Furthermore, RCT mediated by some particles, such as SUVs (see Results) and HDL-like complexes (see References 13 and 181318 ), can produce metabolic consequences that may not be benign. to access the full features of the site or access our. B. Specific lipoproteins are identified in the upper left-hand plot. Thus, RCT by these synthetic SUVs was associated with events that are entirely consistent with known molecular consequences of cellular cholesterol enrichment: sterol-responsive messages in the liver, such as the LDL receptor, were suppressed, and consequently, plasma LDL concentrations rose. Figure 3. First, it has been recently accepted that most human heart attacks are caused by rupture-prone lesions that are rich in lipid and foam cell macrophages.6364 One goal for treatment of preestablished disease is to stabilize these lesions. The third possible explanation is based on the striking quantitative difference in protein adsorption between the two types of vesicles (Fig 2B and Results), which is presumably a result of their distinct surface curvatures.55 Thus, it is conceivable that SUVs, but not LUVs, would avidly strip apoE from VLDL, thereby slowing its clearance from plasma and favoring its conversion to LDL (see Reference 5656 ). Israelachvili et al. It is recommended that you keep a set of syringes to be used only for lipids. 1-800-242-8721 The second possible explanation for the difference in metabolic response to LUVs versus SUVs is based solely on differences in the kinetics of their delivery of cholesterol to the liver. A, Distributions of lipids in whole plasma by particle size. Plots from one animal in each group are shown, with all panels drawn to the same scale.
Selecting this option will search the current publication in context. Preliminary studies indicated that these rocket assays, which are performed in detergent,26 are not affected by the presence of liposomes. Rodrigueza was an International Research Fellow of the American Heart Association and then supported by a Research Fellowship from the Heart and Stroke Foundation of BC and Yukon, Canada. 1P<.01; significant differences between LUV and SUV values. B, Distributions of plasma lipids by particle charge. Lipid Content in Livers of Animals on Day 6 of Treatment With LUVs, SUVs, or Saline. Each data point displays the meanSEM.
Assemble the extruder with filter supports (4 total) and polycarbonate membranes (2 total). mini-extruder from Avanti Polar Lipids. Absolute EC distribution curves were then determined by difference. Dallas, TX 75231 Chem. The identity of each cloned cDNA was confirmed by sequencing, as described in detail by Rea et al.29 For each cloned cDNA, an unlabeled sense mRNA product, to be used as an internal standard, and a labeled antisense mRNA probe were synthesized. liposomes vesicles between difference vesicle liposome The lipid/aqueous solution should result appear cloudy. Enter words / phrases / DOI / ISBN / authors / keywords / etc. and Mitchell and Ninham argue that provided the phospholipid concentration is sufficiently low, lipid aggregates will spontaneously form into SUVs which represent the equilibrium state of the lipid in water. The SUV-mediated increase in LDL concentration was confirmed by agarose gel electrophoresis followed by Sudan black staining,25 which revealed a darker but otherwise unremarkable -band (Fig 2B). Both synthetic mRNAs were designed to contain the partial coding sequence from rabbit, linked to a short sequence from the plasmid. Absolute EC values in each lipoprotein fraction were determined by the difference between the calculated TC and UC in each lipoprotein fraction. Specific lipoproteins are identified in the upper left-hand plot. Radioisotopic studies have suggested that cholesterol of HDL, the apparent natural mediator of RCT,8 is efficiently converted into bile acids by the liver and then excreted.9101112 In contrast, direct measurements of sterol mass showed that accelerated delivery of cholesterol to the liver in vivo by apoE-rich HDL produces no change in biliary output of cholesterol or bile acids, but instead stimulates hepatic acyl-CoA:cholesterol acyltransferase and enhances VLDL secretion.13 Similar results, including LDL receptor suppression, have been reported in other situations involving cholesterol enrichment of hepatic cells.14151617 Moreover, intravenous infusion of apoA-I/PL disks into humans to enhance RCT causes a sustained rise in plasma levels of LDL.18 Taken together, these effects are more consistent with events leading to the promotion, not the inhibition, of atherogenesis. Each data point displays the meanSEM. ANOVA was used to compare the three experimental groups. Small unilamellar phospholipid vesicles (SUVs) are widely used as a model system in which to study the molecular interactions which occur in biological membranes. Notice that the additional UC mobilized into the plasma of LUV-treated animals was mainly confined to the VLDL size range, while the additional UC in the SUV-treated animals was in both the VLDL and LDL size ranges, consistent with the smaller size but greater heterogeneity of SUVs compared with LUVs (see Methods). This procedure was developed in Steve Boxer's laboratory. Both types of PL vesicles did substantially increase the hepatic UC:PL ratio (Table 2; P<.01), consistent with liposomal delivery of cholesterol mass to the liver. This page was last edited on 25 November 2010, at 09:31. Fractionations of plasma from day 6 after repeated injections of LUVs, SUVs, or saline. Rinse the assembled extruder with Millipore water three times to remove any air bubbles. Consistent with prior literature,441 SDSpolyacrylamide gel electrophoresis confirmed that the major protein acquired by both LUVs and SUVs was apoA-I (data not shown). The total area under each curve was normalized to the whole-plasma assay for TC or UC. hydrophobic acids nucleic encapsulation onion molecules liposome wilson Plasma samples on those days were obtained immediately before the injections. Hepatic HMG-CoA reductase message was also significantly suppressed with SUV, but not LUV treatment, and hepatic 7-hydroxylase message showed a similar trend.
