0.0468 0.3331 0.0980 0.0337 0.0059
BMI, body mass index; CI, confidence interval; CMV, cytomegalovirus; HR, hazard ratio.
universal prophylaxis may be a preferable option to preemptive therapy in the management of CMV infection after HTx.30 Nevertheless, only a long-term randomized study comparing universal prophylaxis with preemptive therapy, in which the appearance of CAV would be the primary end point, would confirm this hypothesis. Another significant finding that has come from our study is the protective effect of CCB, mostly diltiazem, in the development of CAV. During the study period, diltiazem was applied to counteract the adverse effects of conventional immunosuppression, namely, hypertension and nephrotoxicity.31 Previous studies have reported that diltiazem slows the development of CAV32 and reduces the incidence of acute rejection.33–35 Moreover, studies with heterotopic
HTx models have shown that diltiazem exerts immunosuppressive and immunomodulating effects. Transmembrane calcium movement plays a critical role in lymphocyte function, and numerous investigators have demonstrated that diltiazem inhibits lymphocyte functions and depresses the immune response.36–38 We speculate whether through any of these mechanisms diltiazem may explain the protective effect against CAV that we have found in our analysis. Other risk factors associated with CAV found in our study were donor age and acute rejection. Donor age has been linked to CAV in different studies.6 Instead, the effect of acute rejection in the appearance of CAV remains controversial.6 In our study, only severe acute rejection seems to be related with the development of CAV.13 A direct causative association of CAV and the observed risk-factors cannot be definitely established because of the retrospective nature of our study. Rejection and CMV infection are known Table 5 Comparison Between Patients With Cardiac Allograft Vasculopathy Grade o 2 and Patients With Cardiac Allograft Vasculopathy Z 2 Following International Society for Heart and Lung Transplantation Criteria According to Cytomegalovirus Infection
Figure 2 Probability of cardiac allograft vasculopathy (CAV)free status during the follow-up in patients with and without cytomegalovirus (CMV) infection done by complementary loglog model.
CAV o 2
CAV Z 2
Variable
(n ¼ 140) No. (%)
(n ¼ 26) No. (%)
p-value
CMV infection Asymptomatic viremia CMV disease High-degree CMV viremia
113 88 25 28
22 13 9 9
0.639 0.996 0.052 0.1
(80.7) (76.5) (17.9) (20)
(84.6) (76.5) (34.6) (34.6)
CAV, cardiac allograft vasculopathy; CMV, cytomegalovirus.
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Figure 3 Predicted probability of cardiac allograft vasculopathy (CAV) development in relation with “high degree antigenemia” done by logistic regression.
to be related; however, rejection and CMV infection both appeared in as independent risk factors for CAV in the different multivariate statistical models we performed. Other factors that have been identified in the literature as CAV risk factors are donor factors, recipient characteristics, use of a ventricular assistance device, or immunosuppressive treatment,12 but these were not related with CAV in our study. Nor did the use of statins39 or angiotensin-converting enzyme inhibitors40 show a protective effect against CAV in our analysis. Our study has some limitations. First, are those that are inherent to a retrospective single-center study, which have reduced external validity and are less generalizable because of potential differences in the management of patients. Nonetheless, in our study, the key variables were collected prospectively and a strict protocol was followed. However, a well-designed prospective study would be needed to define whether CMV contributes directly to the atherosclerotic process in the allograft or instead serves as a marker of immune dysregulation or ongoing inflammation in the HTx population. We used CMV anti-genemia to measure CMV viremia instead of CMV polymerase chain reaction, which was not available in our institution during the study period. The use of a more sensitive and standardized test that is, also, a better tool to quantify CMV viremia would be helpful in better defining the importance of the degree and duration of CMV viremia in the development of CAV. Our data were obtained in a cohort of patients who received intense immunosuppressive therapy with OKT3, which it is no longer used in this setting. We cannot rule out that the influence of CMV infection in the development of CAV in HTx patients may be different when another type of immunosuppression is used. From a statistical viewpoint, we analyzed more than 100 variables influencing CAV development in the risk factor analysis, so we cannot rule out a type I error.
Finally, a comparison of our data with those obtained in a similar analysis including more recent patients receiving modern immunosuppression and more potent anti-viral therapy would reinforce our main findings. We are actually collecting these data from our newer cohort with those patients who received HTx from 2002, but a significant larger follow-up is still needed from this second cohort. Moreover, this comparison would only show whether our current transplantation practices are able or not to eliminate the deleterious effect of CMV in the development of CAV, but not questioning our main findings. In conclusion, following the new ISHLT normalized criteria for the diagnosis of CAV, we have found that CMV disease and asymptomatic CMV viremia are independently associated with the development of CAV in HTx recipients. However, the post-HTx use of CCBs seems to protect against CAV development. Further studies are needed to show if the use of a more aggressive anti-CMV prophylaxis may prevent the appearance of CAV in HTx recipients.
Disclosure statement None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose. The authors are grateful to David Lora-Pablos (Research Institute, Clinical Research Unit, University Hospital 12 de Octubre) for the statistical analyses and to Martin J. Smyth, BA, for revision of the English version of this manuscript. This study was partially supported by the Spanish Cardiovascular Research Networks RIC (Instituto de Salud Carlos III, Ministerio de Economía y Competitividad) and by REIPI (Red Española de Enfermedades Infecciosas). A.G.-R. was supported by a grant from the Instituto de Salud Carlos III (Rio Hortega, Grant No. CN09130).
