Cholesterol Crystalline Domains Research Articles

Eicosapentaenoic acid (EPA) containing phospholipids modulate membrane structure

Background and Aims: Eicosapentaenoic acid (EPA) inhibits membrane lipid oxidation and cholesterol crystalline domain formation compared to other omega-3 fatty acids. These and other atheroprotective benefits of EPA may be mediated by distinct effects on membrane structure as a constituent of phospholipids (PL).

Abstract 784: Eicosapentaenoic Acid, Unlike Other Omega-3 Fatty Acids, Inhibits Membrane Cholesterol Crystalline Domains Under Conditions of Hyperglycemia

Prescription eicosapentaenoic acid (EPA) reduced cardiovascular events in the REDUCE-IT study population, including in patients with diabetes. During hyperglycemia, there is abnormal membrane cholesterol aggregation which has been linked to oxidative stress and plaque instability. EPA at a pharmacologic dose may preserve normal cholesterol distribution as compared to other omega-3 fatty acids (FAs), including docosahexaenoic acid (DHA), eicosatriaenoic acid (ETE) and α-linolenic acid (ALA). Membrane vesicles were prepared from dilinoleoylphosphatidylcholine (DLPC) at a cholesterol-to-phospholipid mole ratio of 0.6:1 and treated with the omega-3 FAs under conditions of hyperglycemia (200 mg/dL). Changes in membrane lipid organization and width were measured using small angle X-ray diffraction approaches. Cholesterol domains were identified by the presence of diffraction peaks corresponding to a unit cell periodicity or width of 34 Å. Results showed that only EPA was able to prevent the formation of cholesterol domains and had a membrane structure characterized by a normal width (54 Å) and cholesterol distribution despite hyperglycemia. But samples containing the other FAs had a biphasic membrane structure containing prominent cholesterol crystalline domains. The relative size of the cholesterol domains was greatest in the order of ALA>DHA>ETE. Thus, EPA preserved normal cholesterol distribution under conditions of hyperglycemia in a manner that was not reproduced with other omega-3 FAs. These data indicate differences between these long chain FAs and support a potential benefit for EPA in reducing atherosclerotic disease.

Eicosapentaenoic acid reduces membrane fluidity, inhibits cholesterol domain formation, and normalizes bilayer width in atherosclerotic-like model membranes

Cholesterol crystalline domains characterize atherosclerotic membranes, altering vascular signaling and function. Omega-3 fatty acids reduce membrane lipid peroxidation and subsequent cholesterol domain formation. We evaluated non-peroxidation-mediated effects of eicosapentaenoic acid (EPA), other TG-lowering agents, docosahexaenoic acid (DHA), and other long-chain fatty acids on membrane fluidity, bilayer width, and cholesterol domain formation in model membranes. In membranes prepared at 1.5:1 cholesterol-to-phospholipid (C/P) mole ratio (creating pre-existing domains), EPA, glycyrrhizin, arachidonic acid, and alpha linolenic acid promoted the greatest reductions in cholesterol domains (by 65.5%, 54.9%, 46.8%, and 45.2%, respectively) compared to controls; other treatments had modest effects. EPA effects on cholesterol domain formation were dose-dependent. In membranes with 1:1 C/P (predisposing domain formation), DHA, but not EPA, dose-dependently increased membrane fluidity. DHA also induced cholesterol domain formation without affecting temperature-induced changes in–bilayer unit cell periodicity relative to controls (d-space; 57Å–55Å over 15–30°C). Together, these data suggest simultaneous formation of distinct cholesterol-rich ordered domains and cholesterol-poor disordered domains in the presence of DHA. By contrast, EPA had no effect on cholesterol domain formation and produced larger d-space values relative to controls (60Å–57Å; p<0.05) over the same temperature range, suggesting a more uniform maintenance of lipid dynamics despite the presence of cholesterol. These data indicate that EPA and DHA had different effects on membrane bilayer width, membrane fluidity, and cholesterol crystalline domain formation; suggesting omega-3 fatty acids with differing chain length or unsaturation may differentially influence membrane lipid dynamics and structural organization as a result of distinct phospholipid/sterol interactions.