Messages in liver samples obtained at day 6 were quantified by RNase protection assay. vesicles olv mlv multilamellar niosomes II. O indicates origin and , migration of an LDL standard.Download figureDownload PowerPoint Human HDL3 was isolated from fresh plasma by sequential ultracentrifugation (1.12
After each injection, LUV- and SUV-injected animals showed large increases in plasma concentrations of unesterified cholesterol, indicating mobilization of tissue stores.
If you are the author of this article, you do not need to request permission to reproduce figures P<.05, P<.01; significantly different from saline control. Size distributions of plasma particles carrying TC and UC were determined by Superose 6HR high-performance gel chromatography (Rainin Instrument Co, Inc), including an on-line post-column analyzer, as previously described.2728 Areas under the peaks were used to calculate the percent distribution of TC and UC corresponding to VLDL, LDL, and HDL size ranges in the elution profiles (Dynamax and Compare Module Software, Rainin Instrument Co, Inc, developed for Macintosh computers). W.V. The inability to form a uniform lipid film can be an indication that the glass vial is not sufficiently clean. Approximately 3 mL of blood was collected from each animal via a medial auricular artery every morning during the study.
These data on hepatic mRNA levels indicate that SUVs, but not LUVs, substantially perturbed liver cholesterol homeostasis. Figure 2. Dry with N2(g). We compared hepatic effects of large (120-nm) and small (35-nm) unilamellar vesicles (LUVs and SUVs), both of which mediate reverse cholesterol transport in vivo but were previously shown to be targeted to different cell types within the liver. In all three panels, SUV values after day 1 were greater than control (P<.01). Also, suppression of CETP is usually followed by increases in HDL EC,59 which we did not see here after LUV injections. Under these conditions, liposomal remnants would not appear in this size range, because vesicle structure is stable in plasma at 300 mg/kg4531 and remnants that form at lower vesicle doses are exclusively PL-apoprotein disks that coelute with HDL.3138 Quantitatively, SUVs increased plasma concentrations of LDL EC to over fourfold compared with saline control (Table 1), whereas injections of LUVs caused a small but statistically insignificant decrease during the study (Fig 1). A, Distributions of lipids in whole plasma by particle size. It is nonetheless surprising that the metabolic effects on the liver of RCT mediated by SUVs in the current study or by apoE-rich particles in a previous study13 appear to be inconsistent with antiatherogenic actions.
After completing 20 extrusions, disassemble the polycarbonate and check for a tear. Normal 3- to 4-kg female New Zealand White rabbits (Hazelton Farms, Denver, Pa) were randomly distributed into three groups (n=4, LUV or saline treatment; n=3, SUV treatment). A. Cornell, J. Middlehurst and F. Separovic, Blood samples were immediately mixed with EDTA for anticoagulation (final concentration, 2 mmol/L in blood) and N-ethylmaleimide to inhibit lecithin:cholesterol acyltransferase (final concentration, 5 mmol/L). Last updated: On day 6, 24 hours after the third injection, hepatic samples were taken for lipid and mRNA analyses. Ideally, the lipids will form a thin, transparent, uniform film over much of the interior glass surface. This question is of substantial relevance for our understanding of membranes and how their dynamics is affected by curvature, a problem that is also of key importance in a number of biological questions. In mice, LUVs are cleared from plasma somewhat more slowly than are SUVs and thereby produce a relatively constant delivery of cholesterol mass to the liver from the time of injection until the bulk of injected material is cleared.6 Similarly, in rabbits, LUVs are cleared with a t1/2 of 27 hours (W.V. 7272 Greenville Ave. Figure 1. Since egg PC is fluid to ~-10C, vesicles of egg PC can be created at room temperature and stored at 4C. Dry the lipids under vacuum for 1 hour. amplification dna isothermal specific rsc circuit vesicles unilamellar molecules giant inside chemical In this study, we sought to determine the effect of liposomal structure on hepatic responses to enhanced RCT mediated by these synthetic particles. use prohibited. 1-800-AHA-USA-1 Triglyceride concentrations in whole plasma were determined by using a commercially available kit (Triglyceride G, Wako Chemicals USA, Inc). Is raising HDL a futile strategy for atheroprotection? While SUVs also have access to Kupffer cells, their sheer number (10 times as many SUVs as LUVs per milligram of PL) appears to saturate the reticuloendothelial system, and so parenchymal cells predominate in their clearance (see Reference 2020 ). In B and C, LUV values were statistically indistinguishable from the corresponding control (saline) values, except for C, day 3 (.01
