Oxidation products of phospholipid-containing delta-9 fatty acids specifically impair the activity of tissue factor pathway inhibitor

In the present study, we explored the active components in oxidized low-density lipoprotein (ox-LDL) that reduce the catalytic activity of tissue factor pathway inhibitor (TFPI), a Kunitz-type protease inhibitor of the extrinsic blood coagulation pathway. The active fraction was extracted from the phospholipid fraction of ox-LDL and separated. The oxidation products of 1- and/or 2-oleoyl phosphatidylcholine (PC) or phosphatidylethanolamine were the most potent compounds, while those of arachidonyl PC possessed only a weak inhibitory effect on the TFPI activity. These oxidized phospholipids associated strongly with rTFPI containing the carboxyl-terminal domain. When rTFPI was incubated with purified oxononanoyl PC (9CHO-PC) and its carboxylic form (9COOH-PC), the catalytic activity was specifically impaired, though neither oxovaleroyl PC (5CHO-PC) nor lyso-phospholipids reduced the TFPI activity. We conclude that the oxidation products of delta-9 unsaturated phospholipid in the lipoproteins are the active components that impair the anti-coagulation activity of TFPI.     

A Pressure-dependent Model for the Regulation of Lipoprotein Lipase by Apolipoprotein C-II

Apolipoprotein C-II (apoC-II) is the co-factor for lipoprotein lipase (LPL) at the surface of triacylglycerol-rich lipoproteins. LPL hydrolyzes triacylglycerol, which increases local surface pressure as surface area decreases and amphipathic products transiently accumulate at the lipoprotein surface. To understand how apoC-II adapts to these pressure changes, we characterized the behavior of apoC-II at multiple lipid/water interfaces. ApoC-II adsorption to a triacylglycerol/water interface resulted in large increases in surface pressure. ApoC-II was exchangeable at this interface and desorbed on interfacial compressions. These compressions increase surface pressure and mimic the action of LPL. Analysis of gradual compressions showed that apoC-II undergoes a two-step desorption, which indicates that lipid-bound apoC-II can exhibit at least two conformations. We characterized apoC-II at phospholipid/triacylglycerol/water interfaces, which more closely mimic lipoprotein surfaces. ApoC-II had a large exclusion pressure, similar to that of apoC-I and apoC-III. However, apoC-II desorbed at retention pressures higher than those seen with the other apoCs. This suggests that it is unlikely that apoC-I and apoC-III inhibit LPL via displacement of apoC-II from the lipoprotein surface. Upon rapid compressions and re-expansions, re-adsorption of apoC-II increased pressure by lower amounts than its initial adsorption. This indicates that apoC-II removed phospholipid from the interface upon desorption. These results suggest that apoC-II regulates the activity of LPL in a pressure-dependent manner. ApoC-II is provided as a component of triacylglycerol-rich lipoproteins and is the co-factor for LPL as pressure increases. Above its retention pressure, apoC-II desorbs and removes phospholipid. This triggers release of LPL from lipoproteins.     

Omega-3 fatty acids and dementia

More than a dozen epidemiological studies have reported that reduced levels or intake of omega-3 fatty acids or fish consumption is associated with increased risk for age-related cognitive decline or dementia such as Alzheimer's disease (AD). Increased dietary consumption or blood levels of docosahexaenoic acid (DHA) appear protective for AD and other dementia in multiple epidemiological studies; however, three studies suggest that the ApoE4 genotype limits protection. DHA is broadly neuroprotective via multiple mechanisms that include neuroprotective DHA metabolites, reduced arachidonic acid metabolites, and increased trophic factors or downstream trophic signal transduction. DHA is also protective against several risk factors for dementia including head trauma, diabetes, and cardiovascular disease. DHA is specifically protective against AD via additional mechanisms: It limits the production and accumulation of the amyloid β peptide toxin that is widely believed to drive the disease; and it also suppresses several signal transduction pathways induced by Aβ, including two major kinases that phosphorylate the microtubule-associated protein tau and promote neurofibrillary tangle pathology. Based on the epidemiological and basic research data, expert panels have recommended the need for clinical trials with omega-3 fatty acids, notably DHA, for the prevention or treatment of age-related cognitive decline—with a focus on the most prevalent cause, AD. Clinical trials are underway to prevent and treat AD. Results to-date suggest that DHA may be more effective if it is begun early or used in conjunction with antioxidants.     

