Low Density Lipoprotein Cholesteryl Ester Research Articles

Structural insights on the deformations induced by various mutations on cholesteryl ester transfer protein

Cholesteryl Ester Transfer Protein (CETP) is a plasma glycoprotein that intervenes the reverse cholesterol transport (RCT) by equimolar exchange of Cholesteryl esters (CE) and Triglycerides (TGs) between anti-atherogenic High-Density Lipoproteins (HDLs) and pro-atherogenic Low-Density Lipoproteins (LDLs) resulting in the increased concentration of CEs in LDL. This is a potential cause for the formation of atherosclerotic plaques in blood vessels leading to fatality. Therefore, blocking the function of CETP has emerged as a novel strategy for suppressing atherosclerotic plaques. The crystal structure of CETP revealed two Cholesteryl esters (CEs) in the hydrophobic tunnel and two phospholipids (PLs) plugged on the concave surface. Previous lipid transfer assay experimental studies have shown a substantial reduction in the neutral lipid transfer in [R201S] and [I443W, V198W] mutants. However, the protein conformational arrangements due to the mutations present in the CETP system leading to a decrease in the transfer rate of neutral lipids is not explored. Thus, I explored the reason behind the decreased transfer rate in mutants using molecular dynamics (MD) simulations and free energy calculations. Resulting evidences show that R201S mutant induces unfavorable bending angle to CETP with a decreased binding efficiency between N-terminal phospholipid of CETP with S201. Also, an unfavorable conformation state of TGs is formed which makes them difficult to transfer across CETP. Likewise, [I443W, V198W] mutant induces unfavorable CE, TG, and bending angle conformation to CETP impeding neutral lipid transfer. Thus, my results provide sufficient insights on the causation for a decreased transfer rate as reported earlier. The detailed understanding obtained here could help in developing a new strategy in preventing the function of CETP by blocking the role of potential hot spot residues.

SR-BI mediates neutral lipid sorting from LDL to lipid droplets and facilitates their formation.

SR-BI binds various lipoproteins, including HDL, LDL as well as VLDL, and mediates selective cholesteryl ester (CE) uptake. HDL derived CE accumulates in cellular lipid droplets (LDs), which also store triacylglycerol (TAG). We hypothesized that SR-BI could significantly facilitate LD formation, in part, by directly transporting LDL derived neutral lipids (NL) such as CE and TAG into LDs without lipolysis and de novo lipid synthesis. SR-BI overexpression greatly increased LDL uptake and LD formation in stably transfected HeLa cells (SR-BI-HeLa). LDs isolated from SR-BI-HeLa contained 4- and 7-times more CE and TAG, respectively, than mock-transfected HeLa (Mock-HeLa). In contrast, LDL receptor overexpression in HeLa (LDLr-HeLa) greatly increased LDL uptake, degradation with moderate 1.5- and 2-fold increases of CE and TAG, respectively. Utilizing CE and TAG analogs, BODIPY-TAG (BP-TAG) and BODIPY-CE (BP-CE), for tracking LDL NL, we found that after initial binding of LDL to SR-BI-HeLa, apoB remained at the cell surface, while BP-CE and BP-TAG were sorted and simultaneously transported together to LDs. Both lipids demonstrated limited internalization to lysosomes or endoplasmic reticulum in SR-BI-HeLa. In LDLr-HeLa, NLs demonstrated clear lysosomal sequestration without their sorting to LDs. An inhibition of TAG and CE de novo synthesis by 90–95% only reduced TAG and CE LD content by 45–50%, and had little effect on BP-CE and BP-TAG transport to LDs in SR-BI HeLa. Furthermore, intravenous infusion of 1–2 mg of LDL increased liver LDs in normal (WT) but not in SR-BI KO mice. Mice transgenic for human SR-BI demonstrated higher liver LD accumulation than WT mice. Finally, Electro Spray Infusion Mass Spectrometry (ESI-MS) using deuterated d-CE found that LDs accumulated up to 40% of unmodified d-CE LDL. We conclude that SR-BI mediates LDL-induced LD formation in vitro and in vivo. In addition to cytosolic NL hydrolysis and de novo lipid synthesis, this process includes selective sorting and transport of LDL NL to LDs with limited lysosomal NL sequestration and the transport of LDL CE, and TAG directly to LDs independently of de novo synthesis.