Overall, our findings indicate that these synthetic particles, LUVs and SUVs, mediate RCT in vivo, though with markedly different regulatory effects on the liver. Having a uniform film of lipids on the interior of the glass vial makes rehydration of the lipids easier. By RNase protection assay, SUVs suppressed hepatic LDL receptor mRNA at day 6 (to 614% of control, meanSEM), whereas LUVs caused a statistically insignificant stimulation. 1997;17:21322139, Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, Nature Clinical Practice Cardiovascular Medicine, Current Treatment Options in Cardiovascular Medicine, Current Opinion in Clinical Nutrition and Metabolic Care, Journal of the American College of Cardiology, Biochimica et Biophysica Acta (BBA) - Biomembranes, Arteriosclerosis, Thrombosis, and Vascular Biology, Large Versus Small Unilamellar Vesicles Mediate Reverse Cholesterol Transport In Vivo Into Two Distinct Hepatic Metabolic Pools, Nanomaterials for the theranostics of obesity, Susceptibility of low-density lipoprotein particles to aggregate depends on particle lipidome, is modifiable, and associates with future cardiovascular deaths, The phenomenon of atherosclerosis reversal and regression: Lessons from animal models, ApoA-IMilano phospholipid complex (ETC-216) infusion in human volunteers.
You do not have JavaScript enabled. Rabbit apoB assays of plasma from day 6 showed a trend toward higher values in the SUV group (0.310.04 AU) than the LUV group (0.160.07 AU). Sometimes excessive pressure can build up in the extruder, and passing the lipid solution through the membrane requires a great deal of force. In all three panels, SUV values after day 1 were greater than control (P<.01). Fractionation data for all particle sizes from the final bleed on day 6 are summarized in Table 1. Subsequent mechanistic studies indicated that dispersed PLs self-assemble into concentric spherical bilayers known as liposomes or vesicles.3 Furthermore, when intravenously injected at sufficient doses, initially cholesterol-free PL vesicles remain intact in the bloodstream and are capable of extracting cholesterol from both lipoproteins and peripheral tissues.24 Thus, these circulating particles act as a sink for cholesterol, which is shuttled to them from tissues by HDL and other small acceptors of cholesterol.24567 Because the liver serves as the predominant organ for the clearance of PL vesicles, it has been suggested that the antiatherogenic effects of these particles result from their ability to act as synthetic mediators of RCT from peripheral tissues to the liver.25. Because vesicles in the bloodstream eventually achieve a molar ratio of UC:PL of about 0.8,6 which is far higher than the ratio of 0.12 found in normal hepatic tissue (Table 2), we anticipated that hepatic uptake of vesicles might increase this ratio in liver. After digestion with RNase, protected mRNA probes were quantified by polyacrylamide gel electrophoresis and then autoradiography using a PhosphorImager (Molecular Dynamics). This may take some time to load. 3P<.01; significantly different from saline control. Dynamical fluctuations of droplet microemulsions and vesicles, M. Gradzielski, M. Bergmeier, M. Muller, and H. Hoffmann, , Novel gel phase: A cubic phase of densely packed monodisperse, unilamellar vesicles, M. Gradzielski, M. Bergmeier, H. Hoffmann, M. Muller, and I. Grillo, , Vesicle gel formed by a self-organization process, Some isotropic mesophases in systems containing amphiphilic compounds, New SANS instrument at the BER-II reactor in Berlin, Germany, R. Gilles, U. Keiderling, P. Strunz, A. Wiedenmann, and H. Fuess, , Silver behenate as a standard for instrumental resolution and wavelength calibration for small angle neutron scattering, The new BerSANS-PC software for reduction and treatment of small angle neutron scattering data, Determination of size distribution from small-angle scattering data for systems with effective hard-sphere interactions, Analysis of classical statistical mechanics by means of collective coordinates, P. Schleger, B. Alefeld, J. Barthelemy, G. Ehlers, B. Farago, P. Giraud, C. Hayes, A. Kollmar, C. Lartigue, F. Mezei, and D. Richter, , The long-wavelength neutron spin-echo spectrometer IN15 at the Institut Laue-Langevin, B. Farago, D. Richter, J. S. Huang, S. A. Safran, and S. T. Milner, , Shape and size fluctuations of microemulsion droplets: The role of cosurfactant, M. Gradzielski, D. Langevin, and B. Farago, , Experimental investigation of the structure of nonionic microemulsions and their relation to the bending elasticity of the amphiphilic film, Bending elasticity of the surfactant monolayer in droplet