Total Synthesis Confirms the Molecular Structure Proposed for Oxidized Levuglandin D₂

Levuglandins (LG)­D₂ and LGE₂ are γ-ketoaldehyde levulinaldehyde derivatives with prostanoid side chains produced by spontaneous rearrangement of the endoperoxide intermediate PGH₂ in the biosynthesis of prostaglandins. Covalent adduction of LGs with the amyloid peptide Aβ_1–42 promotes formation of the type of oligomers that have been associated with neurotoxicity and are a pathologic hallmark of Alzheimer’s disease. Within 1 min of their generation during the production of PGH₂ by cyclooxygenation of arachidonic acid, LGs are sequestered by covalent adduction to proteins. In view of this high proclivity for covalent adduction, it is understandable that free LGs have never been detected <i>in vivo</i>. Recently a catabolite, believed to be an oxidized derivative of LGD₂ (ox-LGD₂), a levulinic acid hydroxylactone with prostanoid side chains, was isolated from the red alga <i>Gracilaria edulis</i> and detected in mouse tissues and in the lysate of phorbol-12-myristate-13-acetate-treated THP-1 cells incubated with arachidonic acid. Such oxidative catabolism of LGD₂ is remarkable because it must be outstandingly efficient to prevail over adduction with proteins and because it requires a unique dehydrogenation. We now report a concise total synthesis that confirms the molecular structure proposed for ox-LGD₂. The synthesis also produces ox-LGE₂, which readily undergoes allylic rearrangement to Δ⁶-ox-LGE₂

Bioactive 4‑Oxoheptanedioic Monoamide Derivatives of Proteins and Ethanolaminephospholipids: Products of Docosahexaenoate Oxidation

Oxidative stress causes lipid-derived oxidative modification of biomolecules that has been implicated in many pathological states. Phospholipids containing polyunsaturated fatty acids are major targets of free radical-initiated oxidation. Phospholipids that incorporate docosahexaenoate (DHA) are highly enriched in important neural structures including the brain and retina, where DHA comprises 40% and 60% of total fatty acids, respectively. Oxidative fragmentation of 2-docosahexaenoyl-1-palmityl-<i>sn</i>-glycerophosphocholine generates esters of 4-hydroxy-7-oxohept-5-enoic acid (HOHA) and 4-keto-7-oxohept-5-enoic acid (KOHA) with 2-lysophosphatidylcholine, HOHA-PC, and KOHA-PC. Covalent HOHA adducts that incorporate the primary amino groups of proteins and ethanolamine phospholipids in carboxyethylpyrrole (CEP) derivatives were detected immunologically with anti-CEP antibodies in human tumors, retina, and blood. Now, we generated an anti-OHdiA antibody to test the hypothesis that KOHA adducts, which incorporate the primary amino groups of proteins or ethanolamine phospholipids in 4-oxo-heptanedioic (OHdiA) monoamide derivatives, are present in vivo. However, whereas the anti-CEP antibody is highly specific and does not cross-react with the OHdiA monoamide epitope, the anti-OHdiA monoamide antibody cross-reacted with CEP epitopes making it of little value as an analytical tool for OHdiA monoamides but suggesting the possibility that OHdiA monoamides would exhibit receptor-mediated biological activity similar to that of CEP. An LC-MS/MS method was developed that allows quantification of OHdiA derivatives in biological samples. We now find that KOHA-PC forms OHdiA monoamide adducts of proteins and ethanolamine phospholipids and that OHdiA-protein levels are significantly higher than OHdiA-ethanloamine phospholipid levels in blood from healthy human subjects, 0.45 μM and 0.18 μM, respectively (<i>n</i> = 3, and <i>p</i> = 0.027). OHdiA monoamide epitopes are angiogenic, causing TLR2-dependent adhesion and tube formation by human umbilical vein endothelial cells. OHdiA monoamide epitopes are only slightly less potent than CEP epitopes that contribute to the pathological angiogenesis of age-related macular degeneration and tumor growth

Low-Density Lipoprotein Has an Enormous Capacity To Bind (<i>E</i>)-4-Hydroxynon-2-enal (HNE): Detection and Characterization of Lysyl and Histidyl Adducts Containing Multiple Molecules of HNE

(<i>E</i>)-4-Hydroxynon-2-enal (HNE), an electrophilic bifunctional cytotoxic lipid peroxidation product, forms covalent adducts with nucleophilic side chains of amino acid residues. HNE-derived adducts have been implicated in many pathophysiological processes including atherosclerosis, diabetes, and Alzheimer’s disease. Tritium- and deuterium-labeled HNE (<i>d</i>₄-HNE) were used orthogonally to study adduction with proteins and individual nucleophilic groups of histidyl, lysyl, and cysteine residues. Using tritium-labeled HNE, we detected the binding of 486 molecules of HNE per low-density lipoprotein (LDL) particle, significantly more than the total number of all reactive nucleophiles in the LDL particle. This suggests the formation of adducts that incorporate multiple molecules of HNE with some nucleophilic amino acid side chains. We also found that the reaction of a 1:1 mixture of <i>d</i>₄-HNE and <i>d</i>₀-HNE with <i>N</i>-acetylhistidine, <i>N</i>-acetyl-Gly-Lys-OMe, or <i>N</i>-acetyl cysteine generates 1:1, 2:1, and 3:1 adducts, which exhibit unique mass spectral signatures that aid in structural characterization. A domino-like reaction of initial 1:1 HNE Michael adducts of histidyl or lysyl nucleophiles with multiple additional HNE molecules forms 2:1 and 3:1 adducts that were structurally characterized by tandem mass spectrometry