Simultaneous Determination of Biliary and Intestinal Cholesterol Secretion Reveals That CETP (Cholesteryl Ester Transfer Protein) Alters Elimination Route in Mice.

Determine the impact of CETP (cholesteryl ester transfer protein) on the route of cholesterol elimination in mice. Approach and Results: We adapted our protocol for biliary cholesterol secretion with published methods for measuring transintestinal cholesterol elimination. Bile was diverted and biliary lipid secretion maintained by infusion of bile acid. The proximal small bowel was perfused with bile acid micelles. In high-fat, high-cholesterol-fed mice, the presence of a CETP transgene increased biliary cholesterol secretion at the expense of transintestinal cholesterol elimination. The increase in biliary cholesterol secretion was not associated with increases in hepatic SR-BI (scavenger receptor BI) or ABCG5 (ATP-binding cassette G5) ABCG8. The decline in intestinal cholesterol secretion was associated with an increase in intestinal Niemann-Pick disease, type C1, gene-like 1 mRNA. Finally, we followed the delivery of HDL (high-density lipoprotein) or LDL (low-density lipoprotein) cholesteryl esters (CE) from plasma to bile and intestinal perfusates. HDL-CE favored the biliary pathway. Following high-fat feeding, the presence of CETP directed HDL-CE away from the bile and towards the intestine. The presence of CETP increased LDL-CE delivery to bile, whereas the appearance of LDL-CE in intestinal perfusate was near the lower limit of detection. Biliary and intestinal cholesterol secretion can be simultaneously measured in mice and used as a model to examine factors that alter cholesterol elimination. Plasma factors, such as CETP, alter the route of cholesterol elimination from the body. Intestinal and biliary cholesterol secretion rates are independent of transhepatic or transintestinal delivery of HDL-CE, whereas LDL-CE was eliminated almost exclusively in the hepatobiliary pathway.

Metabolomic correlates of central adiposity and earlier-life body mass index

BMI is correlated with circulating metabolites, but few studies discuss other adiposity measures, and little is known about metabolomic correlates of BMI from early life. We investigated associations between different adiposity measures, BMI from childhood through adulthood, and metabolites quantified from serum using 1H NMR spectroscopy in 900 British men and women aged 60-64. We assessed BMI, waist-to-hip ratio (WHR), android-to-gynoid fat ratio (AGR), and BMI from childhood through adulthood. Linear regression with Bonferroni adjustment was performed to assess adiposity and metabolites. Of 233 metabolites, 168; 126; and 133 were associated with BMI, WHR, and AGR at age 60-64, respectively. Associations were strongest for HDL, particularly HDL particle size-e.g., there was 0.08 SD decrease in HDL diameter (95% CI: 0.07-0.10) with each unit increase in BMI. BMI-adjusted AGR or WHR were associated with 31 metabolites where there was no metabolome-wide association with BMI. We identified inverse associations between BMI at age 7 and glucose or glycoprotein at age 60-64 and relatively large LDL cholesteryl ester with postadolescent BMI gains. In summary, we identified metabolomic correlates of central adiposity and earlier-life BMI. These findings support opportunities to leverage metabolomics in early prevention of cardiovascular risk attributable to body fatness.

Abstract 364: An NPC1 and NPC2 Independent Pathway Mediates Trafficking of Cholesterol From Lysosomes to the Extracellular Matrix