microemulsions: Determination by a combination of dynamic light scattering and neutron spin-echo spectroscopy, The effect of the charge density of microemulsion droplets on the bending elasticity of their amphiphilic film, L. R. Arriaga, I. Lpez-Montero, F. Monroy, G. Orts-Gil, B. Farago, and T. Hellweg, , Stiffening effect of cholesterol on disordered lipid phases: A combined neutron spin echo + dynamic light scattering analysis of the bending elasticity of large unilamellar vesicles, L. R. Arriaga, I. Lpez-Montero, G. Orts-Gil, B. Farago, T. Hellweg, and F. Monroy, , Fluctuation dynamics of spherical vesicles: Frustration of regular bulk dissipation into subdiffusive relaxation, Undulations and dynamic structure factor of membranes, B. Farago, M. Monkenbusch, K. Goecking, D. Richter, and J. Huang, , Dynamics of microemulsions as seen by neutron spin echo, S. Komura, T. Takeda, Y. Kawabata, S. K. Ghosh, H. Seto, and M. Nagao, , Dynamical fluctuation of the mesoscopic structure in ternary, I. Hoffmann, P. Heunemann, B. Farago, I. Grillo, O. Holderer, M. Pch, and M. Gradzielski, , Structure and dynamics of nanoemulsions: Insights from combining dynamic and static neutron scattering, M. Monkenbusch, O. Holderer, H. Frielinghaus, D. Byelov, J. Allgaier, and D. Richter, , Bending moduli of microemulsions; comparison of results from small angle neutron scattering and neutron spin-echo spectroscopy, Interpreting membrane scattering experiments at the mesoscale: The contribution of dissipation within the bilayer, M. C. Watson, Y. Peng, Y. Zheng, and F. L. H. Brown, , The intermediate scattering function for lipid bilayer membranes: From nanometers to microns, L. R. Arriaga, R. Rodrguez-Garca, L. H. Moleiro, S. Prvost, I. Lpez-Montero, T. Hellweg, and F. Monroy, , Dissipative dynamics of fluid lipid membranes enriched in cholesterol, Liquid dynamics and inelastic scattering of neutrons, Solubilization efficiency boosting by amphiphilic block co-polymers in microemulsions, M. Mihailescu, M. Monkenbusch, J. Allgaier, H. Frielinghaus, D. Richter, B. Jakobs, and T. Sottmann, , Neutron scattering study on the structure and dynamics of oriented lamellar phase microemulsions, Concentration dependence of shape and structure fluctuations of droplet microemulsions investigated by neutron spin echo spectroscopy, T. Foster, T. Sottmann, R. Schweins, and R. Strey, , Small-angle-neutron-scattering from giant water-in-oil microemulsion droplets. Copyright Clearance Center request page. The three injections of SUVs caused 40% to 50% suppression of hepatic mRNA levels for the LDL receptor and HMG-CoA reductase. Also, lipoprotein fractionation revealed an increase in the concentration of HDL EC in SUV-treated animals. If you are an author contributing to an RSC publication, you do not need to request permission Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and published in abstract form (Circulation. Phospholipid content equals phosphatidylcholine plus phosphatidylethanolamine. Williams is an Established Investigator of the American Heart Association/Genentech. After hepatic uptake of this cholesterol, however, SUV-treated animals developed persistently elevated plasma LDL concentrations, which by day 6 had increased to more than four times the values in saline-treated controls.
When liver cells were cholesterol enriched in vivo by a single injection of apoE-rich HDL that was rapidly cleared from plasma into liver13 or by feeding nonhuman primates long term on atherogenic diets,39 similar results were observed, including LDL receptor suppression and lipoprotein oversecretion. In B and C, LUV values were statistically indistinguishable from the corresponding control (saline) values, except for C, day 3 (.01 Delivery of cholesterol to Kupffer cells can be followed by gradual transfer of lipid to parenchymal cells,1953 for example, via the extensions of Kupffer cells that reach down through the space of Disse to make physical contact with parenchymal cells.53 The rate of sterol delivery to the parenchymal cells by transfer from Kupffer cells can be slower than by direct uptake; the chemical form of the sterol may be altered by the Kupffer cells before transfer (see Reference 5454 ); and, on the basis of other pathways for lipid transfer among liver cells,54A the process of transfer from Kupffer to parenchymal cells may be regulated, whereas SUV clearance does not appear to be.520. This increase in HDL EC, however, represented only a small fraction of the total cholesterol mass mobilized by the injected vesicles. Plasma samples were gel filtered through a Superose 6HR column, and eluents were assayed for TC and UC. Thus, depending on hepatic responses, not all agents that enhance RCT will be unambiguously beneficial. American Heart Association, Inc. All rights reserved.