Eicosapentaenoic Acid (EPA), but not other Triglyceride-Lowering Agents, Inhibits Glucose-Induced Changes in Membrane Width and Cholesterol Domain Formation through a Potent Antioxidant Mechanism

Elevated glucose levels are causally related to lipid oxidation and membrane structural changes, including the formation of cholesterol crystalline domains. In this study, we tested the comparative effects of eicosapentaenoic acid (EPA) and other triglyceride-lowering agents (fenofibrate, niacin, and gemfibrozil) on glucose-induced changes in membrane structure. Iodometric approaches were used to measure lipid hydroperoxide (LOOH) formation in vesicles prepared as binary mixtures of dilinoleoylphosphatidylcholine and cholesterol at a cholesterol-to-phospholipid (C/P) mole ratio of 0.6:1. Membrane samples were treated with glucose at 200 mg/dL and subjected to autoxidation at 37°C for 72-96 hr. Small angle x-ray diffraction approaches were used to measure changes in membrane lipid structural organization, including bilayer width (d-space) and cholesterol crystalline domain formation. We found that glucose treatment significantly increased LOOH formation concomitant with a reduction in membrane bilayer width and an increase in cholesterol domain formation. EPA reduced LOOH formation by 90% as compared to glucose treatment alone (p<0.001) and preserved membrane width at 52.6 ± 0.3 A while inhibiting cholesterol domain formation. By contrast, there were pronounced reductions in membrane d-space with fenofibrate (45.4 ± 0.7 A), niacin (46.8 ± 0.9 A) and gemfibrozil (45.0 ± 1.3 A) as compared to vehicle (50.6 ± 1.9 A), all of which were highly significant (p<0.001) and associated with increased lipid oxidation and cholesterol domain formation. These data suggest that EPA inhibits glucose-induced changes in membrane width and cholesterol crystalline domain formation through a potent antioxidant mechanism. The other triglyceride-lowering agents enhanced glucose-induced reductions in membrane width and failed to interfere with cholesterol domain formation or lipid peroxidation. These findings provide insight into novel potentially atheroprotective benefits of EPA that are independent of lipid changes.

Docosahexaenoic Acid (DHA), but not Eicosapentaenoic Acid (EPA), Increases Both Membrane Fluidity and Cholesterol Crystalline Domain Formation in Lipid Vesicles

Elevated free cholesterol levels lead to atherosclerotic-like changes in membrane structure, including cholesterol crystalline domain formation. Omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may differentially affect cholesterol domain formation as a result of their distinct biophysical properties. In this study, we tested the comparative effects of EPA and DHA on lipid oxidation, membrane fluidity, and lipid structural organization. The lipid oxidation effects of each agent (10 μM) were measured in vesicles prepared as binary mixtures of dilinoleoylphosphatidylcholine and cholesterol and exposed to autoxidative conditions for 72 hr. Changes in membrane fluidity and lipid structural organization were measured using diphenylhexatriene (DPH) fluorescence anisotropy and small angle x-ray scattering approaches, respectively, in palmitoyloleoylphosphatidylcholine-enriched membrane vesicles prepared at 50 mol% cholesterol in the absence or presence of each agent at 5-10 mol% and tested over a broad range of temperatures. EPA and DHA were found to reduce lipid oxidation to a similar extent. However, only DHA affected membrane lipid fluidity, reducing DPH apparent rotational correlation values by 20% (19.4 to 15.6 nsec). Notably, DHA also induced cholesterol domain formation and reduced the lipid bilayer unit cell periodicity (d-space) from 57A to 55A with increasing temperature (15-30°C). By contrast, EPA had no effect on cholesterol domain formation and produced larger d-space values (60A to 57A, with increasing temperature) as compared to DHA (p<0.05). These data indicate that EPA and DHA, despite similar antioxidant benefits, had different effects on membrane bilayer width, lipid fluidity, and cholesterol crystalline domain formation. These results suggest that omega-3 fatty acids may differentially affect membrane lipid dynamics and structural organization as a result of their unique phospholipid/sterol interactions.

