Coronary Artery Disease After Heart Transplantation

BACKGROUND

Part one:

Heart transplantation (HTX) is an excellent treatment of end stage heart failure with a mean survival of approximately 15.6 years (1). Long-term survival remains a challenge. With improvement of immunosuppressive therapy, incidences of acute cellular rejection (ACR) have declined, but after the first postoperative year, one of the main causes of death is cardiac allograft vasculopathy (CAV), which is an accelerated form of coronary artery disease (2).

ACR is a well-recognized phenomenon but the diagnosis of antibody-mediated rejection (AMR) has gained acceptance. AMR is associated with greater graft dysfunction, development of CAV and mortality. The diagnosis is based on clinical, histopathologic, immunopathologic and identification of donor-specific antibodies by solid phase assays (3,4). However, AMR is often clinically silent, and the histopathologic and immunopathologic evaluation may be associated with significant inter-observer variation. Identification of donor specific antibodies (DSA) could seem more suitable. In the GRAFT study, we found significantly increased levels of DSA in approximately 25% of HTX patients. They had subclinical reduced graft function, higher previous ACR burden and prevalence of CAV.

Guidelines recommend routinely evaluation of DSA, but the evidence of treating patients with DSA and no pathological findings is poor.

Extracorporeal photopheresis (ECP) inactivates lymphocytes and reduce cellular myocardial infiltration and production of DSA. Although the results for treatment of ACR and AMR appear promising, ECP is limited to a few centers (3,5,6).

The aim of this study is to evaluate the impact of ECP on CAV.

Part two:

The cause of CAV after HTx is unknown. CAV is a diffuse, progressive thickening of the arterial intima that develops in both the epicardial and intramyocardial arteries. Optical coherence tomography (OCT) and virtual histology intravascular ultrasound (IVUS) have revealed multilayered plaques in approximately 50% of patients (7), which likely represent intravascular thrombosis (7,8). Autopsy studies confirm a high prevalence (63-83%) of coronary thrombi (9). Platelets are the cellular mediator of thrombosis, but also play an important role in the immune system. Previous studies indicate that platelets are involved in vascular inflammation and immune cell trafficking in acute graft rejection. Platelet ligand induced binding site-1 (LIBS-1) antibody binding is correlated with CAV progression in HTX patients (10). Hence, platelets may play an important role in CAV progression. Further studies are needed to clarify the relation between platelet function and coronary thrombi, and the effect of aspirin on platelet function in HTX patients.

The aim of this part of the study is to evaluate the relationship between platelet function and the presence of luminal thrombi, and the relation between luminal thrombi and the blood clot formation and degeneration.

Platelet function and blood clot formation:

Ischemic heart disease is caused by atherosclerosis. Rupture of an atherosclerotic plaque causes activation of platelets and the coagulation system, ultimately resulting in a thrombus. Recent reports by IVUS (7) and OCT (8) have revealed high prevalence of complexed layered plaques, which might represent organized thrombus.

Coronary flow velocity reserve:

The coronary flow velocity reserve (CFVR) represents the capacity of the coronary circulation to dilate, following an increase in myocardial metabolic demands. CFVR is defined as the ratio of maximum flow/hyperaemic flow under adenosine infusion to resting blood flow velocity in the epicardial coronary arteries (14).

A CFVR value of 2 discriminates significant from non-significant coronary stenosis.

STUDY DESIGN

Controlled prospective study of all patients with CAV and DSA followed at the Department of Cardiology, AUH, Skejby. Based on our calculation sample size would be 30 patients. A graft age matched group of 30 HTX patients without DSA will be included as controls.

METHODS

Patients are screened for eligibility to be included prior to annual routine examinations and informed consent is obtained. CAG, OCT and advanced echocardiography at rest and during exercise including coronary flow reserve (CFR) measurements at baseline and at 12 months follow-up are performed at Department of Cardiology, Aarhus University Hospital. 34 ml blood will be stored in the biobank. Blood samples before and after 7 days treatment with 75 mg aspirin daily.

If DSA levels >3000 MFI with Luminex analysis, the patient receives ECP treatments at Department of Immunology, Aarhus University Hospital.

Platelet function:

Is evaluated by whole blood platelet aggregometry using Multiplate® Analyzer. This instrument is based on impedance aggregometry, in which the level of platelet aggregation is reflected by changes in impedance between two electrodes. Arachidonic acid (ASPI test), TRAP-6 and adenosine diphosphate (ADP test) are used as agonists to induce platelet aggregation. For verification of aspirin compliance, serum thromboxane B2 levels will be measured.

Platelet turnover parameters are evaluated using automated flow cytometry (Sysmex XN 9000). Platelet count, immature platelet fraction and count, mean platelet volume, platelet distribution width and platelet large-cell-ratio are measured.

Coronary angiography (CAG):

An experienced operator will perform CAG according to routine protocol after HTx. The degree of CAV will be quantified in a blinded fashion according to ISHLT guidelines:

• CAV 0: No stenosis/irregularity
• CAV 1: <70% major branch or <50% left main stem
• CAV 2: >70% major branch or >50% left main stem with normal diastolic graft function
• CAV 3: >70% major branch or >50% left main stem with impaired diastolic graft function.

