Dilinoleoylphosphatidylcholine Research Articles

Synergy of Phospholipid-Drug Formulations Significantly Deactivates Profibrogenic Human Hepatic Stellate Cells.

The pivotal role of hepatic stellate cells (HSCs) in orchestrating the bidirectional process of progression and regression of liver fibrosis makes them an ideal target for exploring new antifibrotic therapies. Essential phospholipids (EPLs), with their polyenylphosphatidylcholine (PPC) fraction, either alone or combined with other hepatoprotective substances such as silymarin, are recommended in hepatic impairment, but a scientific rationale for their use is still lacking. Herein, we compared the ability of EPLs to restore quiescent-like features in HSCs with that of dilinoleoylphosphatidylcholine (DLPC), PPC fraction’s main component. Specifically, we screened at the cellular level the antifibrotic effects of PPC formulations in the presence and absence of silymarin, by using LX-2 cells (pro-fibrogenic HSCs) and by assessing the main biochemical hallmarks of the activated and deactivated states of this cell line. We also proved the formulations’ direct effect on the motional order of cell membranes of adherent cells. LX-2 cells, examined for lipid droplets as a quiescence marker, showed that PPCs led to a more prominent deactivation than DLPC. This result was confirmed by a reduction of collagen and α-SMA expression, and by a profound alteration in the cell membrane fluidity. PPC–silymarin formulations deactivated HSCs with a significant synergistic effect. The remarkable bioactivity of PPCs in deactivating fibrogenic HSCs paves the way for the rational design of new therapeutics aimed at managing hepatic fibrosis.

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.

Bicelle-to-Vesicle Transition of a Binary Phospholipid Mixture Guided by Controlled Local Lipid Compositions: A Molecular Dynamics Simulation Study.

An essential step of nanoliposome formation in an aqueous lipid solution is the transition from discoidal lipid aggregate (bicelle) to vesicle. We investigate here the bicelle-to-vesicle transition of a binary lipid mixture of saturated and unsaturated phosphatidylcholine by performing nonequilibrium molecular dynamics simulations with the coarse-grained representation of di-palmitoyl-phosphatidyl-choline (DPPC) and di-linoleoyl-phosphatidyl-choline (DLiPC). When the DPPC molecules of a stable DPPC bicelle are randomly replaced with the DLiPC molecules, the transition occurs for higher apparent DLiPC concentrations. On the other hand, when the DPPC molecules only in the core region of the bicelle are replaced, the transition occurs even for lower apparent DLiPC concentrations. For the bicelle where the head and tail layers are pure DPPC and DLiPC monolayers, respectively, the side of the DLiPC monolayer becomes the concave surface of bending bicelle. Controlling the local lipid compositions in a binary lipid bicelle has the potential to determine the success of vesicle formation and the direction of bicelle bending. Our findings help explain nanoliposome formation with sonication and give useful information for controlling encapsulation efficiencies of nanoliposomes.

The Arabidopsis thaliana lysophospholipid acyltransferase At1g78690p acylates lysocardiolipins

The Arabidopsis thaliana lysophospholipid acyltransferase At1g78690 acylates a variety of lysophospholipids such as lyso phosphatidylglycerol, lyso phosphatidylethanolamine and lyso phosphatidylserine. Despite di-acylate phosphatidylglycerol being a substrate, overexpression of At1g78690 in Escherichia coli leads to the accumulation of acyl-PG. Here we show that cardiolipin also accumulates in cells overexpressing At1g78690. To help try and explain this observation, we show, using a liquid chromatography mass spectrometry (LC-MS) based assay, that At1g78690 utilizes both mono- and di-lyso cardiolipin as an acyl acceptor. Because At1g78690 shares high homology (∼40%) with the cardiolipin remodeling enzyme tafazzin, we also tested whether At1g78690 was able to catalyze a tafazzin-like transacylation reaction. Di-linoleoyl phosphatidylcholine was used as the acyl donor and mono-lyso cardiolipin was used as the acyl acceptor in a reaction and the reaction was monitored by LC-MS. No transfer of the linoleoyl chains was detected in an At1g78690 dependent manner suggesting that, despite the strong homology, these enzymes catalyze unique reactions.

