Macrophage Depletion Suppresses Cardiac Allograft Vasculopathy in Mice

Cardiac allograft vasculopathy (CAV) is a major source of late posttransplant mortality. Although numerous cell types are implicated in the pathogenesis of CAV, it is unclear which cells actually induce the vascular damage that results in intimal proliferation. Because macrophages are abundant in CAV lesions and are capable of producing growth factors implicated in neointimal proliferation, they are leading end-effector candidates. Macrophages were depleted in a murine heterotopic cardiac transplant system known to develop fulminant CAV lesions. C57BL/6 hearts were transplanted into (C57BL/6 x BALB/c)F(1) recipients, which then received anti-macrophage therapy with intraperitoneal carrageenan or i.v. gadolinium. Intraperitoneal carrageenan treatment depleted macrophages by 30-80% with minimal effects upon T, B or NK cells as confirmed by flow cytometry and NK cytotoxicity assays. Carrageenan treatment led to a 70% reduction in the development of CAV, as compared to mock-treated controls (p = 0.01), which correlated with the degree of macrophage depletion. Inhibition of macrophage phagocytosis alone with gadolinium failed to prevent CAV. Macrophages may represent the end-effector cells in a final common pathway towards CAV independent of T-cell or B-cell alloreactivity and exert their injurious effects through mechanisms related to cytokine/growth factor production rather than phagocytosis.     

大鼠腹腔心脏移植术后移植物血管病模型的建立

目的探讨建立大鼠腹腔心脏移植物血管病动物模型的方法。方法将40只W istar大鼠和40只SD大鼠随机配对分为对照组和实验组,建立大鼠的异位心脏移植;对照组不注射环孢霉素A(C sA),实验组腹腔注射小剂量的C sA,两组分别于移植术后2周和4周切取移植心脏,在光学显微镜下观察移植心脏冠状血管的改变。结果对照组术后2周和4周冠状血管内膜无变化;实验组术后2周冠状血管内膜增厚,4周后冠状血管内膜成同心圆样改变,管腔明显狭窄,出现移植后移植物血管病改变。结论该模型是一种可靠的移植物血管病的动物模型。     

Coronary Flow Reserve by Contrast-Enhanced Echocardiography: A New Noninvasive Diagnostic Tool for Cardiac Allograft Vasculopathy

Noninvasive tests have proven unsatisfactory in cardiac allograft vasculopathy (CAV) diagnosis. We assessed coronary flow reserve (CFR) by contrast-enhanced transthoracic echocardiography (CE-TTE) in heart transplantation (HT). CFR was assessed in the left anterior descending coronary artery in 73 HT recipients (59 male, aged 50 ± 12 years at HT), at 8 ± 4.5 years post-HT. CFR measurements were taken blindly from coronary angiographies. CFR cut points were the standard value of ≤2 and those defined by receiver operating characteristics (ROC) curve analysis. CFR was lower in patients with CAV (2.3 ± 0.7 vs. 3.2 ± 0.5, p < 0.0001). The ≤2 cut point was 100% specific and 38% sensitive. The ≤2.7 cut point, optimal by ROC analysis, was 87% specific and 82% sensitive. Accuracy rose from 71% with the standard ≤2 cut point to 85% with the optimal cut point of ≤2.7. CFR by CE-TTE may offer promise as a novel, easily repeatable and accurate noninvasive tool in CAV detection. However, further longitudinal studies in larger patient cohorts are warranted before widespread adoption can be advocated.     

