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Final Report Summary - COMPLEMENT AND LDL (Complement and atherosclerosis: molecular mechanism of C1 activation by enzymatically modified low-density lipoproteins)

Atherosclerosis is generally considered to be a chronic inflammatory process. Arterial inflammation that precedes development of the atherosclerotic plaque is caused by accumulation of LDL (low density lipoproteins) in the extracellular matrix of the blood vessels. These LDL particles are modified by a number of mechanisms, including enzymatic degradation, aggregation and oxidation.

Complement, a complex system of enzymes and regulators endowed with multiple biological activities (opsonisation, chemoattraction of leukocytes, cell lysis, cell activation), is a major component of innate defence against pathogens. It is also emerging as an important factor in atherosclerosis: complement activation, and formation of the terminal C5b-9 membrane attack complex of complement were shown to occur in atherosclerotic lesions. Recently we have studied the ability of an enzymatically modified form of LDL (E-LDL), obtained by treatment with trypsin and then cholesterol esterase, to activate C1, the complex that triggers activation of the classical pathway of complement. E-LDL is recognised by the C1q subunit of C1 and triggers direct C1 activation, suggesting that complement activation by E-LDL may play a crucial role in the pathogenesis of atherosclerosis.

The objective of this study was to identify the E-LDL component(s) recognised by C1q. In addition to trypsin, plasmin, thrombin, tryptase and matrix metalloprotease-2 each yielded E-LDL particles with high C1-activating efficiency, and the C1 activation extent was strictly dependent on cholesterol esterase treatment in all cases. When incorporated into vesicles, the lipid fraction of E-LDL, but not of native LDL, triggered C1 activation, and activation correlated with the amount of free cholesterol released by cholesterol esterase.

Whereas treatment of E-LDL particles with human serum albumin reduced their free fatty acid content, both cholesterol and free fatty acids were decreased by methyl-beta cyclodextrin, both treatments resulting in a dose-dependent inhibition of the C1-activating ability of the particles. Incorporation of linoleic acid into phosphatidylcholine-containing model vesicles allowed them to both interact with the C1q globular domain and trigger C1 activation, and cholesterol had an enhancing effect in both cases, due to its ability to facilitate incorporation of the fatty acid into the vesicles. Direct evidence that C1q binds E-LDL through its globular domains was obtained by electron microscopy (see attached Figure). The various approaches used in this study all concur in the conclusion that C1 binding to E-LDL involves recognition by C1q of the free fatty acids generated upon CEase treatment.

There is increasing evidence that free fatty acids have the ability to trigger cell apoptosis and it has been shown that exposure of endothelial cells to E-LDL results in programmed cell death. In view of these observations, it is tempting to hypothesize that, through its ability to bind fatty acids on E-LDL particles, C1q may prevent or control their proapoptotic effect. Thus, in addition to its established role in the sensing and clearance of apoptotic cells, C1q may also regulate fatty acid-induced apoptosis. Considering the possible implication of endothelial cell apoptosis in the atherogenesis process, such a property would bear important biological implications.

Our results have important potential implications in the development of new therapeutic strategies against atherosclerosis. Considering that atherosclerosis is a major health problem in developed countries, these data may have a major socio-economical impact.