Neutral Phospholipids Stimulate Na,K-ATPase Activity: A SPECIFIC LIPID-PROTEIN INTERACTION

Membrane proteins interact with phospholipids either via an annular layer surrounding the transmembrane segments or by specific lipid-protein interactions. Although specifically bound phospholipids are observed in many crystal structures of membrane proteins, their roles are not well understood. Na,K-ATPase is highly dependent on acid phospholipids, especially phosphatidylserine, and previous work on purified detergent-soluble recombinant Na,K-ATPase showed that phosphatidylserine stabilizes and specifically interacts with the protein. Most recently the phosphatidylserine binding site has been located between transmembrane segments of αTM8-10 and the FXYD protein. This paper describes stimulation of Na,K-ATPase activity of the purified human α1β1 or α1β1FXYD1 complexes by neutral phospholipids, phosphatidylcholine, or phosphatidylethanolamine. In the presence of phosphatidylserine, soy phosphatidylcholine increases the Na,K-ATPase turnover rate from 5483 ± 144 to 7552 ± 105 (p < 0.0001). Analysis of α1β1FXYD1 complexes prepared with native or synthetic phospholipids shows that the stimulatory effect is structurally selective for neutral phospholipids with polyunsaturated fatty acyl chains, especially dilinoleoyl phosphatidylcholine or phosphatidylethanolamine. By contrast to phosphatidylserine, phosphatidylcholine or phosphatidylethanolamine destabilizes the Na,K-ATPase. Structural selectivity for stimulation of Na,K-ATPase activity and destabilization by neutral phospholipids distinguish these effects from the stabilizing effects of phosphatidylserine and imply that the phospholipids bind at distinct sites. A re-examination of electron densities of shark Na,K-ATPase is consistent with two bound phospholipids located between transmembrane segments αTM8-10 and TMFXYD (site A) and between TM2, -4, -6, -and 9 (site B). Comparison of the phospholipid binding pockets in E2 and E1 conformations suggests a possible mechanism of stimulation of Na,K-ATPase activity by the neutral phospholipid.

Development of ‘leaky’ liposome triggered by radiation applicable to a drug reservoir and a simple radiation dosimeter

Radiation effects on permeability of liposomes with different lipid compositions and the content ratios were investigated using X-rays. Liposomes constituted from an unsaturated phosphatidylcholine (PC), a saturated PC, and cholesterol were selected. As a result, we found that a PC with bis-allylic hydrogen, particularly dilinoleoyl PC (DLOPC), is essential for the radiation sensitivity in permeability, and it can be controlled by changing DLOPC content and dose rate. These characteristics are contributive to a novel pharmaceutical and a new-conceptual radiation dosimeter.

Ternary Lipid Domain Formation using Atomistic Molecular Dynamics Simulations

Lipid rafts are membrane bilayer micro-domains that are thought to compartmentalize cell processes, though their existence has not yet been demonstrated. Understanding phase separation of model membranes containing cholesterol is a crucial step to understanding the properties of lipid rafts. We study the role of cholesterol and lipid tail unsaturation in phase separation behaviour of phosphatidylcholine lipids using atomistic molecular dynamics simulations. We ran a 10-microsecond simulation of a large randomized bilayer of distearoyl-phosphatidylcholine (DSPC), dilinoleoyl-phosphatidylcholine (DLPC), and cholesterol and directly observe domain formation (liquid ordered and liquid disordered coexistence). Phase-separated stripe domains were simulated to determine the free energy for the exchange of a single DSPC and DLPC between the liquid-ordered and liquid-disordered domains. We performed free energy decompositions and also further partition the enthalpic contributions, which we plot over the surface of the bilayer. Interestingly, we found that it is enthalpically favourable for a single lipid of either type (DSPC or DLPC) to be present in the liquid-ordered phase and also that there seems to exist a correlation between lipid tail enthalpy, lipid tail-order parameter, and local occupancy, suggesting certain roles for cholesterol. We determine atomistic-resolution driving forces for domain formation in ternary lipid mixtures.