Molecular Structures of Isolevuglandin-Protein Cross-Links

Isolevuglandins (isoLGs) are stereo and structurally isomeric γ-ketoaldehydes produced through free radical-induced oxidation of arachidonates. Some isoLG isomers are also generated through enzymatic cyclooxygenation. Post-translational modification of proteins by isoLGs is associated with loss-of-function, cross-linking and aggregation. We now report that a low level of modification by one or two molecules of isoLG has a profound effect on the activity of a multi subunit protease, calpain-1. Modification of one or two key lysyl residues apparently suffices to abolish catalytic activity. Covalent modification of calpain-1 led to intersubunit cross-linking. Hetero- and homo-oligomers of the catalytic and regulatory subunits of calpain-1 were detected by SDS–PAGE with Western blotting. <i>N</i>-Acetyl-glycyl-lysine methyl ester and β-amyloid(11–17) peptide EVHHQKL were used as models for characterizing the cross-linking of protein lysyl residues resulting from adduction of iso[4]­LGE₂. Aminal, bispyrrole, and trispyrrole cross-links of these two peptides were identified and fully characterized by mass spectrometry. Aminal and bispyrrole dimers were both detected. Furthermore, a complex mixture of derivatives of the bispyrrole cross-link containing one or more additional atoms of oxygen was found. Interesting differences are evident in the predominant cross-link type generated in the reaction of iso[4]­LGE₂ with these peptides. More aminal cross-links versus bispyrrole are formed during the reaction of the dipeptide with iso[4]­LGE₂. In contrast, more bispyrrole versus aminal cross-links are formed during the reaction of EVHHQKL with iso[4]­LGE₂. It is tempting to speculate that the EVHHQKL peptide–pyrrole modification forms noncovalent aggregates that favor the production of covalent bispyrrole cross-links because β-amyloid(11–17) tends to spontaneously oligomerize

The Oxidative Stress Product Carboxyethylpyrrole Potentiates TLR2/TLR1 Inflammatory Signaling in Macrophages

<div><p>Oxidative stress is key in the pathogenesis of several diseases including age-related macular degeneration (AMD), atherosclerosis, diabetes, and Alzheimer's disease. It has previously been established that a lipid peroxidation product, carboxyethylpyrrole (CEP), accumulates in the retinas of AMD patients. Retinal infiltrating macrophages also accumulate in the retinas of both AMD patients and in a murine model of AMD. We therefore investigated the ability of CEP-adducts to activate innate immune signaling in murine bone-marrow derived macrophages (BMDMs). We found that CEP specifically synergizes with low-dose TLR2-agonists (but not agonists for other TLRs) to induce production of inflammatory cytokines. Moreover, CEP selectively augments TLR2/TLR1-signaling instead of TLR2/TLR6-signaling. These studies uncover a novel synergistic inflammatory relationship between an endogenously produced oxidation molecule and a pathogen-derived product, which may have implications in the AMD disease process and other oxidative stress-driven pathologies.</p></div

4‑Hydroxy-7-oxo-5-heptenoic Acid Lactone Induces Angiogenesis through Several Different Molecular Pathways

Oxidative stress and angiogenesis have been implicated not only in normal phenomena such as tissue healing and remodeling but also in many pathological processes. However, the relationships between oxidative stress and angiogenesis still remain unclear, although oxidative stress has been convincingly demonstrated to influence the progression of angiogenesis under physiological and pathological conditions. The retina is particularly susceptible to oxidative stress because of its intensive oxygenation and high abundance of polyunsaturated fatty acyls. In particular, it has high levels of docosahexanoates, whose oxidative fragmentation produces 4-hydroxy-7-oxo-5-heptenoic acid lactone (HOHA-lactone). Previously, we found that HOHA-lactone is a major precursor of 2-(ω-carboxyethyl)­pyrrole (CEP) derivatives, which are tightly linked to age-related macular degeneration (AMD). CEPs promote the pathological angiogenesis of late-stage AMD. We now report additional mechanisms by which HOHA-lactone promotes angiogenesis. Using cultured ARPE-19 cells, we observed that HOHA-lactone induces secretion of vascular endothelial growth factor (VEGF), which is correlated to increases in reactive oxygen species and decreases in intracellular glutathione (GSH). Wound healing and tube formation assays provided, for the first time, in vitro evidence that HOHA-lactone induces the release of VEGF from ARPE-19 cells, which promotes angiogenesis by human umbilical vein endothelial cells (HUVEC) in culture. Thus, HOHA-lactone can stimulate vascular growth through a VEGF-dependent pathway. In addition, results from MTT and wound healing assays as well as tube formation experiments showed that GSH-conjugated metabolites of HOHA-lactone stimulate HUVEC proliferation and promote angiogenesis in vitro. Previous studies demonstrated that HOHA-lactone, through its CEP derivatives, promotes angiogenesis in a novel Toll-like receptor 2-dependent manner that is independent of the VEGF receptor or VEGF expression. The new studies show that HOHA-lactone also participates in other angiogenic signaling pathways that include promoting the secretion of VEGF from retinal pigmented epithelial cells