We previously reported that cholesterol-enriched macrophages deposit cholesterol into the extracellular matrix and this process depends on ABCA1 and ABCG1. The extracellular cholesterol deposits can be mobilized by HDL, or ApoA-I, the latter dependent on ABCA1-mediated lipidation of ApoA-I. The objective of the current study was to determine the effects of other genes and inhibitors that affect cellular cholesterol trafficking on the deposition of extracellular cholesterol. We used a monoclonal antibody that labels cholesterol microdomains to detect the extracellular cholesterol deposits. Progesterone and U18666A previously were shown to induce accumulation of cholesterol within lysosomes, and we found that these agents also blocked macrophage deposition of extracellular cholesterol. Brefeldin A, an inhibitor of golgi and vesicular trafficking, inhibited extracellular cholesterol deposition. On the other hand, vacuolin, an agent that inhibits the fusion between autophagosomes and lysosomes and thereby induces cellular accumulation of autophagosomes, did not inhibit extracellular cholesterol deposition. To examine the effect of mutant genes known to affect cholesterol trafficking, we examined mutant cultured fibroblasts that were cholesterol-enriched by incubation with LDL plus the LXR agonist, T0901317. Like macrophages, normal cholesterol-enriched fibroblasts deposited extracellular cholesterol that was dependent on ABCA1, because ABCA1-deficient fibroblasts did not deposit extracellular cholesterol. While progesterone and U18666A inhibited fibroblast extracellular cholesterol deposition, surprisingly, fibroblasts deficient in NPC1 or NPC2, which also abnormally accumulate cholesterol within lysosomes during cholesterol enrichment, nevertheless, deposited extracellular cholesterol similar to normal fibroblasts. That lysosomal hydrolysis of LDL cholesteryl ester is involved in extracellular cholesterol deposition was shown by substantially reduced extracellular cholesterol deposition by fibroblasts lacking acid cholesteryl ester hydrolase. Our findings indicate that a pathway not dependent on NPC1 or NPC2 mediates trafficking of cholesterol from lysosomes to the extracellular space.

Routes and mechanisms of post-endosomal cholesterol trafficking: A story that never ends.

Mammalian cells acquire most exogenous cholesterol through receptor-mediated endocytosis of low-density lipoproteins (LDLs). After internalization, LDL cholesteryl esters are hydrolyzed to release free cholesterol, which then translocates to late endosomes (LEs)/lysosomes (LYs) and incorporates into the membranes by co-ordinated actions of Niemann-Pick type C (NPC) 1 and NPC2 proteins. However, how cholesterol exits LEs/LYs and moves to other organelles remain largely unclear. Growing evidence has suggested that nonvesicular transport is critically involved in the post-endosomal cholesterol trafficking. Numerous sterol-transfer proteins (STPs) have been identified to mediate directional cholesterol transfer at membrane contact sites (MCSs) formed between 2 closely apposed organelles. In addition, a recent study reveals that lysosome-peroxisome membrane contact (LPMC) established by a non-STP synaptotagmin VII and a specific phospholipid phosphatidylinositol 4,5-bisphosphate also serves as a novel and important path for LDL-cholesterol trafficking. These findings highlight an essential role of MCSs in intracellular cholesterol transport, and further work is needed to unveil how various routes are regulated and integrated to maintain proper cholesterol distribution and homeostasis in eukaryotic cells.

Evaluation of Antiatherogenic Properties of Ezetimibe Using 3H-Labeled Low-Density-Lipoprotein Cholesterol and 99mTc-cAbVCAM1-5 SPECT in ApoE-/- Mice Fed the Paigen Diet.