4-Hydroxy hexenal derived from dietary n-3 polyunsaturated fatty acids induces anti-oxidative enzyme heme oxygenase-1 in multiple organs

It has recently been reported that expression of heme oxygenase-1 (HO-1) plays a protective role against many diseases. Furthermore, n-3 polyunsaturated fatty acids (PUFAs) were shown to induce HO-1 expression in several cells in vitro, and in a few cases also in vivo. However, very few reports have demonstrated that n-3 PUFAs induce HO-1 in vivo.     

A more detailed fatty acid composition of human lipoprotein(a)--a comparison with low density lipoprotein

Lipoprotein(a)'s (Lp(a)'s) fatty acid composition is partially known for the cholesteryl ester (CE), triglyceride (TG) and total phospholipid (PL) fractions. Individual PLs' fatty acids are unknown. This study sought to confirm and extend existing data and elucidate the individual PLs of Lp(a). For Lp(a) versus LDL, the mole percentage saturated fatty acids comprised 11.3+/-1.3 versus 16.8+/-1.2 (CE) (P<0.05), 43.4+/-5.2 versus 39.2+/-4.0 (TG) (P<0.05), 55.7+/-6.3 versus 54.7+/-5.9 (PL) (P>0.05), 51.9+/-3.5 versus 50.2+/-4.2 (choline-containing phospholipids (PC)) (P>0.05), 40.2+/-4.6 versus 43.1+/-3.9 (ethanolamine-containing phospholipids (PE)) (P>0.05), 73.2+/-7.6 versus 81.2+/-8.2 (sphingomyelin (SPH)) (P<0.05). Linoleic acid was CE's major fatty acid and while palmitic acid was the major fatty acid in all other fractions except PE.     

细菌不饱和脂肪酸合成研究进展

脂肪酸不仅是细菌细胞膜组分，还是许多生物活性物质的合成原料。不饱和脂肪酸(unsaturated fatty acid, UFA)具有更低的相变温度，是细菌调节细胞膜流动性的重要分子，因此UFA合成途径是重要的抗菌药物筛选靶点。细菌可利用厌氧途径合成UFA，其中模式生物大肠杆菌利用经典的FabA-FabB途径合成UFA，但不同细菌中UFA合成的厌氧途径具有多样性，相关催化酶类也不尽相同；细菌还可以利用需氧途径合成UFA，利用脂肪酸脱饱和酶直接将饱和脂肪酸(saturated fatty acid, SFA)转化为不饱和脂肪酸，而不同脱饱和酶会生成不同结构的UFA，在逆境耐受、致病力等多方面发挥重要作用；细菌还可以利用单加氧酶，将脂肪酸合成途径中癸酰酰基载体蛋白(acyl carrier protein, ACP)转化为顺-3-癸烯酰ACP，并最终合成UFA。细菌脂肪酸合成相关的其他酶类在UFA合成或不同种类UFA调节中也发挥着重要作用。本文系统地总结了细菌UFA合成途径与相关酶类的多样性研究进展，旨在为进一步了解细菌UFA合成机制，并以此为靶点开发抗菌药物等方面提供理论支撑。     

Dietary omega-6 fatty acid lowering increases bioavailability of omega-3 polyunsaturated fatty acids in human plasma lipid pools

BackgroundDietary linoleic acid (LA, 18:2n-6) lowering in rats reduces n-6 polyunsaturated fatty acid (PUFA) plasma concentrations and increases n-3 PUFA (eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)) concentrations.ObjectiveTo evaluate the extent to which 12 weeks of dietary n-6 PUFA lowering, with or without increased dietary n-3 PUFAs, alters unesterified and esterified plasma n-6 and n-3 PUFA concentrations in subjects with chronic headache.DesignSecondary analysis of a randomized trial. Subjects with chronic headache were randomized for 12 weeks to (1) average n-3, low n-6 (L6) diet; or (2) high n-3, low n-6 LA (H3–L6) diet. Esterified and unesterified plasma fatty acids were quantified at baseline (0 weeks) and after 12 weeks on a diet.ResultsCompared to baseline, the L6 diet reduced esterified plasma LA and increased esterified n-3 PUFA concentrations (nmol/ml), but did not significantly change plasma arachidonic acid (AA, 20:4n-6) concentration. In addition, unesterified EPA concentration was increased significantly among unesterified fatty acids. The H3–L6 diet decreased esterified LA and AA concentrations, and produced more marked increases in esterified and unesterified n-3 PUFA concentrations.ConclusionDietary n-6 PUFA lowering for 12 weeks significantly reduces LA and increases n-3 PUFA concentrations in plasma, without altering plasma AA concentration. A concurrent increase in dietary n-3 PUFAs for 12 weeks further increases n-3 PUFA plasma concentrations and reduces AA.     