References 1. Stehlik J, Edwards LB, Kucheryavaya AY, et al. The Registry of the International Society for Heart and Lung Transplantation: 29th official adult heart transplant report—2012. J Heart Lung Transplant 2012;31:1052-64. 2. Schmauss D, Weis M. Cardiac allograft vasculopathy: recent developments. Circulation 2008;117:2131-41. 3. Mehra MR, Crespo-Leiro MG, Dipchand A, et al. International Society for Heart and Lung Transplantation working formulation of a standardized nomenclature for cardiac allograft vasculopathy-2010. J Heart Lung Transplant 2010;29:717-27. 4. Prada-Delgado O, Estevez-Loureiro R, Paniagua-Martin MJ, LopezSainz A, Crespo-Leiro MGP. revalence and prognostic value of cardiac allograft vasculopathy 1 year after heart transplantation according to the ISHLT recommended nomenclature. J Heart Lung Transplant 2012;31:332-3. 5. Perez-Sola MJ, Caston JJ, Solana R, Rivero A, Torre-Cisneros J. Indirect effects of cytomegalovirus infection in solid organ transplant recipients. Enferm Infecc Microbiol Clin 2008;26:38-47. 6. Braga JR, Santos IS, McDonald M, Shah PS, Ross HJ. Factors associated with the development of cardiac allograft vasculopathy—a systematic review of observational studies. Clin Transplant 2012;26: E111-24. 7. Vandenbroucke JP, von Elm E, Altman DG, et al. Strengthening the Reporting of Observational Studies in Epidemiology (STROBE): explanation and elaboration. Epidemiology 2007;18:805-35.
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CMV Infection and CAV After HTx
8. Kotton CN, Kumar D, Caliendo AM, et al. International consensus guidelines on the management of cytomegalovirus in solid organ transplantation. Transplantation 2010;89:779-95. 9. Husain S, Mooney ML, Danziger-Isakov L, et al. A 2010 working formulation for the standardization of definitions of infections in cardiothoracic transplant recipients. J Heart Lung Transplant 2011;30:361-74. 10. Stewart S, Winters GL, Fishbein MC, et al. Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection. J Heart Lung Transplant 2005;24:1710-20. 11. Kobashigawa JA, Tobis JM, Starling RC, et al. Multicenter intravascular ultrasound validation study among heart transplant recipients: outcomes after five years. J Am Coll Cardiol 2005;45:1532-7. 12. Crespo-Leiro MG, Marzoa-Rivas R, Barge-Caballero E, PaniaguaMartin MJ. Prevention and treatment of coronary artery vasculopathy. Curr Opin Organ Transplant 2012;17:546-50. 13. Brunner-La Rocca HP, Schneider J, Kunzli A, Turina M, Kiowski W. Cardiac allograft rejection late after transplantation is a risk factor for graft coronary artery disease. Transplantation 1998;65:538-43. 14. Courivaud C, Bamoulid J, Chalopin JM, et al. Cytomegalovirus exposure and cardiovascular disease in kidney transplant recipients. J Infect Dis 2013;207:1569-75. 15. Valantine HA. The role of viruses in cardiac allograft vasculopathy. Am J Transplant 2004;4:169-77. 16. Potena L, Grigioni F, Ortolani P, et al. Relevance of cytomegalovirus infection and coronary-artery remodeling in the first year after heart transplantation: a prospective three-dimensional intravascular ultrasound study. Transplantation 2003;75:839-43. 17. Melnick JL, Petrie BL, Dreesman GR, Burek J, McCollum CH, DeBakey ME. Cytomegalovirus antigen within human arterial smooth muscle cells. Lancet 1983;2:644-7. 18. Melnick JL, Hu C, Burek J, Adam E, DeBakey ME. Cytomegalovirus DNA in arterial walls of patients with atherosclerosis. J Med Virol 1994;42:170-4. 19. Epstein SE, Zhu J, Najafi AH, Burnett MS. Insights into the role of infection in atherogenesis and in plaque rupture. Circulation 2009;119:3133-41. 20. Stassen FR, Vega-Cordova X, Vliegen I, Bruggeman CA. Immune activation following cytomegalovirus infection: more important than direct viral effects in cardiovascular disease? J Clin Virol 2006;35:349-53. 21. Reinhardt B, Mertens T, Mayr-Beyrle U, et al. HCMV infection of human vascular smooth muscle cells leads to enhanced expression of functionally intact PDGF beta-receptor. Cardiovasc Res 2005;67: 151-60. 22. van Dam-Mieras MC, Muller AD, van Hinsbergh VW, Mullers WJ, Bomans PH, Bruggeman CA. The procoagulant response of cytomegalovirus infected endothelial cells. Thromb Haemost 1992;68:364-70. 23. Pawelec G, Derhovanessian E, Larbi A, Strindhall J, Wikby A. Cytomegalovirus and human immunosenescence. Rev Med Virol 2009;19:47-56. 24. Hussain T, Burch M, Fenton MJ, et al. Positive pretransplantation cytomegalovirus serology is a risk factor for cardiac allograft vasculopathy in children. Circulation 2007;115:1798-805.