The addition of ezetimibe, an intestinal cholesterol absorption inhibitor, to statin therapy has recently shown clinical benefits in the Improved Reduction of Outcomes: Vytorin Efficacy International Trial by reducing low-density-lipoprotein (LDL) cholesterol levels more than statin therapy alone. Here, we investigated the mechanisms by which inhibition of intestinal cholesterol absorption might contribute to the clinically observed reduction in cardiovascular events by evaluating its effect on inflammatory plaque development in apolipoprotein E-/- mice. Methods: Apolipoprotein E-/- mice were fed the Paigen diet (1.25% cholesterol, 0.5% cholic acid, and 15% fat) without or with ezetimibe (7 mg/kg/d) for 6 wk. In a first set of mice (n = 15), we intravenously injected 3H-cholesteryl oleate-labeled human LDL to test whether ezetimibe promotes LDL-derived cholesterol fecal excretion. In a second set (n = 20), we used the imaging agent 99mTc-cAbVCAM1-5 to evaluate expression of an inflammatory marker, vascular cell adhesion molecule 1 (VCAM-1), in atherosclerotic plaques. In a third set (n = 21), we compared VCAM-1 expression with 99mTc-cAbVCAM1-5 uptake in various tissues. Results: Mice treated with ezetimibe showed a 173% higher LDL-cholesteryl ester plasma disappearance rate (P < 0.001 vs. control) after 3H-cholesteryl oleate-labeled LDL injection. At 96 h after injection, the hepatic fraction of 3H-tracer was 61% lower in mice treated with ezetimibe (P < 0.001). Meanwhile, LDL-derived 3H-cholesterol excretion in the feces was 107% higher (P < 0.001). The antiatherogenic effect of ezetimibe monitored by 99mTc-cAbVCAM1-5 SPECT showed a 49% reduction in aortic tracer uptake (percentage injected dose per cubic centimeter, 0.95 ± 0.04 vs. 1.87 ± 0.11; P < 0.01). In addition to hypercholesterolemia, the proinflammatory Paigen diet significantly increased VCAM-1 expression with respect to the control group in various tissues, including the aorta, and this expression correlated strongly with 99mTc-cAbVCAM1-5 uptake (r = 0.75; P < 0.05). Conclusion: Inhibition of intestinal cholesterol absorption with ezetimibe promotes antiatherosclerotic effects through increased LDL cholesterol catabolism and LDL-derived cholesterol fecal excretion and reduces inflamed atherosclerotic plaques. These mechanisms may contribute to the benefits of adding ezetimibe to a statin therapy.

Abstract 330: Ezetimibe Prevents Atherogenesis Through Increased Catabolism and Fecal Excretion of LDL-cholesterol and Reduced Atherosclerotic Plaque Inflammation in Apolipoprotein E Knock-out Mice Fed a Paigen Diet

Background: Intestinal cholesterol absorption inhibitor ezetimibe (EZE) added to a statin therapy has demonstrated benefits in the IMPROVE-IT trial by further reducing LDL-cholesterol levels than statin therapy alone. We investigated the mechanisms by which EZE could contribute to cardiovascular events reduction in apolipoprotein E knock-out (apoE ko) mice. Methods: ApoE ko mice were fed a Paigen diet without (control) or with EZE (7mg/kg/day) for 6 weeks. To evaluate the effects of EZE on LDL-cholesterol metabolism and excretion, a first set of mice was injected intravenously with 3 H-cholesteryl oleate labeled human LDL. A second set of mice was used for in vivo SPECT/CT imaging of 99m Tc-cAbVCAM1-5, a single domain antibody directed against the Vascular Cell Adhesion Molecule-1 (VCAM-1), which was used as a marker of inflamed atherosclerotic plaques. The same mice were sacrificed for autoradiography and histology of aortic atherosclerotic plaques. Results: Compared with control, EZE treatment for 6 weeks induced a significant 41% and 65% reduction in plasma total cholesterol levels and atherosclerotic plaque area, respectively. After injection of 3 H-cholesteryl oleate labeled human LDL, mice treated with EZE showed a 173% higher LDL-cholesteryl ester catabolism (p&lt;0.001 vs. control). At time 96 hours after radiolabeled LDL injection, 3 H-tracer hepatic recovery was reduced by 61% with EZE (p&lt;0.001). Meanwhile, LDL-derived 3 H-tracer excretion in the feces was increased by 107% in the fecal cholesterol fraction (p&lt;0.001). Similar trends were observed for hepatic cholesterol levels and fecal cholesterol mass excretion, with a 75% reduction and 99% increase with EZE, respectively (both p&lt;0.001). After intravenous injection of 99m Tc-cAbVCAM1-5, mice treated with EZE also showed a significant 52% reduction in aortic uptake, which was confirmed by significant reduction in tracer uptake in ex vivo biodistribution and autoradiography analysis. Conclusion: EZE promotes anti-atherosclerotic effects through increased LDL-cholesterol catabolism and LDL-derived cholesterol fecal excretion, and reduced inflamed atherosclerotic plaques. These mechanisms may contribute to the benefits of adding EZE to a statin therapy.