Formation of 3D cholesterol crystals from 2D nucleation sites in lipid bilayer membranes: implications for atherosclerosis.

Atherosclerosis is the major precursor of cardiovascular disease. The formation of cholesterol crystals in atherosclerotic plaques is associated with the onset of acute pathology. The cholesterol crystals induce physical injury in the plaque core, promoting cell apoptosis and triggering an increased inflammatory response. Herein we address the question of how cholesterol crystal formation occurs in atherosclerosis. We demonstrate that three-dimensional (3D) cholesterol crystals can undergo directed nucleation from bilayer membranes containing two-dimensional (2D) cholesterol crystalline domains. We studied crystal formation on supported lipid bilayers loaded with exogenous cholesterol and labeled using a monoclonal antibody that specifically recognizes ordered cholesterol arrays. Our findings show that 3D crystals are formed exclusively on the bilayer regions where there are segregated 2D cholesterol crystalline domains and that they form on the domains. This study has potentially significant implications for our understanding of the crucial step in the mechanism by which atherosclerotic lesions form.

Characterization of cholesterol crystalline domains in model and biological membranes using X-ray diffraction.

In this review, we discuss the use of small-angle X-ray diffraction approaches in studying the formation of cholesterol crystalline domains in membranes derived from model and biological sources. Numerous studies have shown that monomeric cholesterol can self-associate and form immiscible, membrane-restricted domains as a result of increased membrane concentration or systematic oxidation of membrane phospholipids. These domains are observed, in an X-ray diffraction pattern, as reflections describing a unit cell periodicity of 34 Å, which is consistent with cholesterol molecules arranged in a tail-to-tail, bilayer conformation. In vascular smooth muscle cells isolated from animal models of atherosclerosis, plasmalemmal cholesterol domain formation is associated with cellular dysfunction, including the disruption of calcium transport mechanisms. Cholesterol domains are also observed in macrophages and precede the deposition of extracellular cholesterol crystals in the atheroma. We have also shown that cholesterol domains can be produced in model membranes following exposure to oxidative stress and other disease-like conditions such as hyperglycemia. By contrast, cholesterol domains appear to be essential to the normal physiology of certain cell groups such as those of the human ocular lens. Cholesterol domains are a prominent structural feature of the lens fiber cell plasma membrane where they are believed to facilitate lens tissue transparency and interfere with various mechanisms of cataract formation. These contrasting biologic effects highlight the complex role that cholesterol domains play in cell plasma membrane structure-function relationships in both health and disease.KeywordsCholesterol DomainsFiber Cell Plasma MembraneFiber CellsCalcium Transport MechanismsTulenkoThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Eicosapentaenoic acid inhibits glucose-induced membrane cholesterol crystalline domain formation through a potent antioxidant mechanism