Dissociable and nondissociable forms of platelet-activating factor acetylhydrolase in human plasma LDL: implications for LDL oxidative susceptibility

Platelet-activating factor acetylhydrolase (PAF-AH) is transported by lipoproteins in plasma and is thought to possess both anti-inflammatory and anti-oxidative activity. It has been reported that PAF-AH is recovered primarily in small, dense LDL and HDL following ultracentrifugal separation of lipoproteins. In the present studies, we aimed to further define the distribution of PAF-AH among lipoprotein fractions and subfractions, and to determine whether these distributions are affected by the lipoprotein isolation strategy (FPLC versus sequential ultracentrifugation) and LDL particle distribution profile. When lipoproteins were isolated by FPLC, the bulk (～85%) of plasma PAF-AH activity was recovered within LDL-containing fractions, whereas with ultracentrifugation, there was a redistribution to HDL (which contained ～18% of the activity) and the d>1.21 g/ml fraction (which contained ～32%). Notably, re-ultracentrifugation of isolated LDL did not result in any further movement of PAF-AH to higher densities, suggesting the presence of dissociable and nondissociable forms of the enzyme on LDL. Differences were noted in the distribution of PAF-AH activity among LDL subfractions from subjects exhibiting the pattern A (primarily large, buoyant LDL) versus pattern B (primarily small, dense LDL) phenotype. In the latter group, there was a relative depletion of PAF-AH activity in subfractions in the intermediate to dense range (d=1.039–1.047 g/ml) with a corresponding increase in enzyme activity recovered within the d>1.21 g/ml ultracentrifugal fraction. Thus, there appears to be a greater proportion of the dissociable form of PAF-AH in pattern B subjects. In both populations, most of the nondissociable activity was recovered in a minor small, dense LDL subfraction. Based on conjugated dienes as a measure of lipid peroxidation, variations in PAF-AH activity appeared to contribute to variations in oxidative behavior among ultracentrifugally isolated LDL subfractions. The physiologic relevance of PAF-AH dissociability and the minor PAF-AH-enriched oxidation-resistant LDL subpopulation remains to be determined.     

Differential effects of unsaturated fatty acids on modulation of endotoxin-induced tissue factor activation in cultured human leukemia U937 cells.

We examined the direct effects of unsaturated fatty acids, oleic (18:1 n-9), linoleic (18:2 n-6), eicosapentaenoic (20:5 n-3) and docosahexaenoic (22:6 n-3) on tissue factor (TF) activity in the human leukemia monocytic U937 cell line. After exposing cells to fatty acids for 16 h, there were no significant effects on either TF activity or its activation induced by bacterial endotoxin (LPS). When the cells were primed with fatty acids for 24 h, 48 h or 72 h, the TF activity remained essentially unchanged. However, the extent of TF-activation induced by LPS depended on the length of priming, and the dose and the degree of unsaturation of the fatty acids to which cells were exposed. After a 72-h priming, 18:1 produced 40-60 per cent elevation in LPS-challenge. In contrast, approximately 20-50 per cent reduction in LPS-challenge was achieved by 18:2, 20:5 and 22:6 at high concentrations. The results suggest that chronic exposure of U937 cells to unsaturated fatty acids leads to modulation of the TF-activation in response to LPS.     