High-oleic canola oil consumption enriches LDL particle cholesteryl oleate content and reduces LDL proteoglycan binding in humans

Oleic acid consumption is considered cardio-protective according to studies conducted examining effects of the Mediterranean diet. However, animal models have shown that oleic acid consumption increases LDL particle cholesteryl oleate content which is associated with increased LDL-proteoglycan binding and atherosclerosis. The objective was to examine effects of varying oleic, linoleic and docosahexaenoic acid consumption on human LDL-proteoglycan binding in a non-random subset of the Canola Oil Multi-center Intervention Trial (COMIT) participants. COMIT employed a randomized, double-blind, five-period, cross-over trial design. Three of the treatment oil diets: 1) a blend of corn/safflower oil (25:75); 2) high oleic canola oil; and 3) DHA-enriched high oleic canola oil were selected for analysis of LDL-proteoglycan binding in 50 participants exhibiting good compliance. LDL particles were isolated from frozen plasma by gel filtration chromatography and LDL cholesteryl esters quantified by mass-spectrometry. LDL-proteoglycan binding was assessed using surface plasmon resonance. LDL particle cholesterol ester fatty acid composition was sensitive to the treatment fatty acid compositions, with the main fatty acids in the treatments increasing in the LDL cholesterol esters. The corn/safflower oil and high-oleic canola oil diets lowered LDL-proteoglycan binding relative to their baseline values (p = 0.0005 and p = 0.0012, respectively). At endpoint, high-oleic canola oil feeding resulted in lower LDL-proteoglycan binding than corn/safflower oil (p = 0.0243) and DHA-enriched high oleic canola oil (p = 0.0249), although high-oleic canola oil had the lowest binding at baseline (p = 0.0344). Our findings suggest that high-oleic canola oil consumption in humans increases cholesteryl oleate percentage in LDL, but in a manner not associated with a rise in LDL-proteoglycan binding.

Minimally oxidized LDL inhibits macrophage selective cholesteryl ester uptake and native LDL-induced foam cell formation

Scavenger receptor-mediated uptake of oxidized LDL (oxLDL) is thought to be the major mechanism of foam cell generation in atherosclerotic lesions. Recent data has indicated that native LDL is also capable of contributing to foam cell formation via low-affinity receptor-independent LDL particle pinocytosis and selective cholesteryl ester (CE) uptake. In the current investigation, Cu(2+)-induced LDL oxidation was found to inhibit macrophage selective CE uptake. Impairment of selective CE uptake was significant with LDL oxidized for as little as 30 min and correlated with oxidative fragmentation of apoB. In contrast, LDL aggregation, LDL CE oxidation, and the enhancement of scavenger receptor-mediated LDL particle uptake required at least 3 h of oxidation. Selective CE uptake did not require expression of the LDL receptor (LDL-R) and was inhibited similarly by LDL oxidation in LDL-R(-/-) versus WT macrophages. Inhibition of selective uptake was also observed when cells were pretreated or cotreated with minimally oxidized LDL, indicating a direct inhibitory effect of this oxLDL on macrophages. Consistent with the effect on LDL CE uptake, minimal LDL oxidation almost completely prevented LDL-induced foam cell formation. These data demonstrate a novel inhibitory effect of mildly oxidized LDL that may reduce foam cell formation in atherosclerosis.

Hexadecenoic Fatty Acid Isomers: A Chemical Biology Approach for Human Plasma Biomarker Development

Hexadecenoic fatty acids are monounsaturated lipid components, which are interesting targets of plasma lipidomic studies and biomarker development. The main positional isomers, palmitoleic (9-cis-16:1) and sapienic acids (6-cis-16:1), have an endogenous origin from palmitic acid, the former being recognized as a component of adipose tissue with signaling activity, whereas the latter is mainly reported as a component of sebum. The trans 16:1 isomers are attributed so far to dietary sources of industrial and dairy fats, whereas the endogenous formation due to the free radical-mediated isomerization can represent an emerging, yet unexplored, pathway connected to cellular stress. Herein, we report a chemical biology approach for the development of hexadecenoic fatty acids as plasma biomarkers, with the first synthesis of 6-trans-16:1 and the efficient analytical setup with unambiguous assignment of 16:1 double bond position and geometry, which was applied to human commercial LDL and plasma cholesteryl esters. Sapienic acid was identified together with its geometrical trans isomer for the first time. The quantitation of hexadecenoic fatty acid isomers evidenced their different levels in the two lipid classes and LDL fractions, making us foresee interesting applications to the metabolic evaluation of fatty acid pathways. These findings open new perspectives for plasma lipidomics involving monounsaturated fatty acids, highlighting future developments for their evaluation in different health conditions including free radical stress.