Cytochrome p450 2C Enzymes Contribute to Peritransplant Ischemic Injury and Cardiac Allograft Vasculopathy

Peritransplant ischemia and reperfusion (I/R) injury contributes to posttransplant vascular dysfunction and cardiac allograft vasculopathy (CAV). We have previously shown that cytochrome p450 (CYP) 2C inhibition significantly reduces I/R-induced myocardial infarction and postischemic vascular dysfunction. In the latter study, pretreatment with sulfaphenazole (SP), a specific inhibitor of CYP 2C, restored postischemic NO-mediated, endothelium-dependent vasodilation and reduced vascular superoxide production. Given the association between I/R injury, early vascular dysfunction and CAV, we hypothesized that CYP 2C may also contribute to the onset of CAV. Lewis-to-Fisher rat heterotopic heart transplants were performed. Donors and recipients were treated with 5 mg/kg SP or vehicle control 1 h prior to surgery. SP did not affect posttransplant morbidity, mortality or weight gain. Coronary blood vessels from rats treated with SP exhibited significantly reduced luminal narrowing and demonstrated a corresponding decrease in smooth muscle cell (SMC) proliferation compared to controls. SP did not reduce diffuse, focal, epicardial, endocardial or perivascular immune infiltration nor did it significantly alter TUNEL positivity in myocardial, endothelial or SMC populations. In conclusion, CYP 2C contributes to SMC proliferation CAV without affecting general immune infiltration.     

Everolimus Versus Mycophenolate Mofetil in Heart Transplantation: A Randomized,Multicenter Trial

In an open‐label, 24‐month trial, 721 de novo heart transplant recipients were randomized to everolimus 1.5 mg or 3.0 mg with reduced‐dose cyclosporine, or mycophenolate mofetil (MMF) 3 g/day with standard‐dose cyclosporine (plus corticosteroids ± induction). Primary efficacy endpoint was the 12‐month composite incidence of biopsy‐proven acute rejection, acute rejection associated with hemodynamic compromise, graft loss/retransplant, death or loss to follow‐up. Everolimus 1.5 mg was noninferior to MMF for this endpoint at month 12 (35.1% vs. 33.6%; difference 1.5% [97.5% CI: –7.5%, 10.6%]) and month 24. Mortality to month 3 was higher with everolimus 1.5 mg versus MMF in patients receiving rabbit antithymocyte globulin (rATG) induction, mainly due to infection, but 24‐month mortality was similar (everolimus 1.5 mg 10.6% [30/282], MMF 9.2% [25/271]). Everolimus 3.0 mg was terminated prematurely due to higher mortality. The mean (SD) 12‐month increase in maximal intimal thickness was 0.03 (0.05) mm with everolimus 1.5 mg versus 0.07 (0.11) mm with MMF (p < 0.001). Everolimus 1.5 mg was inferior to MMF for renal function but comparable in patients achieving predefined reduced cyclosporine trough concentrations. Nonfatal serious adverse events were more frequent with everolimus 1.5 mg versus MMF. Everolimus 1.5 mg with reduced‐dose cyclosporine offers similar efficacy to MMF with standard‐dose cyclosporine and reduces intimal proliferation at 12 months in de novo heart transplant recipients.     

Contemporary Concepts in Prevention and Treatment of Cardiac Allograft Vasculopathy

Cardiac allograft vasculopathy (CAV), is characterized by heterogeneous proliferative thickening of the vascular intima of the cardiac allograft vasculature. Since its presentation is commonly clinically silent, early diagnosis and preventative therapy are critical. Preventative therapy including optimization of immunosuppressive therapy and treatment of comorbidities associated with CAV progression must be initiated early since most of the intimal thickening occurs during the first year posttransplant. Long-term use of calcineurin inhibitors is associated with a high incidence of chronic renal disease and also contributes to hyperlipidemia and hypertension, all of which may exacerbate CAV. In addition, statins, antihypertensive agents and anti-CMV agents all have demonstrated benefits in reducing CAV. Once established, the limited treatment options include nonpharmacologic interventions such as retransplantation, percutaneous coronary interventions, coronary artery bypass grafting, transmyocardial laser revascularization and heparin-induced/mediated extracorporeal LDL plasmapheresis (HELP). As the use of new assessment tools increases our understanding of this disease, better preventative and treatment strategies are evolving.     

