Objective
Maternal immunization with oxidized low-density lipoprotein prior to pregnancy prevents pathogenic in utero programming by gestational hypercholesterolemia, but it is unknown whether gestational hypercholesterolemia and maternal immunization affect similar pathways.
Study Design
A lipidomic approach was used for unbiased plasma eicosanoid profiling in adult offspring of immunized and nonimmunized normocholesterolemic or hypercholesterolemic rabbit mothers.
Results
Gestational hypercholesterolemia was associated with increased levels of some eicosanoids formed by the cyclooxygenase and 12-lipoxygenase pathways only (including thromboxane B 2 , prostaglandin [PG] F 2α , PGE 2 , and PGD 2 ). Immunization of hypercholesterolemic or normocholesterolemic mothers reduced 9 of 14 eicosanoids of the cyclooxygenase pathway, 21 of 23 eicosanoids of the 5- and 12-lipoxygenase pathways (eg, 5-hydroxyeicosatetraenoic acid, hepoxilin B 3 , 12-hydroxyeicosatetraenoic acid), 8 of 19 eicosanoids of the cytochrome P-450 pathway, and all metabolites of the nonenzymatic pathway.
Conclusion
Maternal immunization not only counteracts in utero programming by gestational hypercholesterolemia but reduces a broad range of eicosanoid modulators of immunity and inflammation in offspring.
It is now well established that in utero conditions influence the susceptibility to cardiovascular disease later in life, but the mechanisms involved are largely unknown. Maternal hypercholesterolemia, even if limited to pregnancy, is associated with enhanced formation of fatty streaks in arteries of premature human fetuses and increased atherogenesis in normocholesterolemic children. Diet-induced gestational hypercholesterolemia causes similar atherogenic programming in genetically more homogeneous animal models. Increased atherogenesis in offspring of human beings, rabbits, and mice differs between arterial sites, and may be either spontaneous or require the presence of additional postnatal risk factors to manifest itself. In models resistant to atherosclerosis, eg, rats, maternal high-fat, high-cholesterol diets program reduced vascular reactivity and increased insulin resistance in offspring.
Oxidative stress plays an important role in atherogenic in utero programming. Maternal hypercholesterolemia is associated with increased lipid peroxidation, formation of reactive oxygen species and inflammation, and affects oxidative stress in both the placenta and fetus. Conversely, cholesterol-lowering or antioxidant treatment during pregnancy reduces atherogenic programming, even though antioxidants do not affect maternal cholesterol levels. Protection against oxidative stress may also contribute to the marked reduction of atherosclerosis observed in offspring of rabbits and mice whose mothers were immunized with oxidized low-density lipoprotein (OxLDL) prior to pregnancy. Such immunization gives rise to high-titered antibodies to oxidation-specific epitopes, which form immune complexes with low-density lipoprotein (LDL) particles carrying such epitopes on their apolipoproteins or oxidized phospholipids, and thus eliminate them from the circulation.
Although maternal-fetal cholesterol transport mechanisms have been elucidated. almost nothing is known about the fetal cells that are programmed by hypercholesterolemia or other gestational dysmetabolic conditions, nor about the mechanisms by which developmental programming actually modulates offspring diseases. Proposed mechanisms include epigenetic programming, altered messenger RNA expression or activity of antioxidant enzymes, altered cell differentiation and proliferation induced by the in utero environment alone or in conjunction with inherited genetic traits, altered mitochondrial function, growth restrictions due to protein restrictions, and in utero immune programming. Indeed, maternal OxLDL immunization prior to pregnancy not only reduces atherogenesis in offspring, but also programs B-cell-dependent immune responses in offspring. These include increased splenic presence of specific B-cell populations, increased formation of oxidation-specific IgM antibodies and IgM-LDL complexes, and increased specific IgG and IgM responses in naïve offspring subjected to OxLDL challenge. Although increased titers of such antibodies may have contributed, the substantial reduction of atherosclerosis in offspring of immunized mothers indicates the involvement of other antiatherogenic mechanisms, eg, attenuation of inflammation. Identifying the mechanisms through which gestational hypercholesterolemia and maternal immunization may affect postnatal disease is rendered difficult by the fact that neither the nature of the in utero programming nor the cells involved are known. Systemic factors programmed in utero, such as those modulating immunity and inflammation, are of particular interest, because they may influence not just atherogenesis but also insulin resistance and diabetes.
To determine whether in utero programming affects systemic levels of bioactive lipids in adult offspring, to identify candidate compounds for future investigations of causality, and to establish whether programming by gestational hypercholesterolemia and maternal immunization affects similar cells and pathways, we analyzed plasma levels of eicosanoids in adult rabbit offspring, using a lipidomic approach.