Scavenger receptor-BI is a receptor for lipoprotein(a)

Scavenger receptor class B type I (SR-BI) is a multi-ligand receptor that binds a variety of lipoproteins, including high density lipoprotein (HDL) and low density lipoprotein (LDL), but lipoprotein(a) [Lp(a)] has not been investigated as a possible ligand. Stable cell lines (HEK293 and HeLa) expressing human SR-BI were incubated with protein- or lipid-labeled Lp(a) to investigate SR-BI-dependent Lp(a) cell association. SR-BI expression enhanced the association of both (125)I- and Alexa Fluor-labeled protein from Lp(a). By confocal microscopy, SR-BI was also found to promote the internalization of fluorescent lipids (BODIPY-cholesteryl ester (CE)- and DiI-labeled) from Lp(a), and by immunocytochemistry the cellular internalization of apolipoprotein(a) and apolipoprotein B. When dual-labeled ((3)H-cholesteryl ether,(125)I-protein) Lp(a) was added to cells expressing SR-BI, there was a greater relative increase in lipid uptake over protein, indicating that SR-BI mediates selective lipid uptake from Lp(a). Compared with C57BL/6 control mice, transgenic mice overexpressing human SR-BI in liver were found to have increased plasma clearance of (3)H-CE-Lp(a), whereas mouse scavenger receptor class B type I knockout (Sr-b1-KO) mice had decreased plasma clearance (fractional catabolic rate: 0.63 ± 0.08/day, 1.64 ± 0.62/day, and 4.64 ± 0.40/day for Sr-b1-KO, C57BL/6, and human scavenger receptor class B type I transgenic mice, respectively). We conclude that Lp(a) is a novel ligand for SR-BI and that SR-BI mediates selective uptake of Lp(a)-associated lipids.

Use of mutant 125 I-Perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells

Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study, we generate an (125)I-labeled mutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to (125)I-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol% of total lipids. This 35% threshold is in striking contrast to the 5% threshold previously found for PFO binding to endoplasmic reticulum membranes. The (125)I-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines, we show that this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane.

Abstract 408: Macrophage-mediated Low Density Lipoprotein Modification Reduces Foam Cell Formation

Macrophage metabolism of LDL-derived cholesterol is thought to be a key process regulating the composition and progression of atherosclerotic lesions, including the generation of cholesterol-loaded macrophage foam cells and the lipid-rich necrotic core. Many mechanisms contribute to macrophage endocytosis of LDL, and we recently reported that selective cholesteryl ester (CE) uptake contributes to LDL-induced macrophage cholesterol accumulation. Selective uptake is expected to result in the net removal of cholesteryl ester from LDL; however, the effect of this and other LDL modifications that may occur during incubation with macrophages is not understood. In the current investigation, we pre-incubated LDL for 1-2 days with macrophages in order to promote macrophage-mediated LDL modification. Surprisingly, when macrophage-conditioned LDL was transferred to fresh macrophages, much less cholesterol accumulation resulted compared to cells incubated with unconditioned LDL, as indicated by cholesterol quantification and by neutral lipid staining with Oil Red-O. This difference was not accounted for by changes in uptake of the modified 125I-LDLs, but instead was associated with a large decrease in selective [3H]CE uptake. In contrast, uptake of the non-hydrolyzable CE analog [3H]cholesteryl ether was not reduced, suggesting that reduced uptake in macrophage-conditioned LDL may be due to changes in the hydrolysis of LDL-derived CE or its subsequent metabolism. Cell-mediated LDL oxidation was considered as a possible mechanism for changes in selective CE uptake. However, agarose gel electrophoretic mobility of LDL and analysis of LDL cholesteryl esters indicated that reduced selective uptake and foam cell formation did not depend on extensive oxidative modifications of LDL. These results indicate the importance of selective CE and suggest that macrophage-mediated LDL modification may reduce foam cell formation in some circumstances.

