Abstract
Inflammation is a part of the initial process leading to atherosclerosis and cholesterol crystals (CC), found in atherosclerotic plaques, which are known to induce complement activation. The pentraxins C-reactive protein (CRP), long pentraxin 3 (PTX3), and serum amyloid P component (SAP) are serum proteins associated with increased risk of cardiovascular events and these proteins have been shown to interact with the complement system. Whether the pentraxins binds to CC and mediate downstream complement-dependent inflammatory processes remains unknown. Binding of CRP, PTX3, and SAP to CC was investigated in vitro by flow cytometry and fluorescence microscopy. CRP, PTX3, and SAP bound to CC in a concentration-dependent manner. CRP and PTX3 interacted with the complement pattern recognition molecule C1q on CC by increasing the binding of both purified C1q and C1q in plasma. However, CRP was the strongest mediator of C1q binding and also the pentraxin that most potently elevated C1q-mediated complement activation. In a phagocytic assay using whole blood, we confirmed that phagocytosis of CC is complement dependent and initiated by C1q-mediated activation. The pathophysiological relevance of the in vitro observations was examined in vivo in human atherosclerotic plaques. CRP, PTX3, and SAP were all found in atherosclerotic plaques and were located mainly in the cholesterol-rich necrotic core, but co-localization with the terminal C5b-9 complement complex was only found for CRP. In conclusion, this study identifies CRP as a strong C1q recruiter and complement facilitator on CC, which may be highly relevant for the development of atherosclerosis.
Highlights
Vascular diseases caused by atherosclerosis are the leading cause of death and morbidity worldwide, with ischemic heart disease and stroke accounting for 15 million deaths in 2015 [1]
We investigated the binding of the pentraxins: pentraxin 3 (PTX3), C-reactive protein (CRP), or serum amyloid P component (SAP) to cholesterol crystals (CCs) in vitro by flow cytometry (Figure 1) and fluorescence microscopy (Figure 2). Recombinant PTX3 (rPTX3), purified CRP (pCRP), and purified SAP (pSAP) bound to CC in a concentration-dependent manner (Figure 1A); 100-fold higher concentrations of rPTX3 were necessary to obtain binding than for pCRP and pSAP
Binding of rPTX3 was not inhibited by EDTA, whereas binding of pCRP and SAP was highly calcium dependent and was completely inhibited by addition of EDTA (Figure 1B)
Summary
Vascular diseases caused by atherosclerosis are the leading cause of death and morbidity worldwide, with ischemic heart disease and stroke accounting for 15 million deaths in 2015 [1]. A hallmark of atherosclerotic plaques, found in early atherosclerotic lesions, is deposition of solid phase cholesterol crystals (CCs) [2]. CCs are known to induce inflammation via activation of the complement system [3,4,5,6] leading to a subsequent cytokine release and activation of the nod-like receptor pyrin domain-containing 3 (NLRP3) inflammasome [2, 7,8,9]. Activation initiates a cascade reaction leading to the cleavage of C3 into C3a and C3b and eventually the cleavage of C5 into C5a and C5b and the formation of a terminal C5b-9 complement complex (TCC). Activation results in opsonization and phagocytosis of pathogens or damaged self-structures through deposition of C3b, and proinflammatory signaling induced by C3a and C5a [10]
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