Formation of 4-hydroxynonenal from cardiolipin oxidation: Intramolecular peroxyl radical addition and decomposition

We report herein that oxidation of a mitochondria-specific phospholipid tetralinoleoyl cardiolipin (L 4CL) by cytochrome c and H 2O 2 leads to the formation of 4-hydroxy-2-nonenal (4-HNE) via a novel chemical mechanism that involves cross-chain peroxyl radical addition and decomposition. As one of the most bioactive lipid electrophiles, 4-HNE possesses diverse biological activities ranging from modulation of multiple signal transduction pathways to the induction of intrinsic apoptosis. However, where and how 4-HNE is formed in vivo are much less understood. Recently a novel chemical mechanism has been proposed that involves intermolecular dimerization of fatty acids by peroxyl bond formation; but the biological relevance of this mechanism is unknown because a majority of the fatty acids are esterified in phospholipids in the cellular membrane. We hypothesize that oxidation of cardiolipins, especially L 4CL, may lead to the formation of 4-HNE via this novel mechanism. We employed L 4CL and dilinoleoylphosphatidylcholine (DLPC) as model compounds to test this hypothesis. Indeed, in experiments designed to assess the intramolecular mechanism, more 4-HNE is formed from L 4CL and DLPC oxidation than 1-palmitoyl-2-linoleoylphosphatydylcholine. The key products and intermediates that are consistent with this proposed mechanism of 4-HNE formation have been identified using liquid chromatography–mass spectrometry. Identical products from cardiolipin oxidation were identified in vivo in rat liver tissue after carbon tetrachloride treatment. Our studies provide the first evidence in vitro and in vivo for the formation 4-HNE from cardiolipin oxidation via cross-chain peroxyl radical addition and decomposition, which may have implications in apoptosis and other biological activities of 4-HNE.

Effect of acyl chains of phosphatidylcholines on the pharmacokinetics of menatetrenone incorporated in O/W lipid emulsions prepared with phosphatidylcholines and soybean oil in rats.

Oil-in-water (O/W) lipid emulsions were prepared with phosphatidylcholines (PCs) of various acyl chains and soybean oil (SO) using a microfluidizer system, and the pharmacokinetics of menatetrenone incorporated in these oil particles were examined at the clinical injection volume (0.1 mL kg(-1)) in rats. The plasma half-life of menatetrenone incorporated in the oil particles prepared with SO and dipalmitoylphosphatidylcholine (DPPC) (SO/DPPC) was longer than that prepared with SO and eggyolk phosphatides (EYP) (SO/EYP) by 3 fold, while those of menatetrenone as oil particles prepared with SO and either dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC) or dilinoleoyl phosphatidylcholine (DLoPC) (SO/DLPC, SO/DMPC, SO/DSPC, SO/DOPC and SO/DLoPC, respectively) were similar to that of menatetrenone as SO/EYP. The menatetrenone uptake by the liver was not significantly different from that as SO/EYP in all SO/PCs examined, but the menatetrenone uptake by the spleen as SO/DPPC and SO/DSPC was higher than that as SO/EYP. The menatetrenone uptake by the lungs as SO/DPPC was also higher than that as SO/EYP. These findings suggest that SO/DPPC is a good candidate drug carrier for the prolonged plasma circulation of lipophilic drugs.

Phospholipids block nuclear factor-kappa B and tau phosphorylation and inhibit amyloid-beta secretion in human neuroblastoma cells

Inflammation and oxidative stress have been shown to play a critical role in the pathophysiology that leads to neurodegeneration. Omega-6 phospholipids, e.g. dilinoleoylphosphatidylcholine (DLPC), have been shown to have anti-inflammatory properties and therefore experiments were undertaken to determine whether DLPC can prevent inflammatory neurodegenerative events in the model neuronal cell line, SH-SY5Y. Tumor necrosis factor (TNF-α) and H2O2 activate mitogen-activated protein kinase (MAPK) in SH-SY5Y cells within 5 min and this activation is completely blocked by DLPC (12 μM). DLPC blocks IκBα phosphorylation in the SH-SY5Y cells and prevents the phosphorylation and activation of nuclear factor-kappa B (NF-κB). The phospholipid inhibits induction of MAPK and NF-κB in similar fashion to the MEK1/2-inhibitor, U0126 (10 μM). DLPC completely abolishes TNF-α, H2O2 and lipopolysaccaride (LPS)-induced neuronal tau phosphorylation. Cellular amyloid precursor protein levels are reduced by DLPC and LPS-induced amyloid-β expression and secretion in SH-SY5Y cells are completely blocked by DLPC. Taken together, these data suggest that DLPC can act through MAPK to block neuronal inflammatory cascades and prevent potential pathological consequences in the neuronal metabolism of amyloid and tau proteins.

