Search

Whereas LDL-derived cholesterol accumulates in atherosclerotic lesions, HDL particles are thought to facilitate removal of cholesterol from the lesions back to the liver thereby promoting its fecal excretion from the body. Because generation of cholesterol-loaded macrophages is inherent to atherogenesis, studies on the mechanisms stimulating the release of cholesterol from these cells and its ultimate excretion into feces are crucial to learn how to prevent lesion development or even induce lesion regression. Modulation of this key anti-atherogenic pathway, known as the macrophage-specific reverse cholesterol transport, has been extensively studied in several mouse models with the ultimate aim of applying the emerging knowledge to humans. The present review provides a detailed comparison and critical analysis of the various steps of reverse cholesterol transport in mouse and man. We attempt to translate this in vivo complex scenario into practical concepts, which could serve as valuable tools when developing novel HDL-targeted therapies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Electronegative LDL (LDL(−)), a modified LDL fraction found in blood, induces the release of inflammatory mediators in endothelial cells and leukocytes. However, the inflammatory pathways activated by LDL(−) have not been fully defined. We aim to study whether LDL(−) induced release of the first-wave proinflammatory IL-1β in monocytes and monocyte-derived macrophages (MDM) and the mechanisms involved., Methods: LDL(−) was isolated from total LDL by anion exchange chromatography. Monocytes and MDM were isolated from healthy donors and stimulated with LDL(+) and LDL(−) (100 mg apoB/L)., Results: In monocytes, LDL(−) promoted IL-1β release in a time-dependent manner, obtaining at 20 h-incubation the double of IL-1β release induced by LDL(−) than by native LDL. LDL(−)-induced IL-1β release involved activation of the CD14-TLR4 receptor complex. LDL(−) induced priming, the first step of IL-1β release, since it increased the transcription of pro-IL-1β (8-fold) and NLRP3 (3-fold) compared to native LDL. Several findings show that LDL(−) induced inflammasome activation, the second step necessary for IL-1β release. Preincubation of monocytes with K+ channel inhibitors decreased LDL(−)-induced IL-1β release. LDL(−) induced formation of the NLRP3-ASC complex. LDL(−) triggered 2-fold caspase-1 activation compared to native LDL and IL-1β release was strongly diminished in the presence of the caspase-1 inhibitor Z-YVAD. In MDM, LDL(−) promoted IL-1β release, which was also associated with caspase-1 activation., Conclusions: LDL(−) promotes release of biologically active IL-1β in monocytes and MDM by induction of the two steps involved: priming and NLRP3 inflammasome activation., Significance: By IL-1β release, LDL(−) could regulate inflammation in atherosclerosis.