Coronary Flow Reserve by Transthoracic Echocardiography Predicts Epicardial Intimal Thickening in Cardiac Allograft Vasculopathy

Cardiac allograft vasculopathy (CAV) is the leading cause of morbidity and mortality in heart transplantation (HT). We sought to investigate the role of coronary flow reserve (CFR) by contrast‐enhanced transthoracic echocardiography (CE‐TTE) in CAV diagnosis. CAV was defined as maximal intimal thickness (MIT) assessed by intravascular ultrasound (IVUS) ≥0.5 mm. CFR was assessed in the left anterior descending coronary artery in 22 HT recipients at 6 ± 4 years post‐HT. CAV was diagnosed in 10 patients (group A), 12 had normal coronaries (group B). The mean MIT was 0.7 ± 0.1 mm (range 0.03–1.8). MIT was higher in group A (1.16 ± 0.3 mm vs. 0.34 ± 0.07 mm, p < 0.0001). CFR was 3.1 ± 0.8 in all patients and lower in group A (2.5 ± 0.6 vs. 3.7 ± 0.3, p < 0.0001). CFR was inversely related with MIT (r =?0.774, p < 0.0001). A cut point of ≤2.9, identified as optimal by receiver operating characteristics analysis was 100% specific and 80% sensitive (PPV = 100%, NPV = 89%, Accuracy = 91%). CFR assessment by CE‐TTE is a novel noninvasive diagnostic tool in the detection of CAV defined as MIT ≥0.5 mm. CFR by CE‐TTE may reduce the need for routine IVUS in HT.     

Early Prediction of Cardiac Allograft Vasculopathy and Heart Transplant Failure

Early risk‐prediction is essential to prevent cardiac allograft vasculopathy (CAV) and graft failure in heart transplant patients. We developed multivariate models to identify patients likely to experience CAV, severe CAV, and failure due to CAV, at 1, 5 and 10 years. A cohort of 172 patients was followed prospectively for 6.7 ± 3.9 years. Logistic regression models were developed and cross‐validated using bootstrap resampling. Predictive markers of atherothrombosis (myocardial fibrin deposition, and loss of vascular antithrombin and tissue plasminogen activator) and arterial endothelial activation (intercellular adhesion molecule‐1 expression) were measured in serial biopsies obtained within 3 months posttransplant. Most markers were univariately associated with outcome. Multivariate models showed that loss of tissue plasminogen activator was the dominant and, in most cases, only predictor of long‐term CAV (p < 0.001), severe CAV (p < 0.001), and graft failure due to CAV (p < 0.001). The models discriminated patients having adverse outcomes, had particularly high negative predictive values (graft failure due to CAV: 99%, 99% and 95% at 1, 5 and 10 years) and predicted event incidence and time to event. Early absence of atherothrombotic risk identifies a patient subgroup that rarely develops CAV or graft failure, implying that this low‐risk subgroup could possibly be followed with fewer invasive procedures.     

Differential Effect of Everolimus on Progression of Early and Late Cardiac Allograft Vasculopathy in Current Clinical Practice

Randomized trials showed that mTOR inhibitors prevent early development of cardiac allograft vasculopathy (CAV). However, the action of these drugs on CAV late after transplant is controversial, and their effectiveness for CAV prevention in clinical practice is poorly explored. In this observational study we included 143 consecutive heart transplant recipients who underwent serial intravascular ultrasound (IVUS), receiving either everolimus or mycophenolate as adjunctive therapy to cyclosporine. Ninety‐one recipients comprised the early cohort, receiving IVUS at weeks 3–6 and year 1 after transplant, and 52 the late cohort, receiving IVUS at years 1 and 5 after transplant. Everolimus independently reduced the odds for early CAV (0.14 [0.01–0.77]; p = 0.02) but it did not appear to influence late CAV progression. High‐dose statins were found to be associated with reduced CAV progression both early and late after transplant (p ≤ 0.05). Metabolic abnormalities, such as high triglycerides, were associated with late, but not with early CAV progression. By highlighting a differential effect of everolimus and metabolic abnormalities on early and late changes of graft coronary morphology, this observational study supports the hypothesis that everolimus may be effective for CAV prevention but not for CAV treatment, and that risk factors intervene in a time‐dependent sequence during CAV development.     

