Atherosclerotic and obesity-associated inflammation : focus on cholesterol crystals, complement and coagulation

Abstract

Atherosclerosis is a chronic inflammatory disease of the arterial wall, and a leading cause of death worldwide. An emerging role for the complement system in the pathogenesis of atherosclerosis is demonstrated by the finding that cholesterol crystals, a hallmark of atherosclerotic plaques, are capable of activating the complement system, and thereby promote inflammation. Rupture of atherosclerotic plaques allows its content, including cholesterol crystals, to come into contact with coagulation factors in the blood, as well as with monocytes, which have the ability to express tissue factor. Tissue factor is the main initiator of coagulation, and it is hypothesized that cholesterol crystals can activate coagulation in a complement-dependent manner. Obesity is an independent risk factor for atherosclerosis, and complement and coagulation factors have been found to be elevated in obese individuals. Observations that adipose tissue is the site of production of certain complement components suggest that body weight may influence inflammatory processes. It is therefore of interest to investigate inflammatory factors that might be influenced by weight reduction. In the first part of this project, the expression of several inflammation-related receptors (C3aR, C5aR, C5L2, PAR-2, CD36 and CD14) was measured on peripheral blood leukocytes from obese patients before and after weight loss, using flow cytometry. In the second part of the project, a lepirudin-based whole blood model was used to investigate the effect of cholesterol crystals on the expression of the aforementioned receptors on monocytes and granulocytes, and the involvement of complement was investigated by inhibition of complement at the level of C3 and C5. In the third part of the project, the lepirudin-based whole blood model was used to investigate the effect of cholesterol crystals on coagulation activation, by measuring plasma levels of PTF1.2 and monocyte tissue factor expression following cholesterol crystal stimulation. The involvement of complement was investigated by inhibiting complement at the level of C3 or C5. The main findings in the obesity studies were a marked change on human leukocyte surface expression of C3aR, C5aR, CD36 and CD14, with significantly reduction following weight loss, while C5L2 expression was significantly increased. These data indicates that weight and vi weight reduction might have an impact on the expression levels of the anaphylatoxin receptors, as well as CD36 and CD14. The role of these receptors in inflammation and in atherosclerosis further points to a relationship between obesity, inflammation and atherosclerosis. CC stimulation over time, as studied in a whole blood model, revealed that the expression of the receptors varied both with time, stimuli and between the monocyte and granulocyte populations. A reduction in anaphylatoxin receptor expression was observed initially. While monocytes showed a circulating pattern in general, the granulocyte expression remained generally low. A complement dependency was found particularly for the C5aR receptor expression, and to a lower extent for the C3aR receptor. The expression of C5L2 was in contrast not dependent on complement. Cholesterol crystal stimulation induced coagulation activation as manifested through increased PTF1.2 levels in plasma. The activator of the extrinsic coagulation pathway, tissue factor (TF) appeared to be up-regulated on monocytes after stimulation with CC. These effects were found to be complement-mediated. In conclusion, the present studies showed that expression of the anaphylatoxin receptors (C3aR, C5aR, C5L2), the oxLDL receptor CD36 and the LPS co-receptor CD14 changed after weight reduction. The cholesterol crystals seemed to impact the blood system both on the receptor levels (C3aR, C5aR, C5L2, CD14, CD36, TF) as well as on coagulation activation. The expression of C3aR, C5aR and TF was shown to be complement dependent. These findings demonstrate a further connection between complement, obesity and atherosclerosis.