Cathepsin G (CatG), a serine protease present in mast cells and neutrophils, can produce angiotensin-II (Ang-II) and degrade elastin. Here we demonstrate increased CatG expression in smooth muscle cells (SMCs), endothelial cells (ECs), macrophages, and T cells from human atherosclerotic lesions. In low-density lipoprotein (LDL) receptor-deficient (Ldlr(-/-)) mice, the absence of CatG reduces arterial wall elastin degradation and attenuates early atherosclerosis when mice consume a Western diet for 3months. When mice consume this diet for 6months, however, CatG deficiency exacerbates atherosclerosis in aortic arch without affecting lesion inflammatory cell content or extracellular matrix accumulation, but raises plasma total cholesterol and LDL levels without affecting high-density lipoprotein (HDL) or triglyceride levels. Patients with atherosclerosis also have significantly reduced plasma CatG levels that correlate inversely with total cholesterol (r=-0.535, P<0.0001) and LDL cholesterol (r=-0.559, P<0.0001), but not with HDL cholesterol (P=0.901) or triglycerides (P=0.186). Such inverse correlations with total cholesterol (r=-0.504, P<0.0001) and LDL cholesterol (r=-0.502, P<0.0001) remain significant after adjusting for lipid lowering treatments among this patient population. Human CatG degrades purified human LDL, but not HDL. This study suggests that CatG promotes early atherogenesis through its elastinolytic activity, but suppresses late progression of atherosclerosis by degrading LDL without affecting HDL or triglycerides., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Local acidic areas characterize diffuse intimal thickening (DIT) and advanced atherosclerotic lesions. The role of acidity in the modification and extra- and intracellular accumulation of triglyceride-rich VLDL and IDL particles has not been studied before. Here, we examined the effects of acidic pH on the activity of recombinant human group V secreted phospholipase A(2) (sPLA(2)-V) toward small VLDL (sVLDL), IDL, and LDL, on the binding of these apoB-100-containing lipoproteins to human aortic proteoglycans, and on their uptake by human monocyte-derived macrophages. At acidic pH, the ability of sPLA(2)-V to lipolyze the apoB-100-containing lipoproteins was moderately, but significantly, increased while binding of the lipoproteins to proteoglycans increased >60-fold and sPLA(2)-V-modification further doubled the binding. Moreover, acidic pH more than doubled macrophage uptake of soluble complexes of sPLA(2)-V-LDL with aortic proteoglycans. Proteoglycan-affinity chromatography at pH 7.5 and 5.5 revealed that sVLDL, IDL, and LDL consisted of populations with different proteoglycan-binding affinities, and, surprisingly, the sVLDL fractions with the highest proteoglycan-affinity contained only low amounts of apolipoproteins E and C-III. Our results suggest that in atherosclerotic lesions with acidic extracellular pH, sPLA(2)-V is able to lipolyze sVLDL, IDL, and LDL, and increase their binding to proteoglycans. This is likely to provoke extracellular accumulation of lipids derived from these atherogenic lipoprotein particles and to increase the progression of the atherosclerotic lesions., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Phospholipids are key components of biological membranes and their lipolysis with phospholipase A(2) (PLA(2)) enzymes occurs in different cellular pH environments. Since no studies are available on the effect of pH on PLA(2)-modified phospholipid membranes, we performed 50-ns atomistic molecular dynamics simulations at three different pH conditions (pH 9.0, 7.5, and 5.5) using a fully PLA(2)-hydrolyzed phosphatidylcholine (PC) bilayer which consists solely of lysophosphatidylcholine and free fatty acid molecules. We found that a decrease in pH results in lateral squeezing of the membrane, i.e. in decreased surface area per headgroup. Thus, at the decreased pH, the lipid hydrocarbon chains had larger S(CD) order parameter values, and also enhanced membrane thickness, as seen in the electron density profiles across the membrane. From the lateral pressure profiles, we found that the values of spontaneous curvature of the two opposing monolayers became negative when the pH was decreased. At low pH, protonation of the free fatty acid headgroups reduces their mutual repulsion and accounts for the pH dependence of all the above-mentioned properties. The altered structural characteristics may significantly affect the overall surface properties of biomembranes in cellular vesicles, lipid droplets, and plasma lipoproteins, play an important role in membrane fission and fusion, and modify interactions between membrane lipids and the proteins embedded within them., (2010 Elsevier B.V. All rights reserved.)

Lipovitellin, an egg-yolk lipoprotein, transports lipids in a pocket surrounded by amphiphilic beta-sheets. Its X-ray structure provides possibilities to study interactions between lipophilic beta-sheets and lipids at the atomic level. Here, we studied a 67-residue-long amphiphilic beta-sheet of lipovitellin previously suggested a suitable working model for studies of the lipid-binding behaviour of amphiphilic beta-sheet regions in apolipoprotein B-100 (apoB-100). We performed four molecular dynamics simulations with different starting configurations to define characteristics of the amphiphilic beta-sheet model at a decane-water interface. In each simulation the model beta-sheet bound keenly to the decane layer via its hydrophobic surface. The structural profiles showed unchanged secondary structure of the beta-sheet during the attachment. Also, aromatic side chains, especially tryptophans and tyrosines, mediated the attachment to the hydrophobic layer and influenced the orientation of the decane molecules that are in contact with the beta-sheet. In conclusion, the present simulations reveal high affinity of a lipovitellin-derived amphiphilic beta-sheet to a hydrophobic decane layer. They lay thereby the basis for further studies of the interaction between amphiphilic beta-sheets and lipids in complex molecular systems, like LDL particles, in which the large apoB-100 is the main protein component.

