Polymorphism, Structure, and Nucleation of Cholesterol·H2O at Aqueous Interfaces and in Pathological Media: Revisited from a Computational Perspective.

We revisit the important issues of polymorphism, structure, and nucleation of cholesterol·H2O using first-principles calculations based on dispersion-augmented density functional theory. For the lesser known monoclinic polymorph, we obtain a fully extended H-bonded network in a structure akin to that of hexagonal ice. We show that the energy of the monoclinic and triclinic polymorphs is similar, strongly suggesting that kinetic and environmental effects play a significant role in determining polymorph nucleation. Furthermore, we find evidence in support of various O-H···O bonding motifs in both polymorphs that may result in hydroxyl disorder. We have been able to explain, via computation, why a single cholesterol bilayer in hydrated membranes always crystallizes in the monoclinic polymorph. We rationalize what we believe is a single-crystal to single-crystal transformation of the monoclinic form on increased interlayer growth beyond that of a single cholesterol bilayer, interleaved by a water bilayer. We show that the ice-like structure is also relevant to the related cholestanol·2H2O and stigmasterol·H2O crystals. The structure of stigmasterol hydrate both as a trilayer film at the air-water interface and as a macroscopic crystal further assists us in understanding the polymorphic and thermal behavior of cholesterol·H2O. Finally, we posit a possible role for one of the sterol esters in the crystallization of cholesterol·H2O in pathological environments, based on a composite of a crystalline bilayer of cholesteryl palmitate bound epitaxially as a nucleating agent to the monoclinic cholesterol·H2O form.

Two polymorphic cholesterol monohydrate crystal structures form in macrophage culture models of atherosclerosis.

The formation of atherosclerotic plaques in the blood vessel walls is the result of LDL particle uptake, and consequently of cholesterol accumulation in macrophage cells. Excess cholesterol accumulation eventually results in cholesterol crystal deposition, the hallmark of mature atheromas. We followed the formation of cholesterol crystals in J774A.1 macrophage cells with time, during accumulation of LDL particles, using a previously developed correlative cryosoft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM) technique. We show, in the initial accumulation stages, formation of small quadrilateral crystal plates associated with the cell plasma membrane, which may subsequently assemble into large aggregates. These plates match crystals of the commonly observed cholesterol monohydrate triclinic structure. Large rod-like cholesterol crystals form at a later stage in intracellular locations. Using cryotransmission electron microscopy (cryo-TEM) and cryoelectron diffraction (cryo-ED), we show that the structure of the large elongated rods corresponds to that of monoclinic cholesterol monohydrate, a recently determined polymorph of the triclinic crystal structure. These monoclinic crystals form with an unusual hollow cylinder or helical architecture, which is preserved in the mature rod-like crystals. The rod-like morphology is akin to that observed in crystals isolated from atheromas. We suggest that the crystals in the atherosclerotic plaques preserve in their morphology the memory of the structure in which they were formed. The identification of the polymorph structure, besides explaining the different crystal morphologies, may serve to elucidate mechanisms of cholesterol segregation and precipitation in atherosclerotic plaques.

The Effect of the Phospholipid Bilayer Environment on Cholesterol Crystal Polymorphism.

Cholesterol crystallization from mixtures of unesterified cholesterol with phospholipids and cholesterol esters is believed to be a key event in atherosclerosis progression. Not much is understood, however, about the influence of the lipid environment on cholesterol crystallization. Here we study cholesterol monohydrate crystal formation from mixed bilayers with palmitoyl-oleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC) and sphingomyelin. We show that disordered phospholipids and sphingomyelin stabilize the formation of crystal plates of the triclinic cholesterol monohydrate polymorph, whereas saturated glycerolipids stabilize helical and tubular crystals of the metastable monoclinic polymorph. We followed the subsequent transformation of these helical crystals into the stable triclinic plates. Discovering the relations between membrane lipid composition and cholesterol crystal polymorphism may provide important clues to the understanding of cholesterol crystal formation in atherosclerosis.

The Effect of the Phospholipid Bilayer Environment on Cholesterol Crystal Polymorphism.

Invited for this month's cover are the group of Prof. Lia Addadi at the Weizmann Institute of Science, Israel and collaborators at the Università Degli Studi di Milano, Italy, and the ALBA Synchrotron Light Source, Spain. The front cover shows how cholesterol crystals form in macrophage cells and in lipid bilayers of different compositions. Cholesterol monohydrate stable triclinic crystals form in vitro as rhomb-shaped plates, whereas the monoclinic crystals fold into tubular or helical shapes. Read the full text of the article at 10.1002/cplu.201800632.