Atherosclerosis is a progressive chronic disease with the bidirectional interaction between inflammation and lipids as its hallmark. Indeed, the atherosclerotic lesions are characterized by the accumulation of lipids together with infiltration of immune cells such as monocytes, macrophages, T cells and neutrophils. When cholesterol accumulates in the vessel wall to the level that exceeds macrophage capacity for elimination, precipitation into cholesterol crystals (CC) will occur. Oxidized low-density lipoprotein is endocytosed by CD36 that coordinates the intracellular conversion of this ligand to CC are present at all stages of atherogenesis, and constitute the primary endogenous danger signals that incite plaque inflammation, contributing to the development and progression of atherosclerosis and its complications like myocardial infarction and ischemic stroke. Phagocytosis of CC causes lysosomal damage and this is sensed by the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome, leading to the release of interleukin (IL)-1β. IL-1β is upregulated in atherosclerotic disorders and is associated with disease severity and outcome. the mechanisms of enhanced release of IL-1β as well as the role of CC-induced NLRP3 inflammasome activation in clinical atherosclerosis are not fully understood. A detailed analysis of the relation between complement, NLRP3 signalling pathways and CC in patients with coronary artery disease and carotid atherosclerosis is performed.
Patients with coronary artery disease: The study included only patients with CAD verified by coronary angiography (lesions with ≥50% lumen reduction compared to reference segment). Patients were classified into either stable angina pectoris (SAP) referred to elective coronary angiography with at least one significant coronary stenosis or acute coronary syndrome (ACS).
Patients with carotid plaques: Patients with high-grade internal carotid stenosis (≥70%) or ischemic stroke within the last month were recruited at the Department of Neurology, Oslo University Hospital Rikshospitalet. Patients were classified into either symptomatic group including patients with ischemic stroke within the last month, or asymptomatic group without ischemic stroke.
PBMC were separated from fresh heparinized blood by Isopaque-Ficoll gradient centrifugation using Lymphoprep (Nycomed, Oslo, Norway) or BD Vacutainer CPT (ref 362780 Becton, Dickinson and Company, Franklin Lakes). Data in Fig. 1 include patients with stable angina (SAP, N=21), acute coronary syndrome (ACS, N=25), or healthy donors (controls, N=25) and stored in liquid nitrogen until mRNA analyses. For protein analysis (in Fig. 2), ten patients with ACS and nine healthy donors were recruited, and their PBMCs were used immediately for further in vitro experiments. Cells were maintained in RPMI supplemented with 10% heat inactivated pooled human A+ serum (The Blood Bank, St. Olav's Hospital, Trondheim, Norway) unless otherwise noted. Cells were primed with C5a (1µg/ml), TNF (10 ng/ml) a combination of C5a and TNF or PBS for 2hrs prior to stimulation with CC (500 or 1000 μg/ml) for 16 hrs.
PBMC from ACS patients showed increased transcripts of IL-1β and TNF. PBMC from patients with ACS were highly responsive for priming with combination of C5a and TNF. Higher complement activation was detected in plasma from patients with SAP and ACS. Complement factor C1q and TCC were localized around CC clefts in carotid plaques. High expression of anaphylatoxin receptors were found in advanced human carotid plaques. Carotid plaques showed enhanced sensitivity to C5a priming. Combining C5a and TNF primed carotid plaques for NLRP3 and complement pathway genes.
The ex vivo priming of carotid plaques by C5a+TNF followed by CC activation induced mRNA expression of wide range of inflammatory genes. This was accompanied by enhanced expression of NLRP3, ASC and CASP1 with a corresponding significant increase of IL-1β and IL-18 gene expression and protein release, suggesting that these broad inflammatory responses within carotid plaques are driven by CC and complement activation. We found that monocytes, macrophages and T-cells were detected in plaques, and these cell types are likely to be major contributors to the inflammasome activation. Notably, also within CAD patients, we found an inflammatory response to C5a+ TNF and CC. PMBC from patients with ACS primed with C5a+TNF, and incubated with CC, showed a >2-fold increase in IL-1β release as compared with PBMC from healthy controls. This result is in line with our data showing that PBMC from ACS patients also had upregulated NLRP3 and IL-1β mRNA.
Nevertheless, this study demonstrates a close relationship between complement and CC-induced NLRP3 inflammasome activation in the inflammatory processes both systemically and within the atherosclerotic lesion. Patients with atherosclerosis have increased levels of C3 and C4 in their plasma, which is a risk factor of cardiovascular events. Most complement proteins, including C3 and C4 are acute phase proteins produced in the liver, supporting the idea that atherosclerosis is a systemic low-grade inflammatory disease. The increase in activation products, like C5a, has also been shown to predict cardiovascular events, particularly in patients with advanced atherosclerosis. This increase in activation products, as we showed for components from the whole cascade, is most likely due to release of activation products from the continuously activated atherosclerotic arterial endothelium. Using ex vivo human atherosclerotic plaques together with plasma and PBMC isolated from whole blood of patients with SAP and ACS, we show that this cross talk plays a mechanistic role in the development of atherosclerosis. The pathogenic loop between complement, CC and NLRP3 inflammasomes may represent a promising target for therapy of atherosclerotic disorders.