Photo-oxidations initiated by UV radiation of urocanic acid and its methyl ester in solution, micelles, and lipid bilayers: TYPE I (free radical) or TYPE II (singlet oxygen) mechanisms depend on the medium

Photo-oxidative reactions on methyl linoleate (ML) initiated by UV radiation of urocanic acid (UCA) or methyl urocanate (UCAME) in a non-polar solvent, aqueous sodium dodecyl sulfate (SDS) micelles or ML in lipid bilayers of dimyristoylphosphatidylcholine (DMPC) or of dilinoleoylphosphatidylcholine (DLPC) are investigated to determine medium effects on reaction mechanisms. Experiments include: kinetics of oxygen uptake, effects of radical trapping antioxidants or singlet oxygen quenchers and product analysis of cis,trans to trans,trans ( c,t// t,t) isomer ratios of oxidized linoleate to distinguish free radical, TYPE I and singlet oxygen TYPE II reactions. Irradiation of the system toluene/ML/UCAME reaction occurred by TYPE I according to kinetics studies and inhibiting effects of 2,6-di- t-butyl-4-methoxyphenol (DBHA) and the four typical c,t// t,t isomer ratios formed. Similarly irradiation of the system SDS/ML/UCAME (or UCA) resulted in the TYPE I reaction. The latter system was used to evaluate the antioxidant activities, k inh , showing the relative activities as: Trolox ≥ 2,2,5,7,8-pentamethylhydroxychroman (PMHC) > DBHA. The low k inh values of these antioxidants compared to those in a non-polar solvent are interpreted by quantitative methods of “solvent effects”. Photo-oxidation of the systems DMPC/ML/UCA or DLPC/UCA occurred by singlet oxygen (TYPE II) in contrast to those in solution or micelles according to effects of a singlet oxygen quencher, sodium azide, and lack of inhibition by antioxidants. This contrast is explained by the structure of lipid bilayers where the excited 3n,π* of UCA produces singlet oxygen in the aqueous phase which diffuses into the bilayer phase to cause TYPE II reactions.

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.

The combination of S-adenosylmethionine and dilinoleoylphosphatidylcholine attenuates nonalcoholic steatohepatitis produced by a high-fat diet in rats

In the pathogenesis of non-alcoholic steatohepatitis (NASH), oxidative stress resulting from free radicals generated by cytochrome P4502E1 (CYP2E1) plays a major role suggesting the importance of antioxidants. The objective of this study was to assess in a high-fat diet (HF) rat model the effects of the combination of s-adenosylmethionine (SAMe) plus dilinoleoylphosphatidylcholine (DLPC) in the treatment of NASH. To test the hypothesis that these two antioxidants are beneficial in NASH, male Sprague-Dawley rats were fed five different diets for six weeks: control, HF diet and HF plus SAMe and DLPC or their combination. As expected, the HF diet significantly increased hepatic triacylglycerols and CYP2E1 levels. However, only the combination diet opposed this effect, consistent with different actions of the two antioxidants. Next, 24 additional rats divided in two groups were fed the HF or the HF+SAMe+DLPC diets for 3 weeks. Dietary intake was similar, but liver triacylglycerols dropped from 76.1+/-6.8 to 49.4+/-3.5 mg/g (p=0.002) and hepatic CYP2E1 mRNA decreased after treatment (p=0.01) with a trend for less CYP2E1 protein. This was accompanied by a 41% reduction of hepatic 4-hydroxynonenal (4-HNE) (p=0.008), reflecting control of oxidative stress. Furthermore, the hepatic inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) mRNA and TNF-alpha protein decreased (p=0.05 and p=0.01 respectively) with attenuation of alpha1(I) procollagen mRNA and type I collagen levels (p=0.01 and p=0.02, respectively). We concluded that the combination SAMe+DLPC might be beneficial in NASH by reducing oxidative stress and associated liver injury.