To generate SUVs, 30-mL batches of MLVs were each subjected to three 20-minute cycles of sonication in 50-mL round-bottom Pyrex glass tubes at 0C under nitrogen (power setting=3, duty cycle=50%, Branson stud tip sonifier, VWR Co). If the lipid solution does not become clear and/or it requires very, very little force to pass the lipid solution through the extruder, then it is possible that the polycarbonate membranes have developed a tear. *P<.05, **P<.01; significant differences between LUV and SUV values.Download figureDownload PowerPoint Tissue samples were stored at 70C until isolation of total RNA and lipid analysis. Hepatic mRNA levels for key enzymes in cholesterol homeostasis are shown in Fig 3. Mass values are shown as meanSEM, expressed as microgram lipid per milligram protein in liver (n=4 animals per treatment group). Dry the lipids down with N2(g). Gel filtration revealed that most of the increase in whole-plasma EC was transported by LDL-sized particles (Fig 1C, Table 1, and Fig 2A). The area under the LDL EC peaks in the saline, SUV, and LUV animal were the same, much larger, and slightly smaller, respectively, than on day 1. to permit an approximate estimate of the curvature energy associated with vesicle formation. Consistent with prior literature, the diameters of the LUVs and SUVs generated were found to be 12335 and 3430 nm (meanSD), respectively, determined by quasielastic light scattering using a Nicomp model 370 submicron laser particle sizer, equipped with a 5-mW He-Ne laser (Pacific Scientific).6. These results suggest a proportionately greater increase in LDL EC than in plasma apoB after SUV administration, raising the possibility of increased LDL size, which was confirmed by a slight shift on the EC elution profile (Fig 2A, EC profile after SUV treatment). Fetching data from CrossRef. Because the role of CETP in provoking or preventing atherosclerosis is controversial,606162 the importance in atherogenesis of our finding that CETP mRNA is suppressed after LUV injections is not clear. Arrows indicate intravenous injections of PL vesicles or saline on days 1, 3, and 5. LUVs of 120-nm diameter or slightly larger will not pass easily and are cleared instead by the macrophage Kupffer cells that line liver sinusoids.
Similarly, cholesterol enrichment of hepatocytes in vitro causes suppression of LDL receptors4243 and HMG-CoA reductase,44454647 as well as enhancement of apoB secretion.16434849 The expression of hepatic 7-hydroxylase is stimulated by cellular cholesterol enrichment in rats50 but inhibited in rabbits,1740 consistent with the trend in Fig 3. Figure 2. The EC curve was generated by difference. Each LUV value was significantly different from the corresponding SUV value (P<.01), except for VLDL EC (not significant). In contrast, the three injections of LUVs caused increases, though statistically insignificant, in each of these messages. Bending stiffness of biological membranes: What can be measured by neutron spin echo? You should feel slight resistance when pushing the syringes, and the lipid solution should become transparent (vs. opaque). In contrast, LUVs may be a suitable therapeutic agent, because they mobilize peripheral cholesterol to the liver without suppressing hepatic LDL receptor mRNA and without provoking a subsequent rise in plasma LDL levels. Allow to dry on Kimwipes. We conclude that LUVs and SUVs mobilize peripheral tissue cholesterol and deliver it to the liver, but to distinct metabolic pools that exert different regulatory effects. On the other hand, the small unilamellar vesicles used here have a size between larger vesicles, with dynamics being well described by the Zilman-Granek model and smaller microemulsion droplets which can be described by the Milner-Safran model. The American Heart Association is qualified 501(c)(3) tax-exempt Soc., 1986,81, 163
Unesterified and Esterified Cholesterol Concentrations in Whole Plasma and in VLDL-, LDL-, and HDL-Sized Particles on Day 6. 
Selecting this option will search the current publication in context. Preliminary studies indicated that these rocket assays, which are performed in detergent,26 are not affected by the presence of liposomes. Rodrigueza was an International Research Fellow of the American Heart Association and then supported by a Research Fellowship from the Heart and Stroke Foundation of BC and Yukon, Canada. 1P<.01; significant differences between LUV and SUV values. B, Distributions of plasma lipids by particle charge. Lipid Content in Livers of Animals on Day 6 of Treatment With LUVs, SUVs, or Saline. Each data point displays the meanSEM.
After each injection, LUV- and SUV-injected animals showed large increases in plasma concentrations of unesterified cholesterol, indicating mobilization of tissue stores.
If you are the author of this article, you do not need to request permission to reproduce figures P<.05, P<.01; significantly different from saline control. Size distributions of plasma particles carrying TC and UC were determined by Superose 6HR high-performance gel chromatography (Rainin Instrument Co, Inc), including an on-line post-column analyzer, as previously described.2728 Areas under the peaks were used to calculate the percent distribution of TC and UC corresponding to VLDL, LDL, and HDL size ranges in the elution profiles (Dynamax and Compare Module Software, Rainin Instrument Co, Inc, developed for Macintosh computers). W.V. The inability to form a uniform lipid film can be an indication that the glass vial is not sufficiently clean. Approximately 3 mL of blood was collected from each animal via a medial auricular artery every morning during the study.