Eicosanoids are formed from polyunsaturated fatty acids by enzymatic or nonenzymatic oxygenation. Strictly defined, eicosanoids are derived from 20-carbon fatty acids, but the term is now more broadly used to include related metabolites from virtually all long-chained polyunsaturated fatty acids. Eicosanoids are produced by most cells, typically in response to stimulation, and regulate a wide range of physiological processes through complex interrelated signaling networks. Three major enzymatic pathways lead to eicosanoid production, ie, the cyclooxygenase (COX), lipoxygenase (LOX), and epoxygenase or cytochrome P-450 (CYP) pathways. Arachidonic acid (AA) is a major precursor of eicosanoids, and the 3 pathways are therefore, collectively known as the arachidonate cascade. In addition, autoxidation of polyunsaturated fatty acids through nonenzymatic pathways also generates bioactive lipids commonly used as biomarkers of oxidative stress. Prostanoids, including prostaglandins (PGs) and thromboxanes (TXs), are derived from the COX pathway (the target of nonsteroidal antiinflammatory drugs). Leukotrienes (LTs), lipoxins, and hepoxilins (HXs) are produced by individual or sequential actions of the various LOXs, and hydroxylated and epoxygenated metabolites of AA are formed by the enzymes of the CYP pathway.
We previously developed a liquid chromatography (LC)/mass spectrometry (MS) method for the quantitative analysis of the entire spectrum of eicosanoids. This method is now used to assess the in utero programming effects of gestational hypercholesterolemia and maternal immunization on these modulators of immunity and inflammation, vasoconstriction and platelet aggregation. Plasma samples were from a previous large experiment, which had established that maternal hypercholesterolemia enhances and maternal immunization reduces atherosclerosis in rabbits.
Materials and Methods
Ethics statement
All animal work was carried out under a protocol conforming with all applicable guidelines and approved by the Institutional Animal Care and Use Committee of the University of California San Diego (protocol #S00041).
Experimental design
Plasma eicosanoids were determined in 4 representative subgroups of New Zealand White rabbits (n = 8–10 each, both male and female) from a large study on developmental immune programming. Groups differed only with regard to maternal treatment: (1) offspring of nonimmunized normocholesterolemic mothers (NC); (2) offspring of nonimmunized mothers with diet-induced hypercholesterolemia before and during gestation (HC); (3) offspring of OxLDL-immunized NC (immNC); and (4) offspring of OxLDL-immunized HC (immHC).
Detailed descriptions of diets, antigen preparation, and immunizations, as well as the assays used to assess the immunological effects of immunization in mothers and offspring and to measure aortic atherosclerosis in offspring are provided in Yamashita et al. In brief, mothers fed regular chow were immunized with an equal mixture of homologous malondialdehyde-modified LDL and copper-OxLDL containing a broad spectrum of oxidation-specific epitopes, including oxidized phospholipids, The primary immunization consisted of subcutaneous injections of 200 μg OxLDL per kg body weight in complete Freund’s adjuvant and was followed by 2-3 biweekly intramuscular boosts with the same amount of antigen in incomplete Freund’s adjuvant. Once a marked increase in antibody titer had been confirmed after the last boost, mothers of the HC groups were switched to a diet containing 9% fat, 17% protein, 57% carbohydrate, and 16% fibers, supplemented with 6.5% corn oil (Harlan Teklad, Madison, WI). Initially, 0.15% cholesterol was added to the diet, dissolved in ether. Total plasma cholesterol (TC) was determined after 2 weeks and the cholesterol concentration added to the diet was individually adjusted, if needed to maintain the animal in the target TC range (300–400 mg/dL). Females were then mated and TC during pregnancy was determined after 2.5 weeks. After delivery, all mothers were switched to the same atherogenic diet later fed to offspring (Harlan Teklad diet 7009 supplemented with 1.5% corn oil) containing 4% total fat, 17% protein, 57% carbohydrate, and 16% fibers.
Offspring of all maternal groups were weaned at 4 weeks and then fed the above diet (with individually adjusted cholesterol, if needed to maintain a target TC of 350 mg/dL) for 5 months. Plasma samples used in the present study were obtained under a rigorous operating procedure. Blood was smoothly drawn into EDTA-containing sterile syringes from a rabbit ear vein, centrifuged for 10 minutes at 4°C, aliquoted, frozen, and stored at −80°C until use. Atherosclerosis in the entire aorta was determined at age 6 months. Offspring groups contained roughly equal numbers of males and females, which were analyzed together, because previous studies did not indicate sex differences in atherosclerosis in rabbits. The maternal and offspring cholesterol and atherosclerosis data of the subgroups used for the present study are shown in Figure 1 .