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors

The low-density lipoprotein receptor (LDLR) family of receptors are cell-surface receptors that internalize numerous ligands and play crucial role in various processes, such as lipoprotein metabolism, hemostasis, fetal development, etc. Previously, receptor-associated protein (RAP) was described as a molecular chaperone for LDLR-related protein 1 (LRP1), a prominent member of the LDLR family. We aimed to verify this role of RAP for LRP1 and two other LDLR family receptors, LDLR and vLDLR, and to investigate the mechanisms of respective interactions using a cell culture model system, purified system, and in silico modelling. Upon coexpression of RAP with clusters of the ligand-binding complement repeats (CRs) of the receptors in secreted form in insect cells culture, the isolated proteins had increased yield, enhanced folding, and improved binding properties compared with proteins expressed without RAP, as determined by circular dichroism and surface plasmon resonance. Within LRP1 CR-clusters II and IV, we identified multiple sites comprised of adjacent CR doublets, which provide alternative bivalent binding combinations with specific pairs of lysines on RAP. Mutational analysis of these lysines within each of isolated RAP D1/D2 and D3 domains having high affinity to LRP1 and of conserved tryptophans on selected CR-doublets of LRP1, as well as in silico docking of a model LRP1 CR-triplet with RAP, indicated a universal role for these residues in interaction of RAP and LRP1. Consequently, we propose a new model of RAP interaction with LDLR family receptors based on switching of the bivalent contacts between molecules over time in a dynamic mode.     

NGF blocks polyunsaturated fatty acids biosynthesis in n − 3 fatty acid-supplemented PC12 cells

Regulation of polyunsaturated fatty acid (PUFA) biosynthesis in proliferating and NGF-differentiated PC12 pheochromocytoma cells deficient in n − 3 docosahexaenoic acid (DHA 22:6n − 3) was studied. A dose- and time-dependent increase in eicosapentaenoic acid (EPA, 20:5n − 3), docosapentaenoic acid (DPA, 22:5n − 3) and DHA in phosphatidylethanolamine (PtdEtn) and phosphatidylserine (PtdSer) glycerophospholipids (GPL) via the elongation/desaturation pathway following alpha-linolenic acid (ALA, 18:3n − 3) supplements was observed. That was accompanied by a marked reduction of eicosatrienoic acid (Mead acid 20:3n − 9), an index of PUFA deficiency. EPA supplements were equally effective converted to 22:5n − 3 and 22:6n − 3. On the other hand, supplements of linoleic acid (LNA, 18:2n − 6) were not effectively converted into higher n − 6 PUFA intermediates nor did they impair elongation/desaturation of ALA. Co-supplements of DHA along with ALA did not interfere with 20:5n − 3 biosynthesis but reduced further elongation to 22-hydrocarbon PUFA intermediates. A marked decrease in the newly synthesized 22:5n − 3 and 22:6n − 3 following ALA or EPA supplements was observed after nerve growth factor (NGF)-induced differentiation. NGF also inhibited the last step in 22:5n − 6 formation from LNA. These results emphasize the importance of overcoming n − 3 PUFA deficiency and raise the possibility that growth factor regulation of the last step in PUFA biosynthesis may constitute an important feature of neuronal phenotype acquisition.     

Inhibitory action of polyunsaturated fatty acids on IMP dehydrogenase

We screened the inhibitor of mouse inosine 5'-monophosphate dehydrogenase (IMPDH) type II from natural compounds, and found that a fatty acid, linoleic acid (C18:2), inhibited IMPDH activity. In the C18:2 fatty acid derivatives, all trans-configuration (i.e., linoelaidic acid), ester form, alcohol form, and addition of the hydroxyl group of linoleic acid had no effect on inhibitory activity. Therefore, both parts of a carboxylic acid and an alkyl chain containing cis-type double bonds of fatty acid might be essential for inhibition. Among the various carbon atom lengths and double bonds of fatty acids examined, the strongest inhibitor was C20:2-fatty acid, eicosadienoic acid, and 50% inhibition was observed at a concentration of 16.1 microM. Eicosadienoic acid induced the inhibition of IMPDH activity and was competitive with respect to IMP (K(i)=3.1 microM). For inhibitory effect, the C20-fatty acids ranked as follows: C20:2>C20:3>C20:1> C20:4>C20:5, and C20:0 showed no inhibition. The energy-minimized three-dimensional structures of linear-chain C20-fatty acids were calculated, and it was found that a length of 20.7-22.5A and width of 4.7-7.2A in the fatty acid molecular structure was suggested to be important for IMPDH inhibition. Docking simulation of C20-fatty acids and mouse IMPDH type II, which was homology modeled from human IMPDH type II (PDB code: 1NF7), was performed, and the fatty acid could bind to Cys331, which is a amino acid residue of the active site, competitively with IMP. Based on these results, the IMPDH-inhibitory mechanism of fatty acids is discussed.     

