DEVELOPMENT OF PREDICTION MODELS FOR ALLOGRAFT VASCULOPATHY IN HEART TRANSPLANT RECIPIENTS

SUMMARY Cardiac allograft vasculopathy (CAV) is a limiting factor for the long-term survival of heart transplant recipients1,2. CAV is characterized by the development of diffuse concentric fibromuscular intimal hyperplasia in epicardial and smaller intramyocardial arteries along with focal, eccentric atherosclerotic plaques in the larger epicardial arteries3,4. The development of these lesions may lead to the progressive narrowing of the lumen5. According to the response to injury hypothesis of CAV, these lesions are the result of cumulative endothelial injury induced by alloimmune responses as well as non-immunological risk factors such as ischemia-reperfusion injury, viral infections, and metabolic disorders3,6. Early diagnosis of CAV is essential to implement appropriate prevention and treatment measures. Clinical prediction models of CAV are currently not available and may be useful for non-invasive diagnostic and prognostic purposes. The general aim of this doctoral thesis is to develop diagnostic prediction models for prevalent CAV. The specific central hypothesis of this doctoral thesis is that biomarkers of endothelial homeostasis discriminate between CAV-negative and CAV-positive heart transplant recipients. Endothelial homeostasis reflects the balance between endothelial injury and endothelial repair. In chapter 1, we investigated whether biomarkers related to endothelial injury and endothelial repair discriminate between CAV-negative and CAV-positive heart transplant recipients. Fifty-two patients undergoing coronary angiography between 5 and 15 years after heart transplantation were recruited in this study. Flow cytometry was applied to quantify endothelial progenitor cells (EPCs), circulating endothelial cells (CECs), and circulating endothelial microparticles (CEMPs). Cell culture was used for quantification of circulating EPC number and hematopoietic progenitor cell (HPC) number and for analysis of EPC function. EPC number and EPC function did not differ between CAV-negative and CAV-positive patients. In univariable models, age, creatinine, steroid dose, granulocyte colony-forming units, apoptotic CECs, and apoptotic CEMPs discriminated between CAV-positive and CAV-negative patients. The logistic regression model containing apoptotic CECs and apoptotic CEMPs as independent predictors provided high discrimination between CAV- positive and CAV-negative patients (c-statistic 0.812; 95% CI 0.692-0.932). In a logistic regression model with age and creatinine as covariates, apoptotic CECs (p=0.0112) and apoptotic CEMPs (p=0.0141) were independent predictors (c-statistic 0.855; 95% CI 0.756-0.953). These two biomarkers remained independent predictors when steroid dose was introduced in the model. Taken together, the high discriminative ability of apoptotic CECs and apoptotic CEMPs is a solid foundation for the development of clinical prediction models of CAV. In chapter 2, patients with stable native coronary artery disease (CAD) were compared with heart transplant recipients with CAV. After all, CAV is a particular type of arteriosclerosis with many similarities but also significant differences compared to native CAD. Atherosclerosis in patients with stable native CAD is characterized by the presence of atheromata that contain a lipid core filled with extracellular cholesterol and cellular debris and are covered by a fibrous cap. In contrast, fibromuscular intimal hyperplasia is the most prominent lesion type of CAV and mainly consists of smooth muscle cells and extracellular matrix7. Endothelial injury is assumed to play a key role in the initiation and progression of both native CAD and CAV2,8. In the response-to-injury hypothesis of atherosclerosis of Ross and Glomset, endothelial injury was originally defined as endothelial denudation resulting from focal desquamation of endothelium9,10. Later versions of the response-to-injury hypothesis emphasized endothelial dysfunction rather than denudation8,11. Cellular biomarkers of endothelial injury (CEMPs and CECs) may discriminate between endothelial activation and irreversible endothelial damage. The hypothesis that endothelial injury and circulating platelet microparticles (CPMPs) are distinct in both types of arteriosclerosis was investigated. The geometric mean of the concentration of CECs (CD45- CD31bright VEGFR-2+) was 2.90-fold (p<0.001) and 2.34-fold (p<0.05) higher in patients with stable native CAD (n=80) and with CAV (n=30), respectively, compared to healthy controls (n=25). No significant difference in total, Annexin V negative, and Annexin V positive (apoptotic) CECs was observed between