Toll-like receptor response and circulating cells in cardiovascular disease

Abstract

Atherosclerosis is the underlying cause of coronary artery disease and cerebrovascular disease. Risk prediction of cardiovascular disease and subsequent clinical manifestations are mainly based on models including hypercholesterolemia and smoking, but these models do not always work on an individual level. Therefore, there is an urgent need for biomarkers. Atherosclerosis is considered an inflammatory disease in which innate immune cells like monocytes and neutrophils play a prominent role. Toll-like receptors (TLRs), which are abundantly expressed by these cells, are important receptors for the activation of the immune system. These receptors respond to exogenous ligands derived from pathogens, but also to endogenous triggers. Ligand binding to TLRs results in the transcription of pro-inflammatory and pro-atherogenic mediators like cytokines and increased expression of cell adhesion molecules, but it has also been reported that repetitive TLR stimulation results in attenuated TLR signaling. Leukocyte behavior alters under the influence of multiple factors that are released during vascular or myocardial damage and atherosclerotic development. This suggests that circulating leukocytes are able to sense events occurring in the vascular wall. Therefore, we hypothesized that TLR response might be an interesting marker for cardiovascular disease. Attenuated TLR response has previously been observed in patients undergoing abdominal and vascular surgery. In this thesis we show that reversible ischemia also results in a decreased TLR response. This suggests that acute conditions like ischemia and trauma cause a hypo-response of circulating leukocytes. Furthermore, we have developed an animal model that, hopefully, will provide more insight into the underlying mechanisms of this hypo-response. As atherosclerosis is a chronic inflammatory disease, we hypothesized that this disease would result in a TLR hyper-response. Increased cytokine levels, adhesion molecules expression and TLR expression have previously been associated with cardiovascular disease. In this thesis, we studied whole blood TLR-induced CD11b and L-selectin expression on circulating monocytes and whole blood TLR-induced cytokine release in stable angina patients. Responsiveness was measured after single-dose TLR stimulation, but also activation patterns were analyzed using dose-response curves. These studies showed that circulating leukocytes are indeed hyper-responsive as compared to healthy individuals. But, even though a wide variation was observed among patients, this variation could only minimally be explained by differences in risk factors and atherosclerotic disease severity. TLR response is strictly regulated and multiple factors in the circulation are able to influence this response. Expression levels of monocyte CD14 and plasma levels of β2-glycoprotein I have been suggested to influence TLR response, but no associations were found in our studies. A decreased CD14 expression was even observed in patients as compared to controls. And more importantly, low CD14 expression and high plasma sCD14 were associated with the presence of cardiovascular risk factors and future cardiovascular events. This thesis shows that the potential of TLR-response as biomarker of cardiovascular disease seems limited, but the concept of circulating cells as sources of biomarkers remains interesting and is worth further investigation.