Serum amyloid A (SAA) is a precursor for the amyloid A in AA type of amyloidosis. Distribution of mast cells in tissues is similar to the distribution of amyloid deposits in secondary AA-amyloidosis. Therefore, we studied whether mast cells could be involved in SAA metabolism. Human mast cell line (HMC-1) cells were cultured with recombinant human apoSAA (rhSAA), and the production of tumour necrosis factor (TNF)-alpha and interleukin (IL)-1 beta was determined by ELISA. RhSAA and human SAA (huSAA) were incubated with human chymase, tryptase or with intact human mast cell (huMC) in cultures, and degradation of SAA was followed by gel electrophoresis, liquid chromatography and mass spectrometry. SAA induced dose-dependent production of TNF-alpha and IL-1 beta in HMC-1 cells. Tryptase, chymase, and huMC granules degraded efficiently the SAA protein. Degradation of SAA by tryptase, but not by chymase, released a highly amyloidogenic N-terminal fragment of SAA. Finally, incubation of huMC with rhSAA alone resulted in degradation of SAA and formation of protofibrillar intermediates. These results suggest a pathogenic role for mast cells in AA-amyloidosis.

Low density lipoprotein (LDL) particles are the major cholesterol carriers in circulation and their physiological function is to carry cholesterol to the cells. In the process of atherogenesis these particles are modified and they accumulate in the arterial wall. Although the composition and overall structure of the LDL particles is well known, the fundamental molecular interactions and their impact on the structure of LDL particles are not well understood. Here, the existing pieces of structural information on LDL particles are combined with computer models of the individual molecular components to give a detailed structural model and visualization of the particles. Strong evidence is presented in favor of interactions between LDL lipid constituents that lead to specific domain formation in the particles. A new three-layer model, which divides the LDL particle into outer surface, interfacial layer, and core, and which is capable of explaining some seemingly contradictory interpretations of molecular interactions in LDL particles, is also presented. A new molecular interaction model for the beta-sheet structure and phosphatidylcholine headgroups is introduced and an overall view of the tertiary structure of apolipoprotein B-100 in the LDL particles is presented. This structural information is also utilized to understand and explain the molecular characteristics and interactions of modified, atherogenic LDL particles.

When mast cells from human lungs were stimulated in vitro to degranulate, all of the tryptase secreted was found to be complexed with proteoglycans, three quarters with heparin proteoglycans and one quarter with chondroitin sulphate proteoglycans. Isolation of the tryptase-proteoglycan complexes by fibronectin affinity chromatography and gel filtration on a Sephacryl S-200 column gave the complexes an apparent Mr of 200000, suggesting the presence of heparin and chondroitin sulphate proteoglycans (Mrs=60000) and tryptase (Mr=134000) in a molar ratio of 1:1, equivalent to a mass ratio of about 0.45:1. However, analysis of the total mast cell releasate showed that it contained more proteoglycans (mass ratio of about 2:1) than was needed to complex tryptase. We could demonstrate that the releasate contained two proteoglycan fractions, one complexed (20%) with tryptase and the other not (80%). Incubation of the isolated tryptase-proteoglycan complexes led to rapid monomerisation and inactivation of tryptase, whereas the releasate, containing both complexed and free proteoglycans, retained its tryptase activity for up to at least 18 h. The results indicate that the majority of the proteoglycans secreted by stimulated lung mast cells, although not complexed with the secreted tryptase, are critical for the preservation of its activity.

Isolated rat adipocytes were incubated with serum lipoproteins or lymphchylomicrons which contained 14c-labeled cholesterol. The specific activity of lipoprotein free cholesterol decreased and that of cellular free cholesterol increased linearly up to 7 h. At this time the cell cholesterol specific activity was only 11% of that of medium cholesterol indicating that the rate of exchange was slow. The specific activity of lipoprotein esterified cholesterol remained unchanged while that of cells showed a slight increase suggesting esterification of incorporated free cholesterol. No detectable change of the lipoprotein or cellular cholesterol concentration occurred indicating that the uptake of radioactive free cholesterol was due to exchange without net movement of sterol. The radioactive cholesterol was incorporated into both membrane fraction and the fat droplet of the adipocytes. The rate of cholesterol exchange was temperature-dependent but it was not influenced by the metabolic state of the cells and not by addition of metabolic inhibitors. Trypsin or pronase treatment of the cells were without influence on the rate of the exchange and denaturation of the plasma lipoproteins with formalin increased the rate of exchange. These results indicate that the exchange of cholesterol is a physical chemical process, which is not linked to energy metabolism of the cells, and which is not mediated by either specific lipoprotein receptors on fat cell membranes or pinocytic uptake of lipoproteins. The rate of free cholesterol exchange showed a linear correlation with the concentration of lipoprotein particles in the medium. The relative transfer rate was highest for chylomicrons and decreased in order chylomicron remnants greater than very low density lipoprotein greater than low density lipoprotein greater than high density lipoprotein. A saturation of the system could be obtained only with high density lipoprotein.