patients with native CAD and with CAV. The concentration of Annexin V negative CEMPs (CD144+ CD42a-) was 59.2% (p<0.01) higher in transplant recipients with CAV than in native CAD patients but no difference in Annexin V positive CEMPs was observed. The median value of total CD61+ CPMPs in native CAD patients was 69.4% (p<0.001) and 71.6% (p<0.001) lower compared to healthy controls and transplant recipients with CAV, respectively. These differences were even more pronounced when CD42a+CD31+ CPMPs were quantified. In conclusion, the selective increase of Annexin V negative CEMPs and the absence of a difference in Annexin V positive CECs strongly suggest increased endothelial activation but not endothelial apoptosis in CAV-positive patients compared to stable CAD patients. Use of antiplatelet drugs likely underlies the strikingly lower levels of CPMPs in patients with native CAD. In chapter 3, the relation between high density lipoproteins (HDL) and CAV was investigated. The prevalence and the incidence of CAV have been reported to be increased in heart transplant recipients with decreased high density lipoprotein (HDL) cholesterol levels12-15. The association between HDL cholesterol and CAV may reflect causation but might also be due to residual confounding. One such confounding factor is insulin resistance, which is considered to play a role in the pathogenesis of CAV. A triglyceride/HDL cholesterol ratio of greater than 3 has been recognized as a marker of insulin resistance in overweight subjects16 and constituted a risk factor for CAV and major adverse cardiac events in heart transplant recipients17,18. Remodelling of HDL in heart transplant recipients is significantly affected by a lower activity of cholesterol ester transfer protein, phospholipid transfer protein, and hepatic lipase19,20. Consequently, these patients are characterized by an increased proportion of large HDL particles and reduced pre-ß1-HDL in the presence of normal or even elevated HDL cholesterol levels19,20. These alterations may be partially explained by corticosteroid use21 but may also be potentiated by statin intake22. The modified HDL metabolism and associated compositional changes of HDL particles may lead to an impaired function of these lipoproteins. Reduced HDL function may also occur as a result of ongoing inflammation23. We hypothesized that HDL function may be impaired in these patients and may discriminate between CAV-positive and CAV-negative patients. Cholesterol efflux capacity of apolipoprotein B-depleted plasma was analysed using a validated assay24. The vasculoprotective function of HDL was studied by means of an EPC migration assay. HDL cholesterol levels were similar in heart transplant patients compared to healthy controls. However, normalized cholesterol efflux and vasculoprotective function were reduced by 24.1% (p<0.001) and by 27.0% (p<0.01), respectively, in heart transplant recipients compared to healthy controls. HDL function was similar in patients with and without cardiac allograft vasculopathy (CAV) and was not related to C-reactive protein (CRP) levels. An interaction effect (p=0.0584) was observed between etiology of heart failure before transplantation and steroid use as factors of HDL cholesterol levels. Lower HDL cholesterol levels occurred in patients with prior ischemic cardiomyopathy not taking steroids. However, HDL function was independent of the etiology of heart failure before transplantation and steroid use. The median C-reactive protein (CRP) level was 2.24-fold (p=0.082) higher in patients with CAV than in patients without CAV. In conclusion, HDL function is impaired in heart transplant recipients but is unrelated to CAV-status. In chapter 4, the potential of endothelium-enriched microRNAs (miRNAs) as putative biomarkers for the prediction of CAV was investigated. MiRNAs are small, non-coding, single-stranded RNA sequences that regulate gene expression at the post-transcriptional level. Because miRNAs circulate in remarkably stable forms in blood25,26, they have a significant potential as biomarkers. Several reports indicate that miRNAs may play a role in endothelial homeostasis27,28. In this study, a candidate-based approach using circulating levels of endothelium-enriched miRNAs (miR-21-5p, miR-92a-3p, miR-92a-1-5p, miR-126-3p, miR-126-5p) to predict CAV was evaluated. Circulating levels of endothelium-enriched miRNAs (miR-21-5p, miR-92a-3p, miR-92a-1-5p, miR-126-3p, miR-126-5p) were quantified by real-time RT-PCR. The discriminative ability of logistic regression models was quantified using the concordance statistic (c-statistic). Plasma levels of miR-21-5p, miR-92a-3p, miR-126-3p, and miR-126-5p were 1.86-fold (p=NS), 1.91-fold (p