These data on hepatic mRNA levels indicate that SUVs, but not LUVs, substantially perturbed liver cholesterol homeostasis. Figure 2. Dry with N2(g). We compared hepatic effects of large (120-nm) and small (35-nm) unilamellar vesicles (LUVs and SUVs), both of which mediate reverse cholesterol transport in vivo but were previously shown to be targeted to different cell types within the liver. In all three panels, SUV values after day 1 were greater than control (P<.01). Also, suppression of CETP is usually followed by increases in HDL EC,59 which we did not see here after LUV injections. Under these conditions, liposomal remnants would not appear in this size range, because vesicle structure is stable in plasma at 300 mg/kg4531 and remnants that form at lower vesicle doses are exclusively PL-apoprotein disks that coelute with HDL.3138 Quantitatively, SUVs increased plasma concentrations of LDL EC to over fourfold compared with saline control (Table 1), whereas injections of LUVs caused a small but statistically insignificant decrease during the study (Fig 1). A, Distributions of lipids in whole plasma by particle size. It is nonetheless surprising that the metabolic effects on the liver of RCT mediated by SUVs in the current study or by apoE-rich particles in a previous study13 appear to be inconsistent with antiatherogenic actions. After completing 20 extrusions, disassemble the polycarbonate and check for a tear. Normal 3- to 4-kg female New Zealand White rabbits (Hazelton Farms, Denver, Pa) were randomly distributed into three groups (n=4, LUV or saline treatment; n=3, SUV treatment). A. Cornell, J. Middlehurst and F. Separovic, Blood samples were immediately mixed with EDTA for anticoagulation (final concentration, 2 mmol/L in blood) and N-ethylmaleimide to inhibit lecithin:cholesterol acyltransferase (final concentration, 5 mmol/L). Last updated: On day 6, 24 hours after the third injection, hepatic samples were taken for lipid and mRNA analyses. Ideally, the lipids will form a thin, transparent, uniform film over much of the interior glass surface. This question is of substantial relevance for our understanding of membranes and how their dynamics is affected by curvature, a problem that is also of key importance in a number of biological questions. In mice, LUVs are cleared from plasma somewhat more slowly than are SUVs and thereby produce a relatively constant delivery of cholesterol mass to the liver from the time of injection until the bulk of injected material is cleared.6 Similarly, in rabbits, LUVs are cleared with a t1/2 of 27 hours (W.V. 7272 Greenville Ave. Figure 1. Since egg PC is fluid to ~-10C, vesicles of egg PC can be created at room temperature and stored at 4C. Dry the lipids under vacuum for 1 hour. amplification dna isothermal specific rsc circuit vesicles unilamellar molecules giant inside chemical In this study, we sought to determine the effect of liposomal structure on hepatic responses to enhanced RCT mediated by these synthetic particles. use prohibited. 1-800-AHA-USA-1 Triglyceride concentrations in whole plasma were determined by using a commercially available kit (Triglyceride G, Wako Chemicals USA, Inc). Is raising HDL a futile strategy for atheroprotection? While SUVs also have access to Kupffer cells, their sheer number (10 times as many SUVs as LUVs per milligram of PL) appears to saturate the reticuloendothelial system, and so parenchymal cells predominate in their clearance (see Reference 2020 ). In B and C, LUV values were statistically indistinguishable from the corresponding control (saline) values, except for C, day 3 (.01
Overall, our findings indicate that these synthetic particles, LUVs and SUVs, mediate RCT in vivo, though with markedly different regulatory effects on the liver. Having a uniform film of lipids on the interior of the glass vial makes rehydration of the lipids easier. By RNase protection assay, SUVs suppressed hepatic LDL receptor mRNA at day 6 (to 614% of control, meanSEM), whereas LUVs caused a statistically insignificant stimulation. 1997;17:21322139, Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, Nature Clinical Practice Cardiovascular Medicine, Current Treatment Options in Cardiovascular Medicine, Current Opinion in Clinical Nutrition and Metabolic Care, Journal of the American College of Cardiology, Biochimica et Biophysica Acta (BBA) - Biomembranes, Arteriosclerosis, Thrombosis, and Vascular Biology, Large Versus Small Unilamellar Vesicles Mediate Reverse Cholesterol Transport In Vivo Into Two Distinct Hepatic Metabolic Pools, Nanomaterials for the theranostics of obesity, Susceptibility of low-density lipoprotein particles to aggregate depends on particle lipidome, is modifiable, and associates with future cardiovascular deaths, The phenomenon of atherosclerosis reversal and regression: Lessons from animal models, ApoA-IMilano phospholipid complex (ETC-216) infusion in human volunteers.