1119 25. Lemstrom KB, Bruning JH, Bruggeman CA, et al. Cytomegalovirus infection-enhanced allograft arteriosclerosis is prevented by DHPG prophylaxis in the rat. Circulation 1994;90:1969-78. 26. Lemstrom K, Koskinen P, Krogerus L, Daemen M, Bruggeman C, Hayry P. Cytomegalovirus antigen expression, endothelial cell proliferation, and intimal thickening in rat cardiac allografts after cytomegalovirus infection. Circulation 1995;92:2594-604. 27. Lemstrom K, Sihvola R, Bruggeman C, Hayry P, Koskinen P. Cytomegalovirus infection-enhanced cardiac allograft vasculopathy is abolished by DHPG prophylaxis in the rat. Circulation 1997;95:2614-6. 28. Valantine HA, Gao SZ, Menon SG, et al. Impact of prophylactic immediate posttransplant ganciclovir on development of transplant atherosclerosis: a post hoc analysis of a randomized, placebo-controlled study. Circulation 1999;100:61-6. 29. Valantine HA, Luikart H, Doyle R, et al. Impact of cytomegalovirus hyperimmune globulin on outcome after cardiothoracic transplantation: a comparative study of combined prophylaxis with CMV hyperimmune globulin plus ganciclovir versus ganciclovir alone. Transplantation 2001;72:1647-52. 30. Potena L, Holweg CT, Chin C, et al. Acute rejection and cardiac allograft vascular disease is reduced by suppression of subclinical cytomegalovirus infection. Transplantation 2006;82:398-405. 31. Kelly JJ, Walker RG, d’Apice AJ, Kincaid-Smith P. A prospective study of the effect of diltiazem in renal allograft recipients receiving cyclosporine A: preliminary results. Transplant Proc 1990;22:2127-8. 32. Schroeder JS, Gao SZ, Alderman EL, et al. A preliminary study of diltiazem in the prevention of coronary artery disease in hearttransplant recipients. N Engl J Med 1993;328:164-70. 33. Kunzendorf U, Walz G, Brockmoeller J, et al. Effects of diltiazem upon metabolism and immunosuppressive action of cyclosporine in kidney graft recipients. Transplantation 1991;52:280-4. 34. Delgado JF, Sanchez V, de la Calzada CS, et al. Impact of diltiazem administration and cyclosporine levels on the incidence of acute rejection in heart transplant patients. Transpl Int 2003;16:676-80. 35. Mies S, Massarollo PC, Figueira ER, Leitao RM, Raia S. Lower incidence of liver graft rejection in patients on diltiazem plus cyclosporine therapy. Transplant Proc 1998;30:1437-8. 36. Lapointe N, Chen H, Qi S, Xu D, Daloze P, Dumont L. Immunomodulating effects of second-generation calcium channel blockers on experimental heart transplantation. Eur Surg Res 1999;31:259-66. 37. Dumont L, Chen H, Daloze P, Xu D, Garceau D. Immunosuppressive properties of the benzothiazepine calcium antagonists diltiazem and clentiazem, with and without cyclosporine, in heterotopic rat heart transplantation. Transplantation 1993;56:181-4. 38. Libersan D, Marchand R, Montplaisir S, Chartrand C, Dumont L. Cardioprotective effects of diltiazem during acute rejection on heterotopic heart transplants. Eur Surg Res 1997;29:229-36. 39. Kobashigawa JA, Katznelson S, Laks H, et al. Effect of pravastatin on outcomes after cardiac transplantation. N Engl J Med 1995;333:621-7. 40. Mehra MR, Ventura HO, Smart FW, Collins TJ, Ramee SR, Stapleton DD. An intravascular ultrasound study of the influence of angiotensinconverting enzyme inhibitors and calcium entry blockers on the development of cardiac allograft vasculopathy. Am J Cardiol 1995;75:853-4.
Influence of cytomegalovirus infection in the development of cardiac allograft vasculopathy after heart transplantation Juan F. Delgado, MD, PhD,a Ana García Reyne, MD, PhD,b Santiago de Dios, MD,a Francisco López-Medrano, PhD,b Alfonso Jurado, MD,a Rafael San Juan, MD, PhD,b María José Ruiz-Cano, MD,a M. Dolores Folgueira, MD, PhD,c Miguel Ángel Gómez-Sánchez, MD, PhD,a José María Aguado, MD, PhD,b and Carlos Lumbreras, MD, PhDb From the aCardiology Department; bInfectious Disease Unit; and the cMicrobiology Department, University Hospital 12 de Octubre, Instituto de Investigación Hospital 12 de Octubre, Madrid, Spain.