Unsaturated phosphatidylcholines lining on the surface of cartilage and its possible physiological roles

BackgroundEvidence has strongly indicated that surface-active phospholipid (SAPL), or surfactant, lines the surface of cartilage and serves as a lubricating agent. Previous clinical study showed that a saturated phosphatidylcholine (SPC), dipalmitoyl-phosphatidylcholine (DPPC), was effective in the treatment of osteoarthritis, however recent studies suggested that the dominant SAPL species at some sites outside the lung are not SPC, rather, are unsaturated phosphatidylcholine (USPC). Some of these USPC have been proven to be good boundary lubricants by our previous study, implicating their possible important physiological roles in joint if their existence can be confirmed. So far, no study has been conducted to identify the whole molecule species of different phosphatidylcholine (PC) classes on the surface of cartilage. In this study we identified the dominant PC molecule species on the surface of cartilage. We also confirmed that some of these PC species possess a property of semipermeability.MethodsHPLC was used to analyse the PC profile of bovine cartilage samples and comparisons of DPPC and USPC were carried out through semipermeability tests.ResultsIt was confirmed that USPC are the dominant SAPL species on the surface of cartilage. In particular, they are Dilinoleoyl-phosphatidylcholine (DLPC), Palmitoyl-linoleoyl-phosphatidylcholine, (PLPC), Palmitoyl-oleoyl-phosphatidylcholine (POPC) and Stearoyl-linoleoyl-phosphatidylcholine (SLPC). The relative content of DPPC (a SPC) was only 8%. Two USPC, PLPC and POPC, were capable of generating osmotic pressure that is equivalent to that by DPPC.ConclusionThe results from the current study confirm vigorously that USPC is the endogenous species inside the joint as against DPPC thereby confirming once again that USPC, and not SPC, characterizes the PC species distribution at non-lung sites of the body. USPC not only has better anti-friction and lubrication properties than DPPC, they also possess a level of semipermeability that is equivalent to DPPC. We therefore hypothesize that USPC can constitute a possible addition or alternative to the current commercially available viscosupplementation products for the prevention and treatment of osteoarthritis in the future.

Bilirubin as an antioxidant in micelles and lipid bilayers: Its contribution to the total antioxidant capacity of human blood plasma

The antioxidant capacities, antioxidant activities, k inh, and stoichiometric factors, n, of water-soluble derivatives of bilirubin (BR), BR-human serum albumin (BR-HSA), and BR-ditaurate disodium conjugate (BRC) were determined in aqueous/lipid dispersions of sodium dodecyl sulfate (SDS) micelles/methyl linoleate and in bilayers of dilinoleoylphosphatidylcholine (DLPC) during initiation by water-soluble azo-bis-amidinopropane dihydrochloride (ABAP). The inhibition rate constants for BRC and BR-HSA were similar in micelles ( k inh ≈ 1.3 × 10 4 M − 1 s − 1 ), where n ≈ 2, whereas the k inh for BR-HSA dropped by ½ in bilayers. The dimethyl ester of bilirubin (BRDE) gave a k inh only one-tenth that of the vitamin E analog, pentamethylhydroxychroman (PMHC) in SDS micelles/methyl linoleate when initiated by lipid-soluble azo-bis-2,4-dimethylvaleronitrile (DMVN). Biliverdin hydrochloride (BVHCl) was NOT an effective peroxyl radical-trapping agent in the micellar phase during initiation by ABAP or DMVN containing methyl linoleate but it inhibited oxygen uptake in the aqueous phase. Both BRC and BR-HSA extended the total radical antioxidant parameter (TRAP) of human blood plasma and their contribution to TRAP was in the range of 5–10% of the natural TRAP of blood plasma, depending on the BR content determined in the blood plasma.