Lipid oxidation leads to endothelial dysfunction, inflammation, and foam cell formation during atherogenesis. Glucose also contributes to lipid oxidation and promotes pathologic changes in membrane structural organization, including the development of cholesterol crystalline domains. In this study, we tested the comparative effects of eicosapentaenoic acid (EPA), an omega-3 fatty acid indicated for the treatment of very high triglyceride (TG) levels, and other TG-lowering agents (fenofibrate, niacin, and gemfibrozil) on lipid oxidation in human low-density lipoprotein (LDL) as well as membrane lipid vesicles prepared in the presence of glucose (200mg/dL). We also examined the antioxidant effects of EPA in combination with atorvastatin o-hydroxy (active) metabolite (ATM). Glucose-induced changes in membrane structural organization were measured using small angle x-ray scattering approaches and correlated with changes in lipid hydroperoxide (LOOH) levels. EPA was found to inhibit LDL oxidation in a dose-dependent manner (1.0–10.0µM) and was distinguished from the other TG-lowering agents, which had no significant effect as compared to vehicle treatment alone. Similar effects were observed in membrane lipid vesicles exposed to hyperglycemic conditions. The antioxidant activity of EPA, as observed in glucose-treated vesicles, was significantly enhanced in combination with ATM. Glucose treatment produced highly-ordered, membrane-restricted, cholesterol crystalline domains, which correlated with increased LOOH levels. Of the agents tested in this study, only EPA inhibited glucose-induced cholesterol domain formation. These data demonstrate that EPA, at pharmacologic levels, inhibits hyperglycemia-induced changes in membrane lipid structural organization through a potent antioxidant mechanism associated with its distinct, physicochemical interactions with the membrane bilayer.

Eicosapentaenoic Acid Inhibited Glucose-Induced Membrane Lipid Peroxidation and Cholesterol Crystalline Domain Formation

Eicosapentaenoic Acid Inhibited Glucose-Induced Membrane Lipid Peroxidation and Cholesterol Crystalline Domain Formation

Organization and Dynamics of Cholesterol Crystalline Domains using EPR Spin-Labeling

EPR spin-labeling methods were used to study the organization and dynamics of cholesterol molecules in membranes formed from Chol/PL (cholesterol/phospholipid) mixtures. Membranes made from phospholipids with different cholesterol solubility thresholds (CST) were investigated. It was confirmed using the EPR discrimination methods that cholesterol crystalline domains (CCDs) were present in all of the membrane suspensions when the mixing ratio exceeded the CST. The behavior of phospholipids was monitored with phospholipid analogue spin labels (n-PCs), and cholesterol with the cholesterol analogue spin labels CSL and ASL. Results indicated that phospholipid and cholesterol mixtures can form a membrane suspension up to a mixing ratio exceeding significantly the CST. EPR spectra for n-PC indicated that phospholipids exist in these suspensions in the lipid-bilayer-like structures. Spectral characteristics of n-PCs (spin labels located outside the CCD) change with increase in the cholesterol content up to and beyond the CST. These results present strong evidence that the CCD forms an integral part of the phospholipid bilayer when formed from Chol/PL mixture up to a mixing ratio of 2 to 3. EPR spectra for CSL and ASL in the CCD and the PCD were very similar indicating that in both domains cholesterol exists in the lipid-bilayer-like structures. In the CCD cholesterol molecules are more ordered and sense more rigid environment than in the PCD. This difference is small and can be compared to that induced in the PCD by the ∼10°C decrease of temperature. Thus, cholesterol molecules are unexpectedly dynamic in the CCD what should enhance their active interaction with the PCD. It is suggested that the EPR spin-labeling approach can discriminate the fraction of cholesterol that forms the CCD within the phospholipid bilayer from the fraction that forms the cholesterol structures outside the bilayer.

Discrimination and Characterization of Cholesterol Crystalline Domains using EPR Spin-Labeling: Application to Lens Lipid Membranes

Saturation-recovery EPR at X-band (9.4 GHz) and W-band (94 GHz) was used to discriminate cholesterol crystalline domains (CCDs) in model phospholipid membranes and membranes made of pigs lens lipids. We used dual-probe saturation-recovery EPR approach with cholesterol analogue spin labels, CSL or ASL, and oxygen (hydrophobic) or NiEDDA (polar) relaxation agents. The CCD was present in all of the phospholipid membranes when the cholesterol-to-phospholipid mixing ratio exceeded the cholesterol solubility threshold. Saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. However, in membranes made from nuclear lipids, two domains were detected and were assigned to the bulk phospholipid-cholesterol domain and the CCD, respectively. Profiles of the oxygen transport parameter (oxygen diffusion-concentration product) across these domains contain significant structural information. These profiles also allow calculating the oxygen permeability coefficient across domain. The evaluated upper limit of the oxygen permeability coefficient across the CCD at 37°C (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can form a barrier for oxygen transport in the lens nucleus. The new capabilities in measurements of the oxygen transport parameter at W-band are demonstrated and compared with results obtained at X-band. The loop-gap resonators used for W-band measurements have a sample volume of 30 nL, while the sample volume of the loop-gap resonator used for X-band is 3µL. These specifications ensure feasibility of experiments planned for samples with a limited sample volume. For example, the oxygen transport parameter profiles measured at X-band for lens lipid membranes were obtained for samples prepared from 50 - 100 eyes. Reliable profiles at W-band can be obtained based on samples prepared from one eye.

Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a 2-year-old cow were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation–recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of 6-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454–1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid–cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 °C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.

Glucose promotes membrane cholesterol crystalline domain formation by lipid peroxidation

Oxidative damage to vascular cell membrane phospholipids causes physicochemical changes in membrane structure and lipid organization, contributing to atherogenesis. Oxidative stress combined with hyperglycemia has been shown to further increase the risk of vascular and metabolic diseases. In this study, the effects of glucose on oxidative stress-induced cholesterol domain formation were tested in model membranes containing polyunsaturated fatty acids and physiologic levels of cholesterol. Membrane structural changes, including cholesterol domain formation, were characterized by small angle X-ray scattering (SAXS) analysis and correlated with spectrophotometrically-determined lipid hydroperoxide levels. Glucose treatment resulted in a concentration-dependent increase in lipid hydroperoxide formation, which correlated with the formation of highly-ordered cholesterol crystalline domains (unit cell periodicity of 34 Å) as well as a decrease in overall membrane bilayer width. The effect of glucose on lipid peroxidation was further enhanced by increased levels of cholesterol. Treatment with free radical-scavenging agents inhibited the biochemical and structural effects of glucose, even at elevated cholesterol levels. These data demonstrate that glucose promotes changes in membrane organization, including cholesterol crystal formation, through lipid peroxidation.

Hyperglycemia Promotes Membrane Cholesterol Crystalline Domain Formation Through Lipid Peroxidation: Inhibition with Atorvastatin Metabolite

Insulin resistance and poor glycemic control contribute to atherogenesis through the chemical and structural modification of cell membrane lipids. The direct contribution of glucose to these membrane alterations, however, is not well understood. In this study, small angle x-ray diffraction and spectrophotometry were used to examine the autoxidative effects of glucose on lipid oxidation and structural organization in model membranes comprised of dilinoleoylphosphatidylcholine (DLPC) and cholesterol. Membranes were prepared at cholesterol-to-phospholipid (C/P) mole ratios ranging from 0.2 to 0.8 in order to model physiologic and hyperlipidemic conditions. Changes in membrane lipid organization and unit cell periodicity (d-space) were correlated with lipid hydroperoxide (LOOH) concentration measured at 24 hr intervals. The effects of glucose on lipid peroxidation were more pronounced at elevated levels of membrane cholesterol, with LOOH levels 20% higher at 0.8 C/P than at 0.2 C/P. At 0.6 C/P, glucose treatment resulted in a concentration-dependent increase in LOOH formation as compared to control. These changes corresponded to a reduction in membrane bilayer width (51 A to 49 A) and the progressive formation of highly-ordered cholesterol crystalline domains (d-space value of 34 A). Treatment with atorvastatin hydroxy metabolite, a statin with scavenger antioxidant properties, inhibited the membrane-altering effects of hyperglycemia in a dose-dependent manner, even at elevated cholesterol levels. These data demonstrate that glucose directly stimulates lipid peroxidation and subsequent changes in membrane structure, including the formation of immiscible cholesterol crystalline domains. Insights from this study may also serve as a model for better understanding the membrane structural changes associated with diabetes and related complications.

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55–1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid–cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid–cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid–cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 °C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.