Cholesterol Crystals Activate the Lectin Complement Pathway via Ficolin-2 and Mannose-Binding Lectin: Implications for the Progression of Atherosclerosis

Abstract

Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. We hypothesized that the pattern recognition molecules (PRMs) from the lectin pathway bind CC and function as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC in vitro using recombinant proteins, specific inhibitors, as well as deficient and normal sera. Additionally, we examined the deposition of ficolin-2 and MBL in human carotid plaques by immunohistochemistry and fluorescence microscopy. The results showed that the lectin pathway was activated on CC by binding of ficolin-2 and MBL in vitro, resulting in activation and deposition of complement activation products. MBL bound to CC in a calcium-dependent manner whereas ficolin-2 binding was calcium-independent. No binding was observed for ficolin-1 or ficolin-3. MBL and ficolin-2 were present in human carotid plaques, and binding of MBL to CC was confirmed in vivo by immunohistochemistry, showing localization of MBL around CC clefts. Moreover, we demonstrated that IgM, but not IgG, bound to CC in vitro and that C1q binding was facilitated by IgM. In conclusion, our study demonstrates that PRMs from the lectin pathway recognize CC and provides evidence for an important role for this pathway in the inflammatory response induced by CC in the pathophysiology of atherosclerosis.

Lectin Pathway of Complement Activation Is Associated with Vulnerability of Atherosclerotic Plaques

“…By the functional in vitro assay measuring C3 deposition on mannan, our study suggests that MBL-driven LP activation is associated with plaque instability. This association is further supported by the observation that MBL is present only in ulcerated plaques undergoing rupture, therefore extending previous work reporting MBL binding to intraplaque cholesterol crystals (53), clearly defining that plaque rupture is needed for MBL entry. Thus, although alternative interpretations cannot be excluded, i.e., that MBL is not consumed due to a lack of involvement in the condition, the overall data may indicate the association of MBL with plaque instability.…”

“…As an alternative hypothesis ficolin-2, whose high intraplaque levels correlate with low plaque vulnerability, may be protective. In this case, ficolin-2 could contribute to debris clearance, in line with its function as an opsonin for phagocytosis independent of complement activation (53). …”

Novel Insights into Crystal-Induced Kidney Injury

“…CCs can also induce either passive (physical) or regulated necrosis of the endothelial cells. They are reported to induce endothelial cell injury via complement activation [4]. Furthermore, they kill tubular epithelial cells by activating receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) [5, 6].…”

Mannose-binding lectin 2 (Mbl2) gene polymorphisms are related to protein plasma levels, but not to heart disease and infection by Chlamydia

“…In our study, higher MBL plasma levels were observed in patients with cardiovascular disorders (CAD and HVD) than in the CG, in agreement with the evidence showing that high levels of circulating MBL is a risk factor for ischemic diseases, including myocardial infarction (29). The MBL protein recognizes structures exposed in altered endothelial cells, contributing to tissue damage (33,34). In acute myocardial ischemia, MBL inhibition has been shown to reduce the infarction area by promoting a reduction in neutrophil infiltration and pro-inflammatory gene expression (35).…”