High-capacity selective uptake of cholesteryl ester from native LDL during macrophage foam cell formation

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Recent studies have demonstrated that at high concentrations associated with hypercholesterolemia, native LDL induces macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol from native LDL during foam cell formation has not been clearly defined. Previous reports have suggested that selective cholesteryl ester (CE) uptake might contribute to cholesterol uptake from LDL independently of particle endocytosis. In this study we demonstrate that the majority of macrophage LDL-derived cholesterol is acquired by selective CE uptake in excess of LDL pinocytosis and degradation. Macrophage selective CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. In contrast to CE uptake, macrophages exhibit little selective uptake of free cholesterol (FC) from LDL. Following selective uptake from LDL, CE is rapidly hydrolyzed by a novel chloroquine-sensitive pathway. FC released from LDL-derived CE hydrolysis is largely effluxed from cells but also is subject to ACAT-mediated reesterification. These results indicate that selective CE uptake plays a major role in macrophage metabolism of LDL.

Corrigendum to: Low-density lipoprotein receptor-related protein 1 mediates hypoxia-induced very low density lipoprotein-cholesteryl ester uptake and accumulation in cardiomyocytes

[Cardiovascular Research, Volume 94, Issue 3, 1 June 2012, Pages 469–479; doi:10.1093/cvr/cvs136.] The published version …

Low-density lipoprotein receptor-related protein 1 mediates hypoxia-induced very low density lipoprotein-cholesteryl ester uptake and accumulation in cardiomyocytes

The myocardium accumulates intracellular lipids under ischaemic conditions, and myocardial fat deposition is closely associated with cardiac dysfunction. Our aims were to analyse the effect of hypoxia on low-density lipoprotein receptor-related protein 1 (LRP1) expression in neonatal rat ventricular myocytes (NRVM) and cardiac-derived HL-1 cells and the molecular mechanisms involved in this effect, to determine the role of LRP1 in the very low density lipoprotein (VLDL) uptake by hypoxic cardiomyocytes, and to study the effect of hypoxia on lipoprotein receptor expression and myocardial lipid profile in an in vivo porcine experimental model of acute myocardial infarction. Thin-layer chromatography after lipid extraction showed that VLDL exposure leads to cholesteryl ester (CE) and triglyceride (TG) accumulation in a dose-dependent manner and that hypoxic conditions further increased VLDL-derived intracellular lipid accumulation in HL-1 cells. Knockdown of LRP1 through lentiviral-mediated interfering RNA specifically prevented hypoxia-induced VLDL-CE internalization in HL-1 cells and NRVM. Lipopolysaccharide (LPS)-induced LRP1 overexpression specifically increased VLDL-CE accumulation in NRVM. In addition, using double-radiolabelled [(3)H]CE-[(14)C]TG-VLDL, we found that LRP1 deficiency specifically prevented hypoxia-induced VLDL-[(3)H]CE uptake. Finally, in an in vivo porcine model of infarcted myocardium, ischaemic areas exhibited LRP1 protein up-regulation and intramyocardial CE overaccumulation. Our results demonstrate that hypoxia increases LRP1 expression through HIF-1α and that LRP1 overexpression mediates hypoxia-induced VLDL-CE uptake and accumulation in cardiomyocytes.