Optical coherence tomography (OCT):

OCT acquisition is performed in all three major coronary arteries during CAG by use of Terumo Lunawave system as advised by the manufacturer and according to department standard operating procedure. Analysis is performed in a blinded fashion using the QCU-CMS software (Leiden University Medical Center, NL). Quantitative intimal tissue analysis is performed in areas without advanced plaque morphology. All plaques in the acquired segments are characterized and sized according to luminal presentation and mapped for serial assessment. Macrophage infiltration is quantified as a marker of inflammation. Layered complex plaques are defined as a heterogenic signal in intima with a layered structure.

The patients are divided in three groups according to the extent of CAV by complex layered plaques:

• 0 %
• 0-7 %
• >7 %

Transthoracic echocardiography:

The following parameters will be recorded: 2D ejection fraction, regional wall motion score (17 segment model), left ventricular end diastolic (LVEDV) and systolic volume (LVESV), left atrial volume, tissue Doppler study of the mitral annulus and LV strain and strain-rate. Furthermore, diastolic function will be evaluated by early diastolic myocardial velocity, E/A ratio, EdecT, isovolumetric relaxation time (IVRT) and E/e' ratio. Global longitudinal strain (GLS) will be assessed from two-dimensional images of the apical four-chamber, two-chamber, and long-axis view with an optimized frame rate (50-90 frames/sec).

Coronary Flow Reserve (CFR) by tissue Doppler echocardiography:

The distal LAD is localized and the flow velocity is measured with Doppler using a 6 MHz probe at basal conditions and during adenosine infusion (hyperemia) at 140 µg/kg/min.

Exercise Protocol:

We will measure peak oxygen consumption by cardiopulmonary exercise test (CPX). The patients will perform a multistage symptom limited semi supine cycle ergometer exercise test using GE Healthcare eBike L Ergometer (Wauwatosa, WI 53226 U.S.A.). Workload starts at 0 W and increases by 25 W every 3 minutes. Patients will be encouraged to exercise until exhaustion (Borg >18). Brachial blood pressure will be measured at baseline and every 3 minutes until maximum workload is reached.

ECP:

1. Collection of mononuclear cells by apheresis
2. Addition of 8-methoxyspsoralen (8-MOP) to the cells followed by ultraviolet A (UVA) irradiation
3. Reinfusion of the treated cells.

Collection of mononuclear cells will be performed with Spectra Optia using cMNC or MNC software (TerumoBCT, Lakewood, CO, USA) on 2 consecutive days. With MNC, two chamber collections (21 + 4 mL) are performed providing a fixed product volume of 50 mL. With MNC, collection is performed preferably 1 ml/min until a product volume of 75 mL. Hct is measured on a sample taken from the sample tube of the Spectra Optia kit. The product is diluted with 250 ml NaCl to an end volume of 300 ml. The product bag is sterile connected to the illumination bag and 3 mL 8-MOP (Macopharma, Tourcoing, France) is added to the product. Illumination is performed in Macogenic G2 (Macopharma, Tourcoing, France). The Hct before addition of 8-MOP should not exceed 2 %. Documentation and traceability are maintained by using blood bank IT system (Prosang, Databyrån, Stockholm, Sweden) and ISBT128 labeling.

SAMPLE SIZE CALCULATION - PLATELET FUNCTION

>Sample size calculation based on data from healthy volunteers off-aspirin<

The primary outcome is the difference in platelet aggregation between HTx patients with and without CAV and healthy volunteers. No publications report platelet aggregation in HTx patients but from a previous study we know that the mean platelet aggregation in healthy drug-naive individuals is 1004 aggregation units x minute with a standard deviation of 163 aggregation units x minute using arachidonic acid (AA) as agonist (ASPI-test). Choosing a minimal relevant difference of 150 aggregation units x minute, a significance level (two-sided alpha) of 5%, and a statistical power of 90% (1-β), we have to include 25 patients in each group.

>Sample size calculation based on data from stable CAD-patients on-aspirin<

The primary outcome is the difference in platelet aggregation between HTx patients with and without CAV and patients with stable CAD. From a previous study we know that the mean platelet aggregation in patients with stable coronary artery disease receiving aspirin is 324 aggregation units x minute with a standard deviation of 80 aggregation units x minute using AA as agonist (ASPI-test). Choosing a minimal relevant difference of 70 aggregation units x minute, a significance level (two-sided alpha) of 5%, and a statistical power of 90% (1-β), we have to include 28 patients in each group.

STATISTICS

Patients are divided into three groups according to the severity of CAV assessed by OCT (0%, 0-7% og >7% complex layered plaques). Platelet function are compared between groups. Normally distributed variables will be presented as mean ± SD. Non-parametric statistics and appropriate log-transformation will be performed if assumption of normality is not met. Between-group difference will be tested by Analysis of variance (ANOVA) or Kruskal-Wallis equality-of-populations rank test when appropriate. A two-tailed p-value of <0.05 or less will be considered statistically significant. Platelet variables in HTx patients before and after aspirin are compared to the same variables measured in healthy controls and stable CAD patients. Between-group difference will be tested by students t-test or Wilcoxon-Mann-Whitney test when appropriate.

PERSPECTIVES By combining advanced analyses and expert knowledge from the Department of Cardiology and the research unit at Department of Biochemistry, new knowledge regarding platelet function and ECP treatment in HTx patients will be obtained. Potentially, ECP treatments and medical therapy inhibiting platelets may reduce the development of CAV thus improving quality of life and long-term prognosis for HTx patients worldwide.