Effects of a New Bioactive Lipid-Based Drug Carrier on Cultured Hepatic Stellate Cells and Liver Fibrosis in Bile Duct-Ligated Rats

In the fibrotic liver, hepatic stellate cells (HSC) produce large amounts of collagen and secrete variety of mediators that promote development of fibrosis in this organ. Therefore, these cells are considered an attractive target for antifibrotic therapies. We incorporated the bioactive lipid dilinoleoylphosphatidylcholine (DLPC) into the membrane of liposomes, and then we evaluated its effect on hepatic stellate cell activation and liver fibrosis. To target DLPC-liposomes to HSC, human serum albumin modified with mannose 6-phosphate (M6P-HSA) was coupled to the surface of these liposomes. In vitro, the effects of the carrier were determined in primary cultures of HSC, Kupffer cells, and liver endothelial cells using real-time reverse transcription-polymerase chain reaction. In vivo DLPC-liposomes were tested in bile duct-ligated rats. Targeted M6P-HSA-DLPC-liposomes and DLPC-liposomes significantly reduced gene expression levels for collagen 1alpha1, alpha-smooth muscle actin (alpha-SMA), and transforming growth factor-beta (TGF-beta) in cultured HSC. In fibrotic livers, DLPC-liposomes decreased gene expression for TGF-beta and collagen 1alpha1 as well as alpha-SMA and collagen protein expression. In contrast, M6P-HSA-DLPC-liposomes enhanced expression of profibrotic and proinflammatory genes in vivo. In cultured Kupffer and endothelial cells M6P-HSA liposomes influenced the expression of proinflammatory genes. Both types of liposomes increased hepatocyte glycogen content in fibrotic livers, indicating improved functionality of the hepatocytes. We conclude that DLPC-containing liposomes attenuate activation of cultured HSC. In fibrotic livers, M6P-HSA-mediated activation of Kupffer and endothelial cells probably counteracts this beneficial effect of DLPC-liposomes. Therefore, these bioactive drug carriers modulate the activity of all liver cells during liver fibrosis.

Peroxidation of polyunsaturated phosphatidyl-choline lipids during electroformation

Giant unilamellar vesicles (GUVs) have been utilized both as model systems to study the physico-chemical properties of biomembranes and as host materials for investigating biological processes in microbioreactors. GUVs are commonly formed by an electroformation technique. However, there is a concern that the electric fields applied during electroformation can peroxidize lipid acyl chains, thereby altering the phospholipid composition and material properties of the synthesized vesicles. Here in this paper, we report the effect of electroformation on the extent of peroxidation of a number of polyunsaturated phosphatidyl-choline lipids (PULs). Specifically, we detected peroxidation byproducts (malonaldehydes and conjugated dienes) of the following lipids utilizing UV/Vis spectroscopy: dilinoleoyl phosphatidyl-choline (DLPC) (di-18:2 PC), dilinolenoyl phosphatidyl-choline (DNPC) (di-18:3 PC), diarachidonoyl phosphatidyl-choline (DAPC) (di-20:4 PC), and didocosaheexaenoyl phosphatidyl-choline (DHA) (di-22:6 PC). The results indicate that PC PULs lipids are prone to peroxidation, with increasing unsaturation levels leading to higher levels of peroxidation byproducts. The levels of peroxidation byproducts of DAPC were found to depend linearly on the strength of the electric field, indicating that the observed effects were due to the applied electric field. Lipid peroxidation can affect a number of important membrane properties, including domain formation and mechanical stability. Thus, alteration of the chemical composition of polyunsaturated lipids (PULs) by the electroformation technique can potentially complicate the interpretation of experimental studies that utilize GUVs composed of PULs.

DLPC and SAMe prevent α1(I) collagen mRNA up-regulation in human hepatic stellate cells, whether caused by leptin or menadione

We previously reported that the combination of dilinoleoylphosphatidylcholine (DLPC) and S-adenosylmethionine (SAMe), which have antioxidant properties and antifibrogenic actions, prevented leptin-stimulated tissue inhibitor of metalloproteinase (TIMP)-1 production in hepatic stellate cells (HSCs) by inhibiting H 2O 2-mediated signal transduction. We now show that DLPC and SAMe inhibit α1(I) collagen mRNA expression induced by leptin or menadione in LX-2 human HSCs. We found that DLPC and SAMe prevent H 2O 2 generation and restore reduced glutathione (GSH) depletion whether caused by leptin or menadione. Blocking H 2O 2 signaling through ERK1/2 and p38 pathways resulted in a complete inhibition of leptin or menadione-induced α1(I) collagen mRNA. The inhibition of collagen mRNA by DLPC and SAMe combined is at least two times more effective than that by DLPC or SAMe alone. In conjunction with the prevention of TIMP-1 production, the ability of DLPC and SAMe to inhibit α1(I) collagen mRNA expression provides a mechanistic basis for these innocuous compounds in the prevention of hepatic fibrosis, because enhanced TIMP-1 and collagen productions are associated with hepatic fibrogenesis and their attenuation may diminish fibrosis.