Platelet‐derived growth factor promotes the proliferation of human umbilical cord‐derived mesenchymal stem cells

This study was designed to investigate the effect of platelet‐derived growth factor (PDGF) on the proliferation of human umbilical cord mesenchymal stem cells (UC‐MSCs) and further explore the mechanism of PDGF in promoting the proliferation of UC‐MSCs. The human UC‐MSCs were treated with different concentrations of PDGF, and the effects were evaluated by counting the cell number, the cell viability, the expression of PDGF receptors analyzed by RT‐PCR, and the detection of the gene expression of cell proliferation, cell cycle and pluripotency, and Brdu assay by immunofluorescent staining and Quantitative real‐time (QRT‐PCR). The results showed that PDGF could promote the proliferation of UC‐MSCs in vitro in a dose‐dependent way, and 10 to 50 ng/ml PDGF had a significant proliferation effect on UC‐MSCs; the most obvious concentration was 50 ng/ml. Significant inhibition on the proliferation of UC‐MSCs was observed when the concentration of PDGF was higher than 100 ng/ml, and all cells died when the concentration reached 200 ng/ml PDGF. The PDGF‐treated cells had stronger proliferation and antiapoptotic capacity than the control group by Brdu staining. The expression of the proliferation‐related genes C‐MYC, PCNA and TERT and cell cycle–related genes cyclin A, cyclin 1 and CDK2 were up‐regulated in PDGF medium compared with control. However, pluripotent gene OCT4 was not significantly different between cells cultured in PDGF and cells analyzed by immunofluorescence and QRT‐PCR. The PDGF could promote the proliferation of human UC‐MSCs in vitro. Copyright © 2012 John Wiley & Sons, Ltd.     

Resolution and chromatographic configuration analysis of 2-hydroxy fatty acids

2-Hydroxyoctadecanoic acid was resolved into D and L isomers as salts of 1-phenylethylamine enantiomers The diastereomers of phenylethylamides of 2-hydroxy fatty acids and the corresponding derivatives with protected hydroxy group (acetyl, methyl, trifluoro-acetyl, trimethylsilyl) are well separated by thin-layer or gas-liquid chromatography. This allows a simple microanalysis of configuration and optical purity of 2-hydroxy fatty acids. With this method 2-hydroxy fatty acids from sphingomyelin of the honey-bee were shown to belong exclusively to the D series.     

Omega-3 fatty acids from fish oils and cardiovascular disease

Fish and fish oils contain the omega-3 fatty acids known as eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA). Epidemiological studies have shown an inverse relation between the dietary consumption of fish containing EPA/DHA and mortality from coronary heart disease. These relationships have been substantiated from blood measures of omega-3 fatty acids including DHA as a physiological biomarker for omega-3 fatty acid status. Controlled intervention trials with fish oil supplements enriched in EPA/DHA have shown their potential to reduce mortality in post-myocardial infarction patients with a substantial reduction in the risk of sudden cardiac death. The cardioprotective effects of EPA/DHA are widespread, appear to act independently of blood cholesterol reduction, and are mediated by diverse mechanisms. Their overall effects include anti-arrhythmic, blood triglyceride-lowering, anti-thrombotic, anti-inflammatory, endothelial relaxation, plus others. Current dietary intakes of EPA/DHA in North America and elsewhere are well below those recommended by the American Heart Association for the management of patients with coronary heart disease. (Mol Cell Biochem 263: 217–225, 2004)     

On the mechanism of the so-called uncoupling effect of medium- and short-chain fatty acids