KEYWORDS: cardiac allograft vasculopathy; heart transplantation; cytomegalovirus infection; calcium channel blocker; cardiac angiography
BACKGROUND: Cardiac allograft vasculopathy (CAV) is a major cause of long-term morbidity and mortality after heart transplantation (HTx), whose relationship with CMV infection is uncertain. This study evaluated the influence of CMV infection in the development of CAV. METHODS: We enrolled 166 consecutive HTx recipients who underwent their first transplant from January 1995 to July 2002. All patients received 14 days of intravenous ganciclovir and were prospectively monitored for CMV infection during the first year after HTx. CAV was diagnosed by coronary angiography performed at 1, 5, and 10 years after HTx, following the new criteria of the International Society for Heart and Lung Transplantation. We collected all variables potentially related with the development of CAV. Risk factors were studied using a complementary log-log model. RESULTS: After a median follow-up of 11 years (range, 1–17 years), 72 patients (43%) developed CAV (63.8% CAV1, 15.2% CAV2, 20.8% CAV3). Symptoms secondary to CAV were present in 32% of these patients, and 8% died because of it. In the regression multivariate analysis, independent variables associated with the development of CAV were donor age (hazard ratio [HR], 1.028; 95% confidence interval [CI], 1.002–1.053; p o 0.028), presence of cellular acute rejection Z 2R (HR, 1.764; 95% CI, 1.011–3.078; p o 0.0414), CMV infection (HR, 2.334; 95% CI, 1.043– 5.225; p o 0.0354), and not having been treated with a calcium channel blocker (HR, 0.472; 95% CI, 0.275–0.811; p o 0.0055). CONCLUSIONS: Standardized angiographic criteria show CMV infection is associated with the development of CAV. J Heart Lung Transplant 2015;34:1112–1119 r 2015 International Society for Heart and Lung Transplantation. All rights reserved.
Cardiac allograft vasculopathy (CAV) remains the Achilles heel of heart transplantation (HTx). Current prevalence of Reprint requests: Juan F. Delgado, MD, PhD, Heart Failure and Transplant Unit, Cardiology Department, University Hospital 12 de Octubre, Av. de Córdoba sn., 28041 Madrid, Spain. E-mail address: [email protected]
CAV among HTx patients is 20% at 3 years, 30% at 5 years, and 45% at 8 years after HTx.1 According to the International Society for Heart and Lung Transplantation (ISHLT) Registry, CAV is one of the most important causes of death after HTx, accounting for 10% to 13% of deaths occurring more than 1 year after HTx and may be present in patients who die after the first year due to “graft failure” (16% to 27%).1
1053-2498/$ - see front matter r 2015 International Society for Heart and Lung Transplantation. All rights reserved. http://dx.doi.org/10.1016/j.healun.2015.03.015
Delgado et al.
CMV Infection and CAV After HTx
CAV is a long-term complication of HTx, but most studies of the pathogenesis of CAV are hampered by a very short follow-up and the lack of uniform criteria in its definition. CAV is detected mainly by coronary angiography,2 which is the best screening tool to detect the presence of CAV. However, the different angiographic definitions used make it impossible to draw solid conclusions regarding the influence of a specific factor in the development of CAV. The recently published standardized nomenclature for CAV by the ISHLT will be an important step forward to allow a better comparability between studies3 and a useful tool for prognostic stratification after HTx.4 Several factors have been linked with the development of CAV, including donor and recipient characteristics and post-HTx complications.1 In particular, cytomegalovirus (CMV) infection has been occasionally linked with CAV as a part of the so-called indirect effects in HTx recipients.5 However, a recent meta-analysis of risk factors associated with the development of CAV did not find a conclusive association between CMV infection and CAV.6 Thus, the main objective of our study was to evaluate the influence of CMV infection and other risk factors for the development of CAV.
Methods The Local Ethical Committee approved this investigation.
Study design A single-center, retrospective, observational study from prospectively collected data was designed to evaluate the relationship between CMV infection and CAV. We conducted this study by following the recommendations for observational studies of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement.7
Study population Consecutive patients who received their first HTx from January 1995 to July 2002 and survived at least 1 year were included in the study. Patients were excluded when they underwent repeat HTx, had an inadequate CMV sampling (o90% of the protocolized samples), or did not have an adequate coronary angiographic study. Follow-up continued until death, retransplantation, or until followup ended in December 31, 2011.
Immunosuppressive therapy Induction immunosuppressive therapy included OKT3 (5 mg/day for 14 days) or, after June 2002, 2 doses of basiliximab (20 mg) on Days 1 and 4 after surgery. Methylprednisolone was administered at 500 mg intravenously before surgery and at 125 mg intravenously every 8 hours for 3 doses after the operation, followed by prednisone at 1 mg/kg/day orally, tapered by 0.1 mg/kg on alternate days to 0.2 mg/kg/day and reduced to 0.1 mg/kg/day after 1 year. Azathioprine was administered at 2 mg/kg/day orally. After January 2002, azathioprine was substituted by mycophenolate mofetil (2–3 g/day). As a calcineurin inhibitor we used
1113 cyclosporine A (5 to 8 mg/kg/day) to maintain serum cyclosporine A levels within the range of 250 to 350 ng/ml during the first year and from 100 to 200 ng/ml for the second year and all following.
CMV diagnosis and CMV-related definitions Fourteen CMV anti-genemias were collected during the first 6 months after HTx (Monofluo kit CMV, BioRad, Marnes-la Coquette, France). It was measured in number of total leukocytes pp65 positive/200,000 leukocytes). CMV infection was defined as evidence of CMV replication regardless of symptoms, upon the sole presence of a positive antigenemia test. CMV disease was defined as evidence of CMV infection with attributable symptoms. CMV disease can be further categorized as a viral syndrome with fever, malaise, leukopenia, and thrombocytopenia or as a tissue-invasive disease.8,9 Asymptomatic viremia was diagnosed when a positive antigenemia test was not accompanied by any clinical symptom. Patients with asymptomatic viremia were compared only with patients without CMV infection, and therefore, patients with CMV disease were excluded from this analysis. We defined a “high CMV viremia” if 3 or more consecutive or non-consecutive anti-genemia tests showed more than 10 cells/ 200,000 leukocytes.