A phospholipase A2‐linked receptor for PEDF

Pigment epithelium-derived factor (PEDF), a 50-kDa extracellular serpin, is a neurotrophic and antiangiogenic factor for the retina. Its biological functions are independent from its serine protease inhibition potential. Previously we provided evidence for high affinity PEDF-binding sites and proteins in retina cell-surfaces. Recently a novel human retinal pigment epithelial gene was identified as potential PEDF receptor and termed PEDF-R (pseudonyms: PNLP2, TTS-2.1, ATGL, iPLAζ, desnutrin). It codes for a polypeptide (504 amino acids) with a patatin-like phospholipase (PLA2) domain, and 4 transmembrane, 3 intracellular and 2 extracellular regions. RT-PCR and immunohistochemistry confirmed the PEDF-R expression in the retina. The potential PLA2 activity of PEDF-R was investigated. Recombinant epitope-tagged PEDF-R polypeptide was synthesized in cell-free systems and purified. Using dilinoleoyl phosphatidylcholine as substrate, PEDF-R exhibited potent PLA activity, which was optimum at pH 7.5. Surface plasmon resonance revealed high affinity for the PEDF:PEDF-R interactions (KD = ~3 nM), which decreased significantly with other serpins (maspin, ovalbumin). Preincubation of PEDF-R with increasing concentrations of PEDF stimulated the PLA activity of PEDF-R, but the stimulation did not occur when BSA replaced PEDF. PEDF alone did not show activity, and did not stimulate hog pancreas PLA2. These results demonstrate that the recombinant PEDF-R exhibited PLA activity that liberates fatty acids, an activity that PEDF can stimulate, suggesting a molecular pathway by which ligand/receptor interaction on the cell-surface could generate a cellular signal. Supported by the Intramural Research Program of the NIH, NEI.

DLPC and SAMe combined prevent leptin‐stimulated TIMP‐1 production in LX‐2 human hepatic stellate cells by inhibiting H2O2‐mediated signal transduction

Both dilinoleoylphosphatidylcholine (DLPC) and S-adenosylmethionine (SAMe) have antioxidant properties and antifibrogenic actions. Because H2O2 mediates signal transduction-stimulating liver fibrogenesis, we investigated whether DLPC and SAMe attenuate the production of tissue inhibitor of metalloproteinase (TIMP)-1 by inhibiting H2O2 formation. LX-2 human hepatic stellate cells were treated with leptin with or without DLPC, SAMe or various inhibitors. Leptin-stimulated TIMP-1 mRNA and its protein were diminished by DLPC or SAMe alone, and the response was fully prevented by the combination of DLPC and SAMe. H2O2 was increased while glutathione was decreased; these changes were prevented by AG490, suggesting a Janus kinases (JAK)-mediated process. Up-regulation of leptin receptor and activation of JAK1 and 2 were not affected by DLPC+SAMe, whereas phosphorylation of ERK1/2 and p38 was blocked by DLPC+SAMe or catalase, suggesting an H2O2-dependent mechanism. These treatments also suppressed leptin-stimulated TIMP-1 promoter activity and decreased TIMP-1 mRNA stability, contributing to TIMP-1 mRNA down-regulation. PD098059, an ERK1/2 inhibitor, suppressed TIMP-1 promoter activity, whereas SB203580, a p38 inhibitor, decreased TIMP-1 message stability; both resulted in a partial reduction of TIMP-1 mRNA. As decreased TIMP-1 production may enhance collagen deposition, the combined administration of DLPC+SAMe should be considered for the prevention of H2O2-mediated signaling and the resulting fibrosis.