You do not have JavaScript enabled. Rabbit apoB assays of plasma from day 6 showed a trend toward higher values in the SUV group (0.310.04 AU) than the LUV group (0.160.07 AU). Sometimes excessive pressure can build up in the extruder, and passing the lipid solution through the membrane requires a great deal of force. In all three panels, SUV values after day 1 were greater than control (P<.01). Fractionation data for all particle sizes from the final bleed on day 6 are summarized in Table 1. Subsequent mechanistic studies indicated that dispersed PLs self-assemble into concentric spherical bilayers known as liposomes or vesicles.3 Furthermore, when intravenously injected at sufficient doses, initially cholesterol-free PL vesicles remain intact in the bloodstream and are capable of extracting cholesterol from both lipoproteins and peripheral tissues.24 Thus, these circulating particles act as a sink for cholesterol, which is shuttled to them from tissues by HDL and other small acceptors of cholesterol.24567 Because the liver serves as the predominant organ for the clearance of PL vesicles, it has been suggested that the antiatherogenic effects of these particles result from their ability to act as synthetic mediators of RCT from peripheral tissues to the liver.25. Because vesicles in the bloodstream eventually achieve a molar ratio of UC:PL of about 0.8,6 which is far higher than the ratio of 0.12 found in normal hepatic tissue (Table 2), we anticipated that hepatic uptake of vesicles might increase this ratio in liver. After digestion with RNase, protected mRNA probes were quantified by polyacrylamide gel electrophoresis and then autoradiography using a PhosphorImager (Molecular Dynamics). This may take some time to load. 3P<.01; significantly different from saline control. Dynamical fluctuations of droplet microemulsions and vesicles, M. Gradzielski, M. Bergmeier, M. Muller, and H. Hoffmann, , Novel gel phase: A cubic phase of densely packed monodisperse, unilamellar vesicles, M. Gradzielski, M. Bergmeier, H. Hoffmann, M. Muller, and I. Grillo, , Vesicle gel formed by a self-organization process, Some isotropic mesophases in systems containing amphiphilic compounds, New SANS instrument at the BER-II reactor in Berlin, Germany, R. Gilles, U. Keiderling, P. Strunz, A. Wiedenmann, and H. Fuess, , Silver behenate as a standard for instrumental resolution and wavelength calibration for small angle neutron scattering, The new BerSANS-PC software for reduction and treatment of small angle neutron scattering data, Determination of size distribution from small-angle scattering data for systems with effective hard-sphere interactions, Analysis of classical statistical mechanics by means of collective coordinates, P. Schleger, B. Alefeld, J. Barthelemy, G. Ehlers, B. Farago, P. Giraud, C. Hayes, A. Kollmar, C. Lartigue, F. Mezei, and D. Richter, , The long-wavelength neutron spin-echo spectrometer IN15 at the Institut Laue-Langevin, B. Farago, D. Richter, J. S. Huang, S. A. Safran, and S. T. Milner, , Shape and size fluctuations of microemulsion droplets: The role of cosurfactant, M. Gradzielski, D. Langevin, and B. Farago, , Experimental investigation of the structure of nonionic microemulsions and their relation to the bending elasticity of the amphiphilic film, Bending elasticity of the surfactant monolayer in droplet microemulsions: Determination by a combination of dynamic light scattering and neutron spin-echo spectroscopy, The effect of the charge density of microemulsion droplets on the bending elasticity of their amphiphilic film, L. R. Arriaga, I. Lpez-Montero, F. Monroy, G. Orts-Gil, B. Farago, and T. Hellweg, , Stiffening effect of cholesterol on disordered lipid phases: A combined neutron spin echo + dynamic light scattering analysis of the bending elasticity of large unilamellar vesicles, L. R. Arriaga, I. Lpez-Montero, G. Orts-Gil, B. Farago, T. Hellweg, and F. Monroy, , Fluctuation dynamics of spherical vesicles: Frustration of regular bulk dissipation into subdiffusive relaxation, Undulations and dynamic structure factor of membranes, B. Farago, M. Monkenbusch, K. Goecking, D. 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Monroy, , Dissipative dynamics of fluid lipid membranes enriched in cholesterol, Liquid dynamics and inelastic scattering of neutrons, Solubilization efficiency boosting by amphiphilic block co-polymers in microemulsions, M. Mihailescu, M. Monkenbusch, J. Allgaier, H. Frielinghaus, D. Richter, B. Jakobs, and T. Sottmann, , Neutron scattering study on the structure and dynamics of oriented lamellar phase microemulsions, Concentration dependence of shape and structure fluctuations of droplet microemulsions investigated by neutron spin echo spectroscopy, T. Foster, T. Sottmann, R. Schweins, and R. Strey, , Small-angle-neutron-scattering from giant water-in-oil microemulsion droplets. Copyright Clearance Center request page. The three injections of SUVs caused 40% to 50% suppression of hepatic mRNA levels for the LDL receptor and HMG-CoA reductase. Also, lipoprotein fractionation revealed an increase in the concentration of HDL EC in SUV-treated animals. If you are an author contributing to an RSC publication, you do not need to request permission Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and published in abstract form (Circulation. Phospholipid content equals phosphatidylcholine plus phosphatidylethanolamine. Williams is an Established Investigator of the American Heart Association/Genentech. After hepatic uptake of this cholesterol, however, SUV-treated animals developed persistently elevated plasma LDL concentrations, which by day 6 had increased to more than four times the values in saline-treated controls.