Octanoate applied to rat liver mitochondria respiring with glutamate plus malate or succinate (plus rotenone) under resting-state (State 4) conditions stimulates oxygen uptake and decreases the membrane potential, both effects being sensitive to oligomycin but not to carboxyatractyloside. Octanoate also decreases the rate of pyruvate carboxylation under the same conditions, this effect being correlated with the decrease of intramitochondrial content of ATP and increase of AMP. The decrease of pyruvate carboxylation and the change of mitochondrial adenine nucleotides are both reversed by 2-oxoglutarate. Fatty acids of shorter chain length have similar effects, though at higher concentrations. Addition of octanoate in the presence of fluoride (inhibitor of pyrophosphatase) produces intramitochondrial accumulation of pyrophosphate, even under conditions when oxidation of octanoate is prevented by rotenone. In isolated hepatocytes incubated with lactate plus pyruvate, octanoate also increases oxygen uptake and produces a shift in the profile of adenine nucleotides similar to that observed in isolated mitochondria. It decreases the ‘efficiency’ of gluconeogenesis, as expressed by the ratio between an increase of glucose production and an increase of oxygen uptake upon addition of gluconeogenic substrates (lactate plus pyruvate), and increases the reduction state of mitochondrial NAD. These effects taken together are not compatible with uncoupling, but point to intramitochondrial hydrolysis of octanoyl-CoA and probably also shorter chain-length acyl-CoAs. This mechanism probably functions as a ‘safety valve’ preventing a drastic decrease of intramitochondrial free CoA under a large supply of medium- and short-chain fatty acids.     

Fibroblast growth factor‐2/platelet‐derived growth factor enhances atherosclerotic plaque stability

Increased immature neovessels contribute to plaque growth and instability. Here, we investigated a method to establish functional and stable neovessel networks to increase plaque stability. Rabbits underwent aortic balloon injury and were divided into six groups: sham, vector and lentiviral transfection with vascular endothelial growth factor‐A (VEGF)‐A, fibroblast growth factor (FGF)‐2, platelet‐derived growth factor (PDGF)‐BB and FGF‐2 + PDGF‐BB. Lentivirus was percutaneously injected into the media‐adventitia of the abdominal aorta by intravascular ultrasound guidance, and plaque‐rupture rate, plaque‐vulnerability index and plaque neovessel density at the injection site were evaluated. Confocal microscopy, Prussian Blue assay, Evans Blue, immunofluorescence and transmission electron microscopy were used to assess neovessel function and pericyte coverage. To evaluate the effect of FGF‐2/PDGF‐BB on pericyte migration, we used the mesenchymal progenitor cell line 10T1/2 as an in vitro model. VEGF‐A‐ and FGF‐2‐overexpression increased the number of immature neovessels, which caused intraplaque haemorrhage and inflammatory cell infiltration, eventually resulting in the plaque vulnerability; however, FGF‐2/PDGF‐BB induced mature and functional neovessels, through increased neovessel pericyte coverage. Additionally, in vitro analysis of 10T1/2 cells revealed that FGF‐2/PDGF‐BB induced epsin‐2 expression and enhanced the VEGF receptor‐2 degradation, which negatively regulated pericyte function consistent with the in vivo data. These results showed that the combination of FGF‐2 and PDGF‐BB promoted the function and maturation of plaque neovessels, thereby representing a novel potential treatment strategy for vulnerable plaques.     

Silencing of a lipase maturation factor 2‐like gene by wheat‐mediated RNAi reduces the survivability and reproductive capacity of the grain aphid,Sitobion avenae

Lipase maturation factor (LMF) family proteins are required for the maturation and transport of active lipoprotein lipases. However, the specific roles of LMF2 remain unknown. In this study, a grain aphid lmf2‐like gene fragment was cloned and was highly similar in sequence to a homologous gene in the pea aphid, Acyrthosiphon pisum. An RNAi vector was constructed with this fragment and used for wheat transformation. The expression of the lmf2‐like gene in aphid, as well as the growth and reproduction of the aphids, was analyzed after feeding on the transgenic wheat. There were no significant differences in the expression of the lmf2‐like gene over development. The expression of the lmf2‐like gene was significantly reduced by 27.6% on the fifth day, and 57.6% on the 10th day after feeding. The total number of aphids produced on the transgenic plants was less than the number produced on control plants, and the difference became significant or after 2 weeks. The molting numbers were also reduced in the aphids reared on the transgenic plants. Our findings indicate that lmf2‐like genes may have potential as a target gene for the control of grain aphids and show that feeding aphids with wheat expressing lmf2‐like RNAi resulted in significant reductions in survival and reproduction.