Antiviral therapy All patients, irrespective of their CMV serostatus, received universal CMV prophylaxis with intravenous ganciclovir (5 mg/kg/day) during the first 14 days after HTx as the only anti-CMV prophylactic therapy. Preemptive therapy was not performed during the follow-up, but patients with asymptomatic viremia were monitored closely for signs and symptoms of CMV disease, and when present, intravenous ganciclovir treatment for 14 to 21 days was administered.
Coronary angiographic studies CAV diagnosis was done through a retrospective review of all coronary angiographic and echocardiographic studies performed in every patient by protocol at 1, 5 and 10 years after HTx, following standardized ISHLT nomenclature.3 Coronary angiography was performed using standard techniques after pre-treatment with nitroglycerine. Two expert cardiologists, blinded to the clinical course of patients, examined all angiograms. We defined the presence of CAV as a status Z CAV1.
Risk factor analysis Risk factor data were collected by a retrospective review. More than 100 variables were collected as potential risk factors for CAV, including recipient and donor characteristics, immunosuppression, specific treatments, and complications (acute rejection episodes, severe infections different than CMV, and malignancies). We defined severe infections other than CMV as all bacterial, viral, and fungal infections that needed hospitalization or intravenous anti-biotic treatment and also infections caused by varicella-zoster virus. In addition, we collected classical cardiovascular risk factors before HTx and at 1, 5 and 10 years after HTx, including diabetes, hypertension, smoking status, hypercholesterolemia (defined as cholesterol 4 220 mg/dl) and hypertriglyceridemia (triglyceride
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value 4 200 mg/dl). The real effect of hypertension was difficult to measure after HTx because most patients were receiving antihypertensive drugs, so we measured the effect of anti-hypertensive drugs including angiotensin-converting enzyme inhibitors or calcium channel blockers (CCBs). The same occurred with hypercholesterolemia analysis, so we also analyzed the use of statins. Acute cellular rejection (ACR) surveillance was standardized and performed in all patients. Endomyocardial biopsy specimens were graded according to the 2005 ISHLT classification as 1R, 2R, and 3R.10 We included in the analysis all ACR episodes during the first year after transplantation. Patient survival, CAV-related death, and non-fatal major adverse cardiovascular events (NF-MACE) were analyzed. NFMACE included acute myocardial infarction, congestive heart failure, need for percutaneous cardiac intervention, coronary artery bypass grafting, cardiac defibrillator placement, cerebral strokes, and peripheral vascular disease.11
Statistical analysis The patients’ data were prospectively sampled and stored in a computerized database. They were summarized by descriptive statistics that characterized continuous variables by mean ⫾ standard deviation and categoric variables by proportions. As a first step, a univariate analysis was performed of the association between single putative risk factors and the presences of CAV during follow-up. The chi-square test or Fisher’s exact test for qualitative variables and Student’s t-test or Wilcoxon’s rank test for quantitative variables were used. Because CAV is assessed at discrete time points (1, 5, and 10 years), risk factor analysis (univariate and multivariate) was done using complementary log-log model for interval-censored survival times. This model is analogous to the Cox proportional hazards model with continuous time and is often used to estimate incidence rates at discrete time points while taking into account differential patient follow-up. All continuous and categoric variables that were significant in the univariate analysis with a p o 0.1 were subsequently entered into multivariate analysis. Predicted survival distribution of CAV for CMV was also done using complementary log-log model and was plotted on a graph. We also estimated by logistic regression the predicted probability of developing CAV during the 10 years of follow-up in relation with the intensity of CMV viremia (“high degree viremia”) and we also plotted it on a graph. Statistical significance was defined as a p-value of o0.05. We used SAS/STAT 9 software.
Results Overall, 213 patients received their first HTx at our institution during the study period. We excluded 4 patients who underwent re-HTx, 31 who died during the first year after HTx, 5 whose CMV sampling was inadequate, and 7 without angiographic studies due to vascular access problems. Thus, we finally included 166 HTx recipients. Table 1 describes the main baseline characteristics of the cohort. Most patients received induction therapy with OKT3 and triple therapy with cyclosporine, azathioprine, and prednisone. Median follow-up was 11 years, with an overall mortality of 37%. A NF-MACE event developed in 16% of patients during follow-up.
Table 1
Heart Transplant Baseline Characteristics
Characteristics Age, mean ⫾ SD years Male sex, No. (%) Cytomegalovirus serology, No. (%) Donor positive/recipient negative Donor positive/recipient positive Donor negative/recipient positive Donor negative/recipient negative Immunosuppression, No. (%) Induction therapy OKT3 Basiliximab Maintenance therapy Cyclosporine and steroids Azathioprine Mycophenolate mofetil Follow-up, median (min–max) years NF-MACE, No. (%) Mortality, No. (%) General CAV-associated
Value (N ¼ 166) 53.03 ⫾ 12.47 99 (71) 16 (11.6) 83 (60.1) 34 (24.6) 5 (3.5) 166 (100) 161 (96.9) 5 (3.01) 166 (100) 130 (78.3) 36 (21.6) 11 (1–17) 27 (16.3) 61 (36.7) 6 (3.6)
CAV, cardiac allograft vasculopathy; NF-MACE, non-fatal major adverse cardiovascular events; SD, standard deviation.