Lysosomal Acid Lipase Deficiency Impairs Regulation of ABCA1 Gene and Formation of High Density Lipoproteins in Cholesteryl Ester Storage Disease

ATP-binding cassette transporter A1 (ABCA1) mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, and its expression is regulated primarily by oxysterol-dependent activation of liver X receptors. We previously reported that ABCA1 expression and HDL formation are impaired in the lysosomal cholesterol storage disorder Niemann-Pick disease type C1 and that plasma HDL-C is low in the majority of Niemann-Pick disease type C patients. Here, we show that ABCA1 regulation and activity are also impaired in cholesteryl ester storage disease (CESD), caused by mutations in the LIPA gene that result in less than 5% of normal lysosomal acid lipase (LAL) activity. Fibroblasts from patients with CESD showed impaired up-regulation of ABCA1 in response to low density lipoprotein (LDL) loading, reduced phospholipid and cholesterol efflux to apolipoprotein A-I, and reduced α-HDL particle formation. Treatment of normal fibroblasts with chloroquine to inhibit LAL activity reduced ABCA1 expression and activity, similar to that of CESD cells. Liver X receptor agonist treatment of CESD cells corrected ABCA1 expression but failed to correct LDL cholesteryl ester hydrolysis and cholesterol efflux to apoA-I. LDL-induced production of 27-hydroxycholesterol was reduced in CESD compared with normal fibroblasts. Treatment with conditioned medium containing LAL from normal fibroblasts or with recombinant human LAL rescued ABCA1 expression, apoA-I-mediated cholesterol efflux, HDL particle formation, and production of 27-hydroxycholesterol by CESD cells. These results provide further evidence that the rate of release of cholesterol from late endosomes/lysosomes is a critical regulator of ABCA1 expression and activity, and an explanation for the hypoalphalipoproteinemia seen in CESD patients.

Modified Low Density Lipoproteins Decrease the Activity and Expression of Lysosomal Acid Lipase in Human Endothelial and Smooth Muscle Cells

Lysosomal acid lipase (LAL), the only lysosomal enzyme involved in the hydrolysis of LDL-cholesteryl esters, is a key regulator of cellular cholesterol and fatty acid homeostasis and its deficiency contributes to the pathophysiology of various diseases. In this study, we questioned whether oxidized or glycated LDL, a common occurrence in atherosclerosis and diabetes, affect the activity and expression of LAL in vascular endothelial cells (EC) and smooth muscle cells (SMC). LAL activity and expression were assayed in cultured human EC and SMC exposed to oxidized LDL (oxLDL), (±)9-hydroxyoctadecadienoic acid-cholesteryl ester (HODE), glycated LDL (gLDL), or native LDL (nLDL) as control, in the presence or absence of LXR or PPAR-gamma agonists. We found that LAL activity and expression were significantly down regulated by oxLDL and HODE in EC, and by gLDL in SMC. The LXR agonist T0901317 reversed the decreased LAL expression in modified LDL- or HODE-exposed EC (P<0.001) and in gLDL-exposed SMC, whereas PPAR-gamma agonist rosiglitazone induced a low effect only in EC. In conclusion, modified LDL down regulates LAL expression in human EC and SMC by a process involving the LXR signaling pathway. This is the first demonstration that modified LDL modulate LAL expression, in a cell specific manner.

Effect of Exposure of Human Monocyte-Derived Macrophages to High, versus Normal, Glucose on Subsequent Lipid Accumulation from Glycated and Acetylated Low-Density Lipoproteins

During atherosclerosis monocyte-derived macrophages accumulate cholesteryl esters from low-density lipoproteins (LDLs) via lectin-like oxidised LDL receptor-1 (LOX-1) and class AI and AII (SR-AI, SR-AII) and class B (SR-BI, CD36) scavenger receptors. Here we examined the hypothesis that hyperglycaemia may modulate receptor expression and hence lipid accumulation in macrophages. Human monocytes were matured into macrophages in 30 versus 5 mM glucose and receptor expression and lipid accumulation quantified. High glucose elevated LOX1 mRNA, but decreased SR-AI, SR-BI, LDLR, and CD36 mRNA. SR-BI and CD36 protein levels were decreased. Normo- and hyperglycaemic cells accumulated cholesteryl esters from modified LDL to a greater extent than control LDL, but total and individual cholesteryl ester accumulation was not affected by glucose levels. It is concluded that, whilst macrophage scavenger receptor mRNA and protein levels can be modulated by high glucose, these are not key factors in lipid accumulation by human macrophages under the conditions examined.