CEP Biomarkers as Potential Tools for Monitoring Therapeutics

<div><p>Background</p><p>Carboxyethylpyrrole (CEP) adducts are oxidative modifications derived from docosahexaenoate-containing lipids that are elevated in ocular tissues and plasma in age-related macular degeneration (AMD) and in rodents exposed to intense light. The goal of this study was to determine whether light-induced CEP adducts and autoantibodies are modulated by pretreatment with AL-8309A under conditions that prevent photo-oxidative damage of rat retina. AL-8309A is a serotonin 5-HT_1A receptor agonist.</p> <p>Methods</p><p>Albino rats were dark adapted prior to blue light exposure. Control rats were maintained in normal cyclic light. Rats were injected subcutaneously 3x with 10 mg/kg AL-8309A (2 days, 1 day and 0 hours) before light exposure for 6 h (3.1 mW/cm², λ=450 nm). Animals were sacrificed immediately following light exposure and eyes, retinas and plasma were collected. CEP adducts and autoantibodies were quantified by Western analysis or ELISA.</p> <p>Results</p><p>ANOVA supported significant differences in mean amounts of CEP adducts and autoantibodies among the light + vehicle, light + drug and dark control groups from both retina and plasma. Light-induced CEP adducts in retina were reduced ~20% following pretreatment with AL-8309A (n = 62 rats, <i>p</i> = 0.006) and retinal CEP immunoreactivity was less intense by immunohistochemistry. Plasma levels of light-induced CEP adducts were reduced at least 30% (n = 15 rats, <i>p</i> = 0.004) by drug pretreatment. Following drug treatment, average CEP autoantibody titer in light exposed rats (n = 22) was unchanged from dark control levels, and ~20% (<i>p</i> = 0.046) lower than in vehicle-treated rats.</p> <p>Conclusions</p><p>Light-induced CEP adducts in rat retina and plasma were significantly decreased by pretreatment with AL-8309A. These results are consistent with and extend previous studies showing AL-8309A reduces light-induced retinal lesions in rats and support CEP biomarkers as possible tools for monitoring the efficacy of select therapeutics.</p> </div

CEP selectively enhances Pam3CSK4-signaling in a TLR4-independent manner.

<p>(<b>A, B</b>) BMDMs from either wild-type (WT) or <i>Tlr4−/−</i> mice were co-stimulated as indicated with Pam3CSK4 (1.0 ng/mL) and sham-BSA (10 µg/mL) or CEP-BSA (10 µg/mL) for 9 hours. <i>Tnf</i> or <i>Il12a</i> expression was assessed by qPCR (n = 3). (<b>C, D</b>) Immuno-depletion assays were performed as described in the Methods & Materials section. sham-BSA (1 µg/mL) or CEP-BSA (1 µg/mL) were immuno-depleted with either IgG1-isotype control or anti-CEP antibody. Supernatants were either combined or not with Pam3CSK4 (1.0 ng/mL) for stimulation of BMDMs. <i>Tnf</i> or <i>Il12a</i> expression was assessed by qPCR (n = 3). (<b>E, F</b>) BMDMs were stimulated with the indicated combinations of Pam3CSK4 (1 ng/mL) and either sham-adducted (10 µg/mL) or CEP-adducted (10 µg/mL) BSA, HSA, or transferrin (Tran) for 9 hours followed by qPCR expression analysis of <i>Tnf</i> and <i>Il12</i> (n = 3) *P<0.05. Results are mean +/− SEM.</p

CEP immunoreactivity in rat retina.

<p>A summary is shown of CEP adduct levels in rat retina following blue light exposure with or without pretreatment with AL-8309A (from Western analyses in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076325#pone.0076325.s002" target="_blank">Figure S2</a>). Log10 transformed CEP optical density measurements were normalized to the mean dark control per analysis, outliers were eliminated and average ratios were transformed to linear scale. Δ represents the difference in average ratios between animals groups, the p-values are from the 2-sided t-test and error bars reflect standard deviation. The number of animals assayed in each group is indicated (n).</p

Pretreatment with AL-8309A reduces light-induced CEP immunoreactivity in rat plasma.

<p>A summary is shown of average CEP adduct levels in rat plasma following <i>in </i><i>vivo</i> blue light exposure with or without pretreatment with AL-8309A (from Western analyses in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076325#pone.0076325.s004" target="_blank">Figure S4</a>). Log₁₀ transformed CEP optical density measurements were normalized to the mean dark control per analysis and average ratios transformed to linear scale. Δ represents the difference in average ratios between animals groups, the p-values are from the 2-sided t-test and error bars reflect standard deviation. The number of animals assayed in each group is indicated (n).</p