We analyzed 394 coronary angiographic studies: 166 in the first year after HTx, 126 in the fifth year, and 102 in the tenth year. The reasons for the absence of a coronary angiography in patients who were alive in each study period were medical or surgical conditions that discouraged the procedure (n ¼ 14), severe peripheral vascular disease (n ¼ 8), and patient refusal (n ¼ 8). In 8 patients we did not perform angiography at the fifth year but it was performed in the tenth year and was included in the analysis. As expected, CAV presence increased during the followup. Some degree of CAV was present in 6.6% of studies in the first year, in 25% in the fifth year, and in 57% in the tenth year after HTx (Figure 1). At the end of the follow-up, CAV developed in 72 patients (43%): CAV1 (mild) in 46 patients (63.8%), CAV2 (moderate) in 11 patients (15.2%), and CAV3 (severe) in 15 patients (20.8%). The comparison between the 72 patients who developed CAV during follow-up and the 94 who did not is reported in Table 2. Pre-HTx CMV serostatus did not correlate with the development of CAV. CMV infection and disease were significantly more frequent in patients with CAV and in patients who had the previously defined “high-degree CMV viremia.” The mean value of the area under the curve of CMV viremia was also higher in those patients who developed CAV. The univariate analysis (Table 3) showed that several factors were statistically associated with the development of CAV, including recipient body mass index, donor age, CMV infection, CMV disease, high degree of CMV viremia, ACR Z 2R, hypertriglyceridemia, with the administration of CCB during follow-up a protective factor.
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CMV Infection and CAV After HTx
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Figure 1 Result of coronary angiographies in the first, fifth, and tenth year after heart transplantation. CAV, cardiac allograft vasculopathy; ISHLT, International Society of Heart and Lung Transplantation.
For the multivariate analysis we made different models in which we included 1 CMV-related variable and up to 6 other variables that were statistically significant in the univariate analysis. As reported in Table 4, CMV infection, Table 2
CMV disease, and CMV asymptomatic viremia were significantly associated with the development of CAV. Another risk factor associated with the development of CAV in all of the models was the administration of a CCB. Donor
Comparisons Between Patients With and Without Cardiac Allograft Vasculopathy No CAV
CAV
Variables
(n ¼ 94)
(n ¼ 72)
Recipient age, mean ⫾ SD years Recipient BMI, mean ⫾ SD kg/m2 CAD before HTx, No (%) Donor age, mean ⫾ SD years CMV serology Dþ/R–, No. (%) ACR at first year, No. (%) ACR Z 2R at first year, No. (%) Severe infections different than CMV, No. (%) Severe infections, mean ⫾ SD No. CMV infection, No. (%) Asymptomatic viremia CMV disease High-degree CMV viremia Other risk factors, No. (%) At first year Calcium channel blocker Statin treatment Hypertriglyceridemia At fifth year Calcium channel blocker Statin treatment Hypertriglyceridemia At tenth year Calcium channel blocker Statin treatment Hypertriglyceridemia
54.73 ⫾12.5 23.68 ⫾ 3.5 50 (52.6) 25.36 ⫾ 8.48 8 (12.9) 65 (69.1) 12 (12.98) 54 (57.4) 1.42 ⫾ 1.53 71 (75.5) 60 (72.3) 11 (11.7) 15 (16)
50.80 ⫾ 12.08 24.85 ⫾ 3.54 39 (54.9) 29.89 ⫾ 10.49 6 (13) 59 (81.9) 22 (30.6) 36 (50) 1.3 ⫾ 1.768 64 (88.8) 41 (83.7) 23 (31.9) 22 (30.6)
0.044 0.037 0.769 0.002 0.983 0.06 0.05 0.253 0.633 0.029 0.136 0.001 0.025
85 (90.4) 51 (54.3) 5 (5.3)
52(72.2) 41 (56.9) 8 (11.1)
0.002 0.71 0.166
76 (80.9) 55 (58.5) 5 (5.3)
51 (70.8) 54 (75) 9 (12.5)
o0.0001 0.191 0.06
50 (53.2) 54 (57.4) 5 (5.3)
35 (48.6) 49 (68.1) 18 (25)
0.013 0.47 0.005
p-value
ACR, acute cellular rejection; BMI, body mass index; CAD, coronary artery disease; CAV, cardiac allograft vasculopathy; CMV, cytomegalovirus; D, donor; HTx, heart transplant; R, recipient; SD, standard deviation.
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Table 3 Univariate Analysis for Risk Factors Associated With Cardiac Allograft Vasculopathy Risk factor
HR
95% CI
p-value
Recipient age Recipient BMI Coronary artery disease before HTx Donor age ACR at first year ACR Z 2R at first year CMV infection CMV asymptomatic viremia CMV disease High-degree CMV viremia Diabetes mellitus after HTx Hypertriglyceridemia Treatment with Calcium channel blocker ACE inhibitor Statin
1 1.081 1.180
0.978–1.014 1.009–1.157 0.732–1.901
0.6524 0.0240 0.4885
1.035 1.57 1.730 2.352 1.92 2.258 1.718 1.322 2.070
1.010–1.060 0.845–2.895 1.021–2.932 1.108–4.991 0.885–4.171 1.343–3.795 1.023–2.886 0.783–2.259 1.088–3.924
0.0043 0.1458 0.0375 0.0230 0.092 0.0017 0.0367 0.2824 0.0237
0.390 1 0.910
0.235–0.656 0.604–1.646 0.527–1.587
0.0003 0.9910 0.7451
ACE, angiotensin-converting-enzyme; ACR is acute cellular rejection; BMI, body mass index; CI, confidence interval; CMV, cytomegalovirus; HR, hazard ratio; HTx, heart transplant.