Characteristics of Cardiac Allograft Vasculopathy Induced by Immunomodulation in the Miniature Swine

Purpose: We aimed to develop swine cardiac transplantation model for study of cardiac allograft vasculopathy (CAV) and to characterize the mechanisms of its formation.Methods: Heterotropic cardiac transplantation was performed in swine leukocyte antigen mismatched miniature swine, and CAV was induced by immunomodulation by cyclosporine A (CyA). Histology and immunohistochemistry were performed to identify cellular components of CAV. Fluorescence in situ hybridization (FISH) was developed for detection of 1 and Y-chromosome for identification of cell origin in the female donor to the male recipient heart transplantation model.Results: CAV was successfully developed by immunomodulation of CyA. Severity of CAV revealed more prominent in the distal epicardial coronary arteries than proximal coronary arteries. Phenotype of the SMCs proliferated in the intimal thickening of CAV were mostly embryonal/secretory type. Our new chromosome specific probes for FISH method were useful for discrimination of sex of each cell, and proliferated SMCs were revealed to be mainly donor origin.Conclusion: CAV mimicking human heart transplantation can be developed by appropriate immunomodulation in the swine. In swine CAV, proliferated SMCs seen in the intimal thickening were demonstrated to be from the donor origin.     

The Effect of Everolimus Initiation and Calcineurin Inhibitor Elimination on Cardiac Allograft Vasculopathy in De Novo Recipients: One‐Year Results of a Scandinavian Randomized Trial

Early initiation of everolimus with calcineurin inhibitor therapy has been shown to reduce the progression of cardiac allograft vasculopathy (CAV) in de novo heart transplant recipients. The effect of de novo everolimus therapy and early total elimination of calcineurin inhibitor therapy has, however, not been investigated and is relevant given the morbidity and lack of efficacy of current protocols in preventing CAV. This 12‐month multicenter Scandinavian trial randomized 115 de novo heart transplant recipients to everolimus with complete calcineurin inhibitor elimination 7–11 weeks after HTx or standard cyclosporine immunosuppression. Ninety‐five (83%) patients had matched intravascular ultrasound examinations at baseline and 12 months. Mean (± SD) recipient age was 49.9 ± 13.1 years. The everolimus group (n = 47) demonstrated significantly reduced CAV progression as compared to the calcineurin inhibitor group (n = 48) (ΔMaximal Intimal Thickness 0.03 ± 0.06 and 0.08 ± 0.12 mm, ΔPercent Atheroma Volume 1.3 ± 2.3 and 4.2 ± 5.0%, ΔTotal Atheroma Volume 1.1 ± 19.2 mm³ and 13.8 ± 28.0 mm³ [all p‐values ≤ 0.01]). Everolimus patients also had a significantly greater decline in levels of soluble tumor necrosis factor receptor‐1 as compared to the calcineurin inhibitor group (p = 0.02). These preliminary results suggest that an everolimus‐based CNI‐free can potentially be considered in suitable de novo HTx recipients.     

Strain-Encoded Cardiac Magnetic Resonance for the Evaluation of Chronic Allograft Vasculopathy in Transplant Recipients

The aim of our study was to investigate the ability of Strain-Encoded magnetic resonance imaging (MRI) to detect cardiac allograft vasculopathy (CAV) in heart transplantation (HTx)-recipients. In consecutive subjects (n = 69), who underwent cardiac catheterization, MRI was performed for quantification of myocardial strain and perfusion reserve. Based on angiographic findings subjects were classified: group A including patients with normal vessels; group B, patients with stenosis <50%; and group C, patients with severe CAV (stenosis ≥ 50%). Significant correlations were observed between myocardial perfusion reserve with peak systolic strain (r =−0.53, p < 0.001) and with mean diastolic strain rate (r = 0.82, p < 0.001). Peak systolic strain and strain rate were significantly reduced only in group C, while mean diastolic strain rate and myocardial perfusion reserve were already reduced in group B and A. Myocardial perfusion reserve and mean diastolic strain rate had higher accuracy for the detection of CAV (AUC = 0.95, 95% CI = 0.87–0.99 and AUC = 0.93, 95% CI = 0.84–0.98, respectively) and followed peak systolic strain and strain rate (AUC = 0.80, 95% CI = 0.69–0.89 and AUC = 0.78, 95% CI = 0.67–0.87, respectively). Besides the quantification of myocardial perfusion, the estimation of the diastolic strain rate is a useful parameter for CAV assessment. In combination with the clinical evaluation, these parameters may be effective tools for the routine surveillance of HTx-recipients.     

