Atherosclerosis is a chronic progressive disease characterized by the sub-endothelial accumulation of Low Density Lipoproteins (LDL), cholesterol-esters and cholesterol crystals [1]. Cholesterol crystals are difficult to dissolve by means of known physiological mechanisms and hold pro-inflammatory properties perpetuating the atherogenic process. The combination of these factors facilitates the instability and the eventual rupture of the atheromatous plaque leading to arterial thrombosis. Macrophage cells have a crucial role in LDL metabolism in the atheromatous tissue and the process of cholesterol crystal formation [2]. However, the exact mechanism by which crystals form in atherosclerotic plaques is not yet understood.
Cholesterol crystals are formed by J774 macrophages fed with CE-droplets, presumably intracellularly, from lipid loaded lysosomes [3]. Crystals formed within J774 macrophages are characterized by a needle or plate like appearance. Similar characteristics of cholesterol crystals are observed in intracellular locations of human macrophages and the extracellular matrix of human atherosclerotic lesions. We have studied cholesterol crystals formation in macrophages nucleating from cholesterol nano-domains segregating within the cell plasma membrane or from intracellular locations [4,5].
The main objective of this research is to elucidate the possible mechanisms responsible for cholesterol crystals formation in macrophages. Here we demonstrate that cholesterol crystals are formed within viable J774A.1 macrophages fed with Ac-LDL, implying that the crystallization process may occur during the initial stages of LDL accumulation and is not necessarily associated with or is a product of macrophage cell death. To allow 3D observation and analysis of macrophage foam cells and atherosclerotic tissues under cryogenic conditions, we employ cryo-Focused Ion Beam/Scanning (cryo-FIB-SEM) block face serial imaging [6]. We are currently developing a method comprising cryo-SEM combined with Cathodoluminescence for studying cholesterol crystals formation in macrophage cell culture models (J774A.1/Human monocyte derived macrophages) and rabbit atherosclerotic tissues. Cathodoluminescence signal provided by cholesterol crystals and/or fluorescently labeled cholesterol crystal-specific antibodies is being assessed.
Understanding the underlying mechanisms of cholesterol crystals formation in macrophage model cells and atherosclerotic plaques can provide useful information toward possible therapeutic approaches.
References:
- Kruth, H.S., Lipoprotein cholesterol and atherosclerosis. Current molecular medicine, 2001. 1(6): p. 633-653.
- Kruth, H., Macrophage foam cells and atherosclerosis. Frontiers in bioscience: a journal and virtual library, 2001. 6: p. D429-55.
- Tangirala, R.K., et al., Formation of cholesterol monohydrate crystals in macrophage-derived foam cells. Journal of lipid research, 1994. 35(1): p. 93-104.
- Varsano, N., et al., Development of Correlative Cryo-soft X-ray Tomography and Stochastic Reconstruction Microscopy. A Study of Cholesterol Crystal Early Formation in Cells. Journal of the American Chemical Society, 2016. 138(45): p. 14931-14940.
- Varsano N., et al., Two Polymorphic Cholesterol Monohydrate Crystal Structures Form in Macrophage-derived Foam Relevance to Atherosclerosis PNAS, under revision.
- Vidavsky, N., et al., Cryo-FIB-SEM serial milling and block face imaging: Large volume structural analysis of biological tissues preserved close to their native state. Journal of structural biology, 2016. 196(3): p. 487-495.