age and cellular acute rejection during first year after HTx were not always associated with CAV in the different multivariate models. If we analyzed the probability of developing CAV during follow-up, those patients with CMV infection had a statistically higher probability of developing CAV than patients without CMV infections. Thus, 10 years after transplantation, only 35% of patients with CMV infection were CAV-free compared with 64% of patients without CMV infection (Figure 2). We also analyzed the effect of CMV on the severity of CAV. As can been seen in Table 5, CAV moderate to severe (CAV2 or higher), following the ISHLT score, was more frequent in patients with CMV disease and with a higher degree of anti-genemia, but this difference did not reach statistical significance. High-degree CMV viremia was not seen as statistically significant in the multivariate analysis. However, regarding the probability of developing CAV, it exhibited a lineal relationship with the development of CAV, suggesting that a high CMV viremia may increase the probability of CAV development (Figure 3).
Discussion Our study shows that CMV infection, including asymptomatic replication, is an independent risk factor associated with the development of CAV. We were able to demonstrate it through a special scenario that occurred during the interval we chose for the study: a homogeneous cohort who received intense immunosuppressive therapy, a short course of intravenous ganciclovir prophylaxis, standardized blood sampling to search for CMV replication, and a long-term angiographic follow-up.
Of course, our intention was not to generalize these data with what is currently occurring in HTx programs with a different immunosuppressive therapy and more potent antiviral prevention strategies. However, our data show that CMV infection and disease is definitely associated with CAV when the new, more sensitive ISHLT3 score is used that takes into account milder grades of CAV. This finding is useful for our current transplantation practices because it emphasizes the importance of detecting and avoiding CMV replication to improve the long-term outcome of HTx recipients. The current paradigm for the development of CAV includes the exposure of coronary graft vessels to immune and non-immune pathogenic factors that induce endothelial cell injury, leading to intimal hyperplasia and vascular remodeling.2,12 There are abundant data suggesting that infections may play a role in atherosclerosis, and CMV is one of the pathogens that has been most convincingly implicated.13–16 Several mechanisms have been evoked to explain this association. CMV has been shown to directly infect human vessels,17,18 leading to smooth muscle cell proliferation and migration, uptake of oxidized low-density lipoprotein, release of cytokines and chemokines, and increased procoagulant activity by endothelial cells.19–22 Alternatively, CMV may induce vascular lesions without direct invasion. Viral antigens can trigger an immune response that crossreacts with self-peptides expressed on the vascular wall.19 Finally, CMV is a driver of age-associated immune changes, which lead to a reduction of naive T cells, an expansion of terminally differentiated T cells, and an increase in proinflammatory cytokines.23 The data supporting a role for CMV infection in the pathophysiology of CAV is based on epidemiologic and observational data,24 experimental models,25–27 and therapeutic trials.15,28,29 However, available evidence has not been consistent for different reasons: a limited number of patients, studies handicapped by important heterogeneity, inadequate (usually too-short) follow-up, and heterogeneous definitions of risk factors, CAV, and CMV-related events. Actually, a recent systematic review of observational studies concluded that there is no evidence of a relationship between CMV and CAV.6 More recently, however, the association between CMV replication and atherosclerotic events was proven in kidney transplant patients,14 and previous studies in HTx patients have suggested that the suppression of viral replication could prevent vascular damage.30 Our study shows that not only CMV disease but also asymptomatic viral replication is likewise an independent risk factor. We were not able to probe in the multivariate analysis an independent association between elevated viremia and the development of CAV. However, as is shown in Figure 3, there was a lineal relationship between the degree of CMV viremia and the development of CAV. Our data may have important implications in the clinical management of CMV infection in HTx patients because it strongly suggests that avoiding CMV viremia after HTx might reduce the incidence of CAV. So, in this context,
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CMV Infection and CAV After HTx
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Table 4 Models of Multivariate Analysis That Include One Way of Measuring Cytomegalovirus Infection and Other Risk Factors Associated With Cardiac Allograft Vasculopathy Multivariate analysis Model Model 1 CMV infection Acute rejection Z 2R in the first year Recipient BMI Donor age Calcium channel blocker treatment Model 2 CMV disease Acute rejection Z 2R in the first year Recipient BMI Donor age Calcium channel blocker treatment Model 3 CMV asymptomatic viremia Acute rejection Z 2R in the first year Hypertriglyceridemia Donor age Calcium channel blocker treatment
HR
95% CI
p-value
2.334 1.764 1.066 1.028 0.472
1.043–5.225 1.011–3.078 0.991–1.148 1.002–1.053 0.275–0.811
0.0354 0.0414 0.0816 0.0287 0.0055
1.755 1.337 1.069 1.024 0.457
1.011–3.046 0.695–2.572 0.993–1.152 0.998–1.050 0.267–0.784
0.0414 0.3741 0.0706 0.0606 0.0037
2.428 1.431 2.008 1.033 0.416
1.013–5.819 0.682–3.002 0.0865–4.665 1.002–1.064 0.220–0.786
0.0468 0.3331 0.0980 0.0337 0.0059
BMI, body mass index; CI, confidence interval; CMV, cytomegalovirus; HR, hazard ratio.
