Abstract
Atherosclerosis, the inflammation of artery walls due to the accumulation of lipids, is the most common underlying cause for cardiovascular diseases. Monocytes and macrophages are major cells that contribute to the initiation and progression of atherosclerotic plaques. During this process, an accumulation of LDL-laden macrophages (foam cells) and an alteration in the extracellular matrix (ECM) organization leads to a local vessel stiffening. Current in vitro models are carried out onto two-dimensional tissue culture plastic and cannot replicate the relevant microenvironments. To bridge the gap between in vitro and in vivo conditions, we utilized three-dimensional (3D) collagen matrices that allowed us to mimic the ECM stiffening during atherosclerosis by increasing collagen density. First, human monocytic THP-1 cells were embedded into 3D collagen matrices reconstituted at low and high density. Cells were subsequently differentiated into uncommitted macrophages (M0) and further activated into pro- (M1) and anti-inflammatory (M2) phenotypes. In order to mimic atherosclerotic conditions, cells were cultured in the presence of oxidized LDL (oxLDL) and analyzed in terms of oxLDL uptake capability and relevant receptors along with their cytokine secretomes. Although oxLDL uptake and larger lipid size could be observed in macrophages in a matrix dependent manner, monocytes showed higher numbers of oxLDL uptake cells. By analyzing major oxLDL uptake receptors, both monocytes and macrophages expressed lectin-like oxidized low-density lipoprotein receptor-1 (LOX1), while enhanced expression of scavenger receptor CD36 could be observed only in M2. Notably, by analyzing the secretome of macrophages exposed to oxLDL, we demonstrated that the cells could, in fact, secrete adipokines and growth factors in distinct patterns. Besides, oxLDL appeared to up-regulate MHCII expression in all cells, while an up-regulation of CD68, a pan-macrophage marker, was found only in monocytes, suggesting a possible differentiation of monocytes into a pro-inflammatory macrophage. Overall, our work demonstrated that collagen density in the plaque could be one of the major factors driving atherosclerotic progression via modulation of monocyte and macrophages behaviors.
Highlights
Atherosclerosis is the dominant underlying causation of coronary heart disease and cerebrovascular disease [1] and statistically a major cause of morbidity and mortality worldwide [2,3]
While meaningful contributions have been made towards 3D modeling atherosclerosis in vitro, most studies have focused on co-culture of endothelial cells (ECs), smooth muscle cells (SMCs), and monocytes, in either physiological [18] or synthetic [20] scaffolds, while detailed individual contributions of macrophages still remain unclear
The duration of the treatment was experimentally determined by measuring oxidized low-density lipoproteins (LDL) (oxLDL) uptake at different timepoints, we observed that engulfment of LDL by all cells plateaued from 5 days onwards
Summary
Atherosclerosis is the dominant underlying causation of coronary heart disease and cerebrovascular disease [1] and statistically a major cause of morbidity and mortality worldwide [2,3]. The systematic formation of detrimental atherosclerotic plaques, via monocyte to macrophage differentiation to foam cell formation, which eventually narrows and occludes arteries, is well-reviewed elsewhere [4,5]. Monocytes infiltrate the lesion and differentiate into macrophages, which will uptake lipoproteins, resulting in foam cells that comprise the central core of atheromas that will progressively occlude the vessel. Atherosclerosis is considered a non-resolving inflammatory condition [2,6], akin to chronic wounds that will not resolve towards healing. On account of this close association with wound healing, and evidence that atherosclerotic plaques are vastly populated by pan-macrophage marker CD68+
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