Markers of inducible myocardial ischemia in the systemic circulation

In this thesis, we tested the hypothesis that the presence of inducible myocardial ischemia as a result of blood flow-limiting coronary stenoses in patients with stable coronary artery disease is associated with altered levels of various markers and different molecular pathways detectable in peripheral blood, as opposed to non-ischemic coronary artery disease. In Chapter 1 the rationale of this thesis is described. The importance of the presence of severe coronary stenoses and inducible ischemia is explained and various cellular and molecular mechanisms associated with ischemia or tissue hypoxemia are described, in both clinical and experimental settings. The research presented in this thesis was performed within the framework of the Circulating Cells Study, a project of the Center of Translational Molecular Medicine (CTMM). The goal of the Circulation Cells Study was discovery and validation of novel cell-based biomarkers for risk prediction of secondary manifestations of coronary artery disease. Chapter 2 describes the rationale and study design of the Circulating Cells Study. The remainder of this thesis is divided into 3 parts. Part I describes the tools used for assessment of ischemic coronary disease in this thesis and contains chapters 3 and 4. Ischemic potential of coronary stenoses was assessed by fractional flow reserve (FFR). Chapter 3 provides an overview of the principles and clinical application of this technique. In Chapter 4 we describe the functional syntax score (FSS), which combines anatomical features (scored by the SYNTAX score) and functional severity of coronary artery disease (as measured by FFR). We show that in patients with multivessel coronary artery disease, who are treated by FFR-guided percutaneous coronary intervention (PCI), FSS has better predictive value for the occurrence of future cardiac events than the traditional SYNTAX score. We further attempted to refine the FSS score by incorporating absolute FFR-values in the same study population. However, this did not result in a better predictive value compared to the dichotomous classification of lesions as either FFR-positive or FFR-negative. In Part II, comprising chapters 5 through 9, the relationship between the presence of inducible ischemia and various plasma and cellular markers is investigated. As described in the introduction, the presence of inducible ischemia is associated with increased risk of myocardial infarction and death in patients with coronary artery disease. Simultaneously, biomarker research in patients with coronary artery disease aims to identify biomarkers that can be helpful in predicting the occurrence of future cardiac events. We therefore hypothesized that the presence of locally inducible myocardial ischemia would be associated with altered biomarkers in the systemic circulation. In Chapter 5, we showed that inflammatory cytokines IL-6, IL-8 and TNF-a in plasma and expression of Toll like receptor (TLR) 2 and 4, adhesion molecules CD11b and CD62L, and CD14 on circulating leukocytes are not related to FFR in patients with stable angina, even when extent of ischemic disease is taken into account by FSS. In a control group of healthy subjects, lower levels of IL-6 and TNF-a as well as lower expression of TLR 2 and 4 and CD11b on monocytes and TLR 4 and CD62L on granulocytes, were observed. In Chapter 6, we explored the association between FFR and responsiveness to wholeblood stimulation of TLR 2 and 4 as measured by supernatant levels of IL-6, IL-8 and TNF-a and expression of CD11b, CD62L and CD14 on circulating leukocytes. We observed no differences in TLR responsiveness between FFR-positive patients, FFR-negative patients and healthy controls, except for CD11b on monocytes, which was lower in healthy controls for all conditions. No relation between FSS and TLR responsiveness was observed in FFR-positive patients. In Chapter 7, the effects of FFR-guided percutaneous coronary intervention (PCI) on TLR responsiveness were investigated in a subgroup of 33 patients. We observed decreased responsiveness to TLR2 stimulation 6 weeks after FFR-guided PCI. However, this decrease did not appear to be related to relief of ischemia, nor was there any relationship with extent of ischemic disease, as measured by FSS. In Chapter 8 we investigated the relationship between platelet reactivity and platelet leukocyte complexes in peripheral blood and FFR of 100 patients with stable coronary artery disease. No difference in platelet reactivity was noted, however, the percentage platelet neutrophil complexes were significantly lower in blood samples from patients with at least one coronary stenosis with FFR=0.75. In Chapter 9 we performed an I-Traq proteomic analysis of circulating microvesicles in pooled plasma samples of 35 paired age- and gender-matched FFR-positive and negative patients. This analysis suggests that the microvesicle proteome of patients with stable coronary artery disease and inducible ischemia differs significantly from that of patients without inducible ischemia. By Ingenuity Pathway Analysis ® we identified several possible target molecules, yet to be validated as biomarkers of inducible ischemia. In Part III, comprising chapter 10 through 12, clinical analyses and a general discussion are presented. In Chapter 10, we show that the benefit of FFR-guided PCI versus angiography-guided PCI in patients with multivessel disease is equally present in patients with unstable angina or non-ST-elevation myocardial infarction as in patients with stable angina. Chapter 11 describes the outcome at 9 month follow up of the patients participating in the Circulating Cells Study that were included in the Catharina Hospital Eindhoven. In Chapter 12, a general discussion on the findings of the research described in this thesis is presented and future directions are discussed.