Perfusion Cardiac Magnetic Resonance Imaging as a Rule‐Out Test for Cardiac Allograft Vasculopathy

Cardiac allograft vasculopathy (CAV) is a leading cause of mortality after heart transplantation. Noninvasive imaging techniques used in CAV evaluation have important limitations. In a cross‐sectional study, we investigated perfusion cardiac magnetic resonance (CMR) imaging to determine an optimal myocardial perfusion reserve index (MPR) cutoff for detecting CAV using receiver operating characteristic curve analysis. We evaluated CMR performance using sensitivity, specificity and likelihood ratio analysis. We included 29 patients (mean 5 ± 4 years after transplant) scheduled for coronary angiography with intravascular ultrasound (IVUS) who completed CMR. CAV was defined as maximal intimal thickness (MIT) >0.5 mm by IVUS of the left anterior descending artery. CAV was evident in 19 patients (70%) on IVUS (mean MIT 0.82 ± 0.42 mm). MPR was significantly lower in patients with MIT ≥0.50 mm (1.35 ± 0.23 vs. 1.71 ± 0.45, p = 0.013). There was moderate inverse correlation between MPR and MIT (r = ?0.36, p = 0.075). The optimal MPR cutoff ≤1.68 for predicting CAV showed sensitivity of 100%, specificity of 63%, a negative predictive value of 100%, a positive predictive value of 86% and a positive likelihood ratio of 2.7. An MPR ≤1.68 has high negative predictive value, suggesting its potential as a test to rule out CAV.     

T cells in Cardiac Allograft Vasculopathy Are Skewed to Memory Th-1 Cells in the Presence of a Distinct Th-2 Population

Cardiac allograft vasculopathy (CAV) in heart transplantation (HTx) patients remains the major complication for long-term survival, due to concentric neointima hyperplasia induced by infiltrating mononuclear cells (MNC). Previously, we showed that activated memory T-helper-1 (Th-1) cells are the major component of infiltrating MNC in coronary arteries with CAV. In this study, a more detailed characterization of the MNC in human coronary arteries with CAV (n = 5) was performed and compared to coronary arteries without CAV (n = 5), by investigating MNC markers (CD1a, DRC-1, CD3, CD20, CD27, CD28, CD56, CD68, CD69, FOXP3 and HLA-DR), cytokines (IL-1A, 2, 4, 10, 12B, IFN-γ, and TGF-β1), and chemokine receptors (CCR3, CCR4, CCR5, CCR7, CCR8, CXCR3 and CX3CR1) by immunohistochemical double-labeling and quantitative PCR on mRNA isolated from laser microdissected layers of coronary arteries. T cells in the neointima and adventitia of CAV were skewed toward an activated memory Th-1 phenotype, but in the presence of a distinct Th-2 population. FOXP3 positive T cells were not detected and production of most cytokines was low or absent, except for IFN-γ, and TGF-β. This typical composition of T-helper cells and especially production of IFN-γ and TGF-β may play an important role in the proliferative CAV reaction.     