Eicosanoid extraction
All solvents were of chromatography purity. Eicosanoids used for primary standards in standard curves as well as their deuterated analogs were from Cayman Chemicals (Ann Arbor, MI) and Biomol (Enzo Life Science, Framingdale, NY). The potential formation of eicosanoids during sample collection and processing and the effect of freezing/thawing were tested with a mix of COX and LOX inhibitors. For this purpose, blood was collected from several animals and drawn directly into a mixture consisting of EDTA, the COX inhibitor ketorolac (10 μmol/L final), and the nonspecific LOX inhibitor nordihydroguaiaretic acid (10 μmol/L final). In parallel, blood was collected into EDTA without these inhibitors, and both plasma preparations were subjected to 3 cycles of freezing and thawing. Eicosanoid levels were then examined as outlined below. No significant differences in eicosanoid levels were detected between plasma prepared with or without inhibitors. To test the effect of long-term storage on eicosanoid profiles, identical plasma samples stored at −80°C were analyzed 6 months apart. We did not detect any significant changes in eicosanoid levels attributable to storage. None of the samples showed evidence of hemolysis, which may lead to platelet activation and increase COX eicosanoids. For extraction, plasma was thawed on ice and 0.9 mL were centrifuged at 6000 g for 1 minute to remove any cellular constituents. To the cleared plasma, 100 μL of a cocktail of internal standards consisting of 26 deuterated eicosanoids was added, each at 10 ng dissolved in ethanol. Samples were then purified by solid phase extraction on Strata-X 33μm polymeric reversed phase columns (Phenomenex, Torrance, CA). Columns were activated with methanol, washed with water, and the samples were applied. After washes with 10% methanol, samples were eluted with 1 mL of 100% methanol. The eluent was dried under vacuum, redissolved in 100 μL of buffer A consisting of water:acetonitrile:formic acid = 63:37:0.02 (vol/vol/vol) and immediately used for LC/MS analysis.
Reverse-phase LC and MS
Eicosanoids were separated by reverse-phase LC on a Synergy C18 column (2.1 × 250 mm) (Phenomenex) at a flow rate of 300 μL/min at 50°C. The column was equilibrated in buffer A and 40 μL of sample was injected via a Pal auto-sampler (Leap Technologies, Naperville, IL) set to maintain samples at 4°C to minimize degradation of eicosanoids during queuing for analysis. Samples were eluted with a step gradient to 100% buffer B (acetonitrile:isopropanol = 50:50 (vol/vol) over a period of 20 minutes. The eluted eicosanoids were analyzed using an online tandem quadruple mass spectrometer (ABI 4000 Q-Trap; Applied Biosystems) operated in negative ion mode via multiple reaction monitoring (MRM) with the electrospray voltage set at −4.5 kV, and the turbo ion spray source at 525°C. Collisional activation of eicosanoid precursor ions used nitrogen as a collision gas. Eicosanoids were measured using precursor/product (MRM) pairs, and the MS analysis was divided into periods consisting of 90 seconds each to maximize the duty cycle. The declustering potential and collision energy for each eicosanoid was optimized for maximal signal strength. Eicosanoids were identified by matching their MRM signal and LC retention time with those of pure standards. A complete list of standards, instrument settings, and MRM pairs can be found on www.lipidmaps.org under “resources.” (accessed April 20, 2011).
Quantitation of eicosanoids
Eicosanoids were quantitated by the stable isotope dilution method. Identical amounts of deuterated internal standards were added to each sample and to all the primary standards used to generate standard curves. The use of internal standards neutralizes differences in extraction efficiencies and MS ion suppression. To calculate the amount of eicosanoids in a sample, ratios of peak areas between endogenous eicosanoids and matching deuterated internal eicosanoids were calculated. Ratios were converted to absolute amounts by linear regression analysis of standard curves generated under identical conditions. A total of 140 primary standards and 26 deuterated internal standards were applied to generate standard curves for quantitation. Extraction controls were performed to offset small amounts of unlabeled eicosanoid contaminations in the deuterated internal standards. Final values were normalized to plasma volume and expressed as pmol/mL of plasma. Currently, we can quantitate over 140 eicosanoids at fmol levels.
Statistics
Data were analyzed with SSPS version 16.0 (SPSS Inc, Chicago, IL). Results are expressed as the mean ± SEM. Normal-distributed data were compared by unpaired t test. P values < .05 were considered significant.
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