Abstract
The studies described in this thesis suggest that peptidoglycan (PG) is involved in initiation and progression of atherosclerosis. PG is an antigen that can be found in large amounts in the Gram-positive bacterial wall and only in small amounts in the Gram-negative bacterial wall. PG is able to stimulate the
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Toll-like receptor 2 (TLR2), a receptor that plays an important role in the innate immune system. In the normal situation PG is present in the intestinal mucosal flora, but under certain pathological circumstances PG is able to promote chronic inflammation at non-mucosal sites in the body. IgM, IgG and IgA antibodies against PG are present in serum of healthy subjects. However, in atherosclerotic patients the IgM antibody levels against PG are lower compared to control subjects. Furthermore, IgM antibody levels against PG are negatively associated with intima-media thickness in coronary arteries and therefore with more pronounced human atherosclerotic disease. We found these associations in a cross-sectional study, but these IgM antibody levels against PG are not prognostic markers for development of adverse cardiovascular events after elective coronary angioplasty. We also showed that adventitial fibroblasts and human coronary arteries express TLR2 and that TLR2 stimulation of adventitial fibroblasts increases expression of pro-inflammatory cytokines and chemokines, like IL-1β, IL-6, IL-8 and MCP-1. In addition, in wild-type mice Pam3Cys-SK4 stimulation promotes intimal lesion formation, which is clearly TLR2-mediated since TLR2-/- mice show no significant increase in intimal hyperplasia. In ApoE-/- mice TLR2 stimulation increases development of atherosclerotic plaques. In addition to recognition by TLR2, degradation products of PG can also be recognized by intracellular NOD and Nalp proteins. We found that NOD1 and NOD2 protein expression is significantly increased in human atherosclerotic lesions and in those lesions NOD2 protein is present in macrophages within inflammatory areas and in endothelial cells lining the vessel wall. NOD1 protein expression is associated with the presence of smooth muscle cells (SMC) and collagen, but also with macrophages when SMC are absent. Furthermore, both NOD1 and NOD2 protein levels are inversely associated with matrix metalloproteinase (MMP)-2 levels and positively with MMP-8 and MMP-9 levels. Previous studies from our group showed that PG is present in human atherosclerotic plaques that reveal an inflammatory, vulnerable phenotype and that PG is present in the macrophage-rich regions of those plaques. In this thesis we extend this finding by showing that peripheral blood monocytes that are stimulated with PG increase expression and functionality of integrins on their surface, thereby promoting firm adhesion of the monocytes to endothelial cells and migratory properties. Furthermore, peritoneal macrophages stimulated with PG in vitro show increased homing into atherosclerotic plaques in vivo. These studies not only suggest that PG promotes atherosclerotic lesion development and progression; they also show that PG enhances accumulation of monocytes and/or macrophages at sites of inflammation thereby promoting the formation of vulnerable plaques. These vulnerable plaques are prone to rupture and thereby promote the formation of a thrombus. Such a thrombus eventually might cause a myocardial infarction or stroke.
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