universal prophylaxis may be a preferable option to preemptive therapy in the management of CMV infection after HTx.30 Nevertheless, only a long-term randomized study comparing universal prophylaxis with preemptive therapy, in which the appearance of CAV would be the primary end point, would confirm this hypothesis. Another significant finding that has come from our study is the protective effect of CCB, mostly diltiazem, in the development of CAV. During the study period, diltiazem was applied to counteract the adverse effects of conventional immunosuppression, namely, hypertension and nephrotoxicity.31 Previous studies have reported that diltiazem slows the development of CAV32 and reduces the incidence of acute rejection.33–35 Moreover, studies with heterotopic
HTx models have shown that diltiazem exerts immunosuppressive and immunomodulating effects. Transmembrane calcium movement plays a critical role in lymphocyte function, and numerous investigators have demonstrated that diltiazem inhibits lymphocyte functions and depresses the immune response.36–38 We speculate whether through any of these mechanisms diltiazem may explain the protective effect against CAV that we have found in our analysis. Other risk factors associated with CAV found in our study were donor age and acute rejection. Donor age has been linked to CAV in different studies.6 Instead, the effect of acute rejection in the appearance of CAV remains controversial.6 In our study, only severe acute rejection seems to be related with the development of CAV.13 A direct causative association of CAV and the observed risk-factors cannot be definitely established because of the retrospective nature of our study. Rejection and CMV infection are known Table 5 Comparison Between Patients With Cardiac Allograft Vasculopathy Grade o 2 and Patients With Cardiac Allograft Vasculopathy Z 2 Following International Society for Heart and Lung Transplantation Criteria According to Cytomegalovirus Infection
Figure 2 Probability of cardiac allograft vasculopathy (CAV)free status during the follow-up in patients with and without cytomegalovirus (CMV) infection done by complementary loglog model.
CAV o 2
CAV Z 2
Variable
(n ¼ 140) No. (%)
(n ¼ 26) No. (%)
p-value
CMV infection Asymptomatic viremia CMV disease High-degree CMV viremia
113 88 25 28
22 13 9 9
0.639 0.996 0.052 0.1
(80.7) (76.5) (17.9) (20)
(84.6) (76.5) (34.6) (34.6)
CAV, cardiac allograft vasculopathy; CMV, cytomegalovirus.
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The Journal of Heart and Lung Transplantation, Vol 34, No 8, August 2015
Figure 3 Predicted probability of cardiac allograft vasculopathy (CAV) development in relation with “high degree antigenemia” done by logistic regression.
to be related; however, rejection and CMV infection both appeared in as independent risk factors for CAV in the different multivariate statistical models we performed. Other factors that have been identified in the literature as CAV risk factors are donor factors, recipient characteristics, use of a ventricular assistance device, or immunosuppressive treatment,12 but these were not related with CAV in our study. Nor did the use of statins39 or angiotensin-converting enzyme inhibitors40 show a protective effect against CAV in our analysis. Our study has some limitations. First, are those that are inherent to a retrospective single-center study, which have reduced external validity and are less generalizable because of potential differences in the management of patients. Nonetheless, in our study, the key variables were collected prospectively and a strict protocol was followed. However, a well-designed prospective study would be needed to define whether CMV contributes directly to the atherosclerotic process in the allograft or instead serves as a marker of immune dysregulation or ongoing inflammation in the HTx population. We used CMV anti-genemia to measure CMV viremia instead of CMV polymerase chain reaction, which was not available in our institution during the study period. The use of a more sensitive and standardized test that is, also, a better tool to quantify CMV viremia would be helpful in better defining the importance of the degree and duration of CMV viremia in the development of CAV. Our data were obtained in a cohort of patients who received intense immunosuppressive therapy with OKT3, which it is no longer used in this setting. We cannot rule out that the influence of CMV infection in the development of CAV in HTx patients may be different when another type of immunosuppression is used. From a statistical viewpoint, we analyzed more than 100 variables influencing CAV development in the risk factor analysis, so we cannot rule out a type I error.
Finally, a comparison of our data with those obtained in a similar analysis including more recent patients receiving modern immunosuppression and more potent anti-viral therapy would reinforce our main findings. We are actually collecting these data from our newer cohort with those patients who received HTx from 2002, but a significant larger follow-up is still needed from this second cohort. Moreover, this comparison would only show whether our current transplantation practices are able or not to eliminate the deleterious effect of CMV in the development of CAV, but not questioning our main findings. In conclusion, following the new ISHLT normalized criteria for the diagnosis of CAV, we have found that CMV disease and asymptomatic CMV viremia are independently associated with the development of CAV in HTx recipients. However, the post-HTx use of CCBs seems to protect against CAV development. Further studies are needed to show if the use of a more aggressive anti-CMV prophylaxis may prevent the appearance of CAV in HTx recipients.
Disclosure statement None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose. The authors are grateful to David Lora-Pablos (Research Institute, Clinical Research Unit, University Hospital 12 de Octubre) for the statistical analyses and to Martin J. Smyth, BA, for revision of the English version of this manuscript. This study was partially supported by the Spanish Cardiovascular Research Networks RIC (Instituto de Salud Carlos III, Ministerio de Economía y Competitividad) and by REIPI (Red Española de Enfermedades Infecciosas). A.G.-R. was supported by a grant from the Instituto de Salud Carlos III (Rio Hortega, Grant No. CN09130).
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