Mycophenolate Mofetil Reduces Intimal Thickness by Intravascular Ultrasound After Heart Transplant: Reanalysis of the Multicenter Trial

The mycophenolate mofetil (MMF) trial involved 650 heart transplant patients from 28 centers who received MMF or azathioprine (AZA), both in combination with cyclosporine and corticosteroids. Baseline and 1-year intravascular ultrasound (IVUS) were performed in 196 patients (102 MMF and 94 AZA) with no differences between groups in IVUS results analyzed by morphometric analysis (average of 10 evenly spaced sites, without matching sites between studies). Baseline to first-year IVUS data can also be analyzed by site-to-site analysis (matching sites between studies), which has been reported to be more clinically relevant. Therefore, we used site-to-site analysis to reanalyze the multicenter MMF IVUS data. Results: IVUS images were reviewed and interpretable in 190 patients (99 MMF and 91 AZA) from the multicenter randomized trial. The AZA group compared to the MMF group had a larger number of patients with first-year maximal intimal thickness (MIT) ≥0.3 mm (43% vs. 23%, p = 0.005), a greater decrease in the mean lumen area (p = 0.02) and a decrease in the mean vessel area (the area actually increased in the MMF group, p = 0.03). Conclusion: MMF-treated heart transplant patients compared to AZA-treated patients, both concurrently on cyclosporine and corticosteroids, in this study have significantly less progression of first-year intimal thickening.     

Early Conversion From Calcineurin Inhibitor‐ to Everolimus‐Based Therapy Following Kidney Transplantation: Results of the Randomized ELEVATE Trial

In a 24‐month, multicenter, open‐label, randomized trial, 715 de novo kidney transplant recipients were randomized at 10–14 weeks to convert to everolimus (n = 359) or remain on standard calcineurin inhibitor (CNI) therapy (n = 356; 231 tacrolimus; 125 cyclosporine), all with mycophenolic acid and steroids. The primary endpoint, change in estimated glomerular filtration rate (eGFR) from randomization to month 12, was similar for everolimus versus CNI: mean (standard error) 0.3(1.5) mL/min/1.73² versus ?1.5(1.5) mL/min/1.73² (p = 0.116). Biopsy‐proven acute rejection (BPAR) at month 12 was more frequent under everolimus versus CNI overall (9.7% vs. 4.8%, p = 0.014) and versus tacrolimus‐treated patients (2.6%, p < 0.001) but similar to cyclosporine‐treated patients (8.8%, p = 0.755). Reporting on de novo donor‐specific antibodies (DSA) was limited but suggested more frequent anti‐HLA Class I DSA under everolimus. Change in left ventricular mass index was similar. Discontinuation due to adverse events was more frequent with everolimus (23.6%) versus CNI (8.4%). In conclusion, conversion to everolimus at 10–14 weeks posttransplant was associated with renal function similar to that with standard therapy overall. Rates of BPAR were low in all groups, but lower with tacrolimus than everolimus.     

Coronary Microvascular Dysfunction Correlates With the New Onset of Cardiac Allograft Vasculopathy in Heart Transplant Patients With Normal Coronary Angiography

Coronary microvascular dysfunction is emerging as a strong predictor of outcome in heart transplantation (HT). We assessed the validity of microvascular dysfunction, defined by means of a reduced coronary flow reserve (CFR), as a factor associated with new onset epicardial cardiac allograft vasculopathy (CAV) or death. We studied 105 patients at 4 ± 1 years post‐HT with a normal coronary angiography (CA). New onset CAV was assessed by CA. CFR was assessed in the left anterior descending (LAD) coronary artery by transthoracic Doppler echocardiography and calculated as the ratio of hyperaemic to basal blood flow velocity. A CFR ≤ 2.5 was considered abnormal. Epicardial CAV onset or death was assessed during a follow‐up of 10 years. New onset CAV was diagnosed in 30 patients (28.6%) (Group A), and the CA was normal in the remaining 75 patients (71.4%) (Group B). Group A had reduced CFR compared with group B (2.4 ± 0.6 vs. 3.2 ± 0.7, p < 0.0001). A CFR ≤ 2.5 was independently associated with a higher probability of new onset CAV (p < 0.0001) and a higher probability of death, regardless of CAV onset (p < 0.01). Microvascular dysfunction is independently associated with the onset of epicardial CAV, and associated with a higher risk of death, regardless of CAV onset.