When liver cells were cholesterol enriched in vivo by a single injection of apoE-rich HDL that was rapidly cleared from plasma into liver13 or by feeding nonhuman primates long term on atherogenic diets,39 similar results were observed, including LDL receptor suppression and lipoprotein oversecretion. In B and C, LUV values were statistically indistinguishable from the corresponding control (saline) values, except for C, day 3 (.01To generate SUVs, 30-mL batches of MLVs were each subjected to three 20-minute cycles of sonication in 50-mL round-bottom Pyrex glass tubes at 0C under nitrogen (power setting=3, duty cycle=50%, Branson stud tip sonifier, VWR Co). If the lipid solution does not become clear and/or it requires very, very little force to pass the lipid solution through the extruder, then it is possible that the polycarbonate membranes have developed a tear. *P<.05, **P<.01; significant differences between LUV and SUV values.Download figureDownload PowerPoint Tissue samples were stored at 70C until isolation of total RNA and lipid analysis. Hepatic mRNA levels for key enzymes in cholesterol homeostasis are shown in Fig 3. Mass values are shown as meanSEM, expressed as microgram lipid per milligram protein in liver (n=4 animals per treatment group). Dry the lipids down with N2(g). Gel filtration revealed that most of the increase in whole-plasma EC was transported by LDL-sized particles (Fig 1C, Table 1, and Fig 2A). The area under the LDL EC peaks in the saline, SUV, and LUV animal were the same, much larger, and slightly smaller, respectively, than on day 1. to permit an approximate estimate of the curvature energy associated with vesicle formation. Consistent with prior literature, the diameters of the LUVs and SUVs generated were found to be 12335 and 3430 nm (meanSD), respectively, determined by quasielastic light scattering using a Nicomp model 370 submicron laser particle sizer, equipped with a 5-mW He-Ne laser (Pacific Scientific).6. These results suggest a proportionately greater increase in LDL EC than in plasma apoB after SUV administration, raising the possibility of increased LDL size, which was confirmed by a slight shift on the EC elution profile (Fig 2A, EC profile after SUV treatment). Fetching data from CrossRef. Because the role of CETP in provoking or preventing atherosclerosis is controversial,606162 the importance in atherogenesis of our finding that CETP mRNA is suppressed after LUV injections is not clear. Arrows indicate intravenous injections of PL vesicles or saline on days 1, 3, and 5. LUVs of 120-nm diameter or slightly larger will not pass easily and are cleared instead by the macrophage Kupffer cells that line liver sinusoids.
Similarly, cholesterol enrichment of hepatocytes in vitro causes suppression of LDL receptors4243 and HMG-CoA reductase,44454647 as well as enhancement of apoB secretion.16434849 The expression of hepatic 7-hydroxylase is stimulated by cellular cholesterol enrichment in rats50 but inhibited in rabbits,1740 consistent with the trend in Fig 3. Figure 2. The EC curve was generated by difference. Each LUV value was significantly different from the corresponding SUV value (P<.01), except for VLDL EC (not significant). In contrast, the three injections of LUVs caused increases, though statistically insignificant, in each of these messages. Bending stiffness of biological membranes: What can be measured by neutron spin echo? You should feel slight resistance when pushing the syringes, and the lipid solution should become transparent (vs. opaque). In contrast, LUVs may be a suitable therapeutic agent, because they mobilize peripheral cholesterol to the liver without suppressing hepatic LDL receptor mRNA and without provoking a subsequent rise in plasma LDL levels. Allow to dry on Kimwipes. We conclude that LUVs and SUVs mobilize peripheral tissue cholesterol and deliver it to the liver, but to distinct metabolic pools that exert different regulatory effects. On the other hand, the small unilamellar vesicles used here have a size between larger vesicles, with dynamics being well described by the Zilman-Granek model and smaller microemulsion droplets which can be described by the Milner-Safran model. The American Heart Association is qualified 501(c)(3) tax-exempt Soc., 1986,81, 163
Unesterified and Esterified Cholesterol Concentrations in Whole Plasma and in VLDL-, LDL-, and HDL-Sized Particles on Day 6.